遗传眼病永生化细胞系建立方法

目的　通过遗传眼病患者的外周血或皮肤组织建立永生化细胞系，以保存其遗传资源。方法　永生化Ｂ淋巴细胞系的建立:抽取患者静脉血，分离淋巴细胞，以ＥＢ病毒体外转化Ｂ淋巴细胞为类淋巴母细胞，利用环孢霉素Ａ抑制Ｔ淋巴细胞ＲＮＡ聚合酶Ⅱ活性，防止已转化Ｂ淋巴细胞因受到Ｔ淋巴细胞的攻击而退化，培养后建立永生化Ｂ淋巴细胞系；皮肤成纤维细胞原代培养:利用ＤｉｓｐａｓｅⅡ酶处理离体皮肤组织以分离表皮组织，将其余组织剪碎后贴瓶培养，成纤维细胞可从组织块爬出，生长成为细胞系。结果　永生化Ｂ淋巴细胞系:共完成标本１７例，成功建系１５例，成功率８８．２％；经过转化后Ｂ淋巴细胞悬浮生长，胞浆丰富、形态各异，聚集成团，可无限传代用于实验研究。皮肤成纤维细胞原代培养:共完成标本５例，成功建系３例，成功率６０．０％；皮肤成纤维细胞贴壁生长，呈典型纺锤形，中间饱满，两边细长，５０代以内增殖旺盛可用于实验研究。结论　通过遗传眼病患者的外周血和皮肤组织可建立永生化Ｂ淋巴细胞系和皮肤成纤维细胞系以保存其全部遗传信息，为进一步研究相关遗传性眼病奠定物质基础。

Establishment of immortalized cell lines for genetic eye diseases

Objective To establish immortalized cell lines by acquiring peripheral blood or skin tissues from patients diagnosed with genetic eye diseases , so as to preserve the genetic resources. Methods Immortalized B lymphoblastoid cell lines establishment : The white cells from patients ’ peripheral blood were isolated. then EB virus was used to transform B lymphocytes into B lymphoblastoid cells, and cyclosporin A was used to inhibit T lymphocyte RNA polymerase II activity for preventing the transformed B cells from T lymphocytes attacking, the transformed B lymphoblastoid cells would grow gradually to cell lines. Skin fibroblast cells in primary culture : The skin tissue in vitro was immersed in Dispase n enzyme solution to separate the epidermis and dermis.then the dermal was cut into small pieces and pasted them to the bottom of a cultural vessel.skin fibroblasts would climb out of the tissues and develop to cell line. Results Immortalized B lymphoblastoid cell lines : Totally 17 samples were completed, 15 samples in success .the success rate was 88. 2% ; The transformed B lymphocyte cells grew suspendly with different shapes and the cytoplasm was prominent , every generation could be unlimitedly used for experimental study. Skin fibroblast cells in primary culture :Totally 5 samples were completed, 3 samples in success , the success rate was 60% ; The skin fibroblast cells grew adherently in typical spindle shape, of which the middles were full and both sides were slender ,the skin fibroblast cells showed high proliferation within 50 generations, which could be used in experimental study. Conclusiori The peripheral blood or skin tissues in patients with genetic eye diseases can be used to establish immortalized cell lines and save all genetic information , which laying foundation for the further study.