中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
4期
710-713
,共4页
张新晨%杨维良%徐华锋%王书%吴德全
張新晨%楊維良%徐華鋒%王書%吳德全
장신신%양유량%서화봉%왕서%오덕전
RNA干扰%葡萄糖调节蛋白78%葡萄糖调节蛋白94%胃癌细胞%细胞凋亡
RNA榦擾%葡萄糖調節蛋白78%葡萄糖調節蛋白94%胃癌細胞%細胞凋亡
RNA간우%포도당조절단백78%포도당조절단백94%위암세포%세포조망
RNA interference%Glucose regulative proteins 78%Glucose regulative proteins 94%Gastric cancer cells%Apoptosis
目的 观察利用psiSTRIKETM/葡萄糖调节蛋白78(GRP78)和psiSTRIKETM/GRP94抑制人类胃癌细胞株SGC-7901 GRP78和GRt94基因表达对胃癌细胞活性及凋亡的影响.方法 构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体,采用LipofectamineTM 2000转染试剂进行共转染,将psiSTRIKETM/GRP78和psiSTRIKETM/GRP94同时导入胃癌细胞内,在转染前及转染后72 h通过逆转录-聚合酶链反应(RT-PCR)、间接法免疫荧光技术检测GRP78和GRP94 mRNA及蛋白水平的表达,并设立阴性对照组(只加入转染试剂)比较.设立3组:实验组(共转染组)、阴性对照组(只加入转染试剂)及空白对照组(未加任何处理),在转染72 h后用噻唑蓝(MTT)法检测胃癌细胞活性,流式细胞仪检测胃癌细胞早期凋亡.结果 成功将psiSTRIKETM/GRP78和psiSTRIKETM/GRP94转染至SGC-7901 72 h后,与2个对照组比较,GRP78相对表达量为0.49,GRP94为0.40,与对照组比较的表达明显下降,差异有统计学意义(P＜0.05).MTT结果显示:在转染后48 h及72 h,与2个对照组比较,实验组胃癌细胞生长受到抑制；转染后72 h,实验组具有21.98％的凋亡率,与空白对照组和阴性对照组比较细胞凋亡显著增多,而阴性对照组(6.04％)与空白对照组(1.05％)比较差异无统计学意义(P＞0.05).结论 在体外同时转染psiSTRIKETM/GRP78和psiSTRIKETM/GRP94 72 h后,胃癌细胞株SGC-7901 GRP78及GRP94表达明显降低.转染72 h后胃癌细胞活性明显受到抑制,细胞早期凋亡增多.
目的 觀察利用psiSTRIKETM/葡萄糖調節蛋白78(GRP78)和psiSTRIKETM/GRP94抑製人類胃癌細胞株SGC-7901 GRP78和GRt94基因錶達對胃癌細胞活性及凋亡的影響.方法 構建受控于人RNA聚閤酶Ⅲ啟動子U6的真覈錶達載體,採用LipofectamineTM 2000轉染試劑進行共轉染,將psiSTRIKETM/GRP78和psiSTRIKETM/GRP94同時導入胃癌細胞內,在轉染前及轉染後72 h通過逆轉錄-聚閤酶鏈反應(RT-PCR)、間接法免疫熒光技術檢測GRP78和GRP94 mRNA及蛋白水平的錶達,併設立陰性對照組(隻加入轉染試劑)比較.設立3組:實驗組(共轉染組)、陰性對照組(隻加入轉染試劑)及空白對照組(未加任何處理),在轉染72 h後用噻唑藍(MTT)法檢測胃癌細胞活性,流式細胞儀檢測胃癌細胞早期凋亡.結果 成功將psiSTRIKETM/GRP78和psiSTRIKETM/GRP94轉染至SGC-7901 72 h後,與2箇對照組比較,GRP78相對錶達量為0.49,GRP94為0.40,與對照組比較的錶達明顯下降,差異有統計學意義(P＜0.05).MTT結果顯示:在轉染後48 h及72 h,與2箇對照組比較,實驗組胃癌細胞生長受到抑製；轉染後72 h,實驗組具有21.98％的凋亡率,與空白對照組和陰性對照組比較細胞凋亡顯著增多,而陰性對照組(6.04％)與空白對照組(1.05％)比較差異無統計學意義(P＞0.05).結論 在體外同時轉染psiSTRIKETM/GRP78和psiSTRIKETM/GRP94 72 h後,胃癌細胞株SGC-7901 GRP78及GRP94錶達明顯降低.轉染72 h後胃癌細胞活性明顯受到抑製,細胞早期凋亡增多.
목적 관찰이용psiSTRIKETM/포도당조절단백78(GRP78)화psiSTRIKETM/GRP94억제인류위암세포주SGC-7901 GRP78화GRt94기인표체대위암세포활성급조망적영향.방법 구건수공우인RNA취합매Ⅲ계동자U6적진핵표체재체,채용LipofectamineTM 2000전염시제진행공전염,장psiSTRIKETM/GRP78화psiSTRIKETM/GRP94동시도입위암세포내,재전염전급전염후72 h통과역전록-취합매련반응(RT-PCR)、간접법면역형광기술검측GRP78화GRP94 mRNA급단백수평적표체,병설립음성대조조(지가입전염시제)비교.설립3조:실험조(공전염조)、음성대조조(지가입전염시제)급공백대조조(미가임하처리),재전염72 h후용새서람(MTT)법검측위암세포활성,류식세포의검측위암세포조기조망.결과 성공장psiSTRIKETM/GRP78화psiSTRIKETM/GRP94전염지SGC-7901 72 h후,여2개대조조비교,GRP78상대표체량위0.49,GRP94위0.40,여대조조비교적표체명현하강,차이유통계학의의(P＜0.05).MTT결과현시:재전염후48 h급72 h,여2개대조조비교,실험조위암세포생장수도억제；전염후72 h,실험조구유21.98％적조망솔,여공백대조조화음성대조조비교세포조망현저증다,이음성대조조(6.04％)여공백대조조(1.05％)비교차이무통계학의의(P＞0.05).결론 재체외동시전염psiSTRIKETM/GRP78화psiSTRIKETM/GRP94 72 h후,위암세포주SGC-7901 GRP78급GRP94표체명현강저.전염72 h후위암세포활성명현수도억제,세포조기조망증다.
Objective To study the effect of psiSTRIKETM/glucose regulative proteins 78 (GRP78) and psiSTRIKETM/glucose regulative proteins 94 (GRP94) on the expression of GRP78 and GRP94 in human gastric cancer cell line SGC7901 and its effect on cell proliferation and apoptosis in vitro.Methods PsiSTRIKETM/GRP78 and psiSTRIKETM/GRP94 were transfected to gastric cancer cell by LipofectaminTM 2000.The expression of GRP78 and GRP94 mRNA were analyzed by reverse transcriptasepolymerase chain reaction (RT-PCR) and the protein of GRP78 and GRP94 were detected by immunofluorescence 72h after transfection.Cells proliferation was detected by methyl thiazolium tetrazolium (MTT) and the apoptosis of gastric cancer cell was detected by Flow Cytometry.Results The result of RT-PCR showed that GRP78 (0.49) and GRP94 (0.40)of experimental group were declined obviously compare with control groups.and immunofluorescence showed that the expression of GRP78 and GRP94 of gastric cancer cell were inhibited obviously 72h after transfection.The results of MTT showed that compared with control groups,the proliferation of gastric cancer cell in experiment group was inhibited obviously 72 h after transfection (P ＜ 0.05).The ratio of apoptosis was significantly increased in experiment groups (21.98％) compared with control groups (P ＜ 0.05).There were no statistics significance between negative control group (6.04％) and blank control group (1.05％).Conclusion The siRNA in vitro transcription can reduce the level of GRP78 and GRP94 in SGC-7901 efficiently,and can inhibit the proliferation and increase the ratio of apoptosis of SGC-7901 in vitrv.
