【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(jìn)體外培養(yǎng)的成纖維細(xì)胞凋亡的可能機(jī)制。 方法：體外培養(yǎng)成纖維細(xì)胞并傳代，選用3-10代對(duì)數(shù)生長(zhǎng)期細(xì)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(xì)胞，采用CCK-8法檢測(cè)絲裂霉素刺激后成纖維細(xì)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(duì)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(xì)胞核是否呈現(xiàn)凋亡形態(tài)并計(jì)數(shù)呈現(xiàn)凋亡形態(tài)的細(xì)胞核數(shù)量。流式細(xì)胞儀(Annexin-V/PI雙染法)檢測(cè)特定濃度絲裂霉素刺激后成纖維細(xì)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(xì)胞CHOP，Caspase-12， Caspase-3，BCL-2，BAX等的表達(dá)情況。 結(jié)果：實(shí)驗(yàn)發(fā)現(xiàn)，MMC組成纖維細(xì)胞可見Caspase-12、CHOP、BAX表達(dá)明顯升高，BCL-2表達(dá)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(xì)胞，也可見Caspase-12、CHOP、BAX等表達(dá)明顯升高，BCL-2表達(dá)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(xì)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(xì)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(xì)胞后，再加MMC刺激成纖維細(xì)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(xì)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(xì)胞凋亡的發(fā)生，表明有其他凋亡途徑參與成纖維細(xì)胞凋亡過程。應(yīng)用一定濃度MMC處理可誘導(dǎo)成纖維細(xì)胞凋亡，其機(jī)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(dá)增加，進(jìn)而使凋亡執(zhí)行蛋白caspase-3的表達(dá)水平增加，從而誘導(dǎo)成纖維細(xì)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(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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