本文選題：肝纖維化 + 細(xì)胞治療��； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：目的:建立一種穩(wěn)定的wistar大鼠慢性肝纖維化模型。通過脾臟注射高密度培養(yǎng)骨髓細(xì)胞,觀察移植細(xì)胞能否改善大鼠肝纖維化,并研究其阻止肝臟慢性纖維化進(jìn)展可能的作用機(jī)制,探討高密度培養(yǎng)骨髓細(xì)胞治療慢性肝纖維化的應(yīng)用價值。方法:1.通過四氯化碳(CCl4)腹腔注射,建立wistar大鼠慢性肝纖維化模型;2.將建模成功大鼠隨機(jī)分成三組,高密度培養(yǎng)組(HD)、常規(guī)密度培養(yǎng)組(RD)和PBS組。經(jīng)脾臟分別注射經(jīng)過熒光素CM-Di L標(biāo)記的2×106的骨髓HD細(xì)胞、RD細(xì)胞和200μl PBS;3.血清分析檢測大鼠肝功能指標(biāo);苦味酸天狼星紅(PSR)和Masson染色檢測肝臟中膠原合成與分布;免疫組化檢測肝臟中I型膠原、α-SMA的表達(dá),q RT-PCR檢測組織中α2-procollagen、α-SMA、fibronectin和TGF-β的基因表達(dá);4.組織冰凍切片后行Ⅰ型膠原免疫熒光染色,熒光顯微鏡下觀察CM-Di L陽性的移植細(xì)胞在肝臟組織中的數(shù)量及分布情況;5.肝臟組織切片行Ki-67、CD31免疫組化法染色,觀察細(xì)胞增殖與血管生成;q RT-PCR檢測組織中肝細(xì)胞生長因子(HGF)、血管內(nèi)皮生長因子(VEGF)的基因表達(dá)情況。結(jié)果:1.重復(fù)多次腹腔注射CCl4,可以建立穩(wěn)定的大鼠慢性肝纖維化模型。2.大鼠骨髓細(xì)胞經(jīng)高密度培養(yǎng)后,細(xì)胞高表達(dá)CD31、CD133和KDR,體外成管實(shí)驗(yàn)顯示高密度培養(yǎng)細(xì)胞比普通密度培養(yǎng)細(xì)胞具有更強(qiáng)的成管能力,提示高密度培養(yǎng)的骨髓細(xì)胞富含血管內(nèi)皮前體細(xì)胞。3.造模大鼠血清谷草轉(zhuǎn)氨酶和谷丙轉(zhuǎn)氨酶明顯增高,白蛋白降低,PBS治療組無明顯改善,細(xì)胞移植組血清谷草轉(zhuǎn)氨酶和谷丙轉(zhuǎn)氨酶降低,白蛋白升高,HD組優(yōu)于RD組,提示高密度培養(yǎng)細(xì)胞的作用更好。4.造模大鼠肝臟HE染色發(fā)現(xiàn)肝臟重構(gòu),假小葉形成,細(xì)胞移植后假小葉減少,PSR和Masson染色發(fā)現(xiàn)細(xì)胞外膠原沉積減少。免疫組化分析顯示細(xì)胞移植組肝臟中Ⅰ型膠原和α-SMA的表達(dá)減少,q RT-PCR檢測顯示細(xì)胞移植組α2-procollagen、α-SMA和fibronectin的基因表達(dá)降低,HD組優(yōu)于RD組。5.肝臟組織免疫組化染色顯示HD組中CD31陽性的血管多于RD組,Ki67陽性的增殖細(xì)胞數(shù)多于RD組,肝臟組織q RT-PCR檢測顯示促肝細(xì)胞增殖的HGF和促血管生成的VEGF基因在HD組中表達(dá)明顯高于RD組。結(jié)論:骨髓高密度培養(yǎng)細(xì)胞對緩解大鼠肝纖維化、改善肝功能和促進(jìn)肝臟再生有較好效果,這種簡單、低成本的培養(yǎng)方式可以為骨髓細(xì)胞的未來臨床應(yīng)用提供一種有效的擴(kuò)增方法。
[Abstract]:Objective: to establish a stable model of chronic hepatic fibrosis in wistar rats. High density bone marrow cells were injected into the spleen to observe whether the transplanted cells could improve the hepatic fibrosis in rats and to study the possible mechanism of preventing the progression of chronic hepatic fibrosis. To investigate the value of high density cultured bone marrow cells in the treatment of chronic hepatic fibrosis. Method 1: 1. The chronic hepatic fibrosis model of wistar rats was established by intraperitoneal injection of CCL 4 (CCL 4). The rats were randomly divided into three groups: high density culture group, conventional density culture group and PBS group. Splenic injection of 2 脳 10 ~ 6 bone marrow HD cells labeled with fluorescein CM-Di L and 200 渭 l / L PBS3, respectively. Serum analysis was used to detect liver function, Masson staining and picric acid Sirius) staining were used to detect collagen synthesis and distribution in the liver, and immunohistochemical staining was used to detect the expression of type I collagen, 偽 -SMA expression and 偽 2-procagenolll, 偽 -SMAfibronectin and TGF- 尾 gene expression in the liver of rats. The expression of 偽 2 procagenoll1, 偽 -SMAfifibronectin and TGF- 尾 was detected by immunohistochemistry. The number and distribution of CM-Di L positive transplanted cells in liver tissue were observed under fluorescence microscope. The expression of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) in liver tissue were detected by Ki-67-CD31 immunohistochemical method. The expression of VEGF and VEGF was detected by cell proliferation and angiogenesis Q RT-PCR. The result is 1: 1. Repeated intraperitoneal injection of CCL 4 can establish a stable model of chronic hepatic fibrosis in rats. 2. 2. After cultured with high density, rat bone marrow cells expressed CD31, CD133 and KDR. the in vitro tube forming experiment showed that the cultured cells with high density had stronger ability of tube-forming than those cultured with normal density. These results suggest that high density cultured bone marrow cells are rich in endothelial progenitor cells. 3. Serum glutamic oxaloacetic transaminase and alanine aminotransferase were significantly increased in the model rats, but no significant improvement was found in the PBS treatment group, while the serum alanine aminotransferase and alanine aminotransferase levels were decreased in the cell transplantation group. The serum alanine aminotransferase in HD group was better than that in Rd group. The results suggest that the effect of high density cultured cells is better. 4. Liver remodeling and pseudolobule formation were found by HE staining in the liver of the model rats. PSR and Masson staining showed that extracellular collagen deposition was decreased after cell transplantation. Immunohistochemical analysis showed that the expression of type I collagen and 偽 -SMA in the liver of the transplanted group was decreased. The expression of 偽 2-procollagenase, 偽 -SMA and fibronectin in the transplanted group was lower than that in the Rd group. The expression of 偽 -SMA and 偽 -SMA gene in HD group was higher than that in Rd group. Immunohistochemical staining of liver tissue showed that the number of CD31 positive blood vessels in HD group was more than that in Rd group, and the number of proliferative cells positive in Rd group was more than that in Rd group. The expression of HGF and VEGF in HD group was significantly higher than that in Rd group. Conclusion: bone marrow high density cultured cells have better effect on relieving liver fibrosis, improving liver function and promoting liver regeneration in rats. Low-cost culture can provide an effective method for the future clinical application of bone marrow cells.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575.2

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