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目的研究经尾静脉移植携带生存素(survivin)基因的骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)对小鼠肺纤维化的影响。方法体外构建培养经慢病毒转染绿色荧光蛋白(green fluorescent protein,GFP)及生存素基因的C57BL/6小鼠BMSC-survivin以及经慢病毒转染GFP的C57BL/6小鼠BMSC,并应用流式细胞术比较2种细胞的体外凋亡率。6～8周龄C57BL/6小鼠48只,博莱霉素5 mg/kg气管内灌注制备小鼠肺纤维化模型。随机分为3组:BMSC-survivin组、BMSC组、模型组;造模24 h后,3组分别经尾静脉注射BMSC-survivin、BMSC和生理盐水。每组于第7、28天各处死8只小鼠。肺组织作病理学检查,免疫组织化学法检测肺表面蛋白A的表达;ELISA法测定血清中羟脯氨酸(Hyp)的含量;PCR法检测TGF-β1和MMP-9 mRNA表达情况。结果 BMSC及BMSC-survivin细胞凋亡率分别为(14.466±1.953)%和(7.718±0.493)%,给予BMSC干预的小鼠肺病理改变较对照组轻,BMSC-survivin组、BMSC组、模型组肺组织羟脯氨酸含量第7天分别为(0.106±0.012)、(0.245±0.009)μg/mL和(0.324±0.002)μg/mL,第28天分别为(0.457±0.006)、(0.752±0.012)μg/mL和(1.372±0.035)μg/mL;BMSC-survivin组、BMSC组、模型组肺组织SP-A含量第7天分别为(0.718±0.018)、(0.669±0.013)和(0.581±0.027),第28天分别为(0.433±0.012)、(0.372±0.039)和(0.277±0.013);各时间点,BMSC-survivin组TGF-β1 mRNA、MMP-9 mRNA含量较BMSC组和模型组低,BMSC组较模型组低(P<0.05)。结论外源性的BMSC可以减轻肺纤维化,生存素基因能够抑制BMSC的凋亡,延长BMSC减轻肺纤维化的作用。
Objective To investigate the effect of bone marrow mesenchymal stem cell (BMSC) carrying survivin gene transplanted via tail vein on pulmonary fibrosis in mice. Methods C57BL / 6 mice BMSC-survivin transfected with green fluorescent protein (GFP) and survivin gene by lentivirus and C57BL / 6 mouse BMSC transfected with GFP by lentivirus were constructed and cultured in vitro. Cytometry compared the apoptosis rate of two kinds of cells in vitro. Forty eight C57BL / 6 mice aged 6-8 weeks and bleomycin 5 mg / kg were intraperitoneally injected to prepare mouse model of pulmonary fibrosis. The rats were randomly divided into 3 groups: BMSC-survivin group, BMSC group and model group. After 24 h of modeling, BMSC-survivin, BMSC and normal saline were injected through the tail vein respectively. Eight mice were killed in each group on the 7th and 28th days. The pathological examination of lung tissue was performed. The expression of protein A on the surface of lung was detected by immunohistochemistry. The content of hydroxyproline (Hyp) in serum was measured by ELISA. The mRNA expression of TGF-β1 and MMP-9 was detected by PCR. Results The apoptotic rates of BMSC and BMSC-survivin were (14.466 ± 1.953)% and (7.718 ± 0.493)%, respectively. Compared with control group, BMSC-survivin, BMSC and model group The levels of hydroxyproline in lung tissue were (0.106 ± 0.012), (0.245 ± 0.009) μg / mL and (0.324 ± 0.002) μg / mL on the 7th day, and those on the 28th day were (0.457 ± 0.006) and (0.718 ± 0.018), (0.669 ± 0.013) and (0.581 ± 0.013) μg / mL and (1.372 ± 0.035) μg / mL respectively in the BMSC-survivin group, the BMSC group and the model group ± 0.027) and (0.433 ± 0.012), (0.372 ± 0.039) and (0.277 ± 0.013) at 28th day respectively. Compared with BMSC group and model group, the content of TGF-β1 mRNA and MMP- Group was lower, BMSC group than the model group (P <0.05). Conclusion Exogenous BMSC can reduce pulmonary fibrosis, survivin gene can inhibit apoptosis of BMSC and prolong BMSC to reduce pulmonary fibrosis.