Therapeutic effect of estradiol-pretreated human bone marrow mesenchymal stem cells on liver cirrhosis in mice
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摘要: 目的探讨雌二醇对骨髓间质干细胞(BMSC)的作用及雌二醇预处理的BMSC对肝硬化的治疗效应。方法第3代BMSC分为对照组(未经雌二醇处理)、实验组(不同浓度雌二醇处理),MTT法测雌二醇对其增殖活力的作用;对照组和实验组用H2O2诱导凋亡,正常BMSC为空白组,台盼蓝染色及实时荧光定量PCR(RT-PCR)(测caspase-3)检测雌二醇对BMSC凋亡的影响。46只正常雄性C57BL小鼠分为空白对照组、模型对照组、BMSC及雌二醇处理的BMSC移植组。CCl4腹腔注射造肝硬化模型,12周后BMSC及雌二醇处理的BMSC移植组通过尾静脉注射移植细胞。4周后采血测ALT、AST水平;HE、Masson染色测肝纤维化程度;RT-PCR测Ⅰ型胶原蛋白(collagenⅠ)mRNA表达。满足正态分布和方差齐性的计量资料比较采用方差分析,进一步两两比较采用LSD检验;不满足正态分布和方差齐性的计量资料采用秩转换的Mann-Whitney检验。结果各实验组BMSC增殖活性高于对照组(F=730.08,P=0.000);H2O2促进BMSC凋亡,实验组凋亡率低于对照组(F=258.34,P=0.00...Abstract: Objective To explore the effect of estradiol on human bone marrow mesenchymal stem cells( BMSCs) and the therapeutic effect of estradiol- pretreated BMSCs on liver cirrhosis in mice. Methods Third- passage BMSCs were divided into control( untreated with estradiol) and experimental groups( treated with different concentrations of estradiol); the effect of estradiol on the proliferative activity of BMSCs was measured by MTT assay. Apoptosis was induced by H2O2 in the control and experimental groups of BMSCs,with normal BMSCs as the blank group; the effect of estradiol on apoptosis in BMSCs was tested by trypan blue staining and quantitative real- time PCR of caspase- 3 mRNA. Forty- six normal male C57 BL mice were assigned to control,model,BMSC transplantation,and estradiol- pretreated BMSC transplantation groups. An animal model of liver cirrhosis was established by intraperitoneal injection of CCl4. At 12 weeks after CCl4 injection,estradiol- pretreated and untreated BMSCs were transplanted into mice through the tail vein. At 4 weeks after BMSC transplantation,blood samples were collected for serum alanine aminotransferase( ALT) and serum aspartate aminotransferase( AST) assays. The stage of liver fibrosis was determined by hematoxylin- eosin and Masson staining. Type I collagen( collagen I) expression was analyzed by RT- PCR. Normally distributed categorical data( with homogeneous variances) were analyzed using ANOVA,followed by pairwise comparisons using LSD test; non- normally distributed data( with heterogeneous variances) were analyzed using Mann- Whitney rank- sum test. Results All the experimental groups had higher proliferative activity than the control group( F = 730. 08,P = 0. 000). H2O2 promoted apoptosis in BMSCs,with lower apoptosis rate in the experimental groups than in the control group( F = 258. 34,P = 0. 000). Caspase- 3 mRNA expression was significantly different between the control and experimental groups( F = 50. 29,P = 0. 000). Additionally,there were significant differences in rats between the model,BMSC transplantation,and estradiol- pretreated BMSC transplantation groups in terms of ALT( P = 0. 000 for all),AST( P < 0. 05 for all),fiber ratio( P < 0. 05 for all),and collagen I mRNA expression( P < 0. 05 for all). Conclusion Estradiol promotes the proliferation and reduces H2O2- induced apoptosis in BMSCs. BMSCs can improve liver cirrhosis and their therapeutic effect is enhanced by estradiol pretreatment.
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Key words:
- liver cirrhosis /
- estradiol /
- mesenchymal stem cells /
- collagen type Ⅰ /
- models animal /
- mice,inbred C57BL
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