国际检验医学杂志
國際檢驗醫學雜誌
국제검험의학잡지
INTERNATIONAL JOURNAL OF LABORATORY MEDICINE
2014年
24期
3300-3303
,共4页
胃癌细胞%RNA 干扰%MBP-1 基因%细胞增殖
胃癌細胞%RNA 榦擾%MBP-1 基因%細胞增殖
위암세포%RNA 간우%MBP-1 기인%세포증식
gastric cancer cell%RNA interfering%MBP-1 gene%cell proliferation
目的：探讨 MBP-1（c-myc promter binding protein 1，MBP-1）基因表达沉默对胃癌细胞株 SGC-7901细胞增殖影响。方法实验分3组：空白对照组（未转染胃癌细胞）、阴性对照组（转染错义序列）和干扰组（转染 MBP-1 shRNA）。设计2条针对MBP-1基因的小干扰 RNA 片段及1条阴性对照 siRNA，并构建入 pSIREN-retroQ 质粒。将构建的重组 pSIREN-retroQ 质粒通过 Lipofectamine 2000脂质体转染胃癌 SGC-7901细胞，嘌呤霉素筛选稳转株细胞。Real time PCR 和 Western blot 分别检测MBP-1表达。MTT 法对 MBP-1干扰后 SGC-7901细胞增殖进行检测。结果通过 PCR 扩增阳性克隆及测序，说明已成功构建MBP-1干扰及对照重组 pSIREN-retroQ 质粒。通过 Lipofectamine 2000脂质体将重组质粒转染胃癌 SGC-7901细胞，并通过嘌呤霉素筛选2周，说明已成功构建 MBP-1干扰及对照 SGC-7901稳转株细胞。Real time PCR 检测，干扰组 MBP-1 mRNA 相对表达量与空白对照组相比显著下调（P ＜0.05）。Western blot 检测 MBP-1蛋白表达，干扰组 MBP-1表达量与空白对照组相比也都显著下调。MTT 法检测结果表明，MBP-1干扰组细胞在48、72、96和120 h 增殖能力比空白对照组都有显著的升高（P ＜0.05）。结论下调 MBP-1基因表达能明显促进胃癌细胞 SGC-7901的增殖，从而为胃癌基因治疗提供了新靶点。
目的：探討 MBP-1（c-myc promter binding protein 1，MBP-1）基因錶達沉默對胃癌細胞株 SGC-7901細胞增殖影響。方法實驗分3組：空白對照組（未轉染胃癌細胞）、陰性對照組（轉染錯義序列）和榦擾組（轉染 MBP-1 shRNA）。設計2條針對MBP-1基因的小榦擾 RNA 片段及1條陰性對照 siRNA，併構建入 pSIREN-retroQ 質粒。將構建的重組 pSIREN-retroQ 質粒通過 Lipofectamine 2000脂質體轉染胃癌 SGC-7901細胞，嘌呤黴素篩選穩轉株細胞。Real time PCR 和 Western blot 分彆檢測MBP-1錶達。MTT 法對 MBP-1榦擾後 SGC-7901細胞增殖進行檢測。結果通過 PCR 擴增暘性剋隆及測序，說明已成功構建MBP-1榦擾及對照重組 pSIREN-retroQ 質粒。通過 Lipofectamine 2000脂質體將重組質粒轉染胃癌 SGC-7901細胞，併通過嘌呤黴素篩選2週，說明已成功構建 MBP-1榦擾及對照 SGC-7901穩轉株細胞。Real time PCR 檢測，榦擾組 MBP-1 mRNA 相對錶達量與空白對照組相比顯著下調（P ＜0.05）。Western blot 檢測 MBP-1蛋白錶達，榦擾組 MBP-1錶達量與空白對照組相比也都顯著下調。MTT 法檢測結果錶明，MBP-1榦擾組細胞在48、72、96和120 h 增殖能力比空白對照組都有顯著的升高（P ＜0.05）。結論下調 MBP-1基因錶達能明顯促進胃癌細胞 SGC-7901的增殖，從而為胃癌基因治療提供瞭新靶點。
목적：탐토 MBP-1（c-myc promter binding protein 1，MBP-1）기인표체침묵대위암세포주 SGC-7901세포증식영향。방법실험분3조：공백대조조（미전염위암세포）、음성대조조（전염착의서렬）화간우조（전염 MBP-1 shRNA）。설계2조침대MBP-1기인적소간우 RNA 편단급1조음성대조 siRNA，병구건입 pSIREN-retroQ 질립。장구건적중조 pSIREN-retroQ 질립통과 Lipofectamine 2000지질체전염위암 SGC-7901세포，표령매소사선은전주세포。Real time PCR 화 Western blot 분별검측MBP-1표체。MTT 법대 MBP-1간우후 SGC-7901세포증식진행검측。결과통과 PCR 확증양성극륭급측서，설명이성공구건MBP-1간우급대조중조 pSIREN-retroQ 질립。통과 Lipofectamine 2000지질체장중조질립전염위암 SGC-7901세포，병통과표령매소사선2주，설명이성공구건 MBP-1간우급대조 SGC-7901은전주세포。Real time PCR 검측，간우조 MBP-1 mRNA 상대표체량여공백대조조상비현저하조（P ＜0.05）。Western blot 검측 MBP-1단백표체，간우조 MBP-1표체량여공백대조조상비야도현저하조。MTT 법검측결과표명，MBP-1간우조세포재48、72、96화120 h 증식능력비공백대조조도유현저적승고（P ＜0.05）。결론하조 MBP-1기인표체능명현촉진위암세포 SGC-7901적증식，종이위위암기인치료제공료신파점。
Objective To investigate the effects of c-myc promoter binding protein(MBP-1)gene expression silencing on the pro-liferation in vitro in human gastric cancer cell line SGC-7901.Methods The cells divided into three groups:blank control group (cells without transfecting gastric cancer cell),negative control group(cells transfecting missense sequence)and experimental group (cells transfecting MBP-1 shRNA).Two MBP-1 shRNA sequences and one negative control shRNA sequence were designed,syn-thesized and cloned into pSIREN-retroQ plasma.Then the recombinant plasmids were constructed and transfected into human gas-tric cancer SGC-7901 cells by Lipofectamine 2000.After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was obtained.The expressions of MBP-1 mRNA and protein in SGC-7901 were deter-mined by the real time PCR and Western blot,respectively.The effects of altered expression of MBP-1 on the cell proliferation were measured by MTT cell proliferation assay.Results PCR and sequencing indicated that the recombinant plasmids pSIREN-retroQ was constructed.Then the recombinant plasmids were transfected into human gastric cancer SGC-7901 cells by Lipofectamine 2000. After selection of cells with puromycin for two weeks,stable cell line SGC-7901 expressing gene of MBP-1 specific shRNA was ob-tained.The relative expression level MBP-1 mRNA in the MBP-1 siRNA transfection group was significantly decreased compared with the blank control group(P <0.05).Compared with the blank group,the expression levels of MBP-1 protein in the experimental group also significantly decreased.The proliferation abilities of SGC-7901 cells at 48,72,96,120 h after MBP-1 siRNA transfection were significantly increased compared with the blank control group (P < 0.05 ).Conclusion Down-regulating the expression of MBP-1 can obviously promote the proliferation of human gastric cancer cell line SGC-7901.MBP-1 gene may become the new target of gene therapy for gastric cancer.
