PubMed Health⌕ Search

Biomedical subjects

Ping Wang

Publications and source records attributed to Ping Wang.

At least 91 records · Page 5Linked to original sources
PubMed2006

Function of small hydrophobic proteins of paramyxovirus.

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) and TNF-alpha receptor 1 block rSV5DeltaSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-alpha-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.

Animals↗
PubMed2006

Stromelysin-1 5A/6A and eNOS T-786C polymorphisms, MTHFR C677T and A1298C mutations, and cigarette-cannabis smoking: a pilot, hypothesis-generating study of gene-environment pathophysiological associations with Buerger's disease.

Buerger's disease (BD) etiologies are poorly understood. Beyond smoking cessation, medical-surgical treatments have limited success. We hypothesized that mutations associated with arterial vasospasm (stromelysin-1 5A/6A, eNOS T-786C) and C677T-A1298C methylene tetrahydrofolate reductase (MTHFR) interacted with cigarette-cannabis smoking, reducing vasodilatory nitric oxide (NO), promoting arterial spasm-thrombosis. Of 21 smoking BD patients (14 men [2 siblings], 7 women; 20 white, 1 African-American), compared to 21 age-gender-race matched healthy controls, 5A/6A stromelysin- 1 homozygosity was present in 7 of 21 (33%) BD cases versus 5 of 21 (24%) controls (risk ratio 1.4; 95% confidence interval [CI] 0.5-3.7), and eNOS T-786C homozygosity was present in 3 of 21 (14%) BD cases versus 1 of 21 (5%) controls (risk ratio 3.0; 95% CI 0.3-26.6). C677T MTHFR homozygosity or compound C677T-A1298C heterozygosity was present in 7 of 21 cases (33%) versus 11 of 21 controls (52%) (risk ratio 0.6; 95% CI 0.3-1.3). In 18 patients who stopped and 3 who continued smoking, all stromelysin-1 5A/6A and/or eNOS heterozygotes-homozygotes, superficial phlebitis, lower limb gangrenous ulcers, and intractable ischemic rest pain with arterial occlusion progressed despite conventional medical therapy, threatening amputation. In 15 patients, to increase vasodilatory NO via endothelial NO synthase, l-arginine (15 g/day) was given, along with folic acid (5 mg), vitamin B6 (100 mg), and B12 (2000 mg/day) to optimize homocysteine metabolism and reduce asymmetric dimethylarginine, a NO synthase inhibitor. Unexpectedly quickly and strikingly, within 8 weeks to 8 months receiving l-arginine-folic acid, 11 of 15 treated patients improved with uniform pain reduction, ulcer healing, and in 5, full recovery of previously absent peripheral pulses. In smokers homo/heterozygous for stromelysin-1 5A/6A and eNOS T-786C mutations, we speculate that the development and severity of BD are related to a gene-environment vasospastic interaction with reduced NO-mediated vasodilatation. Increasing NO production by l-arginine while optimizing homocysteine metabolism by folic acid-B6-B12 may have therapeutic benefit. Further blinded, placebo-controlled studies are needed to determine whether our observations can be generalized to larger BD cohorts.

DNA↗
PubMed2006

DNA aptamers that bind to PrP(C) and not PrP(Sc) show sequence and structure specificity.

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.

Animals↗
PubMed2006

Breast milk lactoferrin regulates gene expression by binding bacterial DNA CpG motifs but not genomic DNA promoters in model intestinal cells.

High-affinity binding of DNA by lactoferrin (LF) is an established phenomenon, but the biologic function of this interaction remains unclear. LF is an abundant breast milk protein (12.5-87.5 micromol/L) and is resistant to digestion in the infant gut. Regulation of gene expression by LF appears to be a major activity, particularly in modulating immune responses. We hypothesized that LF binding to DNA is a mechanism of gene regulation and aimed to identify the mechanism and physiologic sites of this activity. Our studies focused on two major biologic compartments of DNA: LF binding to proinflammatory bacterial DNA sequences (CpG motifs) in extracellular compartments and LF binding to genomic DNA promoters in the nucleus. LF 0.5 mmol/L inhibited CpG motif-induced nuclear factor-kappaB (NF-kappaB) activation and interleukin (IL)-8 and IL-12 cytokine gene transcription in B cells. Intestinal epithelial cells were unresponsive to CpG motifs. However, significant LF transferred across M cell-like monolayers, specialized epithelial cells that transcytose intact macromolecules to underlying B-cell follicles in the intestine. LF did not activate gene expression by binding to putative response elements in epithelial and lymphoid cells. Nor did LF bind to putative response elements specifically in gel-shift assays. No nuclear localization of LF was detected in green fluorescent protein (GFP) tagging experiments. We conclude that breast milk LF regulates gene expression by binding CpG motifs extracellularly, with follicular B cells in the infant intestine a likely target.

B-Lymphocytes↗
PubMed2006

Decreased serum free testosterone in workers exposed to high levels of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP): a cross-sectional study in China.

BACKGROUND: Observations of adverse developmental and reproductive effects in laboratory animals and wildlife have fueled increasing public concern regarding the potential for various chemicals to impair human fertility. OBJECTIVE: Our objective in this study was to assess the effect of occupational exposure to high levels of phthalate esters on the balance of gonadotropin and gonadal hormones including luteinizing hormone, follicle-stimulating hormone, free testosterone (fT), and estradiol. METHODS: We examined urine and blood samples of 74 male workers at a factory producing unfoamed polyvinyl chloride flooring exposed to di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) and compared them with samples from 63 male workers from a construction company, group matched for age and smoking status. RESULTS: Compared to the unexposed workers, the exposed workers had substantially and significantly elevated concentrations of mono-n-butyl phthalate (MBP; 644.3 vs. 129.6 microg/g creatinine, p < 0.001) and mono-2-ethylhexyl phthalate (MEHP; 565.7 vs. 5.7 microg/g creatinine, p < 0.001). fT was significantly lower (8.4 vs. 9.7 microg/g creatinine, p = 0.019) in exposed workers than in unexposed workers. fT was negatively correlated to MBP (r = -0.25, p = 0.03) and MEHP (r = -0.19, p = 0.095) in the exposed worker group. Regression analyses revealed that fT decreases significantly with increasing total phthalate ester score (the sum of quartiles of MBP and MEHP; r = -0.26, p = 0.002). CONCLUSION: We observed a modest and significant reduction of serum fT in workers with higher levels of urinary MBP and MEHP compared with unexposed workers.

Adult↗
PubMed2006

Detection of Contarinia nasturtii (Diptera: Cecidomyiidae) in New York, a new pest of cruciferous plants in the United States.

The midge Contarinia nasturtii Kieffer (Diptera: Cecidomyiidae) was first confirmed in North America in Ontario, Canada, in 2000. The insect is now distributed throughout many counties in the provinces of Ontario and Québec. Nearly 1,200 farms in the northeastern United States that grow cruciferous vegetables are at risk for C. nasturtii infestation if this insect were to spread to that region. Over a period of 3 yr (2002-2004), approximately 3,000 ha of crops on 94 farms in western New York State was scouted for C. nasturtii, but none were found. In 2004, 42 experimental pheromone traps were placed in fields of cruciferous vegetables in eight counties. C. nasturtii males were captured at low levels (1-50 per trap / 8 wk) on four farms in Niagara County, but not at any other site. C. nasturtii larvae were found in plant tissue at one of the four farms. Insect specimens were identified by morphological methods, molecular methods, or both. This is the first confirmation of C. nasturtii in the United States, which we believe was made possible by the combined use of pheromone traps, morphological characters of trapped adults, and molecular methods. The early detection in New York presents an opportunity to implement measures to limit the spread and establishment of C. nasturtii across the state and into other regions of the United States.

Animals↗
PubMed2006

[Effects of Guben Hutan Tongmai Recipe on expressions of macrophages and cell adhesion molecules of aortic endothelia in rats with syndrome of phlegm blocking blood vessel].

OBJECTIVE: To observe the effects of Guben Huatan Tongmai Recipe (GBHTTMR), a compound Chinese herbal recipe, on expressions of macrophages and cell adhesion molecules (CAMs) of aortic endothelia in rats with syndrome of phlegm blocking blood vessel, and to explore the pathogenesis of the phlegm-pathogen. METHODS: Fifty normal male Wistar rats, 7-week in age, were randomly divided into five groups: normal control group, untreated group, high-dose GBHTTMR-treated group, low-dose GBHTTMR-treated group and simvastatin-treated group, with 10 rats in each group. Syndrome of phlegm blocking blood vessel was induced in rats of the latter 4 groups by feeding the rats with high lipid diet. Levels of blood lipid were compared among the 5 groups. The expressions of macrophages and CAMs in aortic endothelia were tested by immunohistochemical staining method. RESULTS: The level of blood lipid, and the expressions of macrophages and CAMs showed statistical differences between the normal control group and the untreated group (P<0.01), and between the untreated group and the low-, high-dose GBHTTMR-treated and simvastatin-treated groups as well (P<0.05). CONCLUSIONS: GBHTTMR can decrease the level of serum cholesterol and triglycerides, and increase the level of high density lipoprotein. It also can inhibit the expressions of macrophages, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, P-selectin, and E-selectin.

Animals↗
PubMed2006

[Study on relationship between polymorphism sites of TIM-3 and allergic asthma in a population of adult Hans from Hubei province of China].

OBJECTIVE: To investigate two single nucleotide polymorphism sites of the promoter region in T cells immunoglobulin domain and mucin domain protein-3 (TIM-3) and detect their relationship with allergic asthma in a population of adult Hans from Hubei province of China. METHODS: The polymorphisms were detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allelic specific polymerase chain reaction (ASPCR). The genotype and allele frequencies were calculated and analyzed. RESULTS: The genotype frequencies of CC, CT and TT in -1541 C/T polymorphism were 0.961, 0.039 and 0 respectively in the healthy population and were 0.935, 0.065 and 0 respectively in the allergic asthma population. No significant difference in genotype and alleles frequencies was found between the allergic asthma patients and control subjects (P=0.314, P=0.321). The genotype frequencies of GG, GT and TT in -574 T/G polymorphism were 0.992, 0.008 and 0 respectively in the healthy population and were 0.941, 0.059 and 0 respectively in the allergic asthma population. There was significant difference in genotype and allele frequencies between the allergic asthma patients and control subjects (P=0.046, P=0.048). CONCLUSION: There are polymorphism sites of the promoter region in TIM-3 , and one of these sites, the -574 G/T polymorphism site, may be associated with allergic asthma in the population of adult Hans from Hubei province of China.

Adult↗
PubMed2006

[The advance of research in electro-physiology and chip techniques for olfaction and gustation].

This paper introduces the new research achievement and progress of electro-physiology in olfaction and gustation. Classical implements such as patch-clamp or glass pipette are not appropriate in the dynamic detection of cellular signal transportation. In view of this, we have analyzed the feasibilities and challenges of olfactory or gustatory cell-based biosensors such as field effect transistor (FET) and light addressable potentiometric sensor (LAPS). Finally we present the research work carried our in out lab and a future prospective on the development in this field.

Biosensing Techniques↗
PubMed2006

Adrenomedullin binding protein-1 is downregulated during polymicrobial sepsis in the rat.

We have previously shown that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) in combination maintains cardiovascular stability and reduces mortality in a rat model of sepsis. However, it is unknown whether AMBP-1 is reduced under the septic condition and, if so, whether lipopolysaccharide (LPS) plays a role in down-regulating AMBP-1. To determine this, male adult Sprague-Dawley rats were subjected to either polymicrobial sepsis by cecal ligation and puncture (CLP), or endotoxemia by intraperitoneal injection of LPS (15 mg/kg body weight). In an additional group of animals, LPS neutralizing agent polymyxin B (PMB) was given intramuscularly at 0.5 h before and 9 h after CLP. At 20 h after CLP (i.e. the late stage of sepsis) or endotoxemia, hepatic tissue and blood samples were collected. Hepatic AMBP-1 gene expression along with hepatic and plasma AMBP-1 protein levels were measured by RT-PCR and Western blot analysis, respectively. Our results showed that hepatic AMBP-1 gene expression decreased by 65%, hepatic AMBP-1 protein levels decreased by 72%, and plasma levels of AMBP-1 decreased by 59% at 20 h after CLP. Similar results were also seen in the animals receiving LPS injection. Administration of PMB, however, prevented the downregulation of AMBP-1 expression at 20 h after CLP. Thus, AMBP-1 is downregulated in the late phase of sepsis, and LPS plays a critical role in the reduction of AMBP-1.

Animals↗
PubMed2006

[Effect of small interference RNA on E6, E7 mRNA of human papillomavirus type-18 in cervical cancer cells].

OBJECTIVE: To study the effect of small interference RNA on E6, E7 mRNA of human papillomavirus type-18 (HPV18) in cervical cancer cells. METHODS: The specific HPV18E6 and E7 siRNA synthesized by in virtro transcription. The cell activity was detected by methyl thiazolyl tetrazolium (MTT) assay to determine the most suitable concentration, then cells were transfected into HPV18 cervical cancer cells by oligofectamine. HPV18E6, E7 mRNA level was examined by semi-quantitative RT-PCR, and the cell cycles were analyzed by flow cytometry. RESULTS: The activity of the HeLa cells had an obvious suppression after transfection to 0.57 +/- 0.05 and 0.62 +/- 0.04 compared with the contrast of 0.87 +/- 0.05. The expression of HPV18E6 mRNA level was 0.63 +/- 0.04 before transfection, decreased to 0.53 +/- 0.04, 0.46 +/- 0.02, 0.56 +/- 0.03 evidently after transfection at 24, 48, 72 hours. The expression of HPV18E7 mRNA level was 0.66 +/- 0.03 before transfection and decreased to 0.60 +/- 0.05, 0.52 +/- 0.04, 0.59 +/- 0.02 after transfection at 24, 48, 72 hours. The number of cells in G(2) phase was increased after HPV18E6 siRNA transfection from (1.4 +/- 1.2)% to (66.9 +/- 3.5)%, S phase was declined from (39.4 +/- 0.4)% to (0 +/- 5.5)%, and they were (47.2 +/- 0.5)% and (5.6 +/- 4.2)% in E7 group. CONCLUSION: RNAi exists in HeLa cells, and its effect has specificity.

Apoptosis↗
PubMed2006

[Effects of high concentration manganese on active oxygen production and antioxidant enzyme activities in cucumber leaves under different irradiations].

Effects of high concentration manganese on active oxygen production and antioxidant enzymes in cucumber leaves under natural irradiation and 1/2 natural irradiation were studied. High concentration manganese increased the H2O2 content and O2*- producing rate of cucumber leaves (Fig.1A, B) and caused lipid peroxidation (Fig.1C). Compare to natural irradiation, 1/2 natural irradiation significantly decreased active oxygen production and lipid peroxidation (Fig.1). CAT activities in cytosols and chloroplasts were inhibited by high concentration manganese (Fig.2B), and other antioxidant enzyme activities were enhanced by high concentration manganese (Fig.2A, C-F). Especially activities of APX, DHAR and GR in chloroplasts were greatly stimulated by high concentration manganese under natural irradiation compared to 1/2 irradiation, which may play important roles in scavenging active oxygen species. Antioxidant enzymes in mitochondria showed higher activities under high concentration manganese under natural irradiation, but the differences were not significant under 1/2 natural irradiation (Fig.2A, C-F).

Antioxidants↗
PubMed2006

[Quality standard for Xindi soft capsule].

OBJECTIVE: To establish the quality control standard of Xindi soft capsule. METHOD: Quercetin, kaempferol and isorhamnetin were isolated by TLC with chloroform-ethyl formate-formic acid (5:4:1). The chromatographic separation was performed on a Diamonsil C18 column (4.6 mm x 250 mm, 5 microm). Acetonitrile-water-phosphoric (30:70:0.1) as mobile phase. The flow rate was 1 mL x min(-1) and column temperature was set at 40 degrees C. The UV detection wavelength was set at 254 nm. RESULT: Quercetin, Kaempferol and Isorhamnetin could be identified by TLC. Quercetin showed a good linear relationship at a range of 0.412-1.648 microg, r = 0.999 9, the average recovery was 96.8%, and RSD was 0.9% (n = 6). Kaempferol showed a good linear relationship at a range of 0.021-0.083 microg, r = 0.999 8, the average recovery was 96.9%, and RSD was 2.0% (n = 6). Isorhamnetin showed a good linear relationship at a range of 0.183-0.732 microg, r = 0.999 9, the average recovery was 97.1%, and RSD was 1.6% (n = 6). CONCLUSION: The method is accurate with the good reproducibility and can be used for the quality control of Xindi soft capsule.

Capsules↗
PubMed2006

Suppression of hepatocyte CYP1A2 expression by Kupffer cells via AhR pathway: the central role of proinflammatory cytokines.

The hepatic cytochrome P-450 (CYP) enzyme system provides a major aspect of liver function, yet alterations of CYP in sepsis remain largely unknown. Although we have recently shown that CYP1A2, one of the major isoforms of CYP in rats, is downregulated in sepsis, the underlying mechanism and possible therapeutic approaches warrant further investigation. The aim of this study was to determine whether Kupffer cells (KCs) play any role in suppressing CYP1A2 in the hepatocytes (HCs) and if so, how to modulate CYP1A2 expression in sepsis. To study this, primary KCs and HCs were cultured separately or together with or without transwells. Cells and supernatant samples were collected after various stimulations. Additionally, polymicrobial sepsis was induced in rats by cecal ligation and puncture (CLP) with or without curcumin pretreatment. Liver samples were harvested 20 h post-CLP. The results show that lipopolysaccharide (LPS) did not suppress CYP1A2 in HC or HC/KC coculture with transwells. However, LPS downregulated CYP1A2, aryl hydrocarbon receptor (AhR, a nuclear receptor) and AhR nuclear translocator (Arnt) in coculture without transwells. Anti-TNF-alpha and anti-IL-1beta antibodies attenuated this downregulation. Moreover, elevated hepatic levels of TNF-alpha and IL-1beta post-CLP were decreased by curcumin pre-treatment. This reduction was associated with increased expression of AhR and CYP1A2. These results indicate that KCs-derived proinflammatory cytokines may play an important role in downregulating CYP1A2 in sepsis. The reduction of AhR/Arnt may be the underlying mechanism for such downregulation. Inhibition of proinflammatory cytokines by curcumin may provide a novel approach to modulate the hepatic CYP function in sepsis.

Animals↗
PubMed2006

[Determination of organic acids exuded from plant roots by high performance liquid chromatography].

A simple and highly sensitive method was developed for the determination of organic acids exuded from plant roots by high performance liquid chromatography with ultraviolet (UV) detection. The root exudate was passed through cation and anion exchange resin columns subsequently. The eluant was then concentrated in a rotary evaporator. The residue was dissolved in dilute HClO4 solution at pH 2.1. The separation was performed on a Bio-Rad Aminex HPX-87H sulfonic column at 50 degrees C with an eluent containing 5 mmol/L H2SO4 at a flow-rate of 0.5 mL/min, and the organic acids were detected at a wavelength of 210 nm by a UV detector. Average recoveries for the root exudates were in the range of 82.0%-96.2% and the detection limits were 1-120 microg/L, and the relative standard deviations (RSD) were 0.67%-3.31% for the seven organic acids. The intra-day precisions were 1.2%-4.7%, and inter-day precisions were 3.3%-10.6%. These results demonstrated that the proposed method is simple, sensitive and reliable for the determination of organic acids in plant root exudates, despite the presence of the particularly complex matrix.

Acids↗
PubMed2006

[Interventional effect of behaviour psychotherapy on patients with premature ejaculation].

OBJECTIVE: To investigate the interventional effect of comprehensive behaviour psychotherapy on the ejaculatory latency of premature ejaculation (PE) patients, sexual satisfaction of sexual partners, as well as its influence on the results of clinical treatment. METHODS: Ninety PE patients were randomly divided into a psychological intervention group (n = 45) and a control group (n = 45). Both groups were given medicine therapy, and the former also received comprehensive behaviour psychotherapy for 6 weeks. All the patients were assessed with the Chinese index of sexual function for PE and ejaculatory latency in the vagina, and the clinical efficacy was compared between the two groups. RESULTS: Before treatment, the ejaculatory latency in the vagina was (0.69 +/- 0.25) min and (0.71 +/- 0.19) min respectively in the intervention and the control groups, as compared with (5.87 +/- 0.59) min and (4.76 +/- 0.54) min before treatment, with significant difference (P < 0.01). In the intervention group, the scores in the control of ejaculatory reflex, the sexual satisfaction of the patients and their sexual partners and anxiety or depress in sexual activity in CIPE were higher than in the control group, with significant difference between the two groups (t = 2.12, 2.31, 2.01, 2.24, P < 0.05). The difference in the SAS score after therapy was of significance (P < 0.01). A month after treatment, the effectivity rates of the two groups were 82.9% and 30% respectively, and the difference was significant (P < .01). CONCLUSION: Comprehensive behaviour psychotherapy obviously adds to the clinical efficacy of drugs in the treatment of PE.

Ejaculation↗
PubMed2006

[Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adeno-associated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells].

OBJECTIVES: Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro. METHODS: Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells. RESULTS: The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05). CONCLUSION: RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.

Animals↗