本文選題：大鼠 + 胎肝干細胞��； 參考：《吉林大學》2007年碩士論文

【摘要】： 目前,關于干細胞的研究成為醫(yī)學界的熱點,其可觀前景在于可以作為組織工程的種子細胞,構建人工器官,在臨床器官移植中發(fā)揮重要作用,但是器官移植的障礙還有免疫排斥問題,至今研究很少。關于胎肝干細胞的研究,目前集中在體外分離培養(yǎng)、鑒定及誘導分化,但對其免疫源性的研究國內外未見報道。本研究采用機械剪碎酶消化法分離大鼠胎肝干細胞,進一步的貼壁培養(yǎng)來提高細胞純度,通過細胞免疫熒光化學染色、免疫細胞化學染色、過碘酸-雪夫氏染色及白蛋白檢測等方法對分離培養(yǎng)的大鼠胎肝干細胞進行鑒定,通過免疫細胞化學染色及流式細胞術對其免疫源性做了初步研究,結果如下: 1大鼠胎肝干細胞的形態(tài)觀察 細胞接種24小時后,普通光鏡下可見細胞貼壁,貼壁細胞形態(tài)一致,呈卵圓形,核大,胞質少;48小時觀察細胞為圓形,大小幾乎一致;第5-7天,細胞長成片,呈集落樣,集落由大小不等的致密圓形細胞組成,邊緣清楚;8天細胞鋪展,呈上皮樣。細胞原代或傳代培養(yǎng)至接種第12天時,細胞變大,成圓形或多角形,細胞漿內可見顆粒,生長變得緩慢。 2大鼠胎肝干細胞的鑒定 通過細胞免疫熒光化學染色檢測到大鼠胎肝干細胞甲胎蛋白(alpha fetoprotein, AFP)、細胞角蛋白18(cytokeratin 18, CK18)細胞角蛋白19(cytokeratin 19, CK19)呈陽性表達,成鼠肝細胞都呈陰性;免疫細胞化學染色檢測到胎肝干細胞OV6陽性表達,而成鼠肝細胞陰性表達;胎肝干細胞的過碘酸-雪夫氏染色(periodic acid-schiff stain, PAS染色)陽性比成鼠肝細胞弱,但也說明有糖原物質存在;利用半自動生化分析儀檢測到了白蛋白的存在,這些結果可以證明所得到的細胞為胎肝干細胞。 3大鼠胎肝干細胞的免疫源性檢測 免疫細胞化學染色檢測到胎肝干細胞主要組織相容性復合體(major histocompability complex-1, MHC-1)的表達,但弱于成鼠肝細胞;通過流式細胞術比較了大鼠胎肝干細胞和成鼠肝細胞MHC-1的表達,結果前者31.26%±4.56,后者表達42.94%±3.51,P0.05,有統(tǒng)計學差異。 以上結果表明,用免疫熒光技術分析,分離的細胞純度可達95%以上,所選擇的高度特異的標記物都呈陽性表達,對照成鼠肝細胞陰性表達,說明分離培養(yǎng)的細胞為大鼠胎肝干細胞,其表達MHC-1分子低于成熟肝細胞,提示胎肝干細胞的免疫源性可能較成熟肝細胞低。
[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% 鹵4.56, and the latter was 42.94% 鹵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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R329

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本文編號：1889653