本文選題：間充質干細胞　切入點：肝細胞　出處：《暨南大學》2012年碩士論文

【摘要】：目的 探討人臍帶間充質干細胞（mesenchymal stem cells，MSCs）移植到肝部分切除模型大鼠體內能否存活，并分化為肝細胞，通過相關的實驗方法檢測人臍帶MSCs能否改善肝損傷大鼠的肝功能，進一步為人臍帶MSCs運用于臨床提供動物實驗的研究依據(jù)。 方法 用膠原酶消化法從人的臍帶中分離出干細胞，培養(yǎng)并檢測干細胞的細胞表面標志。取第5代人臍帶MSCs用PKH26標記。將72只SD大鼠隨機分成三組，即實驗組、模型組和對照組，每組24只。三組SD大鼠，用戊巴比妥腹腔注射麻醉。實驗組：雙重結扎大鼠肝臟膈葉的肝蒂,切除肝葉，并將PKH26染色液標記過的細胞懸液注入實驗組大鼠的門靜脈。模型組：按上述方法切除相同比例的肝臟，并予以門靜脈注射等體積的生理鹽水。對照組：僅翻動肝臟，不切肝，予以門靜脈注射等體積的生理鹽水。各組術后取背部皮下注射生理鹽水30ml/kg，補充術中體液的丟失。各組于術后第3、9、15、21天，隨機取6只大鼠，開腹取出大鼠肝臟，觀察人臍帶MSCs能否存活,以及移植細胞能否向肝細胞分化,能否表達肝細胞的標記物白蛋白。檢測指標：1.大鼠血清谷丙轉氨酶、谷草轉氨酶的活性；2.大鼠血清膽汁酸、白蛋白；3.大鼠的肝臟指數(shù)；4.制作肝臟病理切片并對肝臟的病變程度進行病理評分。 結果 人臍帶MSCs能在體外大量擴增，移植到肝部分切除模型大鼠肝臟后細胞能夠存活，并表達肝細胞標記物白蛋白。PKH26標記后細胞在熒光顯微鏡下發(fā)紅色熒光。熒光顯微鏡下可見肝臟冰凍切片中散在分布的標記細胞，免疫熒光染色大多數(shù)標記細胞白蛋白染色陽性，并發(fā)綠色熒光。對照組相關的實驗檢測指標無明顯變化，與實驗組和模型組比較有統(tǒng)計學意義（p0.05）。實驗組和模型組比較，實驗組的ALT、AST的檢測值在第9天明顯低于模型組，TBA的檢測值在第21天明顯低于模型組，ALB檢測值在第15天明顯高于模型組，以上時間點兩組比較有統(tǒng)計學意義（p0.05）。 結論 1.人臍帶間充質干細胞在體外可以成功分離并且培養(yǎng)，，其具有很強的增殖、分化潛能。 2.人臍帶間充質干細胞移植至大鼠體內能夠存活并分化為肝細胞,可改善肝損傷大鼠的肝功能，人臍帶間充質干細胞可作為肝臟細胞移植的重要來源。
[Abstract]:Purpose. To investigate whether human umbilical cord mesenchymal stem cells can survive and differentiate into hepatocytes after partial hepatectomy in rats, and to investigate whether human umbilical cord MSCs can improve liver function in rats with liver injury. Further more, the application of human umbilical cord MSCs in clinical research provides evidence for animal experiments. Method. Stem cells were isolated from human umbilical cord by collagenase digestion, and the surface markers of stem cells were cultured and detected. The fifth passage of human umbilical cord MSCs was labeled with PKH26. 72 SD rats were randomly divided into three groups: experimental group, model group and control group. 24 rats in each group were anesthetized by intraperitoneal injection of pentobarbital. Experimental group: double ligation of liver pedicle of the phrenic lobe of rat liver, resection of liver lobe, The cell suspension labeled with PKH26 staining solution was injected into the portal vein of the experimental group. The model group: the liver was resected in the same proportion according to the above method, and the same volume of normal saline was injected into the portal vein. In the control group, only the liver was turned over, but the liver was not cut off. The rats in each group were injected with saline of the same volume by portal vein, 30 ml / kg of normal saline was injected subcutaneously into the back of each group after operation, and the body fluid was lost during rehydration. Six rats were randomly selected from each group on the 3rd day, 1521 days after operation, and the liver was taken out by laparotomy. To observe whether human umbilical cord MSCs can survive, whether the transplanted cells can differentiate into hepatocytes and whether they can express albumin, the marker of hepatocytes. Detection index: 1. The activity of serum alanine aminotransferase (alt) and aspartate aminotransferase (alt) in rat serum. Albumin 3.The liver index of rats was 4.The pathological sections of the liver were made and the pathological grade of the liver was evaluated. Results. Human umbilical cord MSCs can be expanded in vitro, and the cells can survive after transplantation to the liver of partially resected rat liver. The labeled hepatocyte albumin. PKH26 labeled hepatocytes showed red fluorescence under fluorescence microscope. The distribution of labeled cells in frozen sections of liver was observed under fluorescence microscope, and most of the labeled cells were positive for albumin staining by immunofluorescence staining. Compared with the experimental group and the model group, there was significant difference between the experimental group and the model group, the experimental group and the model group, the experimental group and the model group, the experimental group and the model group, the experimental group and the model group were compared with each other. The detection value of alt AST in the experimental group was significantly lower than that in the model group on the 9th day and the detection value of TBA in the model group on the 21st day was significantly lower than that in the model group on the 15th day. The above time points were significantly higher in the two groups than in the model group. Conclusion. 1. Human umbilical cord mesenchymal stem cells were successfully isolated and cultured in vitro. 2.Human umbilical cord mesenchymal stem cells can survive and differentiate into hepatocytes, which can improve the liver function of injured rats. Human umbilical cord mesenchymal stem cells can be used as an important source of liver cell transplantation.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R657.3;R-332

【參考文獻】

相關期刊論文 前1條

1 徐存拴,public.xxptt.ha.cn,夏民,盧愛靈,李效陽,李永輝,趙緒永,胡軼紅;熱休克蛋白、蛋白水解酶和磷酸酶在大鼠肝再生中的含量和活性變化[J];生理學報;1999年05期

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