Research On The Construction Of Gene-Modified Rat Bone Marrow Mesenchymal Stem Cells By Nerve Growth Factor

Objective:1.To optimize the method for isolation ,cultivation, purification and amplification of rat bone marrow-derived mesenchymal stem cells.2.To infect rat bone marrow-derived mesenchymal stem cells using lentiviral vector with nerve growth factor gene.And then to observe the expression of nerve growth factor in rat bone marrow-derived mesenchymal stem cells.That is to provide the basis for gene therapy inner animals.Methods:1. Whole bone marrow culture and successive adherence method was used to extract, segregate and propagate rat BMSCs in vitro. Rat BMSCs were cultured in low glucose DMEM with 10% fetal bovine serum,and were observed under inverted microscope.Then the BMSCs were purified and expanded through passaging in time, and took some for cryopreservation when passaging cell after the second generation.2. To check the third generation rBMSCs by identifying the surface CD antigens with flow cytometry and then further induce them into adipoblasts and osteoblasts.3. We used lentiviral vector containing NGF gene and containing GFP gene as control group to infect target cell rBMSCs,then observed the infected rBMSCs about the expression of GFP under inverted fluorescence microscope.4. Using lentiviral vector containing GFP gene as control group,we checked the infected rBMSCs about the expression of NGF protein by taking the culture medium supernatant for ELISA.5. Using lentiviral vector containing GFP gene as control group, we checked the infected rBMSCs about the mRNA levels expression of NGF gene by extracting the total RNA for real-time fluorescence quantitative PCR.Results:1. The rBMSCs amplified by whole bone marrow culture growed stably,and presented relatively homogeneous fusiform or fibrous form. Flow cytometry results showed that rBMSCs expressed antigen CD44,CD90,CD106,the symbol of stem cell,and did not or weakly express CD34,CD45 and other symbol of hematopoietic stem cell. They also can be induced into adipoblast and osteoblast,that confirming they are other stem cells different from hematopoietic stem cells.2. In the infected rBMSCs by lentiviral vector, using real-time fluorescence quantitative PCR can detect NGF gene mRNA, and using ELISA can detect NGF protein in the culture medium supernatant.Conclusions:1. We succeeded in isolation,cultivation a great deal of high-purity rat bone marrow mesenchymal stem cells through the whole bone marrow culture, to meet the demand for further research in vitro and in vivo.2. We succeeded in transfer the NGF gene into rat bone marrow mesenchymal stem cells by resorting to lentiviral vector.The infected rBMSCs can secrete NGF protein.That is an ideal gene-modified cells for the next step in vivo gene therapy study.