【摘要】 目的 通过基因转染技术,将核心蛋白聚糖(DCN)转染成纤维细胞,观察DCN在成纤维细胞中的表达,及其表达的DCN对肾间质成纤维细胞特性的影响,探索肾脏疾病基因治疗的途径。方法 在成功构建重组逆转录病毒载体pL(DCN)SN的基础上,采用LipofectAMINE脂质体介导质粒DNA转染逆转录病毒包装细胞PA317,进行逆转录病毒的包装。以NIH 3T3细胞作为指标靶细胞进行病毒滴度测定。重组逆转录病毒在Polybrene的辅佐下感染正常大鼠肾间质成纤维细胞,并应用G418筛选。所得抗G418细胞克隆FB(LDCNSN)应用PCR和逆转录-PCR分析外源基因的整合和表达。采用Western印迹检测转基因细胞FB(LDCNSN)培养细胞上清液中DCN蛋白的检测。应用四甲基偶氮唑蓝(MTT)比色法和流式细胞仪分别测定细胞增殖和细胞周期,观察DCN对肾间质成纤维细胞增殖和细胞周期的影响。结果 重组逆转录病毒载体pL(DCN)SN的质粒DNA经LipofectAMINE介导,转染PA317包装细胞。G418筛选出抗性克隆,扩增。收集含重组逆转录病毒细胞上清液,在Polybrene的辅佐下感染大鼠肾间质成纤维细胞,并获得抗G418的细胞克隆FB(LDCNSN)。PCR和逆转录-PCR分析外源基因DCN的整合和表达,电泳可见1.1kb的条带。Western印迹分析FB(LDCNSN)细胞培养上清液中DCN蛋白质,证实FB(LDCNSN)表达DCN蛋?更多还原

【Abstract】 Objective To construct a retrovirus vector carrying rat DCN cDNA and to investigate the effect of DCN on the behavior of renal interstitial flbroblasts. Methods Retrovirus vector pLXSN was used to construct pL(DCN)SN recombinant vector (carrying rat DCN cDNA with secretive signal peptide). Retrovirus recombinant pL(DCN) SN vector was transfected into PA317 cells by LipofectAMINE. The virus was packaged in PA317 cells. The secreted virus titer was measured by counting the number of formed cell clones of NIH3T3 which had been infected by retrovirus and resistant to G418. The supernatant containing the virus with higher titer was used to infect the normal rats flbroblasts. Integration and expression of DCN gene were analyzed by PCR and RT-PCR in FB(LDCNSN) (fibroblasls conlaining the transfected DCN gene) cells. DCN protein expressed by FB(LDCNSN) was detected by Western blot. To observe the effect of DCN on the behavior of renal interstitial flbroblasts,cellular proliferation and cell cycle were assessed by MTT method and flow cytometry,respectively. Results L(DCN)SN plasmid was transfected to PA317 cells by LipofectAMINE. The cell clones resistant to G418 were selected. After the virus secreted into the supernatant of PA317(LDCNSN) infected the flbroblasts,FB(LDCNSN) cells resistant to G418 were grown and formed cell clones about 3 weeks later. DNA and RNA analysis with DCN primers were showed that there were bands marked 1. 1 kb. It means that the FB(LDCNSN) cells contain the DCN gene. Protein analysis by Western blot showed that there were more DCN protein marked 110 kd in the supernatant of FB(LDCNSN) than that of control cells. DCN protein had an inhibition effect on the proliferation of renal interstitial fibroblasts. TGF-β1(5 ng/ml) could stimulate the proliferation of renal interstitial fibroblasts,but this effect was attenuated when normal renal interstitial fibroblasts were cultured in media of supernatant containing DCN. DCN protein could affect the cell cycle of the fibroblasts. TGF-β1 (5 ng/ml) could increase the percentage of G0/G1 compared with control group,but this effect was also attenuated by adding the supernatant of FB (LDCNSN) cells. These results suggested that the supernatant of FB(LDCNSN) had the effect of blocking or neutralizing the activity of TGF-β1. Conclusions DCN protein expression is increased in renal interstitial fibroblasts infected by retrovirus containing the DCN gene. DCN protein secreted by FB(LDCNSN) has the activity to neutralize the effect of TGF-β1.It may be used to treat the renal interstitial fibrosis by inhibiting the proliferation and changing the cell cycle of renal interstitial fibroblasts stimulated by TGF-β1.更多还原