摘要
目的:探讨hCREG表达与人血管平滑肌细胞增殖的量效关系。方法:构建强力霉素(Dox)调控表达的pRevTRE-hCREG重组载体;分别经G418及潮霉素筛选表达pRevTet-On、pRevTRE-hCREG的人血管平滑肌细胞株-HITASY细胞克隆;RT-PCR鉴定转染细胞克隆中抗性基因和插入基因的表达;Western blotting和免疫共沉淀检测不同浓度的强力霉素诱导后,HITASY细胞内及培养上清中hCREG的表达;流式细胞仪检测不同浓度的强力霉素诱导后,hCREG表达与细胞增殖的关系。结果:成功构建了强力霉素可调控表达的pRevTRE-hCREG重组载体;筛选出稳定表达双抗性基因的HITASY-hCREG细胞克隆;RT-PCR检测证实细胞克隆中G418及插入基因表达均为阳性;Western blotting及免疫共沉淀检测发现随强力霉素诱导剂量的增加(0、0.01、0.1、1mg/L),HITASY-hCREG细胞及培养上清中hCREG表达均呈剂量依赖性增加;流式细胞周期分析提示,hCREG表达增加后,HITASY-hCREG细胞的G1期细胞明显增加。结论:hCREG蛋白通过剂量依赖方式抑制人血管平滑肌细胞的增殖。
AIM： To elucidate the relationship between the dosage of human cellular repressor of E1A - stimulated genes （hCREG） and the proliferation of vascular smooth muscle cells （VSMCs）. METHODS： hCREG cDNA was cloned into a retroviral response vector （pRevTRE） controlled by tetracycline responsive element. The recombinant vector （ pRevTRE - hCREG） was constructed and confirmed by sequence analysis. The pRevTet - On was transferred into human VSMCs - HITASY in vitro and selected by G418. The pRevTRE - hCREG was transferred into pRevTet - On - HITASY clones and selected by hygromycin. The expression of G418 and TRE was detected by RT - PCR analysis. The hCREG expressions in cell lysate and in supernant were evaluated by Western blotting and IP - Western respectively after the HITASY - hCREG cells cultured in medium at different concentrations of doxycycline. The relationship between dosage of hCREG and the proliferation of HITASY cells was assessed by flow cytometry analysis. RESULTS： The recombinant pRevTRE - hCREG was constructed successfully. The fragment inserted was confirmed by sequencing. The pRevTet - On and pRevTRE -hCREG were transferred into human HITASY cells and the clones （HITASY- hCREG） expressed double -resistant genes stably were obtained. RT - PCR identified that HITASY - hCREG cells expressed a 500bp band for G418 and a 899bp band for TRE, respectively. The expressions of hCREG both in lysate and in supernant were upregulated in parallel with the doxcycline increase in a dose dependent manner. Flow cytometry analysis showed that the distribution of G1 stage in HITASY - hCREG cells was also enhanced in accordance with the increase in hCREG expression. CONCLUSION： The expression of hCREG inhibits the proliferation of human VSMCs in a dose dependent manner.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2008年第11期2121-2125,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30070280
No.30570664)
辽宁省科技攻关计划资助项目(No.2007225004-9)
关键词
阻遏蛋白质类
血管平滑肌细胞
细胞增殖
Repressor proteins
Vascular smooth muscle cells
Cell proliferation
