胎盘免疫调节因子对细胞因子诱导的杀伤细胞超微结构及免疫表型的调节作用  

Effect of placental immunoregulatory factor on the ultrastructure and immunophenotype of cytokine-induced killer cells

作　　者：张学荣[1] 蒙怡[1] 舒雨雁[1] 韦敏[1] 石庆秋[1] 徐阳曦[1] 

机构地区：[1]广西医科大学医学科学实验中心蛇毒研究所,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究与临床康复》2007年第23期4461-4465,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广西壮族自治区科技厅资助项目(桂科能0443001-17)~~

摘　　要：目的:通过电子显微镜、流式细胞仪观察经胎盘免疫调节因子处理的细胞因子诱导的杀伤细胞超微结构及免疫表型变化,了解胎盘免疫调节因子的免疫调节活性。方法:实验于2006-02/2007-01在广西医科大学医学科学实验中心重点实验室进行。实验材料:重组人干扰素γ和白细胞介素1α为Peprotech公司产品。重组人白细胞介素2和抗人CD3单克隆抗体为北京远策药业有限公司产品。实验方法:①胎盘免疫调节因子的制备:将新鲜经检人胎盘去筋膜,生理盐水冲净、剪碎匀浆,置-20℃48h后,反复冻融3次,用生理盐水透析,取透析外液,过虑除菌,分装,抽样做有关鉴定实验。②细胞因子诱导的杀伤细胞的制备:抽取健康自愿者静脉血20mL,用淋巴细胞分离液分离外周血单个核细胞,加RPMI1640培养液调整细胞的密度至1×109L-1,放于50mL培养瓶中培养。于当天加入1×106U/L干扰素γ,置于37℃、50mL/LCO2孵育箱中培养24h。次日将悬浮的细胞转移到6孔板培养中,分别加入终浓度为100mg/L的PHA、3×105U/L白细胞介素2、133μg/L抗CD3mAb及1×105U/L白细胞介素1α,每3d半量换液1次,同时补加白细胞介素2至3×105U/L。将培养14d细胞因子诱导的杀伤细胞以5×109L-1接种于24孔板,实验组中加入一定剂量的胎盘免疫调节因子诱导,对照组加入等量生理盐水,继续培养72h。③实验评估:制备电镜标本,观察细胞超微结构。采用流式细胞术测定细胞因子诱导的杀伤细胞CD4+、CD8+、CD3+CD4+、CD3+CD8+、CD25+、HLA-DR、CD3+CD56+、CD80+百分含量。结果:①细胞因子诱导的杀伤细胞表型分析:实验组CD4+、CD8+和CD3+CD4+明显低于对照组[实验组:(8.40±8.15)%,(33.10±4.61)%,(7.98±12.65%);对照组:(39.43±9.16)%,(58.90±11.35)%,(31.30±5.94)%,P<0.05];实验组CD3+CD8+、CD25+表达明显高于对照组[实验组:(30.50±2.56)%,(24.8±6.31)%;对照组:(15.90±7.35)%,(0.56±0.21)%,P<0.05]。②透视电镜下观察AIM： To investigate the immunoregulatory activity of placental immunoreguiatory factor （PIF） by observing the ultrastructure and immunophenotype of cytokine-induced killer calls cultured in medium containing PIF with electron microscope and flow cytometry. METHODS： The experiment was conducted in the Key Laboratory of Medical and Scientific Research Institute of Guangxi Medical University from February 2006 to January 2007. Recombinant human interferon gamma （IFN-γ） and intedeukin-1α （IL-1 α） were purchased from Peprotech company, and recombinant human IL-2 and anti-human CD3 monoclonal antibody （mAb） were products of Beijing Yuance Pharmaceutical Co., Ltd. ①Preparation of PIF： The anadesma was removed from fresh human placenta that had been examined, then washout by normal saline and cut into pieces for homogenate, then placed at -20℃ for 48 hours and freeze thawing for three times, then dialyzed by normal saline. The dialysate was collected, filtered and packaged. Finally, the samples were identified. ②Preparation of cytokine-inducad killer cells： 20 mL venous blood was drawn from the healthy donors, and the peripheral blood mononuclear calls were isolated and added in RPMI1640 culture solution to adjust the call concantration to 1×10^9 L^-1, then cultured in a 50 mL culture flask. 1 ×10^6U/L IFN-γ was added at that day, and then put into the incubation containing 50 mL/L CO2 at 37 ℃ for 24 hours. On the next day, the suspended calls were transferred to the 6-well culture plate, and 100 mg/L PHA, 3×10^5 U/L IL-2, 133 μg/L anti-human CD3 mAb, and lx105 U/L IL-1 oL were added. Half of the solution was changed every 3 days, and supplemented with 2 to 3×10^5 U/L IL^-2. The cytokine induced killer calls that was cultured for 14 days was seeded onto the 24-well plate at concentration of 5×10^9 L^-1. Then the cells were divided into two groups： the experiment group was cultured by medium containing PIF, and the control group was cultured by medium containing normal saline fo

关 键 词：胎盘免疫调节因子 CIK细胞 超微结构 细胞表型 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]