Study On Isolation And Biological Characteristics Of Bama Miniature Pig Bone Marrow Mesenchymal Stem Cell

Stem cell research has been a hot topic in the field of biological science since the emergence of it. As a sort of Adult pluripotent stem cells, Bone marrow mesenchymal stem cells (BMSCs) were easy to obtain and were thought to be the ideal seed cells for stem cell research. Pig was an animal ideal model because the physiological structure, organ size, nutrition metabolism and pathology reaction of it were similar with human. In this experiment, the tissue of bone marrow of Bama miniature pig was used as the material. BMSCs were cultivated by whole bone marrow culture method and identified later. It mainly focuses on the proliferation and biological characteristics of porcine bone marrow mesenchymal stem cells. The results indicated that:1. The cultivation of Bama miniature pig BMSCs originated from Bama Miniature Pig bone marrow tissue in vitro. The best cultivation system of BMSCs was L-DMEM、10%FBS、2mM glutamine、10ng/mL bFGF、10ng/mL EGF and100X penicillin streptomycin mixture. The BMSCs were subcultured16generations and BMSCs had a very strong capacity of self-renewal in vitro.2. The BMSCs were detected by immunofluorescence staining and RT-PCR assay. The result showed that BMSCs specific markers CD29, CD44, CD71, CD73and CD90were positively expressed, however,endothelial cells CD31and hematopoietic cells specific marker CD34were not expressed. That was consistent with the characteristics of mesenchymal stem cells.3. The influences of freezing conservation and resuscitation on viability of the Bama miniature pig were evaluated. The result shows the viability of cells in P3(P>0.05), P9(0.01<P<0.05) and P15(P<0.01). Cell viability decreased after resuscitation, mainly because the mechnical and chemical damages suffered when cryopreserved and recovered.4. Take count of the number of cells of Bama miniature pig BMSC among P3, P9, P15generations that showed a typical "S" shape. Cells growing included latent phase, logarithmic growth phase and stagnate phase. The population doubling time of P3, P9, P15were about39.7h,41.4h and46.2h respectively.5. Colony forming cell assay of BMSCs in different generations showed that the ability of P3, P9, P15were (66.7±5.83)%,(53.2±3.58)%and (28.6±3.36)%. Clonogenic potential of P3is much stronger than those of P15(P<0.01) and P9(0.01<P<0.05). In short, clonality of BMSCs faded with the increasing generation numbers.6. Karyotype analysis of the chromosome number of BMSCs showed that the BMSCs of Bama miniature pig were2n=38and nothing abnormal detected. The ratio of diploid cells was93.2%, indicating that hereditary property of cells cultured in vitro was stable. 7. By Study of multi-directional differentiation potential under the condition of appropriate effect, BMSCs could be induced to differentiate into adipocytes, osteoblasts, chondrocytes, neuron-like cells, Islet cells, liver-like cells and endothelial cells. Objective cells were verified by RT-PCR and immunofluorescence, identifying the plasticity potential of BMSCs, verifying the differentiation potential of BMSCs. Besides, the alteration of the associated genes during induction was studied.