Construction Of Chinese Liver And Lung Cancer Cell CPR Techniques And Preliminary Screening Of Resistant Genes

Drug resistance occurs to the failure of tumor chemotherapy. Therefore, analysing drug resistant mechanism to guide personalized cancer therapy is clinically urgent bottlenecks. The paper used Cyclical Package Rescue(CPR) platform to screen drug resistant genes from Chinese human lung adenocarcinoma cell line named LTEP-a-2 and liver cancer cell line named Bel-7405, which provides the basis for explaining drug resistant mechanism and precise tumor treatment program.The main study results are as follows:1. Given the species difference, Chinese human lung adenocarcinoma cell line named LTEP-a-2 and liver cancer cell line named Bel-7405 were used as materials to construct full-length c DNA libraries, which were expected to represent the characteristic of Chinese lung cancer and liver cancer gene. The both library’s storage capacity reached 107 clones, and the fragment size was between 0.4 kb and 2 kb, which provided a good foundation for gene screening.2. The packaging cells named Phoenix ? 293 T from Phoenix ? retroviral expression kit were used as host cells. The c DNA fragments were inserted between the LTR regions of p LIB, forming a retroviral c DNA library, which was transfected into host cells to be retroviral expression system. Retroviral c DNA libraries were obtained with the titer of 1.0 × 105 copies / μL, meeting the requirement of gene screening storage capacity. Adriamycin(ADM) and the sensitive mouse fibroblasts(NIH-3T3) were used as materials, and retrovirus containing c DNA fragments infected cells, following with ADM intervention, survival cells were collected; p GP and p E-eco plasmids containing retroviral structural genes were co-transfected the survival cells, packing out retrovirus containing c DNA fragments diversity. The whole process formed a method of circulating viral packaging which displayed c DNA fragments in the survival cells, so called CPR platform. Genomic DNA was extracted from the survival cells, following with PCR identification of gene fragments diversity, which verified the feasibility of CPR techniques.3. NIH-3T3 cell line was the study material. CPR displayed the diversity of retroviral c DNA fragments in NIH-3T3 cells; and after the intervention of quantitative ADM drug screening, collected retrovirus containing target genes from the surviving cells. After PCR analysis and identification of the target genes, NIH-3T3 cells were infected with the retroviral containing target genes. The whole process formed CPR circulatory system screening for the target genes. The study obtained seven genes, among which the four genes including the anti-apoptotic Bcl-6 related gene(BAG6), aldehyde dehydrogenase 2(ALDH2), human long non-coding RNA(BCYRN1) and sna R small non-coding RNA(H8iii sna R-A) were associated with ADM resistance.The paper used CPR platform to screen drug resistant gene from Chinese human lung adenocarcinoma cell line LTEP-a-2 and liver cancer cell line Bel-7405, and obtained several ADM resistant genes, which yet needed further identification. Anyway, CPR platform enhanced the method of resistant gene high-throughput screening.