| Objective:(1)To evaluate the reliability of the rat model of mesangial proliferative glomerulonephritis(MsPGN)prepared by anti-Thy1.1 antibody.(2)To study the evolution of Th17,Treg and inflammatory factors in peripheral blood of MsPGN model rats.(3)To initially explore the feasibility of peripheral blood mesenchymal stem cells(PB-MSCs)transplantation in the treatment of MsPGN.Method:(1)Isolation,culture and identification of rat PB-MSCs:SD rats were intraperitoneally administered granulocyte colony-stimulating factor(G-CSF,100μg/kg,once a day),After 5 days of continuous treatment,mononuclear cells in peripheral blood were collected by density gradient centrifugation of rat lymphocyte extract(Ficoll);CD29,CD90,CD44,CD45,CD11b and CD79a were identified by flow cytometry(FCM)on PB-MSCs cultured in P2 generation.(2)Preparation and reliability evaluation of the MsPGN rat model:36 male SD rats(6-8w;180-220 g),a single tail vein injection of 1.29 mg/kg anti-rat CD90(Thy1.1)induced MsPGN model at the 3rd,7th and 14th day,6 rats(a total of 18 rats)were randomly selected at each time point,and the renal tissue was used for paraffin sections.HE and PAS staining was performed,and the proliferation of mesangial cells was marked byα-SMA immunohistochemistry;24 h urine of rats at each time point was collected to measure 24 h urine protein.Urea and creatinine were detected in peripheral blood to evaluate the reliability of the method.(3)Characterization of the evolution of Th17,Treg and inflammatory factors in the peripheral blood of MsPGN rat model:the dynamic changes of Th17(CD4~+IL-17A),Treg(CD4~+CD25~+Foxp3~+)cells in the peripheral blood of the rat model at the corresponding time points were detected by flow cytometry(FCM);The dynamic changes of IL-17A,IL-6,IL-10,TGF-β1 inflammatory factors in peripheral blood were detected by ELISA;The expression of IL-17A,IL-6,IL-10,TGF-β1,RORγT and Foxp3 were detected by RT-qPCR;The expression of IL-17A,Foxp3 in the cortex of the kidney was detected by immunohistochemistry(IHC).(4)Feasibility exploration of PB-MSCs transplantation for the treatment of MsPGN:Eighteen MsPGN rat models were prepared by the above method.After 24 h,PB-MSCs(P3 generation,2×10~6cells per mouse)were transplanted via the tail vein.Six rats were randomly selected for sampling at the 2nd,6th and 13th day(equivalent to day 3,7 after modeling),and the same experimental method was used to detect and analyze the same indicators in kidney tissue and peripheral blood of rats in the stem cell transplantation groupResult:(1)The isolated and enriched PB-MSCs showed uniform morphology,fusiform adherents growth,high expression of CD90,CD29,CD44,but no expression of CD45,CD79a,CD11b.(2)Pathological features of renal tissue:the result of HE and PAS staining showed that mesangial cell proliferation and mesangial matrix increased in mesangial area of renal cortex of MsPGN rat model compared with normal group,and reached the peak at the7th day.The proliferation of mesangial cells and mesangial matrix in the PB-MSCs transplanted group was not significantly changed compared with the model group.α-SMA immunohistochemical of mesangial cells confirmed the proliferation of mesangial cells,and there was no significant changed between PB-MSCs transplantation group and model group.(3)The result of 24 h urinary protein quantification,serum creatinine and urea showed that compared with the normal group,24 h urinary protein and blood urea were higher in MsPGN rat model at three time points(P<0.05),peaked at the 3rd day,and there was no significant difference in the change of serum creatinine(P>0.05);The result of 24 h urine protein quantification,blood urea and creatinine in PB-MSCs transplantation group were not significantly changed from those in model group(P>0.05).(4)The FCM result showed that compared with the normal group,Th17 cells in MsPGN rat model were increased at all three time points and reached the peak at the 3rd day,while Treg cells were lower than normal at all three time points,but the overall change showed an increasing trend(P<0.05);The result of Th17 and Treg cells in PB-MSCs transplantation group were not significantly changed from those in the model group(P>0.05).(5)The ELISA result showed that the expression level of IL-17A,IL-6,TGF-β1 in MsPGN model group was higher compared with normal group(P<0.05),IL-17A peaked at the 3rd day,IL-6 peaked at the 7th day,IL-10 and TGF-β1 gradually increased(P<0.05);There was no significant changed in IL-17A,IL-6,IL-10,TGF-β1 between PB-MSCs transplantation group and model group(P>0.05).(6)The result of RT-qPCR showed that the m RNA expressions of IL-17A,IL-6,IL-10TGF-β1 and RORγT at three time points in MsPGN model group were higher than those in normal group(P<0.05),IL-17A and RORγT peaked at the 3rd day,IL-6 peaked at the7th day,IL-10 and TGF-β1 increased gradually(P<0.05),Foxp3 decreased at the 3rd day and then increased,and reached the peak at the 7th day(P<0.05);The m RNA expression of IL-17A,IL-6,IL-10,TGF-β1,RORγT and Foxp3 in the transplantation group was not significantly changed in the model group(P>0.05).(7)The result of IHC showed that compared with the normal group,IL-17A significantly increased in the mesangial region of the renal cortex in the MsPGN model group,and reached the peak at the 3rd day(P<0.05),Foxp3 decreased at the 3rd day and then increased,finally reached the peak at the 7th day;The expression of IL-17A and Foxp3in PB-MSCs transplantation group showed no significant changed compared with the model group(P>0.05).Conclusion:(1)In the rat MsPGN model prepared by anti-Thy1.1 antibody,Th17 and its related factors IL-17A,IL-6 and RORγT were significantly increased at the beginning of the disease,and with the development of the disease,Treg and its related factors IL-10,TGF-βand Foxp3 were gradually increased,and these changes may be an important pathogenic mechanism of MsPGN immune disorders.(2)The therapeutic effect of PB-MSCs transplanted via tail vein in the MsPGN model rats was not significant. |
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