| Traumatic brain injury (TBI) is a disease of high fatality and mutilation rate, which has brought a lot of burden to the human individual, family and society. For the treatment of traumatic brain injury, people have been committed to the protection of brain tissue damage in order to reduce primary and secondary brain injury. Using stem cells to treat central nervous system damage, is the academic attention of a new strategy for treatment of nerve injury, which has brought a new dawn to the nerve recover after brain trauma. The current focus of the study is the regulation of stem cell therapy for safety and effectiveness. In recent years, researchers found that bone-marrow mesenchymal stem cells (BMSCs) are excellent carrier cells, which are derived from autologous bone marrow, easy to culture in vitro, and the proliferation of fast. And as derived from autologous, the immunogenicity of BMSCs is weak and they could inhibit the mixed lymphocyte reaction. Thus, the problem of tissue typing and immune exclusion does not exist when transplantation, nor is there any ethical issues. Therefore, BMSCs after amplification in vitro are ideal seed cells in tissue engineering area. BMSCs could transform into neurons in vivo or in vitro, and also secrete nerve growth factor which play on the repair of nerve tissue injury, for example, neural growth factor (NGF), brain-derived neurotrophic factor (BDNF) and so on. However, because of their short life cycle BMSCs are unstable to play a stable neuroprotective function.We selected BDNF as the treatment gene related to the repair of brain injury by comparing before scientific studies. Then we establish the rat immortalized BMSCs line by transfection of expression plasmids of exogenous human telomerase reverse transcriptase (hTERT) mediated with the cationic liposome. We further build high BDNF expression vector—Ad5-BDNF, transfect Ad5-BDNF to the hTERT-BMSCs and Proved that BDNF has a role in cell repair of damage in vitro. On this basis, we also use rat fluid percussion brain injury model and the stereotactic equipment to study the biological characteristics and outcome of the transfected cells. The results prove its safety in vivo and the efficacy of treatment for TBI, which establish a foundation for BMSC into the clinical treatment of TBI. This study is divided into three parts. The first part of the study is to get stable rat BMSCs by adherent primary culture method. We Use flow cytometry to detect the surface antigen markers of BMSCs and further detect the differentiation potential of BMSCs into osteoblasts, adipocytes and neural-like cells in vitro. The results show that primary rBMSCs are easy to extract, purification, and amplification in vitro. The Identified markers are consistent with the characteristics of BMSCs. And BMSCs could be induced to fat-like cells, osteoblast-like cells and neuron-like cells in vitro, which implying that they have more potential to differentiate and confirming rBMSCs successful culture.The second part of the study is by transfection of pLXSN plasmid-mediated human telomerase reverse transcriptase (human telomerase reverse transcriptase hTERT) to enhance the activity of BMSCs telomerase. We Use ELISA method to assay telomerase activity and get the immortalized rat BMSCs controlled by hTERT. Meanwhile, by constructing efficient adenovirus-mediated vector—Ad5-BDNF and transfecting Ad5-BDNF complex into hTERT-BMSCs, we demonstrate that BDNF transfected immortalized BMSC in vitro has a certain degree repair of injured neuron cells.In the third part of the study, we selecte the more widely used internationally fluid percussion brain injury model in rats to establish the rat medium traumatic brain injury model with the 1.5-2.0atm condition. At the same time by means of stereotactic cell transplantation technology, we migrate BMSCs co-transfected by hTERT and BDNF gene to damaged rat brain areas, and observe the changes in rat survival, neurologic severity score (NSS), and the brain tissue edema in the magnetic resonance imaging (MRI). Thus, we could evaluate the safety and effectiveness of gene-modified BMSCs treatment on TBI, and establish a good foundation of TBI clinical treatment with BMSCs and BDNF.The total study results show that rat BMSCs are easily isolated, cultured and expanded. Long-term cultured BMSCs can be premature aging and excessive proliferation and other biological characteristics of instability. Transfecting hTERT expression vector—pLXSN-S-hTERT to rat BMSCs could enhance the telomerase activity of BMSCs and maintain long-term subculture in the process. This is a safe and effective method of establishment of immortalized BMSCs line. Ad-BDNF-hTERT-BMSCs can highly expresse BDNF in vivo, which could reduce apoptosis of damaged neurons, improve the survival rate in brain damage rats, and promote the rehabilitation of the neurological function. The results of this study provide an experimental basis for the clinical application of BMSCs and BDNF... |
