| BackgroundRheumatoid arthritis is a highly disabled autoimmune disease.Disease incidence is about 0.34%in China and the total patients are 5 millions.There is a big indirect economic loss because of treatment and loss of work ability.Recent study showed that mesenchymal stem cells have immuno-regulation effect,at the same time MSCs can move to injury and inflammatory area in microenvironment.It can differentiate to dermal layer,mesoblast and endoderm,participating in tissue rebuilding and restoration.All of these bring a hope for RA patients.ObjectiveTo observe the immunologic effect of transplantation of MSCs through studying early and later period of Collagen-induced-arthritis;to observe homing of MSCs in immune organs and inflammatory joints in CIA rats.In this view we discuss the mechanisms of underlying immunomodulating properties and potential applications.MethodsIn the first part,rats MSCs were isolated and expanded from bone marrow cells by density gradient cent rifugation and adhering to the culture plastic,and identified with cell morphology, and the phenotypes were assessed by flow cytometry.In the second part,we established collagen-induced-arthritis rats model immuned by Freund's complete adjuvant and typeⅡcollagen for two times.40 rats were randomly divided into 5 groups,including:early MSCs treatment group(MSCs were transplanted by the time the rats were induced for the first time), later MSCs treatment group(MSCs were transplanted by the time the rats were induced for the second time),normal control group,early CIA control group and later CIA control group.MSCs were injected through tail Venus.Treatment effects were assessed clinically and histologically before sacrificed.All of rats were sacrificed at 42 days after transplantation of MSCs.We detected the proliferation of lymphocyte to ConA and LPS with CCK-8,observed the expression of Foxp3 mRNA using RT-PCR,assayed the level of CD4~+CD25~+ T cell by means of flow cytometry.In the third part,MSCs were labled by PKH-26 and Brdu.64 rats were randomly divided into normal group and CIA group.Every 8 rats were sacrificed at 3,11,30,42 days after transplantation of MSCs.At the end of the experiment,the specimens of thymus gland,spleen, ankle joints were exposed,fixed,decalcified,wrapped and cut into slices.We use confocal laser scanning microscope and immuno-histochemical methed to observe migration and distribution of MSCs in different organs.Results1.Uniform spindle-shaped in appearance and showed active proliferative capacity and had S shape of growth curve.2.The improvement of arthritis index of early and later treatment groups was higher than CIA control group,which showed statistically significant difference(P＜0.05).Arthritis index of early treatment group was lower than later treatment group,which showed statistically significant difference(P＜0.05).3.The percentage of Treg in early and later CIA groups was lower than normal control group and treatment groups,which showed statistically significant difference(P＜0.05).The level of Treg in early MSCs treatment group was higher than the later MSNs treatment group,which showed statistically significant difference(P＜0.05).4.Compared to the normal group and treatment groups,the expression level of Foxp3 mRNA in early and later CIA groups was decreased markedly.While the early MSNs treatment group versus the later treatment group showed no statistically significant difference(P＞0.05).The intensity of Foxp3 mRNA in treatment groups was close to normal group.5.The proliferation level of T cell and B cell was higher in CIA control groups than normal and treatment groups,which showed statistically significant difference(P＜0.05).Compared to the later treatment group,the percentage of T cell was decreased markedly.While the level of B cell in early and later MSNs treatment groups showed no statistically significant difference(P＞0.05).6.The level of TGF-β1 was lower in early and later CIA control groups than normal and treatment groups(P＜0.05).Rats serum TGF-β1 level of the early treatment group was higher than that of the later treatment group,which showed statistically significant difference(P＜0.05). The levels of TNF-αand IL-17,which were increased in CIA control groups,higher than normal and treatment groups(P＜0.05).Compared to the later treatment group,IL-17 in the early treatment group was increased markedly(P＜0.05).TNF-αof treatment groups showed no statistically significant difference(P＞0.05).7.It was found that allogenic MSCs could stay in spleen,thymus gland and joints of CIA rats for a relatively long period(42 days).The quantity of MSCs was higher in CIA group than normal group. Conclusions1.Rats MSCs were isolated and expanded from bone marrow cells.MSCs were uniform spindle-shaped in appearance and showed actively proliferative capacity,with S shape of growth curve. After MSCs were labeled by PKH-26 and Brdu,biological properties were not changed.2.Our results proved that allogenie MSCs transplantation can relieve inflammatory symptoms of joints and pathology of synovial membrane.Moreover,in our experiments,MSCs showed significant immunomodulatory effects,to upregulate the level of Treg in CIA rats,accelerate the expression of Foxp3 mRNA,inhibit the proliferation of T cell and B cell,stimulate secretion of TGF-β,decrease the content of TNF-αand IL-17 in serum.The early treatment group was more effective than the later treatment group.3.It was found that allogenic MSCs can stay in spleen,thymus gland and joints of CIA rats, whatever later or early groups,for a relatively long period(42 days).MSCs stayed in injured tissue and organs preferentially. |
PDF Full Text Request