本文選題：肝纖維化 + 肝星狀細(xì)胞��； 參考：《華北理工大學(xué)》2017年碩士論文

【摘要】：目的肝纖維化(Hepatic fibrosis,HF)是慢性肝病進(jìn)展為肝硬化的必經(jīng)過程,如何逆轉(zhuǎn)HF是阻止肝炎向肝硬化發(fā)展的主要環(huán)節(jié),肝星狀細(xì)胞(Hepatic stellate cell,HSC)活化在發(fā)生HF中起重要作用,誘導(dǎo)其凋亡是防治HF的核心。內(nèi)質(zhì)網(wǎng)應(yīng)激(Endoplasmic reticulum stress,ERS)途徑是一種新的凋亡途徑,衣霉素(Tunicamycin,TM)為ERS誘導(dǎo)劑,鈣蛋白酶抑制劑(N-acetyl-Leu-LeuNorleucinal,ALLN)可阻斷calpain-2,因此本研究應(yīng)用TM和ALLN作用于轉(zhuǎn)化生長因子-β1(Transforming growth factor-β1,TGF-β1)刺激的大鼠HSC,從ERS途徑探討對HSC凋亡的影響,為抗纖維化的研究提供進(jìn)一步的實(shí)驗(yàn)證據(jù)。方法取液氮凍存的大鼠HSC株,復(fù)蘇后接種到完全培養(yǎng)基中培養(yǎng)。采用MTT法選定TM的劑量為2ug/ml,作用時間為24h,ALLN劑量為25u M。同步化之后將HSC分為4組:空白組、TGF-β1組(HSC+TGF-β1)、ALLN+TM組(HSC+TGF-β1+ALLN+TM)、TM組(HSC+TGF-β1+TM)�？瞻捉M用空白培養(yǎng)基培養(yǎng),其余組用10ng/ml TGF-β1處理24h后,TGF-β1組更換空白培養(yǎng)基繼續(xù)培養(yǎng),ALLN+TM組用25u M ALLN預(yù)處理30min,和TM組同時加入2ug/ml TM,避光孵育24h。倒置顯微鏡觀察HSC培養(yǎng)24h、48h、72h后的生長情況以及加入TGF-β1后HSC的形態(tài)改變,MTT法檢測各組HSC增殖情況,吖啶橙-碘化丙啶(AO/PI)雙染法檢測HSC凋亡情況,透射電鏡觀察各組HSC超微結(jié)構(gòu),流式細(xì)胞儀檢測HSC周期變化,激光共聚焦顯微鏡觀察Ca2+濃度改變,免疫組織細(xì)胞化學(xué)染色法檢測calpain-2、caspase-3的表達(dá),Western Blot法檢測α-SMA、GRP78、caspase-9、Bax、Bcl-2、Ⅰ型膠原蛋白的表達(dá)。結(jié)果1形態(tài)學(xué):24h時HSC少量貼壁,48h時細(xì)胞貼壁數(shù)量增加,72h時生長至單層致密;加入TGF-β1后HSC呈長梭形改變,細(xì)胞間隙變大;2細(xì)胞增殖:各組HSC間增殖率的比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),TGF-β1組HSC增殖率明顯高于空白組(P0.05),ALLN+TM組和TM組明顯低于TGF-β1組(P0.05);3細(xì)胞周期:各組間G1期、S期、G2期HSC所占比例的比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),TGF-β1組G1期、G2期所占比例明顯低于空白組(P0.05),S期所占比例則高于空白組(P0.05),ALLN+TM組和TM組G1期細(xì)胞比例明顯高于TGF-β1組(P0.05),S期均明顯低于TGF-β1組(P0.05),G2期變化不顯著(P0.05);4 Ca2+濃度:各組HSC Ca2+濃度比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),空白組與TGF-β1組中Ca2+濃度差異不顯著(P0.05),ALLN+TM組和TM組Ca2+濃度明顯高于TGF-β1組(P0.05),ALLN+TM組Ca2+濃度明顯低于TM組(P0.05);5 AO/PI雙染法:空白組和TGF-β1組大部分呈綠色熒光,ALLN+TM組中綠色熒光仍然占多數(shù),部分呈現(xiàn)橙紅色或橙黃色熒光,TM組橙紅色或橙黃色熒光最多;6透射電鏡:空白組和TGF-β1組HSC染色質(zhì)分布均勻,ALLN+TM組微絨毛脫落,細(xì)胞器崩解成空泡,TM組染色質(zhì)出現(xiàn)明顯邊集,呈“新月形區(qū)域”;7免疫組織細(xì)胞化學(xué)染色法:各組中caspase-3、calpain-2的比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),ALLN+TM組和TM組caspase-3、calpain-2的表達(dá)顯著高于TGF-β1組(P0.05),ALLN+TM組中caspase-3、calpain-2的表達(dá)顯著低于TM組(P0.05);8 Western Blot:TGF-β1組α-SMA表達(dá)明顯高于空白組(P0.05),各組HSC GRP78、caspase-9、Bax、Bcl-2以及Ⅰ型膠原蛋白的表達(dá)差異均有統(tǒng)計(jì)學(xué)意義(P0.05),與TGF-β1組相比,ALLN+TM組和TM組的GRP78、caspase-9及Bax表達(dá)顯著升高(P0.05),Bcl-2、Ⅰ型膠原蛋白表達(dá)明顯降低(P0.05)。結(jié)論1 TM可使HSC內(nèi)Ca2+濃度升高,通過ERS誘導(dǎo)HSC發(fā)生凋亡,降低Ⅰ型膠原的表達(dá)進(jìn)而逆轉(zhuǎn)HF;2阻斷ERS通路中calpain-2可阻抑TM誘導(dǎo)的HSC凋亡作用。
[Abstract]:Hepatic fibrosis (HF) is a necessary process for the progression of chronic liver disease to cirrhosis. How to reverse HF is the main link to prevent the development of liver cirrhosis. The activation of Hepatic stellate cell (HSC) plays an important role in the occurrence of HF, and the induction of apoptosis is the core of the prevention and control of HF. Lum stress, ERS) pathway is a new pathway of apoptosis, and Tunicamycin (TM) is a ERS inducer, and the calsin inhibitor (N-acetyl-Leu-LeuNorleucinal, ALLN) can block calpain-2. Therefore, this study applies TM and ALLN to transforming growth factor beta 1 (beta 1, beta 1). To discuss the effect of HSC apoptosis and provide further experimental evidence for anti fibrosis study. Method the HSC strain of liquid nitrogen cryopreserved rat and inoculated into the complete medium after resuscitation. The dosage of TM was 2ug/ml, the action time was 24h, and ALLN dose was 25U M. synchronization, and HSC was divided into 4 groups: the blank group, TGF- beta 1 groups (HSC+TGF-) Beta 1), group ALLN+TM (HSC+TGF- beta 1+ALLN+TM), group TM (HSC+TGF- beta 1+TM). The blank group was cultured with blank culture medium and the rest group treated 24h with 10ng/ml TGF- beta 1. The TGF- beta 1 group replaced the blank culture medium to continue culture. The growth situation after 72h and the morphologic change of HSC after adding TGF- beta 1, MTT method was used to detect the proliferation of HSC. The apoptosis of HSC was detected by acridine orange and propidium iodide (AO/PI). The ultrastructure of HSC in each group was observed by transmission electron microscope, and the HSC cycle was detected by flow cytometry. The concentration of Ca2+ was observed by laser confocal microscopy, and the concentration of Ca2+ was observed, and the immune tissue cells were observed. The expression of calpain-2 and caspase-3 was detected by chemical staining, and the expression of alpha -SMA, GRP78, caspase-9, Bax, Bcl-2, and type I collagen was detected by Western Blot. Results 1 Morphology: 24h HSC was adhered to a small amount, and the number of cell adherence increased at the time of 48h. The proliferation rate of HSC in each group was statistically significant (P0.05). The proliferation rate of HSC in TGF- beta 1 group was significantly higher than that in the blank group (P0.05), and the ALLN+TM and TM groups were significantly lower than that of the TGF- beta 1 groups (P0.05), and the 3 cell cycle: G1, S, and G2 periods were statistically significant. Compared with blank group (P0.05), the proportion of S phase was higher than that of blank group (P0.05). The proportion of cells in G1 phase of group ALLN+TM and TM group was significantly higher than that of TGF- beta 1 group (P0.05), S stage was obviously lower than TGF- beta 1 group (P0.05), and the G2 phase change was not significant (4). The concentration difference was not significant (P0.05). The concentration of Ca2+ in group ALLN+TM and TM group was significantly higher than that in group TGF- beta 1 (P0.05), and the concentration of Ca2+ in ALLN+TM group was significantly lower than that in TM group (P0.05); 5 AO/PI double staining method: most of the group and TGF- beta 1 groups were green fluorescence. The yellow fluorescence was the most, 6 transmission electron microscopy: the distribution of HSC chromatin in the blank group and TGF- beta 1 groups was evenly distributed, the microvilli in the ALLN+TM group fell off, the organelle collapsed into vacuoles, the chromatin of the TM group appeared "crescent shaped area", and 7 immuno tissue cytochemical staining: the comparison of Caspase-3 and calpain-2 in each group was statistically significant (P0.05), ALLN+T The expression of Caspase-3 and calpain-2 in group M and TM group was significantly higher than that in group TGF- beta 1 (P0.05). The expression of Caspase-3 and calpain-2 in ALLN+TM group was significantly lower than that in TM group (P0.05), and the expression of 8 Western 1 groups was significantly higher than that in the blank group. 0.05), compared with the TGF- beta 1 group, the expression of GRP78, caspase-9 and Bax in group ALLN+TM and TM group increased significantly (P0.05), Bcl-2, type I collagen expression decreased significantly (P0.05). Conclusion 1 TM can increase the concentration of HSC Ca2+. The effect of HSC on apoptosis.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.2

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