Immunoregulatory Effects And Its Mechanism Of Bone Marrow MSCs To GVHD Model Rats

Objective: In order to study the immunomodulatory effects of mesenchymal stem cells(MSCs), this study investigates the effects for MSCs on proliferation stimulated by ConA and killing ability of T cells in vitro. Meanwhile, we approach immunoregulatory effects and its mechanism of bone marrow MSCs to GVHD model rats initially. For the final purpose, this study provides scientific basis for allogeneic hematopoietic stem cell transplantation of MSCs.Methods:1 MSCs from bone marrow cells of rats were isolated and cultured in vitro by method of entire marrow cells adherence. The morphological charactcrization of MSCs was observed under inverted microscope directly or after Wright-Giemsa`s staining. Typical phenotypes of MSCs were identified by flow cytometry(FCM). The multiple differentiation potentials were confirmed by lingage-specific induced differentiation to osteocytes and adipocytes.2 ConA was used as stimuli origin. Effects of MSCs at different ratios(MSCs:T=1:20, 1:10 and 1:5)on proliferation of T cells were determined by MTT method. FCM was used to detect the effects of MSCs at different ratios(MSCs:T=1:20, 1:10 and 1:5)for 24 hours on CD25 expression of T stimulated by ConA.3 We also detect the concentrations of IFN-γ, TNF-α, IL-4 and IL-10 in T cells culture system cultured alone or cocultured with MSCs at different ratios(MSCs:T=1:20, 1:10 and 1:5)for 72 hours by ELISA assay, when 20μg/ml ConA was used as stimuli origin. FCM was used to detect on apoptotic levels of T cells. MTT method was adopted to investigate the killing ability of CTL in vitro after MNC had been induced and cocultured with MSCs(MSCs:MNC =1:5)or cultured alone for 96 hours, when 10μg/ml ConA was used as stimuli origin.4 MSCs from bone marrow cells of SD rats were isolated and cultured in vitro. GVHD model was established after infusing bone marrow cells and MNC from SD rats to Wistar rats, which have been radiated, and then, MSCs were cotransplanted with bone marrow cells at different ratios. The suppre- ssive effects of MSCs on GVHD were studied through general symptoms, lifespan, and pathological changes of target organs.5 Effects of MSCs on concentrations of IFN-γ, TNF-α, IL-4, and IL-10 in serum of peripheral blood were determined by ELISA assay. It was also investigated mRNA level of four kinds of cytokines, as described above, in spleen MNC of recipients peripheral blood by RT-PCR.6 The ratios of CD4~+CD25~+ regulatory T cells in peripheral blood MNC of experimental rats were identified by FCM. Ratios of Foxp3~+ cells in peripheral blood MNC were observed by cell immunology chemical method. Effect of MSCs on cytotoxicity of CTL in vivo was investigated by MTT method after spleen MNC of receipent rats were induced.Results:1 After MSCs of Primary passage were cultured for 72h in vitro, adherenced cells of unhomogeneous morphology were observed and showed a long-spindle shape. The microcolony were near 90% confluence about 10 days, and 90% confluency could be reached at day 7 after passage. Bone marrow MSCs showed potent proliferative ability in vitro. MSCs have the characteristic phenotype with expression of CD44, CD29 and the lack of CD34, CD45. MSCs were able to differentiate into adipocytes and osteocytes in special induction culture system.2 Results of MTT showed that proliferation ability of T cells decreased obviously when cocultured with MSCs, compared with positive control (P<0.01). The proliferation of T cells was inhibited markedly in a dose- dependent manner. CD25 expression of T cells cocultured with MSCs was obviously inhibited when stimulated by 20μg/ml ConA, compared with positive control, and the disparation was meaningful(P<0.01).3 ELISA results showed that the IFN-γand TNF-αconcentration levels in T-MSCs coculture systems were more lower than positive control group that T was cultured alone, but the IL-4 and IL-10 concentration levels were more higher in a dose-dependent manner(P<0.01). Results of FCM revealed that when 20μg/ml ConA was used as stimuli origin for 72h, the apoptosis rates of T cells cocutured with MSCs in earlier period were obviously lower than T cells cutured alone(P<0.01). The apoptosis rate of spleen T cocutured with MSCs in advanced stage at ratio of 1:20 was 4.23±0.65%, compared with group cultured alone(4.63±0.75%), and the disparation was not meaningful (P>0.05). The apoptosis rates of spleen T cocutured with MSCs at ratios of 1:10 and 1:5 were 2.17±0.40% and 1.13±0.21% respectively in advanced stage, which were obviously lower than group cultured alone(P<0.01). After coculturing with MSCs, cytotoxicity of CTL was impaired compared with group cultured alone in vitro(P<0.05).4 It was demonstrated that cotransplantation with MSCs in rats after allogeneic bone marrow transplantation decreased the rate of GVHD obviou- sely, alleviated pathological damagement, and prolonged survival time (P<0.05).5 ELISA results showed that in cotransplantation groups, the levels of IFN-γand TNF-αwere decreased, but the levels of IL-4 and IL-10 were increased markedly in serum of peripheral blood(P<0.05), and this function was also in a dose-dependent manner. The results by RT-PCR were accordan- ce with those by ELISA assay.6 Compared with GVHD group, ratios of CD4~+CD25~+T cells in peripheral blood MNC were increased obviously in a dose-dependent manner in cotransplantation groups by FCM(P<0.05). Compared with GVHD group, ratios of Foxp3~+cells in peripheral blood MNC were increased notably (P<0.05) and cytotoxicity of CTL was impaired in cotransplantation groups in vivo.Conclusion: 1 It is a simple and effective method to obtain rats bone marrow MSCs with high purity and activity by method of entire marrow cells adherence. Their biological characters were identified with stem cells.2 MSCs can inhibit markedly proliferation and CD25 expression of T cells in a dose-dependent manner in vitro.3 The immunomodulatory effects of MSCs on T cells may be related to improving IL-4 and IL-10 expression of T cells, and preventing TNF-αand IFN-γsecretion by T cells. However, the mechanisms of MSCs immunolore- gulation may be not related to apoptosis.4 Donor derived high-dosed MSCs could reduce the incidence and severity of GVHD and prolong the survival time of the recipient rats. The mechanisms of MSCs immunoloregulation may be related to downregulating TNF-αand IFN-γexpression, upregulating IL-4 and IL-10 expression in the serum, and upregulating the ratio of Treg.5 The killing effects of induced CTL on tumor in cotransplantation MSCs groups are lower than that of GVHD group markedly.