神经损伤与功能重建
神經損傷與功能重建
신경손상여공능중건
NEURAL INJURY AND FUNCTIONAL RECONSTRUCTION
2015年
2期
98-101
,共4页
马俊芳%崔博%沈东超%崔丽英
馬俊芳%崔博%瀋東超%崔麗英
마준방%최박%침동초%최려영
cyclinD1%过表达%神经干细胞%细胞周期
cyclinD1%過錶達%神經榦細胞%細胞週期
cyclinD1%과표체%신경간세포%세포주기
cyclinD1%over expression%neural stem cell%cell cycle
目的：针对小鼠 cyclinD1基因构建质粒并进行慢病毒包装，转染小鼠神经干细胞，检测其表达水平。方法：根据 cyclinD1基因信息，采用 DNA 重组技术将 Nestin promoter-Ccnd1基因插入 plenti6慢病毒表达载体，重组获得慢病毒载体 plenti-D1；经测序鉴定后，转染293T 细胞生产病毒液，并检测病毒滴度。设立空白对照组、阴性病毒对照组及过表达慢病毒感染组。将病毒转染小鼠胚胎神经干细胞，经实时荧光定量 PCR 和western blot 法分析转染前后3组 cyclinD1表达情况，MTT 法检测不同 MOI 值对神经干细胞增殖的影响。结果：测序结果证实 cyclinD1基因正确插入载体中，成功构建小鼠 cyclinD1基因过表达载体。实时荧光定量PCR 结果显示过表达慢病毒感染组 cyclinD1 mRNA 较其他2组明显升高； Western Blot 鉴定 cyclinD1蛋白表达成功。 plenti-D1的 MOI 值为10、20、50时均明显促进神经干细胞增殖。结论：cyclinD1基因慢病毒表达载体能感染小鼠胚胎神经干细胞，外源基因稳定表达。 cyclinD1基因过表达能促进神经干细胞的增殖。
目的：針對小鼠 cyclinD1基因構建質粒併進行慢病毒包裝，轉染小鼠神經榦細胞，檢測其錶達水平。方法：根據 cyclinD1基因信息，採用 DNA 重組技術將 Nestin promoter-Ccnd1基因插入 plenti6慢病毒錶達載體，重組穫得慢病毒載體 plenti-D1；經測序鑒定後，轉染293T 細胞生產病毒液，併檢測病毒滴度。設立空白對照組、陰性病毒對照組及過錶達慢病毒感染組。將病毒轉染小鼠胚胎神經榦細胞，經實時熒光定量 PCR 和western blot 法分析轉染前後3組 cyclinD1錶達情況，MTT 法檢測不同 MOI 值對神經榦細胞增殖的影響。結果：測序結果證實 cyclinD1基因正確插入載體中，成功構建小鼠 cyclinD1基因過錶達載體。實時熒光定量PCR 結果顯示過錶達慢病毒感染組 cyclinD1 mRNA 較其他2組明顯升高； Western Blot 鑒定 cyclinD1蛋白錶達成功。 plenti-D1的 MOI 值為10、20、50時均明顯促進神經榦細胞增殖。結論：cyclinD1基因慢病毒錶達載體能感染小鼠胚胎神經榦細胞，外源基因穩定錶達。 cyclinD1基因過錶達能促進神經榦細胞的增殖。
목적：침대소서 cyclinD1기인구건질립병진행만병독포장，전염소서신경간세포，검측기표체수평。방법：근거 cyclinD1기인신식，채용 DNA 중조기술장 Nestin promoter-Ccnd1기인삽입 plenti6만병독표체재체，중조획득만병독재체 plenti-D1；경측서감정후，전염293T 세포생산병독액，병검측병독적도。설립공백대조조、음성병독대조조급과표체만병독감염조。장병독전염소서배태신경간세포，경실시형광정량 PCR 화western blot 법분석전염전후3조 cyclinD1표체정황，MTT 법검측불동 MOI 치대신경간세포증식적영향。결과：측서결과증실 cyclinD1기인정학삽입재체중，성공구건소서 cyclinD1기인과표체재체。실시형광정량PCR 결과현시과표체만병독감염조 cyclinD1 mRNA 교기타2조명현승고； Western Blot 감정 cyclinD1단백표체성공。 plenti-D1적 MOI 치위10、20、50시균명현촉진신경간세포증식。결론：cyclinD1기인만병독표체재체능감염소서배태신경간세포，외원기인은정표체。 cyclinD1기인과표체능촉진신경간세포적증식。
Objective:In order to study the effect of cyclinD1 on neural stem cell proliferation, the cyclinD1 gene overexpression lentiviral vector was constructed and the expression of cyclinD1 in neural stem cells in mice after transfection was tested. Methods: Nestin promoter-Ccnd1 gene was inserted into plenti6 lentiviral expression vec-tors by DNA recombination technique. Lentiviral vector plenti-D1 was established after recombination. Recombi-nant lentiviral vector was detected by DNA sequencing. The plenti-D1 was transfected into 293T cell line. The viruses yielded by 293T cell were transfected into mouse embryonic neural stem cells. The expression of cyclinD1 was detected by real-time PCR and Western Blot analysis after transfection. We studied the effects of different MOI values of plenti-D1 on neural stem cell proliferation by MTT assay. Results: It was confirmed by DNA sequencing that the cyclinD1 gene sequencing was correctly inserted into the vector, and that the cyclinD1 gene overexpression lentiviral vector was successfully constructed. After extracting the infected cells, the real-time PCR showed cy-clinD1 mRNA overexpression was significantly higher than the control groups; Western Blot analysis was used to detect expression of cyclinD1 protein in neural stem cells. The cyclinD1 gene overexpression lentiviral vector was significantly up-regulated in mRNA level or in protein level in neural stem cells. When the MOI was 10, 20, and 50, the viruses significantly promoted the proliferation of neural stem cells. Conclusion: The cyclinD1 gene overex-pression lentiviral vector was successfully constructed and it efficiently up-regulated the expression of cyclinD1 in mouse embryonic neural stem cells. CyclinD1 overexpression can promote the proliferation of neural stem cells.
