ATXN1基因内CAG重复序列拷贝数增加对神经母细胞瘤细胞增殖与凋亡的影响  

Effects of increased copy number of CAG repeats in ATXN1 gene on proliferation and apoptosis of neuroblastoma cells

作　　者：陈诗恺 孙顺昌 CHEN Shikai;SUN Shunchang(Ruijin Hospital North District, Shanghai Jiaotong University School of Medicine, Shanghai 201801, China)

机构地区：[1]上海交通大学医学院附属瑞金医院北院

出　　处：《山东医药》2019年第16期4-7,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(31571294)

摘　　要：目的探讨ATXN1基因内CAG重复序列拷贝数增加对神经母细胞瘤细胞增殖与凋亡的影响。方法首先构建正常ATXN1基因(CAG重复序列拷贝数为29)和突变ATXN1基因(CAG重复序列拷贝数为61),将其插入pLVX-Puro-3xflag-C载体中构建表达载体,表达载体再与pMD2.G及psPAX2共转染人胚肾细胞(293T细胞)产生慢病毒表达载体,分别感染293T细胞,筛选稳定表达的细胞株。通过CCK8实验检测稳定表达细胞株的增殖能力,通过流式细胞仪检测稳定表达细胞株的凋亡率。结果表达突变ATXN1基因的细胞在培养3~5 d时细胞的OD450值均低于表达正常ATXN1基因的细胞(P<0.01)和只转染pLVX-Puro-3xflag-C载体的阴性对照细胞(P<0.01);5 d后表达突变ATXN1基因、正常ATXN1基因、阴性对照细胞凋亡率分别为12.56%、9.06%、8.17%,表达突变ATXN1基因的细胞凋亡率高于表达正常ATXN1基因细胞和阴性对照细胞的凋亡率(P均<0.01)。结论 ATXN1基因内CAG重复序列拷贝数增加可抑制神经母细胞瘤细胞的增殖,促进细胞凋亡。Objective To explore the effects of the expansion of CAG repeats in the ATXN1 gene on proliferation and apoptosis of neuroblastoma cells. Methods Wild-type (29 CAG repeats) and mutant (61 CAG repeats) ATXN1 gene were constructed by PCR cloning, then were inserted into the expression vector pLVX-Puro-3xflag-C.This expression vector,pMD2.G vector, and psPAX2 vector were co-transfected into the human embryonic kidney 293T cells (293T cells) to generate the lentiviruses, which were used to infect the 293T cells, and then we screened out the cell lines with stable expression. The proliferation ability was determined by CKK-8, the apoptosis rate was detected by flow cytometry. Results The OD 450 of the 293T cell line with the mutant ATXN1 gene was lower than that of the cell line with the wild ATXN1 gene ( P <0.01) and the negative control cells only transfected with the pLVX-Puro-3xflag-C vector ( P <0.01) after cells were cultured for 3-5 days. The 293T cell line with the mutantATXN1 gene had higher apoptosis rate (12.56%) than the cell line with the wild ATXN1 gene (9.06%) and the negative control cells (8.17%), with statistically significant difference (all P <0.01) at 5 d after cultivation. Conclusion The expansion of CAG repeats in the ATXN1 gene can inhibit the cellular proliferation and promote the apoptosis.

关 键 词：遗传性脊髓小脑型共济失调 ATXN1基因 CAG重复序列 细胞增殖 细胞凋亡 

分 类 号：R745.1[医药卫生—神经病学与精神病学]