CAJ | 학술논문

目的构建人canstatin克隆载体及重组真核表达载体,观察电脉冲介导骨骼肌内canstatin基因导入对小鼠Lewis肺癌皮下移植瘤生长的影响.方法用一步法RT-PCR钓取胚肝canstatin cDNA,克隆入真核表达载体pEGFP-N1,构建含重组质粒pEGFP-N1/canstatin;将该重组质粒通过电脉冲转入荷瘤Lewis肺癌小鼠肌肉内,应用逆转录PCR、荧光显微镜测量荧光强度、肿瘤称重、肿瘤体积测定及免疫组化的方法检测canstatin基因在体内的表达和抗肿瘤作用.结果获得684 bp人canstatin基因,测序证明canstatin cDNA编码序列与Genbank中报道的序列一致.电脉冲介导的质粒基因导入效率较单纯质粒注射高30倍以上;转canstatin基因后导入部位的canstatin mRNA高表达,目的蛋白表达时间可持续25 d以上.EGFP/Canstatin融合蛋白在导入部位表达并分泌至循环中发挥生物学效应,起到抑制远隔部位肿瘤生长的作用.实验组肿瘤颜色苍白,毛细血管稀少,坏死多,体积小,瘤重抑制率57.7％.结论成功构建了重组真核表达载体pEGFP-N1/canstatin,电脉冲介导的骨骼肌内基因导入法的转移效率较单纯质粒注射高且持续时间长;是一种高效安全,简便经济,可能具有一定临床应用价值的基因导入方法;电脉冲介导骨骼肌内canstatin基因导入对小鼠Lewis肺癌皮下移植瘤生长具有显著的抑制作用.
목적 구건인canstatin극륭재체급중조진핵표체재체,관찰전맥충개도골격기내canstatin기인도입대소서Lewis폐암피하이식류생장적영향.방법 용일보법RT-PCR조취배간canstatin cDNA,극륭입진핵표체재체pEGFP-N1,구건함중조질립pEGFP-N1/canstatin;장해중조질립통과전맥충전입하류Lewis폐암소서기육내,응용역전록PCR、형광현미경측량형광강도、종류칭중、종류체적측정급면역조화적방법검측canstatin기인재체내적표체화항종류작용.결과 획득684 bp인canstatin기인,측서증명canstatin cDNA편마서렬여Genbank중보도적서렬일치.전맥충개도적질립기인도입효솔교단순질립주사고30배이상;전canstatin기인후도입부위적canstatin mRNA고표체,목적단백표체시간가지속25 d이상.EGFP/Canstatin융합단백재도입부위표체병분비지순배중발휘생물학효응,기도억제원격부위종류생장적작용.실험조종류안색창백,모세혈관희소,배사다,체적소,류중억제솔57.7％.결론 성공구건료중조진핵표체재체pEGFP-N1/canstatin,전맥충개도적골격기내기인도입법적전이효솔교단순질립주사고차지속시간장;시일충고효안전,간편경제,가능구유일정림상응용개치적기인도입방법;전맥충개도골격기내canstatin기인도입대소서Lewis폐암피하이식류생장구유현저적억제작용.
Objective To construct human canstatin gene eukaryotic expression vector and investigate the therapeutic effect of intramuscular canstatin gene delivered by electroporation on tumor growth.Methods Canstatin cDNA was amplified from total RNA extracted from fresh fetal liver by reversing transcription polymerase chain reaction (RT-PCR).The canstatin cDNA fragment was in serted into pEGFP-N1 eukaryotic expression vector.The recombination plasmid was delivered to the quadriceps of the mice with Lewis lung carcinomas by electroporation intramuscular.Fluorescence intension measured by fluorescence microscope,reverse-PCR assay,and immunohistochemistry assay were performed to detect the expression of canstatin gene in the muscle and in circulation.The tumor weight and volume were used to detect the biological effects of canstatin gene delivery.Results Recombinant eukaryotic expression vector of recombinant human canstatin was successfully constructed.The canstatin mRNA was significantly increased in the skeletal muscle and intramuscular delivery of canatatin gene by electroporation acquired the expression of enhanced green fluorescent protein (EGFP)/canstatin protein in the circulation and significantly inhibited tumor growth.The percent of the inhibition of tumor weight was 57.7 ％.Conclusions Electroporation mediated gene transfer efficiency in skeletal muscle was compared to simple plasmid injection and lasted for a long time.It was an efficient and safe,convenient and economic,gene transfer methods and might have certain clinical application value.Electroporation mediated canstatin gene transfer in skeletal muscle had obvious inhibitory effect on Lewis lung cancer in mice subcutaneous xenograft tumor growth.