成体肝脏干细胞的体外分离、培养及鉴定

发布时间：2018-03-08 09:23

本文选题：肝癌　切入点：肝脏干细胞　出处：《吉林大学》2006年硕士论文　论文类型：学位论文

【摘要】：肝脏是人体生命的重要器官,具有合成与储存养料,分泌胆汁,解毒和防御等维持生命活动所必需的多种生理功能。然而肝脏又极易受到多种物质(如病毒、化学致癌剂)的损伤,一旦受到损伤或出现病变,将严重影响机体的功能,甚至威胁生命。因此,肝脏保护与肝脏再生的研究,具有非常重要的理论和实际意义。目的:从成人肝癌的癌旁组织中分离及培养肝前体细胞-卵圆细胞。并确定卵圆细胞体外生长和定向分化的条件,为肝干细胞的深入研究奠定基础,最终建立成人肝脏干细胞系。方法:首先将术中所取肝脏癌旁组织经免疫荧光组织化学染色鉴定后证实癌旁组织中存在肝脏干细胞,同时进行体外细胞原代培养,体外培养3周后,经过免疫磁化分离,获得C-kit+细胞,再次经免疫荧光组织化学鉴定,证实体外分离纯化的C-kit+细胞为肝脏前体细胞。结果:术中所取癌旁组织经免疫荧光组织化学染色后可见癌旁组织汇管区存在AFP及CK19双阳性细胞,即卵圆细胞。并以癌旁组织作为肝脏干细胞的来源,体外原代培养3周后,可见细胞呈集落样生长,传代后细胞生长速度加快,呈簇状生长。约传代2-3代后,将细胞经免疫磁化分离,获得C-kit+细胞,再次经免疫荧光组织化学染色,可见分离纯化后的C-kit+细胞共同表达AFP及CK19,具备双向分化潜能,即向成熟干细胞及胆管上皮细胞分化。
[Abstract]:The liver is an important organ of human life. It can synthesize and store nutrients, secrete bile, detoxify and defend vital activities. However, the liver is vulnerable to many kinds of substances (such as viruses). The damage of chemical carcinogens will seriously affect the function of the body and even threaten life once it is damaged or damaged. Therefore, the study of liver protection and liver regeneration has very important theoretical and practical significance. Objective: to isolate and culture liver precursor cells-oval cells from adjacent tissues of adult liver cancer, and to determine the conditions for the growth and directional differentiation of oval cells in vitro, so as to lay a foundation for the further study of liver stem cells. Finally, adult liver stem cell lines were established. Methods: liver stem cells were identified by immunofluorescence histochemical staining in the paracancerous tissues of the liver, and the primary cells were cultured in vitro at the same time. After 3 weeks of culture in vitro, the liver stem cells were separated by immunomagnetization. C-kit cells were obtained and identified by immunofluorescence histochemistry, which confirmed that the purified C-kit cells were liver progenitor cells in vitro. Results: AFP and CK19 double positive cells, i.e. oval cells, were found in the adjacent tissues after immunofluorescence histochemical staining. The paracancerous tissues were used as the source of hepatic stem cells and cultured in vitro for 3 weeks. It can be seen that the cells grow like colony, and the growth rate of the cells increases after passage. After about 2-3 passages, the C-kit cells are separated by immunomagnetic method, and then stained by immunofluorescence histochemistry. It can be seen that the purified C-kit cells co-express AFP and CK19, and have the potential to differentiate into mature stem cells and bile duct epithelial cells.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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