重组质粒pcDNA-HA/ΔNp73α转染对人树突状细胞功能的影响

Effect of the Transfection of Recombinant Plasmid pcDNA-HA/ΔNp73α on Human Dendritic Cell Function

目的研究转染pcDNA-HA/ΔNp73α重组质粒对树突状细胞(DC)功能的影响。方法采取健康人脐带血,分离单个核细胞,置入含有白介素4(IL-4)和粒-巨细胞集落刺激因子(GM-CSF)的RPMI 1640完全培养基中培养。在培养第5、7天时收获DC细胞,加入FITC-anti-CD1a、PE-anti-CD83抗体,采用流式细胞仪检测CD1a、CD83表达情况。将DC分为正常培养至成熟的DC组(空白对照组)、空载体pcDNA-HA转染的DC组(阴性对照组)、ΔNp73α重组质粒转染的DC组(实验组)。分别在转染24、48、72 h后采用荧光显微镜检测转染效率;转染48 h后收获并提取细胞总RNA,采用RT-PCR检测转染后各组ΔNp73αmRNA表达情况;采用Western blotting检测转染DC的ΔNp73α蛋白表达水平;在收获的各组DC细胞中分别加入PE-anti-主要组织相容性复合体(MHC-Ⅱ)、FITC-anti-CD40、PE-anti-CD80单克隆抗体,流式细胞仪检测CD40、CD80、MHC-Ⅱ表达情况;ELISA检测各组DC细胞白介素12(IL-12)分泌水平。结果 CD1a和CD83在培养第7天的表达水平均高于培养第5天(t=16.62、34.75,P<0.001)。DC于第7天诱导成熟。质粒转染DC后,24 h可见特异性绿色荧光蛋白表达,48 h荧光最强,转染效率为38.2%,在48 h后,绿色荧光的表达逐渐减少。实验组在670 bp处可见阳性条带,为ΔNp73αmRNA阳性表达,而空白对照组和阴性对照组未检测到ΔNp73αmRNA的表达。在实验组中检测到ΔNp73α蛋白的表达,而正常对照组和阴性对照组中未检测到ΔNp73α蛋白的表达。各组CD40、CD80、MHC-Ⅱ表达水平比较,差异有统计学意义(F=12.68、41.18、38.95,P<0.05);其中实验组CD40、CD80、MHC-Ⅱ表达水平均高于空白对照组和阴性对照组(P<0.05)。各组IL-12表达水平比较,差异有统计学意义(F=37.43,P<0.001);其中实验组IL-12表达水平高于空白对照组和阴性对照组(P<0.05)。结论从人脐带血中可成功分离DC;重组质粒pcDNA-HA/ΔNp73α转染DC促进其成熟,将有效提高DC的抗原提呈能力。

Objective To investigate the effect of the transfection of recombinant plasmid pcDNA-HA/ΔNp73α on human dendritic cell（DC） function.Methods Umbilical cord blood from healthy people was sampled,and mononuclear cells were separated from it.Then the cells were cultured in RPMI1640 complete medium which contained IL-4 and GM-CSF.On day 5 and day 7,mature DC cells were harvested and were added with FITC-anti-CD1 a and PE-anti-CD83 antibodies,then the expression levels of CD1 a and CD83 were tested by flow cytometry.The DC cells were divided into three groups：blank control group（DC cells cultured to maturity in a normal way）,negative control group（DC cells with pcDNA-HA empty-vector transfection） and experimental group（DC cells with recombinant plasmid ΔNp73α transfection）.Using flurescent microscope,transfection efficiency was tested at 24 h,48 h and 72 h after transfection; total RNA of the cells was harvested and extracted at 48 h after transfection,and ΔNp73α mRNA expression of each group was tested by RT-PCR after transfection;ΔNp73α protein expression level was tested by western blotting; PE-anti-MHC-Ⅱ,FITC-anti-CD40 and PE-anti-CD80 monoclonal antibodies were added to each group,then CD40,CD80 and MHC-Ⅱ expression were tested by flow cytometry; IL-12 level of each group was tested by ELISA.Results The expression levels of CD1 a and CD83 on day 7 were higher than those on day 5（t = 16.62,34.75; P 0.001）.DC cells became mature on day 7.After the transfection,specificity green fluorescent protein expression was noted at 24 h,the expression reached peak at 48 h after transfection with a transfection rate of 38.2%,and the expression gradually decreased after the peak.In experimental group,positive band was found at 670 bp,which indicated the positive expression of ΔNp73α mRNA,while no ΔNp73α mRNA expression was detected in blank control group and negative control group.The protein expression of ΔNp73α was noted in experimental group,while noΔNp73α protein expression was detected in blank control group and negative control group.The three groups were significantly different in the expression levels of CD40,CD80 and MHC-Ⅱ（F = 12.68,41.18,38.95; P 0.05）; experimental group was higher（P 0.05） than blank control group and negative control group in the expression levels of CD40,CD80 and MHC-Ⅱ.The three groups were significantly different in the expression level of IL-12（F = 37.43,P 0.001）; experimental group was higher（P 0.05） than blank control group and negative control group in the expression level of IL-12.Conclusion Dendritic cells could be successfully separated from umbilical cord blood.The transfection of recombinant plasmid pcDNA-HA/ΔNp73α could accelerate the maturity of DC and effectively improve their antigen-presenting ability.