慢病毒载体介导的结缔组织生长因子基因沉默对下游基因的影响*

doi:10.3969/j.issn.1672-5069.2012.03.013

实用肝脏病杂志 ›› 2012, Vol. 15 ›› Issue (3): 212-215.doi: 10.3969/j.issn.1672-5069.2012.03.013

慢病毒载体介导的结缔组织生长因子基因沉默对下游基因的影响^*

彭梅娟, 郝春秋, 张, 野, 谢玉梅, 张, 岩, 白雪帆

710038 西安市第四军医大学唐都医院全军感染病诊疗中心

收稿日期:2011-09-12 出版日期:2016-06-10 发布日期:2016-05-11
通讯作者: 郝春秋,E-mail：haochq@fmmu.edu.cn;
作者简介:彭梅娟女,33岁,主治医师。E-mail：pengmeijuan2007@yahoo.com.cn
基金资助:
国家自然科学基金（No：30571658）

RNA interference mediated by lent viral vector suppresses the expression of connective tissue growth factor and its effect on downstream gene

Peng Meijuan, Hao Chunqiu, Zhang Ye, et al.

The Center of Diagnosis and Treatment for Infectious Diseases of PLA,Tangdu Hospital,Fourth Military Medical University, Xi’an 710038

Received:2011-09-12 Online:2016-06-10 Published:2016-05-11

摘要/Abstract

摘要： 目的利用RNA干扰技术,以慢病毒为载体,介导结缔组织生长因子（CTGF）基因沉默,并观察其对下游基因表达的影响。方法用构建的CTGF小干扰RNA（siRNA）慢病毒转移质粒与包装质粒、包膜蛋白质粒共转染293T细胞,包装成介导CTGF基因沉默的慢病毒载体;感染肝星状细胞系HSC-T6,应用Real-time PCR和Western免疫印迹法筛选基因沉默效率最高的慢病毒载体;再根据绿色荧光蛋白（GFP）表达水平检测感染效率;应用Real-time PCR法检测CTGF基因沉默后HSC-T6中金属蛋白酶组织抑制剂-1（TIMP-1）、TIMP-2和I 型胶原mRNA表达水平。结果包装的慢病毒载体感染HSC-T6细胞的效率高于50%;感染HSC-T6细胞后,CTGF的表达在mRNA和蛋白水平均受到明显抑制,其中TS1具有最佳的抑制效率;HSC-T6细胞CTGF基因沉默后,其TIMP-1、TIMP-2和I型胶原mRNA水平也显著降低。结论包装的慢病毒载体在HSC-T6中具有较好的感染效率和CTGF基因沉默效果,基因沉默后可进一步抑制HSC-T6细胞TIMP-1、TIMP-2和I型胶原的基因表达,这在抗纤维化的研究中具有积极的意义。

关键词: HSC-T6细胞, 结缔组织生长因子, 慢病毒载体, RNA干扰

Abstract: Objective To inhibit the gene expression of connective tissue growth factor （CGTF） by small interfering RNA（siRNA） and observe its influence on downstream gene expression. Methods 293T cells were cotransfected with lent viral transferred plasmid containing siRNA targeted to CTGF,packaging plasmid,and envelope protein plasmid. The lent viral vector was packed to mediate CTGF gene silence and infect hepatic stellate cell line HSC-T6 cells. The lent viral vector with the highest inhibition efficiency was screened by real-time PCR and Western blot. Efficiency of infection was measured by the expression of green fluorescent protein. mRNA levels of tissue inhibitor of metalloproteinase-1 （TIMP-1）,TIMP-2,and collagen I were detected by real-time PCR. Results Efficiency of infection of the packing lent viral vector was more than 50%. After infecting HSC-T6 cells,the expression of CTGF was suppressed on both mRNA and protein level,with the highest inhibition efficiency by TS1. The expressions of mRNA in TIMP-1,TIMP-2,and collagen I were significantly restrained by inhibition of CTGF（P<0.05）. Conclusion The packing lent viral vector in HSC-T6 has a better efficiency of infection and CTGF gene silencing effect. Gene silencing can further inhibit the TIMP-1,TIMP-2 and collagen type I gene expression in HSC-T6 cells. It has an active significance in anti-fibrosis studying.

Key words: HSC-T6 cells, Connective tissue growth factor, lent viral vector, RNA interference

彭梅娟, 郝春秋, 张, 野, 谢玉梅, 张, 岩, 白雪帆. 慢病毒载体介导的结缔组织生长因子基因沉默对下游基因的影响^*[J]. 实用肝脏病杂志, 2012, 15(3): 212-215.

Peng Meijuan, Hao Chunqiu, Zhang Ye, et al.. RNA interference mediated by lent viral vector suppresses the expression of connective tissue growth factor and its effect on downstream gene[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2012, 15(3): 212-215.

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慢病毒载体介导的结缔组织生长因子基因沉默对下游基因的影响^*

RNA interference mediated by lent viral vector suppresses the expression of connective tissue growth factor and its effect on downstream gene

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