High efficient expression of human endostatin in E.coli and its antiangiogensis activity

AIM: To examine the expression of human endostatin in E.coli, produce its fusion protein antibody and observe its biological activity. METHODS: Endostatin gene was amplified by polymerase chain reaction,recombined with plasmid vector pGEX-2T and induced expression with IPTG.The protein activity was tested by endothelial cell proliferation inhibitory assay.Inclusion body crudely purified was used to generate polyclonal antibody to detect its expression at mouse's liver and kidney etc. RESULTS: The protein expressed was 20kD after digestion by thrombin,it appeared the anti-angiogenesis activity and Western blotting indicated the expression of endostatin in liver and kidney of mouse. CONCLUSION: The successful expression of human endostatin and the preparation of polycolonal antibody indicated its potential application in anti-angiogenesis therapy and diagnosis tumors.     

Signal peptide sequence of human interleukin-2 influenced hEndostatin gene expression and protein secretion in HepG2 cells

AIM:The role of human interleukin-2(IL-2) signal peptide sequence in the effect of human Endostatin (hEndostatin) expression and secretion was investigated in HeG2 cells.METHODS:RT-PCR and Western-blotting were conduct to observe mRNA level difference of hEndostatin gene, its protein expression and secretion level difference between with hIL-2 signal peptide sequence and without it.RESULTS:mRNA level of hEndostatin gene in HepG2 (pBlast-hIL2-hEndo) cells was higher than that in HepG2(pBlast-hEndo)(P＜0.05). hEndostatin protein was detected only in HepG2 (pBlast-hIL2-hEndo) cells and its medium, and not in other HepG2 cells and medium.CONCLUSION:Human interleukin-2(IL-2) signal peptide sequence facilitate to increase mRNA level of hEndostatin gene, its protein expression and secretion in HepG2 cells.     

Angiogenesis and its regulation mechanism in S180 transplanted tumor of mice

AIM: To investigate the angiogenesis in the process of sarcoma 180 (S180) tumor transplantation and changes of regulator factors, and explore the possible mechanism. METHODS: The S180 transplanted tumor in the Km mouse was used to detect the tumor angiogenesis by immunohistochemical examination of FⅧ. The levels of VEGF (V) and endostatin (E) in serum and the homogenate of tumor tissue were measured by ELISA and EIA, and the correlation between tumor weight and microvessel count (MVC) and morphology in tumor was also analyzed by multiple ANNOVA method. RESULTS: MVC, the relative count of total vessels and relative total vessel area increased with the development of transplanted S180. VEGF level in tumor tissue were higher at the 10th and 15th day than the 5th day after tumor transplantation. Endostatin in the tumor tissue and serum both reached the highest level at the 15th day, V/E ratio did not changed in this process. Furthermore, MVC, average vessel area and relative total area had a significant correlation with tumor weight. CONCLUSION: MVC increases in the development of S180 transplantation tumor and is related with the tumor weight; the positive regulator of angiogenesis in the tumor tissue is up-regulated during tumor growth, and the regulators in the tumor tissue maintains a relative balance.     

The molecular mechanism of inhibition of murine Lewis lung carcinoma metastasis by weimaining in vivo

AIM: To investigate the anti-metastasis effect of weimaining, extracted from fragopyrum cymosum meissn, a Chinese medicine, on murine Lewis lung carcinoma (3LL). METHODS: The anti-metastasis effect of weimaining in vivo was detected in the grafting lung metastasis model of murine Lewis lung carcinoma. The effects of the drug on the expression of CD34 and E-cadherin were investigated by immunohistochemical staining and RT-PCR. RESULTS: Weimaining effectively inhibited the lung metastasis of 3LL at a concentration of 250 mg·kg^-1·d^-1, significantly suppressed the expression of CD34 and increased the expression levels of E-cadherin protein and mRNA in 3LL cells. CONCLUSIONS: Weimaining inhibits the metastasis of murine Lewis lung carcinoma (3LL) in vivo via increasing the expression of E-cadherin and decreasing microvessel density of tumor tissue.     

Inhibition of tyrosinase gene expression by antisense nucleotide in murine B16 melanoma cells

AIM: To inhibit the expression of tyrosinase gene in murine B16 melanoma cells by antisense nucleotide. METHODS: The antisense recombinant pcDNA3.1(-)-tyr was constructed and was used to infect murine B16 melanoma cells for expression of tyr antisense nucleotide. The effect of antisense nucleotide of tyr on the expression of tyr gene was detected by determination of the activity of tyrosinase and of the production of melanin, Dopa staining and electronic microscope. RESULTS: The tyr antisense recombinant was successfully constructed and injected into murine B16 melanoma cell. The activity of tyrosinase in B16 cells infected with pcDNA3.1 (-)-tyr decreased to 0.0498±0.0036, compared to the tyrosinase activity of 0.0916±0.0132 in the control cells without treatment (P<0.01). The results of Dopa staining and electronic microscopy showed that the production of melanin also decreased significantly compared with control B16 cells. CONCLUSION: tyr antisense nucleotide significantly inhibits the expression of tyr gene in murine B16 melanoma cells.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

Effects of sterigmatocystin on IL-2 and IFN-γ secretion and expression in murine spleen cells in vitro

AIM: To explore the effects of sterigmatocystin (ST) on IL-2 and IFN-γ expression and secretion in murine spleen cells in vitro. METHODS: The secretion and expression of IL-2 and IFN-γ in murine spleen cells after ST pretreatment at five different dosages(0.125 mg/L,0.25 mg/L,0.5mg/L,1 mg/L,2 mg/L) were studied with ELISA and semi-quantitative RT-PCR method, respectively. RESULTS: Pretreatment of murine spleen cells in vitro with ST at five different dosages affected the IL-2 and IFN-γ secretion at protein level and expression at mRNA level of the treated cells. The effects varied dependently to the ST dosage. At relatively lower dosages, ST induced the expression of IL-2 and IFN-γ in murine spleen cells, while at relatively higher dosages, inhibitory effects were found, with the most significant inhibitory effects seen in ST 1 mg/L group. CONCLUSION:ST affected the se-cretion and expression of IL-2 and IFN-γ in treated murine spleen cells.At relatively lower dosage, ST induced IL-2 and IFN-γ secretion and expression, while at relatively higher dosages，inhibitory effects appeared.     

Activation of Akt signal pathway cascades in kidney tissue in murine chronic graft-versus-host disease lupus nephritis and its regulation by prednisone

AIM:To examine whether Akt signal pathway proteins, including Akt, NF-κB and IκBα, are activated in kidney tissue of murine chronic graft-versus-host disease (GvHD) lupus nephritis in vivo, and whether prednisone suppresses activation of them.METHODS:Akt activity and phosphorylated IκBα were detected by Western-blot. Activation of NF-κB was detected by electropheretic mobility shift assay (EMSA). RESULTS:Activity of Akt, NF-κB and phosphorylated IκBα were significantly increased in kidney tissue of murine chronic graft-versus-host disease (GvHD) in 8th week and 12th week after monocell injection, respectively. However, they were no significant elevation in 16th week, when compared with controls. Prednisone treatment significantly prevented the increase in serum anti-dsDNA antibody level, urinary protein excretion and glomerular cell proliferation in GvHD mice, indicating the beneficial effects of prednisone on this model. Prednisone also significantly suppressed the increase in the activities of glomerular Akt, NF-κB and phosphorylated IκBα. CONCLUSION:This study provides the first evidence of marked increase in glomerular Akt-NF-κB signal pathway activities in murine chronic graft-versus-host disease lupus nephritis. The beneficial effect of prednisone on this lupus nephritis model may be partially mediated by the suppression of abnormal Akt- NF-κB activation.     

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

AIM: To investigate the effect of dihydroartemisinin (DHA) on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS: Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester (CFDA-SE) staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3+ T lymphocytes, was measured by flow cytometry (FCM) combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration (［Ca2+］i) of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4+CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS: The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3+T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4+CD25high Treg. CONCLUSION: DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.     

Role of nestin in the maintenance of the structure of podocytes

AIM:To investigate the expression of nestin, a kind of cytoskeletal protein in cultured murine podocytes and the role of nestin in the maintenance of the podocyte structure.METHODS:The immortalized murine podocytes were cultured. The expression of nestin was determined by immunofluorescence. In differentiated podocytes, the expression of nestin was knock-down by RNAi. The effect of nestin knock-down was examined by Western blotting and immunofluorescence. The projections longer than maximal length of the cell body in which the cells transfected with siRNA and control vector that contained nonhomologous oligo were counted, respectively. RESULTS:Nestin siRNA markedly reduced or abolished nestin expression. In cells transfected with nestin siRNA, the percentage of cells with processes was significantly lower than that in cells transfected with control vector (77.0%±6.3% vs 16.0%±4.6%, n=3, P<0.01). CONCLUSION:Nestin may play an important role in maintaining normal function of podocytes.     

Allogeneic antigen-pulsed murine immature CD8α+ dendritic cells prevent T-cells proliferation in vitro

AIM: To explore the optimal condition of predominant immature CD8α⁺ dendritic cells(predo-iDCs) preventing T-cell proliferation pulsed by allogeneic antigen. METHODS: Predo-iDCs, induced with GM-CSF +IL-4 +SCF +Flt3L from SPF healthy C57BL/6 murine bone marrow cells, were pulsed by different doses (0, 2.5, 5, 10 and 20 mg/L)of allogeneic murine splenocyte antigen. Syngeneic T-cells were co-incubated with Ag-pulsed DCs (DCs/T=1∶ 1, 2∶ 1, 4∶ 1) and the T-cell proliferation was measured by MTT. The secretion of cytokines (IFN-γ and IL-10) in the co-incubated supernatants was detected by ELISA. The effect of prevention of T-cell proliferation generated by murine predo-iDCs pulsed by allogeneic antigen was detected. The control derived from mature dendritic cells(mDCs)induced by GM-CSF +IL-4 +TNF-α. RESULTS: The effect of Ag-pulsed predo-iDCs for stimulating T-cell proliferation was the slightest in predo-iDCs/T 1∶ 1 group, compared with that in predo-iDCs/T 2∶ 1 and 4∶ 1 group (P<0.05). The secretion of IFN-γ in mixed lymphocyte reaction(MLR) was significantly lower than the one in mDCs control group, while the secretion of IL-10 was higher than that in control group when low dose of antigen (<2.5 mg/L) was added into MLR. CONCLUSION: Predominant iDCs pulsed by low dose of allogeneic antigen (2.5 mg/L) mixed 1∶ 1 with T-cells is the optimal condition for the prevention of T-cells proliferation.     

Protective effect of the bone marrow cells transfected with multidrug resistance gene on the reconstruction of murine hematopoietic function

AIM: To investigate the protective effect of the bone marrow cells transfected with human multidrug resistance gene (MDR1) on the reconstruction of murine hematopoietic function.METHODS: The mononuclear cells of the bone marrow from donors, BALB/C mice, treated with 5-Fu previously, were isolated and transfected with human multidrug resistance gene in vitro , then transplanted to the tertiary recipients. After lethal irradiation(8.5 Gy) and bone marrow transplantation, the recipients were selected with Taxol 7 mg/kg intraperitoneal injection, VCR 5 mg/kg or DNA 5 mg/kg intravenous injection. The survival rate and blood pictures of mice as well as the integration and expression of target gene MDR1 were studied. RESULTS: The lethal irradiated murine hematopoietic function could be reconstructed and protected from toxicity of high doses Taxol, VCR and DNR selection after reinfusing the hematopoietic progenitor cells containing human multidrug resistance gene (MDR1). The survival rate and survival time of experimental mice were higher than that in the control group. The integration and expression of MDR1 gene in recipients were confirmed by PCR, RT-PCR and FCM. CONCLUSION: The integration and expression of human multidrug resistance gene in recipients may play an important role in the reconstruction and protection of murine hematopoietic function.     

Ca2+-activated chloride channels and airway mucus overproduction in asthma

Ca2+-activated chloride channels (CaCC, CLCA) are a new family of chloride channels discovered recently, which are related to Ca2+-sensitive chloride ions transport, and there were several members identified currently in different biologic bodies. New studies have shown that its member gob-5 in murine （its human counterpart is CaCC1） plays a pivotal role in airway goblet cell metaplasia, mucus overproduction, MUC5AC gene expression as well as airway hyperresponsiveness (AHR) in asthma, and is a key molecule in the induction of murine asthma and an asthma-related gene. Inhibition of their function and/or their signaling pathway may therefore provide a new therapeutic strategy for asthma.     

CNKI引文数据库《园艺学报》高被引频次论文排序

(截至2006年6月,排名前100位)序号被引文献题名被引文献作者被引文献来源被引频次1蔬菜硝酸盐累积的研究———Ⅰ.不同蔬菜硝酸盐沈明珠,翟宝杰,东惠茹,李俊国园艺学报/1982/04266和亚硝酸盐含量评价2温室土壤次生盐渍化的形成和治理途径研究童有为,陈淡飞园艺学报/1991/021323     

Effects of anti-leukemia by antisense c-myc by retrovirus vector

AIM: To study the role of c-myc oncogene in L6565 leukemia oncogenesis and the effects of therapy by inhibition of its expression with antisense c-myc. METHODS: A recombinant retroviral vector containing antisense c-myc of the murine (pGNCas) was constructed and then transfected into PA317 cells by the method of calcium phosphate precipitation. L6565 clone cells were infected with retrovirus particles. Stable integretion of antisense c-myc was shown by PCR. The change of the malignance and phenotype of L6565as were detected by the examination of the growth, morphology, cells cycle, agar assay and expression of c-myc. RESULTS: The shape of most L6565as cells became spherical. The growth of L6565as was inhibited compared to control cells. The analysis of cells cycle: L6565as cells were arrest in G₀/G₁ phase, decreased in S phase. The ability of L6565as cells to form colony in soft agarose was significantly suppressed. c-myc in L6565as cells was lowly expressed. CONCLUSION: (1)c-myc plays a critical role in L6565 leukemia oncogenesis; (2)Inhibition of expression of c-myc makes partly reversion of malignant phenotype of L6565 murine leukemia clone cells.     

Effect of CCK-8 on the B7.1 and B7.2 expressions and costimulation of endotoxemia murine perineral macrophages

AIM: To investigate in vivo effects of CCK-8 on the expressions of B7.1 and B7.2 and the costimulatory activity for T lymphocytes in endotoxemia murine perineral macrophages.METHODS: BALB/c mice were randomly assigned to 8 groups (n=4) injected ip.with NS alone (0.2-0.3 mL/mouse),or with LPS (100 μg/mouse),in the presence or absence of CCK-8 (5 nmol/mouse) and CR1409 (100 μg/mouse),CR2945 (100 μg/mouse).After 12 h,macrophages were purified from the peritoneal exudate.The B7.1/B7.2 expression on purified macrophages was analyzed by flow cytometry and,alternatively,purified macrophages were assayed for macrophage costimulatory activity.ConA was added into the culture medium to stimulate CD4+T cell proliferation.The proliferation was determined by measuring ［³H］-TdR incorporation in a β-scintillation counter.RESULTS: The in vivo administration of CCK-8 resulted in increase of B7.2 expression,but without any influence on B7.1 expression in peritoneal macrophages.CCK-8 also exhibited increased the ［³H］-TdR incorporation in CD4+T cells.However,the in vivo CCK-8 administration reduced both B7.1 and B7.2 expression in LPS-induced endotoxemia murine peritoneal macrophages and the ［³H］-TdR incorporation in CD4+T cells.These effects were consistent with the in vitro effects of CCK-8 on LPS-stimulated peripheral macrophages.CR1409 and CR2945 abolished the above effects of CCK-8.CR1409 was more effective than CR2945.CONCLUSION: CCK-8 enhances macrophage costimulatory activity by upregulating B7.2 expression,and at the same time,reduces LPS-induced costimulatory activity of endotoxemia murine perineral macrophages by downregulating B7.1 and B7.2 expression,which is mediated by CCK1R and CCK2R.CCK1R might be the major receptor responsible for the modulation of CCK-8 on costimulation.     

Differentiation of Sca-1+ cells from murine fetal liver into renal cells in mice with acute renal failure

AIM: To study the effect of acute renal failure (ARF) on the differentiated frequency of Sca-1+ cells from murine fetal liver in irradiated mice. METHODS: The Sca-1+ cells from murine fetal liver were isolated with magnetic cell sorting (MACS) technique, the sex of which was identified by PCR. The 2×104 Sca-1+ cells were transplanted into a lethally irradiated (［60Co］, 8 Gy) inbred female mouse. After 8 weeks, these recipient mice were divided to A, B, and C groups at random (A group: irradiated; B group: ARF; C group: ARF and Sca-1+). The mice in B and C groups were induced to ARF with 50% (V/V) glycerin (11.6 mL/kg). 72 hours later, the mice in C group were injected with the fresh prepared Sca-1+ cells again. 8 weeks later, mice were sacrificed, and their kidneys were taken out, fixed and slices were prepared. Fluorescence in situ hybridization (FISH) of renal slices was performed and the pictures of them were taken and analyzed. RESULTS: The cells containing Y chromosome were found in renal slices from the mice in A, B and C groups, which located in epithelial cells of renal tubules, interstitium, glomeruli, and glomerular margin and increased gradually. The double and encircle zone of Y chromosome cells were found in the slices from the mice in B and C groups separately, which was consist of new renal tubules. The differentiation frequency of Sca-1+ cells in kidney in A, B and C groups were (1.65±0.18)%， (8.58±1.34)% and (18.13±1.91)%， respectively, which showed significant difference between former group and later group (P<0.01). CONCLUSION: The differentiation of Sca-1+ cells from murine fetal liver into renal cells and tissue is promoted by physiological microenvironment of acute damage and regeneration.     

The transdifferentiation of Sca-1+ cells from murine fetal liver

AIM:To explore transdifferentiation potential of Sca-1⁺ cells from murine fetal liver. METHODS:2×10³ of Sca-1⁺ cells from male murine fetal liver were transfused into female mouse irradiated lethally with γ ray from ⁶⁰ Co source (10 Gy) via tail vein. Two months later, FISH and immunohistochemistry were used to detect the situation for transdifferentiating of the donor cells (male cells) in tissues of female recipient mouse. RESULTS:The renal tubular epitheliocyte-like and neurocyte-like cells with Y chromosome were found on the sections of renal and brain tissues from female recipient mice. These cells have phenotype characteristics of RCA⁺/CD₄₅^-F_4/80^- and NueN⁺/CD₄₅^-F_4/80^-, respectively. CONCLUSION:The evidence is provided for Sca-1⁺ cells from murine fetal liver to transdifferentiate into both renal and brain tissue cells.