本文選題：肺腺癌 + 微粒體谷胱甘肽S轉(zhuǎn)移酶　； 參考：《中國(guó)腫瘤生物治療雜志》2017年06期

【摘要】：目的:建立穩(wěn)定過(guò)表達(dá)微粒體谷胱甘肽S轉(zhuǎn)移酶1(microsomal glutathione S-transferase 1,MGST1)基因的肺腺癌SPC-A-1細(xì)胞系,探討MGST1在肺腺癌中的作用及其機(jī)制。方法:重組質(zhì)粒pcDNA3-MGST1和空載體pcDNA3以脂質(zhì)體介導(dǎo)的方法轉(zhuǎn)染至SPC-A-1細(xì)胞中,經(jīng)過(guò)G418篩選穩(wěn)轉(zhuǎn)細(xì)胞系,標(biāo)記為pcDNA3-MGST1細(xì)胞和空載體pcDNA3細(xì)胞。實(shí)時(shí)熒光定量PCR及Western blotting鑒定穩(wěn)轉(zhuǎn)細(xì)胞中MGST1 mRNA和蛋白的表達(dá)情況。MTS法檢測(cè)穩(wěn)轉(zhuǎn)細(xì)胞的活力;流式細(xì)胞儀和Western blotting檢測(cè)H_2O_2誘導(dǎo)下穩(wěn)轉(zhuǎn)細(xì)胞的凋亡率和下游凋亡相關(guān)蛋白水平變化。結(jié)果:酶切鑒定和測(cè)序結(jié)果顯示pcDNA3-MGST1重組質(zhì)粒構(gòu)建成功,并獲得具有G418抗性的穩(wěn)轉(zhuǎn)細(xì)胞株。pcDNA3-MGST1細(xì)胞中MGST1在mRNA和蛋白水平表達(dá)均顯著升高(P0.01),且細(xì)胞活力明顯增加(P0.05)。H_2O_2誘導(dǎo)下,pcDNA3-MGST1細(xì)胞的早期凋亡率明顯低于pcDNA3組[(3.30±0.40)%vs(6.50±0.95)%,P0.05];pcDNA3-MGST1細(xì)胞凋亡相關(guān)蛋白caspase 9、caspase 3、PARP表達(dá)增多,cleaved-caspase 9、cleaved-caspase 3、cleaved-PARP表達(dá)明顯減少。結(jié)論:本研究成功構(gòu)建了穩(wěn)定過(guò)表達(dá)MGST1的SPC-A-1肺腺癌細(xì)胞系,MGST1可能通過(guò)調(diào)節(jié)caspase凋亡通路抑制肺腺癌細(xì)胞的凋亡。
[Abstract]:Aim: to establish a lung adenocarcinoma SPC-A-1 cell line stably expressing microsomal glutathione S transferase 1(microsomal glutathione S-transferase 1 MGST1 gene, and to investigate the role of MGST1 in lung adenocarcinoma and its mechanism. Methods: recombinant plasmid pcDNA3-MGST1 and empty vector pcDNA3 were transfected into SPC-A-1 cells by liposome-mediated transfection. Stable transformed cell lines were screened by G418 and labeled as pcDNA3-MGST1 cells and empty vector pcDNA3 cells. Real-time fluorescence quantitative PCR and Western blotting were used to identify the expression of MGST1 mRNA and protein in stable transfer cells. MTS method was used to detect the activity of stable transfer cells. Flow cytometry and Western blotting were used to detect the apoptosis rate and the level of downstream apoptosis-related proteins induced by H_2O_2. Results: the results of restriction endonuclease digestion and sequencing showed that the recombinant plasmid of pcDNA3-MGST1 was successfully constructed. The expression of MGST1 at mRNA and protein levels was significantly increased in the stable transformed cell line. PcDNA3-MGST1 with G418 resistance, and the apoptosis rate of pcDNA3-MGST1 cells induced by P0.05 + H _ 2O _ 2 was significantly higher than that in pcDNA3 group [3.30 鹵0.40)%vs(6.50 鹵0.95p05] pcDNA3-MGST1 cells apoptosis related eggs were obtained. The expression of cleaved-caspase 3 and cleaved-caspase 3 were significantly decreased in white caspase 9, and the expression of cleaved-caspase 3 was significantly decreased. Conclusion: in this study, we successfully constructed a stable MGST1 overexpression SPC-A-1 lung adenocarcinoma cell line, MGST1, which may inhibit the apoptosis of lung adenocarcinoma cells by regulating the apoptosis pathway of caspase.
【作者單位】： 南方醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院人體解剖國(guó)家重點(diǎn)學(xué)科;昆明醫(yī)科大學(xué)第三附屬醫(yī)院暨云南省腫瘤醫(yī)院腫瘤生物治療中心;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No.U1502222,No.81470005,No.81260307);國(guó)家自然科學(xué)基金重大科研儀器研制項(xiàng)目(No.61427807)~~
【分類號(hào)】：R734.2

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