Efficient Preparation Of Human Umbilical Cord Mesenchymal Stem Cells And Clinical Demonstration Of Systemic Lupus Erythematosus

Objectives:1.To establish a method for the efficient preparation of human umbilical cord mesenchymal stem cells(hUCMSC),to isolate,amplify and identify hUCMSC,and to provide clinical materials.2.To establish a clinical treatment plan for hUCMSC for the treatment of systemic lupus erythematosus(SLE)and to implement clinical treatment of hUCMSC in 10 patients with SLE,providing exemplary technical methods and theoretical basis for the treatment of SLE with hUCMSC.Methods:1.Preparation of hUCMSC The healthy umbilical cords of healthy newborns were taken from the term and decomposed into 1 mm3 tissue blocks.hUCMSC were isolated and amplified by 3 times of adherent method.Cell growth characteristics were observed by inverted phase contrast microscopy.Flow cytometry was used to detect hUCMSC cell surface antigen CD73.The positive expression rate of CD90,CD 105,CD34,and CD45 and the differentiation potential of hUCMSCs by adipogenesis,osteogenic and chondrogenic orientation were analyzed.2.The hUCMSC treatment With the approval of the Provincial Health Planning Commission,20 SLE patients from the Department of Nephrology and Rheumatology,the People's Liberation Army's Kunming General Hospital,who met the inclusion criteria were selected as subjects to participate in the experiment.Informed consent was signed and randomized into a treatment group and a control group,with 10 in each group.name.The control group collected blood on dl as a pre-treatment sample and was intravenously infused with methylprednisolone 15 mg/(kg d).The d4 was changed to oral prednisone 0.5 to 1 mg/(kg d).The 2-week interval was reduced by 2 weeks.The amount of 10%,while intravenous infusion of cyclophosphamide(CTX)1g/time,once every 4 weeks,5 times;the treatment group began intravenous infusion of hUCMSC1x106/Kg,1 time/week,3 times in a row on d6.3.The evaluation of curative effect of hUCMSC At the 2nd,4th,8th,12th,and 24th weeks after the hUCMSC treatment was completed,the clinical symptoms,lupus activity,hematuria,biochemical parameters,and immunological parameters were observed and analyzed.Protein chips were used.The serum proteoglycans were analyzed before and after hUCMSC treatment,and further significant ELISA test was performed to identify the characteristic cytokines with significant differences.Statistical analysis of differences between groups and groups.Results:1.Preparation of hUCMSC After hUCMSC were cultured directly with tissue blocks for 7 days,cells were seen to crawl out of the tissue blocks.Three adherent cultured cells were fibroblast-like and had typical vortex growth characteristics;expression of CD73,CD90,and CD105 in third-generation hUCMSC.The rates were 98.7%,82.6%,and 80.1%,and the expression rates of CD34 and CD45 were 0.17%and 0.92%,respectively.After 2 weeks of adipogenic differentiation,oil red O staining was positive,and 3 weeks after osteogenic differentiation,alizarin red staining.Positive;4 weeks after induction of chondrogenesis,Axin blue staining was positive.2.hUCMSC efficacy(1)Clinical symptoms,signs,and SLEDAI scores:After 2 weeks of treatment,fever disappeared and rash subsided in all cases.After 4 weeks of treatment,the rates of fatigue and fatigue in the treatment group and control group were 80.0%(8 cases)and 70.0 respectively.In%(7 cases),the significant reduction rate of arthralgia was 60.0%(6 cases)and 70.0%(7 cases)respectively,and the complete or partial remission rate was 80%(8 cases)and 70%(7 cases)respectively.No significant difference(P>0.05).The SLEDAI score of the treatment group was significantly lower than before treatment(P<0.05).(2)Changes in laboratory parameters:1 comparison between groups:Serum C3 levels in the treatment group were significantly higher than those in the control group(P<0.05).Intergroup WBC,RBC,PLT,Hb,U-PRO,BUN,Cr,TP,ALB,IgG There was no significant difference between IgA,IgM and C4(P>0.05).Within 2 groups:RBC,Hb,TP and ALB levels in peripheral blood increased,Cr content decreased(P<0.05),U-PRO,GFR,IgA,IgM had no difference(P>0.05);IgG treatment in treatment group After the increase(P<0.05).3 Onset time comparison:ALB and Hb levels in the peripheral blood of the treatment group increased significantly at 2 weeks and 4 weeks respectively,Cr decreased at 2 weeks(P<0.05),and ALB and Hb in the control group were 4 weeks after treatment.After 8 weeks,the level of Cr increased significantly,and Cr decreased after 8 weeks(P<0.05).The levels of TP and RBC in the peripheral blood of both groups increased significantly at 12 and 24 weeks after treatment(P<0.01 and P<0.05).4Immunological indicators:The positive rate of CD3+CD4+ in peripheral blood decreased after treatment,and the NK positive rate increased after treatment.Compared with before treatment,the difference was significant from the 4th and 24th weeks(P<0.05);anti-ANA antibody and anti-antibody The dsDNA antibody titer decreased significantly after treatment(P<0.05).(3)Serum cytokines analysis in the treatment group:protein chip analysis showed that:ICAM-1(CD54),TNF-RII,TIMP-2,TIMP-1,IGFBP-3,IGFBP-6,SCF-R(CD117),BMP-5 were High expression status;BLC(CXCL13),IL-4,BMP-4,EGF,BDNF,?-NGF decreased after treatment,while IL-12p70,AR increased.Quantitative detection of BLC(CXCL13),BDNF,and BMP-4 was performed.The results were consistent with the protein chip,BLC(CXCL13)decreased significantly(P<0.05),BDNF and BMP-4 decreased,but there was no significant difference(P>0.05).Conclusions:1.The use of multiple tissue block repeated direct adherent culture methods to obtain morphology,growth characteristics,cell surface expression markers and differentiation induction functions in accordance with the hUCMSC standard.2.hUCMSC treatment can significantly improve the clinical symptoms and signs of SLE patients,reduce SLEDAI score and autoantibody titers,peripheral blood RBC,Hb,TP,ALB,IgG,complement C3,CD3+CD4+,NK positivity,CD4+/CD8+ The efficacy of the ratio was obvious.Among them,IgG and complement C3,ALB and Hb had obvious advantages in terms of efficacy and onset time.3.Treatment with hUCMSC reduced the levels of BLC(CXCL13),IL-4,BDNF,BMP-4,EGF,and p-NGF in serum and increased IL-12p70 and AR.The results shown that hUCMSC treatment can inhibit the autoimmune response of SLE patients by regulating cytokine expression.