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Clonal Growth of Hum an Acute Leukem ia Cells in Serum -free Methylcellulose Medium

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Journal of Huazhong University of Science and Technology(Medical Sciences) 1999年第3期19卷 190-193页

作者： CHEN Zhichao , ZOU Ping , YOU Yong , LIU Zhongping , XIANG Jianping , YU Dongjiao institute of hematology, Tongji Medical University, wuhan 430030 institute of hematology Tongji Medical University Wuhan 430030

The clonal growth of human acute leukemia cell line (K562) and acute myeloid leukemia cells in the serum free culture (SFC) was studied in order to establish a SFC system which could replace the effects of serum by using semi solid methylcellulose culture technique. Our results showed that the clonal growth of K562 cells in semi solid culture was dependent on exogenous serum. The K562 could be grown in SFC supplemented with 4 major replacing substances. The multifactor and multilevel orthogonal experiment demonstrated that the colony formation was statistically influenced by the 4 replacing substances at various concentrations ( P <0.01). Among them, bovine serum albumin had greatest effect on clonal growth of K562 cells with the optimal concentration being 15 mg/L, followed by transferring, cholesterol and insulin with their optimal concentrations being of 150 mg/L, 7.8 mg/L and 7.0 mg/L respectively. SFC system was formed with the 4 substances at their optimal concentrations. Colony formation of the blast cells in 10 patients with acute myeloid leukemia was observed in this SFC system. There was a heterogeneity of acute myeloid leukemia cells among the 10 patients in response to the growth substances. In SFC system, there was a linear relationship between the number of the clonal formation and the count of the added cells, indicating the colony growth of the cells. Primary acute leukemia cells maintained in SFC system in 10 cases could completely form clones. The colony formation number in some cases in SFC system was more than that of the serum containing culture. The SFC system could partially replace the serum for study of the clonal formation of human leukemia cells.

关键词： leukemia cells serum free culture

The Com prehensive Evaluation on Four Indices of Drug Re-sistance in Acute Myeloid Leukem ia

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Journal of Huazhong University of Science and Technology(Medical Sciences) 1999年第3期19卷 194-197页

作者： CHEN Yan , HE Mingsheng , XIANG Zhifu , WU Yudan, YUE Beibei , YU Dongjiao, LI Huiyu institute of hematology, Xiehe Hospital, Tongji Medical University, wuhan, 430022 institute of hematology Xiehe Hospital Tongji Medical University Wuhan 430022

To study sensitivity of drug resistance indexes and resistance manner in acute myeloid leukemia (AML), MTT drug sensitivity, growth types of CFU L in vitro , Bcl 2 antigen and Bcl 2/Bax ratio and intracellular fluorescence intensity of daunorubicin (DNR) were determined. In 62 cases of AML, the positive coincidence rate was 73 % with MTT test and the negative coincidence rate was 70 %. In 3 commonly used drugs, if one drug showed sensitivity, the coincidence remission rate reached 71 %. In 51 cases of AML, there were 31 patients in the group of complete remission (CR), in which CFU L of 29 patients showed independent growth. CFU L of 2 patients showed no growth. However, there were 20 patients in the group of non remission (NR), in which CFU L of 14 patients showed independent growth. CFU L of 6 patients showed non growth pattern. Statistical analysis showed significant difference ( P <0.05). In 32 cases of AML, the expression rate of Bcl 2 was 59.55 %±19.56 % in drug sensitive group, and one was 77.36 %±11.91 % in drug resistant group, respectively ( P <0.05). At the same time, the ratio of Bcl 2/Bax was 7.50±5.04 in drug sensitive group and one was 14.32±8.99 in drug resistant group, respectively ( P <0.05). In 15 case of clinically drug resistant AML, the fluorescence histogram of DNR showed left shift of main peak (LSMP) in 12 patients. They were diagnosed as classical drug resistance. Meanwhile, 1 patient showed right shift of main peak (RSMP) in 3 patients. They were diagnosed as re growth drug resistance. It is concluded that MTT and CFU L might be used for prediction of drug sensitivity or resistance when patients were on treatment. Bcl 2 and ratio of Bcl 2/Bax might be associated with the prognosis. DNR histogram could be employed for identify the pattern drug resistance. The strength and weakness of these techniques were discussed.

关键词： acute myeloid leukemia drug resistance curative effect

Immunohistochem ical Study of T Lym phocyte Subsets onFrozen Substituted Plastic Em bedding Section of the BoneMarrow from Patients with Myelodysplastic Syndrom e andIts ClinicalIm plication

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Journal of Huazhong University of Science and Technology(Medical Sciences) 1999年第3期19卷 198-202页

作者： LIU Shangqing , LIU Xiufang , ZOU Ping , SONG Shanjun , WANG Bianming institute of hematology, Xiehe Hospital, Tongji Medical University, wuhan 430022 institute of hematology Xiehe Hospital Tongji Medical University Wuhan 430022

An immunohistochemical study of T lymphocyte subsets on frozen substituted plastic embedding bone marrow sections obtained from 10 patients with myelodysplastic syndrome (MDS) was presented. The results of qualitative and quantitative immunohistochemical analysis are as follows: (1) Labile antigens of T lymphocytes were well preserved, thus allowing analysis of distribution of T lymphocyte subsets in situ ; (2) the average number of T 3, T 4 and T 8 lymphocyte of the diffuse infiltrate was about 2 %, 0.4 %, 0.5 %, respectively, of all nucleated cells in bone marrow, and T 4/T 8 of T cells were below 1.0 in patients with MDS; (3) there were cases of RAS showing T lymphocyte aggregation in bone marrow, but no patient exhibited progressive refractory anemia with excess of blasts(RAEB) and RAEB in transformation (RAEBT). These findings indicated that the immunological abnormalities are of importance in the evaluation of pathogenesis and prognosis of MDS.

关键词： immunohistochemistry plastic embedding of bone marrow distribution of T cell subsets in situ myelodysplastic syndrome

Effect of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7

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Asian Pacific Journal of Tropical Medicine 2016年第9期9卷 876-880页

作者： Chang-Jiang Lei Chun Yao De-Ke Li Zhi-Xiong Long Yuan Li Dan Tao Yan-Ping Liou Jiang-Zhou Zhang Ning Liu Tumor laboratory the Fifth Hospital of Wuhan wuhan hematology institute Department of Anesthesiology the Fifth Hospital of Wuhan Oncology Departmen the Fifth Hospita of Wuhan

Objective:To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma ***:Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132 respectively or *** effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry,and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime ***:Compared with the control group,the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly,and the apoptosis ratio increased distinctly(P<0.05).Besides,the affect of the co-transfection group was better than that of the single transfection *** protein levels of GPC3(Glypican-3) and YAP(Yes-associated protein),the target genes transfected only by miR-520c-3p and miR-132,respectively,reduced obviously(P<0.05),which was similar with the co-infected cells,but cells transfected by miR-132 only showed a decrease of ***:The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP,enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.

关键词： MiR-520c-3p MiR-132 Liver cancer Proliferation Apoptosis

Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 199-205页

作者： Min ZHANG, Fang LIU, Wei HE, Yong YOU, Ping ZOU*, and Ling-Bo LIU institute of hematology Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, wuhan 430022, China institute of hematology Disease Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430022 China

To detect a new and more effective way against apoptosis mouse lymphomatic cell line- Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

关键词： transplantation immunology ribozyme Fas Yac-1 apoptosis

Monitoring and Analysis of Chinese Chronic Myeloid Leukemia Patients Who Have Stopped Tyrosine Kinase Inhibitor Therapy

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Current Medical Science 2019年第2期39卷 211-216页

作者： Na SHEN Yong YOU Zhao-dong ZHONG Li MENG Jian-feng ZHOU Ping ZOU Xiao-jian ZHU Hong-xiang WANG Fan-jun CHENG institute of hematology Union HospitalTongji Medical CollegeHtiazhong University of Science and TechnologyWuhan 430022China Department of hematology Tongji HospitalTongji Medical CollegeHuajrong University of Science and TechnologyWuhan 430030China Department of hematology The Central Hospital of WuhanWuhan 430014China

Discontinuation of tyrosine kinase inhibitor(TKI)therapy after achieving a persistent deep molecular response(DMR)is an urgently needed treatment goal for chronic myeloid leukemia(CML)patients and has been included in the National Comprehensive Cancer Network(NCCN)guidelines(version 2.2017)for ***,various studies have confirmed the feasibility of discontinuing TKI *** this study,we analyzed data from 45 CML patients who had discontinued TKI *** analysis was performed to predict factors that were potentially related to treatment-free remission(TFR)and identify the differences between early relapse and late *** of the 45 patients,20 exhibited molecular relapse after a median follow-up of 18 months(range,1-54 months),and the estimated TFR at 24 months was 40%.The univariate analysis revealed that a high Sokal score and interruptions or dose reductions during TKI treatment were the only baseline factors associated with poor *** results indicate that TKI discontinuation could be successfully put into practice in China.

关键词： discontinuation treatment-free remission chronic myeloid leukemia relapse

Methodological Study of Cell Separation with Domestic Immunomagnetic Beads

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2000年第3期20卷 208-209页

作者：陈智超游泳邹萍 institute of hematology Tongji Medical University Wuhan

To establish the method of cell separation with domestic immuomagnetic beads, three methods were investigated. Direct method, SPA method and Biotin Avidin method were applied to separate cell strain Hut 78 and CD4 positive cells. Separation rate of strain Hut 78 was more than 90 % in direct method. Detachment rate with papain was over 95 %. Cell activity was well retained. SPA method and Biotin Avidin methods were also effective, but the direct method was superior to the other two techniques. Before separated by the direct method, CD4 positive cells constituted 46.4 %±6.4 % of mononuclear cells (MNC), but in eliminated suspension there was only 6.2 %±2.3 % CD4 positive cells left. In the separated part, 80.6 %±7.2 % of the cells combined with the beads. It is concluded that the direct method in separating cells had high sensitivity and specificity.

关键词： immunomeganetic beads cell separation

Platelet Fibrinogen in the Patients with Idiopathic BleedingPlatelet Disorder

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2000年第2期20卷 134-136页

作者：杨林花沈迪王爱莲魏文宁刘仲萍杨锐杨焰张玉金 institute of hematology Tongji Medical University Wuhan

The changes of platelet fibrinogen in patients with obscure cutaneous mucosal bleeding and their relation with disease were evaluated. By using flow cytometry and Western blot, the platelet binding site of fibrinogen and the expression of platelet fibrinogen in 8 cases of obscure bleeding were detected. The results showed that platelet surface binding sites of fibrinogen were 65. 38± 3. 62 in the resting state and 65. 25±5. 78 after activated by adenosine diphosphate, showing no significant difference as compared with control group (P>0. 05). The expression of platelet fibrinogen was obviously decreased in 4 patients and increased in one case as compared with control group. It was concluded that the binding sites of fibrinogen on the platelet surface were normal in the patients with iopathic bleeding platelet disorder. The abnormal of platelet fibrinogen might be one of hemorrhagic reasons in partial patients.

关键词： idiopathic platelet disorder bleeding fibrinogen

Bcl-1 Rearrangement and Cyclin D1 Protein Expression in Multiple Myeloma Precursor Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2000年第2期20卷 128-131,136页

作者：刘新月唐泽海邹萍 hematology institute Tongji Medical University Wuhan

The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.

关键词： multiple myeloma BCL-1/IgH rearrangement Clone cell cyclin D1 protein