[1]黄似建,刘畅,秦泽莲**,等.人胸腺素β4RNA干扰载体的构建及其对293T细胞目的基因表达的影响[J].中国微创外科杂志,2009,09(7):658-662.
　Huang Sijian,Liu Chang,Qin Zelian,et al.Construction of Thymosin β4 Shrna Lentivirus and Its Effects on the Expression of Thymosin β4 in 293T Cells[J].Chinese Journal of Minimally Invasive Surgery,2009,09(7):658-662.
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人胸腺素β4RNA干扰载体的构建及其对293T细胞目的基因表达的影响()
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《中国微创外科杂志》[ISSN:1009-6604/CN:11-4526/R]

卷:
09
期数:
2009年7期
页码:
658-662
栏目:
基础研究
出版日期:
2009-08-25

文章信息/Info

Title:
Construction of Thymosin β4 Shrna Lentivirus and Its Effects on the Expression of Thymosin β4 in 293T Cells
作者:
黄似建; 刘畅; 秦泽莲**; 陈莉
北京大学第三医院成形外科，北京100191
Author(s):
Huang Sijian;  Liu Chang;  Qin Zelian;  et al.
Department of Plastic Surgery, Peking University Third Hospital, Beijing 100191, China
关键词:
Thymosin β4; 293T细胞; RNA干扰; 慢病毒
Keywords:
Thymosin β4; 293T cell; RNA interference; Lentivirus
分类号:
R-33
文献标志码:
A
摘要:
目的探讨人胸腺素β4（thymosin β4，TMSB4）基因RNA干扰慢病毒载体的构建，观察病毒感染293T细胞后TMSB4 mRNA和蛋白表达，为进一步研究TMSB4基因的功能提供基础。方法设计特异性干扰人TMSB4基因的小发卡RNA序列，合成含有干扰序列的双链DNA oligo，与经Age I和EcoR I双酶切后的pGCSIL-GFP载体连接产生慢病毒载体。转化感受态大肠杆菌DH5a并对阳性克隆进行PCR扩增测序鉴定。慢病毒载体、包装系统质粒共转染包装细胞293T细胞，产生慢病毒颗粒并测定病毒滴度。将获得的慢病毒感染293T细胞，分别采用实时定量PCR和免疫细胞化学染色方法观察 TMSB4mRNA和蛋白的表达情况，利用生成的 TMSB4 shRNA慢病毒感染原代成纤维细胞。结果感染重组慢病毒的293T细胞与空病毒转染阴性对照细胞的TMSB4 mRNA表达分别为0.56±0.11和1.00±0.06(P=00006)，实验组细胞的敲减率为44%。实验组TMSB4蛋白表达水平0.042±0.007比阴性对照组细胞0.093±0.009的表达降低55%（H=461.342，P<0001）。感染重组慢病毒的原代培养的成纤维细胞与空病毒转染的对照细胞相比，TMSB4蛋白表达水平亦明显降低。结论TMSB4基因干扰慢病毒构建成功并能显著降低TMSB4基因和蛋白在293T细胞中的表达，成功感染原代成纤维细胞使其目的基因蛋白减少，为进一步研究TMSB4基因的功能奠定研究基础。
Abstract:
ObjectiveTo construct a recombinant lentiviral vector for human thymosin β4 (TMSB4) and to test the mRNA and protein expression of TMSB4 in 293T cells after being infected by shRNA lentivirus.MethodsWe designed a specific sequence of small hair RNA targeting TMSB4 gene; the complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was cloned into the pGCSILGFP vector to construct a lentiviral vector. The vector was converted into the competent DH5a coli, which confirmed by PCR and sequencing. Then the viral vector and the packed systemic vector were cotransfected 293T cells to get the lentivirus. The virus titer was determined. Afterwards, in the 293T cells infected with the lentivirus, the expression of TMSB4 was detected by real time PCR and immunocytochemistry. Similarly, a primary fibroblast was also infected with the lentivirus. Results Compared with negative control cells, the mRNA and protein levels of TMSB4 in 293T cells infected with the lentivirus were reduced by 44% (0.56±0.11 vs 1.00±0.06, F=89.673, P<0.01) and 55% (0.042±0.007 vs 0.093±0.009, H=461.342, P<0001) respectively. The expression of TMSB4 was significantly reduced in the primary fibroblasts.Conclusions The expression of TMSB4 in 293T cells and primary fibroblasts is reduced after infection by lentiviral shRNA expression vector targeting human TMSB4, which will be a basis to the further function study of TMSB4.

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备注/Memo

备注/Memo:
*国家自然科学基金资助课题（30471793）**通讯作者
更新日期/Last Update: 2013-09-17