体外培养的实验细胞资源是目前医学与生命科学领域应用最为广泛、最重要的实验工具之一；纵观科学研究发展过程几十年来,对来自人和动物的体外培养细胞的使用率持续稳步增加。医学与生命科学领域已经不可或缺应用体外培养细胞进行的实验。尤其是近年来,重大的科学发现和成果,如转基因克隆、体细胞重编程、非编码RNA乃至最新兴的生物3D打印技术1都无疑要以完善的体外培养细胞资源为基础。当前培养条件改善和培养技术虽不断优化,但与其在科研工作中的重要地位相反,对实验细胞的质量重视程度普遍不足,加之细胞系是活的生命体,在培养、传代过程中始终存在细胞系的种内交叉污染、种间交叉污染、外源因子污染等的风险。成功培养且应用于研究的细胞种类及细胞系也不断增加。很多实验室的操作不够规范。细胞交叉污染的问题愈加明显,交叉污染细胞的使用率增加数倍。毋庸置疑,这种形成了强烈的现实需求与客观状况间的反差,所以使用背景清晰明确的细胞,应成为广大科研工作者的共识,是确保实验研究成功的关键。本研究依托国家实验细胞资源共享平台,平台早先已对于实验细胞的种属鉴定和细菌、支原体污染鉴定做了深入研究,建立了成熟的支原体PCR检测、用于识别人类等细胞的个体起源STR检测分析技术手段。本文试图建立同一个体不同组织起源的鉴定新方法。众所周知,细胞可以通过形态和生长状态对细胞的组织来源进行初步判断,但要准确性分析,现行方法包括采用细胞免疫化学、western blot和基因芯片大通量测序等方法,其比较耗时耗力,成本也高且常常。本研究着眼于组织特异性因子；即利用组织特异性分子在不同组织细胞中表达差异,通过分子生物学手段达到判断鉴别组织细胞的目的。组织特异性因子是由奢侈基因转录,经核内外多种因素调节,驱动组织特异性启动子进而表达的对细胞完成特定独有功能具有重要作用的蛋白。首先,在mRNA水平,根据文献报道与NCBI数据库我们设计了17对组织特异性相关引物,分别针对16个常见的人类组织特异性因子的mRNA保守序列,选用经国家细胞共享平台严格质控的细胞,提取mRNA,反转录为cDNA,以此为模板进行PCR,琼脂糖凝胶电泳分析；对这些引物进行特异性和敏感性的筛选；其中8对引物可用于判断人类组织特异性,以双盲实验对此方法进行验证,用于鉴定人类细胞的组织来源。其中17对组织特异性相关引物中,特异性分子ALB、AFP、SYN、CDH16、SFTPB、LCA、FLT、MUL2的对应引物可有效应用于鉴定细胞是否源自肝、肾、神经、肺、造血、内皮和分泌组织；特异性因子PSA对应引物可广泛在人类各组织细胞中扩增出条带；鉴定其他组织的引物有待进一步研究完善优化此外,我们还初步探讨了利用组织特异性启动子鉴定细胞组织特异性。将组织特异性分子ALB启动子克隆到相关病毒质粒上,启动下游绿色荧光蛋白（EGFP）基因,经质粒构建、病毒扩增、细胞转染、镜下观察和Western Bloting,克隆在病毒载体上ALB蛋白的启动子可成功特异启动下游EGFP表达,对细胞的组织来源进行判断。总之,应用逆转录聚合酶链式反应扩增编码组织特异性分子mRNA的保守序列和通过构建含有组织特异性启动子质粒在培养细胞中转录表达可以用于鉴定待检测细胞的组织来源。本研究为鉴定常见细胞的组织来源,提供新思路和方法 

In vitro cultured continuous cell lines is one of the most widely used research materials for bio-medical sciences and the most important experimental tools. Inspecting the development process of scientific research, for the passing 6 decades, the rate of using cell-cultures from human and animal in vitro is steadily increasing. Especially in recent years, great scientific discoveries and achievements, including transgenic cloning, reprogramming of somatic cells, non coding RNA and the emerging biological 3D printing technology are all areas that have applied cell cultures. Although nowadays great improvement of culture conditions and cultivate technology has achieved, the awareness of quality control of cell lines is just emerging. Since the cells are living "organisms", their culturing conditions are very complicated, growth cycle is long, risks of cross-contamination etc. always exist in the process of passage. As a matter of fact, the use of error-nous, misidentified or cross-contaminated cell lines increased more quickly than ever during the last 2 decades. With the publication of "Cell Identity Crisis—the beginning of an end"internationally and with the complementation of the National Science and Technology infrastructure—The National Platform of Experimental Cell Resource for Sci-Tec (NPECRS) at home, more and more Scientists are now realized that the employment of reliable and authenticated cell lines is the solid base of successful innovation.
The Platform is to provide high quality authenticated cell lines to the Scientific and industrial community. As the headquarter of the NPECRS our center had previously studied and established various SOPs and intensively checked its deposited cell   lines,   including  bacteria,   mycoplasma  detection  and   eradication,   species identification and , STR profiling of human originated cell lines. This paper tries to establish a new method for the identification of the tissue origin of human cells. As everyone knows, the cell can be analyzed by morphology and growth status to estimate its source of tissue, but to be more accurate, immunochemistry, Western blot and gene chip taking high throughput sequence are often employed. For their time consuming, high cost labor intensive and high variation, more easy and convenient methods needs to be established and employed.
This study focused on tissue-specific factors; namely markers specifically expressed in different tissues and cells, by means of molecular biology to determine differential tissue origin of cells. Tissue specific gene transcription factors, coded by the luxury genes, drive tissue-specific promoter, control the cellular specific functional proteins expression. Related method is investigated to determined the tissue origin of cells.
Firstly, at the level of mRNA, according to reports in the literature and the NCBI database we designed 17 pairs of tissue specific primers that were correlated, conserved mRNA sequences for 16 common human tissue specific factors, use cells that had being strictly qualified by the national platform of experimental cell resources for Sci-Tech to extracts mRNA, reverse transcription to get cDNA, as the template for PCR, agarose gel electrophoresis analysis; selection of sensitivity and specificity of these primers; 8 pairs of primers can be used to judge the human tissue specificity, were verified by double blind test, for ensuring its ability of the identification of human cell source of tissue, among 17 tissue specific primers.The primers corresponding the tissue specific molecules ALB, AFP, SYN, CDH16, SFTPB, LCA, FLT, MUL2 can be used for the identification of whether cells are derived from the origin of liver, kidney, lung, nervous, hematopoietic, endothelial and secretory tissue; PSA corresponding primers can be amplified in all the human originated cells. Further optimization is required to improve specificity and sensitivity of primers of other tissues.
In addition, we also explored the use of tissue specific promoter to identification of cell tissue specificity. The tissue specific molecular ALB promoter was cloned and constructed into lentiviral plasmid and could start downstream green fluorescent protein (EGFP) gene expression. After virus amplification and packaging, cells were transfected, microscopically observed and Western Bloting analyzed. The results showed that in hepatic cancer cells EGFP expression was started, indicating that the viral construct with ALB promoter can be successfully used to identify the tissue origin of hepatocyte.
In a word, using reverse transcription polymerase chain reaction and amplification of conserved sequences encoding tissue specific mRNA molecules and taking the building plasmid contains tissue-specific promoter in cultured cells can be used to identify the transcriptional expression and detect cellular source of tissue origin. The study of tissue source origin, for the identification of human cells, provides new ideas and methods.