To Explore The Mechanism About Tonic Drugs In The Treatment Of Psoriasis From The Perspective Of MSCs Regulation In The HaCat Inflammation Model

Research Background:Psoriasis is a common erythema and scaly disease in dermatology,which is refractory,chronic and recurrent.It has become a representative common disease in dermatology.Its main pathological change is the excessive proliferation of keratinocytes,and there are many Inflammatory cell infiltration.At present,the etiology and pathogenesis of psoriasis are not yet clear,but scholars generally support the viewpoint of immune dysfunction,among which KC plays an important role in skin immunity.Stimulated by inflammatory factors and external factors,KC can produce a large number of cytokines,including TNF-?,IL-6,IL-1?,CCL20,and antibacterial peptides.Therefore,this study uses different human recombinant inflammatory factor proteins and Bacterial lipopolysaccharide(LPS)intervenes in HaCat cells,taking HaCat cells as the research object,on the one hand,exploring the biological characteristics and cytokine secretion characteristics of the HaCat inflammation model,and providing a model basis for the in vitro research of psoriasis to further explore silver Pathogenesis of psoriasis.On the other hand,the advantages and limitations of each HaCat inflammation model are analyzed to provide a reference for model selection and establishment of new models.MSCs are a class of stem cells that have the potential for self-renewal and differentiation into osteogenic,adipogenic,and chondrogenic cells.They can exert a strong immune-regulating effect by releasing a variety of soluble immune-regulatory factors,regardless of the autoimmune or allogeneic immunity.Cells,whether they are innate immune cells or acquired immune cells,can play an immunoregulatory role.MSCs therapy is a promising tool for treating many diseases.In this study,AMSCs and inflammatory HaCat cell models were used as research objects.RT-qPCR and cell proliferation experiments were used to investigate the effects of AMSCs and supernatants on the secretion of inflammatory cytokines by inflammatory HaCat cell models.Further exploration of MSCs on silver The regulatory mechanism of KC immune disorder in psoriasis provides a theoretical basis for the clinical use of MSCs in the treatment of psoriasis.Previous studies have found that AMSCs in psoriasis patients are abnormal compared to healthy people.In this study,the effective components of tonic drugs were used to interfere with AMSCs in patients.The main effect of ASIV was on the regulation of inflammatory factor receptors TLR3 and TLR4,and 1FFN can affect inflammatory factors and receptors TGF-?,PD-L1 plays a regulatory role,suggesting that tonic Chinese medicines can play a role in treating psoriasis by regulating the immune function of psoriasis AMSCs.Therefore,in this study,the instructor's commonly used supplementary Chinese medicine extracts for clinical treatment of psoriasis were selected to conduct in vitro mechanism research to explore its repairing effect on the immune regulating function of psoriasis AMSCs,and to provide theoretical basis for clinical application of supplementary Chinese medicine for psoriasis.Purpose(1)To explore the biological characteristics and immune cytokine secretion characteristics of inflammatory HaCat cell models,and to analyze the advantages and limitations of different HaCat inflammatory model stimulating factors,different action concentrations,and different action times.Choose to provide reference data and theoretical basis.(2)To explore the effects of AMSCs and supernatants from healthy people on inflammatory cytokines secreted by the HaCat inflammation model of psoriasis,and to explore the mechanism of AMSCs in treating KC immune disorders of psoriasis.(3)To explore the role of supplemental Chinese medicine monomers ASIV and 1FFN in restoring immune balance to psoriatic AMSCs,and to exert the function of immune regulation of normal AMSCsMethod:(1)The qRT-PCR method was used to detect the secretion characteristics of immune cytokines in HaCat inflammation model under the action time of 24h and 48h using 4 different intervention measures,2-4 action concentrations of each intervention measure;Effects of different interventions on the proliferation of HaCat inflammation model.(2)Isolate and culture healthy AMSCs in vitro until the second generation.Use co-culture of Transwell chamber to co-cultivate AMSCs and HaCat inflammation model.Use qRT-PCR to detect healthy AMSCs and healthy AMSCs supernatant.The influence of immune cytokine secretion characteristics in inflammation model;qRT-PCR was used to detect the characteristics of immune cytokines secreted by healthy AMSCs culture medium,healthy AMSCs treated group and control untreated group HaCat.(3)Isolate and culture AMSCs of patients in vitro and use them until the second generation.Use effective supplements ASIV and 1FFN to intervene psoriatic AMSCs and healthy AMSCs to screen drug intervention concentration.Use qRT-PCR to detect silver after drug intervention.Effect of immune cytokine secretion characteristics of psoriasis AMSCs;qRT-PCR was used to detect the effect of immune cytokine secretion characteristics of healthy AMSCs after drug intervention;CCK-8 test was used to detect the proliferation of psoriatic AMSCs by different drugs mentioned above Impact.Result:(1)When TNF-? stimulates HaCat cells for 24h,the expression of the inflammation model is as follows:TNF-?,IL-1?,IL-1?,IL-8,IL-23 and CCL20 mRNA levels are increased,with significant differences(P<0.05).When TNF-?stimulated HaCat cells for 48 hours,the expression of the inflammation model was as follows:IL-1?,IL-1?,IL-17 and CCL20 mRNA levels were increased,with significant differences(P<0.05).In the case,TNF-?,IL-8 and IL-23 are not consistent with 24h.Considering that the response time of these three is faster,it reaches a peak at 24h,and its peak decreases after 48h.In addition,it may be related to the time of intervention treatment.To ensure the amount of cells at the time of sample collection,the 24h treatment group intervened 24h before the cells were full,the 48h treatment group intervened 48h before the cells were full,and finally selected 24h as the processing time.At the same time,the concentration of human recombinant protein TNF-? was screened at 4 concentrations:10ng/ml,50ng/ml,100ng/ml,and 150ng/ml,and the optimal concentration was finally selected as 100ng/ml.(2)Transwell cell co-culture of AMSCs and HaCat inflammation model,comparing the effects of healthy AMSCs and healthy AMSCs supernatant on inflammatory cytokines secreted by HaCat inflammation model of psoriasis.It was found that in the healthy AMSCs co-culture group,HaCat inflammation model The levels of TNF-?,IL-1?,IL-1?,IL-6,and IL-8 mRNA were reduced,and there were significant differences(P<0.05),thereby regulating the inflammatory response of HaCat cells,suggesting that AMSCs are used to treat local skin lesions.Treatment can serve as a route of administration for biological cell therapy for psoriasis.At the same time,the detection of healthy AMSCs and AMSCs culture medium has a low effect on the level of healthy HaCat inflammatory factors,suggesting that the AMSCs in the healthy AMSCs intervention group can play an immunoregulatory capacity and can exclude the effect of AMSCs culture medium;the AMSCs in the healthy AMSCs supernatant group Exogenous factors are the main reason for aggravating the inflammatory response,and the effect of AMSCs medium is weak.(3)There was a significant difference in the up-regulation of TLR3 and TLR4 when ASIV(60ul/ml)intervention in patients with high concentration group(P<0.05);1FFN(56ul/ml,112ul/ml)in patients with high concentration group interfered with AMSCs There is a significant difference in the up-regulation of TGF-?and PD-L1(P<0.05);ASIV and 1FFN have no effect on the proliferative capacity of AMSCs in patients with psoriasis and do not have cytotoxicity;when healthy AMSCs are interfered with with different concentrations of ASIV and 1FFN There were no significant changes in TLR3,TLR4,TGF-?,and PD-L1(P>0.05).In conclusion:(1)HaCat cells were treated with TNF-? for 24h to establish the optimal method for HaCat inflammation model.The optimal concentration was 100ng/ml,and the most appropriate intervention time was 24h.It had no effect on the proliferation of HaCat cells(2)Healthy AMSCs can reduce the levels of inflammatory factors in the HaCat inflammation model,thereby regulating the inflammatory response of HaCat cells,suggesting that local skin lesion treatment with AMSCs can be used as a biological cell therapy for psoriasis;healthy AMSCs are healthy for health HaCat has low ability to regulate inflammatory factors and has a low immune effect on healthy HaCat.The supernatant of healthy AMSCs cannot reduce the level of inflammatory factors in the HaCat inflammation model.It has no ability to regulate the immune response of HaCat cells,but will aggravate the inflammatory factors.Expression to indicate that exocrine factors of healthy AMSCs have no effect on psoriasis;AMSCs medium has a lower effect on the level of healthy HaCat inflammatory factors,suggesting that AMSCs in the healthy AMSCs intervention group can play an immunoregulatory capacity.(3)Compared with healthy people,immune-related cytokines and immune-receptors are under-expressed in AMSCs of patients with psoriasis,and their immune function is suppressed;For AMSCs in patients with psoriasis,ASIV and 1FFN can regulate the expression of its immune-related factors and receptors,thereby restoring its immune imbalance;in healthy people AMSCs,ASIV and 1FFN have no expression of their immune-related factors and receptors Effect,does not affect its autoimmune balance;compared with healthy people's AMSCs proliferation ability,AMSCs proliferation ability in patients with psoriasis is weakened;ASIV and 1FFN can improve the cell proliferation ability of psoriasis AMSCs.