CAJ | 학술논문

目的建立稳定表达CLN3(ceroid-lipofuscinosis,neuronal 3)和CLN3 1.02 kb缺失突变体基因(CLN3Δex7/8)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CLN 3Δex7/8的SH-SY5Y细胞系,用RT-PCR,Real Time PCR和Western印迹检测CLN3全长及截短基因(CLN 3Δex7/8)的表达,并用Cell Counting Kit-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CLN3Δex7/8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=0.044 527),差异有统计学意义,而过表达CLN 3Δex7/8的空质粒对照组相比,增殖速率无差别(P =0.345 329),差异无统计学意义.CLN3Δex7/8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CLN 3Δex7/8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.
목적 건립은정표체CLN3(ceroid-lipofuscinosis,neuronal 3)화CLN3 1.02 kb결실돌변체기인(CLN3Δex7/8)적은정세포계,연구전장CLN3재인신경모세포류(SH-SY5Y)세포중적촉증식작용급7、8호외현자결실돌변체대세포증식적영향.방법 이용약물Zeocin사선구건은정과표체CLN3전장기인화CLN 3Δex7/8적SH-SY5Y세포계,용RT-PCR,Real Time PCR화Western인적검측CLN3전장급절단기인(CLN 3Δex7/8)적표체,병용Cell Counting Kit-8(CCK8)검측세포증식,비교량충세포계대SH-SY5Y적증식적영향.결과 성공구건료은정과표체CLN3전장기인세포계SH-SY5Y/C화CLN3Δex7/8적세포계SH-SY5Y/Ct,mRNA수평분별과표체43배화124배,단백수평분별과표체1.5배화20배.과표체CLN3전장기인여공질립대조조상비,증식속솔명현제고(P=0.044 527),차이유통계학의의,이과표체CLN 3Δex7/8적공질립대조조상비,증식속솔무차별(P =0.345 329),차이무통계학의의.CLN3Δex7/8대세포적증식몰유명현촉증식작용.결론 CLN3전장기인대세포증식구유명현촉진작용,이과표체CLN 3Δex7/8대세포적증식몰유명현영향,결과제시CLN3기인적7、8호외현자시조공세포증식적관건구역.
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 ( CLN3Δex7/8 ),and to detect the proliferation of CLN3 and CLN3Δex7/8 in SH-SY5Y.Methods After screening culture by Zeocin,the stable SH-SY5Y cells expressing CLN3 and CLN3Δex7/8 were established.The transcription and expression of CLN3 and CLN3Δex7/8 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-SY5Y/C expressing CLN3 stably and cell line SH-SY5 Y/Ct expressing C LN3Δex7/8 stably have been established successfully.The mRNA overexpress of CLN3 and CLN3Δex7/8 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 CLN3Δex7/8 were found to be 1.5 times and 20 times more respectively than the control cell line transfected with vector usingWestern 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 CLN3Δex7/8 was not significantly 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 CLN3Δex7/8 truncated protein has no effect on the growth rate of SH-SY5 Y cell.These results indicate that the exon of 7 and 8 maybe play an important role in regulating proliferation of SH-SYSY cell.