Biological Characteristics And Therapeutic Application Of ADSCs In MRL/lpr Mice

Objective:Systemic lupus erythematosus（SLE）is an autoimmune disease with unclear etiology and pathogenesis,involving multiple systems and organs,which seriously affects the quality of life and life span of patients.The clinical manifestations are skin and mucosal injury,lupus nephritis and other symptoms.Excessive activation of the immune system,increased secretion of antibodies by B cells,decreased number of Foxp3⁺Treg（Forkhea dbox p3 regulatory T cell）and Th17/Treg cell immune imbalance are the key links in the pathogenesis of SLE.Mesenchymal stem cells（MSCs）are a kind of adult stem cells with strong immunomodulatory ability,multi-directional differentiation ability and low immunogenicity.MSCs therapy has been widely used in the treatment of various human diseases.However,it has been reported that SLE is a stem cell disease^[1,2].Due to genetic factors and over-activated immune system,MSCs in SLE patients will have defects such as aging and decreased immune regulation ability.Therefore,allogeneic MSCs are mostly used in clinical experimental studies for cell therapy in SLE patients.The resulting graft rejection may lead to further activation of the body’s immune system and bring more serious damage to patients.Therefore,we want to repair the defects of MSCs in SLE patients by means of molecular biology and apply them to the treatment of SLE.This study will investigate whether adipose mesenchymal stem cells（L-ADSCs）of systemic lupus erythematosus mice（MRL/lpr mice with Fas gene mutation）have abnormal proliferation,senescence,apoptosis,immune regulation and other aspects during in vitro culture.And whether the adipose mesenchymal stem cells（L-ADSCs）of MRL/lpr mice are different from those of C57BL/6J healthy control mice in the treatment of MRL/lpr mice,which lays the research foundation for the subsequent repair of L-ADSCs defects and the application of SLE mice autologous MSCs therapy.Methods:1.Cell identification.L-ADSCs and H-ADSCs were isolated and cultured in vitro.Cell morphology was observed by contrast microscope,and cell differentiation ability was detected by osteogenic,lipogenic and chondrogenic differentiation medium.The expressions of CD29,CD34,CD44,CD45 and CD105 on the cell surface were detected by flow cytometry.2.Cell proliferation capacity.The proliferative ability of H-ADSCs and L-ADSCs cultured in vitro for 0-7 days was detected by CCK-8.3.Cellular senescence.Contrast microscopy was used to observe the morphological differences of ADSCs of the same generation.Western Blotting was used to detect the expressions of p-p65,p16^INK4A,c-Maf and other cycle-related proteins.RT-PCR was used to detect the expressions of age-related genes c-Maf,p15^INK4Band p16^INK4Am RNA.4.Cell apoptosis.The expressions of apoptosis-related genes Bcl-2 and caspase3 of H-ADSCs and L-ADSCs were detected by RT-PCR and Western Blotting.Annexin V-FITC/PI staining was used to analyze the apoptosis-related differences of H-ADSCs and L-ADSCs.5.Migration ability.The migration and healing ability of H-ADSCs and L-ADSCs were investigated by cell scratch test.6.Immune regulatory capacity.The expression of TGF-β,IL-6,p-STAT1,IDO1 and other immunoregulatory related genes were detected by RT-PCR and Western Blotting.7.Animal testing.Fifteen 22-week-old female MRL/lpr lupus mice were divided into control group,H-ADSCs treatment group and L-ADSCs treatment group according to the principle of similar initial mean of urinary protein content,with 5 mice in each group.H-ADSCs or L-ADSCs（1×10⁶/mouse）were transplanted by caudal vein for the first time at 22 weeks of age,and the control group was injected with equal volume of PBS.The second cell transplantation was performed 2 weeks after the first mouse transplantation,and the mice were sacrificed 5 weeks later.The weight changes of mice were recorded weekly,and 24h urine was collected once every two weeks.The protein content in 24h urine was determined by CBB method.The proportion of Th17 and Treg cells in spleen immune cells of mice was detected by flow cytometry,the m RNA expression of spleen immune cytokines was detected by RT-PCR,the pathological changes of kidney were detected by HE and PAS staining,and the changes of urinary protein were detected by CBB method before treatment,2 weeks and 5 weeks after treatment.Results:1.The morphology of ADSCs cultured in vitro showed long spindle shape.It can be induced to differentiate into osteoblasts,adipocytes and chondroblasts.Flow cytometry showed that CD29,CD44 and CD105 were highly expressed,while CD34 and CD45 were low expressed.2.Compared with H-ADSCs,L-ADSCs had lower proliferation capacity in vitro,weak stereocularity,flat morphology,unclear boundary,and more intracellular particle secretions.3.RT-PCR results showed that the expression of L-ADSCs aging gene c-Maf,p15^INK4B,p16^INK4Am RNA was high,the apoptosis-related gene caspase3 was increased,the expression of proinflammatory gene Bcl-2 was decreased,the expression of pro-inflammatory factor IL-6 was lower,and the expression of anti-inflammatory factor TGF-βwas higher.4.Western Blot results showed that the expression of L-ADSCs aging gene p15^INK4B,p16^INK4Am RNA was high,and no significant differences in c-Maf.5.Flow cytometry showed that the proportion of apoptosis and necrosis cells of L-ADSCs was significantly higher than that of H-ADSCs,and the proportion of normal cells was lower than that of H-ADSCs.6.In animal experiments,compared with the control group,the body weight of MRL/lpr mice in H-ADSCs treatment group and L-ADSCs treatment group was significantly increased,and there was no significant difference between the two treatment groups.Spleen inflammatory factors TNF-α,IL-6,IFN-γ,IL-17 were significantly lower than the control group,and anti-inflammatory factor TGF-βwas significantly higher than the control group,but there was no significant difference between the two treatment groups.The levels of IL-6 and Il-17 in serum were lower than those in the control group,and there was no significant difference between the two groups.Flow analysis results showed that,compared with the control group,the proportion imbalance of Th17/Treg in spleen immune cells of MRL/lpr mice decreased after transplantation of H-ADSCs or L-ADSCs,the proportion of Th17 cells decreased and the proportion of Treg cells increased.The proportion of Th17 cells decreased significantly in the H-ADSCs treatment group,and the proportion of Treg increased significantly in the L-ADSCs treatment group,and the difference was statistically significant.HE and PAS staining results of pathological sections showed that,compared with the control group,the infiltration degree of renal interstitial inflammation cells in MRL/lpr mice in H-ADSCs treatment group and L-ADSCs treatment group was significantly reduced,the edema of renal tubular epithelial cells and the dilatation of renal tubular lumen were alleviated,and the number of cells in glomerular capillaries was not significantly different.There is hyperplasia of the mesangial glomerulus,but there was no significant difference between the two treatment groups.In addition,the L-ADSCs treatment group had the least skin injury symptoms.Conclusions:1.Adipose mesenchymal stem cells（L-ADSCs）derived from MRL/lpr mice have poor proliferation ability,earlier aging phenotype,high expression of aging and apoptosis-related genes and proteins,reduced migration ability,and strong expression of immunosuppression-related factors.2.In the treatment of MRL/lpr lupus mice,H-ADSCs and L-ADSCs showed obvious therapeutic effects compared with the control group,but there was no significant difference in efficacy between the H-ADSCs and L-ADSCs treatment groups.