本文選題：間充質(zhì)干細(xì)胞　切入點(diǎn)：肝細(xì)胞　出處：《暨南大學(xué)》2012年碩士論文

【摘要】：目的 探討人臍帶間充質(zhì)干細(xì)胞（mesenchymal stem cells，MSCs）移植到肝部分切除模型大鼠體內(nèi)能否存活，并分化為肝細(xì)胞，通過相關(guān)的實(shí)驗(yàn)方法檢測(cè)人臍帶MSCs能否改善肝損傷大鼠的肝功能，進(jìn)一步為人臍帶MSCs運(yùn)用于臨床提供動(dòng)物實(shí)驗(yàn)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(xì)胞，培養(yǎng)并檢測(cè)干細(xì)胞的細(xì)胞表面標(biāo)志。取第5代人臍帶MSCs用PKH26標(biāo)記。將72只SD大鼠隨機(jī)分成三組，即實(shí)驗(yàn)組、模型組和對(duì)照組，每組24只。三組SD大鼠，用戊巴比妥腹腔注射麻醉。實(shí)驗(yàn)組：雙重結(jié)扎大鼠肝臟膈葉的肝蒂,切除肝葉，并將PKH26染色液標(biāo)記過的細(xì)胞懸液注入實(shí)驗(yàn)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(duì)照組：僅翻動(dòng)肝臟，不切肝，予以門靜脈注射等體積的生理鹽水。各組術(shù)后取背部皮下注射生理鹽水30ml/kg，補(bǔ)充術(shù)中體液的丟失。各組于術(shù)后第3、9、15、21天，隨機(jī)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(xì)胞能否向肝細(xì)胞分化,能否表達(dá)肝細(xì)胞的標(biāo)記物白蛋白。檢測(cè)指標(biāo)：1.大鼠血清谷丙轉(zhuǎn)氨酶、谷草轉(zhuǎn)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(duì)肝臟的病變程度進(jìn)行病理評(píng)分。 結(jié)果 人臍帶MSCs能在體外大量擴(kuò)增，移植到肝部分切除模型大鼠肝臟后細(xì)胞能夠存活，并表達(dá)肝細(xì)胞標(biāo)記物白蛋白。PKH26標(biāo)記后細(xì)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(biāo)記細(xì)胞，免疫熒光染色大多數(shù)標(biāo)記細(xì)胞白蛋白染色陽性，并發(fā)綠色熒光。對(duì)照組相關(guān)的實(shí)驗(yàn)檢測(cè)指標(biāo)無明顯變化，與實(shí)驗(yàn)組和模型組比較有統(tǒng)計(jì)學(xué)意義（p0.05）。實(shí)驗(yàn)組和模型組比較，實(shí)驗(yàn)組的ALT、AST的檢測(cè)值在第9天明顯低于模型組，TBA的檢測(cè)值在第21天明顯低于模型組，ALB檢測(cè)值在第15天明顯高于模型組，以上時(shí)間點(diǎn)兩組比較有統(tǒng)計(jì)學(xué)意義（p0.05）。 結(jié)論 1.人臍帶間充質(zhì)干細(xì)胞在體外可以成功分離并且培養(yǎng)，，其具有很強(qiáng)的增殖、分化潛能。 2.人臍帶間充質(zhì)干細(xì)胞移植至大鼠體內(nèi)能夠存活并分化為肝細(xì)胞,可改善肝損傷大鼠的肝功能，人臍帶間充質(zhì)干細(xì)胞可作為肝臟細(xì)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(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R657.3;R-332

【參考文獻(xiàn)】

相關(guān)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(xué)報(bào);1999年05期

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