Biomedical subjects

Guo-Hua Zhou

Publications and source records attributed to Guo-Hua Zhou.

11 recordsLinked to original sources

PubMed2006

Single-tube-genotyping of gastric cancer related SNPs by directly using whole blood and paper-dried blood as starting material.

AIM: To demonstrate an inexpensive method for typing gastric cancer related single nucleotide polymorphisms (SNPs) using whole blood or paper-dried blood as starting materials. METHODS: PCR amplification is directly carried out from the whole blood or paper-dried blood sample without any DNA extraction step. Before PCR, a blood sample, four primers, and all of biological reagents necessary for PCR were added at a time; After PCR, the amplified products were directly separated by slab gel electrophoresis or microchip CE without any purification. SNP typing was performed by tetra-primer PCR with two inner primers specific to each allele and two outer primers defining the length of allele-specific amplicons. Genotypes were directly discriminated by the size of amplicons specific to each allele, thereby avoiding any post-PCR process. RESULTS: Using a special PCR buffer, inhibitory substances in blood (including the anticoagulant in blood) and filter paper were effectively suppressed; a "true" single-tube-genotyping is thus realized. We successfully determined genotypes IL-1B-511 and IL-1B-31 polymorphisms at the gene IL-1B by using whole-blood and paper-dried blood samples as starting materials respectively. The method is so sensitive that 0.5-1.0 microL of blood sample is enough to give a satisfactory typing results. The genotyping results were confirmed by RFLP-PCR using purified genome DNA, indicating that amplification specificity was not affected by inhibitory components (including coagulants) in blood or filter paper. CONCLUSION: Compared with SNP typing methods based on purified DNA, the proposed method is labor-saving, simple, inexpensive, and less cross-contaminated. It is promising to use this method to type other SNPs.

Cost Control↗

PubMed2006

[Approaches for SNP genotyping].

With the completion of the Human Genome Project (HGP), typing single nucleotide polymorphisms (SNP) has become one of the main tasks in the post-genome era. Consequently, a robust, flexible and cost-effective technique for SNP typing is essential to analyze a large number of SNPs. The latest genotyping technologies and the relative detection platforms were introduced systematically. The principle of SNP typing was described in detail in respect of allele-specific hybridization, restriction enzyme digestion, primer extension and oligonucleotide ligation assay, as well as the genotyping platforms of plate readers, genechips, bead array and mass spectrometry. Moreover, the way to the high-throughput genotyping in the future was briefly discussed.

Genotype↗

PubMed2006

[Adapter-ligation mediated allele-specific amplification (ALM-ASA) for multiplex SNP genotyping].

To establish adapter-ligation mediated allele-specific amplification ("ALM-ASA" for short) for multiplex SNP genotyping, five SNPs, 100C>T, 1661G>C, 1758G>T, 2470T>C and 2850C>T in CYP2D6 gene were used as an example for evaluating the method. Firstly, a preamplification was carried out for producing a long target containing all SNPs of interest. Secondly, the preamplified DNA fragments were digested with a restriction endonuclease to form sticky ends. Thirdly, an adapter was ligated to either end of the digested fragment. One end of the adapter was designed as a sequence sticky to the ends of the enzymatically digested fragments, and the other end had a common sequence. Fourthly, an allele-specific amplification was performed by allele-specific primers and a universal primer in one tube by using the adapter-ligated fragments as templates. Finally, the allele-specific amplification products were separated by agarose gel electrophoresis. Because each tube corresponds to one kind of allele-specific primers, the genotype of an SNP can be easily discriminated by the length of the amplified products in each tube. The products of 5-plex allele-specific amplification can be separated by agarose gel electrophoresis. Five SNPs in the CYP2D6 gene were successfully typed for 20 healthy Mainland Chinese and the results were in agreement with those by RFLP. By ALM-ASA, n+1 primers (n SNP allele-specific primers and a universal primer) can be used for an n-plex PCR amplification; the specificity of PCR is thus enhanced significantly. It is concluded that ALM-ASA can be used for typing multiple SNPs simultaneously.

Alleles↗

PubMed2005

Multiplex SNP typing by bioluminometric assay coupled with terminator incorporation (BATI).

A multiplex single-nucleotide polymorphism (SNP) typing platform using 'bioluminometric assay coupled with terminator [2',3'-dideoxynucleoside triphosphates (ddNTPs)] incorporation' (named 'BATI' for short) was developed. All of the reactions are carried out in a single reaction chamber containing target DNAs, DNA polymerase, reagents necessary for converting PPi into ATP and reagents for luciferase reaction. Each of the four ddNTPs is dispensed into the reaction chamber in turn. PPi is released by a nucleotide incorporation reaction and is used to produce ATP when the ddNTP dispensed is complementary to the base in a template. The ATP is used in a luciferase reaction to release visible light. Only 1 nt is incorporated into a template at a time because ddNTPs do not have a 3' hydroxyl group. This feature greatly simplifies a sequencing spectrum. The luminescence is proportional to the amount of template incorporated. Only one peak appears in the spectrum of a homozygote sample, and two peaks at the same intensity appear for a heterozygote sample. In comparison with pyrosequencing using dNTP, the spectrum obtained by BATI is very simple, and it is very easy to determine SNPs accurately from it. As only one base is extended at a time and the extension signals are quantitative, the observed spectrum pattern is uniquely determined even for a sample containing multiplex SNPs. We have successfully used BATI to type various samples containing plural target sequence areas. The measurements can be carried out with an inexpensive and small luminometer using a photodiode array as the detector. It takes only a few minutes to determine multiplex SNPs. These results indicate that this novel multiplexed approach can significantly decrease the cost of SNP typing and increase the typing throughput with an inexpensive and small luminometer.

DNA Primers↗

PubMed2004

Characterization of recombinant human granulocyte colony stimulating factor (rHuG-CSF) by capillary zone electrophoresis, capillary isoelectric focusing electrophoresis and electrospray ionization mass spectrometry.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) is a hematopietic cytokine that stimulates and regulates the proliferation and differentiation of neutrophils. Glycosylated and non-glycosylated forms of rHuG-CSF cannot be distinguished by traditional biological assays. In addition, it is very difficult to characterize impurities of the same molecular weight in biologicals. In this study, non-glycosylated rHuG-CSF, two glycosylated rHuG-CSF isoforms and their commercial dosages were successfully separated by capillary zone electrophoresis (CZE) using 50mM Tricine containing 20mM NaCl and 2.5mM 1,4-diaminobutane (DAB) at pH 8.0, which could be employed for the qualitative discrimination assay of rHuG-CSF related products. CZE, capillary isoelectric focusing electrophoresis (CIEF), and mass spectrometry (MS) were used to effectively characterize non-glycosylated rHuG-CSF. It was found that proteins in the samples with different pIs in the CIEF profile could not be detected by CZE, while no difference was observed between these proteins and rHuG-CSF. Further analysis by electrospray ionization mass spectrometry with the resolution of 2000 showed that the components with different pIs in the non-glycosylated rHuG-CSF bulk sample are nearly equal in molecular weight. Therefore, it is necessary to combine several modern analytical techniques for quality control to get well-characterized biologicals.

Electrophoresis, Capillary↗

PubMed2004

A gel-free SNP genotyping method: bioluminometric assay coupled with modified primer extension reactions (BAMPER) directly from double-stranded PCR products.

Inexpensive, high-throughput genotyping methods are needed for analyzing human genetic variations. We have successfully applied the regular bioluminometric assay coupled with modified primer extension reactions (BAMPER) method to single-nucleotide polymorphism (SNP) typing as well as the allele frequency determination for various SNPs. This method includes the production of single-strand target DNA from a genome and a primer extension reaction coupled with inorganic pyrophosphate (PPi) detection by a bioluminometric assay. It is an efficient way to get accurate allele frequencies for various SNPs, while single-strand DNA preparation is labor intensive. The procedure can be simplified in the typing of SNPs. We demonstrate that a modified BAMPER method in which we need not prepare a single-strand DNA can be carried out in one tube. A PCR product is directly used as a template for SNP typing in the new BAMPER method. Generally, tremendous amounts of PPi are produced in a PCR process, as well as many residual dNTPs, and residual PCR primers remain in the PCR products, which cause a large background signal in a bioluminometric assay. Here, shrimp alkaline phosphatase (SAP) and E. coli exonuclease I were used to degrade these components prior to BAMPER detection. The specific primer extension reactions in BAMPER were carried out under thermocycle conditions. The primers were extended to produce large amounts of PPi only when their bases at 3'-termini were complementary to the target. The extension products, PPis, were converted to ATP to be analyzed using the luciferin-luciferase detection system. We successfully demonstrated that PCR products can be directly genotyped by BAMPER in one tube for SNPs with various GC contents. As all reactions can be carried out in a single tube, the method will be useful for realizing a fully automated genotyping system.

Alleles↗

PubMed2004

[Construction and screening of human anti-idiotypic single chain antibodies of nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: The potential of anti-idiotypic antibody as a surrogate of tumor antigen for cancer therapy has been demonstrated in clinical investigations. But at present, many anti-idiotypic antibodies are mouse-original antibodies, which can cause human anti-mouse antibody (HAMA) response and decrease the curative effect. The objective of this study was to construct phage human anti-idiotypic antibody library and select beta type anti-idiotypic single chain antibodies bearing the internal image of the nasopharyngeal carcinoma (NPC) associated antigen to overcome human anti- mouse antibody response caused by application of mouse-original anti-idiotypic antibody. METHODS: Peripheral blood mononuclear cells (PBMCs) of patients with NPC were immunized in vitro by anti-NPC monoclonal antibody FC2 and transformed by Epstein-Barr virus (EBV). V(H) and V(L) genes were amplified by RT-PCR and combined to single chain fragments of variable region (scFv) genes. ScFv genes were cloned into vector fUSE5 and transformed into E.coli MC1061 to construct the scFv-displaying phage library. After four rounds of panning with monoclonal antibody (mAb) FC2,the beta type Ab2 scFv were selected by Sandwich ELISA and binding inhibition test. RESULTS: Of 10 NPC patients, 8 patients showed their B cells immunized by FC2 and transformed by EBV produced anti-idiotypic antibodies to NPC. Five types of VH genes and 7 types of V(L) genes were obtained by RT-PCR amplification and then connected to form 14 scFv genes. ScFv genes were transducted into E.coli MC1061. The library capacity was 1.5x10(8) clones. After panning, 270 phage clones were selected randomly and 91 FC2-positive clones were obtained by Sandwich ELISA, the positive ratio was 33.7%. Five clones,which might display beta type Ab2 scFv, were selected by binding inhibition test. CONCLUSION: The strategy for preparing phage anti-idiotypic antibody library and selecting beta type Ab2 scFv by immunization in vitro, EBV transformation, and phage display technique is feasible, which provide a way for preparing cancer vaccine using beta type Ab2 scFv.

Antibodies, Anti-Idiotypic↗

PubMed2002

Transfection of colorectal cancer cells with chemokine MCP-3 (monocyte chemotactic protein-3) gene retards tumor growth and inhibits tumor metastasis.

AIM: To evaluate the possibility of the induction of anti-tumor immune response by transfecting the colorectal cancer cells with chemokine MCP-3 gene. METHODS: Mouse MCP-3 gene was transduced into mouse colorectal cancer cells CMT93 by using of Liposome. G418-resistant clones were selected and the MCP-3 mRNA expression was detected by RT-PCR. The chemotactic activity of MCP-3 in the cell culture supernatant was detected by Chemotaxis assay. The tumorigenicity of wild type CMT93 and CMT93 gene transfectants were detected by in vivo experiments. The immune cell infiltrations in tumor tissue and tumor metastasis were detected histopathologically. RESULTS: MCP-3 mRNA expression was detected by RT-PCR in gene-transfected cells (CMT93/MCP-3), but not in control groups. And MCP-3 secreted in the cell culture supernatant possessed chemotatic activity. The results from in vivo experiments showed that the tumorigenicity of CMT93/MCP-3 had not decreased, but the tumors derived from CMT93/MCP-3 cells grew more slowly than those from CMT93 cells (1.021+/-0.253) cm(2) vs (1.769+/-0.371) cm(2), P<0.05) or CMT93/mock cells (1.021+/-0.253) cm(2) vs (1.680 +/-0.643)cm(2), P<0.05). Histophathological results showed few immune cells infiltrating in the tumor tissue derived from the controls. In the tumor tissue derived from CMT93/MCP-3, infiltrating immune cells increased. In addition, no tumor metastasis was found in all mice inoculated with CMT93/MCP-3 tumor cells. But all mice had tumor metastasis in CMT93 controls and 4 in 5 mice had tumor metastasis in CMT93/mock controls. CONCLUSION: The results suggested that the transfection of chemokine MCP-3 gene could promote the induction of anti-colorectal cancer immunity, but the tumor growth could not be inhibited completely by merely MCP-3 gene transfection.

Animals↗

PubMed1999

Expression of B7 costimulation molecules by colorectal cancer cells reducestumorigenicity and induces anti-tumor immunity.

AIM:To study the tumorigenicity of colorectal cancer cells transfected with B7 gene and the anti-tumor immunity induced by B7 gene modified colorectal cancer cells.METHODS:B7 gene was transfected into mouse colon cancer cell line CMT93.The transfectants were selected in DMEM containing 800mg/L G418, and B7 molecules were detected by immunohistochemistry.Experiments in vivo include: (1)5X10(6) B7(+) CMT93 cells were inoculated into the back of C57BL/6 mice subcutanously to determine their tumorigenicity (n = 4). As control, wild type CMT93 cells were inoculated the same as the experimental group (n= 3). (2) The mice primed by B7(+) CMT93 cells whose tumors vanished were rechallenged with wild type CMT93 to observe the immune protection of these mice against the wild type CMT93 (n = 4). Non-primed 4 native mice inoculated with wild type CMT93 were used as control.With in vivo cytotoxicity assay, the mice were immunized with B7 (+) CMT93 or the wild type CMT93 by intraperitoneal injection (n = 4X2). The spleen cells and the abdominal cavity infiltrating lymphocytes were obtained and cultured for two days. Cytotoxicity of these cells against the B7 gene modified or wild type CMT93 was detected by MTT assay.RESULTS:B7 high expression clones were obtained after the transfection of the B7 gene into CMT93 cells by electroporation. Immunohistochemistry results showed mainly membrance staining and partly cytoplasm staining in B7 gene transfected CMT93 cells. in vivo experiments: (1)After the inoculation of the B7(+) CMT93 cells in the back of C57BL/6 mice, they lost their tumorigenicity greatly (P < 0.01). All the small tumors growing in the early period in the experimental group vanished in one month, and the tumors in control group grew progressively. (2) No tumors were found in all 4 mice primed by B7(+) CMT93 cells after they were rechallenged with wild type CMT93. In the control group all mice had grown tumors (P < 0.05). In vitro cytotoxicity assay, the CTLs induced by B7(+) CMT93 had a higher cytotoxity against the wild type CMT93 than that induced by wild type CMT93 (P < 0.05), and the cytotoxity of CTLs induced by B7(+) CMT93 against B7(+) CMT93 cells was higher than that against wild type CMT93 cells (P < 0.05).CONCLUSION:The results suggest that the expression of costimulation B7 molecules by colorectal cancer cells can decrease their tumorigenicity greatly, and the B7 molecule can augment the activation of the CTLs against colorectal cancer, and it plays an important role in CTL effector function as well.

Journal Article↗

PubMed1999

Study on the quality of recombinant proteins using matrix-assisted laser desorption ionization time of flight mass spectrometry.

AIM:To study the possibility of matrix-assisted laser esorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for controlling the quality of recombinant proteins.METHODS:By using MALDI-TOF MS, the molecular weights and purity of recombinant bioactive proteins were analyzed.RESULTS:The molecular weights and purity were obtained in nine recombinant bioactive proteins, including interleukin 2,tumor necrosis factor alpha, granulocyte macrophage colony stimulating factor, interferon alpha2b, interferon alpha1, erythropoietin, calmodulin and its fragment, and neuronal nitric oxide synthase were obtained. MALDI-TOF MS was also used to assay specific proteins in the mixtures and to characterize the erythropoietin tryptic digests.CONCLUSION:The results showed that MALDI-TOF MS can be employed for the effective quality control of recombinant proteins.

Journal Article↗

PubMed1998

Radioimmunoimaging of colorectal cancer using (99m)Tc labeled monoclonal antibody.

AIM:To determine whether Hb3 and its fragment F(ab')(2) have practical value in radioimmunoimaging of colorectal cancer.METHODS:Intact Hb3 was purified by hydroxylapatite chromatography.The fragment F(ab') (2) was prepared by cold digestion and purified as intact Hb3.Hb3 and its fragment F(ab') (2) were labeled with 99mTc by direct labeling method using SnCl(2) as reducing agent. The radioactive doses ranged from 15 to 40 mCi.The imaging was accomplished by single photon emission computered tomograph (SPECT) with imaging time ranging from 2.5 to 48 hours. In this study, 10 patients were selected. Among them, 7 were administered with intact Hb3, and 3 with F(ab') (2) fragment. All the patients were diagnosed as having colorectal adenocarcinoma.RESULTS:After purification, intact Hb3 and its fragment F(ab') (2) were fit for radioimmunoimaging. The percentage of labeling of (99m)Tc to Hb3 or F(ab') (2) was 80.6%-91.5%. Among the 10 patients, 3 of 7 patients administered with intact Hb3 had positive scans, the other 4 had negative scans, and 2 of 3 patients administered with F(ab') (2)had positive scans, the other 1 had negative scans.CONCLUSION:The results showed that both intact Hb3 and its F(ab') (2) have some practical value in radioimmunoimaging of colorectal cancer, and the effects of imaging with F(ab') (2) was better than that with intact Hb3.

Journal Article↗