The Impacts Of HUC-MSCs On The Differentiation And Immune Function Of B Lymphocytes In Refractory Primary Immune Thrombocytopenia Patients Who Are Involved In Both Qi And Yin Deficiency Type

Objective:Explore the impacts of hUC-MSCs on the differentiation and immune function of B lymphocytes in refractory idiopathic thrombocytopenic purpura patients who are involved in both qi and yin deficiency type.Methods:The rITP patients are armed to the qi and yin deficiency group,non-qi a nd yin group,according to the recruit and group standards,heathy donors are th e cintrol group;Enzyme digestion was the method to extract heathy fetusumbili cal cord mesenchymal stem cells;while density gradient centrifugation approach ed to extracte peripheral blood mononuclear cells. The growth curve of hUC-MSCs was studied by Methyl thiazolyl tetrazolium (MTT) method.Purrty of h UC-MSCs was tested by labels of CD44 and CD31 while the peripheral bloo d mononuclear cells was tested by label of CD19;The concentration of Pokew eed(PWM) and time that were needed to stimulated B lymphocyte into plasm ocyte to secrete IgG and IgM,the ration between hUC-MSCs and B lymphocyt e,the contents of IgG and IgM in cell culture supernatant of three groups wer e analyzed through enzyme-linked immuno sorbent assay (ELISA);Detecting p ositive cells that marked by CD138 with the En Vision immunohistochemistry t o detecte epression of Blimp-1.Results:(1) Adherent cells get by health fetus umbilical cord with enzyme digestion were mainly mesenchymal stem cells and the cells purity was 99.8%.The ratio n of B lymphocytes of peripheral blood mononuclear cells which were extracte d by density gradient centrifugation were 6.9%.(2) PWM stimulated human peripheral blood B develop into plasma cells in a PWM concentration-dependent manner.The antybody IgG and IgM secretion o f all groups increased significantly with the PWM concentration was 30ug/mL when the cells were stimulated 4 days(P=0.033,P=0.010).At the same time, t he were higher than non-qi and yin deficiency type(P=0.013,P=0.008) and hea thy donors type(P=0.001,P=0.004),the content of IgG and IgM were so signific ant between non-qi and yin deficiency and heathy donors type(P=0.077,P=0.58 7).(3)The hUC-MSCs and peripheral blood mononuclear cells co-cultured inhibit ed B cell differentiation and development of plasma cells,thus inhibited IgG an d IgM secretion.① Antibody IgM of qi-yin deficiency type were higher than h eathy donors’while there were no hUC-MSCs(P=0.004),while non-qi and yin d eficiency type’s IgM secretion were no significant with heathy donors’(P=0.58 7);② At the hUC-MSCs/mononuclear cells ratio of 2:10, IgM of qi-yin deficien cy type were declined to heathy donors’when there were no hUC-MSCs(P=0.0 01),non-qi and yin deficiency type’s IgM secretion were no significant with hea thy donors’(P=0.073),the content of IgM were undifferentiated between heathy donors with the other two groups(P=0.055,P=0.51);③ Without hUC-MSCs IgG o fqi-yin deficiency type were higher than heathy donors’(P=0.001),meanwhile the re were no obvious difference of IgG between non-qi and yin dificiency type and heathy donors(P=0.077);④ When hUC-MSCs and mononuclear cells at a 2: 10 ratio,IgG contents of qi-yin deficiency type were declined sharply than non-hUC-MSCs’(P=0.000)and not much difference with heathy donors’(P=0.848), at the same time, IgG secretion of non-qi and yin deficiency type were not obvi ous compared with non hUC-MSCs’(P=0.446),but were higher than heathy dono rs’(P=0.007).(4) The hUC-MSCs inhibited the expression of Blimp-1 protein. The positive rate of CD138 labeled cells of non-qi and yin dficiency type and qi and yin d ficiency type rITP patients apparent declined obviously at the ratio 2:10 of hU C-MSCs/mononuclear cells(P=0.000,P=0.026),and compared to heathy donors,the positive rate of qi-yin deficiency type were no significant(P=0.076),non-qi and yin deficiency type were higher(P=0.029).Conclusion:The hUC-MSCs co-cultured with peripheral blood mononuclear cells at so me ratio would decline IgM secretion of both qi-yin deficiency type and non-qi and yin deficiency,decline IgG secretion of qi-yin deficiency type,decline po sitive cells rate of CD138 marked cells of qi-yin deficiency type and non-qi a nd yin deficiency type.We supposed that hUC-MSCs worked on rITP patients especilly qi-yin deficiency type patinents,howere it still needs to explore the cl inical treatment of rITP.