中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2013年
3期
191-197
,共7页
王姣琦%杜珍武%高娴绯%吴玫%张玉成%潘颖%王茜%张桂珍
王姣琦%杜珍武%高嫻緋%吳玫%張玉成%潘穎%王茜%張桂珍
왕교기%두진무%고한비%오매%장옥성%반영%왕천%장계진
基因,bcl-2%抗肿瘤药%RNA干扰%基因
基因,bcl-2%抗腫瘤藥%RNA榦擾%基因
기인,bcl-2%항종류약%RNA간우%기인
Genes,bcl-2%Antineoplastic agents%RNA interference%Genes
目的 探讨抑制Bcl-2基因表达对人肺癌A549细胞化疗药物杀伤敏感性及其相关基因mRNA表达的影响.为进一步提高非小细胞肺癌(NSCLC)化疗疗效提供实验依据.方法 构建靶向人Bcl-2基因的微小RNA重组质粒,并转染至A549细胞,设稳定转染的实验组、阴性对照质粒组和空白对照组,运用逆转录聚合酶链反应(RT-PCR)琼脂糖凝胶电泳、实时荧光定量PCR和蛋白质免疫印迹法(Western blot)检测转染细胞中Bcl-2 mRNA转录和蛋白的表达水平,四甲基偶氮唑蓝(MTT)法和流式细胞术分析转染后细胞的增殖和周期变化.MTT法检测干扰后A549细胞对依托泊苷、5-氟尿嘧啶、顺铂、阿霉素、长春新碱、紫杉醇及长春瑞滨等7种化疗药物敏感性的变化.应用RT-PCR及实时荧光定量PCR方法进行核苷酸切除修复交叉互补基因1(ERCC1)、胸腺嘧啶合成酶(TYMS)、Ⅲ型β-微管蛋白(ClassⅢβ-tubulin)、拓扑异构酶2α(TOP2α)等化疗敏感性相关基因mRNA表达的检测.结果 成功构建微小RNA表达载体,稳定转染A549细胞后,实验组Bcl-2mRNA表达(相对表达量为0.002 ±0.001)与阴性对照质粒组(相对表达量为0.104±0.003)相比下降98.1％,蛋白表达水平下调57.6％(t值分别为98.70和7.66,均P＜0.05).流式细胞术测定实验组细胞周期阻滞在Gl期,MTT法检测示实验组细胞生长受到明显抑制,对依托泊苷、顺铂、紫杉醇、长春瑞滨等4种药物的敏感性明显增加[实验组IC50分别为(107.3±0.1) mg/L、(7.7±0.6)mg/L、(11.5±1.9) mg/L和(10.8±1.6) mg/L,阴性对照质粒组IC50分别为(145.8±0.1) mg/L、(60.7±1.4) mG/L、(80.6±1.7) mg/L和(20.6±1.7) mg/L],差异均有统计学意义(t值分别为655.33,108.04.82.16和12.48,均P＜0.05).此外,Bcl-2表达下调还使实验组ERCC1、TYMS、TOP2α基因的mRNA转录下降,与阴性对照质粒组相比分别下调99.6％、92.9％、96.1％(t值分别为689.79,689.37和768.04,均P＜0.05),但实验组ClassⅢβ-tubulin基因mRNA转录(1.154 ±0.008)升高,超过阴性对照质粒组(0.520 ±0.009)的122％(T-=160.07,P＜0.05).结论 靶向性抑制抗凋亡相关基因Bcl-2的表达可提高A549细胞对依托泊苷、顺铂、紫杉醇、长春瑞滨等化疗药物的敏感性,下调ERCC1、TYMS、TOP2α基因的表达.
目的 探討抑製Bcl-2基因錶達對人肺癌A549細胞化療藥物殺傷敏感性及其相關基因mRNA錶達的影響.為進一步提高非小細胞肺癌(NSCLC)化療療效提供實驗依據.方法 構建靶嚮人Bcl-2基因的微小RNA重組質粒,併轉染至A549細胞,設穩定轉染的實驗組、陰性對照質粒組和空白對照組,運用逆轉錄聚閤酶鏈反應(RT-PCR)瓊脂糖凝膠電泳、實時熒光定量PCR和蛋白質免疫印跡法(Western blot)檢測轉染細胞中Bcl-2 mRNA轉錄和蛋白的錶達水平,四甲基偶氮唑藍(MTT)法和流式細胞術分析轉染後細胞的增殖和週期變化.MTT法檢測榦擾後A549細胞對依託泊苷、5-氟尿嘧啶、順鉑、阿黴素、長春新堿、紫杉醇及長春瑞濱等7種化療藥物敏感性的變化.應用RT-PCR及實時熒光定量PCR方法進行覈苷痠切除脩複交扠互補基因1(ERCC1)、胸腺嘧啶閤成酶(TYMS)、Ⅲ型β-微管蛋白(ClassⅢβ-tubulin)、拓撲異構酶2α(TOP2α)等化療敏感性相關基因mRNA錶達的檢測.結果 成功構建微小RNA錶達載體,穩定轉染A549細胞後,實驗組Bcl-2mRNA錶達(相對錶達量為0.002 ±0.001)與陰性對照質粒組(相對錶達量為0.104±0.003)相比下降98.1％,蛋白錶達水平下調57.6％(t值分彆為98.70和7.66,均P＜0.05).流式細胞術測定實驗組細胞週期阻滯在Gl期,MTT法檢測示實驗組細胞生長受到明顯抑製,對依託泊苷、順鉑、紫杉醇、長春瑞濱等4種藥物的敏感性明顯增加[實驗組IC50分彆為(107.3±0.1) mg/L、(7.7±0.6)mg/L、(11.5±1.9) mg/L和(10.8±1.6) mg/L,陰性對照質粒組IC50分彆為(145.8±0.1) mg/L、(60.7±1.4) mG/L、(80.6±1.7) mg/L和(20.6±1.7) mg/L],差異均有統計學意義(t值分彆為655.33,108.04.82.16和12.48,均P＜0.05).此外,Bcl-2錶達下調還使實驗組ERCC1、TYMS、TOP2α基因的mRNA轉錄下降,與陰性對照質粒組相比分彆下調99.6％、92.9％、96.1％(t值分彆為689.79,689.37和768.04,均P＜0.05),但實驗組ClassⅢβ-tubulin基因mRNA轉錄(1.154 ±0.008)升高,超過陰性對照質粒組(0.520 ±0.009)的122％(T-=160.07,P＜0.05).結論 靶嚮性抑製抗凋亡相關基因Bcl-2的錶達可提高A549細胞對依託泊苷、順鉑、紫杉醇、長春瑞濱等化療藥物的敏感性,下調ERCC1、TYMS、TOP2α基因的錶達.
목적 탐토억제Bcl-2기인표체대인폐암A549세포화료약물살상민감성급기상관기인mRNA표체적영향.위진일보제고비소세포폐암(NSCLC)화료료효제공실험의거.방법 구건파향인Bcl-2기인적미소RNA중조질립,병전염지A549세포,설은정전염적실험조、음성대조질립조화공백대조조,운용역전록취합매련반응(RT-PCR)경지당응효전영、실시형광정량PCR화단백질면역인적법(Western blot)검측전염세포중Bcl-2 mRNA전록화단백적표체수평,사갑기우담서람(MTT)법화류식세포술분석전염후세포적증식화주기변화.MTT법검측간우후A549세포대의탁박감、5-불뇨밀정、순박、아매소、장춘신감、자삼순급장춘서빈등7충화료약물민감성적변화.응용RT-PCR급실시형광정량PCR방법진행핵감산절제수복교차호보기인1(ERCC1)、흉선밀정합성매(TYMS)、Ⅲ형β-미관단백(ClassⅢβ-tubulin)、탁복이구매2α(TOP2α)등화료민감성상관기인mRNA표체적검측.결과 성공구건미소RNA표체재체,은정전염A549세포후,실험조Bcl-2mRNA표체(상대표체량위0.002 ±0.001)여음성대조질립조(상대표체량위0.104±0.003)상비하강98.1％,단백표체수평하조57.6％(t치분별위98.70화7.66,균P＜0.05).류식세포술측정실험조세포주기조체재Gl기,MTT법검측시실험조세포생장수도명현억제,대의탁박감、순박、자삼순、장춘서빈등4충약물적민감성명현증가[실험조IC50분별위(107.3±0.1) mg/L、(7.7±0.6)mg/L、(11.5±1.9) mg/L화(10.8±1.6) mg/L,음성대조질립조IC50분별위(145.8±0.1) mg/L、(60.7±1.4) mG/L、(80.6±1.7) mg/L화(20.6±1.7) mg/L],차이균유통계학의의(t치분별위655.33,108.04.82.16화12.48,균P＜0.05).차외,Bcl-2표체하조환사실험조ERCC1、TYMS、TOP2α기인적mRNA전록하강,여음성대조질립조상비분별하조99.6％、92.9％、96.1％(t치분별위689.79,689.37화768.04,균P＜0.05),단실험조ClassⅢβ-tubulin기인mRNA전록(1.154 ±0.008)승고,초과음성대조질립조(0.520 ±0.009)적122％(T-=160.07,P＜0.05).결론 파향성억제항조망상관기인Bcl-2적표체가제고A549세포대의탁박감、순박、자삼순、장춘서빈등화료약물적민감성,하조ERCC1、TYMS、TOP2α기인적표체.
Objective To investigate the effects of miRNA-mediated down-regulation of the Bcl-2 gene on the chemotherapeutic sensitivities and mRNA transcriptions of sensitivity associated genes in human lung adenocarcinoma cell line A549 cells,and therefore to provide experimental data for improving the chemotherapeutic effects on non-small cell lung cancer (NSCLC).Methods The miRNA recombinant plasmid targeting to human Bcl-2 gene was designed,synthesized and stably transferred into A549 cells by lipofectin technique as the experiment group.The transcription of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) by agarose gel electrophoresis,real-time PCR,and the protein level of Bcl-2 was measured by Western blot to confirm the function of miRNA plasmid.The cell proliferation was examined by methyl thiazolyl tetrazolium (MTT)assay.Cell cycle was measured by flow cytometry.Drug sensitivities of A549 cells to etoposide,5-fluorouracil,cisplatin,adriamycin,vincristine,paclitaxel and navelbine were analyzed by MTT assay.The mRNA expressions of excision repair crosscomplementing gene 1 (ERCC1),thymidylate synthase(TYMS),Class Ⅲ β-tubulin,topoisomerase 2 alpha (TOP2α)genes were detected by RT-PCR and real-time PCR.Results The recombinant miRNA plasmid was successfully synthesized and stably transferred into A549 cells.The transcription of Bcl-2 mRNA dramatically decreased by 98.1％ in the experiment group(RQ =0.002 ± 0.001) compared to that in the negative control group(RQ =0.104 ± 0.003) by real-time PCR (t =98.70,P ＜ 0.05) ; and the protein level of Bcl-2 in the experiment group decreased by 57.6％ by Western blot(t =7.66,P ＜ 0.05).The cell cycle profile showed that the low expression of Bcl-2 gene led to A549 cell cycle arrest at G1-phase.The results of MTT showed that the growth of A549 cells in the experiment group was markedly inhibited.The sensitivities of A549 cells to etoposide,cisplatin,paclitaxel,and navelbine were significantly enhanced [IC50 values in the experiment group were(107.3 ±0.1) mg/L,(7.7 ±0.6) mg/L,(11.5 ± 1.9) mg/L and(10.8 ± 1.6) mg/L; IC50 values in the negative control group were(145.8 ± 0.1) mg/L,(60.7 ±1.4) mg/L,(80.6 ± 1.7) mg/L and (20.6 ± 1.7) mg/L],the respective t values being 655.33,108.04,82.16 and 12.48,all P ＜0.05.The mRNA level of ERCC1,TYMS,and TOP2o genes in the experiment group decreased by 99.6％,92.9％ and 96.1％ respectively,but Class Ⅲ β-tubulin mRNA increased by 122％ compared to the negative control group(1.154 ± 0.008,0.520 ± 0.009),the respective t values being 689.79,689.37,768.04 and 160.07,all P ＜ 0.05.Conclusion Targeting to inhibit antiapoptotic mitochondrial gene Bcl-2 expression in A549 cells specifically decreased the mRNA of ERCC1,TYMS,and TOP2o genes,and significantly increased the sensitivities of A549 cells to chemotherapeutic agents such as etoposide,cisplatin,paclitaxel and navelbine.
