1.The observation of tear ferning in conjunctivochalasis
Min-Hang XIANG ; Xing-Ru ZHANG ; Rui-Xio CAI ; Qing-Sang LI ; Ya-Min RAO ;
Ophthalmology in China 1993;0(01):-
Objective To evaluate tear ferning changes of conjunctivochalasis.Design Prospective case study series.Partici- pants 30 patients(60 eyes)of conjunctivochalasis and normal subjects were selected.Methods The subjects were observed with gen- eral ophthalmic examination and tear fern test(TFT).Tear ferning was classified into 4 types.TypeⅠand TypeⅡare normal.TypeⅢand TypeⅣare abnormal.Main Outcome Measures The type of tear feming.Results TFT showed that tear ferning was de- creased in conjunctivochalasis group(TypeⅢand TypeⅣoccupied 61.7%).The difference between conjunctivoehalasis and normal control group was significant(P
2.Interaction between osteoblast of bone marrow microenvironment with leukemia cells
Liming LIN ; Shuying CHEN ; Kejing TANG ; Huan LI ; Zheng TIAN ; Min WANG ; Qing RAO
Journal of Leukemia & Lymphoma 2014;23(12):707-711
Objective To elucidate the interaction between osteoblast of bone marrow microenvironment and leukemia cells,and to investigate the role of osteoblast in the leukemia cells survival and apoptosis and the influence of leukemia cells on the osteoblast.Methods Leukemia cells from AML1-ETO9a-Rac1 mouse leukemia model and osteoblast cells were used.The ratio of GFP+ leukemia cells that co-cultured with or without osteoblast was detected by FACS.In addition,the apoptosis level of leukemia cells was detected by flow cytometry by PI and Annexin Ⅴ labeling.Activation level of PARP was determined by Western-blot.Real-time PCR (RT-PCR) was utilized to detect the mRNA level of TPO,N-cadherin,OPN and Ang1 in osteoblast which was separated from leukemic mice.Results The ratio of GFP+ cells in AE9a-Rac1 leukemia cells co-cultured with osteoblast cell was significantly higher than that of AE9a-Rac1 leukemia cells cultured alone.The apoptotic level of AE9a-Rac 1 leukemia cells cultured alone was significant higher than that of AE9a-Rac 1 leukemia cells in co-culture system.Western blot showed that activated level of PARP in AE9a-Rac1 leukemia cells co-cultured with osteoblast was lower than that cultured alone.RT-PCR result showed that TPO and N-cadherin mRNA levels in primary osteoblast separated from leukemic mice were higher than that from normal mice.Ang1 and OPN mRNA levels of osteoblast from leukemia mice were lower.Conclusion Osteoblast cell can support the survival and inhibit the apoptosis of leukemia cells.Leukemia cells can influence the functions of osteoblast by microenvironment associated cytokines production.
3.Expression of Rho GTPase family member RhoA and CDC42 protein in patients with acute leukemia
Jiying WANG ; Jianwei ZHANG ; Yan LI ; Kejing TANG ; Yanzhong WANG ; Min WANG ; Haiyan XING ; Zheng TIAN ; Qing RAO
Journal of Leukemia & Lymphoma 2010;19(6):327-330
Objective To detect the proteins levels of RhoA and CDC42 in bone marrow mononucleated cells (BMMC) of patients with primary acute leukemia,and further determine the role of abnormal interactions between hematopoietic progenitor and bone marrow microenvironment on abnormal behaviors of leukemia cells. Methods BMMC samples were separated from 54 primary acute leukemia patients and 22 normal donors and the cell lysis samples were prepared. RhoA and CDC42 proteins were determined by Western blotting. Independent pair T test was conducted to evaluate whether the differences in RhoA and CDC42 expression were statistically significant between leukemia patients and normal donors. Spearman was applied in analyzing the correlation between expression of RhoA and CDC42 proteins and clinical characters of patients. Results RhoA and CDC42 proteins level of primary acute leukemia patients was significantly higher than that of normal samples. Especially, patients with M2,M3 and M5 subtypes exhibited significant higher RhoA proteins levels and M3 subtype exhibited significant higher CDC42 protein levels. Conclusion RhoA and CDC42 protein levels of primary acute leukemia patients are significantly higher than that of normal donors. This result suggests that RhoA and CDC42 associated efficient migration of leukemia cells could be implicated in abnormal interaction of leukemic cell with bone marrow microenvironment.
4.The effects of different transcripts of AML1 on the transactivation of M-CSF-R gene.
Qing ZHANG ; Min WANG ; Hai-yan XING ; Qing RAO ; Jian-xiang WANG
Chinese Journal of Hematology 2005;26(11):641-644
OBJECTIVETo observe the effects of AML1A and AML1B, two splicing isoforms of AML1, on the transactivation of macrophage colony-stimulating factor receptor (M-CSF-R), and explore the mechanism of hematopoietic stem cell committed differentiation and leukemogenesis.
METHODSThe expressive plasmids of AML1A and AML1B were constructed, and co-transfected into CV-1 cells with a luciferase reporter plasmid containing M-CSF-R promoter. The transactivity of M-CSF-R promoter was assayed by luminometer.
RESULTSAML1B exhibited a distinct transactivity to M-CSF-R promoter with a sequence-specificity and dosage-dependent manner. AML1A showed no any transactivity but antagonized the effect of AML1B, causing marked reduction of M-CSF-R expression.
CONCLUSIONAn intact structure of AML1 is necessary for transactivation of M-CSF-R. AML1A may interfere with the transactivation of AML1B, and play a key role in the fine regulation of committed differentiation of hematopoietic cell.
Animals ; Cell Differentiation ; genetics ; Cells, Cultured ; Core Binding Factor Alpha 2 Subunit ; genetics ; Gene Expression Regulation, Leukemic ; Genetic Vectors ; Haplorhini ; Hematopoietic Stem Cells ; cytology ; Kidney ; cytology ; Plasmids ; genetics ; Receptor, Macrophage Colony-Stimulating Factor ; genetics ; Transcriptional Activation ; Transfection
5.Expression of HtrA2 and WT1 genes in acute myeloid leukemia.
Xiao-Yan LI ; Qing ZHANG ; Yan LI ; Tian YUAN ; Zheng TIAN ; Ke-Jing TANG ; Min WANG ; Qing RAO ; Ying-Chang MI
Journal of Experimental Hematology 2012;20(1):1-6
Objective of this study was to detect the expression of HtrA2 and WT1 mRNA in acute myeloid leukemia (AML) and investigate the relationship of their expression levels with clinical variates and correlation between them. The expression levels of HtrA2 and WT1 were measured by RQ-PCR in bone marrow cells in 104 newly diagnosed AML patients and leukemia cell lines (K562, HL-60, NB4, Kasumi-1, U937), and the relationship between expression level and clinical parameters (age, sex, WBC count, diagnosis and prognosis) was investigated. The results showed that (1) the expression of HtrA2 gene in newly diagnosed AML was lower than that of the normal controls (P < 0.01), while expression of WT1 gene in newly diagnosed AML was higher than that of the normal controls (P < 0.01), the expression levels of HtrA2 and WT1 genes both did not correlate with age, sex and WBC counts of patients. There were no significant difference of HtrA2 gene expression between different NCCN prognosis group, while WT1 gene expression in better-risk group was significantly lower than that in intermediate-risk group (P = 0.003). The HtrA2 expression level rose after treatment in both CR group and non-CR group (P < 0.05), while WT1 expression level significantly decreased after treatment only in CR group (P < 0.01). Negative correlation between HtrA2 and WT1 expression was also observed (r = -0.249, P = 0.011). It is concluded that the low expression of HtrA2 and high expression of WT1 are closely related with occurrence and development of acute leukemia, so up-regulating expression of HtrA2 and interfering expression of WT1 may become the targets for leukemia therapy in the future.
Adolescent
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Adult
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Aged
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Cell Line, Tumor
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Female
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High-Temperature Requirement A Serine Peptidase 2
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Humans
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Leukemia, Myeloid, Acute
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genetics
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metabolism
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Male
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Middle Aged
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Mitochondrial Proteins
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genetics
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metabolism
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Serine Endopeptidases
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genetics
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metabolism
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WT1 Proteins
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genetics
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metabolism
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Young Adult
6.Effect of tyrosine kinase inhibitor Imatinib mesylate on proliferation, differentiation and apoptosis of Kasumi-1 leukemia cell line.
Li-Hong WANG ; Qing RAO ; Min WANG ; Jian-Xiang WANG
Chinese Journal of Hematology 2005;26(8):449-452
OBJECTIVETo explore the effect of Imatinib mesylate on proliferation, differentiation and apoptosis of leukemic Kasumi-1 cells bearing c-kit mutation.
METHODSKasumi-1 cells were treated with Imatinib at different concentrations in culture. Cell proliferation was assayed by MTT assay, expressions of c-kit antigen, surface myeloid antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining and agarose gel electrophoresis. Western blot was used to analyze the level of c-kit protein tyrosine phosphorylation.
RESULTSImatinib treatment caused a time- and dose-dependent inhibition of the cell proliferation, with a 72 h IC50 of 4.45 micromol/L. Imatinib treatment induced a decrease in the mean fluorescence value of c-kit antigen, a progressive decline in S-phase cell fraction and an increase in G0/G1 cells. Treatment with 5.00 micromol/L of imatinib for 72 h induced an increase in expression of myeloid surface protein CD11, CD13 and CD15, and for 24 h induced an increase in early apoptosis cells [from 9.04% to 86.84% (P < 0.05)]. The apoptosis ladder was observed on agarose gel electrophoresis on 5-day treatment. Tyrosine phosphorylation level of c-kit protein was decreased by Imatinib treatment.
CONCLUSIONTyrosine kinase inhibitor Imatinib mesylate treatment could inhibit proliferation of Kasumi-1 cells which bear a c-kit mutation, induce differentiation, apoptosis and G0/G1 cells accumulation.
Apoptosis ; drug effects ; genetics ; Benzamides ; Cell Differentiation ; drug effects ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Imatinib Mesylate ; Mutation ; Piperazines ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Pyrimidines ; pharmacology
7.Study on signal transduction pathway in differentiation and apoptosis of leukemia cells induced by heat shock protein inhibitor.
Wen-Juan YU ; Qing RAO ; Min WANG ; Zheng TIAN ; Zhi-Fang XU ; Jian-Xiang WANG
Chinese Journal of Hematology 2007;28(10):677-680
OBJECTIVETo explore the signal transduction pathway in the differentiation and apoptosis of leukemia cells induced by heat shock protein 90 (HSP90) inhibitor 17-Allyl amide-17-demethoxygeldanamycin (17AAG).
METHODSKasumi-1 cells were treated with increasing concentrations or exposure time of 17AAG. The total kit protein (CD117), phosphorylated kit protein and its downstream signaling molecules were measured by Western blot analysis. Mutated kit protein from control and 17AAG-treated Kasumi-1 cells was immunoprecipitated and immunoblotted for associated chaperones.
RESULTSTotal kit protein and kit activity were decreased in 17AAG treated cells, but c-kit mRNA level was not. Total AKT protein and phospho-AKT, as well as phospho-STAT3 were rapidly down-regulated in Kasumi-1 cell after treatment with 17AAG. There was no change in total STAT3 protein. Immunoprecipitation showed that 1 microM 17AAG treatment for 1 hour caused kit associated HSP90 decrease and HSP70 increase.
CONCLUSION17AAG-induced apoptosis of Kasumi-1 cells is associated with a decline in Asn822Lys mutated kit protein level and phosphorylated kit, and with a downregulation in its downstream activated signaling molecules involved in proliferation. AKT is a client protein of HSP90. The changes of kit associated HSP90 and HSP70 satisfy the circulation mode of molecular chaperone complex.
Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Differentiation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Heat-Shock Proteins ; metabolism ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-kit ; metabolism ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Cells, Cultured
8.Effects of AML1-ETO on transcription activity of p21WAF1/CIP1 gene promoter.
Hui WEI ; Xiang-rong LIU ; Hang LIU ; Qing RAO ; Min WANG ; Jian-xiang WANG
Chinese Journal of Hematology 2007;28(8):545-548
OBJECTIVETo observe the effects of AML1-ETO fusion gene on the transcription activity of p21WAF1/CIP1 gene. And to explore the enhancement of leukemia pathogenesis of AML1-ETO.
METHODSThe luciferase reporter plasmids of p21WAF1/CIP1 gene promoter were constructed, and co-transfected into CV-1 cells with AML1-ETO, AML1b and AML1a expression plasmids. The trans-activity of p21WAF1/CIP1 gene promoter was assayed by luminometer.
RESULTSAML1-ETO exhibited a distinct inhibition activity of p21WAF1/CIP1 gene promoter with a sequence-specificity and dosage-dependent manner. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (19 +/- 4)% compared to control group, when 1000 ng pCMV5-AML1-ETO plasmid was used. AML1b and AMLla showed less inhibition activity. The trans-activity of p21WAF1/CIP1 gene promoter decreased to (61 +/- 16)% and (59 +/- 16)% compared to control group, respectively, when 1000 ng plasmid was used.
CONCLUSIONAML1-ETO exhibits more inhibition activity of p21WAF1/CIP1 gene promoter than AML1b and AMLla, results from recruiting transcription co-repression complex efficiently by ETO. Based on previous researches, the effects of exogenous AML1-ETO on p21WAF1/CIP1 gene promote may be dependent on the type of cell lines.
Animals ; Cell Line ; Core Binding Factor Alpha 2 Subunit ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Haplorhini ; Oncogene Proteins, Fusion ; genetics ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; RUNX1 Translocation Partner 1 Protein ; Transcription, Genetic ; Transfection
9.Construction and expression of single chain variable fragments (ScFv) against human CD19 antigen.
Sen CHEN ; Qing RAO ; Jian-Xiang WANG ; Min WANG
Chinese Journal of Biotechnology 2005;21(5):686-691
The genes encoding for the light and heavy chain variable regions were cloned by RT-PCR from a murine monoclonal hybridoma cell line, which could produce monoclonal antibody to recognize CD19 antigen on human B lymphocyte. Then fused the light and heavy chain variable regions together by a short peptide linker containing 15 amino acid (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant anti-CD19- ScFv was subcloned into the expression vector pET28a and induced to be expressed by IPTG in E. coli BL21. SDS-PAGE and Western blot analysis showed that the recombinant anti-CD19-ScFv gene was expressed in E. coli BL21. ScFv expression was in the form of an inclusion bodies and the purified fusion protein was obtained after a series of purification steps including cell break, inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding. Flow cytometry analysis showed that the ScFv can react with human CD19 antigen. In conclusion, recombinant anti-CD19-ScFv gene has been successful constructed and expressed in E. coli BL21, which could provide a basic study for the future target therapy to the B lymphoid leukemia and B lymphoma.
Antibodies, Monoclonal
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biosynthesis
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genetics
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Antigens, CD19
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immunology
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Humans
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Immunoglobulin Fragments
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biosynthesis
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genetics
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immunology
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Immunoglobulin Variable Region
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biosynthesis
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genetics
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immunology
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Recombinant Proteins
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biosynthesis
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genetics
10.The heat shock protein 90 inhibitor induces apoptosis and differentiation of Kasumi-1 and its mechanisms.
Wen-juan YU ; Qing RAO ; Min WANG ; Zheng TIAN ; Xiang-rong LIU ; Dong LIN ; Jian-xiang WANG
Chinese Journal of Hematology 2005;26(12):728-731
OBJECTIVETo explore the effect of 17-allylamide-17-demethoxygeldanamycin (17AAG), a heat shock protein 90 (HSP90) inhibitor, on the growth, differentiation and apoptosis of leukemic Kasumi-1 cells.
METHODSKasumi-1 cells were treated with 17AAG at different concentrations in suspension culture. Cell proliferation was analysed by MTT assay, expression of myeloid-specific differentiation antigen and cell cycle by flow cytometry, cell apoptosis by annexin V staining, agarose gel electrophoresis and flow cytometry. KIT protein was analysed by Western blot and c-kit mRNA by RT-PCR.
RESULTS17AAG treatment caused a dose-dependent inhibition of the cell proliferation with the IC(50) of 0.62 micromol/L. A dose-dependent increase in early apoptosis occurred at 24 hours treatment and in late apoptosis at 48 hours treatment. 17AAG induced a time- and dose-dependent increase in expression of myeloid cell surface protein CD11b and CD15, a progressive decline in S-phase cell fraction and an increase in G(0)/G(1) cells. When Kasumi-1 cells were incubated with 1 micromol/L of 17AAG, KIT protein began to decrease at 2 hours and KIT protein could hardly be detected at 20 hours, but c-kit mRNA was not decreased.
CONCLUSION17AAG treatment of Kasumi-1 cells could lower KIT protein expression, inhibit cell proliferation, induce cell partial differentiation, apoptosis and accumulation in G(0)/G(1) phase.
Apoptosis ; drug effects ; Benzoquinones ; pharmacology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; RNA, Messenger ; genetics