CAJ | 학술논문

目的构建含有人微管蛋白辅助因子A(TBCA)的真核表达载体并观察其对肾癌细胞株786-0增殖的影响.方法构建真核表达载体pcDNA3.0-Flag-TBCA和pCMV-TBCA-IRES-EGFP,并进行酶切、测序检验、脂质体转染细胞后利用Western blot法检测蛋白表达.以四唑氮化合物(MTS)法绘制细胞生长曲线.结果成功构建了真核表达载体pcDNA3.0-Flag-TBCA和pCMV-TBCA-IRES-EGFP.转染后TBCA在细胞中表达明显升高(P＜0.05).但在转染后第3、4、5、6天,重组质粒组吸光度值分别为0.52±0.04、0.85 ±0.04、1.20±0.08、1.61 ±0.11;与对照组比较无统计学意义(P＞0.05).结论 TBCA的过表达载体构建成功,但在786-0细胞中过表达TBCA后对并不能对细胞增殖产生明显影响.
목적 구건함유인미관단백보조인자A(TBCA)적진핵표체재체병관찰기대신암세포주786-0증식적영향.방법 구건진핵표체재체pcDNA3.0-Flag-TBCA화pCMV-TBCA-IRES-EGFP,병진행매절、측서검험、지질체전염세포후이용Western blot법검측단백표체.이사서담화합물(MTS)법회제세포생장곡선.결과 성공구건료진핵표체재체pcDNA3.0-Flag-TBCA화pCMV-TBCA-IRES-EGFP.전염후TBCA재세포중표체명현승고(P＜0.05).단재전염후제3、4、5、6천,중조질립조흡광도치분별위0.52±0.04、0.85 ±0.04、1.20±0.08、1.61 ±0.11;여대조조비교무통계학의의(P＞0.05).결론 TBCA적과표체재체구건성공,단재786-0세포중과표체TBCA후대병불능대세포증식산생명현영향.
Objective To construct eukaryotic expression vector of human tubulin cofactor A (TBCA) and to study the effect of it on proliferation of renal cell carcinoma cell line 786-0.Methods The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed and transfected into 786-0 cells by liposome mediate method.TBCA protein level in 786-0 cells was detected by Western blotting.The effects of TBCA on proliferation of 786-0 cells were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method.Results The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed correctly,which was confirmed by restriction endonuclease digestion and DNA sequencing analysis.Western blotting indicated that TBCA was highly expression in transfected 786-0 cells (P ＜ 0.05).At 3,4,5 and 6 d after transfection,the recombinant plasmid group absorbance values were 0.52 ± 0.04,0.85 ± 0.04,1.20 ± 0.08,1.61 ± 0.11 ; no statistical significance compared with the control group (P ＞ 0.05).The colony forming efficiency in the recombinant plasmid group was higher than other groups.Conclusion The eukaryotic expression vectors of TBCA was constructed correctly and TBCA should not promote the proliferation of renal cell carcinoma cells.