In Vitro Reprogramming Study Of Mouse Liver Cancer Cells By Combinational Use Of Oct4, Klf4, C-myc And Sox2Genes

Embryonic stem cells (ES cells) have the ability of self-renewing and tremendous potential of multiplex differentiation, which provides us with very precious resources in development, genetics and pathogenesis of disease and replacement therapy. Scientists from Japan and USA in vitro reprogrammed mouse (August,2006) and human (November or December,2007) somatic cells into induced pluripotent stem cells (iPS cells)The technique of iPS cells introduces some genes into mouse and human somatic cells by retrovirus-, lentivirus-and adenovirus-mediated transfer, followed by reprogramming these somatic cells into ES cell-like cells, i.e., iPS cells. In this research field, scientists have got many achievements, including reprogramming somatic cells of mouse, Rhesus monkey and human into iPS cells in vitro, and the generation of individual-specific or disease-specific human iPS cell lines. Against this background, scientists try to reprogram tumor cells into iPS cells by iPS cell technology. Human melanoma cells and prostate cancer cells were successfully in vitro reprogrammed into iPS cells by introducing miR302gene into these human cancer cells. After that. both mouse melanoma cells and chronic myeloid leukemia cells were in vitro induced to iPS cells by Oct4、Sox2、c-Myc and Klf4genes. The mentioned-above achievements support the possibility that in theory, one somatic cells could be reprogrammed to another somatic cells with certain factors under certain culture conditions.In this study, I plan to in vitro reprogram mouse hepatocellular carcinoma cells (Hep1-6) by the combinational use of Oct4, Sox2, Klf4and c-Myc genes.Methods:1. Lentiviral vector of pLentG-KOSM indetified by enzyme digestion and PCR1) pLentG-KOSM identified by enzyme digestion:pLentG-KOSM was cut by Not I and Xba I, respectively.2) Identification of pLentG-KOSM by PCR:primer pairs pl/p2, p3/p4, p5/p6and p7/p8(specific for Oct4, Klf4, c-Myc and Sox2, respectively) were employed to amplify Oct4, Klf4, c-Myc and Sox2from pLentG-KOSM, respectively.2. Production of lentivirus carrying pLentG-KOSMAccording to standard protocol from Invitrogen, lentivirues carring pLentG-KOSM were produced, followed by confirming the successful production of lentivirus harboring pLentG-KOSM by EGFP assay.3. Reprogramming Hepl-6cells by lentivirus-mediated gene transfer of Oct4, Klf4, c-Myc and Sox2Hepl-6cells were infected with the lentiviruses harboring pLentG-KOSM.12h after infection, the infected medium was replaced with293T cell medium.Infected Hepl-6cells were replaced on the feeder with fresh mouse ES cell medium, following by changing the medium every day until the colonies become big enough to be picked up.4. Establishment and characterization of the reprogrammed cells1) Observing colony formation2) Detection of alkaline phosphatase (AP) activity3) Detection of the expression of ES cell marker genes by RT-PCR4) Detection of the expression of ES cell marker genes by immunocytochemistry (ICC)5) The karyotype analysis of reprogrammed cells6) EB-based differentiation of reprogrammed cells7) Teratoma formation by transplanting reprogrammed cells into nude miceResults:1. pLentG-KOSM indetified by enzyme digestion and PCR pLentG-KOSM was confirmed to be right by enzyme digestion and PCR.2. Production and titer datermination of lentivirus carrying pLentG-KOSM Viruses were successfully produced and the titers were beyond1×106IU/ml.3. Reprogramming Hepl-6cells by lentivirus-mediated gene transfer. Colonies firstly become visible approximately in the5th day after the lentiviral infection. They become large enough to be picked up.4. Establishment and characterization of the reprogrammed cells1) Reprogrammed cells had morphological characteristics of ES cell-like colony and showed the expressuion of alkaline phosphatase2) Alkaline phosphatase (AP) staining was positive3) Reprogrammed cells could not express ES cell-specific marker genes4)3coloies expressed ES cell-specific markers:Oct4(+), Sox2(+) and Nanog(+)5) Karyotype analysis of reprogrammed cells haven’t been finished6) Cells from3coloies formed embryoid bodies (EBs) in vitro7) Cells from3coloies could not form teratomas in vivoConclusion:1) Master technique for in vitro reprogramming Hepl-6cells by lentivirus-mediated gene transfer.2) Reprogrammed cells had some characterization of ES cells, but could not form teratomas in vivo.3) Comparaed to Hepl-6cells, the malignancy degree of the reprogrammed cells derived from Hepl-6cells significantly decrease.