The Experimental Study On CVF Inhibiting Acute Rejection Of Heart Allotransplantation In Mice

Objective and Background: Heart transplantation has been an important treatment for the end-stage heart failure. However, the acute rejection is a major barrier to the therapeutic effects of heart transplantation. Immunodepressant such as CsA and glucocorticoid such as medrol have been used to treat acute rejection with toxic and adverse reaction in clinic. It can injure the liver and kidney, depress the immunity and lead to infection and tumor. The immunosuppressive drugs by effecting on T cell can't suppress acute rejection completely and induce immunotolerance by now. So it's significant to find a new immunosuppressive drug. CVF (cobra venom factor) is considered a new kind immunodepressant as known to depress hyperacute rejection in xenotransplantation by consuming complement. But its inhibitory effect on acute rejection remains ambiguous. Our antephase experiments showed that CVF prolonged cardiac allograft survival time of rats and reduce the T cell infiltration and pathological lesion, which indicated CVF can suppress heart allograft acute rejection. Here we plan to establish a cervical heterotopic heart transplantation model in BALB/c-C57 inbred strain mice for further research. This is a high homozygosis model which suit the medicine reagent and monoclone antibody originated from mice. Then we plan to verify the suppressive effect of CVF on acute rejection and T cell activation. It may profit the clinic application for CVF by researching the mechanisms.Methods:1. BALB/c mice hearts were cervical heterotopically transplanted into C57 mice to establish a heart transplantation model in inbred strain mice by using Cuff technique. In all 64 case models, recipients were randomly allocated to control group and experimental group. The control group treated with saline; the experimental group mice treated with CVF 50μg/kg intravenously in orbital sinus on preoperative 24 hours and inject CVF 20μg/kg per trid on postoperative till cardiac grafts stop beating.2. The transplanted hearts in twelve mice in each group were harvested on 3 d, 5 d and 7 d after grafting for histopathological Studies. Rejection grade was checked l by HE. The expressions of MHC-Ⅱ, B7-1 and B7-2 were checked by immunohistochemistry. The survival time of transplanted hearts in the remanent 20 cases of each group were observed. The transplanted hearts'viability was assessed by direct neck palpation of the transplanted hearts. 8 cases in each group were chosen to measure (CH50) the serum complement activity on postoperative 1 hr, 6 hr, 12 hr, 2 d, 4 d, 6 d, 8 d.3. The pathological results were analyzed using the image analysis program (Image-Pro(?) Plus version 6.0) and all data were analyzed using SPSS 13.0 software.Results:1. The cervical heterotopic heart transplantation model was successfully established in BALB/c-C57 inbred strain mice. The operation can be completed within 60 to 70 min. The time of hot ischemia and cold ischemia were 2 to 5min and 20 to 25min, rep. The survival ratio of recipients after operation was 96%.2. The complement activity (%) of control group was higher significantly, compared with experimental's (p<0.01).The complement activity in control group keep on the high level all along. The complement activity in experimental group (CVF-treated) decrease to 50% of normal level on postoperative 1 hr; 10% on postoperative 6 hr, almost 0 on postoperative 2 d; retained at 20% on postoperative 4 d, and sustain at below 50% on postoperative 6 d; recovered approximately to normal level on postoperative 8 d.3. The results of survival analysis indicated that cardiac grafts survival time in the experimental group was 24.03±1.39 days vs. 7.91±0.35 days in control group. CVF can prolong significantly the survival tine of heart allograft (p<0.01).4. The histological study of HE stain showed that all cardiac grafts in control group occurred aboveⅡgrade acute rejection on postoperative 3 d, aboveⅢgrade acute rejection on postoperative 5 d, andⅣgrade acute rejection on postoperative 7 d. The cardiac grafts in experimental group didn't occur acute rejection on postoperative 3 d and 5 d. There only two cases occurredⅠacute rejection on postoperative 7 d. The results of immunohistochemistry showed that MHC-Ⅱantigen, B7-1 and B7-2 were mainly expressed on antigen-presenting cell (APC); Also, the vascular endothelial cell expressed MHC-Ⅱantigen in the acute rejection. The expression level of above-mentioned antigen and factor on integer or on single cell in experiment group were significantly lower than that in control group. There was a significant difference between tow groups (p<0.01). The expression extent were directly correlated with the grade of acute rejection.Conclusions:1. The cervical heterotopic heart transplantation model which we established in BALB/c-C57 inbred strain mice is an ideal animal model in studying on heart transplantation.2. CVF can suppress effectively the cardiac allograft acute rejection and prolong the survival time of heart allogrft in mice (p<0.01).3. CVF can decrease the expression of antigen MHC-Ⅱon APC and VEC, costimulatory molecules B7-1 and B7-2 expressed on APC(p<0.01).The mechanism is still to be explored.