Immunoregulatory Effects Of Mesenchymal Stem Cells In Lupus-Prone Mice (NZBW F1 Mice)

Objective To investigate Immunoregulatory effects of mesenchymal stem cells(MSCs) on T-lymphocytes of NZBW F1 mice in vitro.To explore the therapeutic effects of mesenchymal stem cells(MSCs) transplantation in NZBW F1 mice.Methods(1) Applied 15 NZBW F1 mice as observed objectives,they were divided into two groups randomly:24-week-old group and 32-week-old group.Coomassie brilliant blue method was used to detect 24-hour proteinuria every week.At the age of 24 weeks or 32 weeks,kidneys and spleens were abtained from mice,kidneys were dyed by PAS and the variation of renal pathology were observed.Splenic lymphocytes were isolated and cultured.Under ConA or LPS stimulating,the immunophenotypes(CD69,CD28 and CD86) of lymphocytes were annlyzed by flow cytometry,the T-lymphocytes' apoptosis was also detected by flow cytometry.(2)Human bone marrow mesenchymal stem cells were separated from bone marrow of healthy human.Splenic Iymphocytes were isolated from 24-week NZBW F1 mice.Under ConA stimulating,T-lymphocytes were treated with MSCs accorded to different proportion(1:0.01,1:0.02,1:0.1),the proliferation of T-lymphocytes was assessed by MTT colorimetry.FCM was used to analyze the apoptosis and surface markers-CD69,CD28.The IL-10 and IFN-γmRNA expressions of T-lymphocytes were detected by real-time quantitative PCR.(3) Human MSCs were isolated and expanded in vitro.NZBW F1 mice were devided into transplantation group and control group randomly.Human MSCs were transplanted into transplantation group through tail vein at the age of 24 weeks,and DMEM were injected into control group.24-hour proteinuria was detected with Coomassi brilliant blue method every week. All the mice were sacrificed after 8 weeks,anti-dsDNA antibody were detected by ELISA and the pathology of kidney were observed. Lymphocytes were isolated and cultured from spleens.Under ConA stimulating,the surface markers(CD69/CD28) of T-lymphocytes were annlyzed by flow cytometry,the T-lymphocytes' apoptosis was also detected by FCM.Results(1) It was found that the expression of CD69,CD28 of T-lymphocytes and CD69 of B-lymphocytes in 24-week-old group were significantly higher than that in 32-week-old group(P＜0.05),no difference of the expression of CD86 of B-lymphocytes had been observed(P＞0.05).The apoptosis of T-lymphocytes of 24-week-old group is lower than that of 32-week-old group(P＜0.05).24-hour proteinuria of NZBW F1 mice increased along with age.The renal pathology at 32-week-old group were notably impaired.(2) In all experiments,with the presence of human MSCs splenic T-lymphocytes resulted in a statistically significant decrease of their proliferative activities as dose-dependent manner(P＜0.05).When the proportion of T and MSCs are 1:0.02 and 1:0.1,MSCs can inhibit the apoptosis of T-lymphocytes(P＜0.05),and MSCs can promote the mRNA expression of IL-10 and inhibit mRNA expression of IFN-γ(P＜0.05).When the proportion of T and MSCs is 1:01,no significant effects on apoptosis and cytokines of T-lymphocytes were observed(P＞0.05).No inhibitory effects on CD69 and CD28 of T-lymphocytes were observed in all group treated with MSCs(P＞0.05).(3) Mice that received transplantation had a significant improvement compared with the control group in the observation of pathology in kidney, proteinuria and anti-ds-DNA level(P＜0.05).In comparation with control group,it is higher in transplantation group of expression of IFN-γmRNA, and IL-10 mRNA in transplantation group notably lower(P＜0.05).No difference of CD69 and CD28 expression was observed between transplantation group and control group(P＞0.05).Conclusions(1) Compared with 32-week-old group,the lymphocytes activation increased, apoptosis decreased in 24-week-old group,and kidneys in 32-week group were impaired significantly,which may be assoiated with their autoimmune stage.(2) Human MSCs could inhibit the proliferative activities and apoptosis of splenic T-lymphocytes and change the mRNA expression of T cells differentiation-related cytokines,which may play an important role in its immunoimpressive effects.(3) Human MSCs transplantation is an effective treatment for NZBW F1 mice.MSCs transplantation can decrease the proteinuria and lower the IL-10 mRNA expression.It also can ease the pathology of kidney in NZBW F1 mice.It may be an effective method for treating SLE.