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ABSTRACT: To establish a method for induced and amplified porcine monocyte-derived immature dendritic cells(imDCs).The aim of this study was to assess the pCTLA4-Ig gene-modified porcine imDCs could benefit for prevent the proliferation of xenogeneic spleen cells.
Poricne peripheral blood mononuclear cells(PBMC) were cultured with rpGM- CSF(30 microg/L) and rpIL-4(20 microg/L). To evaluate the suspending cells by transmission electronic microscope and flow cytometer. Porcine imDCs were transduced with adenovirus mediated pCTLA4-Ig gene. The mixed lymphocyte reaction(MLR) was used to examine the proliferation of spleen cells of SD rat to gene-modified imDCs.
With rpGM-CSF and rpIL-4 induced porcine PBMC for 4-5 days, it was exhibited typical morphological characteristics and immunological phenotype of imDCs with high expression of SLA-DR, CD172a and low expression of CD80/CD86 on the cellular surface. The gene-modified imDCs were able to amplified the pCTLA4-Ig and IDO gene by RT-PCR. The stimulate index of gene-modified imDCs group were markedly inferior to that in unmodified imDCs group(P<0.05).
The pCTLA4-Ig gene-modified imDCs can significantly inhibit the proliferation of xenogeneic spleen cells, and there might was correlated with the expression of IDO reduce the immune response of xenogeneic antigen.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 05/2010; 26(5):434-7.
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Yong Zhang, Yi Lü,
Xu-feng Zhang,
Liang Yu,
Chang Liu,
Ni Zhang,
Hao-hua Wang,
Zhen Wan,
Zhan-tao Xie,
Liang-shuo Hu,
Han-xiang Zhan
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ABSTRACT: To screen and identify the peptides that specifically bind to CD13 on monocytes.
The phages capable of specific binding to CD13 were screened in the phage-displayed 12-peptide library. The affinity of the selected phages with CD13 was verified with enzyme-linked immunosorbent assay (ELISA). The sequences of the peptides bound to the phages were deduced according to the phage DNA sequences, and the functional peptides aligned using the BLASTP on the Website NCBI were synthesized. To analyze the biological function of the screened peptides, the location of the peptides bound to THP-1 cells was detected using immunofluorescence assay. The blocking effect of WM15 on the peptide binding to THP-1 cells was assessed by immunofluorescence assay.
The phages that specifically bound to CD13 were effectively enriched to approach saturation after 4 rounds of panning. The recovery rate in the fourth round was 30 times that in the first round. Twenty selected phages were verified by ELISA, and the signals of 10 phages were higher than the control. The sequences of the peptides P9 and P7 showed 83% and 100% identity with those of human cytomegalovirus (HCMV) UL38 and UL105, respectively. The peptides bound to the cell membrane of THP-1 cells as shown by immunofluorescence assay. The binding of the peptides P9 and P7 to THP-1 cells was blocked by CD13-specific monoclonal antibody WM15 at different levels.
Two peptides (P7 and P9) that can specifically bind to CD13 have been screened successfully, and these two peptides show specific binding to CD13 on the membrane of THP-1 cells.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 04/2010; 30(4):827-30.
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ABSTRACT: To Package the recombinant lentivirus containing the fused gene of SP-TAT-Apoptin to infect HepG2 cell and measure the efficiency of apoptosis.
The eukaryotic expression vector of SP-TAT-Apoptin fused gene and other packaging plasmids were transfected into 293FT cells by Lipofectamine(TM);2000 reagent. The supernatant of the cultured 293FT cells was harvested and virus titration was determined by real time PCR. The expression of the fused gene of SP-TAT-Apoptin in 293FT cells infected by the recombinant lentivirus was examined by immunofluorescence histochemistry method. At the same time, the apoptosis rates of the HepG2 cells infected by the recombinant lentivirus were determined by using flow cytometer.
The 293FT cells infected by the recombinant lentivirus could express the fused protein SP-TAT-Apoptin. Anexin-V PI assay showed that SP-TAT-Apoptin carried by the recombinant lentivirus could cause the HepG2 cell apoptosis, and its apoptosis rate was significantly more than paired control group and SP-TAT-Apoptin carried by liposomes only.
The recombinant lentivirus of SP-TAT-Apoptin is successfully packaged and it can induce HepG2 cells to apoptosis.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2010; 26(4):310-2.
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ABSTRACT: To investigate the effects of small interfering RNA (siRNA) knockdown of forkhead box M1 (FoxM1) on the proliferation and invasion capacities of human hepatocellular carcinoma MHCC-97H cells in vitro.
The expression levels of FoxM1 in human hepatocellular carcinoma samples, adjacent non-hepatocellular carcinoma liver samples and MHCC-97 cell lines were detected by RT-PCR and Western blotting. FoxM1 siRNA was transfected into MHCC-97H cells with Lipofectamine 2000. Cell growth was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell cycle analysis was performed by flow cytometry. Protein expression levels were evaluated by Western blotting. Anchorage-independent growth and the invasive potency of MHCC-97H cells were measured by soft agar colony formation and a transwell cell invasion assay, respectively.
FoxM1 was over-expressed in hepatocellular carcinoma samples compared to adjacent non-hepatocellular carcinoma liver samples. FoxM1 siRNA was successfully transfected into MHCC-97H cells, resulting in the significant inhibition of FoxM1 mRNA and protein expression. Down-regulation of FoxM1 inhibited cell proliferation, caused cell cycle arrest, and decreased invasion of MHCC-97H cells. Compared with control and mock groups, the FoxM1 siRNA transfected cells showed decreased protein expressions of cyclin B1 and cyclin D1, whereas p27 protein expression was increased. Down-regulation of FoxM1 reduced the expression of matrix metalloproteinase-2 (MMP-2) and urokinase plasminogen activator (uPA).
FoxM1 is functionally involved in hepatocellular carcinoma cell proliferation and invasion and is a potential target for hepatocellular carcinoma therapy.
Acta Pharmacologica Sinica 02/2010; 31(3):361-6. · 1.95 Impact Factor
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ABSTRACT: The bile duct cannot be repaired or reconstructed in one stage after 24 h of bile duct injury due to significant inflammation. Even if the bile duct can be repaired, anastomosis is difficult because of the extremely technical nature of the procedure. The traditional method of anastomosis utilizes screw thread. This would increase the inflammatory response, delay anastomotic healing, and lead to the increase in the failure rate. To reconstruct the biliary-enteric continuity under the circumstance of severe inflammation after bile duct injury, we invented a new type of anastomotic apparatus (sutureless magnetic stent) for cholangiojejunostomy. The objective of this study was to evaluate the effect of a new type of sutureless magnetic biliary-enteric anastomosis stent, which was used to reconstruct the biliary-enteric continuity in one stage. The reconstruction was conducted under the circumstance of severe inflammation after acute bile duct injury in dogs.
We used a model of acute bile duct injury and bile peritonitis in dogs. The sutureless magnetic biliary-enteric anastomosis stents was used to reconstruct the biliary-enteric continuity in one stage under the circumstance of a bile duct with severe inflammation. The effect of stents was observed. Cholangiography and anastomotic histology were examined at 1 mo and compared with traditional manual anastomosis.
Anastomotic stents were used to reconstruct the biliary-enteric continuity in one stage in dogs. No anastomotic leak or infection occurred. Cholangiography showed that the anastomosis was unobstructed. Histological examinations showed that the anastomosis healed well, the inflammatory reaction was small, and collagen fibers lined up in order. There was high incidence of bile leakage in the conventional suture group. Cholangiography showed that anastomotic stenosis was high. Histological examination showed that there was more extensive inflammation around the anastomosis and the collagen fibers were disorganized.
It was safe and feasible to use the new type of anastomosis stent to reconstruct the biliary-enteric continuity in one stage under the circumstance of severe bile duct inflammation after bile duct injury in dogs.
Journal of Surgical Research 09/2008; 148(2):136-42. · 2.25 Impact Factor
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ABSTRACT: CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamin 2000-mediated transfer method. The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected. And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells, which lays a foundation for further research on the relationship between CCK and tumor.
Journal of Huazhong University of Science and Technology 07/2007; 27(3):278-80. · 0.38 Impact Factor
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ABSTRACT: The aim of the current study was to investigate the role of interleukin (IL)-2, interferon (IFN)-gamma, IL-4 and IL-10 in a concordant hamster-to-rat cardiac xenotransplantation model.
A hamster-to-rat cardiac transplantation was performed using SD rats as recipients of Golden Syrian hamster hearts. A total of 60 SD rats were divided into four groups and treated as follows: control group (n = 15); splenectomy group (n = 15); CsA group (n = 15); CsA + splenectomy group (n = 15). Levels of IL-2, IFN-gamma, IL-4 and IL-10 were measured by enzyme linked immunosorbent assay (ELISA). Sera were harvested at different time points in each group: day 1, and 3 as well as the day the xenograft stopped beating in the control group and CsA group; day 1, 3, 7, 14 and 30 in the splenectomy group and CsA + splenectomy group. The expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined by immunohistochemical analysis of the xenograft after cardiac xenotransplantation.
Serum levels of IL-2 and IFN-gamma were upregulated in untreated (day 3) and splenectomy-treated animals (day 7) compared to CsA + splenectomy treated animals (day 7). IL-10 was upregulated in long-term survival recipients following splenectomy + CsA. Neither P-selectin nor ICAM-1 expression was detected in long-term survival xenografts.
Serum IL-2 and IFN-gamma were elevated following acute vascular rejection. Serum IL-10 was correlated to immunosuppression and protective effects in long-term survival rats following concordant cardiac xenotransplantation.
Chinese medical journal 02/2007; 120(2):145-9. · 0.86 Impact Factor
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ABSTRACT: Pancreatic cancer in the head is frequently accompanied by jaundice and high bile acid level in serum. This study focused on the direct effects of bile acids on proliferation and ultrastructural alteration of pancreatic cancer.
Pancreatic cancer cell lines PANC-1, MIA PaCa-2 and PGHAM-1 were explored in this study. The cell lines were cultured in media supplemented with certain bile acids, CA, DCA, LCA, TCDC, TDCA and GCA. Their influence on cell growth was measured with MTT assay after 72 h of incubation. Cell cycles of PANC-1 cells in 40 microM of bile acids media were analyzed by flow cytometry. Ultrastructural alteration of PANC-1 cells induced by DCA was observed using scanning and transmission electron microscope (SEM and TEM).
At various concentrations of bile acids and incubation time, no enhanced effects of bile acids on cell proliferation were observed. Significant inhibitory effects were obtained in almost all media with bile acids. DCA and CA increased the percentage of G0+G1 phase cells, while GCA and TDCA elevated the S phase cell number. After 48 h of incubation in DCA medium, PANC-1 cells showed some structural damages such as loss of their microvilli and vacuolization of organelles in cytoplasm.
Bile acids can reduce proliferation of pancreatic cancer cells due to their direct cytotoxicity. This result implies that elevation of bile acids in jaundiced serum may inhibit pancreatic cancer progression.
World Journal of Gastroenterology 01/2004; 9(12):2759-63. · 2.47 Impact Factor
