Primary Study On The Biological Characteristic Of Human Umbilical Cord Mesenchymal Stem Cell And Its Therapeutic Effects On The Rats Of Osteoporosis

OBJECTIVE:1. To establish the methods of isolation and expansion of human umbilical cord derived mesenchymal stem cells (hUC-MSCs).2. Studying the biological characteristics of hUC-MSCs and its capacity of adipogenic and osteogenic induction in vitro.3. To find out the feasibility of healing the osteoporosis (OP) rats by transplanting the hUC-MSCs by intravenous injection.METHOD:1. Obtain the term infant umbilical cord samples and apply the enzymatic or the tissue culture method to separate and purify hUC-MSCs.2. Detecct the cell surface markers by flow cytometry; Observe the morphology; Describe the growth curve with MTT method, describe the biological characteristics of hUC-MSCs in vitro.3. Induce the hUC-MSCs differentiation into adipocytes and osteoblasts and detect the induced cells by histochemistry staining.4. The treatment of hUC-MSCs on RA induced rats:the female SD rats were randomly divided into four groups (control group (A), model control group (B), hUC-MSCs transplantation group (C) and Xianling Gubao group (D).Except A group administrated with 0.5%CMC-Na, other groups administrated with RA.At the same time, Xianling Gubao and hUC-MSCs were administrated; C group were adiministrated with 2X 106 hUC-MSCs A and B group were administrated with distilled water. The serum biochemical indicators, biomechanics characteristics were measured and microstructure of bone were also observed.RESULTS:1. We can get 80%fusion hUC-MSCs during 14-20 days both by tissue culture and enzymatic methods The proliferation became faster after passage and the cells after passage three(P3) presented a homogeneous population of spindle fibroblast-like cells. More than 90%of P3 cells expressed the CD44, CD29, CD 105, but the CD28, CD31, CD34, CD45, CD40, CD86 and HLA-DR were negative.80% of the cells were in G0/G1. The assays in vitro demonstrated the P3 cells exhibited osteogenic and adipogenic cells differentiation potential.2. Effect of hUC-MSCs on RA simulated OP rats:(1). In bone biochemical measurement of the serum:The serum Ca2+, ALP and StrACP of C group were significantly higher than B group (p<0.05 or p<0.01); but no found significant difference between D and B group.(2). In BMD measurements of the right femur group C were significantly higher than in group B (p<0.05);(3). In right femoral shaft three-point bending test, the max load,the elastic load, the rigidity and max deformation of group C were significantly higher than in group B (p< 0.05 orp<0.01);(4). In SEM observation, collagenous fiber with regular arrangement in group A; collagen fibers displaced by absorption laeuna in group B; Collagenous fibers were much more regular in group C.CONCLUSION:1. The hUC-MSCs were obtained from the human umbilical cord by tissue or enzymic method; the cells shared the same characteristics MSC. The hUC-MSCs were multipotential cells.2. Bone formation and bone mass decreased, bone microstructure destructive in RA administrated rats. RA simulated OP were a high turnover OP. The rats OP model were successfully established.3. The hUC-MSCs reduced the serum Ca2+, ALP and StrACP level of OP rats, improved the femur microstructure. The BMD and biomechanic function also enhanced.4. The hUC-MSCs has a significant effect on RA simulated OP rats. The cells can promote bone formation and reduce bone loss, and improve the microstructure.5.