A Study On A Temperature-sensitive Stem Cell Line Transplantation Combined With Mild Hypothermia Treatment For Traumatic Brain Injury

Objective:To study the proliferation and differentiation character of a temperature-sensitive stem cell line cultured at33℃and37℃respectively for establishing a basement on stem cells transplantation into injured brain area during hypothermia treatment. The brain derived neurotrophic factor was transferred into UCMSCs for promoting the stem cells differentiation into neuron and enhance neuromotor function after head injury.Methods:Human umbilical cord mesenchymal stem cells were isolated and expanded in culture, and certificated by flow cytometry analysis. After transfecting plasmid containing temperature-sensitive simian virus40large T-antigen (tsSV40LT) into umbilical cord mesenchymal stem cells (UCMSCs), PCR method was used to detect gene integration. Then we cultured these UCMSCs modified by tsSV40LT in33℃and37℃incubators respectively. Cell growth curves were draw, cell cycle was detected by flow cytometry, then telomerase activation analysis by PCR-ELISA telomerase detect kit, and cell cycle regulating protein, including cyclin Dl, cyclin dependent kinase2(CDK2) and P16, detected by western blot. Serum-dependent experimental and soft agar colony assay were used to examine the tumorigenesis of the stem cells. Recombinant adenovirus-mediated brain derived neurotrophic factor gene transferred into human-derived umbilical cord mesenchymal stem cells (UCMSCs). Enzyme linked immunosorbent assay (ELISA) was used to test the expression level of BDNF, and immunofluorescence assay to detect the NSE and GFAP expression, which were characteristic for neuron and glia respectively. After TBI models in thymic mice were established, the stem cells were transplated into injured area. Proliferating cell nuclear antigen expression and in situ apoptosis were detected by immunofluorescence method.Results:The tsSV40LT gene was integrated into UCMSCs successfully. When cultured at33℃incubator, the new cell line displayed high proliferation activity, strong tolerance to low nutrient conditions, and strong cell clone ability, as well as its telomerase activation, highly expressed cyclin D1and CDK2, and lowly expressed P16, meanwhile, highly nestin, lowly NSE and GFAP. But when cultured at37℃ incubator, the cell line showed a completely converse profile. The BDNF expression achieved its peak at72h after gene transfer, and on the meanwhile, the proportion of the NSE-positive cells was increased significantly, but GFAP-positive cells decreased. In vivo, the results showed that the brain tissue during hypothermia treatment more highly expressed PCNA, and existed lower cell apoptosis, than normothermia group. The mouse obtained a better score of nerve function. In the site of BDNF high expression, the apoptosis cell number was much minor.Conclusion:1) The stem cell line modified by tsSV40LT is highly temperature-sensitive, and its proliferation activity can be regulated effectively, and that supports the clinical application of stem cells transplantation into injured brain area during hypothermia treatment.2) When cultured at33℃incubator, the new cell line displayed strong tolerance to low nutrient conditions, and strong cell clone ability. But when cultured at37℃incubator, the cell line showed a converse profile, and that supports that the temperature may control cell proliferation of the temperature-sensitive stem cell line.3) When transplanted into injured brain area, a vast majority of stem cells were dead because of the low-nutrition, high-toxic environment. But at a lower temperature environment (33℃), the quantity of survival cells was significantly increased, cell apoptosis was lower, and the scores of mouse nerve function was better.4) The stem cells modified by BDNF gene may promote the differentiation into neuron, not glia, and enhance neuromotor function after head injury.