科创中国

AIM: To compare the expression level of Fas gene and Bcl-2 gene in gastric cancer cells SGC7901 and gastric cancer multidrug resistant cells (MDR) SGC7901/ VCR, to transduce Fas cDNA and Bcl-2 antisense nucleic acid into SGC7901/ VCR cells respectively, and to observe the expression of two genes in transfectants and non-transfectants as well as their drug sensitivity. METHODS: Eukaryotic expression vector pBK-Fas cDNA and pDOR anti-Bcl2 were constructed and transfected into SGC7901/ VCR cells by lipofectamine, respectively. Northern blot and Western blot were used to detect the expression of mRNA and protein in SGC7901/ VCR and SGC7901 cells and transfectants, and drug sensitivity of transfectants for VCR, CDDP and 5-FU was analyzed with MTT assay. RESULTS: After gene transfection, 80 for Fas and 120 for antisense Bcl-2 drug-resistant clones were selected from 2 × 10 ⁵ cells, transfection rate being 0.04% and 0.06%. Two clones of SGC7901 Fas/ VCR cells and SGC7901 anti Bcl-2/ VCR cells were randomly selected for further incubation. Hybridization results showed that the expression level of Fas mRNA and protein in SGC7901/ VCR cells was much lower, but that of Bcl-2 mRNA and protein was higher than that in SGC7901 cells. The expression of Fas mRNA and protein in SGC7901 Fas/ VCR cells was higher, and of Bcl-2 mRNA and protein was lower in SGC7901 anti Bcl-2/ VCR cells than that in non-transfectants. MTT assay showed that transfectants were more sensitive to VCR, CDDP, 5-FU than non-transfectants. CONCLUSION: Bcl-1 gene displayed high expression while Fas gene had low expression in drug resistant gastric cancer cells. Expression of Bcl-2 protein was effectively blocked in SGC7901 anti Bcl-2/ VCR cells by gene transfection. In contrast, the expression of Fas mRNA and protein in SGC7901 Fas/ VCR cells increased. Fas gene and Bcl-2 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to chemotherapeutic drugs. These results suggest cell apoptosis plays an important role in the mechanism of MDR, and enhancing apoptosis might reverse MDR.

Xiao Bing;Shi Yong-Quan;Zhao Yan-Qiu;You Han;Wang Zuo-You;Liu Xian-Ling;Yin Fang;Qiao Tai-Dong;Fan Dai-Ming

World Journal of Gastroenterology

1998

The gastric cancer associated antigen McAb MG7-Ag was detected by means of a newly established method, termed immuno-PCR. A McAb recombinant DNA chimeric molecule was made which possesses bispecific binding affinity for antigen that had been immobilized on microtiter wells and the segment of the attached DNA was amplified by PCR. The antigen of gastric cancer cell line KATO III was monitored by this method. Analysis of PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 20 cells to be detected readily and reproducibly. Immuno-PCR showed a 10⁴ enhancement in detection sensitivity compared with ELISA assay. When the same numbers of cells (2 x 10⁶/ml) were immobilized and then the serial diluted chimeric molecule was added, 3.8 x 10^-14 moles and 3.0 x 10^-11 moles were needed to give positive results with the immuno-PCR and ELISA assay, respectively. Therefore, immuno-PCR could give an enormous amplification capability with good specificity, and has a sensitivity much higher than any existing techniques for antigen detection.

Ren-jun ;Fan D.;Zhou S.

Chinese Journal of Oncology

1994

Aim: To develop the single chain variable fragment of MG 7 murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. Methods: mRNA was isolated from MG 7 -producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG 7 recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATOIII of highly expressing MG 7 -binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG 7 ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG 7 ScFv. Finally, the relative molecular mass of soluble MG 7 ScFv was measured by SDS-PAGE. Results: The V H , V L and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 10 ⁶ and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG 7 antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG 7 ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG 7 ScFv was 32. Conclusion: The MG 7 ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Yu Z. C.;Ding J.;Nie Y. Z.;Fan D. M.;Zhang X. Y.

World Journal of Gastroenterology

2001

Aim: To follow the principles of evidence based medicine to reach the integrated results of these studies. Methods: Twenty-one papers of case-control studies were selected, including 11 on gastric cancer, 7 on precancerous lesion of stomach and 3 on lymphoma of stomach. Meta analysis was used to sum up the odds ratios (OR) of these studies. Results: H. pylori vs gastric cancer (intestinal and diffuse type): the odds ratio from the fixed effect model is 3.0016 (95% Cl: 2.4197-3.7234, P < 0.001). H. pylori vs precancerous lesion of stomach: a random effect model was used to calculate the summary odds ratio and its value is 2.5635 (95% Cl: 1.8477-3.5566, P < 0.01). H. pylori vs lymphoma of stomach: though the quantity of literature is too small to make Meta analysis, the data of these 3 studies show that lymphoma of stomach is highly associated with H. pylori infections. Conclusion: Since it had been revealed that H. pylori infection pre-exists in gastric carcinoma and precancerous lesions, the results of Meta analysis present a strong evidence to support the conclusion that H. pylori infection is a risk factor for gastric carcinoma.

Xue F. B.;Xu Y. Y.;Wan Y.;Pan B. R.;Ren J.;Fan D. M.

World Journal of Gastroenterology

2001

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. Immunodotting 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 363bp and 321bp, respectively. Conclusion: We have successfully developed the soluble MG7 scFv which possessed strong antigen-binding affinity.

Yu Zhao-Cai;Ding Jie;Pan Bo-Rong;Fan Dai-Ming;Zhang Xue-Yong

World Journal of Gastroenterology

2002

Clinical scientists from eight European countries and China gathered in the ancient Chinese capital of Xi'an on April 26-28, 2001 to discuss collaboration on a modern approach to gastric cancer prevention. Participants at the First Sino-European Workshop on Immunogenetics and Pathogenesis of Gastric Cancer presented their most up-to-date research results on topics ranging from epidemiology and immune mechanisms to Helicobacter pylori and vaccine development. Researchers then formed groups with their Chinese or European counterparts to plan future research endeavors which will benefit Chinese and European populations alike. After 3 years of organization between the Institute of Digestive Diseases of the Fourth Medical University in Xi'an, China and the Laboratory of Immunogenetics, VU University Medical Center in Amsterdam, the first workshop came into being under the joint sponsorship of the Commission of the European Union, National Natural Science Foundation of China and the Institute of Digestive Diseases, Xi'an, China. As gastric cancer is the most prevalent malignant tumor in China, the workshop was of special significance to the Chinese researchers and to the Chinese population in general. During the workshop, presentations on the epidemiology of gastric cancer showed that this disease is in fact common the world over: it is the second most common cancer next to lung cancer and about 1 million new cases were diagnosed in 2000. Three-quarters of the cases of gastric cancer occur in Asia, and approximately 80% of these cases are in China and Japan. Genetic factors and environmental factors such as diet and H. pylori infection play a role in gastric carcinogenesis. As a recognized cause of gastric cancer, H. pylori was the subject of various presentations ranging from immunological studies, molecular analysis of strains and pathogenesis to vaccine development. Specific areas of discussion included bacterial-epithelial interactions in H. pylori infection, epidemiology in China, global distribution of vacA and cagA genotypes, new evidence for host factors, nonsteroidal antiinflammatory drugs and H. pylori as independent risk factor for gastric cancer, new diagnostic techniques for H. pylori using serum levels of pepsinogen I, and autoimmune processes in corpus atrophy. Vaccine development using a variety of strategies against H. pylori was the subject of an entire session of talks. Oral immunization with urease with Escherichia coli heat labile enterotoxin was shown to be safe and immunogenic in humans as a mucosal adjuvant. Results of a study using attenuated Salmonella typhimurium as a vehicle for DNA-mediated immunization in mice were also presented. A final presentation discussed an ongoing trial comparing strain variability in the vacA and cagA gene sequences and disease expression between H. pylori infection in Europe and China. Researchers also discussed the role of IL1 gene family and TNF gene polymorphisms in gastric pathology and various immune mechanisms involved in gastric cancer, such as down-regulation of NFκB, IL-1 and IL-1RA, cyclooxygenase signalling, and identification of MGAg antibodies. An interactive discussion followed each presentation and ideas and suggestions were provided. According to specialty, the presenters were then assigned to groups of four or five to make plans for joint research projects. A number of international and Chinese observers were present, including representatives from the European Commission, the World Health Organization and the Chinese National Center for Biotechnology Development, and offered input on the financial feasibility of such projects. © 2002 Prous Science. All rights reserved.

Wu K.;Crusius J. B.A.;Shivananda S.;Fan D.;Pe\u00f1a A. S.

Drugs of Today

2002

OBJECTIVE: To study the mechanisms of multidrug resistance (MDR) mediated by human 67 000 laminin receptor (LR) with a relative molecular mas of 67 000 in gastric cancer cells. METHODS: Antisense RNA expression vector corresponding to LR precursor (LRP) was constructed by DNA recombinant technique, and transferred into gastric cancer MDR cells SGC7901/VCR with Lipofect AMINE. Western blot was employed to determine the LR expression level in gastric cancer cells. The sensitivity of gastric cancer cells to chemotherapeutic drugs was evaluated with MTT assay. Flow cytometry was used to analyze the cell cycle and to assess the mean fluorescence intensity of intracellular adriamycin in gastric cancer cells. RESULTS: Western blotting analysis demonstrated a decreased expression level of LR in SGC7901/VCR cells transfected with LRP antisense RNA expression vector. In comparison with the gastric cancer cells with out transfection or transfected with invalid vector, LR down-regulated transfectants (SGC7901/VCR-anLRP) showed higher sensitivity to vincristine, adriamycin, 5-fluodrouracil and cisplatin, and increased accumulation and retention of adriamycin. Cell cycle analysis suggested G1 block and spontaneous apoptosis of SGC7901/VCR-anLRP cells. CONCLUSION: LR might take part in mediation of MDR in gastric cancer cells through interfering with drug accumulation and cell apoptosis.

Shi Yongquan;Zhai Huihong;Wang Xin;Ning Xiaoxuan;Zhao Yanqiu;Fan Daiming

Zhonghua Yi Xue Za Zhi

2002

Aim: To investigate the expression level of ZNRD1 gene in gastric cancer cells SGC7901 and gastric cancer MDR (multidrug resistant) cells SGC7901/VCR, and to observe the drug sensitizing and proliferation effect of ZNRD1 antisense nucleic acid transduction on SGC7901/VCR cells. Methods: Amplification of sequences encoding ZNRD1 from SGC7901/VCR cDNA by PCR. The levels of ZNRD1 mRNA expression were demonstrated using semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Eukaryotic expression vector pcDNA3.1-anti ZNRD1 was constructed and transfected into SGC7901/VCR cells by lipofectamine. Immunochemical method was used to detect the expression of protein in SGC7901/VCR cells and transfectants. The cell cycle alteration and the intracellular adriamycin (ADM) accumulation were observed by FACS. Growth curve and drug sensitization of cells for vincristine (VCR) were analyzed with MTT assay. Results: We cloned the open reading frame of full-length ZNRD1. The expression of ZNRD1 showed higher in SGC7901/VCR than in SGC7901 cells. The antisense ZNRD1 drug-resistant clones were selected after gene transfection. Immunochemical results showed that the expression level of ZNRD1 protein was lower in anti ZNRD1-SGC7901/VCR cells than that in non-transfectants. Comparing to SGC7901/VCR and pcDNA3.1-SGC7901/VCR, anti ZNRD1-SGC7901/VCR showed gradually accumulated in G 1 phase, with a concomitant decrease of cell population in S phase. FACS also suggested intracellular ADM accumulation increased 2fold in SGC7901/VCR cells after transfected with antisense ZNRD1. MTT assay showed that transfectants cells proliferation was lagged and more sensitive to VCR than non-transfectants. Conclusion: ZNRD1 gene displayed highly expression in VCR resistant gastric cancer cells. Expression of ZNRD1 protein was effectively blocked in anti ZNRD1-SGC7901/VCR cells by gene transfection. ZNRD1 antisense nucleic acid transfection sensitized drug resistant gastric cancer cells to VCR, increased ADM accumulation and inhibited the cells proliferation. ZNRD1 antisense RNA transduction could reverse the MDR of human drug-resistant gastric cancer cell SGC7901/VCR to a degree.

Zhang Yu-Mei;Zhao Yan-Qiu;Pan Yang-Lin;Shi Yong-Quan;Jin Xiao-Hang;Yi Hui;Fan Dai-Ming

World Journal of Gastroenterology

2003

OBJECTIVE: To investigate the differential expression of RPL6/Taxreb107 between drug-resistant gastric cancer cell line SGC7901/ADR and gastric cancer cell line SGC7901 as well as its correlation with multiple-drug resistance (MDR) in gastric cancer cells. METHODS: Total RNA was extracted from SGC7901 and SGC77901/ADR, with internal control RT-PCR, Northern blot, gene cloning and expression, construction of eukaryotic expression vector, gene transfection by electroporation. The accumulation and retention of ADR in transiently transfected cell was detected by flow cytometry. RESULTS: The internal control RT-PCR and Northern blot showed high RPL6/Taxreb107 expression in SGC7901/ADR cell line. Sense and antisense eukaryonic expression vectors demonstrated by double enzyme digestion were successfully transfected into gastric cancer cell line SGC7901 and SGC7901/ADR respectively by electroporation. The accumulation and retention of ADR detected 48 hours after transfection showed that RPL6 gene had shown effect on drug resistance in gastric cancer cell. CONCLUSION: The high expression of RPL6/Taxreb107 in drug resistant gastric cancer cell shows its correlation with multiple-drug resistance in gastric cancer.

Du Jing-ping;Jin Xiao-hang;Shi Yong-quan;Cao Yun-xin;Zhao Yan-qiu;Liu Chang-Jiang;Yin Fang;Hu Wen-hua;Chen Bao-jun;Qiao Tai-dong;Fan Dai-ming

Zhonghua Zhong Liu Za Zhi Chinese Journal of Oncology

2003

We have efficiently generated the first monoclonal antibody (MAb) against the human ZNRD1 protein, a transcription-associated protein, consisting of two zinc ribbon domains. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-ZNRD1fusion protein or purified 6 x His-OS-9 fusion protein as a control. One MAb named H6 (IgG1), effective in detecting the recombinant and cellular protein, was characterized by ELISA and Western immunoblotting. Thus, it binds to native ZNRD1 protein and should be useful in studies of ZNRD1 protein function and expression. By Western immunoblotting analysis of 16 patients with gastric cancer using the MAb, we found ZNRD1 protein was more overexpressed in incision margin than in carcinoma tissue. The results showed that ZNRD1 might be a novel modifier in gastric carcinogenesis.

Hong Liu;Zhang Yumei;Han Shuang;Wang Jin;Shi Yongquan;Pan Yanglin;Liu Na;Zhang Xiaoyin;Fan Daiming

Hybridoma and Hybridomics

2004