LncRNA Gm13568 regulates the activation of A1 astrocytes and affects the EAE process in mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of long non-coding RNA (lncRNA) Gm13568 on the activation of A1 astrocytes and the progress of experimental autoimmune encephalomyelitis (EAE) in mice.Methods:A recombinant lentiviral vector (LV-Inhibit-Gm13568) carrying astrocyte-specific promoter of glial fibrillary acidic protein (GFAP) was established to inhibit the function of endogenous Gm13568. A control vector (LV-ctrl) was established as well. The recombinant vectors were packaged. C57BL/6 mice were injected with 1×10 7 transforming units of viral suspension via the tail vein and 7 d after the injection, myelin oligodendrocyte glycoprotein 35-55 (MOG 35-55) was used to establish the mouse model of EAE. Four groups, PBS group, EAE group, LV-ctrl+ EAE group and LV-Inhibit-Gm13568+ EAE group, were included in this study. Clinical signs of the mice were monitored daily in a double-blinded manner. The mice were sacrificed 23 d after the EAE model was established and the spinal cord tissues were collected. The expression of Serping 1, C3, Srgn and H2-T23 at mRNA level was detected by real-time PCR. Changes in the expression of IL-6, TNF-α, macrophage chemotactic protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) were measured. Western blot was used to investigate the expression of GFAP and Notch1 in spinal cord tissues and the phosphorylation of signal transduction and transcription activator 3 (STAT3). The expression of Notch1 intracellular domain (NICD) and GFAP in spinal cord tissues was detected by immunofluorescence. Furthermore, the infiltration of inflammatory cells and the demyelination of spinal cord were observed using HE and Luxol fast blue (LFB) staining methods. Results:Compared with PBS group, A1 astrocytes were activated and Notch1 expression was significantly up-regulated in EAE group and LV-ctrl+ EAE group. The clinical score of mice in LV-Inhibit-Gm13568+ EAE group was decreased from an average score of 3.5 to less than 1 on 23 d after antigen induction and the clinical symptoms were alleviated as compared with the mice in LV-ctrl+ EAE group. Meanwhile, the activation of A1 astrocytes was down-regulated, and the production of inflammatory cytokines and chemokines was also reduced. The expression of Notch1, GFAP and NICD at protein level and the phosphorylation of STAT3 were significantly reduced. Moreover, the infiltration of inflammatory cells and demyelination of spinal cord tissues were alleviated significantly.Conclusions:LncRNA Gm13568 might regulate the activation of A1 astrocytes via the Notch1/STAT3 pathway, thus affecting the production of inflammatory cytokines and chemokines and participating in the process of EAE.

Report on recent discontinuation of tyrosine kinase inhibitors and alternative approaches to eradicating the chronic myeloid leukemia stem cells in the 55th ASH annual meeting / 白血病·淋巴瘤

Tyrosine kinase inhibitor (TKI) may significantly improve the treatment outcome in chronic myeloid leukemia (CML).It is the most frequent question about whether the patients with durable complete molecular response (CMR) can safely discontinue TKI treatment without relapse.This has focused attention on the strategies to eradicate residual CML cells,especially the CML stem cells,which should result in long term leukemia-free survival and permanent cure.Here,the progress on discontinuation of TKI therapy and alternative approaches to eradicating CML stem cells in the 55th ASH annual meeting is reviewed.

Effects of kirenol on bovine type II collagen-induced rat lymphocytes in vivo and in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of kirenol on bovine type II collagen (CII)-specific lymphocytes in vivo and in vitro, and explore the mechanism of kirenol-induced immunosuppression in antigen-specific lymphocytes.</p><p><b>METHODS</b>Twenty-four Wistar rats were randomized into control group, collagen-induced arthritis (CIA) model group, kirenol group (2 mg/kg), and prednisolone group (2 mg/kg). After CII injection, the rats in the latter two groups received intragastric administration of kirenol and prednisolone for 30 days, and the spleens and draining lymph nodes of the rats were harvested to prepare single cell suspensions for measurement of the cytokine levels using ELISA. In the in vitro experiment, the lymphocytes from the control rats, with or without 20 µg/ml CII treatment in the presence of 0-80 µg/ml kirenol, were evaluated for cell proliferation and apoptosis using [(3)H]-thymidine incorporation and flow cytometry, respectively.</p><p><b>RESULTS</b>Compared with those in CIA group, IFN-γ and TNF-α production was significantly reduced in splenocyte culture supernatant of kirenol group (P<0.05 and P<0.01, respectively), and the level of IL-10 and IL-4 was up-regulated (P<0.05 and P<0.01, respectively); IFN-γ and TNF-α secretion by the cultured lymph node cells (LNCs) significantly decreased (P<0.05 and P<0.001, respectively) and IL-10 and IL-4 production increased (P<0.05, P<0.001) in kirenol group. In the in vitro experiment, kirenol treatment caused obvious suppression of CII-induced LNC proliferation and dose-dependently induced antigen-specific apoptosis of the splenocytes and LNCs.</p><p><b>CONCLUSION</b>Kirenol treatment reduces pro-inflammatory cytokine secretion, increases anti-inflammatory cytokine production, inhibits cell proliferation and induces apoptosis of CII-specific lymphocytes in vitro, suggesting the potential of kirenol as an immunosuppressant.</p>

Apoptosis-associated Gene PDCD5 Expression in Esophageal Cancer in Xinjiang Kazakh and Han Nationality / 中国肿瘤临床

Objective: To study the expression of PDCD5 mRNA and its significance in esophageal cancer in Xinjiang Kazakh and Han nationality. Methods: RT-PCR was used to detect the mRNA of PDCD5 in 40 cases of esophageal cancer (18 cases of Kazakh, 22 cases of Han). Results: The positive rate of PDCD5 mRNA expression in 40 samples of esopha-geal cancer tissue, adjacent tissue, and normal tissue was 80.0% (32/40), 80.0% (32/40) and 87.5% (35/40), respectively, with no significant difference (P>0.05). The Ods of cancer tissues, adjacent tissues, and normal tissues were 0.7644± 0.1444, 0.9341 ±0.1631 and 1.8703±0.4767, respectively. The expression of PDCD5 was significantly increased in cancer tissues compared with that in normal tissues (P<0.05). The expression level of PDCD5 mRNA was not significantly correlat-ed with the degree of differentiation and lymph node metastasis. Conclusion: No significant difference was found in PDCD5 mRNA expression in esophageal cancer between Xinjiang kazakh and Han nationality (P>0.05). The expression of PDCD5 is not correlated with the degree of differentiation, depth of invasion or lymph node metastasis. Detection of PDCD5 mRNA expression in esophageal cancer tissues may provide valuable information for patient prognosis.

The appraisal in diagnosis of active pulmo-tuberculosis of rCFP-10/ESAT-6 fusion protein by ELISA / 中华微生物学和免疫学杂志

Objective To sensitize the T-cell in the peripheral blood of the active tuberculosis pa-tients by rCFP-10/ESAT-6 fusion protein, phytohaemaggiutinin(PHA) and physiologic saline, and to detect the IFN-γ to approach the significance of the tuberculosis infection. Methods One hundred and eleven pa-tients were diagnosed by clinical definite, 292 undergraduate students were chosen by X-ray and PPD-selec-fion as volunteers. 3.0 ml of blood was taken from each volunteer, rCFP-IO/ESAT-6, PHA and physiologic saline were added into each 1.0 ml, respectively. The A valule and antibody of IFN-γ were assayed by ELISA. Results Treated with rCFP-10/ESAT-6 group: the A value average of patients group was 1. 3885±0.6236, students group was 0.2944±0.0917. Intergroup t'=16.4259, P<0.05, set>0.42 as cut-off, the positive rate of patients group was 93.58%, students group was 13.07%. Treated with PHA group: the A value average of patients group was 1.2463±0.5541, of which the other was 0.5613±0.064, t'=19.1797,P<0.05. Treated with physiologic saline group:the A value average of patients group was 0.0772±0.0444,of which the other was 0.0290±0.0235,t'=13.9487,P<0.05. All had significant deviation. The antibody positive rate of the patients group was 66.36%, the students group was 7.19%. Conclusion rCFP-10/ESAT-6 as specific antigen, the sensitivity of IFN-γ release assay by ELISA is above 90%. No matter specific or non-specific disposal, the active tuberculosis patients have higher IFN-γ, release level and antibody than the control group.

Establishment of human IFN-gamma in vitro release assay and its application in tuberculosis diagnosis / 生物工程学报

This study aimed to establish human IFN-gamma (hIFN-gamma) in vitro release assay and to apply it in diagnosis of human tuberculosis. Human IFN-gamma gene was cloned and expressed in Escherichia coli. The recombinant hIFN-gamma was purified and used as immunogen to immunize mice and rabbits respectively. Monoclonal and polyclonal antibodies were respectively developed and a sandwich ELISA was established. The heparized whole blood from 111 active tuberculosis patients and 292 clinical healthy controls were collected. The blood was stimulated with tuberculosis specific fused antigen ESAT-6/CFP-10 and the plasma was collected for IFN-gamma detection. The sensitivity for tuberculosis diagnosis was 95.5%, whereas the positive detection rate for the healthy controls was 16.7%. There was a significant difference between the patients and healthy controls (P<0.01) indicating that this assay had a high sensitivity and specificity, and thus could be promising in tuberculosis diagnosis.

Expression of novel apoptosis-related protein PDCD5 in granulosa cells of polysystic ovary syndrome / 北京大学学报(医学版)

Objective: To detect the novel apoptosis-related protein PDCD5 expression in granulosa cells of polysystic ovary syndrome(PCOS) and normal ovary, and explore the pathogenesis of PCOS. Methods:The granulosa cells were collected from 30 cases of PCOS and normal ovary in IVF-ET. Expression of PDCD5 was detected by flow cytometry; immunofluorescence and immunohistochemistry. Cell apoptosis was detected by Propidium Iodide (PI) staining. Results: The number of hypodiploidy cells associated with apoptosis in granulosa cells of PCOS was greater than that of the normal control. PDCD5 protein expression in PCOS granulosa cells was significantly higher than that in normal ovary(P

Preparation and identification of monoclonal antibodies against SARS-CoV putative protein X4 / 北京大学学报(医学版)

Objective: To obtain monoclonal antibodies against putative protein X4 of SARS-CoV for further study of the structure and function of protein X4. Methods: Balb/c mice were immunized with recombinant Protein X4, hybridoma cell lines secreting monoclonal antibodies against protein X4 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by Western blotting and Immunofluorescecence assay. Results: Three hybridoma cell lines (8A5, 8H6 and 4A2) stable in secreting specific monoclonal antibodies were successfully obtained. They could bind specifically to protein X4 proved by Western blotting. All of them belonged to the IgG2a isotype proved by antigen mediated ELISA. Indirect Immunofluorescence assay indicated that they could specifically bind to protein X4 expressed in SARS-CoV infected Vero E6 cells. Conclusion: Monoclonal antibodies of high specificity against protein X4 have been successfully prepared, which laid the foundation for the further study of protein X4.

The effects of Zhiling capsule on the proliferation inhibition and apoptosis induction in K562 cell line via caspase-3 pathway / 中国药理学通报

Aim To investigate the effects of Zhiling capsule (ZLJN) on the proliferation inhibition and apoptosis induction in K562 cell line.Methods According to the different components of ZLJN,K562 cells were treated respectively with tradtional Chinese medicine,Western medicine and ZLJN compound groups.The cell viability and colony formation were observed by MTT assay and colony formation assay respectively.Apoptotic cells were detected by Annexin V-FITC/PI staining and DNA fragmentation assay.Caspase-3 activity was detected by flow cytometry,and pro-caspase-3 was detected by Western blot.Results Treated with drug,K562 cell growth and cell colony formation were significantly inhibited.Apoptosis occurring in the early stage was identified by Annexin V-FITC/PI staining.Typical DNA ladder was seen from gel electrophoresis and apparent apoptotic peaks were observed by flow cytometer.The level of caspase-3 activity increased after the treatment,while the level of pro-caspase-3 decreased.Conclusion ZLJN can efficiently inhibit proliferation and induce apoptosis in K562 cells,which may be related with the up-regulation of caspase-3 activity.

Effects of Zhiling capsule on apoptosis and expressions of hTERT,CD44 in small cell lung cancer cell line NCI-H446 / 中国药理学通报

Aim To explore the mechanisms of the apoptosis induction and the effects of adhesion suppression of Zhiling capsule (ZLJN) in small cell lung cancer cell line NCI-H446.Methods According to the different components of ZLJN,NCI-H446 cells were treated with traditional Chinese medicine,western medicine and ZLJN composite groups.Apoptotic cells were tested by light microscopy,Hochest33258 staining method.The mRNA and protein expressions of bcl-2,bax and hTERT were analyzed by RT-PCR and Western blot respectively.The expressions of CD44 were detected by flow cytometry.Results After NCI-H446 cells were treated with different drug groups,The morphological changes of apoptotic cells were found by light microscopy and Hochest33258 staining method.The mRNA and protein expressions of bcl-2 were down-regulated while the expressions of bax were up-regulated compared to the control groups(P

Nuclear translocation of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis / 北京大学学报(医学版)

Objective: To study the expression and localization of apoptosis-related protein TFAR19 in TF-1 cells undergoing apoptosis. Methods: Using monoclonal antibody against TFAR19, the expression level and cell localization of TFAR19 were examined by fluorescence microscope, confocal laser scan microscope(CLSM) and flow cytometry. Simultaneously, we also analyzed the relationship of TFAR19 protein with phosphatidylserine (PS) externalization and cell nuclear DNA fragmentation. Results: The level of TFAR19 proteins expressed in TF-1 cells treated with GM-CSF withdrawal was significantly increased compared with normal TF-1 cells, then translocated rapidly from cytoplasm to the nucleus of cells. Appearance of TFAR19 in the nucleus of apoptotic cells preceded the detection of PS externalization and DNA fragmentation. Conclusion: Nuclear translocation of TFAR19 protein is one of the earliest events of cell apoptotic process. These data provided a new clue to further approach to the biological function of TFAR19 and study of cell apoptosis.

Expression of TFAR19 in Apoptotic Processes of Jurkat Cells Induced with Various Methods / 中国实验血液学杂志

A new apoptosis-related gene TFAR19 was recently cloned. A primary functional analysis indicates its role in the apoptotic process of TF-1 cells. To clarify TFAR19 was involved in which apoptotic pathway, the changes in TFAR19 expression were observed during the apoptotic processes of Jurkat cells induced with various methods: serum deprivation, VP-16 treatment and Fas McAb activation. Then TFAR19 expression in Jurkat cells was detected with flow cytometry and Western blot, and TFAR19 mRNA with RT-PCR. Our results showed that expression of TFAR19 in Jurkat cells was increased at 12 hours after serum deprivation, 2 hours after VP-16 treatment and 2 hours after Fas McAb activation, respectively. These observations suggested that TFAR19 was involved in the apoptotic process of Jurkat cells induced with serum deprivation, DNA destruction and death receptor activation. It might take effect in the early apoptotic process. TFAR19 is a participant of the "final common pathway" in the apoptotic pathway.

Apoptosis of HL60 cells induced by F951,a novel bcl-2 gene antisense oligodeoxynucleotide / 中国药理学通报

Aim To observe the apoptosis of human leukemic cells induced by F951, a novel bcl-2 antisense oligodeoxynucleotide. Methods HL60 cells were cultured with F951 in variant doses. Apoptotic cells were detected by flow cytometry, DNA ladder, electron microscope observation and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Results After HL60 cells were treated for 48 hours, mitochondria apoptotic cells can be detected at the ratio of 3.00% in the untreated group, 13.57% in the FNS control group and 30.95%, 38.08%, 52.55% with 5, 10, 20 ?mol?L-1 F951 respectively; the ratio of cells with caspase activity was 0.08% in the untreated group, 0.14% in the FNS control group and 43.68%, 60.54%, 80.37% with 5, 10, 20 ?mol?L-1 F951 respectively. Typical DNA ladder was seen from gel electrophoresis in F951 treated groups, and more apparent effect in the aspect of inducing DNA ladder had been observed with the improvement of F951 concentration. Detected through TUNEL and electron microscope, apoptotic cells in untreated group and FNS control group can only be found by chance, but very commonly seen in F951 treated groups and more frequently with the improvement of F951 concentration. Conclusion F951 can induce cell apoptosis in HL60 cells. Such effect is achieved through the inhibition of bcl-2 gene expression by F951 which initialize apoptosis passageway in consequence.

Effect of F951,a novel bcl-2 antisense oligodeoxynucleotide,on human leukemia transplanted subcutaneously in nude mice / 中国药理学通报

Aim To study the inhibitory effects of F951,a novel bcl-2 antisense oligodeoxynucleotide,on expression of bcl-2,growth of tumor and survival time of nude mice transplanted subcutaneously with acute myeloid leukemia.Methods HL-60 cells with high expression of bcl-2 were proliferated in vitro.The models of the nude mice with HL-60 cells were established by subcutaneous transplantation with drugs directly injected.The effects of F951 and F951 with low dose Ara-c on growth of tumor and survival time of mice with tumor were observed.The expressions of bcl-2 mRNA in the tumors implanted were detected by fluorescent quantitation RT-PCR.The morphologic structure of tumor tissues was assayed by light microscope.Results After each group mice with tumors were treated for 14 days,the volume,the weight of tumor and the bcl-2 mRNA expression of tumor tissue were shown respectively as follows: NS control group(15.17?3.40)cm3、(12.69?0.92)g、9.79?104 Copies??g-1;FNS group(15.91?3.77)cm3,(12.38?1.21)g;8.31?104 Copies??g-1;Ara-C group(1.24?0.55)cm3,(2.32?0.49)g,2.59?104 Copies??g-1;F951 group(2.6?1.55)cm3,(3.53?0.67)g;1.01?103 Copies??g-1;F951+Ara-C group(0.62?0.48)cm3,(1.05?0.63)g,9.5?102 Copies??g-1.The data above showed that F951 could downregulate the expression of bcl-2 in nude mice with HL-60 cells xenograft and inhibit growth of tumor.The growth of tumor of F951 group was reduced,and the inhibitory rate was 72.18%,there was significant difference comparing control groups with NS and FNS(P

Down-regulation of Bcl-2 expression and inhibition of HL60 cells proliferation by F951, a novel Bcl-2 antisense oligodeoxynucleotide / 中国药理学通报

Aim To investigate whether F951,a novel Bcl-2 antisense oligodeoxynucleotide,down-regulates Bcl-2 expression in HL60 cells and inhibits HL-60 cells proliferation.Methods HL60 cells were cultured with F951 in variant doses.The proliferation of HL60 cells was assayed by MTT and Typan Blue exclusion test.Expression of Bcl-2 protein and its mRNA was measured by FACS and RT-PCR respectively.The apoptotic cells were detected by DNA ladder.Results After HL60 cells were treated with F951 in 5,10,20 ?mol?L~(-1) doses respectively for 1～5 days,they showed apparent inhibition of proliferation.With the improvement of the concentration of F951 and the prolongation of the time of treatment, F951 showed stronge effect in the aspect of inhibiting the HL60 cells proliferation.It was determined with MTT method that the inhibition rates of HL60 cells treated with 5,10,20 ?mol?L~(-1) F951 were 20.56%, 37.66%, 54.11% respectively. F951 significantly down-regulated the expression of Bcl-2 mRNA and protein in the HL60 cells.Typical DNA ladder was seen from gel electrophoresis. With the improvement of the concentration of F951,it showed more apparent effect in the aspect of inducing DNA ladder.Conclusion F951 can inhibit cells proliferation through down-regulating Bcl-2 gene expression and promoting cells apoptosis in HL60 cells.

The role of TF-1 cell apoptosis-related gene 19 in systemic lupus erythematosus / 中国病理生理杂志

AIM: To clarify the role of new apoptosis-related gene, TF-1 cell apoptosis-related gene 19(TFAR19), in the pathogenesis of systemic lupus erythematosis (SLE) and the relationship between TFAR19 and SLE. METHODS: DNA Ladder detection, Western blotting, immunological fluorescence method, ELISA and so on were used to test if ultraviolet B(UVB) could induce HaCaT cell apoptosis and TFAR19 expression. RESULTS: HaCaT cell apoptosis could be detected after 24 hours of 30 mj/cm 2 UVB irradiation. Also, we found that in active SLE patients, the TFAR19 antibody was increased, but not significant compared to the normal control. CONCLUSION: TFAR 19 is involved in the process of UVB induced ketatinocyte line HaCaT apoptosis and SLE pathogenesis.

The effect of antisense bcr - abl oligonucleotides on the growth of K562 cells / 中国病理生理杂志

AIM:To study the effect of bcr - abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chronic myelogencous leukemia (CML) gene therapy. METHODS: Cells were exposed to oligomers, observed by inverted microscope. Cells inhibitory rate were determined by 0.4% trypan blue exclusion. CFU - K562 were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. RESULTS:K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than 5?mol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h. There was significant inhibition of cell proliferation in a rang of cells number from 1 ? 10-4/mL to 5 ? 10-4/mL after treatment with 10?mol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA. CONCLUSION: bcr - abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.