| 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 passing6decades, 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 biological3D 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 last2decades. 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 designed17pairs of tissue specific primers that were correlated, conserved mRNA sequences for16common 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;8pairs 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, among17tissue specific primers.The primers corresponding the tissue specific molecules ALB, AFP, SYN, CDH16, SFTPB, LCA, FLT, MUL2can 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. |
