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

Abstract

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. Key words: Antigens, CD36; Platelets; Herpesvirus 4, human; Cell line; Lymphoblast cell lines