摘要
目的探讨中药延缓免疫衰老的作用和机制。方法清洁级健康雄性SD大鼠50只,分为4月龄(4 mon)、24月龄(24 mon)、24月龄加PDTC处理(24 mon+PDTC)、24月龄加Ica(icariin,Ica)干预(24 m+Ica)和24月龄加Ica干预再加PDTC处理(24 mon+Ica+PDTC)五组,每组10只。自21月龄满开始,24 mon+Ica组和24 mon+Ica+PDTC组大鼠予Ica灌胃,24 mon组和24 mon+PDTC组给予等量蒸馏水灌胃,均连续3个月。24 mon+PDTC组和24 mon+Ica+PDTC组大鼠在灌胃结束前1 w每2 d腹腔注射PDTC,按200 mg/kg体重分2次注射。处死大鼠分别提取大鼠脾CD4+T淋巴细胞,用流式细胞仪检测细胞凋亡率,电泳迁移率(EMSA)检测细胞NF-κB的活性,Western印迹法检测细胞P65蛋白表达水平。结果 24 mon组大鼠脾CD4+T淋巴细胞凋亡率明显高于4 mon组和24 mon+PDTC组(P<0.05),而24 mon+Ica组细胞凋亡率显著降低(P<0.05);与24 mon+PDTC组比较,24 mon+Ica+PDTC组脾CD4+T淋巴细胞凋亡率明显降低(P<0.05)。与4 mon组大鼠相比,24 mon组大鼠脾CD4+T淋巴细胞中p65蛋白表达显著下调(P<0.01);与24 mon组比较,24 mon+Ica组p65蛋白表达明显上调(P<0.01),24 mon+PDTC组则显著下调(P<0.05)。与24 mon+PDTC组比较,24 mon+Ica+PDTC组p65蛋白表达明显增强(P<0.05)。24 mon组大鼠脾CD4+T淋巴细胞NF-κB的活性明显低于4 mon组(P<0.01);与24 mon组比较,24 mon+Ica组NF-κB的活性增强(P<0.01),24 mon+PDTC组NF-κB活性则降低(P<0.01)。相关分析表明,CD4+T淋巴细胞NF-κB活性与细胞凋亡率呈负相关(r=-0.75,P<0.01)。结论 NF-κB通过抑制淋巴细胞凋亡参与免疫衰老的调控;Ica可能通过提高NF-κB的活性和诱导P65高表达来抑制淋巴细胞过度凋亡从而延缓免疫衰老。
Objective To construct retrovirus vectors carrying IL-17 or siRNA-IL-17 genes with Thy1.1 gene and determine the infected ability of the retrovirus vectors to diabetogenic BDC2.5 T cells.Methods The IL-17 cDNA and thy1.1 full-length cDNA were subcloned into MIT（MSCV-IRES-Thy1.1） retrovirus vector,and the siRNA-IL-17,U6 promoter and Thy1.1 full-length cDNA were also inserted into retrovirus vector of pMND-BANSHEE.The recombined vectors transfected 293 packaging cells by DNA calcium phosphate coprecipitation.The virus supernatant which infected pre-activated spleen cells from NOD/BDC mice were collected.After incubation,the IL-17 expression in diabetogenic T cells was detected.Results By flow cytometry,retrovirus vectors carrying IL-17 or siRNA-IL-17 genes were constructed successfully.After infection of IL-17 or siRNA-IL-17 retrovirus to pre-activated primary NOD/BDC spleen T cells,the percentages of Thy1.1＋/Thy1.2＋ double positive cells were（0.6±0.3）%,（7.2±2.4）%,（6.8±2.6）%,（6.4±2.4）% and（4.6±1.8）% in Mock,MIT empty vector,Bcl-2 positive control,IL-17 p and siRNA-IL-17 vector groups,respectively.After the retrovirus with IL-17 infecting BDC2.5 T cells in vitro,the expression of IL-17 was significantly higher than that of siRNA-IL-17 group and control（both of P0.01）.Infected IL-17 retrovirus to either non-activated or activated T cells respectively in vitro,the expression of IL-17 was distinctly higher than that of siRNA-IL-17 groups and control（both of P0.01）.Moreover,the expression of IL-17 in the T cells activated groups was higher than that of non-activated T cells groups（P0.01）.On the other hand,the expression of IL-17 was markedly reduced in the groups of infection siRNA-IL-17,and no difference to the control（both of P0.01）.Conclusions The IL-17 expression of IL-17 diabetogenic BDC2.5 T cells in vitro is higher than that of the transgenic cells of siRNA-IL-17.It is concluded the retrovirus vectors can be used as am important tool to transfer a foreign gene into T cells efficiently.
出处
《中国老年学杂志》
CAS
CSCD
北大核心
2010年第20期2938-2941,共4页
Chinese Journal of Gerontology
基金
中国博士后科学基金(No.20060400578)
上海市科委科研计划项目(No.10JC1404800
No.09410705800)
