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 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 (e)xplore inhibition of endothelial progenitor cells (EPCs) against hepatic vein thrombosis after allogeneic bone marrow transplantation (BMT).Methods Balb/c mice were randomly divided into three groups: (1) BMT group [Balb/c mice were injected intravenously with 5 × 106 bone marrow cells after total body irradiation (TBI)]; (2) EPCs co-transfusion with bone marrow cells group: 5 × 105 EPCs were infused into recipient mice simultaneously; (3) Normal control group.Liver index was detected on the day 0,5,10,15 and 20 after transplantation.Hepatic vein thrombosis,hepatic cells and vascular endothelial damage were observed under the light microscopy after H&E staining.The injury of liver cells,liver veins,hepatic sinusoidal endothelial cells (SECs)and platelet adhesion conditions were observed under a transmission electron microscope (TEM).The proportion of activated platelets and TNF-α concentration in peripheral blood were detected by using flow cytometry.Results On the day 0,5,10,15 and 20 after transplantation,the proportion of activated platelets,liver index and TNF-α concentrations in BMT group and EPCs co-transfusion group showed an upward trend,peaked on the 15th day,and then decreased.However,they were still significantly higher than those in normal control group (P＜0.05).The above parameters in EPCs co-transfusion group at each time point were significantly lower than those in BMT group (P＜0.05).As compared with BMT group,platelet adhesion decreased,hepatic vein thromboses were reduced,hepatocyte swelling and necrosis were alleviated,and liver damage repaired rapidly in EPCs co-transfusion group.Conclusion EPCs co-transfusion with bone marrow cells could inhibit the hepatic veins thrombosis and ameliorate liver damage significantly.

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 study the repair function of united endothelial progenitor cells (EPC)transplantation on injured liver endothelium by bone marrow transplantation (BMT) conditioning.Methods C57BL/6 mice were divided into four groups randomly: normal control group, without any treatment; irradiation alone group, administered a total body irradiation(TBI) pretreatment, without BMT; (3) BMT alone group: C57BL/6 mice were infused with bone marrow mononuclearcells (MNC) 5 × 106/only through caudal vein not more than 4 h after the same TBI pretreatment as the irradiation alone group; united transplantation group: receiving the same way as the BMT alone group, but C57BL/6 mice were infused with EPC 5 × 105/only at the same time. Two, 4, 7, 14, and 21 days after the TBI, the changes of the liver weight were observed regularly. The histopathological examination of liver was done at the 4th, 7th, 14th, and 21st day after the TBI. Results In irradiation alone group, BMT alone group and united transplantation group the liver weight began to increase significantly on the day 2 and peaked at 14th day after the TBI, and the peaks were respectively (1.65±0. 15) times (P＜0. 05), (1.61 ±0.06) times (P＜0.05), and (1.11 ±0.40)times (P＜0. 05) of those in normal control group. At the day 14, the liver weight in irradiation alone group, BMT alone group and united transplantation group began to decrease, and on the day 21 the liver weight in united transplantation group had been completely restored to normal level, however the liver weight in irradiation alone group and BMT alone group were still significantly heavier than that in normal control group (P＜0. 05). Liver histopathological examination revealed that there were obvious sinusoidal endothelial cells (SEC) injury, hepatocyte edema and severe inflammatory cell infiltration in irradiation alone group, and on the day 7 the hepatocyte edema and necrosis were significantly worse than before, and almost no alive SEC were found. On the day 14 the injury of SEC in BMT alone group was lighter than before, but on the day 21 the injury had not returned to normal. On the day 7 the injury of SEC, hepatocyte edema and necrosis were alleviated in united transplantation group as compared with irradiation alone group and BMT alone group, and on the day 14 the injury had returned to normal basically. Conclusion The transplantation conditioning could damage recipient liver endothelium and the injury would persist, and united EPC infusion could repair the injured SEC following BMT.

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.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

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

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.