Objective　To study the viability and histological change of encapsulated rat hepatocytes after being transplanted into abdominal cavity of rat. Methods　The two-step collagenase perfusing method was used to separate hepatocytes from Wistar rat liver. The separated hepatocytes were purified with Percoll density gradient centrifugation and encapsulated by the alginate-barium method. Then the purified hepatocytes were transplanted into abdominal cavity of SD rats (group 1) and the encapsulated hepatocytes were transplanted into abdominal cavity of SD rats (group 2) and Wistar rats (group 3). At different time points post-transplantation, trypan blue stain exclusion was used to determine the viability of recovered hepatocytes. The histological changes of transplanted microencapsulated hepatocytes was examined using HE stain. Results　Twenty-four h after transplantation, the viability of hepatocytes between group 1 and group 2 showed significant difference (P<0.01), but there was no significant difference between group 2 and group 3 (P>0.05). At day 4 and day 7 after transplantation, the viability of hepatocytes showed significant difference between group 1 and group 2, and group 2 and group 3 (P<0.01). At day 14 after transplantation, no significant difference was found in the viability of hepatocytes between group 2 and group 3 (P>0.05). From day 4 post-transplantation, fibrosis overgrowth was found around some microencapsules, and it was more obvious in group 2 than in group 3. Conclusions　 Microencapsulation can provide protection to transplanted hepatocytes from host immunorejection, and thus increase the viability of hepatocytes post transplantation. The existence of inadequately encapsulated microencapsule cause the fibrosis overgrowth around these capsules, resulting in ischemia and subsequent necrosis of the hepatocytes and decreasing hepatocyte viability.

Objective　To investigate the infection of hepatitis virus and spirochete in renal transplant donors and recipients to study the relationship between infection and human/kidney survival rate following renal transplantation. Methods　A total of 361 donors and 300 recipients were investigated on infection of HBV, HCV, HGV, CMV, EBV, HSV, HIV and RPR. Results　Of the 361 donors, 31 cases (8.6!%), 9 cases (2.5!%) and 2 cases (0.6!%) were found having HBV, HCV, HGV infection respectively. In the 231 recipients, the percentage of CMV, EBV, HSV, HIV and RPR carriers was 16.9!%, 11.7!%, 16.0!%, 0.4!% and 0.8!% respectively. Among the 300 grafting recipients, the infective rate of HBV, HCV and HBV plus HCV was 68.7!%, 34.7!% and 25.0!% respectively. Forty patients were randomly selected from the 300 patients, it was found that 10 (25.0!%) patients were positive for anti-HGV, 10 (25.0!%) for HGV and HBV, 5 (12.5!%) for all HGV, HBV and HCV. The percentage of CMV, EBV, HSV, HIV, RPR carriers among the 300 recipients was 49.0!%, 32.7!%, 42.0!%, 0 and 0.3!% respectively. Conclusion　Viral infectious status of the donors and recipients before operation might contribute to the occurrence of viral infection in the recipients after transplantation.

Objective To systematically evaluate the effect of routine insertion of double-J stents to prevent major urological complications(MUCs)in kidney transplant recipients.Methods Medline,Embase,Cochrane Library,and Chinese Biomedicine database were searched to locate relevant randomized controlled trials(RCT).Data extraction and assessment of methodologic quality were performed independently by two reviewers.Meta-analysis was performed by Revman 5.0 software.Results Ten RCT(including 1616 patients)were identified.By comparing the routine stent group with the no stent group,the meta-analysis showed:(1)incidence of urine leak,urinary obstruction and UTI was 4 times lower,6 times lower,increased by 52 % respectively(P＜0.0001);(2)Patient and graft survival,rate of acute rejection,delayed graft function and hematuria were of no significant difference.In subgroup analysis,it was found:(1)Compared with the no stent group,the group in which stent duration was≤ 4 weeks had a lower incidence of MUCs and a higher incidence of UTI;meanwhile,the group in which stent duration was ＞ 4 weeks had a much lower incidence of MUCs and the rate of UTI was increased without significant difference;(2)In the RCT of which urethral catheter duration was ＜ 5 days,there were no significant differences between the two groups in MUCs and UTI.In the RCT of which urethral catheter duration was ≥5 days,the stent group had a lower incidence of MUCs and a higher incidence of UTI.Conclusion Routine stenting reduces the incidence of MUCs.Although the double-J stent increases the risk of UTI,it seems that UTI doesn't affect the outcome of transplantation.The stent duration should be within 4 weeks.For the stent recipients,the longer duration of urethral catheter,the lower incidence of MUCs,the higher incidence of UTI;thus,it is up to clinicians to decide the optimal duration of urethral catheter.Long term prescription of 480 mg cotrimoxazole once daily,from the operation day till after stent removal,effectively reduces the risk of UTI associated with stent placement.

Objective To study the immunologic and pathologic features of an accelerated rejection model of renal allotransplantation in presensitized monkeys.Methods The accelerated rejection model of renal allotransplantation was established in presensitized monkeys,which received donor skin transplantation in advance(n=3).The changes of donor specific antibody(DSA)levels in the recipient monkeys before/after skin and kidney transplantation were measured.The kidney grafts were examined for routine pathology,antibody and complement depositions,various lymphocyte subsets infiltration by HE staining,immunofluorescence,or immunohistochemistry.Results All renal allografts in 3 presensitized monkeys developed accelerated rejection within 4 days.In 2 presentized monkeys,the levels of DSA and their mediated complement-dependent cytotoxicity(CDC)were significantly increased after skin transplantation,and further markedly elevated at the time of kidney graft rejection.In the rejected renal grafts,massive C3,C4,C5b-9 and IgG deposits with few lymphocytes infiltration were found.Typical pathologic changes included severe arterionecrosis,thrombosis,interstitial hemorrhage,and infiltration of neutrophils.In the rest one presentized monkey,the levels of DSA and CDC were only marginally increased,and the pathological changes of the rejected renal graft were characterized mainly by the injury of renal tubules.Conclusion Presensitization by donor skin transplantation could elevate the levels of DSA and CDC in recipient monkeys,which resulted in severe antibody-mediated acute humoral rejection in most of the following renal transplants.

Objective To improve arterial anastomosis method for renal transplantation model in rats.Methods Male Fisher and Lewis rats were used as kidney donors and recipients respectively,and left kidneys were harvested in situ.Revascularizations of renal artery were fashioned end-to-end by the modified sleeve anastomosis.The renal artery was placed in the orthotopic position and sutures were inserted in order to place the feeding vessel into the receiving vessel.One invaginating suture was placed starting outside the donor's renal artery wall,approximately 2 mm from the free edge,then passing through the free edge of the recipient renal artery,and lastly passing again through the donor's renal artery wall out alongside the point of entry.The sutures Were tied so that the recipient renal artery was drawn inside the donor's renal artery.Thereafter,one external stitch was placed opposite to the invaginating suture passing through the overlapped free distal edge and the adventitia of the recipient renal artery,and the suture the opposite side using the same technique.The renal veins and ureters were anastomosed using end-to-end interrupted suture technique.Results Twenty cases of rat renal transplantation were performed.The transplantation procedures took totally between 70-90 min.The time for arterial anastomosis was approximately(4.6 ± 0.6)mint the mean time for anastomosis of the renal vein in 20 grafts was(11.8 ± 1,2)min.and ureter was(12.2 ± 1.4)mia The successful rate of the model was 95 % at the 5th day.Conclusion New end-to-end technique which incorporatesa modification of the sleeve anastomosis is the safest way to perform a revascularization of renal artery.This suggests that the technique is feasible and reliable.

Objective To evaluate the suppressive effect of CDS+ CD28-regulatory T cells(Treg)in vivo from spontaneous tolerance models on the acute rejection responses in rat liver transplantation.Methods Spontaneous tolerance models of inbred rat liver transplantation were established.CDS+ CD28-Treg isolated from recipients of spontaneous tolerance models were adoptively transferred into the recipients with acute rejection responses one day before the operation.The survival time and histological changes were observed in the adoptive-transferred models.Results CDS+ CD28-Treg from spontaneous tolerance models(LEW→DA)prolonged the survival time of the recipients in acute rejection models(LEW→BN)(from 14.0±2.2 days to 24.0 ± 3.0 days,P=0.0049),and the severity of rejection was alleviated in liver pathology.Conclusion CDS+ CD28-Treg from spontaneous tolerance models can suppress the acute rejection responses in rat liver transplantation,with antigen-specific properties.

Objective To explore the clinical therapy for cyclosporine A(CsA)-induced gingival overgrowth (GO) and the pathological changes in gingival overgrowth tissues.Methods Nine cases of CsA-induced GO after renal transplantation were subjected to periodontal non-surgical treatment and surgical treatment.Under light and electron microscopy,the pathological changes in CO tissues were observed.Results The bleeding index(BI) and the plaque index(PLI) of patients were declined after periodontal treatment.GO recurred in 2 patients 6 months later and happened to recur in all 9 patients 12 months later(GOD≤1).At 18th month after transplantation,an obvioUS GO(GOD≥2)occurred in one patient,and re-operation was done to cut hyperplastic gingiva.At 48th month during the observation period,GO existed continuously but no more than 2 in GOD.There were 3 other patients who had their GO(GOD≥2)at 24th month after peridental treatment and re-operation was carried out to remove the hyperplasic gingivaL Under a light microscope,epithelial pegs constituted of basal cells and prickle cells elongated and presented as cancellation structure;spinus layer thickened:hyperkeratosis or parakeratosis occurred in cuticular layer where inflammatory cells infiltrated:collagen increased in proper layer.Under the transmission electron microscopy,the volume of fibroblasts in hyperplastic gingival tissues was increased,rough endoplansmic reticula in the intracytoplasm were abundant and expanded slightly,and there were a few of the apoptotic fibroblasts in the early stage.Conclusion BI and PLl were declined in patients taking CyA for a long-term who were subjected to periodontal and surgical treatments.GO recurred in some patients.The proliferation and differentiation of fibroblasts was not observed in hyperplastic gingival tissues.

Objective To establish the method for isolation and culture of porcine Sertoli cell sand detect the expression of the immune privilege-related molecules in the cultured cells. MethodsTestes were aseptically removed from the 10-to 15-days old Large White piglets. Testes were decap-sulated, minced and then digested with 0.2% (W/V) collagenase type V, 0.25% (W/V) trypsin and 0.05% (W/V) DNaseI. In the primary isolation, Sertoli cells were cultured at 37℃with 5% CO2.Inverted phase contrast microscopy and HE staining were used to observe the morphology of Sertoliceils, and the Sertoli cells were identified under an electron microscope. Viability and apoptosis ofcultured cells were measured with the AnnexinV-PI staining by flow cytometry. The expression of sox9, FasL, TGF-β and clusterin in Sertoli cells was detected by RT-PCR. The viability of long-termcultured Sertoli cells was assayed by MTT. Results In the cultured total cells, Sertoli cells accountedfor more than 90%. The apoptosis rate and mortality of Sertoli cells was (2.61±0.96)% and (2.12±0.74)% respectively. RT-PCR revealed that the expression of sox9, TGF-β and clusterin in theSertoli cells was strongly positive, but FasL was weakly positive. Viability of cultured cells measuredby MTT conformed that the sertloli cells could survive more than 21 days. Conclusion The isolationand culture methods of the neonatal porcine Sertoli cells was established. Under the culturedconditions, the Sertoli cells can express the immune privilege molecules and successfully survive at least 21 days.

Objective To investigate the effects of down-regulating uncoupling protein-2 (UCP-2) expression on acute damage to fatty liver cells and explore a new target for the donor liverwith steatosis. Methods Primary fatty liver cells were isolated from C57BL/6J-ob/ob transgenic miceby two-step collagenase perfusion method. RNAi lentivirus vector targeting mouse UCP-2 gene wasused to knock down the UCP-2 gene in the steatosis hepatocytes (the experimental group). Emptylentivirus vector was transfected into the steatosis hepatocytes cells as the control group. Under thefluorescence microscopy, the transfection efficiency was tested. Real time PCR was used to determinethe effect of RNAi. After the transfected cells were treated with TNF-α for 24 h, apoptosis wasanalyzed by flow cytometry using PI staining. Activation of caspase3 was detected by Western blot.Resalts The expression of UCP-2 gene was inhibited effectively, and the knockdown rate of UCP-2gene was 75%. The apoptosis rate in the experimental group was (4.97±0.25)%, significantlylower than in the control group [(21.13±1.28)%, p<0.05 ]. Activation 'of caspase3 in theexperimental group was also weaker than in the control group. Conclusion Inhibiting the expression ofUCP-2 can attenuate the injury of fatty liver cells.

Objective To study the molecular mechanisms of hepatoeyte regeneration following different cold preservation (CP) durations after rat partial liver transplantation. Methods Mate inbred Lewis rats were used as donors and recipients. Donor liver was kept in 4℃ UW solution for 1 h (coldisehemia 1 h group, CI 1 h group), 8 h (CI 8 h group) and 16 h (CI 16 h group) and then implantedorthotopieally. 50% liver graft transplantation model was established by ligating the left portion ofmedian lobe, left lateral lobe and caudate lobe with 3-O silk suture prior to reperfusion. Survival rate ofeach group and hepatoeyte regeneration were recorded after grafting. Reverse transcription-polymerasechain reaction was used to detect the expression of IL-6 and TNF-α in the liver tissues. Western blotanalysis was done to measure STAT3 activation in the liver. Immunohistoehemistry was conducted toanalyze the expression of cyclin D1 and hepatocyte replication with BrdU uptake in the graft. ResultsOperative success rate in all groups was 100%. Compared with CI 1 h group, the TNF-α and IL-6expression (F=67.45 for TNF-a comparison, P<0.05 and F=287.73 for IL-6 comparison,P<0.05 respectively) in 8 h CI and 16 h CI groups was markedly increased after partial grafttransplantation. STAT3 activity in 8 h C1 and 16 h C1 groups was also significantly increased ascompared with that in 1 h CI group. Cyclin D1 expression in 8 CI group was demonstrated withcytoplasmic and nuclear staining at 24 h after transplantation. Grafts in 16 h CI group showed largeareas with no cyclin D1 expression. Number of hepatocytes with BrdU positively stained neclei in 8 hCI group was more than that in 16 h C1 group at 24 h after transplantation (t=19.40, P<0.05).Conclusion Hepatocytes regeneration was present following rat partial transplantation in the graftspreserved for limited time, which may be regulated by TNF-α/IL-6' STAT3/ Cyelin D1/DNAsynthesis pathways; Hepatocytes could not respond to early signals for liver graft regeneration when50%liver graft preserved for 16 h.