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Objective:To explore the microRNA profile changes in the development of NKT cells.Methods: Differently developmental stage of NKT cells in mouse thymus were sorted by flow cytometry.Total RNA were extracted,reversely transcribed and pre-amplified.TaqMan low density microRNA assay and single real-time PCR were applied to detect the expression changes of microRNAs in the developmental process of NKT cells.Results: There were total 92 microRNAs whose expression changed significantly during the development and maturation of NKT cells.Among them,increasly expressed microRNAs were 71,including 36 microRNAs whose expression continuously increased;decreasly expressed microRNAs were 21,including 12 microRNAs whose expression continuously decreased.In addition,single real-time PCR analysis showed that the expression of Let-7f,miR-150,miR-24,miR-29 increased,while the expression of miR-223 and miR-155 decreased during the development and maturation of NKT cells.Conclusion: NKT development and maturation is accompanied by expression changes of large amount of microRNAs,indicating that specific microRNA regulates NKT development and function.
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Objective:To investigate the effect of miR-150 gene deletion on the breeding and hematologic parameters of mice. Methods:The nest litter size,wean rate and weight changes of miR-150 knock out (miR-150ko) and C57BL/6J mice were com-pared. The hematology indexes were analyzed by automated blood cell counter, the serum biochemical parameters were analyzed by automatic biochemical analyzer. Results:The nest litter size and wean rate of miR-150ko mice were significantly decreased compared with that of C57BL/6J mice. The number of total white blood cells,intermediate cells,neutrophils,and the percentage of neutrophils and intermediate cells were significantly increased in miR-150ko mice compared with that of C57BL/6J mice. However,the number and per-centage of platelets and lymphocytes decreased significantly in miR-150ko mice. In addition,the levels of serum glucose and TC were in-creased significantly in miR-150ko mice compared with that of C57BL/6J mice. Conclusion: miR-150 gene deletion impairs the breeding and has complex impact on hematologic parameters of mice.
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Objective To explore changes of the myeloid derived suppressor cell (MDSC), regulatory T cell (Treg), traditional T cell, and their mechanisms in lung tumor mice. Methods Twenty C57BL/6 mice were randomly divided into the experimental and the normal control groups. The experimental group was injected with Lewis lung cancer cells (LLC, 100μL 1 × 106) subcutaneously to prepare the lung tumor model mice, the normal control group was given the same amount of saline (NC). Spleen cells were obtained from LLC and NC groups. Flow cytometry was used to detect the ratio and number changes of MDSC, Treg, CD4+and CD8+T cells in the lung tumor of mice. CD4+and CD8+T cell apoptosis were detected by Annexin-Ⅴstaining, and their proliferation were detected by 5-bromine deoxidization uracil nucleoside (BrdU) incorporation. Results Compared with normal control mice, the ratio and number of MDSC in spleen increased significantly in LLC group (P<0.01), in addition, the ratio of CD4+Foxp3+Treg in CD4+T cells and their number in spleen increased significantly in LLC group. However, the ratio and number of CD4+and CD8+T cells in spleen decreased significantly in LLC group (P<0.05). The proliferation of CD4+and CD8+T cells decreased significantly in LLC group compared with that of NC group (P<0.05), while the apoptosis of CD8+T cells increased significantly (P<0.05). Conclusion MDSC and Treg cells increase in lung tumor model mice, which inhibit proliferation of CD4+and CD8+T cells and promote apoptosis of CD8+T cells.
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Objective:To explore the changes of number of CD4+ CD25 + foxp3 + regulatory T cells (Treg)and natural killer cells (NK)in the peripheral immune organs and tumor tissue of the murine models of lung cancer,and to clarify their effects on the development of lung cancer.Methods:The C57BL/6 mice were divided into Lewis lung carcinoma cells (LLC)injection group and normal control group.The mice in LLC injection group were injected with LLC subcutaneously in the armpit to establish the tumor models,while the mice in normal control group were injected with the same amount of saline.The number of CD4+ CD25 + T cells,CD4+ CD25 + foxp3 + Tregs in the spleen,lymph nodes and lung cancer tissues,and the number of NK cells in the spleen tissue were labeled by cell surface or intracellular antibody staining,and detected by flow cytometry.Results:The ratios of CD4+ CD25 + T cells to CD4+ T cells,foxp3+ cells to CD4+ CD25 + T cells,and the number of CD4+ CD25 + foxp3 + Treg in the spleen and lymph nodes of the mice in LLC injection group were increased significantly compared with normal control group (P <0.05 or P <0.01).Moreover,the ratios of CD4+ CD25 + T cells to CD4+ T cells and foxp3 + cells to CD4+ CD25 + T cells in the tumor tissue were significantly higher than those in the spleen and lymph nodes of the mice in LLC injection group.However,the ratio of NK cells in the spleen tissue of the mice in lung cancer group was significantly decreased compared with normal control group (P <0.05).Conclusion:The increase of ratio and the number of Treg cells and the decrease of ratio of NK cells in the main immune organs of lung cancer mice may promote the development of tumor and inhibit the immune response to cancer cells in vivo .
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Objective:To investigate the change of nature killer T cell(NKT)and myeloid derived suppressor cells(MDSC) during the development of mouse lung tumor.Methods:Lung tumor mouse models were made by subcutaneous injection of Lewis lung tumor cells( LLC) ,peripheral blood leukocytes were extracted from mouse tail blood at different time points after LLC injection.NKT and MDSC were detected by flow cytometry after relative antibody staining.Results:With the increasing volume of lung tumor,the ratio of NKT cells decreased gradually,while the ratio of MDSC increased gradually in the peripheral blood of LLC-injected mice.Both NKT and MDSC showed significantly changes in LLC-injected mice compared with that of normal control mice.Conclusion:NKT and MDSC in LLC-injected mice show opposite changes during the development of lung tumor,so,they can be used as potential monitoring index for lung tumor development.
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Objective:To investigate the changes of CD4+T cells,CD8+T cells and myeloid derived suppressor cells( MDSC) in immune organs of lung cancer mice.Methods: Lung cancer mouse models were made by subcutaneously injection of Lewis lung cancer cell line( LLC).The CD4+T cell,CD8+T cell and MDSC were detected by flow cytometry.Results:Compared to that of normal control mice,the ratio and numbers of CD4+and CD8+T cell were significantly decreased in the spleen and lymph nodes of lung cancer mice.The number of CD4+T cells had no significant change while CD8+T cells decreased in the bone marrow of lung cancer mice.However,the ratio and number of MDSC were significantly increased in the bone marrow,spleen and lymph nodes of lung cancer mice compared to that of normal control mice.Conclusion: The development of lung cancer leads to decreased T cells and increased MDSC;which may induce immune tolerance to tumor cells.
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Objective To explore the role of zinc finger protein (ZNF)185 in the proliferation of human glioma cells. Methods Human glioma tissues and tumor adjacent tissues were obtained from glioma patients diagnosed pathologically in Tangshan Gongren Hospital from January 2011 to December 2013. Total protein was extracted from different tissues. The ZNF185 expression was detected by Western-blot assay. Total RNA was extracted from tumor adjacent tissues. ZNF 185 cod?ing sequence was obtained by RT-PCR and inserted into pEGFPC2 plasmid to construct the ZNF185 expression vector. Li?pofactamine2000 was used to transfect the ZNF185 expression vector to human glioma cell SF767. pEGFPC2 blank vector transfected SF767 cells were used as control. Changes of cell cycle were analyzed by flow cytometry, and cell proliferation was analyzed by MTT assay. Results The expression of ZNF185 decreased significantly in human glioma tissues compared to that of tumor adjacent tissues(P<0.01). The over expression of ZNF185 in SF767 resulted in the increased proportion of cell cycle G0-G1phase, but decreased proportion of S phase(P<0.05). Furthermore, the cell proliferation was significantly inhibited in the ZNF185 over-expressed SF767 cells compared with that of blank vector transfected cells(P<0.05). Con?clusion ZNF185 plays an inhibitory role in cell proliferation of human brain glioma cells.
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Objective To explore the effect of p21Waf1/Cip1 methylation changes on the process of cellular senescence .Methods Bisulfite sequencing was used to analyze the methylation changes of p21Waf1/Cip1 in the process of cellular senescence;p21Waf1/Cip1 ex‐pression was detected by RT‐PCR and Western‐blot ;Middle‐aged 2BS cells was treated by 5‐aza‐CdR and cellular senescence was detected by MTT and SA‐β‐Gal staining .Results Bisulfite sequencing analysis of p21Waf1/Cip1 promoter showed that CpGs were methylated by 1 .25% in the young 2BS cells ,by 27 .27% in the middle‐aged 2BS cells ,while only by 0 .64% in the senescent cells . The expression of p21Waf1/Cip1 was low in the young(28 PD) 2BS cells ,it increased first(35 PD) but decreased later in the middle‐aged(42 PD) cells .In the senescent 2BS cells ,p21Waf1/Cip1 expression was further increased .5‐aza‐CdR treatment resulted in de‐creased growth rate but increasedβ‐Gal staining of middle‐aged 2BS cells .Conclusion The process of cellular senescence is regula‐ted by the status of p21Waf1/Cip1 methylation ,and p21Waf1/Cip1 demethylation accelerates cellular senescence .
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To explore the effects of microRNA-150 deletion on the development and homeostasis of regulatory T cells (Treg),γδT cells,NK and NKT cells.Methods:microRNA-150 knockout mice were used and microRNA-150 expression was detected by Real-time PCR.The numbers of Treg ,γδT,NK and NKT cells in the thymus and spleen of normal control and microRNA-150 knockout mice were detected by Flow cytometry.Cell apoptosis was detected by Annexin V staining , and cell proliferation was detected by 5-Bromo-2-deoxyUridine ( Brdu ) incorporation.Results: microRNA-150 deletion did not affect the development and homeostasis of regulatory T cells (Treg) andγδT cells.However,microRNA-150 deletion resulted in a significant reduction of the NK and thymic NKT cell number.In addition, microRNA-150 deleted NK and NKT cells showed an arrested developmental and maturational phenotype with a reduced expression of NK 1.1 and CD122.Moreover , cell apoptosis was significantly increased in microRNA-150 deleted NK and thymic NKT cells ,while a lower cell proliferation rate was shown in the microRNA-150 deleted NK but not NKT cells.Conclusion: CD122 may play an important role in the development and homeostasis of mouse NK and NKT cells regulated by microRNA-150.
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Objective To investigate the clinical efficacy of transthoracic echocardiography (TTE) in catheter interventional treatment of congenital heart disease. Methods 57 patients with congenital heart disease were selected by preoperative TTE screening and then received Amplatzer occluder interventional treatment under X-ray monitoring. 23 of them were atrial septal defect (ASD), 29 were ventricular septal defect (VSD), 4 were patent ductus arteriosus (PDA), and one was VSD complicated with ASD. Results All the patients were treated successfully. The occlusion effect was observed by follow-up immediately after the procedure, and one week, one month, three months, six months, and one year after the procedure. The position of the occluder did not change, the surrounding of the occluder has no residual shunt. 3 cases of ASD and 2 of VSD were failed to plugged. Conclusions TTE has important clinical values in selection of the patients with indication , intraoperative detection of the release of Amplatzer, and postoperatve assessment of the efficacy.
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Objective:To explore the effect of Zinc finger protein 185 (ZNF185) on the migration of human gliocytoma cells. Methods: ZNF185 protein expression in human gliocytoma and its adjacent normal tissues was detected through Western blot. The ZNF185 gene was cloned and transfected into the human gliocytoma cell line SF767. ZNF185 cells were located through immunofluo-rescence staining, and cell migration was analyzed using the cell scoring method. Results:CZNF185 protein expression was lower in the human gliocytoma cell line than in normal para-neoplastic tissues. ZNF185 protein was mainly expressed in the cytoplasm and cell membrane of the human gliocytoma cells and was co-located with F-actin. ZNF185 overexpression repressed the migration of human gliocytoma cells. Conclusion:ZNF185 represses the migration of human gliocytoma cells by binding to F-actin.