基于SV40LT基因转染的产前遗传学诊断质控细胞株构建方法的研究

It is known that the SV40LT can immortalize the almost all human cells, including cells in skin, gastric mucosa, nasopharynx, tracheal epithelium, and fetal lung fibroblasts, we infer that SV40LT can immortalize homologous amniotic fluid cells. The study intends to construct SV40LTag-pcDNA3.1(-) recombinant clones. Liposomal transfection method will be used to introduce into primary amniotic fluid cells, transfected cells will be screened with G418 to get positive clones. RT-PCR and Southern blot will be used to detect the expression of SV40LT. Thus we will establish the regular construction method of amniotic cell lines. Thus we can establish the construction method of amniotic cell lines for quality control with chromosomal abnormalities which can survive in vitro permanently and amplify infinitely based on this method. This method is the basis for the industrial preparation of quality control cells for prenatal genetic diagnosis. For rare diseases that are difficult to diagnosis, their amniotic fluid cells can be replicated to the desired amount and used directly as quality controls in molecular genetic diagnosis. The several kinds of rare disease cells that are difficult to diagnosis can be mixed in a desired ratio to get mosaic cells for quality control. The mosaic cells can be used for the periodic evaluation of technical quality of the cellular tests and the genetic diagnosis such as amniotic fluid cell culture, chromosome preparation and karyotype analysis. These cells can also be shared for the diagnosis of rare cases. Since there is no ideal quality control materials, the quality control cannot be carried out for prenatal genetic diagnosis, it is a big problem for quality control department for many years, now it can be solved with this method.

已知SV40LT基因能使皮肤、胃粘膜、鼻咽、气管上皮等人类细胞永生化，推断SV40LT基因能使同源的胎儿羊水细胞永生化。本项目拟重组SV40LTag-pcDNA3.1(-)克隆，以脂质体转染法导入原代羊水细胞并筛选阳性克隆，以RT-PCR和Southern blot检测SV40LT基因的表达，以此建立胎儿羊水细胞株的构建方法，进而建立染色体异常羊水质控细胞株的常规构建方法，以使每例易于误诊的疑难罕见病例的羊水细胞复制成所需细胞数量后，作为可再生利用的产前遗传学诊断的质控物，并进一步将数种易于误诊的病例细胞按所需比例配成嵌合型质控细胞后，用于羊水细胞培养、染色体制备和核型分析等的室内质控和室间质评物，以及用于疑难罕见病例的资源共享，以解决困扰多年的因无理想质控物而无法开展产前遗传学诊断质量监管的难题。

本项目根据SV40LT基因能使皮肤、胃粘膜、鼻咽和气管上皮等人类细胞永生化的文献报道，推断SV40LT基因同理能使同源的胎儿羊水细胞永生化，并通过实验研究证明该推断的成立。本项目构建了重组载体SV40LTag-pcDNA3.1(-)并转染胎儿羊水细胞，鉴定、制备了多种染色体异常羊水细胞系，建立了出生缺陷细胞库进行收藏，并试用于全国染色体非整倍体产前诊断及产前分子诊断的室间质评，为有限出生缺陷资源的可再生利用、易误诊病例的资源共享、质控细胞株的产业化以及产前遗传学诊断质量控制的开展奠定基础。在执行课题的期限内发表论文7篇，其中SCI论文6篇，获得授权国家发明专利34项，授权实用新型专利3项，申请国家发明专利数42项，主编并由人民卫生出版社出版《产前诊断—染色体检查入门手册》。更重要的是，为将产前诊断剩余废弃的羊水细胞转化为干细胞治疗的临床应用，例如经进一步研究将产前诊断后剩余的羊水细胞转化为新冠病毒病干细胞治疗的临床应用奠定了基础。