| BackgroundAutoimmunity disease (AID), one of the refractoriness diseases, is seriously harmful to humanity health. Currently, no curing medicine has been developed yet. Immunosuppressive drug therapy is the present method of treating AID. Although its therapeutic effect is confirmed, it can only alleviate the symptoms, and cannot prevent the occurrence and development of the disease. Furthermore, its toxicity and side effects are severe, making it unsuitable for long term application. Therefore it is urgent to develop some highly effective, non-toxic immunomodulating drugs. There is a lot of clinical practice of Traditional Chinese materia medica in treating AID. TCM incurs small toxic side effect and good curative effect, and has been used in clinic practice already. Thus TCM has become an important domain for the immunomodulator development.Bone marrow mesenchymal stem cells (MSCs), as a kind of non-hematopoietic tissue stem cells, are contained in bone marrow and have the capacity of self-renewal, high proliferation and multilineage differentiation. Recent researches have shown that beside supporting the survivor of hematopoietic stem cells in vitro and promoting reconstitution of hematopoietic function in vivo, MSCs could also prevent graft-versus-host disease (GVHD) in bone marrow transplantation, which indicates that MSCs have the immunoregulatory function. The main mechanism of the immunoregulatory effect of MSCs is that they could suppress proliferation of T-lymphocyte in response to stimuli. Because of their immunoregulatory function, MSCs can be used widely in applications of treating AID and GVHD. It is proved that the autologous or allogeneic bone marrow transplantation would exert curative effect on AID. Marrow stem cell transplantation could be a new method of treating AID such as rheumatic arthritis.According to TCM, restoration of the balance of mechanism in the human body is the best way to health. So maintainance of the organism endogenous normal immunoloregulation could be an ideal model of study on the effects of Chinese materiamedica on the immunoregulation of MSCs. This article observes the intervention of the Chinese materia medica on MSCs, to explore the immunological regulation mechanism of Chinese materia medica.Methods and ResultsThis experiment is based on the reaction system of rat lymphocyte proliferation stimulated by PHA, PMA,ConA and LPS. We observe the influence of rats' MSCs on lymphocyte activation and proliferation in vitro, to confirm whether rat MSCs has suppressed the proliferation of lymphocyte. Then, through RT-PCR and ELIS A method, we discuss the possible mechanism of the MSCs influence. Based on the discussion, we observe the intervention of some active components or simple substance of Chinese materia medica, and investigate into the possible immunoloregulation mechanism of Chinese materia medica.1.Isolation, culture, and identification of rat MSCs in vitro.The tibias and femurs were dissected from SD rat and the marrow in the tibias and femurs was taken out with PBS. Sticking wall methods were used to obtain rat MSCs. The primary cells were nurtured in L-DMEM, the passage cells nurtured in conditioned medium. The shape of most of the MSCs is fusiform, and few are round. The surface antigens were checked with immunohistochemical, and it was positive for CD44, CD54 and collagen I . Primary propagation took eight to ten days, while the growth of passage cells took only four to six days with 80% confluens. MSCs had a strong capacity of proliferating and could reach a total numbers of lxio8 after five passages. The adherence time of MSCs is very short after passage;95% of the MSCs are adhered in 10 hours, with high survival rate. The following experiment uses the passage three or last. This experiment established a kind of simply, effective, economically culture method of rat MSCs in vitro.2.Determination the suitable dose of PHA, PMA, ConA and LPS as well as the cell density of lymphocyte for the cell proliferation.Through rat lymphocyte activation and proliferation experiment with different dose of PHA, PMA, ConA, or LPS, and different cell densities of lymphocyte, the researcher determines the approproate dose of PHA, PMA, ConA, or LPS and cell density of lymphocyte used in the following experimental study. The outcome indicated that when the lymphocyte density is 6xl06cells/ml, and PHA, PMA, ConA and LPS dose is 40, 1, 5, 20 Hg/ml respectively, the stimulus effect turns out to be the best.3. Exper i ment on MSCs suppress i on on a 11 ogene i c I ymphocyte pro I i f erat i on.Based on the MSCs layers with different densities: 10%, 20%, 40% of the quantity oflymphocyte, added PHA, PMA, ConA, or LPS in the cultivate system, co-cultivation 72h, detect the proliferation of lymphocyte and the suppress rate of proliferation. Simultaneously the inhibitory action of the MSCs culture supernatant is observed. The results showe that rat MSCs could suppress the lymphocyte proliferation causesd by PHA, PMA, ConA, but not by LPS. The inhibitory is in positive correlation with the quantity of MSCs. The culture supernatant of MSCs also presents the inhibition ability, which correlates with its concentration.4. The intervention of Chinese materia medica on MSCs suppression on the proliferation of lymphocyte.Based on the quantity of MSCs 20% of lymphocyte, MSCs were pretreated by Chinese materia medica for 24h, then lymphocyte and PHA, PMA, ConA were added to the culture system. After co-cultivation of 72h, the proliferation of lymphocyte and the suppress rate of proliferation were detected. The results showed that Astragaloside could weaken MSCs immunosuppressive function, while Quercetin, Curcumine and Q-C strengthen the immunosuppressive activity of MSCs.5. The preliminary study on Chinese materia medica's intervention mechanism on MSCs restrain on the proliferation of lymphocyte.MSCs' TGF-pl, IFN-ymRNA and lymphocyte's IL-lOmRNA were tested, by co-cultivation of MSCs with RT-PCR, to illuminate the mechanism of MSCs' restrain on the proliferation of lymphocyte, to detect whether mRNA had changed or not after processed with Chinese materia medica, and to conjecture the intervention mechanism of Chinese materia medica's immunosuppress on MSCs according to the test results.ELISA was used to detect the density of TGF-pi excreted by MSCs in the up-layer culture liquid, according that to speculate MSCs'restrain lymphocyte proliferation had something to do with the TGF-pi that excreted by MSCs, and at the same time to detect the change of the quantities of IL-10 in the culture liquid of MSCs and lymphocyte coculture system, to speculate whether MSCs' restrain proliferation of lymphocyte had something to do with The Suppressive Cytokines excreted by coculture system. Explored whether the expression of the cytokine above had altered after Chinese materia medica's treat, and study the mechanism of intervention action of Chinese materia medica.RT-PCR assay show that MSCs expressed TGF-pi but not IFN-gamma mRNA. After Astragaloside processed, the quantities of TGF-pl expression reduced, but the quantities of TGF-pi expression raised, After processed by Quercetin, Curcumine and Q-C. RT-PCR analysis revealed pronounced up-regulation of IL-10 mRNA levels in lymphocyte coculrured with MSCs. When MSCs was pretreated with Astragaloside, and thencocultured with lymphocyte, which down-regulation IL-10 mRNA levels compared with untreated MSCs, but up-regulation by treated with Quercetin, Curcumine and Q-C.The ELISA results revealed that Astragaloside could decrease the TGF-pi secretion of rMSCs and the IL-10 secretion of co-culture system, and Quercetin, Curcumine and Q-C could increase the level of IL-10 secreted by co-culture system and TGF-pi of rMSCs.The current studies demonstrates that:1 .Rat MSCs can suppress proliferation of T-lymphocyte stimulated by PHA, PMA, ConA in a dose-dependent manner and the suppressive effect is not MHC restricted.2.Rat MSCs suppressive effect is positive correlation with the level of TGF-pl in rMSCs culture supernatant.3.There is positive correlation with the suppressive effect of rat MSCs and the level of IL-10 in co-culture system supernatant.4.Astragaloside could weaken the suppressive effect of MSCs by up-regulated the levels in TGF-pi excreted by MSCs. IL-10 secreted by co-culture system was decreased obviously after MSCs treated with Astragaloside. Quercetin, Curcumine and Q-C could increase the secretion of TGF-pi by MSCs, and IL-10 by co-culture system.
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