稳定过表达CLN3的SY5Y细胞增殖功能加强  

SY5Y cells stably- expressing CLN3 have better proliferating ability than wild type cells

作　　者：刘静[1] 杨萱[1] 迟洪滨[2] 吴丹[1] 吴白燕[1] 

机构地区：[1]北京大学医学部基础医学院医学遗传学系,100191 [2]北京大学第三医院,100191

出　　处：《国际遗传学杂志》2012年第3期133-138,168,共7页International Journal of Genetics

基　　金：国家自然科学基金（30900802）;高等学校博士学科点基金（20090001120102）

摘　　要：目的建立稳定表达CLN3（ceroid—lipofuseinosis，neuronal3）和CLN31．02kb缺失突变体基因（CLN3^△ex7/8）的稳定细胞系，研究全长CLN3在人神经母细胞瘤（SH—SY5Y）细胞中的促增殖作用及7、8号外显子缺失突变体对细胞增殖的影响。方法利用药物Zeocin筛选构建稳定过表达CLN3全长基因和CLN3^△ex7/8的SH—SY5Y细胞系，用RT-PCR，RealTimePCR和Western印迹检测CLN3全长及截短基因（CLN3^△ex7/8）的表达，并用CellCountingKit-8（CCK8）检测细胞增殖，比较两种细胞系对SH—SY5Y的增殖的影响。结果成功构建了稳定过表达CLN3全长基因细胞系SH—SY5Y／C和CLN3^△ex7/8的细胞系SH—SY5Y／Ct，mRNA水平分别过表达43倍和124倍，蛋白水平分别过表达1．5倍和20倍。过表达CLN3全长基因与空质粒对照组相比，增殖速率明显提高（P=0．044527），差异有统计学意义，而过表达CLN3^△ex7/8的空质粒对照组相比，增殖速率无差别（P=0．345329），差异无统计学意义。CLN3^△ex7/8对细胞的增殖没有明显促增殖作用。结论CLN3全长基因对细胞增殖具有明显促进作用，而过表达CLN3^△ex7/8对细胞的增殖没有明显影响，结果提示CLN3基因的7、8号外显子是调控细胞增殖的关键区域。Objective To establish a human neuroblastoma （SH-SY5Y）cell line, which can stably-express human ceroid-lipofuscinosis, neuronal 3 （ CLN3 ） and 1. 02 kb deletion within CLN3 （CLN3^△ex7/8） , and to detect-the proliferation of CLN3 and CLN3^△ex7/8 in SH-SY5Y. Methods After screening culture by Zeocin, the stable SH-SY5Y cells expressing CLN3 and CLN3^△ex7/8 were established. The transcription and expression of CLN3 and CLN3^△ex7/8 were identified by RT-PCR, Real Time PCR and Western blotting. To compare the proliferation of SH-SY5Y/C and SH-SY5Y/Ct using Cell Counting Kit-8 （CCK-8）. Results The cell line SH-SY5 Y/C expressing CLN3 stably and cell line SH-SY5 Y/ Ct expressing CLN3^△ex7/8 stably have been established successfully. The mRNA overexpress of CLN3 and CLN3^△ex7/8 were 43 times and 124 times more respectively than the control cell line transfected vector using method of Real Time PCR. The overexpression, at protein level, of CLN3 and CLN3^△ex7/8 were found to be 1. 5 times and 2 0 times more respectively than the control cell line transfected with vector using Western blot. The cell growth rate of overexpressing the full length of CLN3 was significantly higher than empty vector control （ P = 0. 044 527 ）. The cell grouth rate of overexpressing CLN3^△ex7/8 was not signifi- cantly different from empty vector control （ P = 0. 345 329 ） . Conclusion The full length of CLN3 protein results in an increase in cell growth rate of SH-SYSY cell and the CLN3^△ex7/8 trnneated protein has no effect on the growth rate of SH-SY5Y cell. These results indicate that the exon of 7 and 8 maybe play an important role in regulating proliferation of SH-S＇Y5Y cell.

关 键 词：SH-SY5Y细胞 CLN3 CLN3^△ex7/8 细胞系构建 增殖 

分 类 号：R748[医药卫生—神经病学与精神病学]