B7基因修饰的白血病细胞外泌体的抗白血病效应

doi:10.3969/j.issn.1674-8115.2025.02.001

摘要/Abstract

摘要：

目的·探究高表达B7分子（共刺激分子CD80和CD86）的白血病细胞外泌体在荷瘤小鼠中的免疫治疗效应。方法·L1210白血病细胞株转染含有CD80和CD86基因的慢病毒载体，从而获得高表达CD80和CD86分子的白血病细胞，分离其培养上清液获得高表达CD80和CD86分子的外泌体LEX-8086。应用透射电子显微镜（电镜）和Western blotting分析LEX-8086的生物学特征。建立DBA/2-白血病荷瘤小鼠模型，将荷瘤小鼠分为LEX-8086组和PBS组，LEX-8086组分别在肿瘤细胞接种后7 d和12 d尾静脉注射LEX-8086 500 μL（150 μg/mL），PBS组同时注射等体积PBS；观察2组小鼠的体质量、肿瘤体积、肿瘤质量及生存期差异。应用流式细胞术分析2组小鼠脾脏和淋巴结免疫细胞的变化特点。应用Western blotting和实时荧光定量PCR（qPCR）检测2组小鼠脾脏M1型和M2型巨噬细胞标志物的表达情况。结果·从高表达CD80和CD86分子的L1210细胞上清液中分离获得LEX-8086，透射电镜显示为直径约100 nm的盘状囊泡结构，Western blotting结果显示其表达外泌体特异性标志物。动物实验结果显示，LEX-8086组较PBS组，体质量无明显改变，肿瘤体积和肿瘤相对质量比均显著下降且生存期显著延长（均P<0.05）。流式细胞术结果显示，LEX-8086组淋巴结、脾脏内自然杀伤细胞比例、CD4⁺T细胞比例均显著升高（均P<0.05），而CD8⁺ T细胞仅在淋巴结内显著升高（P=0.012）。此外，流式细胞术结果显示LEX-8086组小鼠脾脏中M1型巨噬细胞比例显著升高（P=0.003），M2型巨噬细胞比例无明显变化。Western blotting和qPCR同样显示，LEX-8086组小鼠脾脏中M1型巨噬细胞标志物白细胞介素-6和肿瘤坏死因子α mRNA和蛋白表达水平均显著升高（均P<0.05）。结论·共刺激分子CD80和CD86高表达的白血病细胞外泌体能够诱导一定的治疗性抗白血病效应，该效应可能是通过提高NK细胞、M1型巨噬细胞、CD4⁺ T细胞和CD8⁺ T细胞的比例实现的。

关键词: 白血病, 外泌体, 共刺激分子, 基因修饰, 免疫治疗

Abstract:

Objective ·To investigate the immunotherapeutic effects of exosomes derived from leukemia cells with high expression of B7 molecules (co-stimulatory molecules CD80 and CD86) in tumor-bearing mice. Methods ·L1210 leukemia cells were transfected with lentiviral vectors containing the CD80 and CD86 genes, and thereby the leukemia cells with high expression of CD80 and CD86 molecules were obtained. Exosomes LEX-8086 with high expression of CD80 and CD86 molecules were then isolated from the culture supernatant. The biological characteristics of LEX-8086 were analyzed by using transmission electron microscopy (TEM) and Western blotting. A DBA/2-leukemia tumor-bearing mouse model was established. The tumor-bearing mice were divided into LEX-8086 group and PBS group. The LEX-8086 group received intravenous injections of 500 μL LEX-8086 (150 μg/mL) via the tail vein 7 d and 12 d after tumor cell inoculation, while the PBS group received injections of equal volumes of PBS at the same time points. Differences in body weight, tumor volume, tumor mass, and survival of the mice between the two groups were observed. Flow cytometry was used to analyze the changes in the immune cells in spleens and lymph nodes of the mice in both groups. Western blotting and real-time fluorescent quantitative PCR (qPCR) were used to detect the expression of markers for M1 and M2 macrophages in the spleens of the mice in both groups. Results ·The exosomes LEX-8086 were isolated from the supernatant of L1210 cells overexpressing CD80 and CD86 molecules. TEM revealed that LEX-8086 exhibited a disc-shaped vesicular structure with a diameter of approximately 100 nm. Western blotting demonstrated the expression of exosome-specific markers. In the animal experiments, compared with the PBS group, the body weight of the LEX-8086 group showed no significant change, while both the tumor volume and the relative tumor mass ratio decreased significantly, and the survival was significantly prolonged (all P<0.05). Flow cytometry results indicated that the proportions of natural killer (NK) cells and CD4⁺ T cells in the lymph nodes and spleens of the LEX-8086 group significantly increased (all P<0.05); as for CD8⁺ T cells, they only increased in the lymph modes (P=0.012). Moreover, flow cytometry results showed that the proportion of M1 macrophages in the spleens of mice in the LEX-8086 group was significantly elevated (P=0.003), while the proportion of M2 macrophages remained unchanged. Western blotting and qPCR also revealed that the mRNA and protein expression levels of M1 macrophage markers interleukin-6 and tumor necrosis factor-α in the spleens of mice in the LEX-8086 group increased significantly (all P<0.05). Conclusion ·Exosomes derived from leukemia cells with high expression of co-stimulatory molecules CD80 and CD86 can induce a therapeutic anti-leukemic effect, which may be achieved by increasing the proportions of NK cells, M1 macrophages, CD4⁺ T cells, and CD8⁺ T cells.

Key words: leukemia, exosome, costimulatory molecule, gene modification, immunotherapy

中图分类号:

R557

张涤凡, 王明慧, 赵洁, 万江波, 黄方, 郝思国. B7基因修饰的白血病细胞外泌体的抗白血病效应[J]. 上海交通大学学报（医学版）, 2025, 45(2): 129-137.

ZHANG Difan, WANG Minghui, ZHAO Jie, WAN Jiangbo, HUANG Fang, HAO Siguo. Anti-leukemia effect of B7 gene-modified leukemia cell-derived exosomes[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(2): 129-137.

图/表 9

表1 qPCR检测的引物序列

Tab 1 Primer sequences for qPCR

Gene	Forward primer (5'→3')	Reverse primer (5'→3')
Cd206	GGGTTGCTATCACTCTCTATGC	TTTCTTGTCTGTTGCCGTAGTT
Il-10	GACTTTAAGGGTTACCTGGGTTG	TCACATGCGCCTTGATGTCTG
Il-6	ACTCACCTCTTCAGAACGAATTG	CCATCTTTGGAAGGTTCAGGTTG
Tnf-α	ACCAGCAGATGGGCTGTACC	GCGGAGAGGAGGCTGACTTT
Gadph	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG

图1 L1210-CD8086细胞和外泌体LEX-8086的生物学特征Note：A/B. Flow cytometry was used to assess the expression of CD80 (A) and CD86 (B) in L1210 and L1210-CD8086 cells. C. The L1210 cells transfected with lentiviral vectors were observed under a fluorescence microscope (×200). D. Exosomes LEX-8086 were observed by TEM (×20 000). E. Exosome-specific markers were detected by Western blotting. ①P<0.001.

Fig 1 Biological characteristics of L1210-CD8086 cells and exosomes LEX-8086

图2 2组DBA/2-白血病荷瘤小鼠的体质量和生存期变化Note：A. Changes in body weight of the DBA/2-leukemia tumor-bearing mice of the two groups. B. The overall survival curves of the DBA/2-leukemia tumor-bearing mice of the two groups.

Fig 2 Changes in body weight and survival time of DBA/2-leukemia tumor-bearing mice in the two groups

图3 2组DBA/2-白血病荷瘤小鼠的肿瘤体积和质量Note：A. Tumor volume growth curves of the DBA/2-leukemia tumor-bearing mice of the two groups. B. Typical gross tumor samples from the two groups of mice. C. Relative tumor mass ratios of the two groups.①P=0.025.

Fig 3 Tumor volume and tumor weight of DBA/2-leukemia tumor-bearing mice in the two groups

图4 流式细胞术检测2组DBA/2-白血病荷瘤小鼠淋巴结和脾脏中NK细胞比例Note： A. The proportions of NK cells in the lymph nodes of the two groups. B. The proportions of NK cells in the spleens of the two groups. ①P=0.015, ②P=0.003.

Fig 4 Proportions of NK cells in the lymph nodes and spleens of DBA/2-leukemia tumor-bearing mice in the two groups determined by flow cytometry

图5 流式细胞术检测2组DBA/2-白血病荷瘤小鼠淋巴结和脾脏中CD4+ T细胞和CD8+ T细胞比例Note： A. The proportions of CD4+ T cells and CD8+ T cells in the lymph nodes of the two groups. B. The proportions of CD4+ T cells and CD8+ T cells in the spleens of the two groups. ①P<0.001, ②P=0.012, ③P=0.036.

Fig 5 Proportions of CD4+ T cells and CD8+ T cells in the lymph nodes and spleens of DBA/2-leukemia tumor-bearing mice in the two groups determined by flow cytometry

图6 流式细胞术检测2组DBA/2-白血病荷瘤小鼠脾脏中M1型和M2型巨噬细胞比例Note： A. The proportions of M2 macrophages in the spleens of the two groups. B. The proportions of M1 macrophages in the spleens of the two groups. ①P=0.003.

Fig 6 Proportions of M1 and M2 macrophages in the spleens of DBA/2-leukemia tumor-bearing mice in the two groups determined by flow cytometry

图7 RT-qPCR检测2组DBA/2-白血病荷瘤小鼠脾脏中巨噬细胞标志物mRNA的表达水平Note： The expression levels of Cd206 (A), Il-10 (B), Il-6 (C), and Tnf-α (D) mRNA in the spleens of the two groups. ①P=0.013, ②P=0.035.

Fig 7 mRNA expression levels of macrophage markers in the spleens of DBA/2-leukemia tumor-bearing mice in the two groups detected by RT-qPCR

图8 Western blotting检测2组DBA/2-白血病荷瘤小鼠脾脏中IL-6和TNF-α的蛋白表达水平Note： A. Typical bands of IL-6 and TNF-α in Western blotting. B/C. Quantitative results of IL-6 (B) and TNF-α (C). ①P<0.001.

Fig 8 Protein expression levels of IL-6 and TNF-α in the spleens of DBA/2-leukemia tumor-bearing mice in the two groups detected by Western blotting

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