免疫复合物AtgG刺激小鼠肾小球系膜细胞增殖过程中Hint2基因的表达和作用研究

The expression and function of Hint2 genes during the proliferation of mouse MSCs stimuated by the immune complex AIgG

目的观察免疫复合物AIgG刺激小鼠肾小球系膜细胞（GMCs）后不同增殖状态下Hint2-mRNA的表达量变化情况及Hint2蛋白过表达对免疫复合物AIgG刺激的小鼠GMCs不同增殖状态下的影响。方法（1）采用CCK-8法检测免疫复合物AIgG刺激体外培养小鼠GMCs增殖的量-效和时-效关系。（2）采用SYBRGreenReal—timePCR技术检测不同浓度的AIgG（0、1000、2000tJg／m1）刺激小鼠GMCs增殖后的Hint2-mRNA表达量的变化情况。（3）依次构建Hint2-Pmdl9克隆载体及Hint2-MigRl表达载体，采用磷酸钙共沉淀法将MigRl和Hint2-MigRl质粒分别成功转染小鼠GMCs。（4）将MigRl和Hint2-MigRl质粒分别转染小鼠GMCs后，并用流式细胞术将两者转染效率调整至一致，分别予不同浓度的AIgG（0、1000、2000μg／ml）刺激小鼠GMCs36h后，再用流式细胞术检测两者转染细胞绿色荧光蛋白（GFP）表达的阳性率。结果（1）AIgG对体外培养小鼠GMCs具有明显的促增殖作用，以2000μg／ml组作用最强。（2）Hint2-mRNA的表达量与小鼠GMCs的增殖状态呈负相关。（3）MigRl和Hint2-MigRl质粒通过磷酸钙共沉淀法均能成功转染小鼠GMCs，于荧光倒置显微镜下可见GFP阳性的细胞，FCM检测两者转染效率分别为38．22％和20．44％。（4）FCM检测不同浓度的AIgG刺激转染小鼠GMCs中GFP的阳性率：Hint2蛋白过表达组和对照组水平的差异均无统计学意义。结论Hint2-mRNA的表达量与小鼠GMCs的增殖状态呈负相关；单独Hint2蛋白过表达对小鼠GMCs的增殖状态未见明显影响。

Objective To observe Hint2-mRNA expression after the immune complex AIgG stimulated proliferation of mouse GMCs,and the influence of Hint2 protein over-expression on the immune complex AIgG stimulated proliferation of mouse GMCs. Methods Using CCK-8 method, test the relationship between dosage and potency, time and potency of immune complex AIgG stimulating the in vitro cultured mouse GMCs proliferation.And then the SYBR Green Real-time PCR technology is used to detect AIgG of different concentrations （0 μ g/ml, 1 000 μ g/ml, 2 000 μ g/ml） stimulating mouse GMCs to proliferate and the change of Hint2-mRNA expression. After successive construction of Hint2-Pmd19 cloning vector and Hint2-MigR1 expression vector, respectively transfect the mouse GMCs with MigRI and Hint2-MigRI plasmids using calcium phosphate co-precipitation method. Finally After respective transfection of MigRI and Hint2-MigRI plasmids on mouse GMCs, inoculate on the 24-well plate after adjusting the transfection efficiencies to the same by flow cytometry. It is distributed with different concentrations of AIgG （0 μg/ml, 1 000 μ g/ml, 2 000 μ g/ml） stimulating the mouse GMCs for 36 hours. Then detect the positive incidence of transfected cell GFP expression by flow cytometry. Results （1）The immune complex AIgG showed its distinguished effect of promoting proliferation of in vitro cultured mouse GMCs. The proliferation effect should be strongest if in the 2 000 μ g/ml dosage group and reach to the peak after 36 hours. （2）SYBR Green Real-time PCR results suggest that after different concentrations of AIgG （0 μg/ml, 1 000 μg/ml, 2 000 μ g/ml） stimulating mouse GMCs to proliferate, the higher the cell PI value is, the lower the Hint2-mRNA expression. （3）MigR1 and Hint2-MigR1 plasmids can manage to transfect mouse GMCs by means of calcium phosphate co-precipitation. The inverted fluorescence microscope can show cells expressing GFP. FCM is used to detect the transfection efficiencies respectively as 38.62% and 20.44%. （4）GFP positive rate of mouse GMCs transfected is detected by means of FCM with different concentrations of AIgG （0μg/ml, 1 000μ g/ml, 2 000μg/ml） stimulus： Hint2 protein over-expression groups respectively as 9.99%, 7.99% and 8.69% with contrast groups as 9.23%, 8.47% and 8.61%, revealing no oblivious differences between. Conclusion Expression of Hint2-mRNA is in negative correlation with the proliferation status of mouse GMCs,and the single Hint2 protein over-empression has no obvious influence on the proliferation state of mouse GMCs. However, it will indirectly adjust the apoptosis of mouse GMCs possibly by enhancing the sensitivity of GMCs on other apotosis-related genes.