脐带间充质干细胞对急性肝衰竭的修复及移植途径的优化

发布时间：2018-06-16 14:18

本文选题：脐带间充质干细胞 + 急性肝衰竭　；参考：《重庆医科大学》2013年硕士论文

【摘要】：目的：探讨脐带间充质干细胞对急性肝衰竭大鼠的治疗作用，并优化移植治疗途径。方法：采用组织块贴壁法收集hUCMSCs，流式细胞术检测细胞表面标志物。清洁级健康SD大鼠48只（6-8周龄，体重180-220g，雌雄不论），采用完全随机对照法均分为4组：经尾静脉注射移植治疗组，经肝叶注射移植治疗组，模型对照组，空白对照组。急性肝衰竭动物模型采用一次性腹腔注射2.5ml/kg CCL4橄榄油（1：1）混合溶液建立，，24h后移植治疗组分别经尾静脉和肝叶注射hUCMSCs细胞悬液；治疗后0h、24h、48h、72h、96h和1w收集血清分析肝功能恢复情况；治疗后3d、1w、2w留取肝脏标本，观察病理学恢复情况，用Real-time PCR检测鼠肝中人CK8、CK18和AFP基因mRNA转录水平，免疫组化法检测人CK18的表达。结果： 1、hUCMSCs通过贴壁培养法能成功从脐带中分离并在体外大量扩增，流式细胞仪鉴定符合MSCs表面标志物特性。 2、肝功能和肝脏病理学结果显示：移植治疗组肝功能指标TBil和ALT含量较模型对照组有明显恢复（P0.05），肝细胞再生程度提高，炎细胞减少，肝脏病理学修复作用增强。 3、Real-time PCR结果显示：在造模后1w、2w时，经尾静脉移植治疗组和经肝叶注射移植治疗组的肝组织中，CK8、CK18和AFP基因mRNA的相对转录量与模型对照组相比差异有统计学意义（P0.05），提示转录量明显增加。 4、免疫组化结果提示：经肝叶注射移植治疗组和经尾静脉移植治疗组中，移植后的hUCMSCs在鼠肝中诱导分化后表达人肝细胞相关蛋白CK18，随移植时间的延长，分化后的细胞从汇管区向中央静脉区迁移扩散。 5、经尾静脉和经肝叶注射移植治疗组的肝功能、移植细胞分化程度比较差异无统计学意义（P0.05）。结论： 1、贴壁培养法能从脐带中成功分离培养出hUCMSCs。 2、采用hUCMSCs移植治疗急性肝衰竭大鼠模型后，能促进肝功能和肝脏病理学修复。 3、移植后的hUCMSCs自身能分化为具有肝细胞功能的类肝样细胞，表达人肝细胞相关标志物。 4、经尾静脉与经肝叶注射移植治疗组疗效相似，且前者更易于应用和推广。
[Abstract]:Aim: to investigate the therapeutic effect of umbilical cord mesenchymal stem cells (CBMSCs) on rats with acute liver failure and to optimize the therapeutic approach. Methods: hUCMSCswere collected by tissue mass adherence method, and cell surface markers were detected by flow cytometry. Forty-eight healthy SD rats (6-8 weeks old, weight 180-220 g) were randomly divided into 4 groups: caudal vein transplantation group, liver lobe injection treatment group, model control group and blank control group. The animal model of acute hepatic failure was established with a single intraperitoneal injection of 2.5ml/kg CCL4 olive oil 1: 1) for 24 hours, the transplantation group was injected with hUCMSCs cell suspension through caudal vein and liver lobe respectively, and the serum was collected for the recovery of liver function at 0 h, 24 h, 48 h, 72 h, 96 h and 1 week after treatment. Liver samples were collected 3 days after treatment for 1 week and the pathological recovery was observed. Real-time PCR was used to detect the mRNA transcription level of human CK8, CK18 and AFP genes in the liver, and the expression of human CK18 was detected by immunohistochemical method. Results: 1hUCMSCs were successfully isolated from umbilical cord by adherent culture and expanded in vitro. The results of liver function and liver pathology showed that the contents of TBil and alt in transplantation group were significantly higher than those in model control group (P 0.05), and the degree of hepatocyte regeneration was increased. The inflammatory cells decreased and the pathological repair of liver was enhanced. The results of Real-time PCR showed that: 1 week after modeling, The relative transcription of CK8CK18 and AFP gene mRNA in the liver tissue of the tail vein transplantation group and the liver lobe injection treatment group was significantly different from that of the model control group, indicating that the transcription quantity was significantly increased. 4. 4%, the immunoreactivity of CK8 CK18 and AFP mRNA in the liver tissue was significantly higher than that in the model control group (P < 0.05). The immunohistochemical results showed that: in the liver lobe injection group and the tail vein transplantation group, Human hepatocyte associated protein CK18 was expressed in the transplanted hUCMSCs after induced differentiation in the rat liver, and the expression of CK18 protein was observed with the prolongation of the transplantation time. The differentiated cells migrated and diffused from the catchment area to the central venous area. There was no significant difference in the degree of differentiation of the transplanted cells between the caudal vein group and the transhepatic lobe injection group (P 0.05). Conclusion: 1. The method of adherent culture can successfully isolate and culture hUCMSCs.2 from umbilical cord. After transplantation of hUCMSCs, the rat model of acute liver failure can be treated. It can promote liver function and liver pathological repair. 3. After transplantation, hUCMSCs can differentiate into hepatoid cells with hepatocyte function. Expression of human hepatocyte markers. 4. The therapeutic effect of transcaudal vein was similar to that of liver lobe injection, and the former was easier to be applied and popularized.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R575.3

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