| Purpose: The characteristics of mesenchymal stem cells (MSCs) involving the multipotent differentiation, the repair of tissue injury and the capability of immune regulation bring the new application prospect for the treatment of autoimmune diseases such as rheumatoid arthritis (RA). However, it has shown that the controversies over MSCs for the therapy of RA from many previous experimental studies. We investigated the influence of inflammatory fluid in RA patients to exogenetic MSCs in vitro experiments, and discussed the biological characteristics changes of MSCs under RA inflammatory environment.Method:1 Isolation, culture and identification of bone marrow MSCs:14 cases of bone marrow samples were collected in this study. Five of them were treated by simple adherence screening method, and the other nine cases were treated by the density gradient centrifugation associated with adherent screening method. The cells were isolated, cultured and passaged, and the capability of osteoinduction differentiation was detected. The growth curve of P3 MSCs was rendered and the cells of P3 generation were used for subsequent experiment.2 Blood serum of patients with RA had an effect on proliferation of MSCs: There were 12 cases of patients with RA and four cases of healthy individuals in our study. MSCs with the same cells (4000 per pore) were inoculated in the 96-pore plate. The culture medium of experimental group was respectively added with blood serum of the concentration of 10%, 30% in patients with RA, and the culture media of blood serum of healthy individuals with the corresponding concentration were served as the control group. Preparation of three pores was selected for each specimen and each concentration. Five days after cultivation, the absorbance value of each pore was detected by MTT method.3 MSCs involved by blood serum of patients with RA had an effect on proliferation of T cells: MSCs with the same cells (4000 per pore) were inoculated in the 96-pore plate. The culture media of experimental group were added with blood serum of the concentration of 30% in patients with RA, and the culture media of blood serum of healthy individuals with the same concentration were served as the control group. Five days after cultivation, the peripheral blood (10ml) of RA patients at active stage was respectively collected and then CD3+ T lymphocytes were separated and acquired. MSCs of the experimental group and the control group were co-cultivated with CD3+ T cells. We also set separate MSCs group and T-cell training group. And then CD3+ T lymphocytes were stimulated with PHA. After 68 hours, the absorbance value of each pore was detected by MTT method. Inhibition rate of the proliferation of T cells by MSCs was calculated.4 Joint fluid of patients with RA had an effect on the proliferation of MSCs:MSCs with the same cells (4000 per pore) were inoculated in the 96-pore plate. The culture medium of experimental group was respectively added with joint fluid of patients with RA with the proportion of 10%, 30%, 50%, and the simple culture medium was served as control. Five days after cultivation, the absorbance value of each pore was detected by MTT method.5 Joint fluid of patients with RA had an effect on the apoptosis of MSCs: MSCs with the same cells (105 per pore) were inoculated in the six-pore plate. The culture medium of experimental group was added with joint fluid of patients with RA with the proportion of 50%, and the simple culture medium was served as control. Five days after cultivation, the cells of each group were digested and collected, and then apoptosis of cells of each group was detected by apoptosis assay kits and flow cytometer. 6 Joint fluid of patients with RA had an effect on the osteoinduction of MSCs:MSCs with the same cells (105 per pore) were inoculated in the six-pore plate. The culture medium of experimental group was added with joint fluid of patients with RA with the proportion of 50%, and the simple culture medium was served as control. Five days after cultivation, MSCs were added by complete medium of osteoinduction and the osteoinduction process was stoped seven days later (medium completely change every three days). Identification was adopted with calcium nodules staining (alizarin red staining), and the expression of osteopontin gene (specific products of osteoblasts) was detected by RT-PCR method.Result:1 Growth characteristics and morphological observation of MSCs: MSCs were cultivated in only one of five samples by simple adherence screening method, whereas MSCs were successfully isolated to receive among the nine cases treated by the density gradient centrifugation associated with adherent screening method. The bottom of bottle had been covered over 80% through the primary culture for 7-12 days. Rapid proliferation of MSCs was shown after passaged. For generation of P3, the bottom of bottle had been covered over 80% during 5-7 days. The cell proliferation began to slow down in P5 generation, and then the ageing phenomenon including larger cell bodies associated with the increasing processes appeared. The growth curve of P3 MSCs had shown that the incubation period of 1-3 days, the logarithmic growth of 3-5 days, and the platform period of 5-7 days later. Two weeks after osteoinduction, red mineralized nodules were visible with alizarin red staining.2 Blood serum of patients with RA had an effect on proliferation of MSCs: After the impact of blood serum with the concentration of 10% in patients with RA on MSCs, the absorbance value was respectively 0.28±0.044 for experimental group, 0.305±0.01 for the control group (t=0.939, P=0.366). For the concentration of 30% of blood serum, the absorbance value was respectively 0.327±0.092 for experimental group, 0.437±0.164 for the control group (t=1.58, P=0.14).3 MSCs involved by blood serum of patients with RA had an effect on proliferation of T cells: The absorbance value was respectively 0.534±0.022 for experimental group involved by blood serum of patients with RA, 0.712±0.069 for co-cultured group with T cells. The absorbance value was respectively 0.465±0.019 for blood serum of healthy individuals, 0.71±0.083 for healthy individuals co-cultured with T cells, and 0.508±0.053 for simple T cells group. The difference was not statistically significant for experimental group comparison with healthy control group (t=0.417, P=0.717). The proliferation of T cell were suppressed by MSCs from both experimental group and control group.4 Joint fluid of patients with RA had an effect on the proliferation of MSCs: After the influence of joint fluid of patients with RA to MSCs, the absorbance value was respectively 0.234±0.029 for joint fluid with the concentration of 10%, 0.227±0.065 for concentration of 30%, 0.194±0.059 for concentration of 50%, and 0.269±0.047 for the control group. There was no significant difference between experimental group and the control group (F=3.44, P=0.028). As to experimental group itself, the difference was only signifcant between the control group and group with concentration of 50% (P=0.003). The proliferation of MSCs was inhibited when concentration of joint fluid of patients with RA was 50%.5 Joint fluid of patients with RA had an effect on the apoptosis of MSCs: There was no significant difference of apoptosis ratio between experimental group and the control group. For MSCs followed by the effect of joint fluid of patients with RA, apoptosis ratio was respectively 41.53% in experimental group and 45.5% in the control group. There was no significant difference between them (χ2=0.256, P=0.613).6 Joint fluid of patients with RA had an effect on the osteoinduction of MSCs: During the influence of joint fluid with concentration of 50% for five days, more and bigger red calcium nodules of experimental group were seen by alizarin red staining in seven days after osteoinduction. Futhermore, the expression of osteopontin gene for MSCs affected by joint fluid of patients with RA was significant in seven days after osteoinduction, however, that of the control group was still rarely.Conclusion:1 The bone marrow MSCs can be effectively isolated and cultured by the density gradient centrifugation associated with adherent screening method.2 Blood serum of RA patients at active stage would not restrain the proliferation of external MSCs, and it also won't reduce the inhibition role of MSCs to the proliferation of T lymphocyte.3 The growth of MSCs was inhibited by joint fluid of patient with RA with concentration of 50%. However, increase in apoptosis of MSCs did not occur in this setting.4 The condition of the joint fluid of RA patients would accelerate MSCs differentiation into osteoblasts faster.
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