CAJ | 학술논문

目的构建针对多效蛋白(pleiotrophin,PTN)基因的小干涉RNA(siRNA)慢病毒表达载体并观察其对人小细胞肺癌H446细胞株PTN表达抑制及对细胞生长和凋亡的影响.方法设计4对针对PTN基阒的小发夹RNA(shRNA)序列,与Plvthm载体连接,构建慢病毒表达载体LV(lentiviral)-shPTN,连接产物转化到DH5α感受态细胞,阳性克隆测序鉴定.再用LV-shPTN与pRsvREV、pMDlg-pRRE、pMD2G三种质粒共转染293T细胞,小量包装与纯化产生慢病毒颗粒.感染H446细胞后,用荧光定量PCR及Western blot法检测PTN的表达.将包含对PTN基因十扰效率最高的序列慢病毒载体进行大量包装、纯化及病毒滴度测定,将包装好的病毒感染H446细胞.实验分为正常未干扰细胞组、阴性对照组、PTN抑制组、化疗组及PTN抑制加化疗组,四甲基偶氮唑盐比色法观察细胞生长,流式细胞仪分析细胞凋亡.结果测序结果与构建慢病毒载体的预期结果一致,对PTNmRNA的最高抑制效率为72%,对PTN蛋白的最高抑制效率为59%.大量包装与纯化后病毒滴度为1×10~8 TU/ml.PTN基因抑制组H446细胞活力明显低于正常未干扰细胞组和阴性对照组,基因抑制联合化疗组较单纯基因抑制组及化疗组细胞活力下降,凋亡率增高,并具有浓度依赖性.结论构建PTN RNA干涉载体,转染后可有效降低H446细胞FIN的转录和表达,抑制H446细胞的生长并促进其凋亡,有望成为小细胞肺癌基因治疗的可选择的方法之一.
목적 구건침대다효단백(pleiotrophin,PTN)기인적소간섭RNA(siRNA)만병독표체재체병관찰기대인소세포폐암H446세포주PTN표체억제급대세포생장화조망적영향.방법 설계4대침대PTN기격적소발협RNA(shRNA)서렬,여Plvthm재체련접,구건만병독표체재체LV(lentiviral)-shPTN,련접산물전화도DH5α감수태세포,양성극륭측서감정.재용LV-shPTN여pRsvREV、pMDlg-pRRE、pMD2G삼충질립공전염293T세포,소량포장여순화산생만병독과립.감염H446세포후,용형광정량PCR급Western blot법검측PTN적표체.장포함대PTN기인십우효솔최고적서렬만병독재체진행대량포장、순화급병독적도측정,장포장호적병독감염H446세포.실험분위정상미간우세포조、음성대조조、PTN억제조、화료조급PTN억제가화료조,사갑기우담서염비색법관찰세포생장,류식세포의분석세포조망.결과 측서결과여구건만병독재체적예기결과일치,대PTNmRNA적최고억제효솔위72%,대PTN단백적최고억제효솔위59%.대량포장여순화후병독적도위1×10~8 TU/ml.PTN기인억제조H446세포활력명현저우정상미간우세포조화음성대조조,기인억제연합화료조교단순기인억제조급화료조세포활력하강,조망솔증고,병구유농도의뢰성.결론 구건PTN RNA간섭재체,전염후가유효강저H446세포FIN적전록화표체,억제H446세포적생장병촉진기조망,유망성위소세포폐암기인치료적가선택적방법지일.
Objective To construct a siRNA lentiviral expressing vector targeting PTN (pleiotrophin) gene in human small cell lung cancer H446 cells and to study the RNAi effect on tumor growth and apoptosis.Methods Four pairs of small hairpin RNA specific for PTN were designed,synthesized and cloned into the Plvthm vector.The resulting lentiviral vector containing shPTN was confirmed by DNA sequencing named LV-shPTN.293T cells were co-transfected with LV-shPTN,pRsv-REV,pMDlg-pRRE and pMD2G,then packed and purified slightly to produce lentivirus.After infecting H446 cells with recombinant lentivirus,PTN expression was determined by real-time RT-PCR and Western blot Finally the selected lentiviral vector was packed and purified with best interference efficiency in large scale and the titer of virus was determined.The packed virus was used to infect H446 cells,and then the experiment was divided to a normal cell group,a negative control group,a PTN interference group,a chemotherapy group and a combined group with RNAi and chemotherapy.The effect on cell growth and apoptosis of H446 cells infected with high titer virus was analyzed using MTT and FCM.Results DNA sequencing analysis confirmed that shPTN lentiviral vector was successfully established as expected with interference efficiency as high as 72% and 59% at the mRNA and protein level.The titer of concentrated virus was 1×10~8 TU/ml.Compared to normal cells and control group,the cell viability of PTN interference group was decreased.Compared to RNAi group and chemotherapy group alone,the combined group with RNAi and chemotherapy showed less cell viability and a higher apoptotic rate in a concentration independent manner of the virus.Conclusion PTN RNAi vector construction and H446 cell transfection effectively reduced the PTN transcription and expression,inhibited the growth and promoted the apoptosis of tumor cells.This method may become a useful therapeutic strategy for small cell lung cancer overexpressing PRIN.