沉默转化生长因子β1对肝星状细胞-T6生长及增殖的影响
网络出版日期: 2014-09-26
基金资助
国家自然科学基金(81273918);重庆市中医药科技项目(2012-2-63);军队中医药科研专项课题(2010ZYZ231)
Effects of silencing TGFβ1 on growth and proliferation of hepatic stellate cell line T6
Online published: 2014-09-26
Supported by
National Natural Science Foundation of China,81273918;Project of Traditional Chinese Medicine Science and technology of Chongqing,2012-2-63;Specific Project of Traditional Chinese Medicine of Chinese PLA,2010ZYZ231
目的 观察沉默转化生长因子β1 (TGFβ1)对肝星状细胞(HSC)-T6生长及增殖的影响。方法 用具有高效感染力的TGFβ1 shRNA慢病毒液感染HSC-T6细胞,以未经感染或经空病毒感染的HSC-T6细胞作对照。倒置荧光显微镜下观察HSC-T6细胞绿色荧光蛋白(GFP)的表达变化;CCK-8法检测TGFβ1 shRNA慢病毒对HSC-T6细胞生长及增殖的影响;RT-PCR和Western blotting分别检测HSC-T6细胞TGFβ1及增殖细胞核抗原(PCNA)基因及其编码蛋白的表达,并予以定量分析。结果 经TGFβ1 shRNA慢病毒感染的HSC-T6细胞显著表达GFP。CCK-8法检测结果显示:经TGFβ1 shRNA慢病毒感染的HSC-T6细胞的生长及增殖慢于对照细胞,培养48 h后差异有统计学意义(P<0.01)。RT-PCR和Western blotting检测结果显示:TGFβ1 shRNA慢病毒能有效沉默HSC-T6细胞TGFβ1,并下调其核抗原PCNA基因及其编码蛋白的表达,与对照细胞的差异有统计学意义(P<0.01)。结论 沉默TGFβ1能明显抑制HSC-T6的生长及增殖,并下调其核抗原PCNA的表达。
张荣华 , 闫国和 , 汪国建 , 等 . 沉默转化生长因子β1对肝星状细胞-T6生长及增殖的影响[J]. 上海交通大学学报(医学版), 2014 , 34(9) : 1315 . DOI: 10.3969/j.issn.1674-8115.2014.09.012
Objective To observe the effects of silencing transforming growth factor β1 (TGFβ1) on the growth and proliferation of hepatic stellate cell line T6 (HSC-T6). Methods The TGFβ1 shRNA lentiviral vector particles with high infectivity were transfected into HSC-T6 cells. The controls were HSC-T6 cells that were not infected or infected by empty virus. The expression of green fluorescent protein (GFP) of the HSC-T6 cells was observed under the inverted fluorescence microscope. The effects of TGFβ1 shRNA lentivirus on the growth and proliferation of HSC-T6 cells were detected by the CCK-8. The expressions of the gene and encoding protein of TGFβ1 and proliferating cell nuclear antigen (PCNA) of HSC-T6 cells were detected by the RT-PCR and Western blotting, respectively. The quantitative analysis was then conducted. Results HSC-T6 cells transfected by TGFβ1 shRNA lentivirus significantly expressed GFP. The results of CCK-8 showed that the growth and proliferation of HSC-T6 cells transfected by TGFβ1 shRNA lentivirus were slower than those of controls and the differences were statistically significant after being cultured for 48 h (P<0.01). The results of RT-PCR and Western blotting revealed that the TGFβ1 shRNA lentivirus effectively silenced the TGFβ1 and down-regulated the expressions of gene and encoding protein of PCNA of HSC-T6 cells. The differences of transfected cells and controls were statistically significant (P<0.01). Conclusion Silencing TGFβ1 can effectively inhibit the growth and proliferation of HSC-T6 cells and down-regulate the expression of PCNA.
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