Construction of a murine model of chronic graft-versus-host disease by haplo-identical spleen cell infusion / 中华器官移植杂志

Objective For providing experimental platform of chronic graft-versus-host disease (cGVHD),to establish a mouse model by haplo-identical spleen cell infusion.Methods The donor male mice (Balb/cH-2d) and the recipient (Balb/c C57BL/6) F1 H2-d/b (CB6F1) female mice were randomly divided into four groups:3 experimental groups injected with 3 107,6 107 and 9 107 spleen cells,respectively,while the control group received RPMI 1640 solution.H-2d and H-2b were checked to analyze the chimerism in bone marrow cells.Body mass,figure,cutaneous manifestation and survival of recipient mice were observed and scored every 3 days.Pathologic changes of target organs were observed and scored.Results Injection of 6 107 and 6 107 splenocytes in the recipient mice resulted in a chronic disease with a low level of parental cell engraftment steadily.As compared with 3 107 group,the incidence of cGVHD in 6 107 and 9 107 groups were significantly increased (P ＜0.01).But there was no significant difference between 6 107 and 9 107 groups (P＞0.05).Conclusion A murine model of cGVHD after haplo-identical spleen cell infusion of donor is successfully established by injection of 6 107 and 9 107 spleen cells.

The homing of transplanted bone marrow mesenchymal stem cells to the thymus / 中国组织工程研究

BACKGROUND:Bone marrow mesenchymal stem cels have low immunogenicity and can induce immune tolerance. At present, the mechanism of immune regulation of bone marrow mesenchymal stem cels is not completely understood. It has been rarely reported whether the bone marrow mesenchymal stem cels can migrate to the thymus after transplantation. OBJECTIVE:To observe the distribution and survival of bone marrow mesenchymal stem cels in the thymus of aging rats after transplantation. METHODS: Bone marrow mesenchymal stem cels cultured in vitrowere transfected by adenovirus vectors expressing green fluorescent protein. Transfected bone marrow mesenchymal stem cels were injected into the portal vein of aging rats. At days 3, 7, 14, 21 after transplantation, the survival of bone marrow mesenchymal stem cels homing to the thymus was observed under fluorescence microscope. At day 3 after transplantation, thymus tissues were taken and stained with hematoxylin-eosin for pathological observation. RESULTS AND CONCLUSION:Green fluorescent protein-labeled bone marrow mesenchymal stem cels had a strong green fluorescence at days 3 and 7 after transplantation, and the cel contour was clear. There was no significant difference in the mean absorbance values at days 3 and 7 (P> 0.05). Expression of green fluorescent protein was weakened significantly at days 14 and 21 compared with that at day 3 (P < 0.05). At 3 days after transplantation, the transplanted bone marrow mesenchymal stem cels were clearly visible in the thymus, and acute rejection was not observed. The results show that bone marrow mesenchymal stem cels can migrate to the damaged thymus tissue through the blood circulation, and can survive at least 1 week.

Repairing effect of bone marrow mesenchymal stem cells on lung injury in aging rat models / 中国组织工程研究

BACKGROUND:Studies have shown that exogenous bone marrow mesenchymal stem cells can settle down in lung tissue, participate in long regeneration, but few studies concerned the repair of aging lung injury. OBJECTIVE:To observe the effect of bone marrow mesenchymal stem cells on lung injury induced by D-galactose. METHODS:A total of 30 Sprague-Dawley rats were equal y divided into three groups at random:control group, aging model group and celltreatment group. To establish the aging rats, 10 rats each in the aging model group and celltreatment group were daily subcutaneously injected with D-galactose for 4 months. 3×106 bone marrow mesenchymal stem cells were transplanted via caudal vein in the celltreatment group, once a week, for 4 weeks. cellmedium of equal dose was added in the control and aging model groups. Bone marrow mesenchymal stem cells were transfected by lentiviral vectors expressing green fluorescent protein to determine the implantation of bone marrow mesenchymal stem cells in rat lung. Superoxide dismutase activity and malondialdehyde content in rat lung were measured in each group. The difference in rat lung structure was observed using hematoxylin-eosin staining in each group. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells marked by green fluorescent protein were implanted in rats, migrated towards lung tissue and survived. Compared with aging model group, superoxide dismutase activity was apparently increased, but malondialdehyde content was obviously diminished in the celltreatment group. In each group, histopathological sections revealed that normal pulmonary alveolus was damaged in the aging model group, showing enlarged air cavity and emphysema. Lung injury was evidently repaired inthe celltreatment group. Results suggested that bone marrow mesenchymal stem cells could repair lung injury in aging rats, and exert anti-aging effects.

Effects of curcumin on proliferation and apoptosis of CD34+CD38-KG1 a cells and its synergetic effect with busulfan / 西安交通大学学报(医学版)

Objective To investigate the effects of curcumin on proliferation and apoptosis of CD34+CD38-KG1a cells and its synergetic effect with busulfan on CD34+CD38-KG1a cells.Methods The expressions of CD34 and CD38 on the surface of KG1a cells and the effect of curcumin on the cell cycle and apoptosis in CD34+CD38-KG1a cells were detected by flow cytometry.MTT assay was used to analyze curcumin’s inhibitory effects on proliferation and synergistic effect with busulfan on CD34+CD38-KG1a.Clone formation rate was measured by methylcellulose colony-formation assay.Morphological changes of apoptotic cells were observed with the inverted optical microscope.The expression of Bcl-2 at the protein level was detected by Western blot.Results The percentage of CD34+CD38-KG1a was (98.2±3.2)% in KG1a cells.Curcumin could inhibit the proliferation in time-and dose-dependent manners and reduce the colony-formation ability of CD34+CD38-KG1a.The coefficient of drug interaction between curcumin and busulfan was less than 1.CD34+CD38-KG1a cells were arrested in the G0/G1 phase by decreasing S phase cells.Meanwhile,curcumin induced the apoptosis of CD34+CD38-KG1a cells. Apoptotic cells became bigger than normal ones,with unclear cell structure and rough edge of cell membrane.The expression of Bcl-2 at the protein level was down-regulated by curcumin.Conclusion Curcumin inhibited the proliferation of CD34+CD38-KG1a cells by reducing colony-formation ability,arresting cells in the G0/G1 phase and inducing apoptosis.Besides,there was a synergistic effect between curcumin and busulfan in CD34+CD38-KG1a cells.The down-regulated expression of Bcl-2 at the protein level may be associated with curcumin-induced apoptosis of CD34+CD38-KG1a cells.

Haploidentical hematopoietic stem-cell transplantation for acute myeloid leukemia in first relapse after complete remission by standard induction chemotherapy / 中华器官移植杂志

Objective To investigate the therapeutic effects of haploidentical hematopoietic stem-cell transplantation (Haplo-PBSCT) for acute myeloid leukemia in first relapse after complete remission by standard induction chemotherapy. Methods Eighty-nine cases of AML in first relapse after complete remission by standard DA/Hi-Ara-C regimens induction chemotherapy were evaluated retrospectively. Fiftythree cases were grafted by haplo-PBSCT and 26 cases were treated with iDA/Mid-Ara-C or MA/ Mid- Ara-C agents. Results The second remission rate in haplo-PBSCT group and continuous chemotherapy group was 86. 7 % (46/53 cases) and 38. 1% (9/23 cases) respectively (P＜0. 01). Survival postprogression (SPP) at 36th month was 43. 4 % (23/53 cases) in haplo-PBSCT group and 11.5 % (3/26 cases) in continuous chemotherapy group (P ＜ 0. 05). Conclusion Haplo-PBSCT could significantly increase the second remission rate and prolong the survival time of patients with acute myeloid leukernia in first relapse after complete remission by standard induction chemotherapy.

Haploidentical hematopoietic stem cells transplantation for the treatment of medulloblastoma One case report / 中国组织工程研究

BACKGROUND: Some reports demonstrated that, autoallergic or hematopoietic stem cells transplantation combined with chemotherapy received good outcomes in treating medulloblastoma, which can prolong survival time of patients. However, whether haploidentical hematopoietic stem cells transplantation can treat medulloblastoma remains poorly understood.OBJECTIVE: To firstly report a patient receiving haploidentical hematopoietic stem cells transplantation for treating medulloblastoma.METHODS: A terminal cancer patient with bone matastases successively received six lymphocyte transfusions from an unrelated donor combined with chemotherapy and three haploidentical hematopoietic stem cell transplantations.RESULTS AND CONCLUSION: The patient presented erythra, accompanied by fever, diarrhea and yellow brown liquid stools, which was considered as graft versus host disease, and treated by urbason, gammaglobulin, CellCept, Prograf, Basiliximab (anti-CD25 antibody), Infliximab (anti-tumor necrosis factor α antibody), effective antibacterial and supportive treatments. After that, the erythra and diarrhea were remised. But the patient died from cerebral hemorrhage. Allogeneic lymphocyte transfusion can kill or damage tumor cells, improve life quality, but the outcome is restrained for patient with a high tumor burden. Immunosuppressant, such as anti-CD25 antibody and anti-tumor necrosis factor α antibody should be timely used in consideration of allogeneic hematopoietic stem cell transplantation.

Bone marrow mesenchymal stem cell transplantation for treating irradiation-induced injury to the rat kidney / 中国组织工程研究

BACKGROUND:Bone marrow mesenchymal stem cells (BMSCs) can differentiate into renal paranchymal cells including renal intercapillary cells.BMSCs can repair kidney structure and function after damage.OBJECTIVE:To investigate renal histology and function changes following BMSC transplantation in a rat model of radiation-inducad damage.DESIGN,TIME AND SETTING:The cytological in vitro study was performed at the Laboratory of the Department of Hematology,Zhujiang Hospital from January to October 2009.MATERIALS:A total of 35 clean male Sprague Dawley rats were selected.Of them,20 were used to prepare BMSCs.The remaining was randomly assigned to a normal control,model and cell transplantation groups,with 5 in each group.METHODS:Rat BMSCs were incubated by the whole bone marrow method.When 90% cells were confluent,BMSCs were digested in trypsin for subculture.In the model and cell transplantation groups,rats were used to establish radiation-induced models,and then underwent X-ray general irradiation,at a dose of 500 cGy/min,100 cm from the target,6 Gy each,once per week,for consecutively 3 weeks.24 hours following irradiation,BMSCs of 3 passage were collected at logarithmic phase.In the cell transplantation group,1 mL cell suspension was infused into the rat caudal vein,containing 3×10~6 cells,totally three times.In the normal control and model groups,rat caudal vein received an equal volume of saline.MAIN OUTCOME MEASURES:The following parameters were measured:results of hematoxylin-eosin staining;serum and kidney malondialdehyde (MDA) content and superoxide dismutase (SOD) activity;serum creatinine and urea nitrogen levels.RESULTS:Kidney histopathology demonstrated thin renal cortex,increased number of mesenchyme,uneven renal corpuscle,and disordered structure of renal glomerulus,narrow renal glomerular vessel,partial disappeared capsular space,and degeneration and sclerosis of some glomerulus in the model group.In the cell transplantation group,renal cortex became thick,with clear structure;interstitial hyperemia and edema was significantly relieved;many complete renal corpuscles were observed;partial renal glomerulus presented degeneration and sclerosis;significant capsular space could be seen.Oxygen free radical examination results showed that compared with the model group,SOD activity was significantly higher (P ＜ 0.05),MDA levels were significantly lower (P ＜0.05) in the cell transplantation group.Renal function examination results demonstrated that compared with the model group,serum creatinine and urea nitrogen levels were significantly reduced in the cell transplantation group (P ＜ 0.05).CONCLUSION:BMSC transplantation can effectively treat renal radiation injury and improve renal function.

The effects of mesenchymal stem cells on the aging kidneys in rats / 中华内科杂志

Objectives To study the effect of mesenchymal stem cells on the aging rat kidney and to explore the underlying mechanism. Methods Rat models of senile kidney were built with hypodermic injection of D-galactose daily. Injections of MSCs of 3 × 10<'6>were given to each rat through vena caudalis and CFSE was used as a tracing label to detect the distribution of MSCs in vivo. After 24 h, rats were dissected and their kidneys were frozen for section. MSCs were observed with Fluophot and quantitative analysis of the various parameters of kidney was performed under a light microscope with B12000 image analysis system.The contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and kidney were measured wtih hydroxylamine and chromatometry. The expression of VEGF and P16 mRNA in kidney tissue was detected with real-time PCR and Western blotting. Results MSCs was found homing in the rat kidney,and the glomerular size, sklerosis-rate and the average cell count of glomerulus in the treated group were different from those of the model group (P<0.05). In the treated group, the activity of SOD was significantly higher and the content of MDA was lower in serum and kidney than that in the model control group (P<0.05). The expression of VEGF mRNA and protein in the kidneys of MSCs group increased significantly as compared with the model group (P<0.05). The expression of P16 mRNA and protein in the kidney of MSCs group decreased significantly compared with the model group (P<0.05). Conclusion MSCs can increase the expression of VEGF while decrease the expression of P16, so as to play a key role in the anti-aging on rat kidney.

Staphylococcal protein A immunoadsorption plus nonmyeloablative chemotherapy with CD34+ autologous peripheral blood stem cell transplantation in the treatment of three cases of refractory systemic lupus erythematosns / 中华皮肤科杂志

Objective To observe the clinical efficacy of staphylococcal protein A immunoadsorption plus nonmyeloablative chemotherapy with CD34+ autologous peripheral blood stem cell transplantation in the treatment of refractory systemic lupus erythematosus (SLE). Methods Three patients with active SLE were enrolled into this study. All patients were diagnosed with lupus nephritis by renal biopsy and poorly responded to routine therapy. Before transplantation, patients were given 6 sessions of immunoadsorption apheresis using columns of staphylococcal protein A-silica with an interval of 3 days; each session processed 3 L plasma and a total of 18 L plasma was processed over the 6 treatments. Three days following the immunoadsorption apheresis, the mobilization of stem cells was realized by intravenous cyclophosphamide at a dose of 2 g per square meter of body surface area and subcutaneous recombinant human granulocyte colony-stimulating factor (G-CSF) at a dose of 5 g per kilogram of body weight per day for 5 days. Then, peripheral blood raonoclonal cells were obtained by CS-3000 Cell Separator, and passed through the Clini Macs CD34+ cell selection device, with the final concentration of CD34+ cells being 2.6×106, 2.1×106 and 2.4×106 per kilogram of body weight respectively, and that of CD3+ cells being 3×105, 2.1×105, and 2.0×105 per kilogram of body weight, respectively, in these three patients. The conditioning regimen consisted of oral fludarabine of 50 mg/d for 5 days plus intravenous pig anti-human thymocyte immunoglobulin (ATG) at a daily dose of 90 mg/kg for 5 days. After 72-hour treatment with ATG, the frozen stem cells were infused back to the patients. Clinical manifestations and lupus-correlated immune parameters were compared in patients at baseline and after transplantation. Results Following immunoadsorption apheresis, an obvious decrease was observed in the level of serum anti-dsDNA, antinuclear antibody and IgG antibodies, while an increase in the level of serum complement 3. All patients achieved the reconstruction of hemopoiesis 2-3 days after the transplantation. Also, an apparent clinical remission was achieved with the SLEDAI score being less than 3. Six months after the transplantation, serum anti-dsDNA and antinuclear antibodies as well as urine protein were undetectable, the level of complement 3 reached the normal range, and renal function was restored. Conclusions Staphylococcal protein A immunoadsorption plus nonmyeloablative CD34+ autologous peripheral blood stem cell transplantation are effective and safe for refractory SLE, but the long-term effect remains to be connfirmed by further studies.

2-methoxyestradiol-induced apoptosis in U937 cells through generation of reactive oxygen species / 吉林大学学报(医学版)

Objective To investigate the effect of 2-methoxyestradiol(2-ME) on U937 myeloid leukemia cell line and its mechanism.Methods The experiment was divided into control group(myeloid leukemia U937 cell in RPMI 1640 culture medium with equal DMSO),2-ME-treated group,NAC-treated group,and 2-ME+NAC-treated group.The cytotoxicity was analyzed by MTT assay.Apoptosis and cellular nitric oxide(NO) were detected by flow cytometry using annexin V and NO sensor dye.Superoxide anion was measured with a fluorescent plate reader by DHE.Results Viabilities of U937 cells treated with 2-ME(0.25,0.50,1.00,and 2.00 ?mol?L-1) for 48 h were gradually reduced to 0.68?0.05,0.28?0.07,0.18?0.07,and 0.11?0.04,respectively.The differences were significant compared with control group(1.00?0.05)(P0.05).2.00 ?mol?L1 2-ME also significantly increased the mean fluorescence of NO sensor dye and superoxide anions in U937 cells compared with control group(P0.05).An markedly increase in apoptotic cells was detected after 2-ME treatment for 3 h(6.78%?1.01% vs 1.59%?0.12%,P

Antileukemic activity of donor-derived NK cell in mice in vivo / 吉林大学学报(医学版)

0.05). The survival time of the second control group and experimental groups was much longer than that of the first control group (P

Low expression of MHC class I-related chain A and B reduces NK cell-mediated lysis in nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP / 中国肿瘤生物治疗杂志

Objective: To analyze the expression of HLA class Ⅰ molecules and MHC classⅠ related chain A and B (MICA/MICB) in human nasopharyngeal carcinoma cell line (CNE2) and multi-drug resistant nasopharyngeal carcinoma cell line (CNE2/ DDP), and to assess their influence on NK cell-mediated lysis.Methods: Expression of HLA classⅠ molecules and MICA/MICB on the surface of CNE2 and CNE2/DDP cell lines was analyzed by flow cytometry. Cytotoxicity of NK cells (isolated from 3 healthy persons) against CNE2 and CNE2/DDP cells were detected by LDH releasing assay at different effect-to-target cell ratios (E∶T). In blocking experiments, anti-MHC class Ⅰ monoclonal antibody (mAb) (W6/32, a pan anti-HLA class Ⅰ antibody) and anti-MHC class I chain related molecules mAb (BAMO-1, specificly against MICA and MICB) were added to the target cells at a E∶T ratio of 10∶1. Results:It was found that the expression of HLA class Ⅰ molecules and MICA/MICB on CNE2 was higher than that on CNE2/DDP(P

Effect of donor's activating KIR on prognosis after haemopoietic stem cell transplantation / 中华器官移植杂志

Objective To investigate the effect of donors' activating killer cell immunoglobulin-like receptor (aKIR) on recipients prognosis post haemopoietic stem cell transplantation (HSCT).Methods Fifty-nine cases of related HLA full matched HSCT were studied. Sequence specific primer polymerase chain reaction (SSP-PCR) was used to type donors' KIR. Virus,bacterium,and fungi infection as well as bleeding,relapse,survival were observed in the recipients post transplantation. Relationship between aKIR and clinical indexes mentioned above were analyzed.Results Donors' aKIR showed no beneficial effects on recipients' virus and bacterium infection. Furthermore,the incidence of fungi infection was increased in HSCT if donors expressed KIR3DS1 (?2= 4.804 ,P= 0.028 ). No significant difference was found in survival curve between donor aKIR positive HSCT or negative one (KIR2DS1+/-: P= 0.651 ; KIR2DS2+/-: P= 0.847 ; KIR3DS1+/-: P= 0.341 ). No effect of donor's aKIR on relapse was observed,too.Conclusion In Guangdong Han,so far as related HLA full matched HSCT is concerned,no improvement in recipient prognosis due to donor's aKIR is verified.

Effect of Ly49A transfected lymphocytes on graft versus host disease and graft versus leukemia effect after allogeneic bone marrow transplantation / 中华器官移植杂志

Objective To observe the effect of Ly49A transfected lymphocytes on graft versus host disease (GVHD) and graft versus leukemia (GVL) effect post allogeneic bone marrow transplantation. Methods Ly49A gene was transfected into lymphocytes of C57BL/6 mice by retrovirus and the expression rate of Ly49A receptor was detected by flow cytometer. The murine model of allogeneic acute GVHD was established in this way: the donor was C57BL/6(H 2 b). The recipient was BALB/c(H 2 d), which was injected EL9611 cells. After the irradiation (TBI, 60 Co 9.0 Gy) the recipient accepted the transplantation of mixed spleen cells and bone marrow cells to establish the GVHD model. The effects of Ly49A transfected spleen cells on GVHD and GVL post allogeneic bone marrow transplantation were detected. Results The expression rate of Ly49A receptor was ( 42.20 ? 4.87 )?% for lymphocytes transfected with pLXSN Ly49A; ( 18.67 ? 2.48 )?% for lymphocytes transfected with pLXSN and ( 18.73 ? 3.82 )?% for untransfected control respectively. In the case of allo BMT (C57BL/6 H 2 b→BALB/c H 2 d), survival time was ( 6.50 ? 2.41 ) days for the irradiation group; ( 20.90 ? 2.88 ) days for cyclophosphomide therapy group; ( 17.10 ? 4.65 ) days for bone marrow cells mixed with lymphocytes transplantation group; ( 17.40 ? 5.32 ) days for bone marrow cells mixed with pLXSN transfected lymphocytes transplantation group; ( 35.20 ? 12.52 ) days for bone marrow cells mixed with Ly49A transfected lymphocytes transplantation group, which was much longer than the survival time of any of the above group ( P = 0.000 ). Conclusion The Ly49A transfected lymphocytes transplantation could alleviate GVHD and retain GVL effect on acute GVHD model post allo BMT.

Induction of tolerance in MHC haploidentical bone marrow transplantation in mice by combination of Tju103 and CTLA4-Ig / 中华器官移植杂志

Objective To observe the effect of combination of Tju103 and CTLA4-Ig on engraftment, graft versus host disease (GVHD), graft versus leukemia (GVL) and anti-infection post major histocompatibility complex (MHC) haploidentical bone marrow transplantation in mice in order to seek an effective access to transplantation with less GVHD and more potential GVL and anti-infection.Methods In the presence of the recipient's antigen (normal CB6F1, H-2 bd) as a stimulus for induction of specific immune tolerance, T cells from the MHC haploidentical donors (C57BL/6, H-2 b) were first in vitro cultured with Tju103 and CTLA4-Ig, then were transfused with the donors' bone marrow cells into the preconditioned recipients. At last, the effect of combination of Tju103 and CTLA4-Ig on hematopoietic rebuilding, GVHD, GVL and anti-infection was observed in compared with CsA, Tju103 and CTLA4-Ig as controls.Results The only irradiated group (group A): All the mice (10 mice) died of failure of hematopoiesis within 11 days post irradiation, of which most (8 mice) died within 4～7 days post transplantation. The CTX-treated leukemia group (group B): All the mice (10 mice) died of leukemia within 16～23 days post leukemia cells infusion (11～18 days post BMT). CTX treatment prolonged the survival time. The only transplanted group (group C): The mice began to die from day 16 post transplantation, and all (10 mice) died of GVHD within 3 weeks. The CsA prophylaxis group (group D): 4 mice died within 8～22 days after transplantation, of which one died of leukemia, two died of infection and one died of GVHD, and the remaining 6 survived over 30 days post transplantation. The Tju103 treated group (group E): 4 mice died within 9～26 days post transplantation, of which one died of leukemia, one died of infection and two died of GVHD, and the remaining 6 mice survived over 30 days post transplantation. The CTLA4-Ig treated group (group F): 3 mice died within 14～23 days after transplantation, of which one died of infection and two died of GVHD, and the remaining 7 survived over 30 days post transplantation. The Tju103/CTLA4-Ig treated group (group G): one died of GVHD on day 19 after transplantation, and the remaining 9 mice survived over 30 days post transplantation. Conclusions CsA, Tju103 or CTLA4-Ig alone could prolong survival time and reduce incidence and degree of GVHD severity. But CTLA4-Ig could spare much ability of GVL and anti-infection while Tju103, just like CsA, couldn't. Combination of both was the most favorable way for transplantation with the most remarkable efficiency on prolongation of survival time and reduction of GVHD.

Application of FBC conditioning regimen in HLA haplotype peripheral blood stem cell transplantation / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the influence of decreasing conditioning regimen intensity on the engraftment of HLA haplotype peripheral blood stem cell transplantation.</p><p><b>METHOD</b>Twelve patients with leukemia, including 4 in complete remission, whose HLAs were full matched with donors, and 8 with refractory leukemia, whose HLAs were mismatched, were transplanted with G-CSF mobilized allogeneic peripheral blood stem cells after conditioned with a regimen consisting of fludarabine (30 mg/m(2) x 6 days), busulfan (4 mg/kg x 2 days) and cyclophosphamide (30 approximately 60 mg/kg x 2 days) (FBC). Donor lymphocytes were infused at day + 30, + 60 and + 90 after transplantation, respectively. Hematopoietic reconstitution was observed. Engraftment was documented by the analysis of short tandem repeats with polymerase chain reaction (STR-PCR).</p><p><b>RESULT</b>Patients in HLA haplotype group received a mean number of 4.87 x 10(8)/kg donor mononuclear cells (MNC), with CD(34)(+) cells of 4.58 x 10(6)/kg and patients in HLA identical group a mean number of 4.85 x 10(8)/kg MNC with CD(34)(+) cells of 4.47 x 10(6)/kg. The mean time of white blood cell count more than 1.0 x 10(9)/L was 14 (10 approximately 18) days in HLA matched patients and 29 (11 approximately 90) days in HLA haplotype group. One three locus mismatched patient failed to engraft, but auto-hematopoiesis was recovered on day + 50. Full donor chimerism was observed in all patients except one with mixed chimera. The mixed chimera was converted into full donor chimera after three times donor lymphocyte infusion. One each died from severe acute GVHD, severe VOD and severe chronic GVHD in HLA haplotype group, and one from chronic GVHD in HLA identical group.</p><p><b>CONCLUSION</b>Patients survived engraftment was not influenced by decreasing conditioning intensity as in this regimen. Haplotype stem cells could be engrafted durable in recipients by this regimen combined with donor lymphocyte infusion.</p>

Effect of Ly49A transfected mouse spleen cells on graft versus host disease and graft versus leukemia after haploidentical allogeneic bone marrow transplantation in mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To observe the effect of Ly49A transfected mouse spleen cells on graft versus host disease (GVHD) and graft versus leukemia (GVL) effect after haploidentical allogeneic bone marrow transplantation in mice.</p><p><b>METHODS</b>Ly49A gene was transfected into spleen cells of C57BL/6 mice by retrovirus and the expression rate of Ly49A receptor was evaluated by flow cytometry. The murine model of haploidentical allogeneic acute GVHD was established by using C57BL/6(H - 2b) mouse as donor, and (BALB/c x C57BL/6) F1(H - 2d/b) (CB(6)F(1)) mouse as the recipient which was injected EL9611 cells before transplantation. After irradiation (TBI, (60)Co 10.5 Gy), the recipient received mixed graft of spleen cells and bone marrow cells to establish a GVHD model. The effects of Ly49A transfected spleen cells on GVHD and GVL post haploidentical allogeneic bone marrow transplantation were detected with this model.</p><p><b>RESULTS</b>The expression rate of Ly49A receptor was (42.20 +/- 4.87)%, (18.67 +/- 2.48)% and (18.73 +/- 3.82)% for pLXSN-Ly49A, pLXSN transfected and untransfected spleen cells respectively. Among haploidentical allo-BMT (C57BL/6(H - 2b)-->CB6F1(H - 2d/b)) groups, the survival time was (7.80 +/- 3.36) days for irradiation group; (21.70 +/- 2.87) days for cyclophosphomide therapy group; (29.40 +/- 6.43) days for mixed bone marrow cells and spleen cells transplantation group; (29.10 +/- 7.39) days for mixed bone marrow cells and pLXSN transfected spleen cells transplantation group and (45.00 +/- 12.38) days for mixed bone marrow cells and Ly49A transfected spleen cells transplantation group, which was much longer than that of any other groups (P = 0.000).</p><p><b>CONCLUSION</b>The Ly49A transfected spleen cell transplantation could alleviate GVHD and retain GVL effect in the acute GVHD model post haploidentical allo-BMT.</p>

Expression of human G6PD gene in K562 cells mediated by retroviral vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>This study aimed to investigate the feasibility of gene therapy for severe G6PD deficiency.</p><p><b>METHODS</b>The recombinant retroviral vector bearing normal human G6PD cDNA was constructed and transferred into the erythroleukemia cell line K562. Author identified the integration of NeoR gene in the targeted cellular DNA by means of specific PCR. Quantitative method was used to measure the expression of G6PD in the targeted cells.</p><p><b>RESULTS</b>Construction of the recombinant retroviral vector was successfully established. PCR indicated the integration of NeoR gene in the targeted genomic DNA of the cells. The vector was also shown to be capable of expressing the foreign gene compared to the control (P<0.01).</p><p><b>CONCLUSIONS</b>The recombinant retroviral vector is competent for transferring and expressing the G6PD gene.</p>

Cloning and Regulating Expression of Human CD34 TRS / 中国肿瘤生物治疗杂志

Objective: To clone the 5＇-flanking region of the human CD34 gene containing transcriptional regulatory sequence (TRS). Methods: According to the registered 5＇-flanking region of CD34 gene, two pairs of primers were designed and net-PCR was used to amplify 661 bp long TRS of CD34 gene. The CD34 TRS fragment was cloned into reported plasmid pEGFP-1. The role of the regulating the specific expression of recombinant plasmid pCD34 EGFP in hematopoietic and non-hematopoietic cells was observed. Results: Restrictive endonuclease identification and DNA sequencing provedthat the CD34 promoter cloned was consistent with the sequence reported to a large extent. It could induce the EGFP gene to express in hematopoietic cell line K562 specifically, while has no effect on hepatocellular carcinoma cell HepG-2. Conclusion: The cloned CD34 gene TRS has the effect of regulating gene expression specifically. The study established the fundament for the construction of specific gene expression vector used in hematopoietic system cells.

Effects of Allogeneic Transplantation of T Helper-2 Cells on Graft-versus-Host Disease and Graft-versus-Leukemia Effect / 中国实验血液学杂志

This study was undertaken to explore whether the graft-versus-host-disease could be decreased and graft-versus-leukemia effect be retained by transplantation of allogeneic T helper-2 (Th2) cells. T cells from C57BL/6(H-2b) mice were incubated and polarized with rmIL-4, Con A and ionomycin in vitro, and then, the T cells were mixed with marrow cells and transplanted into recipient BALB/c(H-2d) mice bearing erythroleukemia cells. The occurence of GVHD and GVL effect was observed. The results showed that the mean survival time in the groups of untreated control, cyclophosphamide treatment, marrow and spleen T cell transplantation and marrow and Th2 cell transplantation was 10.6 +/- 1.3, 18.7 +/- 4.2, 22.7 +/- 7.4 and 36.9 +/- 10.8 days, respectively. In untreated control and cycophosphamide treatment groups, all of ten mice died from leukemia. Nine of ten mice died from GVHD in marrow and spleen T cells transplantation group. In marrow and spleen Th2 cell transplantation group, three of ten mice died from GVHD, and GVHD was not occurred in the other seven mice, and there was no any evidence of leukemia in two mice on 50 days after transplantation. It was concluded that tranplantation with polarized Th2 cells could relieve GVHD, and at the same time retain the GVL effect.