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

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。

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

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 prolifera-tion 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 lentivi-ral plasmids (pLVTHM);(2) AP cells were successfully infected by recombinant lentivirus, which was obtained by each plasmid co-transfection into HEK 293t cells, together with the plasmids pS-PAX2 and pMD2. G;(3) The expression level of Tβ10 mRNA in the infected AP cells was signifi-cantly 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 ob-tained preliminary results for the role of Tβ10 in AP cells.