【摘要】：目的探討AQP9基因過表達(dá)對(duì)順鉑誘導(dǎo)肝癌SMMC-7721細(xì)胞凋亡的影響及其可能的機(jī)制。方法將重組慢病毒LV-AQP9(以LV-GFP作為對(duì)照)導(dǎo)入SMMC-7721細(xì)胞中,構(gòu)建SMMC-7721/LV-AQP9重組細(xì)胞。激光共聚焦顯微鏡觀察重組慢病毒LV-AQP9在SMMC-7721中的感染效率,實(shí)時(shí)熒光定量PCR和蛋白質(zhì)印跡法檢測AQP9在SMMC-7721中表達(dá)情況。CCK-8法檢測不同濃度順鉑(DDP)處理肝癌SMMC-7721細(xì)胞后24h、36h、48h細(xì)胞的增殖抑制率并選取最佳順鉑處理濃度和時(shí)間。順鉑分別處理轉(zhuǎn)染前后的肝癌SMMC-7721細(xì)胞。實(shí)驗(yàn)分組:GFP組為空載體慢病毒轉(zhuǎn)染的SMMC-7721細(xì)胞;AQP9組為含過表達(dá)AQP9基因慢病毒載體轉(zhuǎn)染的SMMC-7721細(xì)胞;GFP+DDP組為順鉑處理的空載體慢病毒轉(zhuǎn)染的SMMC-7721細(xì)胞;AQP9+DDP組為順鉑處理的含過表達(dá)AQP9基因慢病毒載體轉(zhuǎn)染的SMMC-7721細(xì)胞。采用AnnexinⅤ/7AAD雙標(biāo)流式細(xì)胞術(shù)和DAPI染色檢測各組細(xì)胞的凋亡情況。用蛋白質(zhì)印跡法檢測AQP9,GRP78以及Caspase-12的蛋白表達(dá)水平。結(jié)果激光共聚焦顯微鏡觀察到重組慢病毒LV-AQP9在SMMC-7721細(xì)胞中轉(zhuǎn)染效率達(dá)90%以上,且AQP9 mRNA和AQP9蛋白表達(dá)水平在SMMC-7721細(xì)胞中均顯著增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.01)。順鉑作用SMMC-7721細(xì)胞24h后的半數(shù)抑制率(IC50)為5ug/mL。四組細(xì)胞的凋亡率之間都有顯著差異,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。采用多因素方差分析AQP9和順鉑對(duì)人肝癌細(xì)胞對(duì)凋亡的影響,其結(jié)果顯示:AQP9過表達(dá)明顯促進(jìn)細(xì)胞凋亡(FAQP9=71.391.780,P=0.000);DDP顯著誘導(dǎo)肝癌細(xì)胞凋亡(FCDDP=361.682,P=0.000);AQP9可顯著增強(qiáng)順鉑誘導(dǎo)肝癌細(xì)胞凋亡,具有協(xié)同作用(FAQP9×CDDP=26.681,P=0.001)。AQP9+DDP組細(xì)胞GRP78和cleaved Caspase12蛋白表達(dá)顯著高于GFP組、AQP9組、GFP+DDP組,差異有統(tǒng)計(jì)學(xué)意義(P值均0.01),而GFP組和AQP9組間無明顯統(tǒng)計(jì)學(xué)差異。結(jié)論AQP9過表達(dá)能夠增強(qiáng)DDP誘導(dǎo)肝癌SMMC-7721細(xì)胞發(fā)生凋亡,增強(qiáng)其對(duì)化療的敏感性。其機(jī)制可能是AQP9通過內(nèi)質(zhì)網(wǎng)應(yīng)激信號(hào)通路細(xì)胞凋亡途徑,發(fā)揮與DDP的協(xié)同作用促進(jìn)凋亡的發(fā)生。
[Abstract]:Objective to investigate the effect of AQP9 gene overexpression on cisplatin induced apoptosis of hepatoma SMMC-7721 cells and its possible mechanism. Methods Recombinant lentivirus LV-AQP9 (LV-GFP as control) was transfected into SMMC-7721 cells to construct SMMC-7721 / LV-AQP9 recombinant cells. Laser confocal microscopy was used to observe the infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721. The expression of AQP9 in SMMC-7721 was detected by real-time fluorescence quantitative PCR and Western blot. CCK-8 assay was used to detect the proliferation inhibition rate of SMMC-7721 cells treated with different concentrations of cisplatin (DDP) for 24 h or 36 h or 48 h, and the optimal concentration and time of cisplatin treatment were selected. Cisplatin was used to treat SMMC-7721 cells before and after transfection. SMMC-7721 cells transfected with empty vector lentivirus were divided into two groups: SMMC-7721 cells transfected with empty vector lentivirus and SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. The GFP DDP group was treated with cisplatin and treated with cisplatin in SMMC-7721 cells transfected with empty vector lentivirus-transfected SMMC-7721 cells. SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. Apoptosis was detected by Annexin V / 7 AAD double flow cytometry and DAPI staining. The protein expression levels of AQP9, GRP78 and Caspase-12 were detected by Western blot. Results the transfection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was more than 90%, and the expression of AQP9 mRNA and AQP9 protein were significantly increased in SMMC-7721 cells (P0.01). The half inhibition rate (IC50) of SMMC-7721 cells treated with cisplatin for 24 hours was 5ug- mL. There were significant differences in apoptosis rate among the four groups (P0.05). The effects of AQP9 and cisplatin on apoptosis of human hepatoma cells were analyzed by multivariate variance analysis. The results showed that the overexpression of AQP9 significantly promoted apoptosis (FAQP9 71.391.780 P0. 000) DDP significantly induced apoptosis of hepatoma cells (FCDDP 361.682P0.000) AQP9 significantly enhanced cisplatin induced apoptosis in hepatoma cells. The expression of GRP78 and cleaved Caspase12 protein in AQP9 DDP group was significantly higher than that in GFP group (P < 0.01), but there was no significant difference between GFP group and AQP9 group. Conclusion the overexpression of AQP9 can enhance the apoptosis of SMMC-7721 cells induced by DDP and enhance its sensitivity to chemotherapy. The mechanism may be that AQP9 plays a synergistic role with DDP in promoting apoptosis through endoplasmic reticulum stress signaling pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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