本文選題：臍帶間充質(zhì)干細(xì)胞 + 急性肝衰竭�。� 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：探討臍帶間充質(zhì)干細(xì)胞對急性肝衰竭大鼠的治療作用，并優(yōu)化移植治療途徑。 方法：采用組織塊貼壁法收集hUCMSCs，流式細(xì)胞術(shù)檢測細(xì)胞表面標(biāo)志物。清潔級健康SD大鼠48只（6-8周齡，體重180-220g，雌雄不論），采用完全隨機對照法均分為4組：經(jīng)尾靜脈注射移植治療組，經(jīng)肝葉注射移植治療組，模型對照組，空白對照組。急性肝衰竭動物模型采用一次性腹腔注射2.5ml/kg CCL4橄欖油（1：1）混合溶液建立，，24h后移植治療組分別經(jīng)尾靜脈和肝葉注射hUCMSCs細(xì)胞懸液；治療后0h、24h、48h、72h、96h和1w收集血清分析肝功能恢復(fù)情況；治療后3d、1w、2w留取肝臟標(biāo)本，觀察病理學(xué)恢復(fù)情況，用Real-time PCR檢測鼠肝中人CK8、CK18和AFP基因mRNA轉(zhuǎn)錄水平，免疫組化法檢測人CK18的表達(dá)。 結(jié)果： 1、hUCMSCs通過貼壁培養(yǎng)法能成功從臍帶中分離并在體外大量擴(kuò)增，流式細(xì)胞儀鑒定符合MSCs表面標(biāo)志物特性。 2、肝功能和肝臟病理學(xué)結(jié)果顯示：移植治療組肝功能指標(biāo)TBil和ALT含量較模型對照組有明顯恢復(fù)（P0.05），肝細(xì)胞再生程度提高，炎細(xì)胞減少，肝臟病理學(xué)修復(fù)作用增強。 3、Real-time PCR結(jié)果顯示：在造模后1w、2w時，經(jīng)尾靜脈移植治療組和經(jīng)肝葉注射移植治療組的肝組織中，CK8、CK18和AFP基因mRNA的相對轉(zhuǎn)錄量與模型對照組相比差異有統(tǒng)計學(xué)意義（P0.05），提示轉(zhuǎn)錄量明顯增加。 4、免疫組化結(jié)果提示：經(jīng)肝葉注射移植治療組和經(jīng)尾靜脈移植治療組中，移植后的hUCMSCs在鼠肝中誘導(dǎo)分化后表達(dá)人肝細(xì)胞相關(guān)蛋白CK18，隨移植時間的延長，分化后的細(xì)胞從匯管區(qū)向中央靜脈區(qū)遷移擴(kuò)散。 5、經(jīng)尾靜脈和經(jīng)肝葉注射移植治療組的肝功能、移植細(xì)胞分化程度比較差異無統(tǒng)計學(xué)意義（P0.05）。 結(jié)論： 1、貼壁培養(yǎng)法能從臍帶中成功分離培養(yǎng)出hUCMSCs。 2、采用hUCMSCs移植治療急性肝衰竭大鼠模型后，能促進(jìn)肝功能和肝臟病理學(xué)修復(fù)。 3、移植后的hUCMSCs自身能分化為具有肝細(xì)胞功能的類肝樣細(xì)胞，表達(dá)人肝細(xì)胞相關(guān)標(biāo)志物。 4、經(jīng)尾靜脈與經(jīng)肝葉注射移植治療組療效相似，且前者更易于應(yīng)用和推廣。
[Abstract]:Aim: to investigate the therapeutic effect of umbilical cord mesenchymal stem cells (CBMSCs) on rats with acute liver failure and to optimize the therapeutic approach. Methods: hUCMSCswere collected by tissue mass adherence method, and cell surface markers were detected by flow cytometry. Forty-eight healthy SD rats (6-8 weeks old, weight 180-220 g) were randomly divided into 4 groups: caudal vein transplantation group, liver lobe injection treatment group, model control group and blank control group. The animal model of acute hepatic failure was established with a single intraperitoneal injection of 2.5ml/kg CCL4 olive oil 1: 1) for 24 hours, the transplantation group was injected with hUCMSCs cell suspension through caudal vein and liver lobe respectively, and the serum was collected for the recovery of liver function at 0 h, 24 h, 48 h, 72 h, 96 h and 1 week after treatment. Liver samples were collected 3 days after treatment for 1 week and the pathological recovery was observed. Real-time PCR was used to detect the mRNA transcription level of human CK8, CK18 and AFP genes in the liver, and the expression of human CK18 was detected by immunohistochemical method. Results: 1hUCMSCs were successfully isolated from umbilical cord by adherent culture and expanded in vitro. The results of liver function and liver pathology showed that the contents of TBil and alt in transplantation group were significantly higher than those in model control group (P 0.05), and the degree of hepatocyte regeneration was increased. The inflammatory cells decreased and the pathological repair of liver was enhanced. The results of Real-time PCR showed that: 1 week after modeling, The relative transcription of CK8CK18 and AFP gene mRNA in the liver tissue of the tail vein transplantation group and the liver lobe injection treatment group was significantly different from that of the model control group, indicating that the transcription quantity was significantly increased. 4. 4%, the immunoreactivity of CK8 CK18 and AFP mRNA in the liver tissue was significantly higher than that in the model control group (P < 0.05). The immunohistochemical results showed that: in the liver lobe injection group and the tail vein transplantation group, Human hepatocyte associated protein CK18 was expressed in the transplanted hUCMSCs after induced differentiation in the rat liver, and the expression of CK18 protein was observed with the prolongation of the transplantation time. The differentiated cells migrated and diffused from the catchment area to the central venous area. There was no significant difference in the degree of differentiation of the transplanted cells between the caudal vein group and the transhepatic lobe injection group (P 0.05). Conclusion: 1. The method of adherent culture can successfully isolate and culture hUCMSCs.2 from umbilical cord. After transplantation of hUCMSCs, the rat model of acute liver failure can be treated. It can promote liver function and liver pathological repair. 3. After transplantation, hUCMSCs can differentiate into hepatoid cells with hepatocyte function. Expression of human hepatocyte markers. 4. The therapeutic effect of transcaudal vein was similar to that of liver lobe injection, and the former was easier to be applied and popularized.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R575.3

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