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Expert forum
 Current situation, problems and directions of high-risk pregnancy research in China LIN Yi 2022, 42 (4):  403-408.  doi: 10.3969/j.issn.1674-8115.2022.04.001 Abstract ( 133 )   HTML ( 145 )   PDF (1151KB) ( 71 )   With the progressive initiation of the two- and three-child policy in China, and under the influence of various factors in society, the proportion of high-risk pregnancy has increased, especially those linked to advanced age conception (more than 35 years old). In pathological pregnancy, the incidence of recurrent miscarriage and preterm birth remains high. The diagnosis and treatment of common pregnancy complications such as preeclampsia and eclampsia are lagging behind. Diseases that cause maternal death have changed. The cases of deep vein thrombosis causing maternal deaths have increased. Expanding floating population increases the risk of adverse pregnancy outcomes. The widespread use of assisted reproduction has led to high risk of adverse pregnancy outcomes. The increase in the number of elder mothers has led to high risk in fetal birth defects. All of these have led to a new challenge in terms of maternal safety and care, urging scientists and clinicians to establish a more accurate risk assessment standard for pregnant women, especially for the elderly pregnant women, implement whole pregnancy management and standardized diagnosis and treatment, standardize the treatment for abortion, and establish a triple monitoring system for identifying maternal arterial rhythm, maternal heart rate and fetal heart rate. Finally, a hospital-home monitoring and warning system will be set up based on the Internet of things and information automation. This paper describes the current status, problems and directions of high-risk pregnancy research under the background of the new population policy, and provides new strategies for the protection of maternal and child health.
High-risk pregnancy column
 Analysis of risk factors of hysterectomy caused by intractable postpartum hemorrhage HU Jianing, ZHANG Jinwen, LIU Xiaorui, CHEN Cailian, LIN Yi 2022, 42 (4):  409-414.  doi: 10.3969/j.issn.1674-8115.2022.04.002 Abstract ( 95 )   HTML ( 145 )   PDF (1004KB) ( 57 )   Objective·To investigate the risk factors and related preventive measures of intractable postpartum hemorrhage which leads to emergency peripartum hysterectomy.Methods·The clinical data of 110 934 pregnant women treated in the International Peace Maternal & Child Health Hospital, Shanghai Jiao Tong University School of Medicine from January 2014 to December 2020 were analyzed retrospectively. According to the treated time, pregnant women in the 2015?2017 year-period (n=48 984) and 2018?2020 year-period (n=45 262) were selected as the research objects to analyze the incidence of emergency peripartum hysterectomy. Besides, a total of 108 pregnant women with intractable postpartum hemorrhage were selected as the research objects. The patients were divided into the uterine-removed group (n=22) and the uterine-preserved group (n=86) according to whether they had hysterectomy. The clinical data of the two groups were compared and analyzed by univariate analysis and multivariate Logistic regression model, and the risk factors of emergency peripartum hysterectomy were explored.Results·Compared with the 2015?2017 year-period, the incidence of emergency peripartum hysterectomy decreased in the 2018?2020 year-period (P=0.039). Meanwhile, compared with the uterine-preserved group, the pregnant women in the uterine-removed group were older, with more than 2 pregnancies, monocyesis, pregnancy complications (placenta previa, placenta implantation, scarred uterus, etc.), amniotic fluid embolism and greater postpartum hemorrhage (all P<0.05). Multivariate Logistic regression analysis showed that the patients' age and the volume of postpartum hemorrhage were independent risk factors for emergency peripartum hysterectomy caused by intractable postpartum hemorrhage (P<0.05).Conclusion·The older the patients with intractable postpartum hemorrhage, the greater volume of postpartum hemorrhage, which is easy to lead to emergency peripartum hysterectomy. Clinical monitoring and intervention should be carried out as soon as possible to reduce the patients' incidence of emergency peripartum hysterectomy.
 Analysis of risk factors for postpartum thrombotic disease ZHU Yueyue, ZHANG Jinwen, MA Ruixiang, CHEN Cailian, LIN Yi, LIU Xiaorui 2022, 42 (4):  415-421.  doi: 10.3969/j.issn.1674-8115.2022.04.003 Abstract ( 115 )   HTML ( 151 )   PDF (1196KB) ( 51 )   Objective·To explore the high-risk factors and provide guidance for the prevention of postnatal thromboembolism.Methods·Based on a retrospective case-control design, a total of 45 262 electronic cases of women giving birth in the International Peace Maternal & Child Health Hospital, Shanghai Jiao Tong University School of Medicine from 2018 to 2020 were excavated. The case group was 60 parturients diagnosed with postpartum venous thrombosis or pulmonary embolism, while the control group was 45 202 parturients who were normal. Multivariate logistic regression analysis was used to find the risk factors of postpartum thrombosis.Results·The incidence of postpartum venous thrombosis was 130/100 000 and pulmonary embolism was 46/100 000. The incidence of postpartum thrombotic disease showed a trend of increasing year by year (P<0.05). The left lower extremity was the main site of postpartum thrombosis. In this study, the average age of the mothers at delivery was 33.52±4.79 years old in the case group and 31.35±4.01 years old in the control group. The proportion of preterm birth in the case group was 2.05 times that of the control group, and the proportion of pre-pregnancy overweight and obese was 1.94 times that of the control group. Multivariate Logistic regression analysis showed that the elder age (aOR=1.10, 95%CI 1.04?1.17), the smaller the gestational age (aOR=0.88, 95%CI 0.78?0.99), less than a bachelor's degree (aOR=2.24, 95%CI 1.20?4.18), elective caesarean (aOR=6.68, 95% CI 2.56?17.41), emergency caesarean (aOR=14.40, 95%CI 5.37?38.63) and pre-pregnancy overweight and obesity (aOR=1.91, 95%CI 1.04?3.49) all increased the risk of postpartum thrombosis.Conclusion·Maternal age, education level, gestational age, overweight and cesarean section were independent risk factors for postpartum thrombosis. The most prominent risk factor was emergency cesarean section. The occurrence of postpartum venous thrombosis was associated with a variety of risk factors and can lead to maternal death. Clinicians need to assess the risk of thrombosis in time, and take preventive measures as soon as possible for women with high risk factors to reduce the risk of postpartum thrombosis.
 Neuropsychiatric effects of gestational diabetes mellitus in adult offspring in C57BL/6J mice WU Xiafei, FANG Jie, QI Hongbo, YU Xinyang 2022, 42 (4):  422-432.  doi: 10.3969/j.issn.1674-8115.2022.04.004 Abstract ( 119 )   HTML ( 2 )   PDF (5175KB) ( 33 )   Objective·To investigate the influence of gestational diabetes mellitus (GDM) on the neuropsychiatric function of C57BL/6J adult offspring mice.Methods·C57BL/6J mice aged 8 weeks were randomly divided into the GDM group and the control group. GDM model was induced by a high-fat diet (HFD). Fasting glucose, oral glucose tolerance test (OGTT), and insulin tolerance test (ITT) of the pregnant mice were tested to verify whether the model was successfully induced. The offspring were fed with a standard diet for 18 weeks, and then underwent open field test (OFT), elevated plus maze (EPMT), elevated zero maze test (EZMT), forced swimming test (FST), tail suspension test (TST), and sucrose preference test (SPT) to test the emotional behaviors of the offspring mice. The hippocampus was collected after the behavioral experiments for histological verification. Hematoxylin-eosin (H-E) staining and silver staining were used to determine the structure and morphology of the offspring hippocampus of the GDM group, and immunofluorescence staining was used to detect the number of neuron marker and astrocyte marker positive cells. Real time quantitative PCR (RT-qPCR) was used to detect the expression of dopamine (DA), 5-hydroxytryptamine (5-HT), and brain-derived neural factor (BDNF), cAMP response element-binding protein (CREB) relative genes (Drd1, Htr2a, Bdnf and Creb1) in the hippocampus. One-way ANOVA was used for comparison among multiple groups, and independent-sample t-test was used to analyze differences between two groups.Results·The GDM model induced by HFD in C57BL/6J mice showed that blood glucose levels and AUC were significantly increased at each time point of OGTT, and insulin tolerance was decreased, confirming that the GDM model was successfully established. The results of OFT, EPMT and EZMT in the progeny showed no statistical difference between the GDM group and the control group. The FST, TST and SPT displayed that the immobility time was observably increased, and the percentage of sucrose preference was markedly reduced in the GDM group, and the difference in F1 female mice was more significant (P=0.000). The expression of Drd1, Htr2a and Bdnf was reduced in the GDM group compared with the control group. Analysis of H-E and silver staining results showed that there was no difference in the structure of the hippocampus between the two groups, but the number of neurons and astrocytes decreased in the GDM group.Conclusion·GDM is associated with neuropsychiatric disorders in adult offspring of C57BL/6J mice, which mainly manifest as depression tendency instead of anxiety tendency.
Innovative research team achievement column
 Application of CRISPR/Cas9-mediated gene editing system to studying the regulation of T-bet in B cells HAN Xiaxia, GU Shuangshuang, DAI Dai, SHEN Nan 2022, 42 (4):  433-442.  doi: 10.3969/j.issn.1674-8115.2022.04.005 Abstract ( 118 )   HTML ( 3 )   PDF (4499KB) ( 43 )   Objective·To study the regulation of transcription factor T-bet in antibody isotype switching via the optimized CRISPR/Cas9-mediated gene editing system in primary B cells.Methods·In order to demonstrate the regulation of T-bet in antibody isotype switching, the transcriptome data of in vitro-derived B cells from public database GEO was analyzed. The retroviral small guide RNA (sgRNA) vector was constructed to deliver sgRNA targeting Tbx21 (T-box 21, encoding T-bet), and retroviral packaging cell line Plat-E was used for retrovirus production. The retroviruses with sg-Tbx21 plasmids were harvested and concentrated. Primary B cells isolated from Cas9 transgenic mice were activated in vitro by synergistic stimulation of anti-B cell receptor antibody, anti-CD40 antibody and a Toll-like receptor agonist, and then infected with concentrated retroviruses with sg-Tbx21. The genome DNA was extracted from the infected cells isolated by flow cytometry, and then PCR amplification was performed for target gene sequence. ICE CRISPR analysis tool was used to calculate the gene knockout efficiency. The edited B cells were induced to plasma cells in vitro and the antibody isotypes of IgM, IgG2c, and IgG3 production in the culture supernatant were assessed by ELISA. Independent sample t-test was used to analyze the difference of antibody levels in the supernatant between the sg-NC editing cells and sg-Tbx21 editing cells.Results·① According to the analysis of GEO database, interferon-γ induced the expression of Tbx21 in B cells, and T-bet expression promoted antibody isotype switching-related pathways. ② The retroviral infection efficiency was improved in primary B cells after in vitro activation, which were infected by the concentrated high titer viruses. ③ The sg-Tbx21 plasmids were delivered to Cas9 transgenic B cells by retroviruses, which led to T-bet knockout efficiency of about 59%. ④ Cultured in the plasma cells-polarizing condition, the Tbx21 edited B cells secreted less IgG2c into the culture supernatant (P=0.000), while the levels of IgM and IgG3 did not significantly change.Conclusion·The efficiency of sgRNA delivery can be improved by concentrating retroviruses and activating primary B cells through synergistic stimulation, and Tbx21 gene can be knocked out in primary B cells of Cas9 transgenic mice; it is proved that T-bet knockout hinders the production of antibody isotype IgG2c through this system.
Basic research
 Negative-stain electron microscopic study of the nucleosome remodeling and deacetylase complex DUAN Yujuan, HUANG Jing 2022, 42 (4):  455-463.  doi: 10.3969/j.issn.1674-8115.2022.04.007 Abstract ( 85 )   HTML ( 5 )   PDF (3787KB) ( 30 )   Objective·To study the structure of human nucleosome remodeling and deacetylase complex (NuRD complex) with negative-stain electron microscopy to obtain the profile information of human NuRD complex.Methods·Full length MBD3 (methyl-CpG binding domain protein 3) and GATAD2A (GATA zinc finger domain containing 2A) constructs were cloned into the pMLink vectors with an amino terminal 10×His tag and a carboxyl terminal 3×Flag affinity tag, respectively. Proteins were expressed in human Expi293F cells grown in suspension cultures by using polyethylenimine as the transfection reagent, and were isolated via affinity purifications with Ni-NTA and anti-DYKDDDDK (Flag) G1 affinity resin sequentially. The complex was further purified by Superose 6 Increase 5/150 gel filtration chromatography to improve the homogeneity, identified by Western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and was then studied by negative-stain electron microscopy and single particle analysis. The existing atomic structural model of the subcomplex (7AO9, 5FXY) in Protein Data Bank (PDB) was automatically docked into the generated structural model by the UCSF Chimera software to predict the localization of multiple protein components in the structural model.Results·The Flag-tagged MBD3 and His-tagged GATAD2A proteins could effectively pull down the other endogenous components of the NuRD complex through two-step affinity purifications. The high-purity complex was obtained by gel filtration chromatography and confirmed as the NuRD complex by LC-MS/MS and Western blotting identification. Preliminary study on the three-dimensional (3D) structure of the NuRD complex was carried out with negative-stain electron microscopy and single particle analysis, which revealed that the NuRD complex was in the obvious shape of a long asymmetrical rod. The 3D structural model of the human NuRD complex was finally obtained by further 3D refinement at an overall resolution of 17 ? (1 ?=0.1 nm). The existing atomic structural model (PDB: 7AO9, 5FXY) was automatically docked into the negative staining structural model, and the localizations of MTA1/2/3 (metastasis-associated protein 1/2/3), HDAC1/2 (histone deacetylase 1/2), RBBP4/7 (retinoblastoma-binding protein 4/7) and MBD2/3 (methyl-CpG-binding domain protein 2/3) subunits in the NuRD complex were preliminarily confirmed.Conclusion·A low-resolution negative-staining structural model of human NuRD complex is obtained by single particle analysis.
 Involvement of miRNA target genes in esophageal squamous cell carcinoma through ubiquitination based on GEO database JIN Bu, YUAN Ying, CHEN Wanyu, XU Hudong, HUANG Xiaolei, HE Jialu, YU Hong 2022, 42 (4):  464-471.  doi: 10.3969/j.issn.1674-8115.2022.04.008 Abstract ( 118 )   HTML ( 6 )   PDF (3774KB) ( 57 )   objective·To screen new potential pathogenic genes in the course of esophageal squamous cell carcinoma.Methods·The data of miRNA chips GSE122497 and GSE164174 were obtained by GEO database. The differential mirnas of esophageal cancer patients and healthy people were screened by GEO2R, and the intersection was obtained by Venn Diagram Webtool. Target Scan tool and DIANATOOLS tool were used to predict the target genes of differential miRNA, and the intersection of predicted target genes of the two tools was used for gene function analysis. The protein interactions of target genes were analyzed by using STRING tool and Hub genes were selected.Results·① A total of 108 differential miRNAs were detected in the chip. ② After taking the intersection of Target Scan tool and DIANATOOLS tool, there were 1 354 different target genes. ③Gene Ontology (GO) analysis showed that gene functions of differential target genes were significantly enriched in biological processes such as DNA template transcriptional regulation and RNA polymerase Ⅱ promoter transcriptional regulation, as well as RNA polymerase Ⅱ core promoter proximal sequence specific DNA binding, protein binding, metal ion binding and other molecular functions. It mainly existed in nucleus, cytoplasm, cytoplasm and other cellular components. In descending order of the number of enriched genes, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the above genes were significantly enriched in tumor pathway, MAPK signaling pathway, stem cell pluripotency regulation signaling pathway, etc. ④ There were 1 326 nodes and 2 300 edges in the protein interaction network, and the average node connectivity was 3.47. The selected Hub genes were SMAD specific E3 ubiquitin protein ligase 2 (SMURF2), β-transducin repeat containing E3 ubiquitin protein ligase (BTRC), SMAD specific E3 ubiquitin protein ligase 1 (SMURF 1), ubiquitin conjugating enzyme E2 D1 (UBE2D1), and cullin 2 (CUL2). Previous studies have shown that the above genes are related to ubiquitination.Conclusion·SMURF2, BTRC, SMURF1, UBE2D1 and CUL2 genes screened from GEO database may be involved in the disease process of esophageal squamous cell carcinoma through the ubiquitin of targeted regulatory proteins.
 Role of circulating and infiltrating B cells in immune microenvironment of colorectal cancer by multi-omics data profiling GONG Qiyu, CHEN Lei 2022, 42 (4):  472-481.  doi: 10.3969/j.issn.1674-8115.2022.04.009 Abstract ( 122 )   HTML ( 3 )   PDF (4884KB) ( 54 )   Objective·To explore the role of circulating and infiltrating B cell subsets in the immune microenvironment of colorectal cancer and discover the potential molecular-driving mechanisms behind the above transformation. Then to analyze the interaction patterns between B cell subsets and other immune cells in the microenvironment, including the function analysis of communicating molecules. All these steps assist to find possible regulatory ways to augment the anti-tumor effect of B cells.Methods·Paired tumor, normal and peripheral blood tissues were collected from three patients with colorectal cancer. A total of 9 samples were applied to prepare the single-cell suspension. Then all immune cells were sorted by flow cytometry. 10X Genomics platform was applied for single-cell transcriptome and B cell receptor repertoire (BCR) sequencing. Bioinformatic tools such as Seurat etc. were taken to analyze scRNA and BCR data. The downstream analysis includes differential analysis of B cell in tumor versus normal tissues, cell interaction analysis in the immune microenvironment and regulatory networks of B cell subsets.Results·CD20+B cells were significantly increased in tumor versus normal tissues, especially germinal center B cells (GCB), which showed positively clonal amplification and high mutation level. Besides, GCB showed the tendency of transformation into memory B cells. On the contrary, plasma cells significantly reduced their activity in tumor, which was dominated by the IgA class. In addition, comparing with the immune microenvironment in normal tissues, GCB in tumor tissues are more closely connected with T cells and other immune cells, and the communication molecule pairs that positively regulate immune function were significantly enriched.Conclusion·The importance of GCB in the immune microenvironment of colorectal cancer is greatly increased. And they exert a positive anti-tumor effect through the up-regulated heavy-chain mutation level and their close association with surrounding immune cells.
Clinical research
 Analysis of lipoprotein subclasses of family with familial hypertriglyceridemia based on VAP method HE Zhijie, HE Jinchun, ZHANG Yanpei, WANG Yaodong, WANG Zhanke 2022, 42 (4):  482-489.  doi: 10.3969/j.issn.1674-8115.2022.04.010 Abstract ( 83 )   HTML ( 3 )   PDF (1455KB) ( 24 )   Objective·To analyze the characteristics of lipoprotein subclasses of family members with familial hypertriglyceridemia (FHTG).Methods·A FHTG family with 13 members meeting the diagnostic criteria of the FHTG family was collected at the First Hospital of Lanzhou University. They were divided into the high triacylglycerol (TAG) group (7 persons) and the control group (6 persons) according to whether the level of TAG of the family members increased (≥2.26 mmol/L). Through the face-to-face investigation, age, gender, height, weight, body mass index (BMI), waist circumference, living habits and medical history and other general information of each member were collected. Based on the survey results, a family tree was drawn. After 8?12 h in abrosia, peripheral venous blood samples of the family members were collected for biochemical testing. The vertical auto profile (VAP) was used to determine the serum lipoprotein subclasses of the study subjects, including TAG, total cholesterol, low-density lipoprotein cholesterol (LDL-C), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), very low-density lipoprotein 3 (VLDL3), high-density lipoprotein cholesterol (HDL-C), high-density lipoprotein 2 (HDL2), high-density lipoprotein 3 (HDL3), intermediate-density lipoprotein, remnant-like lipoprotein particle, lipoprotein a and non-high-density lipoprotein. The LDL pattern was obtained by VAP method based on the analysis of LDL max time value. The vertical lipoprotein profile (VLP) was used to determine the serum low-density lipoprotein particle (LDL-P) of the study subjects. The levels of lipoprotein subclasses between the control group and the high TAG group were compared by independent sample t test.Results·Compared with the control group, the high TAG group in FHTG pedigree had remarkably higher levels of height, weight, BMI and waist circumference (P=0.022, P=0.000, P=0.001, P=0.000). There was statistical significance in gender difference between the two groups (P=0.029), but there was no significant difference in age (P=0.561). Compared with the control group, the high TAG group had remarkably higher levels of TAG, VLDL, VLDL3 and LDL-P (P=0.003, P=0.033, P=0.020, P=0.043), and remarkably lower level of HDL-C, HDL2 and HDL3 (P=0.000, P=0.001, P=0.001). The LDL pattern in the high TAG group was small and dense, while the LDL pattern in the normal control group was mixed or large and sparse. There were no significant differences between other indicators.Conclusions·In addition to the TAG and HDL-C commonly used in clinical practice, VLDL, VLDL3, LDL-P, small and dense LDL, HDL2 and HDL3 are expected to become the indicators for FHTG diagnosis and treatment.
 Correlation between apnea hypopnea index and arteriosclerosis in patients with obstructive sleep apnea hypopnea syndrome complicated with hypertension TANG Biwen, BAI Yaya, HU Yueliang, CHAO Huijuan, WANG Qian, ZUO Junli 2022, 42 (4):  490-495.  doi: 10.3969/j.issn.1674-8115.2022.04.011 Abstract ( 79 )   HTML ( 5 )   PDF (974KB) ( 37 )   Objective·To explore the effect of apnea hypopnea index (AHI) on arterial stiffness in patients of obstructive sleep apnea hypopnea syndrome (OSAHS) complicated with hypertension.Methods·The patients admitted to Ruijin Hospital,Shanghai Jiao Tong University School of Medicine, from July 2018 to December 2020 for polysomnography monitoring were enrolled, and divided into mild to moderate OSAHS group and moderate to severe OSAHS group according to AHI (5 times/h < AHI < 15 times/h or AHI≥15 times/h). Clinical and biochemical parameters were collected. 24-hour ambulate blood pressure and carotid-femoral pulse wave velocity (c-fPWV) were measured. Logistic regression analysis was performed between AHI and cardiovascular risk factors including age, body mass index (BMI), nocturnal systolic blood pressure, nocturnal diastolic blood pressure, low-density lipoprotein, fasting blood glucose, estimated glomerular filtration rate (eGFR), using of statin and hypoglycemic drugs.Results·A total of 365 patients were enrolled with an average age of (49.1±12.8) years. Three hundred and twenty-six (89.3%) were male and average BMI was (28.1±3.8) kg/m2. BMI, c-fPWV, fasting blood glucose and glycosylated hemoglobin (HbA1c) in moderate to severe OSAHS group (n=257) were significantly higher than those in mild to moderate OSAHS group (n=108)(P<0.05), and there was a significant positive correlation between AHI and c-fPWV (R2=0.047, P<0.001). In patients of OSAHS complicated with hypertension (n=291), c-fPWV, fasting blood glucose and HbA1c in moderate to severe OSAHS group (n=216) were significantly higher than those in mild to moderate OSAHS group (n=75) (P<0.05). After adjusting for age, BMI, AHI, nocturnal systolic blood pressure, nocturnal diastolic blood pressure, low density lipoprotein, fasting blood glucose, eGFR, medication of statin and hypoglycemic drugs, the Logistic regression model showed that AHI (OR=1.032, 95% CI 1.009?1.055, P=0.006), age (OR=1.078, 95%CI 1.021?1.138, P=0.007), and nocturnal systolic blood pressure (OR=1.058, 95% CI 1.010?1.109, P=0.017) were independent risk factors for c-fPWV elevation.Conclusion·Elevated AHI increases the risk of arterial stiffness in the patients of OSAHS complicated with hypertension.
 Diagnostic value of PSA, TAP and MACC1 expression in blood of patients with prostate cancer WANG Hui, ZHAO Ying, WEN Lirong, CAO Jun, YANG Jiping, YUAN Yongming 2022, 42 (4):  496-501.  doi: 10.3969/j.issn.1674-8115.2022.04.012 Abstract ( 114 )   HTML ( 3 )   PDF (1080KB) ( 33 )   Objective·To investigate the expression and diagnostic value of prostate specific antigen (PSA), tumor abnormal protein (TAP) and colon cancer metastasis related gene 1 (MACC1) in the blood of patients with prostate cancer.Methods·One hundred and seven patients with prostate cancer (case group) admitted to Dahua Hospital, Xuhui District, Shanghai, from January 2019 to December 2020 were retrospectively analyzed, and 60 healthy people (control group) who underwent physical examination in the hospital in the same period were selected. The levels of blood PSA, TAP and MACC1 in the two groups, as well as their correlation with Gleason score and T stage, were analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the value of various indexes in the diagnosis of prostate cancer.Results·The levels of blood PSA, TAP and MACC1 in the case group were significantly higher than those in the control group (P<0.05). With the increase of Gleason grade, the levels of blood PSA, TAP and MACC1 increased gradually. With the progress of T stage, the levels of blood PSA, TAP and MACC1 increased gradually. The levels of PSA and MACC1 in the patients with lymph node metastasis were significantly higher than those without lymph node metastasis, but the levels of TAP in the patients with lymph node metastasis were significantly lower than those without lymph node metastasis (P<0.05). The analysis results of ROC curve showed that the AUC of PSA in the diagnosis of prostate cancer was 0.764, the sensitivity was 86.12%, the specificity was 88.63%, and the cut-off value was 6.01 μg/L. The AUC of TAP in the diagnosis of prostate cancer was 0.796, the sensitivity was 88.18%, the specificity was 89.58%, and the cut-off value was 135.62 μm2. The AUC of MACC1 in the diagnosis of prostate cancer was 0.873, the sensitivity was 78.46%, the specificity was 80.10%, and the cut-off value was 37.80 pg/mL. The AUC (0.941), sensitivity (93.15%) and specificity (94.08%) of combined detection were higher than those of any single detection (P<0.05). Spearman correlation analysis showed that the blood PSA, TAP and MACC1 levels were positively correlated with Gleason score (r=0.648, 0.513, 0.501; P=0.000), and with T stage (r=0.616, 0.537, 0.542; P=0.000).Conclusion·Blood PSA, TAP and MACC1 are closely related to Gleason score and T stage of prostate cancer, which has some value in the diagnosis of prostate cancer, and the combined detection of three items has higher diagnostic value.
 Construction and evaluation of a prediction model for liver injury induced by chemotherapy for breast cancer XIA Kunjian, DENG Linlin, WANG Lin 2022, 42 (4):  502-509.  doi: 10.3969/j.issn.1674-8115.2022.04.013 Abstract ( 91 )   HTML ( 5 )   PDF (1187KB) ( 30 )   Objective·To analyze the risk factors of liver injury induced by chemotherapy for breast cancer, and to construct and evaluate a prediction model of liver injury induced by chemotherapy.Methods·Breast cancer patients hospitalized at the Second Affiliated Hospital of Nanchang University from April 2019 to September 2021 who received anthracycline combined with cyclophosphamide sequential paclitaxel ± trastuzumab chemotherapy were enrolled in the study, and were divided into liver injury group and non-injury group. The risk factors of liver injury induced by chemotherapy were analyzed by χ2 test and binary Logistic regression, and the prediction model was established. The receiver operating characteristic (ROC) curve was used to evaluate the predictive performance of the regression model, and then the predictive model was externally validated.Results·Two hundred and seven patients with breast cancer met the inclusion criteria in this study, and sixty-nine patients with liver injury (33.3%). Univariate analysis showed that the difference of chemotherapy regimen (χ2=44.851, P=0.000), hepatitis B virus infection (χ2=16.682, P=0.000), previous history of hypertension (χ2=13.211, P=0.004), tumor TNM staging (χ2=14.422, P=0.001), previous history of diabetes (χ2=4.839, P=0.028), low albumin level before chemotherapy (χ2=10.073, P=0.002) and elevated triacylglycerol (χ2=39.367, P=0.000) were statistically significant between the two groups. Binary Logistic regression showed that chemotherapy regimen (OR=4.734, 95%CI 1.687?13.283, P=0.003), hepatitis B virus infection (OR=4.530, 95%CI 1.806?11.366, P=0.001), tumor TNM stage Ⅲ (OR=5.304, 95%CI 1.802-15.608, P=0.002), previous history of diabetes (OR=3.041, 95%CI 1.196?7.729, P=0.019), and low albumin level before chemotherapy (OR=3.744, 95%CI 1.413?9.920, P=0.008) were independent risk factors for liver injury caused by chemotherapy. logit（P）=-3.471+$1.511?X1+1.112?X2+1.320?X3+0.755?X4+0.691?X5+0.973?X6+1.258?X7+0.741?X8+1.668?X9+1.555?X10$. The area under the ROC curve was 0.874, the standard error was 0.024, 95%CI 0.827?0.922, P=0.000. External validation showed that the specificity of the prediction model was 86.9%, the sensitivity was 76.9%; the positive predictive value was 71.4%, the negative predictive value was 89.8%; the accuracy was 83.9%; the Kappa value was 0.624, the standard error was 0.091, P=0.000.Conclusion·This Logistic regression model has high predictive performance and has certain reference value for breast doctors to predict whether patients have liver damage.
Techniques and methods
 Construction and evaluation of IgG antibody Fc combinational mutation library with mammalian cell surface display system ZHANG Mi, LIU Shujun, LI Fubin, ZHANG Yan 2022, 42 (4):  510-517.  doi: 10.3969/j.issn.1674-8115.2022.04.014 Abstract ( 77 )   HTML ( 1 )   PDF (1276KB) ( 31 )   Objective·To establish an efficient method to obtain a high throughput mammalian cell surface display library of IgG antibody Fc combined mutations based on the screening results of the single point mutation library and the introduction of combined mutations by multiple degenerate primers.Methods·Multiple degenerate primers were designed according to the mutated amino acids at 233?240 of EU numbering with high affinity to Fcγ receptor IIB screened by fluorescence-activated cell sorting from the single point mutation library; the DNA fragments containing combined mutations were obtained through PCR by using the degenerate primers mixed with equal proportion, or according to the proportion of mutation diversity; the recombinant enzyme was used to ligate the fragment to the vector in one step and then transformed into E. coli DH5α, and the plasmid library was obtained. The plasmid library was used to transfect Pheonix cells to prepare retroviruses, and then 3T3 cells were infected with the retroviruses to obtain mammalian cell library that expressed Fc mutations on the surface. Flow cytometry was used to analyze the transfection efficiency of Pheonix cells to package retroviruses and the infection efficiency of 3T3 cells by the retroviruses. The quality of the plasmid library and the cell library was evaluated by next generation sequencing (NGS).Results·According to the point mutation library screening results, 32 degenerate primers were designed, which could cover all desired combined mutations, and the actual library diversity was 3.6 times that of the expected library (41 600/11 520). The results of NGS showed that the plasmid library obtained by using proportionally mixed primers according to the primer diversity had better homogeneity and integrity, and could cover 99.58% of the expected combinational mutations (11 472/11 520). Sanger sequencing results showed that 3 sequences among 10 sequences were the expected mutations, which were consistent with the diversity calculated during degenerate primers design. Flow cytometry showed that the transfection efficiency of Pheonix cells was 51.6%, and the infection efficiency of 3T3 cells was 47.4%. NGS results showed that the cell library could cover 85.92% of the expected combinational mutations (9 898/11 520).Conclusion·Based on the amino acid mutations screened from the single point mutation library, multiple degenerate primers were designed, and then through PCR, plasmid transfection and retrovirus infection, an IgG Fc combined mutation plasmid library and mammalian cell surface display library with more than 85% coverage of the expected combined mutations can be obtained efficiently at low cost.
 Establishment and application of an in vitro detection method for antibody gene point mutation, insertion and deletion LUO Simin, YEAP Lengsiew, HAO Qian 2022, 42 (4):  518-527.  doi: 10.3969/j.issn.1674-8115.2022.04.015 Abstract ( 95 )   HTML ( 1 )   PDF (2945KB) ( 34 )   Objective·To establish an in vitro method for rapidly detecting mutations, including point mutation, insertion and deletion events in the variable (V), diversity (D) and joining (J) regions of antibody genes in the process of somatic hypermutations caused by activation-induced cytidine deaminase (AID). The above method was used to detect the effects of two DNA polymerase β (Polβ) inhibitors on the mutations in the VDJ regions of antibody genes.Methods·CH12F3 cells were treated with lentivirus infection (i.e. treatment group), and the CH12F3 cells before infection were used as control group. Western blotting was used to detect the expression level of AID in the two groups of cells. The high-throughput sequencing libraries were constructed, and the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes in the two groups of cells were analyzed by bioinformatics. CH12F3 cells were treated with different concentrations of Polβ inhibitors [2', 3'-dioxycytidine (DDC) and 5- methoxyflavone (5-MF)], and the effects of the two inhibitors on cell proliferation were measured by Trypan blue exclusion method. CH12F3 cells (i.e. experimental group) were treated with the selected DDC concentration or 5-MF concentration, respectively; the cells treated with 0.9% NaCl or DMSO were recorded as the control group of the above experimental group, respectively. The four groups of cells before and after lentivirus infection were detected by the above established methods, and finally high-throughput sequencing was carried out to analyze the effects of the two inhibitors on the frequencies of point mutation, insertion and deletion in the VDJ regions of antibody genes.Results·Western blotting results showed that compared with CH12F3 cells in the control group, the expression of AID in CH12F3 cells in the treatment group was higher; and the high-throughput sequencing results showed that there were a large number of point mutations in the VDJ regions of antibody genes of CH12F3 cells in the treatment group, and its frequencies of insertion and deletion were also higher than those of the control group (both P=0.000). Trypan blue exclusion method showed that the optimal concentration of DDC and 5-MF for CH12F3 cells was 100 μmol/L and 25 μmol/L, respectively. The high-throughput sequencing results showed that compared with the 0.9% NaCl control group, the point mutation frequencies in the VDJ regions of antibody genes were decreased after cells were treated by 100 μmol/L DDC (P=0.000), and its 1 bp deletion frequencies decreased slightly (P=0.009); compared with DMSO control group, the frequencies of point mutation (P=0.000), longer than 1 bp insertion and deletion (both P=0.000) in the VDJ regions of antibody genes were decreased after cells were treated by 25 μmol/L 5-MF.Conclusion·The in vitro detection method established in this study can be used to analyze the AID-induced mutation events of VDJ regions of antibody genes. The Polβ inhibitors DDC and 5-MF can inhibit the mutations in the VDJ regions of antibody genes induced by AID.
Review
 Classification and research progress of corneal neurotization WU Yue, ZHANG Jiaying, WANG Wei, LI Jin 2022, 42 (4):  528-534.  doi: 10.3969/j.issn.1674-8115.2022.04.016 Abstract ( 129 )   HTML ( 3 )   PDF (2216KB) ( 46 )   The corneal nerve originates from the trigeminal nerve, which mainly innervates the corneal sensation. Therefore, corneal nerve injury and decrease of corneal sensation occur when the trigeminal nerve is damaged. Neurotrophic keratopathy (NK) is a disease related with alterations in corneal nerves, leading to corneal epithelial defects, ulcer and perforation. However, mild NK is mostly manifested as a slight decrease in corneal sensation and instability of the tear film, so it is quite insidious in clinical practice. At the same time, the clinical treatment methods for promoting corneal reinnervation and restoring corneal sensation are usually relatively simple. Currently, medical treatment, non-surgical interventions and surgical interventions are the treatments for NK thus promoting corneal nerve and sensation recovery. In addition to maintaining the stability of the ocular surface, the main purpose of medical treatment is anti-infection and anti-inflammation, and to prevent further damage to the cornea. Novel biological agents are also under clinical trials such as platelet-rich plasma, autologous serum and recombinant human nerve growth factor. Non-surgical intervention which mainly includes therapeutic contact lenses, medical tape and botulinum toxin injections aims to reduce corneal exposure and protect the cornea. With regard to surgical interventions, in addition to the conventional procedures aimed at repairing corneal defects and reducing the exposed area, there is also a new procedure called corneal nerve transplantation, which is aimed at improving corneal sensation and corneal innervation. Corneal nerve transplantation is a new surgical method that is directly targeted at improving corneal sensation and innervation. It is an ideal surgical method for patients with loss of corneal subbasal nerve fibers due to trigeminal nerve injury. In conclusion, medical treatment, non-surgical intervention and surgical neurotization are not separated in clinical practice. This article summarizes the progress of the application of the above three types of treatments in order to provide a reference for the future clinical selection of appropriate treatment modalities and comprehensive reconstruction of corneal sensation according to the course and different severity of disease development.
 Resistance mechanisms and overcoming strategies of the third-generation EGFR-TKI in non-small cell lung cancer LU Wenqing, MENG Zhouwenli, YU Yongfeng, LU Shun 2022, 42 (4):  535-544.  doi: 10.3969/j.issn.1674-8115.2022.04.017 Abstract ( 96 )   HTML ( 2 )   PDF (1338KB) ( 51 )   Epidermal growth factor receptor (EGFR) gene is the most common driver gene of non-small cell lung cancer. Tyrosine kinase inhibitors (TKIs) targeting EGFR mutations are the first-line treatment choice for patients with EGFR mutations. Although three generations of drugs have been widely used in clinical practice, unavoidable secondary resistance and primary resistance to some treatment-naive patients still pose great challenges to the long-term use of EGFR-TKIs. Resistance mechanism of the first- and second-generation EGFR-TKIs are well studied, including T790M mutation, MET amplification, ERBB2 amplification, IGF1R up-regulation, AXL activation, etc. The third-generation TKIs can overcome the most common T790M mutation that the first two generations bring in, but with the increasingly widespread clinical use, their drug resistance problem is also attracting widespread attention, and the related mechanisms and overcoming strategies are still under study. Mechanisms can be divided into EGFR-dependent and EGFR-independent ones, involving target-gene mutation, bypass signaling activation, phenotypic plasticity, epigenetic regulation, inhibitory immune microenvironment and so on. The fourth-generation TKIs, combination therapy and immunotherapy are all potential modalities after drug resistance.
 Research progress of ketogenic diet regulating intestinal microbiome in the treatment of diseases WU Ya, YIN Jun 2022, 42 (4):  545-550.  doi: 10.3969/j.issn.1674-8115.2022.04.018 Abstract ( 106 )   HTML ( 4 )   PDF (838KB) ( 60 )   Ketogenic diet is a high-fat, very low-carbohydrate, appropriate amount of protein and other nutrients diet. It was first used to treat children's epilepsy. With the continuous development of medicine, ketogenic diet has been gradually used in the adjuvant treatment of other diseases, but its specific mechanism is not clear. Recent studies have shown that ketogenic diet can affect the diversity and quantity of intestinal flora, which is of great significance to the health of the host. Studies have also shown that ketogenic diet can increase the abundance of Akkermansia muciniphila (A. muciniphila) and Parabacteroides in mouse intestine, resulting in decreased subset of gamma-glutamylated amino acids and increased ratio of hippocampal gamma-aminobutyric acid and glutamate, which could reduce the frequency of seizures. Elevated abundance of A. muciniphila can also improve cerebrovascular function, and then reduce the risk of Alzheimer's disease. Besides, ketogenic diet can also decrease the abundance of Bifidobacterium in mouse intestine, resulting in the decrease of intestinal lactic acid level and pro-inflammatory T helper 17 cell (Th17 cell) level, which could improve cognitive and memory function and treat obesity, respectively. This article reviews the therapeutic effects and possible mechanisms of ketogenic diet on the regulation of intestinal flora in neurological diseases (epilepsy, Alzheimer's disease, multiple sclerosis and autism), metabolic diseases (diabetes mellitus and obesity), and tumors.
Case report
 Hb M-Boston: a case report and overview WANG Yefei, ZHANG Yuemin, WU Beiying, XIA Wenquan 2022, 42 (4):  551-556.  doi: 10.3969/j.issn.1674-8115.2022.04.019 Abstract ( 83 )   HTML ( 8 )   PDF (1394KB) ( 54 )   A 29-year-old male with cyanosis had low oxygen saturation (SaO2 89.3%) with normal echocardiography and chest X-ray findings. The patient's peripheral blood was analyzed by routine blood analysis and screening tests for hemolysis, and hemoglobin detection was performed by high performance liquid chromatography (HPLC). The polymerase chain reaction (PCR) and reverse dot blot (RDB) technique were used to detect 17 common β-thalassemia gene mutations and non-deletional α-thalassemia in Chinese. Gap-PCR combined with agarose gel electrophoresis was used to detect deletional α-thalassemia. PCR and DNA sequencing for α- and β-globin gene (HBA1, HBA2 and HBB) were simultaneously performed. Both of the routine blood analysis and hemolysis screening tests were with normal findings. The HPLC pattern showed a peak with 8.1% of area at 4.53 min position (S window). Gene analysis showed a heterozygous mutation of HBA2 c.175C>T, Hb M-Bostonα58 His>Tyr. Hb M is a kind of Hb with the structural variant that stabilizes heme iron in the oxidized (ferric) state. It can be confused with other causes of methemoglobinemia, like genetic alterations in methemoglobin reductase enzyme systems of red cells. The heterozygotes of Hb M-Boston with typical lifelong cyanosis and good prognosis is really unusual in Chinese population, which does not require any treatment.