【摘要】：目的：通過內(nèi)質(zhì)網(wǎng)應(yīng)激途徑探討絲裂霉素C促進體外培養(yǎng)的成纖維細胞凋亡的可能機制。 方法：體外培養(yǎng)成纖維細胞并傳代，選用3-10代對數(shù)生長期細胞。分別使用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的絲裂霉素刺激體外培養(yǎng)的硬膜外瘢痕成纖維細胞，采用CCK-8法檢測絲裂霉素刺激后成纖維細胞的存活率，得出半數(shù)致死濃度。然后在半致死濃度下，按處理方法不同分成8組：對照組、caspase-8抑制劑預(yù)處理組、caspase-9抑制劑預(yù)處理組、caspase-8+caspase-9抑制劑預(yù)處理組、MMC組、 caspase-8抑制劑預(yù)處理+MMC組、 caspase-9抑制劑預(yù)處理+MMC組、 caspase-8抑制劑預(yù)處理+caspase-9抑制劑預(yù)處理+MMC組。Hoechst33342染色法觀察特定濃度絲裂霉素刺激后成纖維細胞核是否呈現(xiàn)凋亡形態(tài)并計數(shù)呈現(xiàn)凋亡形態(tài)的細胞核數(shù)量。流式細胞儀(Annexin-V/PI雙染法)檢測特定濃度絲裂霉素刺激后成纖維細胞的凋亡率。提取總蛋白質(zhì)，觀察特定濃度絲裂霉素刺激后成纖維細胞CHOP，Caspase-12， Caspase-3，BCL-2，BAX等的表達情況。 結(jié)果：實驗發(fā)現(xiàn)，MMC組成纖維細胞可見Caspase-12、CHOP、BAX表達明顯升高，BCL-2表達減少；使用Caspase-8和Caspase-9抑制劑分別和聯(lián)合預(yù)處理后，再用MMC刺激成纖維細胞，也可見Caspase-12、CHOP、BAX等表達明顯升高，BCL-2表達減少，但升高幅度與單用MMC刺激組比較差異無顯著意義。MMC可明顯降低成纖維細胞的存活率，差異具有顯著意義(*P0.05)，使用Caspase-8、Caspase-9抑制劑分別和聯(lián)合預(yù)處理后，再加MMC刺激成纖維細胞，其存活率較單用MMC組上升，差異具有顯著意義（#P0.05）；聯(lián)合使用Caspase-8、Caspase-9抑制劑預(yù)處理成纖維細胞后，再加MMC刺激成纖維細胞，較分別使用Caspase-8、Caspase-9抑制劑預(yù)處理后再加MMC刺激，其成纖維細胞存活率上升，差異具有顯著意義（P0.05）。 結(jié)論：阻斷線粒體和死亡受體途徑，并不能阻斷成纖維細胞凋亡的發(fā)生，表明有其他凋亡途徑參與成纖維細胞凋亡過程。應(yīng)用一定濃度MMC處理可誘導(dǎo)成纖維細胞凋亡，其機制可能是通過內(nèi)質(zhì)網(wǎng)應(yīng)激途徑使凋亡蛋白CHOP、Caspase-12、BAX等表達增加，進而使凋亡執(zhí)行蛋白caspase-3的表達水平增加，從而誘導(dǎo)成纖維細胞凋亡。
[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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

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