人类白细胞抗原A表达沉默对大鼠脑内人细胞移植排斥反应的调节

Role of human leukocyte antigen A silencing in regulating immune rejection against human fibroblasts grafted in the brain of rats

背景:移植物主要组织相容性抗原复合物在移植排斥反应中起关键作用。用慢病毒介导的人类白细胞抗原RNA干扰技术可以降低T淋巴细胞的细胞毒作用和抗体介导的溶细胞作用,但尚未见相关体内研究报道。目的:拟用携带人类白细胞抗原A小分子干扰RNA序列的慢病毒感染人胚肺成纤维细胞,再将其移植到帕金森大鼠纹状体,观察人类白细胞抗原A表达沉默对移植排斥反应的调节。设计、时间及地点:细胞基因工程体内实验,于2005-09/2008-01在首都医科大学神经科学研究所完成。材料:清洁级12周龄雌性SD大鼠33只,用于建立帕金森动物模型。原代培养的人胚肺成纤维细胞取材于12周流产人胚胎,由北京京北医院提供。293T为贴壁依赖型成上皮样细胞。慢病毒载体系统由pSicoR,pCMV-VSV-G和psPAX2三质粒组成,为美国Addgene公司产品。携带HLA-A2 cDNA的pcDNA I/Amp-HLA质粒由比利时Ludwig肿瘤研究所Pierre van Bruggen教授惠赠。方法:以携带HLA-A2 cDNA的pcDNA I/Amp-HLA质粒为模板PCR扩增HLA-A2 cDNA编码区序列,构建人类白细胞抗原A融合蛋白表达载体pEGFP-Flag-HLA,转染293T细胞。用HLA-A2编码区序列设计小分子干扰RNA4个待选靶点序列,分别起始于编码区的第257,350,833,251位碱基,构建表达小分子干扰RNA慢病毒载体。用带有Flag标签的人类白细胞抗原AcDNA表达载体转染293T细胞后,再进行不同靶点病毒直接感染。33只帕金森模型大鼠随机分为2组,实验组大鼠纹状体植入人类白细胞抗原A小分子干扰RNA病毒感染的人胚肺成纤维细胞,对照组植入带有小分子干扰RNA对照序列和绿色荧光蛋白病毒感染的人胚肺成纤维细胞。主要观察指标:免疫组织化学染色检测脑内移植物人类白细胞抗原A、CD4和CD8的表达,用抗人细胞核的阳性表达评价移植细胞的存活情况。结果:与对照组比较,实验组移植后4d、2周时人类白细胞抗原A阳性细胞均较少(t=3.301,2.631,P<0.01,0.05),6周时无明显变化;CD4阳性细胞仅移植后6周明显减少(t=2.269,P<0.05);移植后4dCD8阳性细胞无明显变化,2周及6周时明显减少(t=2.333,P<0.05);抗人细胞核阳性细胞仅移植后2周明显减少(t=2.952,P<0.05)。结论:人类白细胞抗原A表达敲减可以抑制移植排斥反应,但并不能显著延长移植物的存活时间。

BACKGROUND： Major histocompatibility complex of grafts plays an important role in rejection. Lentivirus-mediated human leukocyte antigen （HLA） RNA interfering technique can reduce cytotoxicity of T lymphocyte and antibody-mediated cytolytic effect To date, no related reports are found. OBJECTIVE： To transfect human embryonic lung fibroblast using lentivirus-based small interfering RNA of HLA-A, which is transplanted in the striatum of Parkinson disease rats to explore effects of HLA-A silencing on immune rejection against human fibroblasts grafted in the rat brain. DESIGN, TIME AND SETTING： Cell gene engineering in vivo trial was performed at Beijing Institute for Neuroscience, Capital Medical University from September 2005 to January 2008. MATERIALS： Thirty-three 12-week-old female SD rats of clean grade were selected for Parkinson disease model. Primarily cultured human embryonic lung fibroblast was derived from 12-week human abortion embryo （Beijing Jingbei Hospital）. 293T cells were of adherence dependent epithelioid cells, Lentivirus vector system consisted ofpSicoR, pCMV-VSV-G and psPAX2 （Addgene, USA）. pcDNA 1/Amp-HLA plasmid carrying HLA-A2 cDNA was graciously provided by Professor Pierre van Bruggen （Ludwig Tumor Institute, Belgium）. NETGIDS： HLA-A2 cDNA coding region sequence was amplified by PCR with pcDNA I/Amp-HLA plasmid carrying HLA-A2 cDNA as template to construct HLA-A fusion protein expression vector pEGFP-Flag-HLA to transfect 293T cells. Four target sequences of small interfering RNA were designed based on HLA-A2 coding region sequence from the 257^th, 350^th, 833^th, and 251^st basic radicals to construct lentivirus-based small interfering RNA. 293T cells were transfected using Flag-labeled HLA-A cDNA expression vector, followed by direct infection of four targets. Thirty-three rats were randomly divided into 2 groups： in experimental group, human embryonic lung fibroblasts treated with lentivirus-based small interfering RNA of HLA-A were grafted into the striatum of Parkinson disease rats; in control group, human embryonic lung fibroblasts treated with small interfering RNA and green fluorescent protein were grafted. MAIN OUTCOME MEASURES： lmmunohistochemistry was used to evaluate HLA-A expression in the grafted cells, CD4 and CD8 expression in the host brain, and survival of the grafted cells using anti-human nuclei （HN） antibody. RESULTS： Compared with the control group, HLA-A-positive cells were decreased in the experimental group 4 days and 2 weeks after grafting （t =3.301.2.631, P 〈 0.01, 0.05）, but showed no obvious changes at 6 weeks; CD4-positive cells were decreased at 6 weeks （t=2.269, P 〈 0.05）; CD8 showed no significant changes at 4 days, while decreased at 2 and 6 weeks （t =2.333, P 〈 0.05）. HN expression significantly decreased at 2 weeks （t =2.952, P 〈 0.05）. CONCLUSION： HLA-A knock-down could reduce graft rejection, but the survival time of the grafted cells could not be lengthened significantly.