Biomedical subjects

Zu-Hong Lu

Publications and source records attributed to Zu-Hong Lu.

10 recordsLinked to original sources

PubMed2007

Construction of an antibody microarray based on agarose-coated slides.

The antibody microarray, a high-throughput multiplex immunoassay method, has become a significant tool for quantitative proteomics studies. We describe here the strategies for optimizing the condition of antibody microarray building based on agarose-coated slides. In this study, modified glass slides were robotically printed with capture antibodies against monocyte chemoattractant protein 1 (MCP-1), then dilutions of the cytokine were applied to the arrays, and the protein was detected with biotin-labeled antibody coupled with Cy3-conjugated streptavidin. Thus a protein profiling microarray based on sandwich immunoassay has been established. Various factors in the production of antibody microarrays were analyzed: the capture antibody concentrations, shelf life of the postprinting slides, blocking buffers, and reproducibility of the system. A calibration curve with a correlation coefficient of 0.9995 was established which suggested that the matrix can retain arrayed proteins in near-quantitative fashion. The results revealed high signal uniformity and reproducibility with regard to intra-array (1.3%) and the interarray (8.7%) variation at the capture antibody concentration of 125 microg/mL. Besides, the printed arrays could be stored for at least two months without any apparent change of the performance parameters.

Antibodies↗

PubMed2006

[A new method for 5, 10-methylenetetrahydrofolate reductase single nucleotide polymorphisms genotyping used to study susceptibility of hematological malignancy].

The aim of this study was to set up a new method for 5, 10-Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNP) genotyping, and to investigate the hereditary susceptibility of hematological malignancy. Prepared an aimed gene microarray based on cDNA microarray theory, dual-color fluorescence hybridization was used to detect SNP loci, and DNA sequencing was performed to confirm the results. The MTHFR C677T SNP loci of 157 controls and 127 patients with hematological malignancies (30 multiple myeloma, 28 non-Hodgkin's lymphoma, 22 acute lymphoblastic leukemia, 40 acute myeloid leukemia, 7 chronic myeloid leukemia) from Jiangsu province were detected. The results showed that after overlapping, homozygous wild type, heterozygote type and homozygous mutant type yielded green, yellow and red fluorescence, respectively. DNA sequencing validated these results. The allele frequency of 677C and 677T in patients and controls were 58.7% and 66.9%, 41.3% and 33.1% respectively, showing statistically significant difference (chi2 = 4.077, P = 0.043). 677TT genotype showed a significantly higher risk of MM (OR = 4.21; 95% CI = 1.50 - 11.83; P = 0.006). It is concluded that this microarray-based method is accurate, high-throughput and inexpensive, suitable for SNP genotyping in a large number of individuals. C677T polymorphisms influence the risk of hematological malignancies. 677TT genotype is susceptive to MM.

Adult↗

PubMed2005

Resetting blood pressure by a closed-loop implanted chip system in normotensive rats.

A closed-loop implanted chip system was designed to control blood pressure without using drugs. The chip system instantaneously reset blood pressure by stimulating the left aortic depressor nerve according to the feedback signals of arterial blood pressure. The relationship between pressure signals and frequency of stimulation was identified in vitro and in vivo, and the efficiency of the chip system was evaluated in normal anesthetized Wistar rats. To determine whether the depressor effect of the chip was primarily independent on the bradycardia induced by the resetting, the effects of methyl atropine (1.5 g/kg, iv.) and bilateral vagotomy on depressor effect induced by the chip system were determined, respectively. The results indicated that the chip system worked well. The frequency of stimulus linearly increased following the elevation of pressure from 70 to 160 mm Hg. The frequency of the stimulus reached its maximum (100 Hz) when pressure exceeded 160 mm Hg, and the stimulation stopped when MAP was below 70 mm Hg. There were significant decreases in mean arterial pressure (MAP, -20.0+/-4.4 mm Hg) and heart rate (HR, -43.0+/-10.5 bpm) during the resetting in rats. After resetting, both MAP and HR recovered in a minute without any significant rebound. Pretreatment with either methyl atropine or bilateral vagotomy abolished the bradycardia effect but produced no significant effect on hypotension. The results demonstrated that the chip system successfully reset blood pressure in rats, and that the hypotension induced by the chip system was primarily independent on the bradycardia effect.

Anesthesia↗

PubMed2005

Detection of p16 hypermethylation in gastric carcinomas using a seminested methylation-specific PCR.

Aberrant DNA methylation of a CpG site is among the earliest and most frequent alterations in various tumors including gastric carcinoma. The aim of this study is to detect tumor-associated aberrant hypermethylation of the p16 gene from 60 gastric tumor and corresponding normal tissues using a seminested methylation-specific PCR (MSP). The results indicated that hypermethylation of the p16 gene could be detected in 80% (48/60) of the gastric tumor samples from the first PCR. However, the frequency increased significantly to 86.7% (52/60) of the gastric tumor samples after the second PCR. These results show that this technique increases the sensitivity of detecting p16 hypermethylation from tumor samples. Furthermore, the aberrant methylation of p16 was observed in all of the stages, confirming that this epigenetic alteration is an early event during gastric carcinogenesis. Clinicopathologic parameters such as age, sex, and histological differentiation of GC were not significantly associated with the methylation status.

Cyclin-Dependent Kinase Inhibitor p16↗

PubMed2005

[Using protein chips to study mechanism underlying reversion of drug resistance in leukemia cells in tetrandrine alone or in combination with droloxifene].

The aim of this study was to investigate the mechanism to reverse the drug resistance of leukemia cells in tetrandrine (Tet) alone or in combination with droloxifen (Drol) by using protein chips and to lay the theoretical basis for the clinical applications. Three monoclonal antibodies against P-glycoprotein (P-gp), the multidrug resistance-associated protein (MRP1) and the breast cancer resistance protein (BCRP) were immobilized onto the agarose gel film-coated glass slides. Protein chips were prepared respectively from K562/A02 cells cultured for 12, 24 and 48 hours with Tet alone or in combination with Drol. The results showed that Tet alone or in combination with Drol could decrease only the expression of P-gp in a time-dependent manner, the effect for 48 hours as follows: Tet + Drol 82.620 +/- 3.227; Tet alone 86.440 +/- 2.906; Drol alone 87.230 +/- 2.049; control 93.670 +/- 2.748 (P < 0.05). However, down-regulation of P-gp by K562/A02 cells cultured with Tet alone or in combination with Drol began at 24 hours (Tet + Drol 85.270 +/- 3.095; control 93.670 +/- 2.748, P < 0.05). The results were coincident with that of FCM. It is concluded that Tet and Drol can downregulate the expression of P-gp in the time-dependent way. There is a significant difference between Tet alone and Tet combined with Drol at 24 hours (P < 0.05). The expression of MRP1 and BCRP are not closely correlated with the reversal mechanism of Tet and Drol, and which may be involved in the mechanism of this combination to reverse multidrug resistance in leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Microarray-based method for detecting methylation changes of p16(Ink4a) gene 5'-CpG islands in gastric carcinomas.

AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer. METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP). RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP. CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.

Base Sequence↗

PubMed2004

[A novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology].

Multiplex amplifiable probe hybridization (MAPH) has recently been developed to detect gene copy number changes in total genome in several genetic disease. Here we reported a novel oligonucleotide arrays-based multiplex amplifiable probe hybridization technology for DNA fragment copy number measurement. A set of amplifiable probes were prepared by locus-specific forward and reverse primers synthesized with the T7 and T3 promoter sites at their respective 5'-ends to the interest DNA fragments and purified by Qiagen PCR products purification kit. The set of probes were then hybridized with genome DNA immobilized on the nylon membrane. The selective probes after MAPH were collected with streptavidin coated magnetic beads and amplified by a biotin labeled universal primers. The biotinylated PCR products were then intended for hybridization to the corresponding oligonucleotide arrays consisted of 10 exons of DMD gene probes and positive,negative control probes immobilized on glass slides. After hybridization, the slides were washed by hybridization buffer and stained by streptavidin-Cy3. The hybridization fluorescence images were scanned by the ScanArray software and further quantitatively analyzed by the ImageJ software. Here we used oligonucleotide array technology to replace the agarose gel analysis for detecting the biotinylated PCR products so that extendable ability of parallel assay in our method had greatly been improved. One control female, one control male and one DMD patient were tested in this study. The results demonstrate the feasibility of high-throughput detection and relative quantification of DNA fragment copy numbers changes in total genome DNA combination of MAPH and high-density oligonucleotide arrays in single reaction.

Gene Dosage↗

PubMed2004

[The progress in complex homeobox domains].

The proteins with 'homeobox' (HOX) domain, a large family of DNA binding, play an important role in embryonic development, gene regulation, cell differentiation and neurogenesis. Some other domains are found in the proteins together with HOX, such as PAX, POU, LIM, OAR, ELK, SIX, bZIP, PHD-finger, Engrailed, hexapeptide domains. The HOX combined with other domains that associate with DNA binding or protein interactions are called 'complex homeobox'. Recent reports indicate that 'complex homeobox' genes also take part in tumorigenesis by the way of gene fusions and deregulation. The progress of 'complex homeobox' in types, structures and functions have been summarized in this review.

Animals↗

PubMed2003

Amperometric hydrogen peroxide biosensor with sol-gel/chitosan network-like film as immobilization matrix.

A new type of sol-gel/organic hybrid composite material based on the cross-linking of natural polymer chitosan with (3-aoryloxypropyl) dimethoxymethylsilane was developed for the fabrication of an amperometric H(2)O(2) biosensor. The composite film was used to immobilize horseradish peroxidase (HRP) on a gold disk electrode. The properties of sol-gel/chitosan and sol-gel/chitosan-HRP films have been carefully characterized by atomic force microscopy and Fourier transform infrared. By using fluorescent label, a protein density on sol-gel/chitosan has been calculated to be 3.14 x 10(12) moleculescm(-2). With the aid of catechol mediator, the biosensor had a fast response of less than 2 s with linear range of 5.0 x 10(-9)-1.0 x 10(-7) mol l(-1) and a detection limit of 2 x 10(-9) mol l(-1). Its current response shows a typical Michaelis-Menten mechanism. The apparent Michaelis-Menten constant K(M)(app) is found to be 1.30 micromol l(-1). The activation energy for enzymatic reaction is calculated to be 8.22 kJ mol(-1). The biosensor retained approximately 75% of its original activity after about 60 days of storage in a phosphate buffer at 4 degrees C.

Biosensing Techniques↗

PubMed1997

Preparation of Unilamellar Immunoliposomes by Passing Reversed Micelles through Oil-water Interface.

A novel method for the preparation of unilamellar immunoliposomes is introduced. In this method, the aqueous phase is first encapsulated into reverse micelles passing through the oil-water interface, where the monolayer of lecithin embedded with antibody has been formed to self-assemble into immunoliposomes. The main advantages of this method are that the procedure of preparation is simple with high encapsulation yield and it is favorable for large scale production. As shown by negative staining electronic micrograph, the immunoliposomes are unilamellar and 100-500 nm in size. The UV spectra of immunoliposomes solution and lysis assay show that sheep anti-human IgG has been coated on liposomes.

Journal Article↗