Goldenhar综合征家系及散发病例永生细胞系的建立

Establishment of immortalized lymphoblastoid cell lines of the Goldenhar syndrome family and sporadic cases

背景:利用EB病毒转化外周血B淋巴细胞建立各种疾病,特别是罕见疾病永生细胞库,已成为当今永久保存全基因组的首选方法。目的:采用EB病毒转化淋巴细胞技术,建立Goldenhar综合征家系及散发病例外周血B淋巴细胞永生细胞系。设计、时间及地点:单一样本观察,于2008-03/10在中南大学组织学与胚胎学系实验室完成。对象:湖南怀化地区的一个Goldenhar综合征家系中目前存活的3代20人及4例Goldenhar综合征散发病例及其父母。方法:收集Goldenhar综合征家系20人及4例散发病例及其父母外周血样品进行淋巴细胞分离,以EB病毒转化淋巴细胞,并加入环孢素A抑制T淋巴细胞生长,每个血样采用两线同时培养,转化后进行细胞株的冻存和复苏,检测其建系前后的遗传稳定性。主要观察指标:光镜观察B淋巴细胞的转化情况。细胞冻存后复苏成功率及染色体G带分析情况。结果:培养1周左右,可在倒置显微镜下观察到B淋巴细胞明显母细胞化,细胞明显增大,细胞周边可见明显的胞质突起,形状各异,聚集成团。约15d后,可见白色聚集生长的细胞团,1个月左右细胞增殖形成生长密集的细胞集落。共建成Goldenhar综合征患者外周血永生细胞株8例,建株成功率100%,父母等家庭成员建系成功率为95.8%(23/24)。所有建成的细胞系冻存1,3,6个月后复苏成功率为100%,经染色体G显带分析未见异常,表明建立细胞株的方法稳定。结论:实验成功建立了Goldenhar综合征患者永生细胞系。

BACKGROUND：At present,the first choice method of eternally preserving a whole genome is to establish immortalized cell lines of various diseases,especially rare diseases,using Epstein-Barr virus for transforming B lymphocytes,.OBJECTIVE：To establish the immortalized lymphoblastoid cell lines from a family and sporadic patients with Goldenhar syndrome.DESIGN,TIME AND SETTING：A single sample observation was performed in the laboratory of the Department of Histology and Embryology,Central South University（Changsha,China）from March to October in 2008.PARTICIPANTS：The peripheral blood samples were collected from 20 surviving members in three generations of a Goldenhar syndrome family in Huaihua of Hunan province,4 sporadic cases and their parents.METHODS：The peripheral blood samples were collected to isolate B lymphocytes,followed by transformation with Epstein-Barr virus to establish immortalized cell lines.The cyclosporine A was also added to inhibit T lymphocytes growth.Each blood sample was divided into two groups to be cultured simultaneously.Then,the transformed cells were cryopreserved and then resuscitated.Their genetic stability was detected by Karyotype analysis before and after the establishment of the cell lines.MAIN OUTCOME MEASURES：The transformation of B lymphocytes was observed under a light microscope.The recovery rate of the immortalized cell lines after cryopreservation was calculated and the chromosomes were analyzed through G-banding staining.RESULTS：After about 1-week culture,it was seen under the inverted microscope that B lymphocyte blastogenesis appeared and lymphocytes were enlarged.There were visible cytoplasmic prominences,with various shapes,gathering around the cells.About 15 days later,a few white cell colonies were visible with the naked eyes.And about 1 month later,the cells proliferated and formed dense colonies.Finally,a total of 8 immortalized cell lines from the patients with Goldenhar syndrome were established successfully,with the success rate of 100%.Among all the subjects,the success rate of establishing immortalized cell lines was 95.8 %（23/24）.The recovery rates of all the established immortalized cell lines were 100% after 1,3 or 6 months of cryopreservation.Moreover,the genetic integrity of the cell lines was stable according to the Karyotype analysis through G-banding staining.All of these findings show that this is a reliable method to establish a cell line.CONCLUSION：The immortalized cell lines from Goldenhar syndrome patients were established successfully in this study.