本文選題：非酒精性脂肪肝病 + 肝星狀細(xì)胞株T6　； 參考：《延邊大學(xué)》2017年碩士論文

【摘要】：目的:本實(shí)驗(yàn)通過(guò)測(cè)定抑瘤素M(oncostatinM,OSM)對(duì)大鼠肝星狀細(xì)胞株T6(hepatic stellate cell line T6,HSC-T6)的活化及增殖的影響,以及抑瘤素MⅡ型受體(oncostatinM receptor β,OSMRβ)在膽堿缺乏氨基酸(cho-line-deficient-L-amino acid-defined,CDAA)飼料誘導(dǎo)的大鼠非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)模型肝臟中的表達(dá),探討NAFLD發(fā)病的可能機(jī)制。方法:體外實(shí)驗(yàn)采用HSC-T6細(xì)胞,HSC-T6細(xì)胞培養(yǎng)1天、4天、7天后提取蛋白質(zhì)用Western blotting測(cè)定α-平滑肌激動(dòng)蛋白(α-smooth muscle actin,α-SMA)和OSMRβ的表達(dá)。在96板孔中加入3×103個(gè)/孔HSC-T6細(xì)胞適應(yīng)性培養(yǎng)4小時(shí)后,分別加入0、5、10、20ng/ml的大鼠OSM培養(yǎng)24小時(shí)和48小時(shí),再添加10μl Cell Counting Kit-8(CCK-8)試劑2個(gè)小時(shí)后在450nm波長(zhǎng)測(cè)光密度(OD)值。體內(nèi)實(shí)驗(yàn)選取6周齡,體重160g±20g的SD雄性大鼠30只。適應(yīng)性喂養(yǎng)7天后,隨機(jī)平均分為正常對(duì)照組和模型組。正常組給予普通飼料;模型組給予CDAA飼料,實(shí)驗(yàn)周期為12周。實(shí)驗(yàn)結(jié)束時(shí),乙醚麻醉并處死大鼠后取其肝右葉,用于制作病理標(biāo)本及目的蛋白檢測(cè)。用免疫組化法檢測(cè)肝組織切片中α-SMA的表達(dá)情況。用Western blotting法測(cè)定肝臟OSMRβ的表達(dá)。結(jié)果:1.HSC-T6細(xì)胞培養(yǎng)1天、4天和7天,隨著培養(yǎng)時(shí)間的延長(zhǎng),HSC-T6細(xì)胞表達(dá)α-SMA和OSMRβ水平逐漸升高(P0.05)。2.添加OSM可促進(jìn)HSC-T6細(xì)胞增殖(P0.05),其促進(jìn)作用呈劑量和時(shí)間依賴性。3.CDAA組肝臟的α-SMA 水平較正常對(duì)照組升高(n=15,P0.05),同時(shí)CDAA組肝臟表達(dá)OSMRβ,且高于正常對(duì)照組(n=15,P0.05)。結(jié)論:1.HSC-T6 細(xì)胞表達(dá) OSMRβ。2.OSM促進(jìn)HSC-T6細(xì)胞活化和增殖。3.隨著CDAA誘導(dǎo)大鼠肝纖維化,肝臟表達(dá)OSMRβ增加,提示OSM通過(guò)與HSC的OSM受體結(jié)合促進(jìn)肝臟炎癥反應(yīng)和纖維化,在NAFLD發(fā)生發(fā)展中發(fā)揮作用。
[Abstract]:Objective: to investigate the effect of tumor suppressor Mncostatin OSMon on the activation and proliferation of rat hepatic stellate cell line T6(hepatic stellate cell line T6 (HSC-T6). And the expression of oncostatin M receptor 尾 -OSMR 尾) in the liver of non-alcoholic fatty liver disease model induced by choline deficiency (cho-line-deficiency L-amino acid-defineded-defined CDAAA) in rats with non-alcoholic fatty liver disease (NAF LDD) was studied to explore the possible mechanism of NAFLD. Methods: HSC-T6 cells were cultured in HSC-T6 cells for 1 day, 4 days and 7 days later. The expression of 偽 -smooth muscle activin (偽 -SMA) and OSMR 尾 were measured by Western blotting. After 4 hours of adaptive culture of 3 脳 103 / well HSC-T6 cells in 96 plate holes, rat OSM was cultured for 24 hours and 48 hours, respectively, and then 10 渭 l Cell Counting Kit-8 CCK-8 reagent was added for 2 hours, then the value of 450nm wavelength photometric density (450nm) was measured. The results were as follows: (1) after 4 hours of adaptive culture, 3 脳 10 3 / well HSC-T6 cells were cultured for 24 hours and 48 hours, respectively, and 10 渭 l Cell Counting Kit-8 CCK-8 reagents were added for 2 hours. In vivo, 30 SD male rats of 6 weeks old and weight of 160 g 鹵20 g were selected. After 7 days of adaptive feeding, they were randomly divided into normal control group and model group. The normal group was given normal diet and the model group was given CDAA forage for 12 weeks. At the end of the experiment, the rats were anesthetized and killed. The expression of 偽 -SMA in liver sections was detected by immunohistochemical method. The expression of OSMR 尾 in liver was determined by Western blotting assay. Results 1. The expression of 偽 -SMA and OSMR 尾 in HSC-T6 cells increased gradually with the increase of culture time. Addition of OSM could promote the proliferation of HSC-T6 cells in a dose-and time-dependent manner. 3. The level of 偽 -SMA in the liver of the CDAA group was higher than that of the normal control group. Meanwhile, the expression of OSMR 尾 in the liver of the CDAA group was higher than that of the normal control group. Conclusion the expression of OSMR 尾. 2. OSM in HSC-T6 cells promotes the activation and proliferation of HSC-T6 cells. As CDAA induced liver fibrosis, the expression of OSMR 尾 in the liver increased, suggesting that OSM promoted the inflammation and fibrosis of the liver by binding to the OSM receptor of HSC, and played an important role in the pathogenesis and development of NAFLD.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R575

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