大鼠脐带间充质干细胞对肝细胞增殖、凋亡及功能的影响

发布时间：2018-04-16 05:37

本文选题：脐带间充质干细胞 + 原代肝细胞　；参考：《苏州大学》2014年硕士论文

【摘要】：目的探讨肝衰竭大鼠血清对脐带间充质干细胞（UMSCs）表面免疫学标记CD86的影响。了解UMSCs对大鼠原代肝细胞增殖、凋亡与功能的影响。方法采用胶原酶和胰酶两步法分离UMSCs，采用贴壁法培养、纯化；观察细胞形态特征及生长状态；流式细胞仪检测UMSCs细胞表面分子标记CD90、CD105；将达70%～80%融合的第3代UMSCs随机分为正常培养基组，正常血清组（含10%正常大鼠血清培养基），肝衰竭血清组（含10%肝衰竭大鼠血清培养基）。以上措施干预培养24h后用流式细胞仪定量监测细胞表面免疫学标记CD86的变化。用胶原酶灌注法分离大鼠肝细胞，肝细胞与UMSCs应用Transwell共培养。原代大鼠肝细胞分为三组：UMSCs组、大鼠原代肝细胞组及UMSCs与大鼠原代肝细胞共培养组，分别于共培养第1d、3d、5d、7d通过MTT法检测各组细胞增殖能力；UMSCs与肝细胞共培养组、肝细胞组，采用脂多糖（LPS）诱导肝细胞凋亡，于共培养24h后采用流式细胞仪检测细胞凋亡率；收集各组细胞共培养第1d、3d、5d培养上清液，ELISA法检测上清液中白蛋白含量。结果 1．采用胶原酶和胰酶两步酶法能获得大量的单个UMSCs，在体外可以稳定的生长、扩增。流式细胞仪检测结果显示，分离得到的细胞稳定表达相关抗原标记CD90、CD105，表达率为99.7%、99.5%。2．不同组诱导培养UMSCs24小时后，正常培养基组、正常血清组，肝衰竭血清组细胞表面CD86表达率为0.5%，0.6%，0.4%。相比差异无统计学意义（P＞0.05）。3．（1）UMSCs与原代大鼠肝细胞共培养组在第1d、3d、5d、7d的OD值大于相应时间点的UMSCs组OD值与原代大鼠肝细胞组OD值之和（P＜0.05）。（2）诱导肝细胞凋亡后，流式细胞仪检测阳性对照组、阴性对照组及UMSCs密度为1*104/ml、1*106/ml与肝细胞共培养组凋亡率分别为93.3%、54.2%、59.7%、72.9%，相比差异有统计学意义（P0.05）。（3）UMSCs无白蛋白分泌能力，UMSCs与大鼠原代肝细胞共培养组培养上清液中白蛋白水平第1d、3d、5d均较原代大鼠肝细胞组明显升高（P0.05）。结论胶原酶和胰酶两步消化法是一种简单、可靠的分离UMSCs的方法；大鼠UMSCs稳定表达相关抗原标记CD90、CD105。经肝衰竭大鼠血清干预培养UMSCs后CD86仍不表达。UMSCs在体外可以促进肝细胞增殖，抑制其凋亡，，并具有增强白蛋白分泌的功能。
[Abstract]:PurposeTo investigate the effect of serum from rats with liver failure on CD86 labeled by UMSCs on the surface of umbilical cord mesenchymal stem cells (UMSCs).To investigate the effects of UMSCs on the proliferation, apoptosis and function of rat primary hepatocytes.MethodUMSCs were separated by collagenase and trypsin, cultured and purified by adherent method; morphological characteristics and growth status of UMSCs cells were observed; CD90 CD105, a molecular marker on the surface of UMSCs cells, was detected by flow cytometry; and the third generation of UMSCs, which fused up to 70%, was randomly divided into normal medium group.Normal serum group (containing 10% normal rat serum medium), liver failure serum group (containing 10% liver failure rat serum medium).After 24 hours of culture, flow cytometry was used to quantitatively monitor the changes of immuno-labeled CD86 on cell surface.Rat hepatocytes were isolated by collagenase perfusion and co-cultured with UMSCs with Transwell.The primary rat hepatocytes were divided into three groups: the primary rat hepatocytes group, the primary rat hepatocyte group and the co-culture group of UMSCs and rat primary hepatocytes. The proliferation ability of each group was detected by MTT assay on the 1st day, 3d after incubation, and 5 days after co-culture, respectively.Hepatocyte apoptosis was induced by lipopolysaccharide (LPS), the apoptosis rate was detected by flow cytometry after 24 h co-culture, and the albumin content in supernatant was detected by Elisa.Result1.A large number of single UMCs can be obtained by using collagenase and trypsin two-step enzymatic method, which can grow and amplify stably in vitro.The results of flow cytometry showed that the stable expression of CD90 and CD105 was detected by flow cytometry, and the expression rate of CD90 and CD105was 99.70.2.After induction and culture of UMSCs24 in different groups, the expression rate of CD86 on the cell surface of normal medium group, normal serum group and serum group of liver failure was 0.5% and 0.64% respectively.There was no significant difference between 0.05).3.(1)UMSCs and primary rat hepatocyte coculture group. The OD value of the co-cultured rat hepatocytes on the 1st day was higher than that of the UMSCs group and the primary rat hepatocyte group on the 1st day (P < 0.05), and the positive control group was detected by flow cytometry after the OD value of the UMSCs group was higher than that of the primary rat hepatocyte group (P < 0.05), and the total OD value of the primary rat hepatocyte co-culture group was significantly higher than that of the control group (P < 0.05).The apoptotic rates of the negative control group and the UMSCs density of 1 / 104 / ml / ml and the hepatocyte co-culture group were 93.3% and 54.2%, respectively. The difference was significant compared with the control group (P 0.05 / ml) and the rat primary hepatocyte co-culture group (P < 0.05). The albumin level in the supernatant of the primary rat hepatocyte co-culture group was significantly higher than that in the control group (P < 0.05) and the rat primary hepatocyte co-culture group (P < 0.05), respectively, and the level of albumin in the supernatant was significantly higher than that in the control group (P < 0.05).Compared with the primary rat hepatocyte group, the level of P0. 05 was significantly increased at 1 d, 3 d and 5 d.ConclusionThe two-step digestion of collagenase and trypsin is a simple and reliable method for the separation of UMSCs and the stable expression of CD90 and CD105 in rat UMSCs.In vitro, CD86 could promote the proliferation of hepatocytes, inhibit their apoptosis and enhance the secretion of albumin.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.3;R457.7

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