血小板CD36缺失个体永生化淋巴母细胞株的建立及验证

Establishment and verification of immortalized lymphoblastic cell lines derived from platelets CD36 deficient individuals

目的探讨血小板CD36缺失个体永生化淋巴母细胞株的构建及验证方法。方法选择2012年1月至2018年1月经相关检测确定为血小板CD36缺失的8例个体为研究对象。其中,6例为于南宁中心血站参与献血的无偿献血者,1例为广西923医院收治的血小板输注无效(PTR)患者,1例为广西壮族自治区妇幼保健院收治的胎儿/新生儿同种免疫血小板减少症(FNAIT)患儿母亲。全部受试者年龄为15~66岁,研究前期经流式细胞术与血小板抗原单克隆抗体特异性免疫固定试验(MAIPA)及CD36基因分型等技术的检测,鉴定为血小板CD36缺失者,其中1例为CD36基因538T>C突变杂合子,3例为380C>T突变杂合子,1例为329-330del突变纯合子,3例为329-330del突变杂合子。采集受试者肝素抗凝血5 mL,并且分离淋巴细胞。采用EB病毒(EBV)与环孢素处理血小板CD36缺失个体的外周血淋巴细胞,获得永生化淋巴母细胞株,稳定传代后冻存,并且对这些细胞株进行复苏活性与支原体检测,同时进行CD36基因的测序验证。本研究遵循的程序符合2013年修订版《世界医学会赫尔辛基宣言》的要求,征得受试者的知情同意,并与之签署知情同意书。结果①受试者外周血淋巴细胞经EBV与环孢素处理后,继续培养7 d后,细胞母细胞化,细胞边缘多刺,部分细胞凝集呈团状。持续更换全培养液约1个月,细胞大量增殖,生长旺盛,肉眼可见较多克隆球形成,成功获得CD36缺失永生化淋巴母细胞株。② CD36缺失永生化淋巴母细胞株传代20代后,冻存于液氮;全部CD36缺失永生化淋巴母细胞株均复苏成功,倒置相差显微镜下观察细胞株生长状态良好。③ CD36缺失永生化淋巴母细胞株支原体检测结果示阴性。④对CD36缺失永生化淋巴母细胞株DNA进行PCR扩增测序结果显示,其与建株前血小板CD36缺失个体的CD36基因型完全一致,没有发生基因突变。本研究建立的CD36缺失永生化淋巴母细胞株基因类型包括CD36基因538T>C突变杂合子(1例),380C>T突变杂合子(3例),329-330del突变纯合子(1例),329-330del突变杂合子(3例)。结论CD36缺失永生化淋巴母细胞株传代稳定,血小板CD36缺失个体的基因能够稳定地被保存,并且为CD36基因相关的血小板免疫学等方面研究提供永久性实验材料。

Objective To investigate the establishment and verification of immortal lymphoblastic cell lines from platelets CD36 deficient individuals. Methods From January 2012 to January 2018, eight individuals with platelet CD36 deficiency were selected as the study subjects. Among them, six cases were blood donors from Nanning Blood Center, one case was patient with platelet transfusion refractoriness (PTR) hospitalized in Guangxi 923 Hospital, and 1 case was mother of a child with fetal/neonatal alloimmune thrombocytopenia (FNAIT) hospitalized in Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. All the subjects′ ages ranged from 15 to 66 years. The subjects were identified as platelet CD36 deficiency by flow cytometry, monoclonal antibody immobilization of platelet (MAIPA) and CD36 genotyping. Among them, one case was a CD36 mutant heterozygote of 538T>C, three cases were mutant heterozygote of 380C>T, one case was mutant homozygous of 329-330del, and 3 cases were mutant heterozygote of 329-330del. Volume of 5 mL heparin anticoagulant blood was collected from participants and their lymphocytes were isolated. Epstein-Barr virus (EBV) and cyclosporine were used to treat peripheral blood lymphocytes of participants with platelet CD36 deficiency to obtain immortal lymphoblast cell lines, which were frozen after stable passages. Resuscitation activity and mycoplasma detection of these cell lines were performed, and the CD36 gene was sequenced for verification. The procedure of this study is accordance with the requirement of the revised World Medical Association Declaration of Helsinki in 2013. Informed consent was obtained from each participant. Results ① After EBV and cyclosporine treatment, the peripheral blood lymphocytes of the participants were cultured for 7 d, and blastoformation of the cells with prickly edges were observed, and some of the cells were agglutinated in clusters. After continuous replacement of the complete culture medium for about 1 month, a large number of cells proliferated and grew vigorously, and more clonal spheres could be seen to form by naked eyes, and immortalized lymphoblastic cell line was successfully obtained.② CD36 deficient immortalized lymphoblastic cell lines were subcultured for 20 generations, then frozen in liquid nitrogen. All of them were successfully resuscitated, and the cell lines were observed growing well under inverted phase contrast microscope.③ Mycoplasma test results of the CD36 deficient immortalized lymphoblastic cell lines showed negative.④ DNA of CD36 deficient immortalized lymphoblastic cell lines were amplified and sequenced by PCR, and comparison results showed that the DNA of immortalized lymphoblastic cell lines were identical to those of individuals with CD36 deficiency, and no gene mutation occurred. The genotypes of CD36 deficient immortalized lymphoblastic cell lines established in this study included 1 case of CD36 gene 538T>C mutant heterozygote, three cases of 380C>T mutant heterozygotes, one case of 329-330del mutant homozygote and 3 cases of 329-330del mutant heterozygotes. Conclusions CD36 deficient immortalized lymphoblastic cell lines passaged stably, the genotypes of these platelets CD36 deficient individuals are permanently preserved and can be used as long-term experimental reliable materials for the study of CD36 related platelet immunology.