人PKD2基因慢病毒的构建及其对常染色体显性遗传性多囊肾病小鼠肾脏上皮细胞多囊蛋白-2和Wnt/β-catenin信号通路的修复作用  

Construction of human PKD2 lentivirus to rectify polycystin-2 expression and Wnt/β-catenin signaling pathways in the Pkd2-null cell lines

作　　者：孟佳林[1] 徐雨辰[1] 沈旭峰 李奥 樊松[1] 郝宗耀[1] 吴冠青 梁朝朝[1] 

机构地区：[1]安徽医科大学第一附属医院泌尿外科安徽省多囊肾诊疗与转化医学中心,230032

出　　处：《中华泌尿外科杂志》2018年第1期62-68,共7页Chinese Journal of Urology

基　　金：国家自然科学基金（81630019,81673402,81400757）

摘　　要：目的构建携带人PKD2基因的重组慢病毒载体，并探讨该载体对Pkd2缺失小鼠肾脏上皮细胞中多囊蛋白-2（PC2）的表达，以及对下游Wnt/β-catenin信号通路的作用。 方法2016年8月至2017年2月采用XbaⅠ和XhoⅠ酶将PKD2基因从已有pcDNA3.1-TM-PKD2质粒中切出，并插入慢病毒空载体pLV-sfGFP_2A_Puro，测序验证后获得重组慢病毒载体pLV-sfGFP-PKD2。然后，将重组慢病毒载体与包装质粒共脂质体转染293T细胞，包装病毒，获得病毒浓缩液。按照实验组（pLV-sfGFP-PKD2病毒感染）、对照组（pLV-sfGFP病毒感染）和空白组（未感染）进行分组，分别处理携带Pkd2基因的B3、D3细胞（Pkd2＋/－）和Pkd2缺失的B2、E8细胞（Pkd2－/－），采用蛋白质印迹法、免疫荧光染色法检测PC2表达情况。最后，选取B3和B2细胞，利用增殖细胞核抗原（PCNA）荧光染色检测细胞增殖活性，实时荧光定量PCR检测PKD2下游Wnt/β-catenin信号通路的变化。 结果重组慢病毒载体酶切后凝胶电泳结果显示插入片段与PKD2基因一致，基因测序结果显示PKD2插入方向正确。经pLV-sfGFP-PKD2病毒感染后，实验组B2和E8细胞的PC2表达量分别为0.668±0.013和0.763±0.021，空白组B3和D3细胞的PC2表达量分别为0.687±0.015和0.776±0.008，差异均无统计学意义（P＝0.164，P＝0.409）。实验组B2细胞增殖活性（0.573±0.010）明显低于空白组（0.848±0.031），差异有统计学意义（P〈0.01）；与空白组B3细胞（0.585±0.017）比较差异无统计学意义（P＝0.369）。实时荧光定量PCR检测结果显示，实验组B2细胞中的Wnt7a和β-catenin基因表达量分别为0.037±0.005和0.554±0.008，与空白组B3细胞的0.037±0.004和0.571±0.013比较差异均无统计学意义（P＝0.969，P＝0.640）。 结论本研究成功构建携带人PKD2基因并具有感染能力的重组pLV-sfGFP-PKD2慢病毒，慢病毒可重建PKD2基因缺失的小鼠肾脏上皮�ObjectiveTo establish PKD2 gene recombinant lentivirus and to investigate its restorative effects on polycystin-2 and Wnt/β-catenin signaling pathways in Pkd2-null cell lines. MethodsFrom August 2016 to February 2017, PKD2 gene was cleaved from the pcDNA3.1-TM-PKD2 plasmid and was inserted into the pLV-sfGFP_2A_Puro by restriction enzymes XbaⅠ and XhoⅠ. The recombinant pLV-sfGFP-PKD2 plasmid was sequenced to verify a correct construction. Then we obtained recombinant lentiviruses by co-transfecting 293T cells with recombinant plasmid and packaging plasmids. B3/D3 （Pkd2＋ /-） and B2/E8 （Pkd2-/-） cell lines were used to evaluate the effectiveness of lentivirus, they were divided into experimental group, control group, blank group and treated with pLV-sfGFP-PKD2 virus, pLV-sfGFP virus or culture media, respectively. The expression of PC2 was detected by Western blotting and immunofluorescence staining. Finally the cell proliferation was evaluated by detecting of proliferating cell nuclear antigen. The changes of Wnt/β-catenin signaling pathway were evaluated by real-time quantitative PCR. ResultsEnzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-sfGFP-PKD2 was constructed successfully. After infected with pLV-sfGFP-PKD2 virus, the expression of PC2 in the experimental group B2 and E8 cells（0.668±0.013, 0.763±0.021） was restored to the normal level, compared with control group B3 and D3 cells, respectively （0.687±0.015, P=0.164; 0.776±0.008, P=0.409）. The proliferative activity in experimental group B2 cells（0.573±0.010） was significantly lower than that in control group B2 cells （0.848±0.031, P〈0.01）, and was returned to the level of blank group B3 cells （0.585±0.017, P=0.369）. Reexpression of PKD2 in experimental group B2 cells also reduced the expression of Wnt7a, β-catenin, back to blank group B3 cells′ level（0.037±0.005 vs.0.037±0.004, P=0.969; 0.554±0.008 vs. 0.571±0.013, P=0.64）. ConclusionsThe recombinant pLV-sfG

关 键 词：多囊肾 常染色体显性 WNT/Β-CATENIN信号通路 转基因 慢病毒 

分 类 号：R692[医药卫生—泌尿科学]