Study On Proliferation Capability And Differentiation Potency Of Bone Marrow-derived Mesenchymal Stem Cells From Rheumatoid Arthritis Patients

Objectives By comparing the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) from rheumatoid arthritis (RA) patients and normal human to discuss the existence of RA patients with BMSCs defects or variation, whether can been used in autologous transplantation, or RA itself is a stem cell disease associated with the incidence of RA.Methods BMSCs were obtained from eight cases of RA patients and healthy volunteers bone marrow. The cells were isolated cultured by gradient centrifugation and adherence cultivation, and the surface markers of BMSCs were identitied by flow cytometer. Firstly, BMSCs reproductive activity of the two groups were compared: the auxodrome and cell cycle of BMSCs were measured respectively, then the doubling generation time of cell colony and proliferation index were calculated in order to see whether there was a difference of the proliferation capability between the two groups. Further more the hTERT were measured. Besides, potentiality of cell differentiation of the two groups were compared: BMSCs from the third passage(P3) were grown in complete medium and in chondrocyte induction culture solution seperately. The growth of the cultured BMSCs was observed. Immunohistochemistry was applied to detect the expression of collagen typeâ…¡.The synthetic proteoglycans were detected by toluidine blue staining.Results BMSCs from the two groups were similar in cell morphology. By flow cytometry, the cells expressed CD44 and CD71, but not CD34 and CD45. Comparison of doubling time and proliferation index of two groups's BMSCs in P3 and the sixth passage(P6) showed that the proliferative capacity of P6 cells were lower than P3. The differences of doubling time and proliferation index of two groups's BMSCs in P6 did not have statistical significance, but the differences of P6 had statistical significance. Determination of two populations of hTERT differences had statistical significance. 14 days after two groups of cells cultured in the cartilage cells medium, it showed that toluidine blue staining and II collagen immunohistochemistry were positive. It confirmed the formation of cartilage cells.Conclusion Density gradient centrifugation is an effective way to obtain human bone marrow mesenchymal stem cells. They were stable growth and phenotype homogeneous in vitro after the screening of adherent cultured cells. Two groups has no significant differences in cell morphology. The proliferative capacity of P6 cells of the two groups has declined obviously than the cells at P3. It proved that aging situation occurred in P6 cells. In addition, the proliferative capacity of two groups at P3 were no significant difference, but BMSCs proliferation lags behind the normal group in RA patients at P6, which inferred that the senescence of the BMSCs from RA patients were earlier than healthy donor. The determination results of hTERT showed that the difference between the two groups had statistical significance, and it proved that the premature aging may be associated with hTERT expression. The reduction of hTERT expression can not maintain telomere length, so the life span of cells shorten, then enter the senescence and apoptosis in advance. The experiments also show that the BMSCs from RA patients can differentiate into cartilage cells. So it might that the differentiation ability of the BMSCs from RA patients had no obvious defects.