梅花鹿Thymosin beta 10基因RNAi慢病毒载体的构建及其对鹿茸干细胞增殖的影响

Construction of RNAi Lentiviral Vector for Thymosin beta 10 and Effects of Its Introduction on Proliferation of Antler Stem Cells in Sika Deer

利用慢病毒干扰系统,对梅花鹿生茸区骨膜干细胞(Antlerogenic periosteum cells,AP细胞)Thymosin beta 10(Tβ10)基因进行RNA干扰(RNA Interference,RNAi),并初步研究该基因对细胞增殖的影响。结果表明:试验设计的3条针对梅花鹿Tβ10基因的shRNA序列与载体质粒p LVTHM连接成功,与p SPAX2、p MD2.G质粒共转染HEK 293T细胞,获得的重组慢病毒成功感染了AP细胞;荧光定量PCR检测结果表明,RNA干扰后Tβ10基因的mRNA水平下调,MTT试验结果显示Tβ10基因的下调可以抑制AP细胞增殖。试验成功构建了针对梅花鹿Tβ10基因RNAi载体,并成功干扰了Tβ10基因在AP细胞中的表达,基因下调最高可达35.6%,并初步确定Tβ10基因下调可抑制AP细胞的增殖。

Thymosin beta 10（ Tβ10） gene of antlerogenic periosteum cells of Chinese sika deer was interfered by using RNAi lentiviral vector system,and effects of the system introduction on proliferation of antler stem cells were primarily studied. The results showed that：（ 1） Three sequences of small interfering RNAs,targeting Tβ10 gene of sika deer,were successfully ligated into the lentiviral plasmids（ pLVTHM）;（ 2） AP cells were successfully infected by recombinant lentivirus,which was obtained by each plasmid co-transfection into HEK 293 t cells,together with the plasmids p SPAX2 and pMD2. G;（ 3） The expression level of Tβ10 mRNA in the infected AP cells was significantly decreased through RT-PCR measurement,and the interference of Tβ10 played a role in the suppression of proliferation of AP cells through MTT assay. Therefore,in this study we successfully interfered Tβ10 gene in the AP cells with the efficiency of 35. 6% compared to the control,and obtained preliminary results for the role of Tβ10 in AP cells.