OX40Ig修饰大鼠脂肪间充质干细胞的实验研究

Experimental study on OX40Ig modified rat adipose derived mesenchymal stromal cells

目的构建含OX40融合蛋白(^(OX40Ig))基因修饰的大鼠脂肪间充质干细胞(ADSCs)即ADSCs^(OX40Ig),探讨其在体外对淋巴细胞的免疫调节作用。方法构建pcDNA3.1(+)/^(OX40Ig)融合基因表达载体,采用核转染技术转染大鼠ADSCs,Western blot检测ADSCs中^(OX40Ig)表达水平。ADSCs与大鼠异基因淋巴细胞共培养后分为对照组(反应细胞+刺激细胞)、ADSCs组(反应细胞+刺激细胞+ADSCs)及ADSCs^(OX40Ig)组(反应细胞+刺激细胞+ADSCs^(OX40Ig)),MTT法检测淋巴细胞增殖抑制率,逆转录聚合酶链反应(RT-PCR)检测淋巴细胞内干扰素(IFN)-γ、白细胞介素(IL)-10及转化生长因子(TGF)-βmRNA表达。结果经测序鉴定验证,pcDNA3.1(+)/^(OX40Ig)质粒构建成功,转染ADSCs后^(OX40Ig)蛋白呈高表达;与ADSCs组比较,ADSCs^(OX40Ig)显著增强异基因淋巴细胞增殖抑制率(P<0.05);对照组、ADSCs组及ADSCs^(OX40Ig)组的IFN-γmRNA相对表达水平依次降低,而IL-10、TGF-βmRNA相对表达水平依次升高(P<0.05)。结论ADSCs^(OX40Ig)较单纯ADSCs能更好地发挥对异基因淋巴细胞的免疫调节作用。

Objective To construct OX40 Ig genetically modified rat adipose derived mesenchymal stromal cells(ADSCs^(OX40Ig)),and to investigate its immunomodulatory effect on lymphocytes in vitro.Methods The fusion gene expression vector of pcDNA3.1(+)/OX40 Ig was constructed and transfected into rat ADSCs by nuclear transfection.The expression of OX40 Ig in ADSCs was measured by Western blot assay.The transfected and untransfected ADSCs were co-cultured with rat allogeneic lymphocytes,and were divided into the control group(responder+stimulator),the ADSCs(responder+stimulator+ADSCs)group,and the ADSCs^(OX40Ig)(responder+stimulator+ADSCs^(OX40Ig))group.The proliferatory activity of lymphocytes was detected by MTT method.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect interferon(IFN)-γ,interleukin(IL)-10 and transforming growth factor(TGF)-βmRNA levels in lymphocytes.Results The sequencing confirmed that pcDNA3.1(+)/OX40 Ig plasmid was successfully constructed,and OX40 Ig was highly expressed in transfected ADSCs.Compared with ADSCs,the proliferation inhibition rate of allogeneic lymphocytes was significantly increased in ADSCs^(OX40Ig)(P<0.05).The expression levels of IFN-γmRNA decreased gradually,but the expression levels of IL-10 and TGF-βmRNA increased gradually in the control,ADSCs and ADSCs^(OX40Ig)groups(P<0.05).Conclusion Compared with ADSCs along,ADSCs^(OX40Ig)has better immunomodulatory effects on allogeneic lymphocytes.