| Objective:To study the therapeutic effect and the underlying mechanism of treatment of MRL/lpr mice with fetal and neonatal murine peripheral blood (FNPB) obtained from C57BL/6pregnant mice plus reduced-dose radiation (fractioned irradiation)as conditioning.Methods:1. FNPB cells were collected form C57BL/6pregnant mice, with MNC isolated by density gradient centrifugation and the number of which was counted. Then the MRL/lpr mice at the age of20weeks were infused with the MNC.2. The MRL/lpr mice in all groups were followed for GVHD manifestation including body weight, body position, locomotor activity and hair changes as well as stools and urination (defecation).3. We analyzed the mortality, body weight of MRL/lpr mice (at20,24,28and32weeks of age) and the spleen indexes of MRL/lpr mice (at32weeks of age), determined serum concentrations of anti-ds-DNA antibodies and ANA of MRL/lpr mice (at20,24and32weeks of age) and serum IL-17and TGF-β of MRL/lpr mice (at32weeks of age) by ELISA, determined serum creatinine concentration of MRL/lpr mice (at20,24and32weeks of age) by fully automatic biochemistry analyzer, detected urine protein of MRL/lpr mice (at20,24,28and32weeks of age) using coomassie brilliant blue, analyzed Th1,Th2, Th17, Treg and CD8+T cells of MRL/lpr mice (at32weeks of age) by flow cytometry. MRL/lpr mice at32weeks of age were put to death for pathological section of renal tissues and H.E staining followed by microscopic analysis. The microRNA-145expression in renal vascular smooth muscle cells was detected by in situ hybridization.Results:1. The cell viability was greater than97%if MNC concentration in FNPB was9.5×106/ml.2. In the transplantation group, all the mice had lively survival until being putting to death at32weeks of age then which presented with no GVHD manifestation such as alopecia, diarrhea, bleeding, wasting or other typical symptoms typical of GVHD. However, in the radiation group, at24days of radiation, the hair at head, neck and back of the mice became shallow and turned gray then turned white by55days.3. There were no statistically significant differences between the control group and the radiation group in survival rate (P>0.05); however, the survival rate in the transplantation group increased significantly as compared with control group and radiation group (all P<0.05). 4.(1) At32weeks of age, both the radiation group and the transplantation group showed significant reduction in spleen index as compared with the control group; however, differences between the radiation group and the transplantation group were not significant (P>0.05).(2) At the age of20and24weeks, no statistically significant differences were noted across the three groups in mean weight, serum anti-ds-DNA antibody, ANA and creatinine (all P>0.05). At the age of28weeks, no statistically significant differences were noted across the three groups in mean weight (all P>0.05). At the age of32weeks, the mean weight in both the radiation group and the transplantation group were all significantly higher than in the control group (all P<0.05), however, the mean weight was slightly higher in the transplantation group than in the radiation group (P>0.05); at the age of32weeks, serum anti-ds-DNA antibody and ANA in both the radiation group and the transplantation group were all significantly lower than in the control group (all P<0.05), yet no statistically significant differences were noted between the radiation group and the transplantation group in terms of serum anti-ds-DNA antibody and ANA (all P>0.05); at the age of32weeks, serum creatinine in the transplantation group was significantly lower than in the control group or radiation group (all P<0.05) and the serum creatinine in the radiation group was slightly lower than in the control group (P>0.05).(3) At the age of20weeks, no statistically significant differences were noted across the three groups in24-hour urine protein level (all P>0.05). At the age of24and28weeks, the24-hour urine protein levels in the radiation group and the transplantation group were significantly lower than in the control group (all P<0.05). At the age of32weeks, no statistically significant differences were noted between the radiation group and the control group in24-hour urine protein level (P>0.05); the24-hour urine protein level in the transplantation group was significantly lower than in the control group or the radiation group (all P <0.05).(4) The time for white cells to increase from the lowest level to1.0×109/L was significantly shortened in the transplantation group as compared with the radiation group (P<0.05); moreover, the lowest value of platelets in the transplantation group was higher than in the radiation group (P<0.05); significantly less time was needed for platelets to decrease to the lowest level and or to increase from lowest to100×109/L for the radiation group than for the radiation group (all P<0.05) and significantly less time was needed for hemoglobin level to increase from the lowest to100g/L in the transplantation group than in the radiation group (P<0.05).(5) Compared with the control group, the radiation group and the transplantation group at the age of32weeks showed significant reduction in the percentage of CD4+cells, significant increase in the percentage of CD8+T cells and significant reduction in the ratio of CD4/CD8(all P<0.05); moreover, the percentage of Th1cells increased significantly, the percentage of Th2cells decreased significantly and the ratio of Th1/Th2increased significantly in both the radiation group and the transplantation group as compared with the control group (all P<0.05). However, in the transplantation group as compared with the radiation group, Thl cells and CD8+T cells increased slightly while Th2cells and CD4+T cells decreased slightly in number (all P>0.05).(6) Compared with the control group, both the radiation group and the transplantation group at the age of32weeks showed significant increase in the percentage of Treg cells, significant reduction in the percentage of Th17cells and significant increase in the ratio of Treg/Thl7(all P<0.05). However, no significant differences were noted between the radiation group and the transplantation group with regard to Treg cells, Th17cells and the ratio of Treg/Th17(all P>0.05).(7) Compared with the control group, the radiation group and the transplantation group at the age of32weeks showed significant increase in serum TGF-β concentration but significant decrease in serum IL-17concentration (all P<0.05) yet the differences between the radiation group and the transplantation group were all not statistically significant (P>0.05).(8) In MRL/lpr mice, there was expression of microRNA-145in renal vascular smooth muscle cells, yet no marked expression of microRNA-145was noted in either intima or tunica adventitia of renal vessels.(9) Compared with the control group and the radiation group, the expression of microRNA-145 in renal vascular smooth muscle cells of the transplantation group showed significant increase (all P<0.05); as compared with the control group, the radiation group showed mild increase in the expression of microRNA-145in renal vascular smooth muscle cells (P>0.05).(10) Marked hyperplasia of mesangial cells and mesangial matrix was noted in the control group, yet mild focal hyperplasia of mesangial cells and mesangial matrix was noted in the radiation group and the transplantation group; in the control group, there was marked vacuolar degeneration of the renal tubules and shedding of brush border; in the radiation group, the capillary loops in some capillaries had poor patency and there was vacuolar degeneration in some of the renal tubules; in the transplantation group, the capillary loops had good patency and there was vacuolar degeneration in just a few of the renal tubules.Conclusions:1. FNPB infusion therapy showed an excellent GVA effect and good safety in treatment of MRL/lpr mice without inducing marked GVHD manifestation.2. FNPB infusion therapy facilitated recovery of hematopoietic system in MRL/lpr mice with radiation as conditioning and it had immunoregulatory function in MRL/lpr mice.3. There was expression of microRNA-145in renal vascular smooth muscle cells of MRL/lpr mice, and FNPB therapy resulted in increased expression of microRNA-145in renal vascular smooth muscle cells. |
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