The Study Of Rabbit Bone Marrow Mesenchymal Stem Cells Separation Culture And Directional Differentiation

Mesenchymal stem cells(MSCs) originating from mesoblast, resides mainly in mammalian bone marrow, and coexists with hematopoietic stem cells(HSCs). Functionally bone marrow mesenchymal stem cells(BMSCs) not only exerts the crucial role of supporting hemopoiesis by participating in tne formation of hematopoietic microenvironment, but they can be cultured and proliferated easily in vitro, autoplastic transplantation and have differentiation multipotentials into multiple cell lineages of the three germ layers, such as osteoblast, chondrocyte, adipocyte, tendon cell, muscle cell and neuron. BMSCs with undifferentiated or after genetic manipulation would be one of tne ideal seed cells source for cell/gene therapy and tissue engineering in future. The aim of this research is to illuminate the biological property and the potential on inducation to adipocyte of the rabbit bone marrow BMSCs and establishes expansion and culture condition for rabbit BMSCs, so as a model to apply BMSCs to diseases cell or gene therapy in future. Therefore, the isolation, culture, proliferation, identification and growth characteristics and in vitro differentiation of one month white rabbit of Japan BMSCs were assayed and researched, systematically. It found an experimental base for further investigation and using of mesenchymal stem cells. The results are as follows:1. In our test, the rabbit BMSCs were isolated from rabbit femur and tibia bone marrow by red blood cell lysis method and adherence-dependent growth characters to tissue culture plates, and sub-cultured to the 7th passage successfully. Observe pattern of BMSCs with Inverted phase contrast microscope. The result shows that MSCs were adherent cells with a similar fibroblastic spindle-shaped morphology during different passages. MSCs show fibroblast-like morphology from primary passage to passage 7 before treatment, the growth curve of different generation of MSCs indicated that the activity of BMSCs declined with the passage of cells. Rabbit MSCs can be isolated and cultured in vitro successfully. It put the foundation for further studies.2. Basic medium and serum concen-tration for BMSCs culture were optimized with MTT (3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-di-phenytetrazoliumromide) reduction assay. In this experiment, DMEM/F12+ 20%FBS was the best system for rabbit BMSCs in vitro. MTT assay showed that proliferation promotion effect of bFGF nearly reached the peak at 80μg/L(P<0.01). Compared with the negative control group, significant proliferation promotion effect was detected in the bFGF groups from 72h(P<0.01). Basic fibroblast growth factor can promote proliferation of rabbit BMSCs in vitro, 80μg/L is good concentration for in vitro culture which can be used as the best culture condition of rabbit BMSCs proliferation in vitro.3. Dexamethasone(DEX), insulin(In) could induce rabbit BMSCs to differentiate into the adipocytes. After induced with induction of lipid vacuoles gradually, and their shape from long fusiform cells to round or polygonal, and Oil Red O stain positive. That established a model for BMSCs differentiate into the adipocytes. The potential of adipocytes differentiation was an important criterion for distinguishing the BMSCs from hematopoietic stem cells.