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1.
Chinese Journal of Hematology ; (12): 366-372, 2023.
Article in Chinese | WPRIM | ID: wpr-984631

ABSTRACT

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.


Subject(s)
Humans , U937 Cells , RUNX1 Translocation Partner 1 Protein , Leukemia/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Oncogene Proteins, Fusion/genetics , Leukemia, Myeloid, Acute/genetics
2.
China Tropical Medicine ; (12): 886-2023.
Article in Chinese | WPRIM | ID: wpr-1005159

ABSTRACT

@#Abstract: Objective To investigate the correlation of serum exosomal microRNA (exomiR)-27a with drug resistance and adverse outcomes in pulmonary tuberculosis (PTB), to provide new evidence for the development of anti-tuberculosis treatment. Methods From May 2018 to June 2020, 326 patients with PTB in the Eighth Affiliated Hospital of Xinjiang Medical University were selected and divided into 228 patients with active TB (active group) and 98 patients with latent TB infection (latent group), and 100 healthy subjects were included as controls. The serum exosomes of all subjects were extracted and identified by transmission electron microscopy, nanoparticle particle size analyzer and flow cytometry. The expression level of serum exomiR-27a was detected by real-time quantitative PCR. The diagnostic value of serum exomiR-27a in PTB was analyzed by receiver operating characteristic (ROC) curve. Drug resistance in patients with active PTB was recorded after standard treatment. The effect of serum exomiR-27a on the incidence of adverse outcomes was analyzed. Results The characterization of serum exosomes confirmed that exosomes were successfully isolated from serum. Compared with the control group 0.92(0.63, 1.17), the expression of serum exomiR-27a was up-regulated in the active group 1.55(1.18, 2.09) and the latent group 1.27 (0.96, 1.65), and the expression of exomiR-27a in the active group was higher than that in the latent group (P<0.05). PTB patients with high serum exomiR-27a expression (≥1.50) had poorer chest radiography and a higher proportion of chronic hepatitis comorbidities (P<0.05). ROC curve analysis showed that the area under the curve (AUC) of serum exomiR-27a distinguishing control and active group was 0.838 (95%CI: 0.795-0.880) and the AUC of serum exomiR-27a distinguishing normal and latent group was 0.766 (95%CI: 0.701-0.830). Patients in the active group received standard antituberculosis therapy (≥6 months). The expression level of serum exomiR-27a in drug-resistant patients was higher than that in sensitive patients (P<0.001), and the expression level of serum exomiR-27a in multi-drug resistant tuberculosis patients was higher than that in single-drug resistant tuberculosis patients (P<0.001). Logistic regression analysis showed that elevated serum exomiR-27a was an independent predictor of PTB resistance (P<0.001). High expression of serum exomiR-27a indicated a higher incidence of adverse outcomes (log rank=15.725, P<0.001). Conclusions High expression of serum exomiR-27a is helpful in the diagnosis and prediction of adverse outcomes of PTB.

3.
Chinese Journal of Hematology ; (12): 279-286, 2022.
Article in Chinese | WPRIM | ID: wpr-929636

ABSTRACT

Objective: To construct chimeric antigen receptor (CAR) T cells targeting CD52 (CD52 CAR-T) and validate the effect of CD52 CAR-T cells on CD52-positive leukemia. Methods: A second-generation CD52-targeting CAR bearing 4-1BB costimulatory domain was ligated into a lentiviral vector through molecular cloning. Lentivirus was prepared and packaged by 293 T cells with a four-plasmid system. Fluorescein was used to label cell surface antigens to evaluate the phenotype of CD52 CAR-T cells after infection. Flow cytometry and ELISA were used to evaluate the specific cytotoxicity of CD52 CAR-T cells to CD52-positive cell lines in vitro. Results: ①A pCDH-CD52scFv-CD8α-4-1BB-CD3ζ-GFP expressing plasmid was successfully constructed and used to transduce T cells expressing a novel CD52-targeting CAR. ②On day 6, CD52-positive T cells were almost killed by CD52-targeted CAR-T post lentivirus transduction [CD52 CAR-T (4.48 ± 4.99) %, vs Vector-T (56.58±19.8) %, P=0.011]. ③T cells transduced with the CAR targeting CD52 showed low levels of apoptosis and could be expanded long-term ex vivo. ④The CD52 CAR could promote T cell differentiation into central and effector memory T cells, whereas the proportion of T cells with a CD45RA(+) effector memory phenotype were reduced. ⑤CD52 CAR-T cells could specifically kill CD52-positive HuT78-19t cells but had no killing effect on CD52-negative MOLT4-19t cells. For CD52 CAR-T cells, the percentage of residual of HuT78-19t cells was (2.66±1.60) % at an the E:T ratio of 1∶1 for 24 h, while (56.66±5.74) % of MOLT4-19t cells survived (P<0.001) . ⑥The results of a degranulation experiment confirmed that HuT78-19t cells significantly activated CD52 CAR-T cells but not MOLT4-19t cells[ (57.34±11.25) % vs (13.06± 4.23) %, P<0.001]. ⑦CD52 CAR-T cells released more cytokines when co-cultured with HuT78-19t cells than that of vector-T cells [IFN-γ: (3706±226) pg/ml, P<0.001; TNF-α: (1732±560) pg/ml, P<0.01]. Conclusions: We successfully prepared CD52 CAR-T cells with anti-leukemia effects, which might provide the foundation for further immunotherapy.


Subject(s)
Humans , CD52 Antigen , Cell Line, Tumor , Immunotherapy, Adoptive/methods , Lentivirus/genetics , Leukemia , Receptors, Antigen, T-Cell , Receptors, Chimeric Antigen/genetics
4.
Chinese Journal of Hematology ; (12): 376-382, 2022.
Article in Chinese | WPRIM | ID: wpr-929571

ABSTRACT

Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.


Subject(s)
Humans , B7-H1 Antigen/pharmacology , Leukemia, Myeloid, Acute/metabolism , Sialic Acid Binding Ig-like Lectin 3/pharmacology , T-Lymphocytes
5.
Chinese Journal of Hematology ; (12): 370-375, 2022.
Article in Chinese | WPRIM | ID: wpr-929570

ABSTRACT

Objective: To investigate the prognostic significance of interferon regulatory factor 9 (IRF9) expression and identify its role as a potential therapeutic target in acute promyelocytic leukemia (APL) . Methods: The gene expression profile and survival data applied in the bioinformatic analysis were obtained from The Cancer Genome Atlas and Beat acute myeloid leukemia (AML) cohorts. A dox-induced lentiviral system was used to induce the expression of PML-RARα (PR) in U937 cells, and the expression level of IRF9 in U937 cells treated with or without ATRA was examined. We then induced the expression of IRF9 in NB4, a promyelocytic leukemia cell line. In vitro studies focused on leukemic phenotypes triggered by IRF9 expression. Results: ①Bioinformatic analysis of the public database demonstrated the lowest expression of IRF9 in APL among all subtypes of AML, with lower expression associated with worse prognosis. ②We successfully established a PR-expression-inducible U937 cell line and found that IRF9 was downregulated by the PR fusion gene in APL, with undetectable expression in NB4 promyelocytic cells. ③An IRF9-inducible NB4 cell line was successfully established. The inducible expression of IRF9 promoted the differentiation of NB4 cells and had a synergistic effect with lower doses of ATRA. In addition, the inducible expression of IRF9 significantly reduced the colony formation capacity of NB4 cells. Conclusion: In this study, we found that the inducible expression of PR downregulates IRF9 and can be reversed by ATRA, suggesting a specific regulatory relationship between IRF9 and the PR fusion gene. The induction of IRF9 expression in NB4 cells can promote cell differentiation as well as reduce the colony forming ability of leukemia cells, implying an anti-leukemia effect for IRF9, which lays a biological foundation for IRF9 as a potential target for the treatment of APL.


Subject(s)
Humans , Cell Differentiation , Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Promyelocytic, Acute/genetics , Oncogene Proteins, Fusion/metabolism , Phenotype , Tretinoin/therapeutic use , U937 Cells
6.
Chinese Journal of Hematology ; (12): 229-234, 2022.
Article in Chinese | WPRIM | ID: wpr-929562

ABSTRACT

Objective: This study aimed to create a type of CAR-T cells that targets LMP1 antigen and study its immunotherapeutic effect on LMP1-positive hematological malignancies. Methods: To generate LMP1 CAR-T cells, a plasmid expressing LMP1 CAR was created using molecular cloning technology, and T cells were infected with LMP1 CAR lentivirus. The effects of LMP1 CAR-T cells on specific cytotoxicity against LMP1-positive tumor cell lines infected with the EB virus had been confirmed. Results: ① LMP1 protein expressing on EB virus-positive lymphoma cells surface was verified. ② The LMP1 CAR-expressing plasmid was created, and LMP1 CAR-T cells were obtained by infecting T cells with a lentivirus packaging system, with an infection efficiency of more than 80% . ③LMP1 CAR-T cells have a 4∶1 effect-to-target ratio in killing LMP1-positive lymphoma cells. The killing effect of LMP1 CAR-T cells on Raji cells was enhanced after 48 h of coculture, but there was no significant killing effect on Ramos, which are LMP1-negative lymphoma cells. ④After coculture with LMP1-positive lymphoma cells at a ratio of 1∶1 for 5 h, the degranulation effect was enhanced. The proportion of CD107a(+) T cells in the LMP1 CAR-T cell treatment group was significantly higher than that in the vector-T cell group [ (13.25±2.94) % vs (1.55±0.05) % , t=3.972, P=0.017]. ⑤After coculture with LMP1-positive lymphoma cells, the proportion of CD69(+) and CD25(+) T cells in the LMP1 CAR-T cell group was significantly higher than that in vector-T cell group [ (7.40±0.41) % vs (3.48±0.47) % , t=6.268, P=0.003; (73.00±4.73) % vs (57.67±2.60) % , t=2.842, P=0.047]. ⑥After coculture with LMP1-positive lymphoma cells, cytokine secretion in the LMP1 CAR-T cell group was higher than that in the vector-T cell group [interferon-gamma: (703±73) ng/L vs (422±87) ng/L, t=2.478, P=0.068; tumor necrosis factor-alpha: (215±35) ng/L vs (125±2) ng/L, t=2.536, P=0.064]. Conclusion: In this study, we found that the LMP1 protein is only found on the surface of the EBV-positive tumor cell. Simultaneously, we created an LMP1 CAR-expressing plasmid and obtained LMP1 CAR-T cells by infecting T cells with a lentivirus packaging system. Furthermore, we demonstrated that LMP1 CAR-T cells could specifically kill LMP1-positive tumor cells in vitro. The degranulation and activation effects of LMP1 CAR-T cells were enhanced after coculture with LMP1-positive tumor cells, indicating a potential clinical application.


Subject(s)
Humans , Cell Line, Tumor , Herpesvirus 4, Human , Lentivirus , Lymphoma/therapy , Receptors, Chimeric Antigen/genetics , T-Lymphocytes , Viral Matrix Proteins
7.
Journal of Leukemia & Lymphoma ; (12): 412-418, 2022.
Article in Chinese | WPRIM | ID: wpr-953980

ABSTRACT

Objective:To investigate the clinical characteristics, diagnosis, treatment and prognosis of acute leukemia (AL) with NUP98-DDX10 fusion gene-positive.Methods:The clinical data of 2 AL patients with NUP98-DDX10 fusion gene-positive who admitted to Blood Diseases Hospital, Chinese Academy of Medical Sciences in April 2020 and February 2021, respectively were retrospectively analyzed. Transcriptome gene sequencing was used to detect fusion gene, and the fusion gene fragment was amplified by using reverse transcription polymerase chain reaction (RT-PCR), and Sanger sequencing was used to clarify sequences. The clinical and experimental indicators characteristics were analyzed and the relevant literatures were reviewed.Results:According to the clinical diagnosis, 1 patient was diagnosed as acute myeloid leukemia M 5 (AML-M 5) and 1 patient was diagnosed as acute leukemia of ambiguous lineage, not otherwise specified (ALAL-NOS). The AML-M 5 patient presented with severe coagulation abnormalities, and fulfilled the diagnostic criteria for diffuse intravascular coagulation (DIC) at the initial visit. Transcriptome sequencing of 2 patients showed NUP98-DDX10 fusion gene- positive. RT-PCR confirmed that sequencing results identified 2 different splice fusion modes: one was NUP98 exon 14 fused with DDX10 exon 7(usually called "type Ⅱ"), the other was NUP98 exon 14 fused with DDX10 exon 13, which was never reported and named as "type Ⅳ". From 1997 to 2018, a total of 16 cases with NUP98-DDX10 related hematologic neoplasms were reported in the literature. A summary analysis of 16 cases added with 2 patients in our center included 13 males and 5 females with median age 31.5 years (0.08-61 years). The median overall survival was 12 months (1-46 months). Conclusions:A novel fusion gene NUP98-DDX10 transcriptome is identified in ALAL-NOS patient. Hematological malignancies with NUP98-DDX10 are very rare. They respond poorly to conventional treatment and require allogeneic hematopoietic stem cell transplantation (allo-HSCT) to improve the prognosis.

8.
Chinese Journal of Orthopaedics ; (12): 1377-1386, 2020.
Article in Chinese | WPRIM | ID: wpr-869091

ABSTRACT

Objective:To explore the method and curative effect evaluation of repairing and rebuilding the long segmental bone defect after resection of long bone malignant tumors in adolescents.Methods:Reviewed our experience in 10 patients. Among them, 4 were male and 6 were female. The age was 10.2 years old (range, 6-16 years old). Including 7 cases of osteosarcoma, 3 cases of Ewing's sarcoma; 2 cases of humerus tumors, 7 cases of femoral tumors, and 1 case of tibial tumors-who presented between April 2015 and October 2019, in which the proportion of bone defect account for 71.4%-89.3% of the total length of diseased bone after tumor resection, with an average value of 80.9%. Considering the epiphyseal growth of young people, on the premise of ensuring enlarged tumor resection, we use traditional fibula autograft and prosthesis combined with steel plate, 3D printing design to retain the super-prosthesis of the epiphysis, and 3D printing bone trabecular was processed for the sake of making it grow successfully into the binding part of prosthesis and bone as we expect, simultaneously, the design of the short-force arm retaining the bilateral epiphysis can assit the steel plate and interlocking screws to enhance the stability.Results:The operation was completed successfully, and there were no complications such as nerve and blood vessel injury during the operation. After follow-up of 4-8 months, with an average follow-up of 22.9 months. One patient's review of X-ray film after 2 years of the operation showed that the transplanted fibula had a sign of bone resorption, and the internal fixation was in it's original position without obvious abnormalities. Two cases' auxiliary steel plates had fractured within one and a half to two years after surgery. With replacing the fractured steel plates, the internal fixation was good until now. According to the evaluation standards of the International Society of Limb Salvation: 2 cases of humerus osteosarcoma were respectively scored 23 points, 26 points, an average of 24.5 points; 4 cases of femoral osteosarcoma and 3 cases of femoral Ewing's sarcoma respectively scored 26 points and 25 points, 19 points, 30 points, 27 points, 24 points, 28 points (average, 25.6 points); 1 case of tibial osteosarcoma scored 26 points; all obtained relative satisfying treatment results.Conclusion:The combination of 3D printing and traditional machining techniques to produce an individualized long segmental prosthesis that preserves the epiphysis which applicated in the limb salvage treatment of bone malignant tumors can achieve excellent therapeutic effect.

9.
Chinese Journal of Hematology ; (12): 804-811, 2019.
Article in Chinese | WPRIM | ID: wpr-796967

ABSTRACT

Objective@#To construct the BCMA-CAR using the B-cell maturation antigen (BCMA) specific ligand APRIL as antigen binding region and to validate the effect of BCMA-CAR modified T cells (BCMA-CAR-T) on myeloma cells.@*Methods@#The BCMA-CAR was constructed using the BCMA specific ligand APRIL as antigen binding domain and 4-1BB as the costimulatory domain. The specific cytotoxicity against BCMA+ myeloma cell lines and primary multiple myeloma (MM) cells in vitro were evaluated. In addition, BCMA+ myeloma xenograft mouse model was established to assess the anti-tumor effect of BCMA-CAR-T cell therapy in vivo.@*Results@#BCMA-CAR-T cells could specifically kill BCMA+ myeloma cell lines (For BCMA-CAR-T cells, BCMA+ cells are almost undetectable in the E∶T ratio of 1∶4) and MM patients’ bone marrow mononuclear cells (the proportion of residual cells in BCMA-CAR-T and vector-T groups was 16.0% vs 66.85%, P=0.003) with significant degranulation (CAR-T and vector-T cells cocultured with MM1.S, H929 and U266 had degranulation levels of 33.30% vs 5.62%, 16.97% vs 2.95% and 25.87% vs 2.97%, respectively, P<0.001) and cytokines release (P<0.01) in vitro. In a human BCMA+ myeloma xenograft mouse model, BCMA-CAR-T cells could significantly prolong the survival of mice (The median survival time of mice treated with BCMA-CAR-T and vector-T cells was 87.5 days and 67.5 days, respectively, P<0.001) .@*Conclusion@#The ligand-based BCMA-CAR-T cells could be a promising strategy for BCMA+ multiple myeloma treatment.

10.
Journal of Experimental Hematology ; (6): 233-238, 2019.
Article in Chinese | WPRIM | ID: wpr-774330

ABSTRACT

OBJECTIVE@#To explore the effect of damage of bone marrow stroma cells induced by chemotherapeutic drug on the function of normal hematopoitic cells.@*METHODS@#Senescence cells were detected by flow cytometry after SA-β-gal staining; real-time PCR was used to detect the expression of a serial molecules in bone marrow stromal cell line OP9 cells; the expression of γ-H2AX was determined by flow cytometry after histone γ-H2AX staining; the colony forming ability of hematopoietic cells was tested by colony formation assay.@*RESULTS@#The percentage of senescence cells in OP9 cells after DNR treatment was 2.24 times as much as that in untreated OP9 cells (P<0.05). Compared with normal OP9 cells, the expression levels of IL-6 and TNF-alpha in DNR-treated OP9 cells increased by 2.73 times (P<0.01) and 0.56 times (P<0.01), and the expression levels of N-cadherin, alpha smooth muscle actin (alpha-SMA), angiopoietin1 (Angpt1) and osteopontin (OPN) decreased by 69.54%(P<0.01),63.90%(P<0.01),87.41%(P<0.01)and 42.78%(P<0.01)respectively. After the co-culture with DNR-treated OP9 cells, the colony formation of normal hematopoietic cells decreased by 47.10% than that co-cultured with untreated OP9 cells (P< 0.05), meanwhile, the percentage of γ-H2AX+ cells in normal hematopoietic cells increased by 2.19 times (P<0.05).@*CONCLUSION@#After treatment with DNR, the senescence cell number of OP9 cells sgnificantly increases; the expression of TNF-α and IL-6 is up-regulated, while the expression of α-SMA, Angpt-1 and OPN is down-regulated as compared with normal OP9 cells. In addition, after co-culture of DNR-treated OP9 cells with normal hematopoietic cells, the colony formation ability of hematopoietic cells decreases and the genome instability of hematopoietic cells increases as compared with normal hematopoietic cells.


Subject(s)
Animals , Mice , Bone Marrow , Bone Marrow Cells , Cells, Cultured , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells , Mesenchymal Stem Cells , Stromal Cells
11.
Chinese Journal of Clinical Oncology ; (24): 190-194, 2019.
Article in Chinese | WPRIM | ID: wpr-754399

ABSTRACT

Objective: To analyze the application and significance of three-dimensional (3D) printing in the surgical simulation and plan-ning for pelvic malignant tumors before the actual surgery, surgical resection, and postoperative resection effect analysis. Methods: A retrospective analysis was performed for 20 cases of primary pelvic malignant tumors in 11 male and 9 female patients with a mean age of 39.1 years (range, 14-60 years) who were admitted to Xinjiang University of Medical Science and Technology between January 2014 and January 2018. The tumors included osteosarcoma in 7 patients, chondrosarcoma in 12, and Ewing's sarcoma in one. Accord-ing to the Enneking pelvic classification, 10 cases involved the pelvicⅡarea; 6, the pelvicⅢarea; 6, the pelvicⅠandⅡareas; and 8, the pelvic I andⅣareas. According to whether or not a 3D-printed osteotomy guide plate was used, the cases were divided into a 3D-printed guide plate group and a conventional group, with 10 cases in each group. Operation time, blood loss volume, function score, and linear distance deviation were compared between the two groups. Results: The operation was completed successfully with com-plete excision in 20 patients. The operation time in the 3D-printed guide plate group (4.4~1.4 h) was not statistically significantly dif-ferent from that in the control group (4.5~1.4 h; P=0.83). No significant difference in intraoperative blood loss volume was found be-tween the 3D-printed guide plate (1,980~1,158 mL) and control (2,785~1,784 mL; P=0.16) groups. The postoperative Musculoskele-tal Tumor Society score (20.4~0.98) in the 3D-printed guide plate group showed no significant difference from that in the convention-al group (20.2~1.29; P=0.78). The mean linear difference between the 3D-printed guide plate group (3.8~1.9 cm) and the conven-tional group (8.7~1.0) was statistically significant (P=0.001). Conclusions: The 3D-printed group was not significantly different from the conventional group in terms of operation time, blood loss volume, and function score but had better surgical resection accuracy than the conventional group.

12.
Journal of Experimental Hematology ; (6): 970-975, 2019.
Article in Chinese | WPRIM | ID: wpr-771853

ABSTRACT

OBJECTIVE@#To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment.@*METHODS@#The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme,glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry.@*RESULTS@#Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P<0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P<0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P<0.001); the expression of GPX7 decreased by 65.7% (P<0.001); the expression of GPX8 decreased by 24.7% (P<0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P<0.05).@*CONCLUSION@#After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.


Subject(s)
Apoptosis , Daunorubicin , Mesenchymal Stem Cells , Oxidative Stress , Reactive Oxygen Species
13.
Chinese Journal of Hematology ; (12): 98-104, 2018.
Article in Chinese | WPRIM | ID: wpr-806125

ABSTRACT

Objective@#To investigate the spectrum of gene mutations in adult patients with B-acute lymphoblastic leukemia (B-ALL), and to analyze the influences of different gene mutations on prognosis.@*Methods@#DNA samples from 113 adult B-ALL patients who administered from June 2009 to September 2015 were collected. Target-specific next generation sequencing (NGS) approach was used to analyze the mutations of 112 genes (focused on the specific mutational hotspots) and all putative mutations were compared against multiple databases to calculate the frequency spectrum. The impact of gene mutation on the patients’ overall survival (OS) and recurrence free survival (RFS) was analyzed by the putative mutations through Kaplan-Meier, and Cox regression methods.@*Results@#Of the 113 patients, 103 (92.0%) harbored at least one mutation and 29 (25.6%) harbored more than 3 genes mutation. The five most frequently mutated genes in B-ALL are SF1, FAT1, MPL, PTPN11 and NRAS. Gene mutations are different between Ph+ B-ALL and Ph- B-ALL patients. Ph- B-ALL patients with JAK-STAT signal pathway related gene mutation, such as JAK1/JAK2 mutation showed a poor prognosis compared to the patients without mutation (OS: P=0.011, 0.001; RFS: P=0.014,<0.001). Patients with PTPN11 mutation showed better survival than those without mutation, but the difference was not statistically significant (P value > 0.05). Besides, in Ph+ B-ALL patients whose epigenetic modifications related signaling pathway genes were affected, they had a worse prognosis (OS: P=0.038; RFS: P=0.047).@*Conclusion@#Gene mutations are common in adult ALL patients, a variety of signaling pathways are involved. The frequency and spectrum are varied in different types of B-ALL. JAK family gene mutation usually indicates poor prognosis. The co-occurrence of somatic mutations in adult B-ALL patients indicate the genetic complex and instability of adult B-ALL patients.

14.
Journal of Sun Yat-sen University(Medical Sciences) ; (6): 215-219, 2018.
Article in Chinese | WPRIM | ID: wpr-712936

ABSTRACT

[Objective]To investigate the role of microRNA-145/Smad interacting protein 1(SIP 1)in VEGF-C-enhanced cervical cancer metastasis.[Methods]Cervical cancer cell line SiHa cells were cultured and treated with VEGF-C to observe its effect on the expression of miR-145 and SIP1. After transfection with specific SIP1 siRNA,the invasion number of cultured cells were calculated by transwell chamber assay.[Results]Treatment with VEGF-C(100 ng/mL)for 12 h,24 h and 48 h all reduced miR-145 expression,with the expression abundance of(82.4±6.4)% (P<0.05),(72.5±7.2)%(P<0.01),and(60.6±9.6)%(P<0.001),respectively,when compared to control.Meanwhile, the same treatment with VEGF-C also increased SIP1 protein expression,with the expression abundance of(142.4 ± 16.5)%(P<0.05),(183.6 ± 11.4)%(P<0.01)and(220.8 ± 15.7)%(P<0.001),respectively. The transfection of miR-145 mimic significantly impaired VEGF-C effect on SIP1 expression. Finally,VEGF-C promoted SiHa cell invasion,which was largely inhibited by the tranfection of SIP siRNA with the inhibitory rate of(56.6±10.3)%(P<0.01).[Conclusion]VEGF-C downregulates miR-145,thus increases SIP1 expression and promotes cervical cancer cell invasion,which may contributes to cervical cancer malignant progression.

15.
Journal of Experimental Hematology ; (6): 1589-1597, 2018.
Article in Chinese | WPRIM | ID: wpr-773051

ABSTRACT

OBJECTIVE@#To screen the differentially expressed proteins at the early stage of K562 cells treated with meisoindigo by using tandem mass tags (TMT)-based proteomics technology, and to explore the mechanism for meisoindigo-inducing apoptosis.@*METHODS@#The half inhibitory concentration (IC) of mesoindigo on K562 cells was determined by CCK8. The flow cytometry was used to assay the apoptosis of K562 cells treated by meisoindigo or DMSO. Total proteins were extracted from the cells treated with 0.2% DMSO (control) or 20 μmol/L meisoindigo (Test) for 2 hours. Then, the TMT-labeling HPLC-MS/MS was used to identify and quantify the peptides and their abundance, all the tests were repeated for 3 times. The Mascot software was used to identify the proteins; the GO annotations, enrichment and cluster analysis were used to analyze the differentially expressed proteins.@*RESULTS@#Meisoindigo-induced K562 cell apoptosis in a dose-dependent manner (r=0.98), 5 544 proteins were identified, 4792 of which were quantified. The protein with expression difference>1.5-folds in Test group accoanted for 8, out of which the expression of 4 proteins were up-regulated and 4 were down-regulated. The differentially expressed proteins mainly associated with reactive oxygen species (ROS).@*CONCLUSION@#Several proteins including DDIT4 were found to have dramatic changes in the early stage of K562 cells treated with meisoindigo by using quantitative proteomics technology. The ROS metabolic process may play important roles in meisoindigo-inducing apoptosis of K562 cells.


Subject(s)
Humans , Apoptosis , Indoles , K562 Cells , Proteomics , Tandem Mass Spectrometry
16.
Chinese Journal of Hematology ; (12): 956-961, 2017.
Article in Chinese | WPRIM | ID: wpr-809588

ABSTRACT

Objective@#To investigate the feasibility of multiplex real-time RT-PCR with fluorescent probes in early screening of Ph-like acute lymphoblastic leukemia (ALL) and analyze the clinical feature and prognos.@*Method@#A total of 118 adult B-ALL patients diagnosed between October 2010 and March 2016 were enrolled in this study. Multiplex RT-PCR was used to detect the Ph-like ALL related fusion gene and CRLF2 expression in 58 BCR-ABL and MLL rearrangement negative patients. The clinical features, treatment response and prognosis were analyzed in Ph-like fusion gene positive and/or CRLF2 over-expression patients.@*Result@#Among 58 patients, 9 patients (9/58, 15.5%) showed Ph-like ALL related fusion genes positive and 10 patients (10/58, 17.2%) showed CRLF2 over-expression. There were statistical differences in age, WBC count, immunophenotypes, cytogenetics and risk stratification among Ph-like fusion gene positive or CRLF2 over-expression patients, Ph+ patients, MLL+ patients and B-other patients. The 2-year overall survival rates were 65%, 47%, 64% and 74% respectively among these four groups (P=0.043) . The 2-year relapse free survival rates were 51%, 39%, 62% and 70% respectively among these four groups (P=0.010) .@*Conclusion@#Routine screening of Ph-like ALL by multiplex RTPCR is feasible.

17.
Journal of Experimental Hematology ; (6): 1621-1626, 2017.
Article in Chinese | WPRIM | ID: wpr-278773

ABSTRACT

<p><b>OBJECTIVE</b>To explore the effect of c-FLIP expression on drug resistance of Kasumi-1 leukemia cells and its mechanisms.</p><p><b>METHODS</b>Tet-on inducible system was used to construct the conditional expression vector of c-FLIP by cloning the c-FLIP gene into lentivirus vector pLVX-Tight-Puro, then the Kasumi-1 cells were transfected with lentivirus pLVX-Tight-Puro-c-FLIP. The expression of c-FLIP was induced by doxycycline(Dox) for different time and doses, and verified by qRT-PCR and Western blot. On the basis of the overexpression of c-FLIP, the Kasumi-1-c-FLIP cells were treated with CH11 and PB in order to induce apoptosis, and the Giemsa staining was used to show the apoptotic cell morphology.</p><p><b>RESULTS</b>qRT-PCR and Western blot showed the overexpression of c-FLIP, the CH11 and PB can induce Kasumi-1 cell apoptosis, while the c-FLIP overexpression weakened this effects. Western blot showed that the c-FLIP blocked the caspase-8 activation.</p><p><b>CONCLUSION</b>The overexpression of c-FLIP inhibits the apoptosis caused by CH11 and PB, and leads to drug resistance in leukemia cells.</p>

18.
Journal of Experimental Hematology ; (6): 668-672, 2017.
Article in Chinese | WPRIM | ID: wpr-271939

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the role of asymmetric division in leukemia cells through detection of expression and asymmetric division of Numb in differentiated and undifferentiated K562 cells.</p><p><b>METHODS</b>Firstly, Hemin was used to induce K562 cell differentiation, and the expression of Numb was detected by the real-time quantitative RT-PCR and flow cytometry. After K562 cells were synchronized by nocodazole, the Numb protein was labeled by immunohistochemical staining, followed by the determination of the terminally differentiated cells through confocal microscopy. The fluorescence intensity was calculated by Image J software, and the cell division pattern was analyzed on the basis of the fluorescence intensities of Numb in 2 divided daughter cells.</p><p><b>RESULTS</b>Compared with the undifferentiated K562 cells, the level of Numb mRNA expression increased 2.3 times (P<0.001). The ratio of Numb positive cells was(67.37±5.01)% in differentiated K562 cells, while that was (43.97±5.72)% in undifferentiated K562 cells (P<0.01). Compared with undifferentiated K562 cells, the ratio of cells with asymmetric division in differentiated K562 cells increased 18.3%, the percentage of cells with symmetry self-renewal reduced 49.7%(P<0.001) and that with symmetry differentiation increased 32%(P<0.001).</p><p><b>CONCLUSION</b>In differentiated K562 cells, expression of Numb and proportion of cells with asymmetric division were higher than that in undifferentiated cells. With the differentiation of leukemia cells, the proportion of cells with asymmetrical division increases, and the proportion of cells with symmetrical self-renewal decreases. The stemness of leukemia cells is maintained mainly through the symmetrical self-renewal.</p>

19.
Journal of Preventive Medicine ; (12): 682-686, 2016.
Article in Chinese | WPRIM | ID: wpr-792524

ABSTRACT

Objective To explore the effect of lactoferrin fortified formula milk on growth and blood erythrocyte indices among infants.Methods In this prospective multi -center controlled intervention study,a total of 260 infants were selected from six maternal and children's health care hospitals in the area.All subjects were divided into two groups with the sequence of outpatient:lactoferrin fortified formula milk group (fortified group,FG,containing lactoferrin 38 mg/100 g milk and iron element 4mg/100 g milk)and no lactoferrin fortified milk (control group,CG,containing lactoferrin 0 mg/100 g milk and iron element 4.2 mg/100 g milk)for 3 months.The levels of weight,height and head circumference and the concentration of hemoglobin (Hb),serum ferritin (SF),red blood cell count (RBC),hematocrit(Hct),mean corpuscular volume (MCV ), mean corpuscular hemoglobin (MCH ), mean corpuscular hemoglobin concentration (MCHC),red blood cells volume distribution width (RDW)and HAZ,WAZ and WHZ were recorded before and after intervention,respectively.Results A total of 213 (including 115 in FG,and 98 in CG)infants were included in the intervention trial and all measurement of biochemical indicators were detected.There were no significant difference in the average amount of daily intake of formula milk (94.3 ±9.8 g vs.88.2 ±8.7 g for FG and CG,P >0.05)and iron element (3.8 ±0.4 mg vs.3.7 ±0.6 mg for FG and CG,P >0.05).The average amount of daily intake of lactoferrin for infants in FG group was 35.8 ±3.7 mg.The levels of weight,WAZ,WHZ,Hb,SF,MCV and MCHC of infants in FG after intervention were all significantly higher than those of infants in CG [weight,(8723 ±245)g vs.(8558 ±214)g;WAZ,(1.02 ±0.31)vs.(0.44 ±0.18);WHZ,(0.98 ±0.31)vs.(0.41 ±0.12);Hb,(125.5 ±15.4)g/L vs. (116.9 ±13.1)g/L;SF,(44.7 ±17.2)μg/L vs.(31.6 ±18.4)μg/L,but significantly lower for the prevalence of anemia (4.35% vs.7.14%)and iron deficiency (13.91% vs.24.49%)(P <0.05).Conclusion The intervention of lactoferrin fortified formula milk presented significant effects on iron metabolic homeostasis and erythrocyte indices of infant with breastfeeding.

20.
Journal of Experimental Hematology ; (6): 1856-1862, 2016.
Article in Chinese | WPRIM | ID: wpr-332597

ABSTRACT

<p><b>OBJECTIVE</b>To reconstruct a human bone marrow niche in immunodeficiency mouse (NOD/SCID) so as to provide a model for observing the effect of abnormal BM niche on the occurence and development of leukemia.</p><p><b>METHODS</b>Human platelet lysate(HPL) was obtained by repeated freezing and thawing of concentrated platelet. Bone marrow-derived mesenchymal stem cells were cultured in α-minimal essential medium (α-MEM) containing 10% HPL or 10% FBS. The morphology, cell phenotype, multilineage differentiation potential in vitro and proliferation capacity between the mesenchymal stem cells cultured with HPL or FBS were compared. The human bone marrow formation capacity of HPL-cultured MSC was observed. The MSC was seeded on β-TCP scaffolds for 12h, then the MSC-coated scaffold were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12 week, the scaffolds were harvested from the mice, then fixed, paraffin-embedded and stained for HE.</p><p><b>RESULTS</b>Whether cultured in the presence of HPL or FBS, the MSC all displayed a spindle-shaped fibroblast-like morphology; the flow cytometry analysis revealed no obvious differences in cell immunophenotype in this 2 groups; they all have the ability to differentiate towards osteoblasts, adipocytes, and chondrocytes in vitro. However, the mesenchymal stem cells cultivated with HPL-contained medium showed stronger proliferation capacity and higher activity to differentiate towards osteoblasts. Mesenchymal stem cells cultivated with HPL still have in vivo bone-forming capacity.</p><p><b>CONCLUSION</b>HPL cultured MSC have stronger proliferation capacity and potential of differentiate towards osteoblasts, HPL-cultured MSC also can reconstruct humanized bone marrow niche in murine host.</p>

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