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Chronic granulomatous disease (CGD) in children is a rare primary immunodeficiency disorder that can lead to life-threatening infections and inflammatory complications. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is increasingly being used to treat severe CGD in children. We conducted a multicenter retrospective analysis of children with CGD who were treated with allo-HSCT at four pediatric hematopoietic stem cell transplant centers in China from September 2005 to December 2019. The study included a total of 171 patients (169 males and 2 females). The median age at the time of transplantation was 6.1 (0-16.4) years. Among them, 154 patients had X-linked recessive inheritance caused by CYBB gene mutations, 12 patients were autosomal recessive, 1 patient had DNAH11 and HYDIN gene mutations, and 4 patients had no gene mutations. The median follow-up period was 36.3 (1.9-79) months. All participating patients were applied to myeloablative conditioning (MAC) regimens. The rates of OS, EFS, and GEFS within three years were 87.5%, 85.3%, and 75.2%, respectively. The total graft failure and the total mortality rate were 5.3% and 11.1%. The cumulative incidence of acute GVHD was 53.8% and the incidence of chronic GVHD was 12.9%, The incidence of chronic GVHD was higher for patients who received unrelated donor cord blood stem cell transplantation (UD-CB) (P = 0.001). Chronic GVHD and coinfections are the risk factors for OS and EFS in patients with CGD after receiving allo-HSCT. UD-CB is a risk factor for EFS and the presence of pneumonia before transplantation is a risk factor for OS. In conclusion, through this study, we have demonstrated that allo-HSCT has excellent efficacy in the treatment of CGD in children, especially, RD-haplo is associated with a lower rate of graft failure incidence and mortality than the treatment modalities of other donor type. Therefore, allo-HSCT is strongly recommended when a well-matched donor is available. If a well-matched donor is not available, the HLA-mismatched donor should be carefully evaluated, and the conditioning regimen modified accordingly.
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Doença Enxerto-Hospedeiro , Doença Granulomatosa Crônica , Transplante de Células-Tronco Hematopoéticas , Masculino , Criança , Feminino , Humanos , Adolescente , Estudos Retrospectivos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , Doença Granulomatosa Crônica/complicações , Doença Enxerto-Hospedeiro/etiologia , Doadores não Relacionados , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , China , Condicionamento Pré-TransplanteRESUMO
Objective: X-linked chronic granulomatous disease (X-CGD) is a rare primary immunodeficiency disease characterized by phagocyte dysfunction. It is caused by genetic mutations in the CYBB gene, predominantly affecting males. However, a small number of female carriers can also present with the disease due to biased X chromosome inactivation.1 This study aims to enhance the understanding of X-CGD in a rare case of an infant and young woman and provide insights into its diagnosis and treatment. Methodology: This study utilized various methods to investigate X-CGD in children and their parents. These methods included assessing neutrophil respiratory burst function, measuring gp91phox protein expression, analyzing chronic granuloma enzyme levels, conducting whole exon gene analysis, and evaluating X chromosome inactivation. Additionally, hematopoietic stem cell transplantation was performed using haploidentical donors from immediate family members. Results: The children in this study were found to be carriers of the CYBB gene mutation, and their neutrophil respiratory burst function was abnormal with no expression of the gp91phox protein. X chromosome inactivation analysis revealed a rate of 99.5%. Following hematopoietic stem cell transplantation, there was successful engraftment of granulocytes and megakaryocytes, with normalization of gene and enzyme examinations. Conclusion: The findings of this study highlight the importance of considering X-CGD in the diagnosis of children and women presenting with granulomatous disease. Furthermore, the use of hematopoietic stem cell transplantation was shown to achieve significant therapeutic effects in the treatment of X-CGD. Further research is warranted to explore early diagnostic strategies for X-CGD and to optimize the use of hematopoietic stem cell transplantation in managing the disease. Early diagnosis and intervention can lead to improved outcomes for patients with X-CGD. This study contributes to the understanding of X-CGD and its treatment by demonstrating the possibility of X-CGD in female carriers and the efficacy of hematopoietic stem cell transplantation. These findings emphasize the importance of early diagnosis and highlight the potential for successful outcomes in the management of X-CGD.
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INTRODUCTION: This retrospective study aimed to compare a range of conditioning regimens in children with severe aplastic anemia (SAA) undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) at the Seventh Medical Center of PLA General Hospital between January 2008 and June 2017. METHODS: Patients were categorized into the Bu (Bu + Flu + Cy + ATG-F regimen) and control (Flu + Cy + ATG-F) groups, with a median follow-up time after HSCT of 3.5 (range, 3.1-6.2) and 3.7 (3.2-5.9) years in the Bu and control groups, respectively. RESULTS: No differences were observed between the two groups regarding the median time of peripheral blood neutrophil and platelet engraftment (p = 0.538 and p = 0.491); the 28-day engraftment rates of neutrophils were similar (p = 0.199), although higher for platelets with Bu (p = 0.044). Additionally, graft failure was 0% and 20.0% in the Bu and control groups, respectively (p = 0.004). In both groups, the incidence of grades III-IV (or grades II-IV) acute graft-versus-host disease (GVHD) and chronic GVHD was not significantly different (p > 0.05). Moreover, the 3-year overall survival and failure-free survival did not show significant differences (p = 0.670 and p = 0.908). DISCUSSION: In children with SAA undergoing allo-HSCT, conditioning regimen with Bu + Flu + Cy + ATG-F is capable of enhancing the myeloablation effect, promoting donor hematopoietic stem cell engraftment, and reducing the graft failure rate. Furthermore, it does not increase the incidence of complications, including GVHD.
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Anemia Aplástica , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Criança , Humanos , Bussulfano/uso terapêutico , Estudos Retrospectivos , Anemia Aplástica/terapia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Condicionamento Pré-Transplante , CiclofosfamidaRESUMO
AIMS: To compare the efficacy of hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA)-haploidentical related donors (RD) and unrelated donors (URD) in the treatment of leukemia. METHODS: Ninety-three leukemia patients underwent allogeneic HSCT were divided into two groups: 51 cases of RD-HSCT and 42 cases of URD-HSCT. In the RD-HSCT group, a preconditioning regimen with fludarabine (Flu) + busulfan (Bu) + cytosine arabinoside (Ara-C) was used in 42 patients and total body irradiation (TBI) + Flu + Ara-C was used in the remaining 9. RESULTS: In the URD-HSCT group, a modified preconditioning regimen with Bu + cyclophosphamide was used in 35 patients, while the other 7 patients underwent treatment with TBI + Flu. After transplantation, the occurrence rate of grade II-IV acute graft-versus-host disease (GVHD) was 46.0 and 51.2% in the two groups. Likewise, the rate of chronic GVHD was 46.0 and 63.4%, respectively. No significant differences in the occurrence of acute and chronic GVHD were detected between the two groups. The differences in early-stage infection rate after transplantation, recurrence rate, 3-year survival rate, and disease-free survival rate between the two groups were not significant. CONCLUSION: HLA-haploidentical RD-HSCT with enhanced preconditioning and administration of immunosuppressants showed a clinical efficacy similar to that of URD-HSCT against leukemia, without the risk of increased infection and GVHD.
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Antígenos HLA/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia/terapia , Doadores não Relacionados , Adolescente , Adulto , Criança , China/epidemiologia , Feminino , Doença Enxerto-Hospedeiro/epidemiologia , Doença Enxerto-Hospedeiro/mortalidade , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/mortalidade , Humanos , Masculino , Pessoa de Meia-Idade , Condicionamento Pré-TransplanteRESUMO
The treatment of pediatric severe aplastic anemia (SAA) with allogeneic hematopoietic stem cell transplantation (allo-HSCT), presents major challenges including the risks of graft failure, septic complications, and graft-versus-host disease (GVHD). Additive infusions of human umbilical cord derived mesenchymal stem cell (hUC-MSC) may be administered to improve patient survival. We retrospectively examined 37 pediatric patients with SAA who received allo-HSCT and subsequent infusions of hUC-MSC suspension at a dose of 1.0 × 10(6 )/kg. The times and doses of hUC-MSC infusions were increased in patients with severe GVHD. All patients received hUC-MSC infusions. The median time to post-transplantation neutrophil count of greater than 0.5 × 10(9 )/L was 14 days (range, 11-20 days) and time to post-transplantation platelet count of greater than 20 × 10(9 )/L was 19 days (14-29 days). The overall frequency of acute GVHD (aGVHD) was 45.9% (17/37). These aGVHD episodes occurred at a median time of post-transplantation 47 days (15-83 days). The frequency of chronic GVHD (cGVHD) was 18.9% (7/37); cGVHD developed from aGVHD in 10.8% (4/37) of patients. The GVHD-associated mortality rate was 18.9% (7/37) and aGVHD-specific mortality rate was 8.1% (3/37). The median overall survival time was 35 months (9-67 months) and the three-year overall survival rate was 74.2% (28/37). Seven patients died of GVHD, one patient died of a severe invasive fungal infection, and one patient died of renal failure. In conclusion, post-transplantation hUC-MSC infusions seemed to be safely infused in children with SAA who have previously received allo-HSCT.
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Anemia Aplástica/cirurgia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Aloenxertos , Contagem de Células Sanguíneas , Transplante de Medula Óssea/estatística & dados numéricos , Criança , Pré-Escolar , Transplante de Células-Tronco de Sangue do Cordão Umbilical/estatística & dados numéricos , Feminino , Sobrevivência de Enxerto , Doença Enxerto-Hospedeiro/diagnóstico , Doença Enxerto-Hospedeiro/epidemiologia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas/estatística & dados numéricos , Humanos , Lactente , Masculino , Transplante de Células-Tronco Mesenquimais/estatística & dados numéricos , Micoses/etiologia , Micoses/mortalidade , Transplante de Células-Tronco de Sangue Periférico/estatística & dados numéricos , Complicações Pós-Operatórias/epidemiologia , Insuficiência Renal/etiologia , Insuficiência Renal/mortalidade , Estudos Retrospectivos , Sepse/etiologia , Sepse/mortalidade , Taxa de Sobrevida , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
Hematopoietic stem cell transplantation (HSCT) is curative in patients with primary immunodeficiency syndrome. The safety and efficacy of HSCT as a therapeutic option for primary immunodeficiency diseases (PID) have been studied by many research groups. The purpose of our study was to perform a bibliometric analysis of research on HSCT for the treatment of PID, to assess research trends in this field, and note future research priorities. The Web of Science Core Collection (WOSCC) was used to identify relevant publications. VOSviewer and CiteSpace software were used to analyze bibliometric parameters, such as yearly records, authors, grouped authors, countries, institutions, categories and keywords. There are 602 relevant records for the last decade (2013-2022). The top 5 productive authors and high-quality paper journals are listed. Reference co-citations analysis demonstrated recent research trends were "inborn errors of immunity," "gene editing," and "enteropathy." Research on HSCT for the treatment of PID has increased rapidly in the last decade, and bibliometrics are valuable for researchers to obtain an overview of hot categories, academic collaborations and trends in this study field.
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Transplante de Células-Tronco Hematopoéticas , Doenças da Imunodeficiência Primária , Humanos , Bibliometria , Edição de Genes , PesquisadoresRESUMO
It has been established that the tumor microenvironment (TME) has a crucial role in enabling tumors to evade from host immune responses. Previous studies demonstrated that tumor cells are not only able to reshape immune milieu at the tumor site, but also exert systemic effects, which has been demonstrated to be important for metastasis. At present, how the peripheral immune environment change in the tumor-bearing host is unclear. The present study identified a number of changes in the proportions of lymphocyte subpopulations and the levels of cytokines in patients with NSCLC, which may provide a preliminary profile of the immune environment in the peripheral blood of patients harboring a tumor. These findings expand on the present knowledge on how tumors can alter the immune system to benefit its growth and metastasis, which may provide a potential novel strategy for immunotherapy.
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OBJECTIVE: To construct cell adhesion mediated drug resistance (CAM-DR) model based on Acute lymphocyte leukemia bone marrow stromal cells(BMSC) for further studying drug resistance of leukemia. METHODS: Firstly, we adhesively cultured Jurkat cell strain of human leukaemia lymphocyte with the matrix cell radiated by (60)Co to construct the model of CAM-DR, then evaluated the model in morph and construction by scanning electron microscope. The IC50 of DNR on Jurkat cell was examen by MTT and the concentration and distribution of DNR in the cell was detected by flow cytometry. RESULTS: When the Jurkat cells were cultured with BMSC for 24 h, we found that Jurkat cells had adhesioned the bone marrow stromal cell layer by parapodium and some of them had nichd the mesh consisted by the confluence of BMSC. At the point of 48 h, some Jurkat cells had migrated to underlayer of BMSC, and Jurkat cells were nichd the mesh of BMSC like nidi. The accumulation and distribution of DNR in the Jurkat cells were not affected in the model, but the reaction of Jurkat cells to DNR were significantly inhibited. CONCLUSION: The model of CAM-DR based on Acute lymphocyte leukemia bone marrow stromal cells was successfully built.
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Células da Medula Óssea/patologia , Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Estromais/patologia , Adolescente , Adulto , Antibióticos Antineoplásicos/farmacologia , Células da Medula Óssea/ultraestrutura , Adesão Celular , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Daunorrubicina/farmacologia , Feminino , Humanos , Concentração Inibidora 50 , Células Jurkat , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Células Estromais/ultraestrutura , Células Tumorais CultivadasRESUMO
This study was aimed to investigate the safety and effectiveness of tumor-ablative Chemotherapy combined with low intensity conditioning regiment BUCy/TBICy for patients with hematologic malignancies receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical data of 30 patients with hematologic malignancies received above-mentioned therapeutic method from January 2012 to January 2013 was analyzed retrospectively, and the engraftment, GVHD, infection, conditioning-related toxicity, relapse and survival rates were evaluated. All the patients signed the informed consent before transplantation. The median follow-up duration was 20.5 (16.3-27.3) months. The results indicated that all the patients had been engrafted successfully. One year overall survival (OS) and disease-free survival (DFS) rates were 93.3% and 83.3% respectively. No conditioning-related toxicity occurred. The incidences of II-IV grade aGVHD was 37.9%, among which incidence of III-IV grade aGVHD was 3.4%; incidence of extensive cGVHD was 13.8%. So far, 1 case relapsed, 1 case displayed graft rejection, and poor function of graft occurred in 1 case, death occurred in 2 cases(6.7%). It is concluded that tumor-ablative chemotherapy combined with low intensity-modified BUCy/TBICy is safe and effective in allogeneic hematopoietic stem cell transplantation for hematologic malignancies, and it is useful to reduce relapse of hematologic malignancies after transplantation.
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Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Condicionamento Pré-Transplante , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Condicionamento Pré-Transplante/efeitos adversos , Condicionamento Pré-Transplante/métodos , Transplante Homólogo/efeitos adversos , Resultado do Tratamento , Adulto JovemRESUMO
We recently reported that Cx43 expression and gap junction intercellular communication (GJIC) between acute leukemic bone marrow stromal cells (BMSCs) were deficient, which could recovery after effective chemotherapy. However, the exact role of GJIC in the hematopoietic microenvironment in leukemic cell death and proliferation is not clear. We show here that following transfection with the Cx43 gene, GJIC function was increased between leukemic BMSCs. Furthermore, compared with leukemic cells alone, the proliferation and apoptosis of leukemic cells co-cultured with BMSCs were inhibited, the percentage of G0-phase cells was higher; and expression of p53 increased and bax decreased. However, after co-culturing leukemic cells with Cx43-modified BMSCs, the number of proliferative and spontaneously apoptotic Jurkat cells increased; the percentage of G0-phase cells decreased; the expression of p53 decreased; and bax increased. Compared with Jurkat cells co-cultured with BMSCs and Jurkat cells alone, the sensitivity of leukemic cells co-cultured with Cx43-modified BMSCs to chemotherapeutics increased. Our data suggests that GJIC between leukemia BMSCs is one of the impact factor to the proliferation, apoptosis and drug sensitivity of co-cultured leukemic cells. Up-regulating its function can inhibit the protective effects of leukemic BMSCS and enhance the efficacy of therapies in hematologic malignancies.
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Apoptose , Células da Medula Óssea/patologia , Conexina 43/genética , Leucemia Mieloide Aguda/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Estromais/patologia , Adulto , Western Blotting , Células da Medula Óssea/metabolismo , Comunicação Celular , Ciclo Celular , Proliferação de Células , Técnicas de Cocultura , Feminino , Citometria de Fluxo , Junções Comunicantes , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
The hematopoietic inductive microenvironment (HIM) is where hematopoietic stem/progenitor cells grow and develop. Hematopoietic stromal cells were the key components of the HIM. In our previous study, we had successfully cultured and isolated human cord blood-derived stromal cells (HUCBSCs) and demonstrated that they could secret hemopoietic growth factors such as GM-CSF, TPO, and SCF. However, it is still controversial whether HUCBSCs can be used for reconstruction of HIM. In this study, we first established a co-culture system of HUCBSCs and cord blood CD34(+) cells and then determined that using HUCBSCs as the adherent layer had significantly more newly formed colonies of each hematopoietic lineage than the control group, indicating that HUCBSCs had the ability to promote the proliferation of hematopoietic stem cells/progenitor cells. Furthermore, the number of colonies was significantly higher in vascular cell adhesion molecule-1 (VCAM-1)-modified HUCBSCs, suggesting that the ability of HUCBSCs in promoting the proliferation of hematopoietic stem cells/progenitor cells was further enhanced after having been modified with VCAM-1. Next, HUCBSCs were infused into a radiation-damaged animal model, in which the recovery of hematopoiesis was observed. The results demonstrate that the transplanted HUCBSCs were "homed in" to bone marrow and played roles in promoting the recovery of irradiation-induced hematopoietic damage and repairing HIM. Compared with the control group, the HUCBSC group had significantly superior effectiveness in terms of the recovery time for hemogram and myelogram, CFU-F, CFU-GM, BFU-E, and CFU-Meg. Such differences were even more significant in VCAM-1-modified HUCBSCs group. We suggest that HUCBSCs are able to restore the functions of HIM and promote the recovery of radiation-induced hematopoietic damage. VCAM-1 plays an important role in supporting the repair of HIM damage.
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Transplante de Células/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Células Estromais/citologia , Veias Umbilicais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Proliferação de Células , Técnicas de Cocultura , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Plasmídeos/metabolismo , Células-Tronco/citologiaRESUMO
OBJECTIVE: To explore the feasibility and safety of conditioning regimen containing fludarabine (Flud) for haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Preparative regimen containing Flud 40 mgxm(-2)xd(-1) on day -7 to -3 in place of cyclophosphamide (CTX) for haploidentical HSCT was given to 35 patients with hematologic malignancies (4 standard risk, 16 high risk, 15 relapse with no remission). All donors received rhG-CSF followed by HSC harvest. One patient received peripheral blood HSCT (PBSCT), one bone marrow transplantation (BMT), and the others BM combination with PBSCT. The regimen-associated side effect, engraftment, incidence of graft-versus-host disease (GVHD) and disease-free survival (DFS) probabilities were observed. RESULTS: All patients achieved sustained, full donor-type engraftment. Thirty-four patients obtained primary durable engraftment, and 1 who rejected graft from his mother obtained successful durable engraftment after the second graft from his father. The cumulative incidence of grade III-IV acute GVHD and chronic GVHD was 12.1% and 31.7%, respectively. With a follow-up duration of 8-25 months, 6 patients were dead, in which 3 died of relapse, 2 of acute GVHD, 1 of fungal infection, none died of regimen-associated side effect. The other 29 patients remained alive and DFS probability was 79.7%. CONCLUSION: Flud based conditioning regimens for haploidentical HSCT is safe and feasible, which reduces regimen-associated side effect, with no increasing the rate of relapse and infection, and decreases the incidence of aGVHD.
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Transplante de Células-Tronco Hematopoéticas , Condicionamento Pré-Transplante/métodos , Vidarabina/análogos & derivados , Adolescente , Adulto , Criança , Pré-Escolar , Ciclofosfamida , Estudos de Viabilidade , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Masculino , Pessoa de Meia-Idade , Condicionamento Pré-Transplante/efeitos adversos , Transplante Homólogo , Vidarabina/efeitos adversos , Vidarabina/uso terapêutico , Adulto JovemRESUMO
This study was aimed to investigate the effect of vcam-1 gene-modified human umbilical cord blood derived stromal cells (CBDSCs) on hematopoietic regulation so as to establish the experimental foundation for further study. The target gene vcam-1 was cloned into the shuttle plasmid with the report gene GFP. The recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-l, and the recombinant adenoviral vector ad-vcam-1-gfp was confirmed after transfection with CBDSCs. The results indicated that two fragments of about 9 kb and 2 kb were obtained after digestion of recombinant plasmid pAdTrack-vcam-1 with NotIand XhoI, and single fragment of 600 bp was obtained after amplification with PCR; two fragments of about 31 kb and 4 kb were obtained after digestion of recombinant plasmid pad-vcam-1-gfp with PacI, which suggested a successful homologous recombination. The expression of vcam-1 gene in ad-vcam-1-gfp transfected CBDSCs could be detected by immunocytochemistry, RT-PCR and fluorescent microscopy. It is concluded that the recombinant adenoviral vector ad-vcam-1-gfp has been constructed successfully, and the expression of vcam-1 is up-regulated in CBDSCs transfected by gene ad-vcam-1-gfp.
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Adenoviridae/metabolismo , Sangue Fetal/citologia , Proteínas de Fluorescência Verde/metabolismo , Células Estromais/citologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adenoviridae/genética , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
This study was purposed to investigate the connexin 43 (Cx43) expression level in acute leukemia bone marrow stromal cells (ABMSCs) and normal bone marrow stromal cells (NBMSCs), and to explore the difference in communicating functions between these cells. The Cx43 expression levels of ABMSCs and NBMSCs were detected by using immunohistochemistry and computer gray scale assay, and the difference of gap junction intercellular communication (GJIC) was examined through dry transfer technique. The results showed that expression level of Cx43 in ABMSCs was lower than that in NBMSCs and its function of GJIC in ABMSCs was also weaker than that in NBMSCs. It is concluded that cell-cell communication function is lowered in ABMSCs.
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Células da Medula Óssea/metabolismo , Comunicação Celular/fisiologia , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Leucemia/metabolismo , Células Estromais/metabolismo , Doença Aguda , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Humanos , Células Estromais/citologia , Células Tumorais CultivadasRESUMO
OBJECTIVE: To find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition. METHODS: The HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days. RESULTS: (1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05). CONCLUSION: (1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.