| Objective In this study, retrovirus carrying PLNCX2-HA-CD40 Ig gene was used for its local transfection into kidney of guinea pig and immune tolerance was induced by blocking the CD40-CD40 L co-stimulatory pathway; at the same time, immunosuppressor―sinomenine was applied to inhibit the proliferation of CD4+ cells in peripheral blood of the recipients, so as to observe their combined effect on expression of Bcl-2/Bax protein within rejection in renal xenotransplantation from guinea pig to SD rat, and thus to provide new method and strategy for prevention of rejection in renal xenotransplantation.Methods 40 male SD rats(with an age of 12- 14 weeks) and 40 male guinea pigs(with an age of 12- 14 weeks) were selected and randomly divided into 4 groups(10 SD rats and 10 guinea pigs in each group). They were: Group A: transfection with empty retrovirus + normal saline; group B: transfection with empty retrovirus + sinomenine; group C: local transfection with retrovirus carrying CD40Ig; group D: local transfection with retrovirus carrying CD40 Ig + sinomenine. Kidneys of guinea pigs locally transfected with retrovirus carrying CD40 Ig were transplanted to the neck of SD rats. After transplantation, survival times of the recipients in each group and their transplanted kidney were observed. 48 hours after transplantation, blood from caudal vein of the recipients was obtained to detect the serum creatinine value and at the same time, the 48-hour urine volume was detected; western blotting was used to detect the transfection efficiency of CD40 Ig in the right kidney; expression of Bcl-2/Bax protein in transplanted kidney was detected by western blotting and immunohistochemical techniques. Grades of rejection in transplanted kidneys of rats in each group were observed by using HE staining.Results 1. Measurements on the serum creatinine value and the urine volume48 hours after transplantation, blood from caudal vein of the recipients was obtained to detect the serum creatinine value: values in group B and group C were respectively(9.20±0.83) mg/L and(9.20±0.73) mg/L; values in both of them were significantly lower than the value of(15.00±1.00)mg/L in group A(P<0.05); however, group D had the lowest value of(4.10±0.81)mg/L and there was significant difference when it was compared to each of the other 3 groups(P<0.05).48-hour urine volume: values in group B and group C were respectively(11.80±1.09)ml and(11.50±1.12)ml; values in both of them were significantly higher than the value of(6.40±0.55)ml in group A(P<0.05); however, group D had the highest value of(18.90±0.80)ml and there was significant difference when it was compared to each of the other 3 groups(P<0.05).2. Survival times of the transplanted kidney and the recipients Survival times of the transplanted kidney and the recipients in group A were(5.60±0.89)days and(6.60±0.89) days, respectively; Survival times of the transplanted kidney in group B and group C were respectively(10.06±0.89) days and(10.90±0.83)days, and survival times of the recipients in group B and group C were respectively(12.60±0.89) days and( 12.90±0.83) days; survival times of the transplanted kidney and the recipients in both of them were longer than those in group A(P<0.05); group D had the longest survival times of the transplanted kidney and the recipients ―( 14.60±0.83) days and(17.50±0.83)days, respectively, and there was significant statistical difference when it was compared to each of the other 3 groups(P<0.05).3. Western blotting After kidneys from guinea pigs were transplanted to the neck of SD rats, western blotting was used to detect the expression of CD40 Ig in right kidneys transplanted in each group. Expression of CD40 Ig could be detected in group C and group D, but couldn’t be observed in group A and group B. Expression of Bax protein can be seen in transplanted kidneys in each group, with the highest expression in group A; Expressions of Bax protein in group B and group C were lower than that in group A, while there was no significant difference between group B and group C(P> 0.05); expression of Bax protein in group D was significantly lower than that in each of the other three groups(P <0.05). Expression of Bcl-2 protein could be seen in transplanted kidneys in each group, with the lowest expression in group A; expressions of Bcl-2 protein in group B and group C were higher than that in group A, while there was no significant difference between group B and group C( P> 0.05); expression of Bcl-2 protein in group D was significantly higher than that in each of the other three groups(P <0.05).4. HE staining HE staining was performed to pathological sections from transplanted kidneys in each group and results showed that: after HE staining, a large number of lymphocytic infiltrations could be seen in tissues from transplanted kidneys in group A, with a pathological grade of IIB-II according to Banff09 classification of renal allograft, prompting that there was intense rejection of the transplanted kidney. Lymphocytic infiltration could also be seen in group B and group C, but weaker than that in group A, with a pathological grade of IIA-III according to Banff09 classification of renal allograft. However, with a pathological grade of IA-IIB according to Banff09 classification of renal allograft, group D had a significant lower grade than each of the other three groups.5. Immunohistochemical detection Expressions of Bcl-2/Bax in transplanted kidneys in each group were detected by immunohistochemical techniques:Expression of Bax in tissues from transplanted kidney in group A was stained as brown, indicating a strongly positive expression of Bax, with a strongly positive rate of 100%; the strongly positive rates regarding expression of Bax in tissues from transplanted kidney in group B and group C were 60% and 40%, respectively; group D had a strongly positive rate of 20%(1 had strong positive reaction, while the rest 4 had weak positive reaction).Expression of Bcl-2 in tissues from transplanted kidney in group A was weakly positive; the strongly positive rates regarding expression of Bcl-2 in tissues from transplanted kidney in group B and group C were 40% and 60%, respectively; group D had a strongly positive rate of 80%(1 had weak positive reaction, while the rest 4 had strong positive reaction).Conclusions 1. CD40 Ig into kidney of guinea pig plays a role in prevention of rejection; it can decrease the expression of Bax, increase the expression of Bcl-2 and prolong the survival time of transplanted kidney.2. With strong immunosuppressive effect, sinomenine can inhibit the rejection in renal xenotransplantation of rats, decrease the expression of Bax, increase the expression of Bcl-2 and prolong the survival time of transplanted kidney.3. There are synergistic anti-rejection effects of CD40 Ig and sinomenine; joint application of retrovirus carrying CD40 Ig and sinomenine can significantly reduce the rejection in renal xenotransplantation of rats, decrease the expression of Bax, increase the expression of Bcl-2 and prolong the survival time of transplanted kidney. |
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