CAJ | 학술논문

目的：构建甲胎蛋白（AFP）介导的以沉默胰岛素样生长因子1型受体（IGF1R）和表达野生型p53（Wtp53）的重组慢病毒，探讨该慢病毒载体对肝癌干细胞CD45－CD90＋双靶点基因系统（ IGF1R和Wtp53）和对体内肝癌生长的抑制作用。方法从肝癌细胞 Hep3B 系中分离出肝癌干细胞 CD45－CD90＋细胞亚群，用携带 AFP?Wtp53?pPRIME?miR30?shRNA?IGF1R融合基因的慢病毒进行体外转染。利用RT?PCR和Western blotting检测IGF1R、WtP53在细胞内的表达，将荷人肝癌的移植瘤裸鼠随机分为空白对照组（腋下接种未感染任何病毒的CD45?CD90＋细胞）、空载体对照组（腋下接种感染空载体慢病毒pPRIME的CD45－CD90＋细胞）和实验组（腋下接种感染空载体慢病毒AFP?WtP53?pPRIME?miR30?shRNA?IGF1R的CD45－CD90＋细胞），检测各组的IGF1R表达，微血管密度和细胞凋亡程度。结果成功构建anti?AFP 介导的慢病毒；qRT?PCR和Western blotting检测显示，anti?AFP介导的慢病毒能表达Wtp53并同时干扰IGF1R的表达。体内实验显示，与空白对照组和空载体对照组相比，实验组肿瘤的潜伏期较长，肿瘤体积较小，瘤组织的IGF1R水平、微血管密度较低，但凋亡指数较高，差异有统计学意义（ P＜0?05）。结论 anti?AFP介导的慢病毒可高效特异性转染肝癌干细胞CD45－CD90＋双靶点基因系统，起到杀伤肝癌干细胞及抑制肝癌体内生长的作用。
목적：구건갑태단백（AFP）개도적이침묵이도소양생장인자1형수체（IGF1R）화표체야생형p53（Wtp53）적중조만병독，탐토해만병독재체대간암간세포CD45－CD90＋쌍파점기인계통（ IGF1R화Wtp53）화대체내간암생장적억제작용。방법종간암세포 Hep3B 계중분리출간암간세포 CD45－CD90＋세포아군，용휴대 AFP?Wtp53?pPRIME?miR30?shRNA?IGF1R융합기인적만병독진행체외전염。이용RT?PCR화Western blotting검측IGF1R、WtP53재세포내적표체，장하인간암적이식류라서수궤분위공백대조조（액하접충미감염임하병독적CD45?CD90＋세포）、공재체대조조（액하접충감염공재체만병독pPRIME적CD45－CD90＋세포）화실험조（액하접충감염공재체만병독AFP?WtP53?pPRIME?miR30?shRNA?IGF1R적CD45－CD90＋세포），검측각조적IGF1R표체，미혈관밀도화세포조망정도。결과성공구건anti?AFP 개도적만병독；qRT?PCR화Western blotting검측현시，anti?AFP개도적만병독능표체Wtp53병동시간우IGF1R적표체。체내실험현시，여공백대조조화공재체대조조상비，실험조종류적잠복기교장，종류체적교소，류조직적IGF1R수평、미혈관밀도교저，단조망지수교고，차이유통계학의의（ P＜0?05）。결론 anti?AFP개도적만병독가고효특이성전염간암간세포CD45－CD90＋쌍파점기인계통，기도살상간암간세포급억제간암체내생장적작용。
Objective To construct the alpha?fetroprotein(AFP) mediated lentivirus in order to silence the expression of in?sulin like growth factor 1 receptor ( IGF1R) and over?express p53 ( Wtp53) , and to explore the effect of lentivirus on the double target gene system ( IGF1R and Wtp53) of liver cancer stem cells CD45-CD90+ and growth of liver cancer in vivo. Methods Liver cancer stem cells CD45-CD90+ were isolated from Hep3B cells. The lentivirus carrying the fusion genes of AFP?Wtp53?pPRIME?miR30?shR?NA?IGF1R were used for in vitro transfection. Western blotting and RT?PCR were used to detect the expressions of wtp53 and IGF1R in the cells. The transplanted tumor model was established in BALB?C nude mice and then randomly divided into the blank control group ( axillary inoculation with CD45-CD90+ cells) , empty vector control group ( axillary inoculation with CD45-CD90+ cells infected with empty vector pPRIME) and experimental group ( axillary inoculation with CD45-CD90+ cells infected with AFP?Wtp53?pPRIME?miR30?shRNA?IGF1R) . The IGF1R expression, microvessel density and apoptosis in each group were detected. Results The anti?AFP mediated lentivirus was successfully constructed. The results of qRT?PCR and Western blotting showed that the recombinant lenti?virus can over?express Wtp53 and interfere the expression of IGF1R. In vivo experiments showed that in comparison with the blank con?trol group and empty vector control group, there were longer incubation period, smaller tumor volume, lower level of IGF1R and mi?crovessel density in tumor tissue and higher apoptosis index in experimental group( P<0?05) . Conclusion Anti?AFP mediated lenti?virus targeted double target gene system of CD45-CD90+ cells presents high efficiency and specificity, playing a role in the growth of liver cancer stem cells and inhibits the growth of liver cancer cells.