衣霉素对肝星状细胞凋亡的调控作用

发布时间：2018-05-12 09:23

本文选题：肝纤维化 + 肝星状细胞　；参考：《华北理工大学》2017年硕士论文

【摘要】：目的肝纤维化(Hepatic fibrosis,HF)是慢性肝病进展为肝硬化的必经过程,如何逆转HF是阻止肝炎向肝硬化发展的主要环节,肝星状细胞(Hepatic stellate cell,HSC)活化在发生HF中起重要作用,诱导其凋亡是防治HF的核心。内质网应激(Endoplasmic reticulum stress,ERS)途径是一种新的凋亡途径,衣霉素(Tunicamycin,TM)为ERS诱导剂,钙蛋白酶抑制剂(N-acetyl-Leu-LeuNorleucinal,ALLN)可阻断calpain-2,因此本研究应用TM和ALLN作用于转化生长因子-β1(Transforming growth factor-β1,TGF-β1)刺激的大鼠HSC,从ERS途径探讨对HSC凋亡的影响,为抗纤维化的研究提供进一步的实验证据。方法取液氮冻存的大鼠HSC株,复苏后接种到完全培养基中培养。采用MTT法选定TM的剂量为2ug/ml,作用时间为24h,ALLN剂量为25u M。同步化之后将HSC分为4组:空白组、TGF-β1组(HSC+TGF-β1)、ALLN+TM组(HSC+TGF-β1+ALLN+TM)、TM组(HSC+TGF-β1+TM)。空白组用空白培养基培养,其余组用10ng/ml TGF-β1处理24h后,TGF-β1组更换空白培养基继续培养,ALLN+TM组用25u M ALLN预处理30min,和TM组同时加入2ug/ml TM,避光孵育24h。倒置显微镜观察HSC培养24h、48h、72h后的生长情况以及加入TGF-β1后HSC的形态改变,MTT法检测各组HSC增殖情况,吖啶橙-碘化丙啶(AO/PI)双染法检测HSC凋亡情况,透射电镜观察各组HSC超微结构,流式细胞仪检测HSC周期变化,激光共聚焦显微镜观察Ca2+浓度改变,免疫组织细胞化学染色法检测calpain-2、caspase-3的表达,Western Blot法检测α-SMA、GRP78、caspase-9、Bax、Bcl-2、Ⅰ型胶原蛋白的表达。结果1形态学:24h时HSC少量贴壁,48h时细胞贴壁数量增加,72h时生长至单层致密;加入TGF-β1后HSC呈长梭形改变,细胞间隙变大;2细胞增殖:各组HSC间增殖率的比较差异有统计学意义(P0.05),TGF-β1组HSC增殖率明显高于空白组(P0.05),ALLN+TM组和TM组明显低于TGF-β1组(P0.05);3细胞周期:各组间G1期、S期、G2期HSC所占比例的比较差异均有统计学意义(P0.05),TGF-β1组G1期、G2期所占比例明显低于空白组(P0.05),S期所占比例则高于空白组(P0.05),ALLN+TM组和TM组G1期细胞比例明显高于TGF-β1组(P0.05),S期均明显低于TGF-β1组(P0.05),G2期变化不显著(P0.05);4 Ca2+浓度:各组HSC Ca2+浓度比较差异有统计学意义(P0.05),空白组与TGF-β1组中Ca2+浓度差异不显著(P0.05),ALLN+TM组和TM组Ca2+浓度明显高于TGF-β1组(P0.05),ALLN+TM组Ca2+浓度明显低于TM组(P0.05);5 AO/PI双染法:空白组和TGF-β1组大部分呈绿色荧光,ALLN+TM组中绿色荧光仍然占多数,部分呈现橙红色或橙黄色荧光,TM组橙红色或橙黄色荧光最多;6透射电镜:空白组和TGF-β1组HSC染色质分布均匀,ALLN+TM组微绒毛脱落,细胞器崩解成空泡,TM组染色质出现明显边集,呈“新月形区域”;7免疫组织细胞化学染色法:各组中caspase-3、calpain-2的比较差异有统计学意义(P0.05),ALLN+TM组和TM组caspase-3、calpain-2的表达显著高于TGF-β1组(P0.05),ALLN+TM组中caspase-3、calpain-2的表达显著低于TM组(P0.05);8 Western Blot:TGF-β1组α-SMA表达明显高于空白组(P0.05),各组HSC GRP78、caspase-9、Bax、Bcl-2以及Ⅰ型胶原蛋白的表达差异均有统计学意义(P0.05),与TGF-β1组相比,ALLN+TM组和TM组的GRP78、caspase-9及Bax表达显著升高(P0.05),Bcl-2、Ⅰ型胶原蛋白表达明显降低(P0.05)。结论1 TM可使HSC内Ca2+浓度升高,通过ERS诱导HSC发生凋亡,降低Ⅰ型胶原的表达进而逆转HF;2阻断ERS通路中calpain-2可阻抑TM诱导的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.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.2

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