中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2011年
2期
91-96
,共6页
陈建勇%罗斌%郭晓白%万里晖
陳建勇%囉斌%郭曉白%萬裏暉
진건용%라빈%곽효백%만리휘
自杀基因%基因,STAT3%结肠癌%细胞,HCT116%细胞,HUVEC
自殺基因%基因,STAT3%結腸癌%細胞,HCT116%細胞,HUVEC
자살기인%기인,STAT3%결장암%세포,HCT116%세포,HUVEC
Suicide gene%Genes,STAT3%Colorectal carcinoma%Cells,HCT116%Cells,HUVEC
目的 构建含CDglyTK基因和靶向干扰信号转导和转录激活因子3(STAT3)基因的真核表达载体,探讨多基因联合治疗对结肠癌细胞的抑制作用.方法 采用聚合酶链反应(PCR)方法,扩增大肠杆菌胞嘧啶脱氨酶(CD)基因和带状疱疹病毒胸苷激酶(TK)基因,经测序、酶切、连接、转化、提取筛选的重组质粒.采用酶切和PCR电泳法鉴定所构建质粒.构建靶向STAT3基因shRNA真核表达质粒.将重组质粒转染HCT116和HUVEC细胞,采用RT-PCR检测CDglyTK基因表达和干扰STAT3基因的效果.采用Western blot法检测干扰STAT3蛋白表达的水平.四甲基偶氮唑蓝(MTT)法检测前药对转染了重组质粒pEGFP/CDglyTK、pEGFP/STAT3 siRNA的HCT116和HUVEC细胞的抑制率.结果 重组质粒pEGFP/CDglyTK经限制性酶切、PCR电泳法鉴定分析显示质粒构建正确.RT-PCR法从转染了重组质粒pEGFP/CDglyTK的HCT116和HUVEC细胞中扩增出CDglyTK片段.RT-PCR和Western blot结果显示,转染了pEGFP/STAT3 siRNA的HCT116细胞株的STAT3基因表达水平降低.MTT法检测显示,前药对HCT116细胞的pEGFP/CDglyTK组的抑制率为(63.72±0.64)%,高于对照组(P＜0.05);pEGFP/STAT3 siRNA组抑制率为(47.02±0.39)%,低于pEGFP/CDglyTK组(P＜0.05),但比对照组抑制率高(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA组抑制率为(85.10±0.17)%,高于pEGFP/CDglyTK组和pEGFP/STAT3 siRNA组(P＜0.05).前药对HUVEC细胞的pEGFP/CDglyTK组的抑制率为(70.24±0.33)%,高于对照组(P＜0.05);pEGFP/STAT3 siRNA组抑制率为(46.32±0.15)%,低于pEGFP/CDglyTK组(P＜0.05),但高于对照组(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA组抑制率为(87.10±0.24)%,高于pEGFP/CDglyTK组和pEGFP/STAT3 siRNA组(P＜0.05).结论 重组质粒pEGFP-CDglyTK和pEGFP/STAT3 siRNA对HCT116和HUVEC细胞均有抑制效果.转导双自杀基因CD/TK与靶向于扰STAT3基因联合作用,可以明显提高前药对HCT116和HUVEC细胞的杀伤效果.
目的 構建含CDglyTK基因和靶嚮榦擾信號轉導和轉錄激活因子3(STAT3)基因的真覈錶達載體,探討多基因聯閤治療對結腸癌細胞的抑製作用.方法 採用聚閤酶鏈反應(PCR)方法,擴增大腸桿菌胞嘧啶脫氨酶(CD)基因和帶狀皰疹病毒胸苷激酶(TK)基因,經測序、酶切、連接、轉化、提取篩選的重組質粒.採用酶切和PCR電泳法鑒定所構建質粒.構建靶嚮STAT3基因shRNA真覈錶達質粒.將重組質粒轉染HCT116和HUVEC細胞,採用RT-PCR檢測CDglyTK基因錶達和榦擾STAT3基因的效果.採用Western blot法檢測榦擾STAT3蛋白錶達的水平.四甲基偶氮唑藍(MTT)法檢測前藥對轉染瞭重組質粒pEGFP/CDglyTK、pEGFP/STAT3 siRNA的HCT116和HUVEC細胞的抑製率.結果 重組質粒pEGFP/CDglyTK經限製性酶切、PCR電泳法鑒定分析顯示質粒構建正確.RT-PCR法從轉染瞭重組質粒pEGFP/CDglyTK的HCT116和HUVEC細胞中擴增齣CDglyTK片段.RT-PCR和Western blot結果顯示,轉染瞭pEGFP/STAT3 siRNA的HCT116細胞株的STAT3基因錶達水平降低.MTT法檢測顯示,前藥對HCT116細胞的pEGFP/CDglyTK組的抑製率為(63.72±0.64)%,高于對照組(P＜0.05);pEGFP/STAT3 siRNA組抑製率為(47.02±0.39)%,低于pEGFP/CDglyTK組(P＜0.05),但比對照組抑製率高(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA組抑製率為(85.10±0.17)%,高于pEGFP/CDglyTK組和pEGFP/STAT3 siRNA組(P＜0.05).前藥對HUVEC細胞的pEGFP/CDglyTK組的抑製率為(70.24±0.33)%,高于對照組(P＜0.05);pEGFP/STAT3 siRNA組抑製率為(46.32±0.15)%,低于pEGFP/CDglyTK組(P＜0.05),但高于對照組(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA組抑製率為(87.10±0.24)%,高于pEGFP/CDglyTK組和pEGFP/STAT3 siRNA組(P＜0.05).結論 重組質粒pEGFP-CDglyTK和pEGFP/STAT3 siRNA對HCT116和HUVEC細胞均有抑製效果.轉導雙自殺基因CD/TK與靶嚮于擾STAT3基因聯閤作用,可以明顯提高前藥對HCT116和HUVEC細胞的殺傷效果.
목적 구건함CDglyTK기인화파향간우신호전도화전록격활인자3(STAT3)기인적진핵표체재체,탐토다기인연합치료대결장암세포적억제작용.방법 채용취합매련반응(PCR)방법,확증대장간균포밀정탈안매(CD)기인화대상포진병독흉감격매(TK)기인,경측서、매절、련접、전화、제취사선적중조질립.채용매절화PCR전영법감정소구건질립.구건파향STAT3기인shRNA진핵표체질립.장중조질립전염HCT116화HUVEC세포,채용RT-PCR검측CDglyTK기인표체화간우STAT3기인적효과.채용Western blot법검측간우STAT3단백표체적수평.사갑기우담서람(MTT)법검측전약대전염료중조질립pEGFP/CDglyTK、pEGFP/STAT3 siRNA적HCT116화HUVEC세포적억제솔.결과 중조질립pEGFP/CDglyTK경한제성매절、PCR전영법감정분석현시질립구건정학.RT-PCR법종전염료중조질립pEGFP/CDglyTK적HCT116화HUVEC세포중확증출CDglyTK편단.RT-PCR화Western blot결과현시,전염료pEGFP/STAT3 siRNA적HCT116세포주적STAT3기인표체수평강저.MTT법검측현시,전약대HCT116세포적pEGFP/CDglyTK조적억제솔위(63.72±0.64)%,고우대조조(P＜0.05);pEGFP/STAT3 siRNA조억제솔위(47.02±0.39)%,저우pEGFP/CDglyTK조(P＜0.05),단비대조조억제솔고(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA조억제솔위(85.10±0.17)%,고우pEGFP/CDglyTK조화pEGFP/STAT3 siRNA조(P＜0.05).전약대HUVEC세포적pEGFP/CDglyTK조적억제솔위(70.24±0.33)%,고우대조조(P＜0.05);pEGFP/STAT3 siRNA조억제솔위(46.32±0.15)%,저우pEGFP/CDglyTK조(P＜0.05),단고우대조조(P＜0.05);pEGFP/CDglyTK+pEGFP/STAT3 siRNA조억제솔위(87.10±0.24)%,고우pEGFP/CDglyTK조화pEGFP/STAT3 siRNA조(P＜0.05).결론 중조질립pEGFP-CDglyTK화pEGFP/STAT3 siRNA대HCT116화HUVEC세포균유억제효과.전도쌍자살기인CD/TK여파향우우STAT3기인연합작용,가이명현제고전약대HCT116화HUVEC세포적살상효과.
Objective The aim of this study was to construct a recombinant plasmid carrying fusion suicide gene CDglyTK and RNA interference eukaryotic expressing vector targeting to STAT3,and to investigate the effect of double suicide gene combined with RNAi targeting to STAT3 on HCT116 and HUVEC cells in vitro. Methods The CD and TK were cloned by polymerase chain reaction(PCR),and fusion gene CDglyTK was inserted into plasmid pEGFP after DNA sequence analysis,enzyme digestion and ligation.The recombinant plasmid was analyzed by PCR amplification and electrophoresis and enzyme digestion.DNA sequences containing small hairpin structure targeting to STAT3 were synthesized and inserted into the vector.The CDglyTK gene expressions in HCT116 and HUVEC cells were examined by reverse transcription-polymerase chain reaction(RT-PCR) after transfection of HCT116 and HUVEC cells.The inhibitory effect of RNA interference vector targeting to STAT3 was analyzed by RT-PCR and Western blot.The effects of 5-FC and GCV on HCT116 and HUVEC cells trnsfected with the recombinant plasmids were detected by MTT staining. Results The results of restriction enzyme digestion and PCR amplification and electrophoresis showed that the recombinant pEGFP/CDglyTK was constructed correctly.The mRNA expression of gene CDglyTK was detected in HCT116 and HUVEC cells which transfected with the recombinant plasmid.The results of RT-PCR and Western blot showed that the RNA interference expression vector targeting to STAT3 effectively inhibited the expression of STAT3 in HCT116 cells.The results of MTT test showed that the inhibition ratio of group pEGFP/CDglyTK was(63.72 ± 0.64 )%,significantly higher than that of control group(P ＜0.05 ).The inhibition rate of group pEGFP/STAT3 siRNA was(47.02 ±0.39 )%,which was lower than that of group pEGFP/CDglyTK(P ＜0.05 ),and higher than that of control group(P ＜0.05 ).The inhibition rate of group pEGFP/CdglyTK + pEGFP/STAT3 siRNA was(85.10 ±0.17)%,significantly higher than those of groups pEGFP/CDglyTK and group pEGFP/STAT3 siRNA(P ＜0.05 ).Meanwhile,in HUVEC cells,the inhibition rate of group pEGFP/CDglyTK was(70.24 ± 0.33 ) %,significantly higher than that of the control group(P ＜0.05 ).The inhibition rate of group pEGFP/STAT3 siRNA was(46.32 ±0.15 )%,significantly lower than that of group pEGFP/CdglyTK(P ＜0.05 ),and higher than that of the control group(P ＜0.05 ).The inhibition rate of group pEGFP/CdglyTK + pEGFP/STAT3 siRNA was(87.10 ± 0.24)%,significantly higher than those of groups pEGFP/CDglyTK and pEGFP/STAT3 siRNA(P ＜0.05).Conclusion The recombinant plasmids pEGFP-CDglyTK and pEGFP/STAT3 siRNA have inhibitory effect on HCT116 and HUVEC cells.The killing effects of double suicide gene combined with RNAi targeting to STAT3 are much better than those of single gene therapy.
