中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2011年
1期
16-18,22
,共4页
ras蛋白质类/遗传学/代谢%肿瘤抑制蛋白质类/遗传学%遗传载体%肝肿瘤/代谢%细胞凋亡
ras蛋白質類/遺傳學/代謝%腫瘤抑製蛋白質類/遺傳學%遺傳載體%肝腫瘤/代謝%細胞凋亡
ras단백질류/유전학/대사%종류억제단백질류/유전학%유전재체%간종류/대사%세포조망
Ras proteins/GE/ME%Tumor suppressor proteins/GE%Genetic vectors%Liver neoplasms/ ME%Apoptosis
目的 构建pcDNA3.1-RASSF1真核表达载体,并检测其对肝癌细胞系HepG2凋亡的影响.方法 应用聚合酶链反应技术从人RASSF1cDNA中扩增出RASSF1基因后,以内切酶Xho Ⅰ和EcoR Ⅰ进行双酶切,将其克隆入用相同酶处理的载体pcDNA3.1;将重组质粒pcDNA3.1-RASSF1转染肝癌HepG2细胞,应用Western blot法检测RASSF1的表达水平;用Annexin V/PI法检测细胞的凋亡情况.结果 酶切和测序结果表明,重组质粒pcDNA3.1-RASSF1构建成功;Western blot法结果表明转染pcDNA3.1-RASSF1后HepG2细胞中RASSF1表达升高;Annexin V/PI法用流式细胞术检测凋亡,空白组、pcDNA3.1组和pcDNA3.1-RASSF1组HepG2细胞的凋亡率分别为(5.8±0.42)%、(7.48±0.68)%和(35.1±3.15)%.结论 真核表达载体pcDNA3.1-RASSF1构建成功;RASSF1蛋白在HepG2细胞中高表达,并促进细胞凋亡.
目的 構建pcDNA3.1-RASSF1真覈錶達載體,併檢測其對肝癌細胞繫HepG2凋亡的影響.方法 應用聚閤酶鏈反應技術從人RASSF1cDNA中擴增齣RASSF1基因後,以內切酶Xho Ⅰ和EcoR Ⅰ進行雙酶切,將其剋隆入用相同酶處理的載體pcDNA3.1;將重組質粒pcDNA3.1-RASSF1轉染肝癌HepG2細胞,應用Western blot法檢測RASSF1的錶達水平;用Annexin V/PI法檢測細胞的凋亡情況.結果 酶切和測序結果錶明,重組質粒pcDNA3.1-RASSF1構建成功;Western blot法結果錶明轉染pcDNA3.1-RASSF1後HepG2細胞中RASSF1錶達升高;Annexin V/PI法用流式細胞術檢測凋亡,空白組、pcDNA3.1組和pcDNA3.1-RASSF1組HepG2細胞的凋亡率分彆為(5.8±0.42)%、(7.48±0.68)%和(35.1±3.15)%.結論 真覈錶達載體pcDNA3.1-RASSF1構建成功;RASSF1蛋白在HepG2細胞中高錶達,併促進細胞凋亡.
목적 구건pcDNA3.1-RASSF1진핵표체재체,병검측기대간암세포계HepG2조망적영향.방법 응용취합매련반응기술종인RASSF1cDNA중확증출RASSF1기인후,이내절매Xho Ⅰ화EcoR Ⅰ진행쌍매절,장기극륭입용상동매처리적재체pcDNA3.1;장중조질립pcDNA3.1-RASSF1전염간암HepG2세포,응용Western blot법검측RASSF1적표체수평;용Annexin V/PI법검측세포적조망정황.결과 매절화측서결과표명,중조질립pcDNA3.1-RASSF1구건성공;Western blot법결과표명전염pcDNA3.1-RASSF1후HepG2세포중RASSF1표체승고;Annexin V/PI법용류식세포술검측조망,공백조、pcDNA3.1조화pcDNA3.1-RASSF1조HepG2세포적조망솔분별위(5.8±0.42)%、(7.48±0.68)%화(35.1±3.15)%.결론 진핵표체재체pcDNA3.1-RASSF1구건성공;RASSF1단백재HepG2세포중고표체,병촉진세포조망.
Objective To construct pcDNA3.1 RASSF1 eukaryotic vector and observe the influence of RASSF1 on the apoptosis of hepatocarcinoma cell line HepG2. Methods RASSF1 gene was amplifled from human RASSF1 cDNA by polymerase chain reaction (PCR) and cloned into pcDNA3.1. The recombinant plasmid pcDNA3. 1 RASSF1 was transfected into hepatocarcinoma HepG2 cell line. The expression of RASSF1 was examined by Western blot. The influence of RASSF1 on the cell apoptosis was measured by Annexin V/PI assay. Results DNA enzyme digestion and sequencing results showed that recombinant plasmid pcDNA3. 1-RASSF1 was successfully constructed. RASSF1 protein was overexpressed in HepG2 cell line transfected with pcDNA3. 1-RASSF1 plasmid. The apoptosis rate of blank, pcDNA3. 1 and pcDNA3. 1-RASSF1 group was (5.8 ±0.42)%, (7.48 ±0.68)% and (35. 1 ±3. 15)%, respectively.Conclusion The pcDNA3. 1- RASSF1 eukaryotic vector was successfully constructed, RASSF1 protein overexpression could induce apoptosis in HepG2 cell line.
