Objective To investigate the aim antigen coursing the hyperacute rejection of xenotransplantation. Methods Documents about hyperacute rejection in xenotransplantation were reviewed and summarized in detail. Results Pig is thought to be one of the ideal donors of xenotransplantation, but the major obstacle is hyperacute rejection mediated by complement that is activated though human serum. ?-Gal is recognized as the major antigen and its expression is controlled by ?-1,3 galactosyltransferase. Immunoabsorption of pre-exsisted antibody, enzymatic digestion of ?-Gal, knockout ?-GT gene and transgenic technology have been used to solve this problem. Even so, there remain other antigens which can combine with natural antibodies in human serum, such as, 40?10 3 molecule in erythrocyte, 210?10 3, 10 5?10 3 and 50?10 3 antigen in pig embryo brain cell, etc. Conclusion ?-Gal is the major antigen which course the hyperacute rejection. Besides ?-Gal, many nonalpha-gal need further investigation.

Objective To observe the effect of bortezomib on acute graft-versus-host disease (aGVHD) in an aGVHD model of mice and investigate the related mechanism. Methods Male C57BL/6( H-2Kb)mice were used as donors and female Balb/c (H-2Kd) mice used as recipients. Balb/c mice received total body irradiation (TBI) by 7.0 Gy X-radiation, and randomly divided into five groups. normal (group A), TBI (group B), TBI + bortezomib (group C), TBI + bone marrow cells (BMC) + spleen cells (SC) (group D) and TBI + bortezomib + BMC + SC (group E). The physical signs and the pathological damage of aGVHD, mean survival time, and chimerism were observed in recipients. The NF-κB p65 levels in nuclei of the liver and small intestine tissues of groups A,B and C were analyzed by Western blot. Results ( 1 ) The clinical aGVHD score in group D was (7.37±0. 32), significantly higher than in group E (5.85 ± 0.40) (P＜0. 05). Histopathology of the gut, liver and skin illuminated that the Ⅲ-Ⅳ degree GVHD occurred in group D. The occurrence of aGVHD in group E was later than in group D. The symptoms and the pathological damage of aGVHD in group E were milder than in group D. The average survival time in group E was significantly longer than that in group D (P＜0.05). The percentage of donor-derived cells in recipient mice was above 90% at day 12 after transplantation; (2) NF-κB p65 levels in nuclei of the liver and small intestine tissues in group B was significantly higher than in group C on the day 1,3 and 5 (P＜0. 05). Conclusion Bortezomib can inhibit the activation and expression of NF-κB,which may be the underlying mechanism for it to relieve aGVHD.

Objective This study aimed to establish the leukemia mouse model by using EL4/DsRed cell line expressing red fluorescent protein (DsRed) and to evaluate the model. Methods After total body irradiation with X-ray of 7.0 Gy, C57BL/6 mice were inoculated 5×106 bone marrow cells mixed different numbers of EL4/DsRed cells via tail vein. The model was evaluated by flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), and histopathology. Results The incidence of leukemia was 100 %. The presence of EL4/DsRed cells was found in liver, spleen, bone marrow and peripheral blood of recipients by FCM two weeks after transplantation. Pathological section revealed that all recipients had several organs infiltration apparently. With the increase in the number of inoculated tumor cells, the survival time of recipients was reduced and the infiltration of leukemia cells in organs was more serious. Conclusion Mouse leukemia model was successfully established when C57BL/6 mouse was intravenously transplanted with ≥5×102 EL4/DsRed cells. The model could be employed usefully in the future research such as the pathogenesis of leukemia and minimal residual disease (MRD).

Objective To explore the different expression of NF-κB in both K562 and its multidrug resistant cell line K562/A02 and discuss the mechanism of muhidrug resistance(MDR). Methods To detect the growing feature of the cells. Flow cytometry was used to analys the difference between the distribution profile of K562/S and K562/A02 cell. MTT colorimetry was used to determine the cytotoxic effect of adramycin, and expression of mdrl gene was detected by semi-quantitative reverse transcriptase poly-merase chain reaction (RT-PCR) in K562 and K562/A02 cells. FACS was used to determine the expression and function of glycoprotein (P-gp) on the cell membrane. Western blotting was used to determine the NF-κB p65protein in nueleus. Results There was a difference between K562 and K562/A02 cells growed in a halfadherent way rather than suspending ones, there were increases in the percentage number of cells at G0/G1 and S phases(P ＜0.05). This was mirrored by a decreasing number of cells within the G2/M phase(P＜0.05). Butthere was no difference in apoptosis rate(P ＞0.05). mdr1 mRNA was detected in K562/A02 cells, in which the expression P-gp was much higher [(94.17±0.89)%:(1.41 ±O.491)%]. NF-κB p65 protein in nucleus was overexpressed in K562/A02 cells. Conclusion The activation of NF-κB signaling pathway may attribute to the formation of MDR in K562/A02 cells.

Objective:To evaluate the efficiency of the tibial coronal alignment after total knee arthro-plasty with the extramedullary cutting system guided by the tibial tubercle and anterior tibial tendon,and to discuss the potential risk factors for the postoperative malalignment.Methods:A retrospective study of 212 primary unilateral TKA surgeries (in 188 patients)in our hospital between June to December in 2014 had been analysed.All the cases were performed by one surgical group.An extramedullary tibial cutting guide system had been used,with a landmark of one third inner-medial tibial tubercle as a proxi-mal anatomy reference,and anterior tibial tendon as a distal marker.The mechanical axis of lower ex-tremity in full-length X-ray was measured before surgery,and the tibial prosthetic coronal alignment was checked two weeks postoperation,evaluating the accuracy of this extramedullary cutting system guided by our method.Results:Since good alignment was defined as an angle within 3 degrees of the perpendicular to the mechanical axis,the tibial component positions were acceptable in 191 knees (90.1%),five knees were in valgus postoperative,and sixteen knees were in varus.There were seventeen(8.7%)in 179 knees with a preoperative varus presented malalignment after surgery,and four in 12 preoperative valgus kneess howed malalignment also,no statistical difference was found by Chi-square test (χ2 =2.778,P=0.096),which cannot define the relationship between the varus or valgus deformity preopera-tion and the malalignmentposition in tibial prosthesis after surgery.Twenty-two knees suffered a severe preoperative deformity as a varus or valgus angle larger than 20 degrees with absolute value of mechanical axis before surgery and tibial prosthetic coronal alignment were 21.95°(20.00°,26.90°)and 1.85° (0.10°,7.10°),showed a significant difference (Z =2.11,P =0.035 )compared with the data [10.65°(0.50°,19.80°)in preoperative mechanical axis and 1.10°(0.00°,4.60°)in the tibial pros-thetic coronal alignment]of 190 knees who presented a mild deformity before surgery(less than 20 de-grees),the result indicated the severe preoperative deformity might be a potential mal-alignment risk fac-tor within this cutting system in TKA surgery.Conclusion:The tibial coronal alignment after total knee arthroplasty could achieve good results with extramedullary cutting guide,by using one third inner-medial tibial tubercle and anterior tibial tendon as a proximal and distal anatomy marker.Postoperative varus might occur in this system,and tibial prosthetic malalignment appeared more often in the patients who suffered a severe deformity before surgery.

Objective To explore the influence of the lentiviral vectors mediated mouse genetic engineering regulatory T cells(Tr) infused after allogeneic bone marrow transplantation(allo-BMT) on graft-versushost disease(GVHD) in mice. Methods Lentivirus-mediated expression of forkhead box P3 (Foxp3) converted CD4 + CD25 - T cells from BALB/c mice into engineered Tr in vitro. An allo-BMT model of BALB/c→C57BL/6 mice was established. After irradiation, the recipients were injected with donor cells along with genetic engineering Tr. Survival time, histopathological analysis, serum levels of inflammatory cytokines were observed after allo-BMT. Results The mean survival times in radiation group, transplantation control group, engineering Tr group and empty vector control group were ( 8.8 ± 0.6 ) d, ( 36.7 ± 2.5 ) d, ( 51.6 ± 4.0 ) d and ( 34.1 ± 2. 3 ) d. The survival time in engineering Tr group was significantly increased as compared to other groups as judged by the log-rank test ( P ＜0.05 ). Histopathological analysis in several target organs( skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD were observed in recipients in engineering Tr group. The serum levels of IFN-γ, IL-2 and TNF-α were all increased after transplantation in above groups. The peaks of concentrations of IFN-γ, IL-2 and TNF-α in engineering Tr group were significantly decreased compared to transplantation control group and empty vector control group at day 21 ( P ＜ 0. 05 ). Conclusion Co-injection of genetic engineering Tr can efficiently prevent recipients from lethal GVHD during allo-BMT in mice by reducing the serum levels of inflammatory cytokines.

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneic bone marrow transplantation (alloBMT) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) transformed CD4 + CD25- T cells from Balb/c mice into engineered Tregs in vitro. An allo-BMT model of Balb/c→C57BL/6 mice was established. The recipients were given lethal X-ray total body irradiation before transplantation.Mice were randomly assigned into five groups and each group contained 10 recipients: (1) The recipients in radiation group were injected with 0.2 ml RPMI 1640; (2) The recipients in leukemia control group were injected with 5 × 106 donor bone marrow cells and 500 mouse T-cell leukemia/lymphoma cells (EL4 cells); (3) The recipients in transplantation control group were injected with 5 × 106 donor bone marrow cells and 5 × 106 splenocytes plus 500 EL4 cells; (4) The recipients in engineering Treg group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 genetic engineering Treg; (5) The recipients in empty vector control group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 empty vector-transduced CD4+ CD25- T cells. Survival time, clinical GVHD score or histopathological analysis (skin, liver and small intestine) were observed after allo-BMT. Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured. Results The mean survival time in radiation group, leukemia control group, transplantation control group,engineering Treg group and empty vector control group was ( 10. 3 ± 1.5), (20. 7 ± 1.9), (26. 0 ±4.3), (49. 0 ± 17. 7) and (24. 4 ± 4. 1 ) days respectively. The survival time in engineering Treg group was significantly prolonged as compared with other groups as judged by the log-rank test (P＜0. 05).Histopathological analysis in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD or leukemia were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased as compared with transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD without affecting GVL activity during allo-BMT in mice.

Objective To determine the conditioning regimen suitable for mice allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Twelve BALB/c mice were randomly divided into 2 equal groups to undergo X-ray irradiation by linear accelerator at the dose of 7.0 Gy (pure X-ray group) or 60Co source irradiation at the dose of 7.0 Gy (pure γ-ray group).Thirty mice were randomly divided into 2 equal groups to undergo X-ray irradiation and then infusion of bone marrow from donor mice via caudal vein (X-ray + transplantation group) or γ-ray and then infusion of bone marrow via caudal vein (γ-ray + transplahtation group).3,5,7,10,15,20,and 30 d later peripheral blood samples were collected to calculate the number of white blood cells (WBCs) and detect the chimeric rates of lymphocytes by flow cytometry.5,10,and 20 d after irradiation 15 mice were killed with their lung,liver,small intestine,spleen,and femurs taken out to undergo pathological examination.Results The survival rates during the period 5-15 days of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group.The pathological changes of organs of the X-ray +transplantation group were all more severe than those of the γ-ray + transplantation group.Since the fifth day after transplantation cells originating from the donor began to appear in the peripheral blood.The chimeric rate of the γ-ray + transplantation group 10 days after transplantation was (95.53± 2.57) %.The chimeric rates 5,10,and 20 days after transplantation of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group (t = 15.263,3.256,P < 0.05).The WBC count of both irradiation groups decreased to the lowest level 5 d later and began to increase 10 days after transplantation and the WBC counts of the γ-ray + transplantation group 10 and 20 days aftertransplantation were both significantly higher than those of the X-ray + transplantation group (t = 3.624,6.695 ,P < 0.05).The chimeric rats of the peripheral lymphocytes 10 and 20 days after transplantation of the γ-ray + transplantation group were both significantly higher than those of the X-ray + transplantation group (t = 12.317,8.295,P < 0.05).The homogeneity rate of transplantation of the γ-ray +transplantation group was better than that of the X-ray + transplantation group.Conclusions As a conditioning regimen in allogeneic hematopoietic stem cell transplantation γ-ray irradiation causes milder injury and accelerated reconstitution of hematopoiesis and immunity,in comparison with X-ray irradiation.

Objective To explore prevention of cyclosporine A (CsA) combined with Cobalt protoporphyrin (CoPP) against murine graft versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods C57BL/6 (H-2Kb) mice were used as donors and BALB/c (H-2Kd) mice as recipients,which were randomly divided into 4 groups.The mice in total body irradiation group (TBI group) were lethally irradiated and injected intravenously with PBS; The mice in Allo-HSCT group (BS group) were lethally irradiated and injected intravenously with bone marrow cells and spleen cells; The mice in CsA intervention group (CsA group) were injected with CsA intraperitoneally after allo-HSCT; The mice in CsA combine with CoPP intervention group (combination group) received both CsA and CoPP intraperitoneally after alloHSCT.Recipients were monitored for condition,survival rate and weight.The liver,small intestine and skin in the recipients were gained and pathological changes of GVHD were assessed.The kidney was stained with Masson staining dye to observe the tissue fibrosis.The expression levels of renal HO-1 mRNA in the recipients were detected.Results In contrast to BS and CsA groups,GVHD degree in combination group was mild,with less reduction and quick recovery of weight.On the day 30 after HSCT,survival rate in BS group was 36.8％,and that in combination group and CsA group was 69.6％ and 53.5％ respectively (P＜0.05).In comparison with BS and CsA groups,pathological changes in combination group were mild,cellular edema and degeneration degree of the liver,small intestine and skin were slight,and few necrosis and infiltrated inflammatory cells were observed.Tubulointerstitial fibrosis hardly occurred in combination group,but it occurred in CsA group abundantly.As compared with BS group,the expression levels of HO-1 mRNA was increased in combination group,while decreased in CsA group (P＜0.05).Conclusion CsA combined with CoPP enhanced the protective effect of CsA against GVHD,moreover,CoPP could alleviate the side effects of CsA,which might be related with up-regulation of the expression levels of HO-1.

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneie bone marrow transplantation (allo-BMT)on graft-versus-host disease (GVHD) in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) converted CD4~+ CD25~- T cells from Balb/c mice into engineered Tregs in vitro.An allo-BMT model of Balb/c→C57BL/6 mice was established.Mice were randomly assigned into four groups:(1) The recipients in engineering Treg group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenoeytes plus 5×10~6 genetic engineering Treg;(2)The recipients in transplantation control group were iniected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytes;(3) The recipients in radiation group were injected with 0.2 ml RPMI 1640;(4)The recipients in empty vector control group were injected with 5×10~6 donor bone marrow cells and 5×10~6 splenocytas plus 5×10~6 empty vector transduced CD4~+ CD25~- T cells.Survival time,clinical GVHD Score or histopathological analysis(skin,liver and small intestine) were observed after allo-BMT.Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured Results The mean survival times in radiation group, transplantation control group,erIgineering Treg group and empty vector control group were (8.8±0.6),(36.7±2.5),(51.6±4.0) and (34.1±2.3)days respectively.The survival time in engineering Treg group was signiticantly prolonged as compared with other groups as judged by the log-rank test(P<0.05).Histopathological ahalysis in several target organs (skin,liver and small intestine)confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased compared to transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD during allo-BMT in mice