大鼠胎肝干细胞的分离培养与鉴定以及免疫源性的研究

发布时间：2018-05-14 22:05

本文选题：大鼠 + 胎肝干细胞　；参考：《吉林大学》2007年硕士论文

【摘要】： 目前,关于干细胞的研究成为医学界的热点,其可观前景在于可以作为组织工程的种子细胞,构建人工器官,在临床器官移植中发挥重要作用,但是器官移植的障碍还有免疫排斥问题,至今研究很少。关于胎肝干细胞的研究,目前集中在体外分离培养、鉴定及诱导分化,但对其免疫源性的研究国内外未见报道。本研究采用机械剪碎酶消化法分离大鼠胎肝干细胞,进一步的贴壁培养来提高细胞纯度,通过细胞免疫荧光化学染色、免疫细胞化学染色、过碘酸-雪夫氏染色及白蛋白检测等方法对分离培养的大鼠胎肝干细胞进行鉴定,通过免疫细胞化学染色及流式细胞术对其免疫源性做了初步研究,结果如下: 1大鼠胎肝干细胞的形态观察细胞接种24小时后,普通光镜下可见细胞贴壁,贴壁细胞形态一致,呈卵圆形,核大,胞质少;48小时观察细胞为圆形,大小几乎一致;第5-7天,细胞长成片,呈集落样,集落由大小不等的致密圆形细胞组成,边缘清楚;8天细胞铺展,呈上皮样。细胞原代或传代培养至接种第12天时,细胞变大,成圆形或多角形,细胞浆内可见颗粒,生长变得缓慢。 2大鼠胎肝干细胞的鉴定通过细胞免疫荧光化学染色检测到大鼠胎肝干细胞甲胎蛋白(alpha fetoprotein, AFP)、细胞角蛋白18(cytokeratin 18, CK18)细胞角蛋白19(cytokeratin 19, CK19)呈阳性表达,成鼠肝细胞都呈阴性;免疫细胞化学染色检测到胎肝干细胞OV6阳性表达,而成鼠肝细胞阴性表达;胎肝干细胞的过碘酸-雪夫氏染色(periodic acid-schiff stain, PAS染色)阳性比成鼠肝细胞弱,但也说明有糖原物质存在;利用半自动生化分析仪检测到了白蛋白的存在,这些结果可以证明所得到的细胞为胎肝干细胞。 3大鼠胎肝干细胞的免疫源性检测免疫细胞化学染色检测到胎肝干细胞主要组织相容性复合体(major histocompability complex-1, MHC-1)的表达,但弱于成鼠肝细胞;通过流式细胞术比较了大鼠胎肝干细胞和成鼠肝细胞MHC-1的表达,结果前者31.26%±4.56,后者表达42.94%±3.51,P0.05,有统计学差异。以上结果表明,用免疫荧光技术分析,分离的细胞纯度可达95%以上,所选择的高度特异的标记物都呈阳性表达,对照成鼠肝细胞阴性表达,说明分离培养的细胞为大鼠胎肝干细胞,其表达MHC-1分子低于成熟肝细胞,提示胎肝干细胞的免疫源性可能较成熟肝细胞低。
[Abstract]:At present, the research on stem cells has become a hot topic in the medical field. Its prospect is that it can be used as seed cells for tissue engineering to construct artificial organs and play an important role in clinical organ transplantation. However, the obstacle of organ transplantation is also the problem of immune rejection, so far little research has been done. The research on fetal liver stem cells is focused on isolation, culture, identification and induction of differentiation in vitro, but its immunogenicity has not been reported at home and abroad. In this study, rat fetal liver stem cells were isolated by mechanical shredding enzyme digestion, and further adherent culture was carried out to improve the purity of the cells. The cells were stained by immunofluorescence staining and immunocytochemical staining. The isolated and cultured rat fetal liver stem cells were identified by periodate and Scheffer's staining and albumin detection. The immunogenicity was studied by immunocytochemical staining and flow cytometry. The results were as follows: Morphological observation of rat fetal liver stem cells 24 hours after inoculation, the cells adhered to the wall under ordinary light microscope. The adherent cells were identical in shape, oval, large nucleus, round in cytoplasm for 48 hours and almost identical in size. The colony was composed of dense round cells of different sizes, and the edges were clear for 8 days. At the 12th day of inoculation, the cells became larger, rounded or polygonal, and the granules in the cytoplasm became slow. Identification of rat fetal liver stem cells The positive expression of alpha feto protein (AFPN), cytokeratin 18(cytokeratin 18 (CK18) and cytokeratin 19(cytokeratin 19 (CK19) in rat fetal liver stem cells were detected by immunofluorescence staining. The positive expression of OV6 in fetal liver stem cells was detected by immunocytochemistry, but the expression of OV6 was negative in adult liver stem cells, and the positive rate of periodate acid-schiff staining (PAS) in fetal liver stem cells was weaker than that in adult rat hepatocytes, but it also indicated that glycogen existed in fetal liver stem cells. The presence of albumin was detected by semi-automatic biochemical analyzer, which proved that the obtained cells were fetal liver stem cells. Immunogenic detection of rat fetal liver stem cells The expression of major histocompability complex-1 (MHC-1), a major histocompatibility complex of fetal liver stem cells, was detected by immunocytochemistry, but weaker than that of rat hepatocytes, and the expression of MHC-1 in rat fetal liver stem cells and rat hepatocytes was compared by flow cytometry. Results the former was 31.26% 卤4.56, and the latter was 42.94% 卤3.51% (P 0.05). The results showed that the purity of the isolated cells was over 95% by immunofluorescence technique, and the highly specific markers were all positive, and the negative expression of hepatocytes was found in the control rats. The results indicated that the cells isolated and cultured were rat fetal liver stem cells, and the expression of MHC-1 was lower than that of mature hepatocytes, suggesting that the immunogenicity of fetal liver stem cells might be lower than that of mature hepatocytes.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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