从内质网应激途径探讨丝裂霉素C诱导成纤维细胞凋亡的机制

发布时间：2019-03-29 08:29

【摘要】：目的：通过内质网应激途径探讨丝裂霉素C促进体外培养的成纤维细胞凋亡的可能机制。方法：体外培养成纤维细胞并传代，选用3-10代对数生长期细胞。分别使用0.001mg/ml、0.005mg/ml、0.01mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml、0.3mg/ml、0.4mg/ml、0.5mg/ml的丝裂霉素刺激体外培养的硬膜外瘢痕成纤维细胞，采用CCK-8法检测丝裂霉素刺激后成纤维细胞的存活率，得出半数致死浓度。然后在半致死浓度下，按处理方法不同分成8组：对照组、caspase-8抑制剂预处理组、caspase-9抑制剂预处理组、caspase-8+caspase-9抑制剂预处理组、MMC组、 caspase-8抑制剂预处理+MMC组、 caspase-9抑制剂预处理+MMC组、 caspase-8抑制剂预处理+caspase-9抑制剂预处理+MMC组。Hoechst33342染色法观察特定浓度丝裂霉素刺激后成纤维细胞核是否呈现凋亡形态并计数呈现凋亡形态的细胞核数量。流式细胞仪(Annexin-V/PI双染法)检测特定浓度丝裂霉素刺激后成纤维细胞的凋亡率。提取总蛋白质，观察特定浓度丝裂霉素刺激后成纤维细胞CHOP，Caspase-12， Caspase-3，BCL-2，BAX等的表达情况。结果：实验发现，MMC组成纤维细胞可见Caspase-12、CHOP、BAX表达明显升高，BCL-2表达减少；使用Caspase-8和Caspase-9抑制剂分别和联合预处理后，再用MMC刺激成纤维细胞，也可见Caspase-12、CHOP、BAX等表达明显升高，BCL-2表达减少，但升高幅度与单用MMC刺激组比较差异无显著意义。MMC可明显降低成纤维细胞的存活率，差异具有显著意义(*P0.05)，使用Caspase-8、Caspase-9抑制剂分别和联合预处理后，再加MMC刺激成纤维细胞，其存活率较单用MMC组上升，差异具有显著意义（#P0.05）；联合使用Caspase-8、Caspase-9抑制剂预处理成纤维细胞后，再加MMC刺激成纤维细胞，较分别使用Caspase-8、Caspase-9抑制剂预处理后再加MMC刺激，其成纤维细胞存活率上升，差异具有显著意义（P0.05）。结论：阻断线粒体和死亡受体途径，并不能阻断成纤维细胞凋亡的发生，表明有其他凋亡途径参与成纤维细胞凋亡过程。应用一定浓度MMC处理可诱导成纤维细胞凋亡，其机制可能是通过内质网应激途径使凋亡蛋白CHOP、Caspase-12、BAX等表达增加，进而使凋亡执行蛋白caspase-3的表达水平增加，从而诱导成纤维细胞凋亡。
[Abstract]:Aim: to explore the possible mechanism of mitomycin C (MMC) promoting apoptosis of fibroblasts cultured in vitro through endoplasmic reticulum (ER) stress pathway. Methods: fibroblasts were cultured and passaged in vitro. Using 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, respectively, The cultured epidural scar fibroblasts were stimulated by mitomycin (0.5mg/ml) in vitro. The survival rate of fibroblasts stimulated by mitomycin was detected by CCK-8 method, and the lethal concentration of 50% was obtained. Then they were divided into 8 groups: control group, caspase-8 inhibitor pretreatment group, caspase-9 inhibitor pretreatment group, caspase-8 caspase-9 inhibitor pretreatment group, MMC group, caspase-8 inhibitor pretreatment group, and caspase-8 inhibitor pretreatment group, then divided into 8 groups: control group, caspase-8 inhibitor pretreatment group, caspase-8 caspase-9 inhibitor pretreatment group, caspase-8 inhibitor pretreatment group, and caspase-8 inhibitor pretreatment group. MMC group was pretreated with caspase-9 inhibitor. Caspase-8 inhibitor pretreatment caspase-9 inhibitor pretreatment MMC group. Hoechst 33342 staining was used to observe whether the fibroblasts showed apoptotic morphology and count the number of nuclei with apoptotic morphology after specific concentration of mitomycin stimulation. Flow cytometry (Annexin-V/PI double staining) was used to detect the apoptosis rate of fibroblasts stimulated by mitomycin. Total protein was extracted and the expression of CHOP,Caspase-12, Caspase-3,BCL-2,BAX in fibroblasts stimulated by mitomycin was observed. Results: the results showed that the expression of Caspase-12,CHOP,BAX was significantly increased and the expression of BCL-2 was decreased in the fibroblasts composed of MMC. After pretreatment with Caspase-8 and Caspase-9 inhibitors, and then stimulated with MMC, the expression of Caspase-12,CHOP,BAX and BCL-2 was significantly increased and the expression of BCL-2 was decreased. However, there was no significant difference between the two groups. The survival rate of fibroblasts was significantly decreased by MMC alone (* P0.05). After pretreatment with Caspase-8,Caspase-9 inhibitor and combined pretreatment, the survival rate of fibroblasts was significantly decreased (* P0.05), and after pretreatment with Caspase-8,Caspase-9 inhibitor or combined treatment, the survival rate of fibroblasts was significantly decreased. The survival rate of fibroblasts stimulated by MMC was significantly higher than that of MMC alone (# P0.05). When fibroblasts were pretreated with Caspase-8,Caspase-9 inhibitor and then stimulated by MMC, the survival rate of fibroblasts was increased compared with that treated with Caspase-8,Caspase-9 inhibitor and then stimulated by MMC. The difference was significant (P0.05). Conclusion: blocking mitochondrial and death receptor pathway can not block the occurrence of fibroblast apoptosis, indicating that other apoptosis pathways are involved in the process of fibroblast apoptosis. Treatment with certain concentration of MMC can induce the apoptosis of fibroblasts. The mechanism may be that the expression of apoptosis protein CHOP,Caspase-12,BAX and other proteins can be increased through endoplasmic reticulum stress pathway, and then the expression level of apoptosis execution protein caspase-3 can be increased. As a result, fibroblast apoptosis was induced.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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