分化抑制因子1重组腺病毒的构建及其动物模型的建立

Construction of the recombinant adenovirus of inhibitor of differentiation 1 and establishment of its animal model

目的:构建分化抑制因子-1(inhibitor 1 of differentiation,Id1)重组腺病毒(recombinant adenovirus,Rad),并建立Id1过表达的小鼠动物模型。方法:在HEK293细胞中将腺病毒重组质粒Ad Easy-Id1进行包装以获得Id1重组腺病毒(RAd-Id1);通过绿色荧光蛋白(green fluorescent protein,GFP)标记法测定腺病毒滴度;通过q RT-PCR和Western blot验证RAd-Id1感染的Hep G2细胞中的Id1过表达效果,用MTS比色法检测RAd-Id1对Hep G2细胞增殖的影响;通过尾静脉注射RAd-Id1的感染方式建立肝组织中过表达Id1的小鼠动物模型,病毒载量梯度实验分PBS空白组、RAd-GFP对照组及RAd-Id1实验组(n=5/组)以测定RAd-Id1感染小鼠的适合载量;病毒感染时间梯度实验每5 d检测1次为1组,共7组(n=5/组)以测定感染小鼠的适合时间;通过Western blot检测小鼠肝组织中Id1的过表达效果及甲胎蛋白(alpha fetal protein,AFP)和癌胚抗原(carcinoembryonic antigen,CEA)的表达水平,再采用ELISA检测小鼠血清中Id1的免疫原性及AFP和CEA的表达情况。结果:成功包装获得RAdId1,测得其滴度为1.5×1011 GFU/m L;RAd-Id1感染Hep G2细胞48 h和72 h后,Id1的转录和蛋白水平均明显升高(P<0.01);96 h时RAd-Id1实验组的MTS检测值较PBS空白组和RAd-GFP对照组(2.00±0.06 vs.1.18±0.05,2.00±0.06 vs.1.10±0.07;F=51.350,P=0.000)明显升高;Western blot结果显示:在RAd-Id1感染的小鼠肝组织中,Id1、AFP和CEA蛋白水平较PBS空白组和RAd-GFP对照组升高;ELISA实验结果表明:血清中AFP和CEA蛋白水平与RAd-Id1的载量呈正相关(rs AFP=0.903,PAFP=0.014;rs CEA=0.964,PCEA=0.002),不同载量RAd-Id1感染小鼠的血清中Id1抗体滴度无显著差异(P>0.05)。结论:成功构建了RAdId1及其介导的小鼠动物模型;RAd-Id1可作为细胞水平上研究Id1对肝癌影响的载体工具;AFP和CEA的升高提示Id1可能参与诱导AFP和CEA的表达。

Objective ：To construct the recombinant adenovirus（Rad） with expressing inhibitor 1 of differentiation（Id1）, and to establish its animal model. Methods：AdEasy-Id1 ,the recombinant adenovirus plasmid which had been constructed previously,was packaged in HEK293 cells to obtain recombinant adenovirus Id1 （RAd-Id1）. Then the titer of virus was determined by green fluorescent protein（GFP） labeling method. The over-expression efficiency of Id1 was measured by qRT-PCR and Western blot in HepG2 cells infected with RAd-Id1, and the effect of Id1 on proliferation of HepG2 cells was detected by MTS assay. Animal model of Id1 expressing in liver tissue was established through the way of tail vein injection of RAd-Id1. In order to determine the suitable load of RAd-Id1 virus for infecting the mice,viral load gradient experiment was divided into PBS control group,RAd-GFP control group and RAd-Id1 experimental group（n=5/group）. Time gradient experiment examined once every 5 days as a group, a total of 7 group（n=5/group）,to explore the appropriate time. The expression of Id1 as well as the changes of alpha fetal protein（AFP） and carcinoembryonic antigen（CEA） in liver tissues were determined by Western blot. The level of Id1 antibody and the concentrations of AFP and CEA in serum were detected by ELISA. Results：Recombinant adenovirus plasmid containing Id1 gene was successfully established,then the corresponding adenovirus were acquired and the measured titer was 1.5 × 10 11 GFU/mL. Western blot and qRT-PCR analysis showed that Id1 mRNA and protein levels significantly increased in HepG2 infected with RAd-Id1 for 48 h and 72 h respectively（P〈0.01）. MTS assay reported that the values in RAd-Id1 group were higher than those in NC group and RAd- GFP group after 96 h（2.00 ± 0.06 vs. 1.18 ± 0.05,2.00 ± 0.06 vs. 1.10 ± 0.07;F=51.350,P=0.000） ;Western blot results showed：when compared with PBS group and RAd-GFP group,the RAd-Id1 group＇s levels of Idt as well as AFP and CEA in liver was higher. ELISA results showed：the concentrations of AFP and CEA in sernm were positively correlated with the load of RAd-Id1 （rs AFP=0.903, P AFP=0.014;rs CEA=0.964,P CEA=0.002）. There was no significant difference among these concentrations of Id1 antibody in serum of mice infected with different load of RAd-Id1 （P〉0.05）. Conclusion：The RAd-Id1 is successfully constructed,which serves as the vector tool to study the effect of Id1 on hepatic neoplasm at the cellular level. Mouse animal model of RAd-Id1 accompanied with the elevation of both AFP and CEA in liver also has been successfully established by tail vein injection,which suggests that the ectopic expression of Id1 may be related to the expression of AFP and CEA, but further studies will be required to determine how the mechanism works.