一个细胞融合实验体系的建立及初步应用

Construction of a cell-cell fusion system and its primary application

目的建立一个细胞融合实验体系,观察凝血酶(thrombin,Th)对Ⅰ型人免疫缺陷病毒(human immunodeficiency virus typeⅠ,HIV-1)包膜糖蛋白(envelope glycoproteins,Env)介导的细胞融合的影响。方法采用携带HIV-1 JRFL株env基因的表达质粒pSV-Ⅲ-JRFL-dCT与携带HIV-1 tat基因的表达质粒pcTat共转染293T细胞,检测到细胞膜表面Env表达后。将pSV-Ⅲ-JRFL-dCT与pcTat分别按1:2、2:3、1:1、2:1、5:1、10:1共转染293T细胞,然后再与Magic 5A细胞进行融合实验,计数融合细胞数目并确定最适宜的融合条件,建立稳定的细胞-细胞融合实验体系。在所建立融合体系基础上利用Th处理Env表达细胞,观察对融合作用的影响。结果pSV-Ⅲ-JRFL-dCT与pcTat按1:1、2:1共转染293T细胞,可建立稳定的细胞-细胞融合实验体系。Th能够增强细胞融合,且融合细胞数随着Th浓度增高而逐渐增加。Th处理10 min与处理30 min比较,融合增强作用更加显著。结论质粒pSV-Ⅲ-JRFL-dCT与pcTat按1:1、2:1共转染293T细胞,与Magic5A细胞进行融合实验可得到稳定的细胞-细胞融合实验体系,Th对HIV-1包膜介导的细胞融合有促进作用。

Objective To construct a cell-cell fusion system and to examine the effect of thrombin on fusion mediated by Human immunodeficiency virus type Ⅰ （HIV-1） envelope glycoproteins （Env）. Methods 293T cells were cotransfected with HIV-1 Env expression plasmid psv-Ⅲ-JRFL-dCT and Tat expression plasmid pcTat. The transfected cells were harvested at different time and HIV-1 Env proteins expressed on the surface of 293T cells were detected by flow cytometry. Then the 293T cells expressing Env proteins were cocuhured with Magic5A cells for 20 h. Fused cells were stained by X-gal as blue cells and were examined by microscopy. When a stable fusion system was constructed, the 293T cells were treated by thrombin and the effect of thrombin was examined by counting the fused cells. Results The cotransfection with psv-Ⅲ-JRFL-dCT and pcTat at 1 ： 1 and 2： 1 could render a stable fusion system. After the treatment on 293T cells expressing Env, Th promoted the fusion effect in a time-dependent manner. Conclusions Via coculturing 293T cells cotransfected with psv-m-JRFL-dCT and pcTat at 1 ： 1 or 2 ： 1 with Magic5A cells, the HIV-1 envelope-mediated cell-cell fusion system has been constructed. The enhancing effect of Th on the fusion has been proven.