Biomedical subjects

Bo-Rong Pan

Publications and source records attributed to Bo-Rong Pan.

At least 37 records · Page 2Linked to original sources

PubMed2003

Development of an oral DNA vaccine against MG7-Ag of gastric cancer using attenuated salmonella typhimurium as carrier.

AIM: To develop an oral DNA vaccine against gastric cancer and evaluate its efficacy in mice. METHODS: The genes of the MG7-Ag mimotope and a universal Th epitope (Pan-DR epitope, PADRE) were included in the PCR primers. By PCR, the fusion gene of the two epitopes was amplified. The fusion gene was confirmed by sequencing and was then cloned into pcDNA3.1(+) plasmid. The pcDNA3.1 (+)-MG7/PADRE was used to transfect an attenuated Salmonella typhimurium. C57BL/6 mice were orally immunized with 1X10(8) cfu Salmonella transfectants. Salmonella harboring the empty pcDNA3.1(+) plasmid and phosphate buffer saline (PBS) were used as negative controls. At the 6th week, serum titer of MG7-Ag specific antibody was detected by ELISA. At the 8th week cellular immunity was detected by an unprimed proliferation test of the spleenocytes by using a ((3)H)-thymidine incorporation assay. Ehrlich ascites carcinoma cells expressing MG7-Ag were used as a model in tumor challenge assay to evaluate the protective effect of the vaccine. RESULTS: Serum titer of antibody against MG7-Ag was significantly higher in mice immunized with the vaccine than that in control groups (0.841 vs 0.347, P<0.01; 0.841 vs 0.298, P<0.01), while in vitro unprimed proliferation assay of the spleenocytes showed no statistical difference between those three groups. Two weeks after tumor challenge, 2 in 7 immunized mice were tumor free, while all the mice in the control groups showed tumor formation. CONCLUSION: Oral DNA vaccine against the MG7-Ag momitope of gastric cancer is immunogenic. It can induce significant humoral immunity against tumor in mice, and the vaccine has partially protective effects.

Administration, Oral↗

PubMed2003

Construction and expression of recombined human AFP eukaryotic expression vector.

AIM: To construct a recombined human AFP eukaryotic expression vector for the purpose of gene therapy and target therapy of hepatocellular carcinoma (HCC). METHODS: The full length AFP-cDNA of prokaryotic vector was digested, and subcloned to the multi-clony sites of the eukaryotic vector. The constructed vector was confirmed by enzymes digestion and electrophoresis, and the product expressed was detected by electrochemiluminescence and immunofluorescence methods. RESULTS: The full length AFP-cDNA successfully cloned to the eukaryotic vector through electrophoresis, 0.9723 IU/ml AFP antigen was detected in the supernatant of AFP-CHO by electrochemiluminescence method. Compared with the control groups, the differences were significant (P<0.05). AFP antigen molecule was observed in the plasma of AFP-CHO by immunofluorescence staining. CONCLUSION: The recombined human AFP eukaryotic expression vector can express in CHO cell line. It provides experimental data for gene therapy and target therapy of hepatocellular carcinoma.

Animals↗

PubMed2003

Detection of anti-HAV antibody with dot immunogold filtration assay.

AIM: To establish a rapid, sensitive and specific immunogold assay for detection of hepatitis A virus infection. METHODS: Rabbit monoclonal antibodies to anti-human IgM and IgG (Dako) were dotted on a nitrocellulose membrane (NCM) respectively to capture the human sera IgM and IgG. Then the captured antibodies would conjugate to HAV antigen, which was revealed by mouse anti-HAV IgG conjugated to gold particles. Final results were assessed by blind method. RESULTS: Sera from 96 patients with acute hepatitis were used for our study. Compared with well-recognized standard (Abbott Laboratory, USA), the sensitivity and specificity of IgM-DIGFA (self-made) were 91.3 % (42/46) and 96.0 % (48/50), and those of IgM-ELISA (Kehua, Shanghai) were 97.8 % (45/46) and 100.0 % (50/50). The identical results were produced from the study with reagents at different conditions, and the study was repeated in 15 negative sera and 10 positive sera. The serum anti-HAV IgG was tested with DIGFA at the same time. In comparison with ELISA, the sensitivity and specificity of DIGFA for IgG anti-HAV were 87.2 % (41/47) and 91.8 % (45/49), respectively. CONCLUSION: This assay can detect anti-HAV IgM and IgG simultaneously, and be done within 3 minutes. The simplicity, rapidity and specificity of the assay were useful for screening and epidemiological study.

Enzyme-Linked Immunosorbent Assay↗

PubMed2003

Cloning of human 15ku selenoprotein gene from H9 T cells.

AIM: To clone human 15ku selenoprotein gene. METHODS: H9 human T cells were cultured in RPMI1640 medium supplemented with 100 mL /L fetal calf serum. mRNA was isolated from the cells. cDNA library was constructed by RT-PCR. The human 15ku selenoprotein gene was obtained by PCR and cloned into T vector and sequenced. RESULTS: A unique cDNA fragment about 1 244 bp was obtained. Sequence analysis identified an open reading frame within the cDNA. The gene had an in-frame TGA, which encoded selenocysteine (Sec), and a 3'-UTR SECIS element, which was required for synthesis of selenoprotein. The predicted protein molecular mass was about 15ku (162 residues). The result was identical with human liver 15ku selenoprotein gene published in Genbank. CONCLUSION: Human 15ku selenoprotein gene can be successfully obtained from T cell line.

Base Sequence↗

PubMed2002

Screening and identification of proteins mediating senna induced gastrointestinal motility enhancement in mouse colon.

AIM: To isolate the proteins involved in pharmacologic action of senna extract (SE) from mouse gastrointestinal tract and to explore the molecular mechanism of gastrointestinal motility change induced by SE. METHODS: SE was administrated to mice by different routes. Gastrointestinal motility of mice was observed using cathartic, gastrointestinal propellant movement experiments and X-ray analysis. Mouse model for gastrointestinal motility enhancement was established through continuous gastric administration of SE at progressively increased dose. At 3 h and week 3, 4, 6 and 10, morphological changes of gastrointestinal tissues were found under light microscope. Ultrastructural changes of intestinal and colonic tissues at week 6 were observed under transmission electron microscope. The colonic proteomic changes in model mice were examined by two-dimension polyacrylamide gel electrophoresis with immobilized pH gradient isoelectric focusing to screen the differentially expressed proteins, and their molecular masses and isoelectric points were determined. Two N-terminal sequences of the samples were also determined by mass spectrometry. RESULTS: SE (0.3g) caused diarrhea after gastric administration in 1-6h and enhanced gastrointestinal propellant (65.1+/-7.5%; 45.8+/-14.6%, P<0.01) in mice, but intramuscular and hypodermic injection had no cathartic effect. X-ray analysis of gastrointestinal motility demonstrated that gastric administration of SE enhanced gastric evacuation and gastrointestinal transferring function. At 3 h and week 3 and 4 after gastric administration of SE, light microscopic examination revealed no apparent change in gastrointestinal mucosal tissues, but transmission electron microscopic examination revealed inflammatory changes in whole layer of intestinal and colonic wall. Twenty differential proteins were detected in the colonic tissues of the model mice by two-dimensional electrophoresis, and the N-terminal amino acid sequences of two proteins were determined. CONCLUSION: SE causes diarrhea and enhances gastrointestinal motility through digestive tract administration. Long-term gastric administration of SE induces inflammatory changes and cell damage in the whole gastrointestinal tract. The differential proteins screened from the colonic tissues of the model mice might mediate the enhancing effect of SE on gastrointestinal motility.

Animals↗

PubMed2002

Differential display of vincristine-resistance-related genes in gastric cancer SGC7901 cell.

AIM: To isolate and clone the vincristine-resistance-related genes in gastric cancer SGC7901 cell line and to clarify the multidrug-resistant molecular mechanism of gastric cancer cells. METHODS: The modified differential-display polymerase chain reaction (DD-PCR) was used to examine differences in the mRNA composition of Vincristine-resistant gastric cancer SGC 7901 cells (SGC7901/VCR), induced by vincristine sulfate versus SGC7901cells. The differentially expressed cDNA fragments were confirmed by reverse Northern analysis, sequencing, BLAST analysis and Northern bolt analysis. RESULTS: The DD-PCR identified that 54 cDNA fragments were preferentially expressed in SGC 7901/VCR cells. When these cDNA fragments were analyzed by reverse Northern blot, twenty were reproducibly expressed at high level in SGC7901/VCR. Sequencing and BLAST analysis revealed that seven of the genes were known genes:ADP-ribosylation factor 4, Cytochrome oxidase subunit II, Ss-A/Ro ribonucleoprtein autoantigen 60kd subunit,ribosomal protein S13, galaectin-8 gene, oligophrenin 1 mRNA, ribosomal protein L23 mRNA; thirteen of the genes were unknown genes. The length and abundance of the four unknown genes were further confirmed by Northern blot analysis. CONCLUSION: The twenty differential known and unknown genes may be related to the vincristine-resistant mechanism in human gastric cancer SGC7901 cell line.

ADP-Ribosylation Factors↗

PubMed2002

Expression and bioactivity identification of soluble MG7 scFv.

AIM: To examine the molecular mass and identify the bioactivity of MG7 scFv for its application as a targeting mediator in gene therapy of gastric cancer. METHODS: Two strongly positive recombinant phage clones screened from MG7 recombinant phage antibody library were separately transfected into E.coli TG1. Plasmid was isolated from the transfected E.coli TG1 and digested by EcoR I and Hind III to examine the length of exogenous scFv gene. Then, the positive recombinant phage clones were individually transfected into E.coli HB2151. The transfectant was cultured and induced by IPTG. Perplasmic extracts was prepared from the induced transfectant by osmotic shock. ELISA was used to examine the antigen-binding affinity of the soluble MG7 scFv. Immunodotting assay was adopted to evaluate the yield of soluble MG7 scFv produced by transfected E.coli HB2151. Western blot was used to examine the molecular mass of MG7 scFv. Finally, the nucleotide sequence of MG7 scFv was examined by DNA sequencing. RESULTS: Two positive recombinant phage clones were found to contain the exogenous scFv gene. ELISA showed that MG7 scFv had strong antigen-binding affinity. Immuodotting assay showed that transfected E.coli HB2151 could successfully produce the soluble MG7 scFv with high yield via induction by IPTG. The molecular mass of MG7 scFv was 30 kDa by western blot. DNA sequencing demonstrated that the VH and VL genes of MG7 scFv were 363 bp and 321 bp,respectively. CONCLUSION: We have successfully developed the soluble MG7 scFv which possessed strong antigen-binding affinity.

Antibody Affinity↗

PubMed2002

mIL-2R, T cell subsets & hepatitis C.

AIM: To study the levels of membrane interleukin-2 receptor (mIL-2R) and T cell subsets in peripheral blood mononuclear cells (PBMC) from patients with hepatitis C and their role in the pathogenesis of hepatitis C. METHODS: The levels of mIL-2R and T cells subsets in PBMC were detected by biotin- streptatividin (BSA) technique before and after stimulation with PHA in 203 patients with hepatitis C with HCV-RNA(+), anti-HCV(+), anti-HCV(-). RESULTS: The total expressive levels of mIL-2R before and after stimulation with PHA(0.03+/-0.01, 0.03+/-0.02, 0.04+/-0.02, 0.36+/-0.03), and T cell subsets in PBMC (0.62+/-0.06, 0.37+/-0.05, 0.35+/-0.07) were all lower in patients with hepatitis C than those in normal controls (0.66+/-0.07, 0.41+/-0.06, 0.31+/-0.05, P<0.01). Among the patients, the levels of mIL-2R were lower in silence than those in situation of PHA inducting (P<0.01). However, the levels of mIL-2R were similar in acute hepatitis C to that in chronic hepatitis C (P>0.05). The levels of CD(3)(+), CD(4)(+), CD(4)(+)/CD(8)(+) were lower and CD(8)(+) was higher in patients with acute and chronic hepatitis C with anti-HCV(+) than those in normal controls (0.62+/-0.06, 0.37+/-0.05, 0.35+/-0.07, 1.18+/-0.30, 0.61+/-0.07, 0.37+/-0.05, 1.39+/-0.33, 0.31+/-0.05, P<0.05-P<0.01). CONCLUSION: The cellular immunity is obviously changed in patients with hepatitis C. The levels of mIL-2R and activation of T cells are closely associated with chronicity of hepatitis C.

Adolescent↗

PubMed2002

Effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats.

AIM: To investigate the effect of L-NAME on nitric oxide and gastrointestinal motility alterations in cirrhotic rats. METHODS: Rats with cirrhosis induced by carbon tetrachloride were randomly divided into two groups, one n =13 receiving 0.5mg.kg(-1) per day of N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, for 10 days, whereas the other group (n =13) and control (n =10) rats were administrated the same volume of 9g.L(-1) saline. Half gastric emptying time and 2h residual rate were measured by SPECT, using (99m)Tc-DTPA-labeled barium sulfate as test meal. Gastrointestinal transition time was recorded simultaneously. Serum concentration of nitric oxide (NO) was determined by the kinetic cadmium reduction and colorimetric methods. Immunohistochemical SABC method was used to observe the expression and distribution of three types of nitric oxide synthase (NOS) isoforms in the rat gastrointestinal tract. Western blot was used to detect expression of gastrointestinal NOS isoforms. RESULTS: Half gastric emptying time and trans-gastrointestinal time were significantly prolonged(124.0 +/- 26.4 min; 33.7 +/- 8.9 min; 72.1 +/- 15.3 min; P<0.01), (12.4 +/- 0.5h; 9.5 +/- 0.3h; 8.2 +/- 0.8h; P<0.01), 2h residual rate was raised in cirrhotic rats than in controls and cirrhotic rats treated with L-NAME (54.9 +/- 7.6%,13.7 +/- 3.2%, 34.9 +/- 10.3%, P<0.01). Serum concentration of NO was significantly increased in cirrhotic rats than in the other groups (8.20 +/- 2.48) micromol.L(-1), (5.94 +/-1.07) micromol.L(-1) and control (5.66 +/- 1.60 micromol.L(-1), P<0.01. NOS staining intensities which were mainly located in the gastrointestinal tissues were markedly lower in cirrhotic rats than in the controls and cirrhotic rats after treated with L-NAME. CONCLUSION: Gastrointestinal motility was remarkably inhibited in cirrhotic rats, which could be alleviated by L-NAME. Nitric oxide may play an important role in the inhibition of gastrointestinal motility in cirrhotic rats.

Animals↗

PubMed2002

Epidemiological survey of cryptosporidiosis in Anhui Province China.

AIM: To provide scientific evidence for prevention and controlling of cryptosporidiosis, the infection of Cryptosporidium parvum and its epidemiological characteristics were studied in some areas of Anhui Province. METHODS: The oocyst of Cryptosporidium parvum in 5421 fresh stool samples from eleven areas of Anhui Province was tested by auramine-phenol stain and improved anti-acid stain respectively. The specific antibody of IgG, IgM and T subsets of 41 patients with positive Cryptosporidium parvum in stools were detected by ELISA and biotin-streptavidin (BSA) respectively. RESULTS: The total infective rate of Cryptosporidium parvum was 1.33% (74/5421). Among them, the positive rates of oocyst in the areas of Huaibei (1.82%) and Fuyang (1.80%) were higher. The positive rates of oocyst in stools of infants, pupils, middle school students, college students, adults, patients with diarrhea, and those with immunodeficiency were 3.15%(28/889), 0.82% (9/1098), 0.82%(9/1092), 0.83%(8/969), 0.85% (9/1095), 2.88%(8/278) and 8.33%(3/36)% respectively. The positive rates of oocyst in infants and the patients with diarrhea and immunodeficiency were significantly higher than those in controls (P<0.01). The positive rate of oocyst in males was similar to that in females (P>0.05). The positive rate of oocyst in urban areas (1.13%) was significantly lower than those in rural areas (1.72%, P<0.01). The positive rates of specific IgG, IgM and IgG+IgM in sera of the patients with positive oocyst in stool were 63.4% (26/41), 17.1% (7/41), 19.5% (8/41) respectively. The number fractions of T subsets of CD(3)(+), CD(4)(+), CD(8)(+) and CD(4)(+)/CD(8)(+) of the patients were 0.66+/-0.07, 0.44+/-0.06, 0.28+/-0.04 and 1.58+/-0.32 respectively. The difference between the patients and the controls was significant (P<0.05). The main manifestations of the patients were subclinical infection, in forms of slight abdominal pain, mild diarrhea, and loose stool. CONCLUSION: There are two infection peaks in infection of Cryptosporidium parvum and its infection can be found more often in infants, patients with diarrhea or immunodeficiency, and in rural areas. Subclinical infection is the main manifestation and might be easily misdiagnosed. When the therapeutic effectiveness is low for diarrhea, the infection of Cryptosporidium parvum should be considered, concerning their age and immune function.

Adolescent↗

PubMed2002

Expression and function of classical protein kinase C isoenzymes in gastric cancer cell line and its drug-resistant sublines.

AIM: To investigate the expression and function of classical protein kinase C (PKC) isoenzymes in inducing MDR phenotype in gastric cancer cells. METHODS: Two cell lines were used in the study: gastric cancer cell SGC7901 and its drug-resistant cell SGC7901/VCR stepwise-selected by vincristine 0.3, 0.7 and 1.0 mg.L(-1), respectively. The expression of classical PKC (cPKC) isoenzymes in SGC7901 cells and SGC7901/VCR cells were detected using immunofluorescent cytochemistry, laser confocal scanning microscope and Western blot. The effects of anti-PKC isoenzymes antibody on adriamycin accumulation in SGC7901/VCR cells were determined using flow cytometric analysis. RESULTS: (1)SGC7901 cells exhibited positive staining of PKC-alpha. SGC7901/VCR cells exhibited stronger staining of PKC-alpha than SGC7901 cells. The higher dosage vincristine selected, the much stronger staining of PKC-alpha was observed on SGC7901/VCR cells. (2)Both SGC7901 and SGC7901/VCR cells exhibited positive staining of PKC-beta I and PKC-beta II with no significant difference. (3) Compared with SGC7901, SGC7901/VCR cells had decreased adriamycin accumulation and retention. Accumulation of adriamycin in SGC7901 was 5.21+/-2.56 mg.L(-1),in SGC7901/VCR 0.3 was 0.85+/-0.29 mg.L(-1), in SGC7901/VCR 0.7 was 0.81+/-0.32 mg.L(-1), and in SGC7901/VCR 1.0 was 0.80+/-0.33 mg.L(-1); Retention of adriamycin in SGC 7901 was 2.51+/-1.23 mg.L(-1), in SGC7901/VCR 0.3 was 0.47+/-0.14 mg.L(-1), in SGC7901/VCR 0.7 was 0.44+/-0.15 mg.L(-1), and in SGC 7901/VCR 1.0 was 0.41+/-0.11 mg.L(-1). (4) Fluorescence intensity presented adriamycin accumulation in SGC7901/VCR cells was increased from 1.14+/-0.36 to 2.71+/-0.94 when cells were co-incubated with anti-PKC-alpha but not with anti-PKC-beta I PKC-beta II and PKCgamma antibodies. CONCLUSION: PKC-alpha, but not PKC-beta I, PKC-beta II or PKCgamma, may play a role in multidrug resistance of gastric cancer cells SGC7901/VCR.

Antineoplastic Agents, Phytogenic↗

PubMed2002

The role of KDR in the interactions between human gastric carcinoma cell and vascular endothelial cell.

AIM: To study the interactions between human gastric carcinoma cell (HGCC) and human vascular endothelial cell (HVEC), and the role of KDR in these interactions. METHODS: Antisense oligodexynucleotide(ASODN) specific to KDR gene was devised and added to the culture medium of HGCC and HVEC. After the action of ASODN, the proliferation of two cells was measured by MTT method. The role of KDR in regulating the proliferation of two kinds of cells was known through observing the effect of ASODN on them. The conditioned mediums (CMs) of HGCC and HVEC were prepared. The CM of one kind of cell was added acting on the other kind of cell, then the cell proliferation was measured by MTT. After the action of ASODN or CM, the cellular expression of KDR gene was detected with in situ hybridization (ISH) for mRNA level and with immunohistochemical staining for protein level. ABC -ELISA was used to detect hVEGF in the CMs of two cells. RESULTS: KDR ASODN could specifically inhibit the proliferation of HGCC and HVEC significantly. The growth inhibitory rate amounted to 55.35 % and 54.83 %, respectively (P<0.01). HGCC and HVEC could secret a certain level of hVEGF(92.06+/-1.69 ng/L, 77.70+/-8.04 ng/L). The CM of HGCC could significantly stimulate the growth(2.70+/-0.01 times) and KDR gene expression of HVEC(P<0.01) while the CM of HVEC could significantly inhibit the growth(52.97+/-0.01 %) and KDR gene expression of HGCC (P<0.01). CONCLUSION: KDR plays a key role in regulating the proliferation of HGCC and HVEC. There exist complicated interactions between HGCC and HVEC. HGCC can significantly stimulate the growth of HVEC while HVEC can significantly inhibit the growth of HGCC. KDR is involved in the interactions between them.

Cell Communication↗

PubMed2002

Expression of sphingosine kinase gene in the interactions between human gastric carcinoma cell and vascular endothelial cell.

AIM: To study the interactions between human gastric carcinoma cell (HGCC) and human vascular endothelial cell (HVEC), and if the expression of sphingosine kinase(SPK) gene was involved in these interactions. METHODS: The specific inhibitor to SPK, dimethyl sphingosine (DMS), was added acting on HGCC and HVEC, then the cell proliferation was measured by MTT. The conditioned mediums (CMs) of HGCC and HVEC were prepared. The CM of one kind of cell was added to the other kind of cell, and the cell proliferation was measured by MTT. After the action of CM, the cellular expression of SPK gene in mRNA level was detected with in situ hybridization(ISH). RESULTS: DMS could almost completely inhibit the proliferation of HGCC and HVEC. The growth inhibitory rates could amount to 97.21 %, 83.42 %, respectively (P<0.01). The CM of HGCC could stimulate the growth of HVEC (2.70+/-0.01, P<0.01) while the CM of HVEC could inhibit the growth of HGCC (52.97+/-0.01 %, P<0.01). There was no significant change in the mRNA level of SPK gene in one kind of cell after the action of the CM of the other kind of cell. CONCLUSION: SPK plays a key role in regulating the proliferation of HGCC and HVEC. There exist complicated interactions between HGCC and HVEC. HGCC can significantly stimulate the growth of HVEC while HVEC can significantly inhibit the growth of HGCC. The expression of SPK gene is not involved in the interactions.

Cell Communication↗

PubMed2000

Expressions of chromogranin A and cathepsin D in human primary hepatocellular carcinoma.

AIM:To determine the expression and clinical significance of chromogranin A and cathepsin D in hepatocellular carcinoma (HCC).METHODS:Double immunofluorescence stain-ing techniques combined with laser confocal scanning microscopy (LSCM) was used to investigate chromogranin A and cathepsin D expressions in 85 HCC patients.RESULTS:Cathepsin D was expressed in 3 normal liver tissues, while in HCC the staining showed regional variation and the fraction of strongly stained cells increased as the tumors became less differentiated and usually clinically more malignant.Cells which showed strong positivity for cathepsin D were present in 71/85(83.5%) cases. Strong expression of cathepsin D in cancer cells was related to histopathological features. They were more common in grade 3-4 (26/28, 92.9%) and grade 2 (46/53, 86.8%) tumors than in grade 1 tumors (1/4, 25.0%)(P <0.01). No significant correlation was found between age and cathepsin D expression. In patients with positive cathepsin D reaction, the mean age was 52.1± 2.8 years (range 32-68 years) and in the group with negative reaction, the mean age was 51.3± 4.5 years (range 28-71 years). No obvious relationship was observed between CgA expression in cancer cells and the histopathological features. The CgA positive rate was 75.0% (3/4) in grade 1, 71.7% (38/53) in grade 2,and 71.4% (20/28) in grade 3-4(P >0.05) tumors. The coexpression of CgA and cathepsin D was found by double labeled immunofluorescence staining techniques. The processing of cathepsin D was disturbed in HCC cells and accumulated in the cells.Cathepsin D had proteolytic activity and autocrine mitogenic effect, suggesting their functions in invasion. These findings demonstrated that the expression of cathepsin D in HCC had prognostic value.CONCLUSION:Chromogranin A and cathepsin D are expressed in a high proportion of HCC and the existence of cathepsin D in HCC might be related to processing of CgA. This is clearly a subject for further studies because of its potential clinical applications.

Journal Article↗

PubMed1999