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1.
Article in Chinese | WPRIM | ID: wpr-828065

ABSTRACT

This present study aimed to explore the molecular mechanism of Erzhi Wan(a prescription of nourishing Yin and toni-fying liver and kidney) in treatment of aging by network pharmacology. The active constituents and target proteins of Erzhi Wan were searched from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP) and PubChem databases respectively. Aging-related genes were searched from Gene and HAGR databases. Based on the Ingenuity Pathway Analysis(IPA), we analyzed the common molecular network, biological pathway and interaction sites between these two parts, and verified some of them by Western blot. Twelve active constituents of Erzhi Wan were screened by TCMSP databases, 69 protein targets were predicted through PubChem, and 148 aging-related genes were found in Gene and HAGR databases. IPA comparison showed that the molecular networks of these two were complex, with diversity of biological functions. The common pathways involved 292 pathways, mainly related to tumors. They acted on hypoxia inducible factor-1α gene(HIF1α), nuclear factor-E2 related factor(Nrf2/NFE2 L2), tumor necrosis factor(TNF) and other sites. Western blot results suggested that Erzhi Wan could down-regulate the expression of HIF1α, with statistical difference(P<0.05). It was concluded that, Erzhi Wan could intervene aging through improving pseudo-hypoxic microenvironment and inflammation. The molecular mechanism of Erzhi Wan in delaying aging was preliminarily revealed, which laid a foundation for further stu-dying the anti-aging mechanism of Erzhi Wan, and also provided a reference for the compatibility mechanism and extended application of Chinese medicine compounds.


Subject(s)
Aging , Drugs, Chinese Herbal , Humans , Medicine, Chinese Traditional , Neoplasms , Proteins , Tumor Microenvironment
2.
Article in Chinese | WPRIM | ID: wpr-802187

ABSTRACT

Gas chromatography (GC) is mainly used to detect the levels of short-chain fatty acids (SCFAs), but with the deepening of research,the drawbacks of GC have become more and more obvious in the fields of food,chemical engineering and clinical application. The analysis on existing research results showed that ultra performance convergence chromatography (UPC2) was appropriate for the analysis of lipid metabolism. The UPC2 is a new kind of chromatographic separation technology developed in recent five years and the level of SCFAs is associated with the research on multiple diseases. Therefore,application of UPC2 in the detection of SCFAs would be helpful for the scholars at home and abroad to carry out deeper researches,and also helpful to guide the treatment for various metabolic disorders. In this paper,the researches on SCFAs in recent ten years were reviewed; the shortcomings of GC and liquid chromatography (LC) in the detection of SCFAs were reviewed; the development process,basic characteristics and research status of UPC2 at home and abroad were introduced; feasibility and innovation of UPC2 in the detection of SCFAs were summarized. Pretreatment methods for UPC2 application to the detection of SCFAs in feces or serum were collected; the problems that should be noticed during the process of sample pretreatment were pointed out; meanwhile, an research outlook on methodology of UPC2 application in the detection of SCFAs was conducted. The effects of extracting solvent,mobile phase,and auxiliaryt solvent on chromatographic behavior as well as the physicochemical property, type and choice of UPC2 chromatographic column were mainly discussed in this paper. In addition, the choices of basic modifier,acid modifier,and salinity modifier were briefly outlined, in order to provide efficient,simple,environmental,and economic detection technologies for the research on SCFAs, and provide better reference solutions for the rapid detection of massive clinical samples.

3.
Article in Chinese | WPRIM | ID: wpr-801855

ABSTRACT

The cure of tumors is a difficulty in the world, and both the quality of life and the survival rate of patients remain low. Therefore, it is very meaningful to find a drug target to inhibit the occurrence and development of tumors. In recent years, autophagy or self-phagocytosis has become a hotspot of medical research. It can remove damaged or excess organelles from cells, be survived from external environmental pressures, and affect the survival, metabolism, differentiation, aging and death of tumor cells. The biological behavioral process plays important roles in remodeling and maintaining the dynamic balance of cell survival, especially in close relations to tumor development. Autophagy is also a double-edged sword in effect on a single tumor cell and the entire tumor. When the autophagy of the tumor cells is abnormal, or the cells are unable to remove the damaged substances in time under the conditions of hypoxia and nutrient deficiency, autophagy is beneficial to the proliferation and survival of the tumor cells. Contrarily, moderate autophagy acts as an inhibitor of tumors and has an anti-tumor effect. Traditional Chinese medicine (TCM) has a long history of controlling tumors, with the advantages of low toxicity and multiple targets. Through overall and local therapies, it has a comprehensive therapeutic effect in cancer. With the deepening of tumor autophagy research, in addition to western medicine researches on tumor autophagy, there are also domestic and foreign researches on the autophagy in single herb and TCM compounds. The latest insights into the molecular mechanism of autophagy have led to the discovery of potential drug targets. At the same time, TCM researches have made some progress in tumor autophagy. The authors review the research progress of autophagy in TCM and the research progress of effect of TCM in regulating tumor autophagy, in the hopes to provide useful reference for effect of TCM in the treatment of autophagy.

4.
Article in Chinese | WPRIM | ID: wpr-798376

ABSTRACT

Shenfutang is a famous prescription used in clinic. It has been used for more than one thousand years, and currently is still widely used in clinic, with a significant effect. Shenfutang was first recorded in the Shengji Collection. It consists of two herbs, namely Ginseng Radix Et Rhizoma and Aconiti Lateralis Radix Praeparata. It can be used to mainly treat syncope and collapse due to sudden collapse of Yang Qi, and the symptoms include disfigurement of the extremities, cold sweats, cold limbs, umbilical and abdominal pain, weak breathing, and slight desire. Ginseng supports healthy Qi, and comforts five organs. Aconitum is good at activating twelve meridians and collaterals. With the effect of returning the yang to rescue the enemy, aconitum can also support yang. Different ratios of ginseng and aconitum are combined for reinforcing Yang of heart, kidney and spleen, so as to treat various syndromes. However, the occurrence and development of diseases are complicated and changeable. Different ratios of Shenfutang may increase the efficacy due to the synergistic effect, or weaken or even lose the original efficacy due to mutual antagonism. Different ratios of ginseng and aconitum can be used for different diseases, such as cardiovascular disease, various types of inflammation, respiratory diseases. In the existing literatures on Shenfutang, there is a lack of systematic summarization for how to adjust the ratios. This paper introduces the effect and mechanism of the combination, and summarizes different ratios of the two herbal ingredients, so as to provide certain reference for the clinical application.

5.
Article in Chinese | WPRIM | ID: wpr-705366

ABSTRACT

OBJECTIVE To investigate the underlyingmechanism on the association of red blood cell and gut microbiota in rats induced by High-Fat Diet(HFD).METHODS A total of 36 male Sprague-Dawley rats (180±20g) were randomly divided into two groups. The control group (n=10) was given a normal chow diet(10% calories of fat),and the High-fat diet group(n=26)was given a HFD(60% calo-ries of fat).We recorded body weight,length and detected serum glucose,serum lipids and insulin ev-ery two weeks.The fresh arterial blood was collected during the experiments and blood gases were measured immediately (Radiometer Medical ApS, Denmark).Thehematocrit (Hct) and partial pressure of oxygen(pO2)were detected by the sensor cassette,following themanufacturer′s instructions.The de-tection method was conductivity measurements and current method, respectively. The feces from ce-cum were analyzed by 16S rRNA gene high-throughput sequencing(Illumina Miseq,USA). RESULTS According to the insulin resistance(IR),body weight and body length,the model group was divided into two small groups.(1)IR group,in which IR,body weight and body length were higher than the control group (P<0.05). (2) un-IR group, body weight and body length were higher than the control group (P<0.05),but the IR was not significantly different.In addition,the levels of hematocrit(Hct),checktotalhe-moglobin (ctHb) and check total blood oxygen content (ctO2) showed significantly increased in the IR group when compared with the control group (P<0.05), however, the pO2was not statistically signifi-cant. Furthermore, we identified that the genus Lactobacillus was moderate positive correlation with Hct,ctHb and ctO2(P<0.05).Compared with the control group,the relative abundance of the Lactoba-cillus was significantly lower in IR group(P<0.05).CONCLUSION The high-fat diet induced rats′local tissue hypoxia under the red blood cell increasing,oxygen partial pressure constant and the reduction of Lactobacillus′abundance might be caused by aerobic oxidation and glycolysis inhibition in the meantime.

6.
Article in Chinese | WPRIM | ID: wpr-705308

ABSTRACT

OBJECTIVE To explore the biomarkers and molecular mechanism of Huanglianjiedu decoction (HJD) on high fat diet-induced experimental atherosclerosis in rats. METHODS SD male rats were randomly dividedinto five groups(n=8):normal control group,model group,and three dosage groups (1.5, 3 and 6 g crude drug per kilogram of body weight). Atherosclerosis was induced by the combination of regular intraperitoneal injection of vitamin D3and high fat diet for 8 weeks. HJD was administered by oral gavage from the third week once per day and until the end of the study.After the final administration, the blood samples were collected for biochemical analyses [total cholesterol (TC), triglycerides (TG), highdensity lipoprotein (HDL-C), low-density cholesterol (LDL-C)] and blood gas analyses(PaO2, PaCO2, pH, ctHb, etc); the abdominal aorta sections were stained with hematoxylin and eosin for histopathology; the liver homogenate were determined for MDA, SOD, OX-LDL, MCP-1 and VCAM-1.The plasma samples were detected using ultraper formance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS).The data of endogenous compounds were preliminarily preprocessed by software Progenesis QI and then analyzed by multivari-ate statistical analysis software EZinfo 2.0 to screen the distinguished biomarkers and the metabolic pathways were analyzed through website http://www.metaboanalyst.ca/. RESULTS Compared with the normal control group,the content of TC,TG,LDL-C,PaCO2,MDA,Ox-LDL,MCP-1 and VCAM-1were significantly increased and HDL-C, PaO2, ctHb and SOD decreased in the atherosclerosis rats. HJD could significantly attenuated the high fat-induced atherosclerosis pathological injury and the above-mentioned indexes (P<0.05). The five groups could be clearly distinguished using the metabolomics method.The administration groups profile exhibited an apparent returning trend from that of the model group and that of the normal control group.Twenty-one endogenous metabolites has been significantly changed in atherosclerosis rats.HJD could remarkably up-regulate 5-L-glutamyl-taurine,L-beta-aspartyl-L-glutamic acid, histidinyl-hydroxyproline, tryptophyl-alanine, 4′-O-methyl-(-)-epicatechin, and down-regulate protoporphyrin IX,azelaic acid,lacto-N-triaose,cinnamoylglycine and 9′-carboxy-alpha-tocotri-enol. CONCLUSION The beneficial effect of HJD in high fat-induced atherosclerosis rats may be due to anti-oxidant and anti-inflammatory. And it is suggested that HJD may affect the model rats through tryptophan metabolism, taurine and hypotaurine metabolism, histidine metabolism, lysine degradation and porphyrin and chlorophyll metabolism pathway.

7.
Article in Chinese | WPRIM | ID: wpr-246129

ABSTRACT

To establish a HPLC method for simultaneously determining plasma concentrations of gastrodin (Gas) and its metabolites hydroxybenzyl alcohol (HBA), puerarin (Pur) and internal standard (IS) p-hydroxyphenylethanol (Tyr) in rats and studying the pharmacokinetic process and interactions of gastrodin and puerarin after single and combined intravenous injection and oral administration. With Tyr as the internal standard, plasma samples were processed with methanol for protein precipitation, supernatant was dried with N2, and residues were re-dissolved with acetonitrile-0.05% phosphoric acid (20: 80). Chromatography was carried out on an Agilent ZORBAX SB-Aq C18 column (4.6 mm x 250 mm, 5 μm), with acetonitrile-0.05% phosphoric acid as the gradient mobile phase for the gradient elution. The UV detector wavelength was set at 221 nm for Gas HBA and IS and 250 nm for Pur. After the single or combined administration of Gas and Pur, their plasma concentrations in rats were detected. WinNonlin 5.2 pharmacokinetic software and SPSS 17. 0 software were used to respectively calculate pharmacokinetic parameters of each group, make a statistical analysis and compare the pharmacokinetic processes of Gas and Pur after the single or combined administration. According to the results, the absolute recoveries between low, media and high concentrations of Gas, HBA and Pur and IS as well as Tyr were more than 77.20%, with a good linearity (r > 0.999 6, n = 5) for Gas, HBA and Pur within concentration ranges of 0.10-101, 0.03-7.58 and 0.05-5.98 mg xL ('1) respectively. The lower limits of quantification for Gas, HBA and Pur were 0.10, 0.03, 0.05 mg x L(-1), respectively. Their in-ra-day and inter-day precisions were less than 12% with the accuracy between 85. 1% -1 10. %. All of the three substances and IS were stable during the whole analysis process. The findings showed significant differences in the main in vivo pharmacokinetic parame-ers in rats (AUC, C.(max) T,½ T.(max) MRT) after the single and combined administration of Gas and Pur. Either after the oral adminis-ration or after the intravenous injection, parameters showed a lower clearance rate ( L) longer mean residence time ( RT) and higher relative bioavailability, especially after the oral administration. Specifically, the relative bioavailability of the combined oral ad-inistration of Pur was 10. 7 times of that of the single administration, while that of Gas was 1. times of that of the single administra-ion. The combined administration of Gas and Pur can promote the absorption, decrease the elimination rate and prolong the mean resi-ence time. The method is simple and accurate and can be applied in the simultaneous determination of plasma concentrations of Gas, HBA and Pur in rats and the pharmacokinetic studies.


Subject(s)
Administration, Oral , Animals , Benzyl Alcohols , Blood , Pharmacokinetics , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Pharmacokinetics , Glucosides , Blood , Pharmacokinetics , Isoflavones , Blood , Pharmacokinetics , Male , Rats , Rats, Wistar
8.
Acta Pharmaceutica Sinica ; (12): 280-284, 2011.
Article in Chinese | WPRIM | ID: wpr-348964

ABSTRACT

This study is designed to investigate the anti-tumor and anti-angiogenesis mechanism of carrageenan oligosaccharides. The effects of carrageenan oligosaccharides on basic fibroblast growth factor (bFGF) induced cell proliferation, heparanase activity and bFGF binding ability were evaluated in human cervical cancer cells (HeLa) and human umbilical vein endothelial cells (HUVEC). Results indicate that, at rational concentrations, carrageenan oligosaccharides showed low cytotoxic effect. At relatively low concentrations (0.2-200 microg x mL(-1)), these oligosaccharides could competitively bind bFGF and inhibit bFGF induced cell proliferation. In these samples, oligo-lambda-carrageenans (dp2-8) were the most potent bFGF antagonists. At concentration of 20 microg x mL(-1), their inhibitory ratio reached to 30%. The heparanase enzyme assay revealed that three kinds of carrageenan oligosaccharides showed different inhibitory activities to two cell lines. For HeLa cell, oligo-lambda-carrageenans showed highest inhibitory effect, but for HUVEC, oligo-kappa-carrageenans (dp9-17) were the best inhibitors. Current observations demonstrated that the biological activities of carrageenan oligosaccharides are closely related to the molecular weight, carbohydrate structure and the content and linking position of sulfur groups. Carrageenan oligosaccharides with high sulfate fraction, 2-8 units saccharide size and suitable molecular structure are able to achieve potent heparin sulfate-like compounds.


Subject(s)
Angiogenesis Inhibitors , Pharmacology , Antineoplastic Agents , Pharmacology , Carrageenan , Pharmacology , Cell Proliferation , Fibroblast Growth Factor 2 , Metabolism , Glucuronidase , Metabolism , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Oligosaccharides , Pharmacology , Protein Binding
9.
Acta Pharmaceutica Sinica ; (12): 521-526, 2011.
Article in Chinese | WPRIM | ID: wpr-348925

ABSTRACT

This study is to investigate the protective effect of astaxanthin against injured hepatocyte L-02 cells induced by sodium azide (NaN3) and reveal the possible mechanisms. Hepatocyte L-02 cells were exposed to 100 mmol.L-1 NaN3 with various concentrations of astaxanthin pre-incubated, then the cell viability was measured by MTT method; The level of reactive oxygen species (ROS) was determined by DCFH-DA method; The changes of mitochondrial membrane potential (MMP) and apoptosis ratio were detected by JC-1 method and Annexin V-FITC/PI double stain method, respectively. Results showed that after cells were exposed to 100 mmol.L-1 NaN3 for 3 hours, the cell viability significantly decreased; ROS level and the percentage of late phase apoptosis increased obviously; MMP was also declined. When cells were pretreated with astaxanthin, the cell damage and late phase apoptosis ratio reduced and MMP was maintained. However, the level of ROS showed insignificant decrease (P>0.05). The beneficial concentration of astaxanthin in improving cell viability and MMP was not in a dose dependent manner and the most effective of which was 0.10 nmol.L-1 (P<0.01). In order to reveal its possible non-antioxidant mechanism, mitochondrial membrane was imitated and H+ transferring function of astaxanthin was also detected by bilayer lipid membrane (BLM) method. Results showed that 2.0% astaxanthin could transfer H+ efficiently. These suggested the mechanisms of astaxanthin in protection of hepatocyte L-02 cells not via its ROS quenching capability but via its H+ transferring function, which improved the mitochondrial function and had the sequence biology effects.


Subject(s)
Antioxidants , Pharmacology , Apoptosis , Cell Line , Cell Survival , Hepatocytes , Cell Biology , Humans , Hydrogen-Ion Concentration , Membrane Potential, Mitochondrial , Protons , Reactive Oxygen Species , Metabolism , Sodium Azide , Toxicity , Xanthophylls , Pharmacology
10.
Acta Pharmaceutica Sinica ; (12): 980-986, 2009.
Article in Chinese | WPRIM | ID: wpr-354608

ABSTRACT

This study is to investigate the effect of fascaplysin on human cervical cancer cells (HeLa) in order to provide insights into the mechanisms of growth suppression involved in fascaplysin-mediated apoptosis. Cytotoxic activity of fascaplysin on HeLa cells was determined using MTT assay, cell cycle analysis, and apoptosis (Annexin V-FITC and PI double staining) studies. The role of the molecules in cell cycle regulation and apoptosis was analyzed by Western blotting and flow cytometry. Fascaplysin markedly inhibited HeLa cells proliferation in a dose-dependent manner, however, did not provoke G1 phase arrest in HeLa cells with downregulation of CDK4, cyclin D1 and CDK4-specific Ser795 pRb phosphorylation. Furthermore, fascaplysin induced significantly apoptosis evidenced by sub-G1 peak and Annexin V-FITC and PI double staining. The molecular mechanism of fascaplysin-induced apoptosis was characterized with the activation of caspase-3, -8, and -9, truncation of Bid, release of cytochrome c into cytosol, and down-regulation of Bcl-2 level. Fascaplysin exhibits anti-proliferation effect towards human cervical cancer HeLa cells through induction of apoptosis via extrinsic death pathway and mitochondrial pathway, but not arresting cell cycle progression at G1 phase. All together, these data sustain our contention that fascaplysin has anticancer properties and merits further investigation as a potential therapeutic agent.


Subject(s)
Apoptosis , Cell Cycle Checkpoints , Cell Proliferation , HeLa Cells , Humans , Indoles , Pharmacology , Mitochondria , Metabolism
11.
Article in Chinese | WPRIM | ID: wpr-814016

ABSTRACT

OBJECTIVE@#To determine the relationship between serum antibody against HPV16 E4 and cervical cancer, and to construct HPV 16 E4 protein expression vector as an antigen to detect its corresponding serum antibody among different populations.@*METHODS@#HPV16 E4 early gene was ligated into pRSET-A expression vector. The constructed plasmids were transformed into BL21 (DE3)cells, and induced to express HPV 16 E4 protein by isopropylthio-beta-D-galactoside (IPTG). The expressed E4 inclusions were denatured, purified through Ni-column, and renatured. After the activity was revealed, antibodies against HPV 16 E4 in the sera from healthy women and patients with chronic cervicitis and cervical cancer were respectively determined by enzyme-linked immunosorbent assay (ELISA) using the fusion protein as the antigen.@*RESULTS@#HPV 16 E4 fusion protein of Mr 15*10(3) was expressed by pRSET-16E4 after IPTG induction. The fusion protein accounted for 30% of the total bacterial proteins and expressed as inclusive body. After purification with Ni-NTA agarose resin, the recombinant protein revealed purity of 95%, and activity of the renatured protein was identified by ELISA. The serum antibody-positive rate of HPV 16 E4 was 10.00%, 39.13% and 28.13%, respectively in 80 healthy women, 46 chronic cervicitis patients, and 32 cervical cancer patients. The antibody-positive rate in cervical cancer patients and chronic cervicitis patients were significantly higher than that in healthy women (P<0.01), while the difference between the antibody-positive rate in cervical cancer patients and chronic cervicitis patients was not significant.@*CONCLUSION@#HPV 16 E4 protein expressed from pRSET-A/BL21 can be used in serological studies on cervical cancer-related HPV infection. Serum antibody against HPV16 E4 is present in a significantly higher percentage in cervical cancer and chronic cervicitis patients than in healthy women.


Subject(s)
Adult , Aged , Antibodies, Viral , Blood , Female , Genetic Vectors , Human papillomavirus 16 , Genetics , Allergy and Immunology , Humans , Middle Aged , Oncogene Proteins, Viral , Genetics , Allergy and Immunology , Papillomavirus Infections , Allergy and Immunology , Virology , Recombinant Fusion Proteins , Genetics , Uterine Cervical Neoplasms , Allergy and Immunology , Virology , Uterine Cervicitis , Virology
12.
Acta Pharmaceutica Sinica ; (12): 474-479, 2008.
Article in Chinese | WPRIM | ID: wpr-277828

ABSTRACT

To study the anti-proliferation effect of lambda-carrageenan oligosaccharides (lambda-CO) on human umbilical vein endothelial cells (HUVECs) and expression of apoptotic relevant genes, the influence of lambda-CO on HUVECs proliferation was measured by MTT assay; apoptotic rate, cell cycle distribution and the level of active caspase-3 of HUVECs were analyzed using flow cytometry; the mRNA level of apoptosis related genes was determined by RT-PCR. At a high concentration of 1 mg x mL(-1), lambda-CO significantly inhibited the endothelial cell proliferation. Annexin-V FITC/PI double stain assay showed that when treated with 0, 0.8, 1 mg x mL(-1) of lambda-CO for 24 h, cell apoptotic rates were (1.67 +/- 1.6)%, (11.48 +/- 2.4)% and (13.81 +/- 2.2)%, respectively, when treated for 48 h, cell apoptotic rates were (2.02 +/- 2.3)%, (13.84 +/- 1.9)% and (38.72 +/- 2.5)%, respectively, cell cycle assay showed the decrease of cells in G0/G1 phase, and increase in S phase. Furthermore, we observed the level of active caspase-3 increased in a dose-dependent manner at 24 th and 48 th. RT-PCR results indicated that mRNA of TNFalpha, p53, caspase-8 and caspase-3 in cells increased after treated with lambda-CO. lambda-CO induce apoptosis of HUVECs in a dose-dependent way and arrests cells at S phase, which mainly due to the up-regulation of apoptotic genes such as TNFalpha, p53, caspase-8, caspase-3 and increase the level of active caspase-3.


Subject(s)
Angiogenesis Inhibitors , Pharmacology , Apoptosis , Carrageenan , Pharmacology , Caspase 3 , Genetics , Metabolism , Caspase 8 , Genetics , Cell Cycle , Cell Proliferation , Cells, Cultured , Endothelial Cells , Cell Biology , Humans , Oligosaccharides , Pharmacology , RNA, Messenger , Metabolism , Tumor Necrosis Factor-alpha , Genetics , Tumor Suppressor Protein p53 , Genetics , Umbilical Veins , Cell Biology
13.
Acta Pharmaceutica Sinica ; (12): 595-600, 2007.
Article in Chinese | WPRIM | ID: wpr-281870

ABSTRACT

This study was designed to evaluate the inhibition effect of lambda-carrageenan oligosaccharides on neovascularization in vitro by chick chorioallantoic membrane (CAM) model and human umbilical vein endothelial cell ( HUVEC). lambda-Carrageenan oligosaccharides caused a dose-dependent decrease of the vascular density of CAM, and adversely affected capillary plexus formation. At a high concentration of 1 mg x mL(-1), this compound inhibited the endothelial cell proliferation, while low concentration of lambda-carrageenan oligosaccharides (< 250 microg x mL(-1)) affected the cell survival slightly (> 95%). Different cytotoxic sensitivity of lambda-carrageenan oligosaccharides in three kinds of cells was observed, of which HUVEC is the most sensitive to this oligosaccharides. The inhibitory action of lambda-carrageenan oligosaccharides on the endothelial cell invasion and migration was also observed at relatively low concentration (150 - 300 microg x mL(-1)) through down-regulation of intracellular matrix metalloproteinases-2 (MMP-2) expression on endothelial cells. Current observations demonstrated that lambda-carrageenan oligosaccharides are potential angiogenesis inhibitor with combined effects of inhibiting invasion, migration and proliferation.


Subject(s)
Angiogenesis Inhibitors , Pharmacology , Animals , Carrageenan , Pharmacology , Cell Movement , Cell Proliferation , Chick Embryo , Chorioallantoic Membrane , Cell Biology , Endothelial Cells , Humans , Matrix Metalloproteinase 2 , Oligosaccharides , Pharmacology
14.
Article in Chinese | WPRIM | ID: wpr-321159

ABSTRACT

<p><b>OBJECTIVE</b>To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput.</p><p><b>METHODS</b>Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated.</p><p><b>RESULTS</b>The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD.</p><p><b>CONCLUSION</b>TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.</p>


Subject(s)
Aged , Alleles , Alzheimer Disease , Genetics , Apolipoproteins E , Genetics , Gene Frequency , Genotype , Humans , Middle Aged , Polymerase Chain Reaction , Methods , Polymorphism, Genetic , Reproducibility of Results
15.
Acta Pharmaceutica Sinica ; (12): 903-907, 2005.
Article in Chinese | WPRIM | ID: wpr-253544

ABSTRACT

<p><b>AIM</b>To evaluate the hepatocyte protective effect of agarohexaose against indirect oxidative stress injury induced by antimycin A.</p><p><b>METHODS</b>Antimycin A was used to induce oxidative injury of human hepatocyte L-02. The oxidative degree in cells was detected by dichlorofluorescin diacetate (DCFH-DA) and the fluorescence generation was recorded by flow cytometer and fluorescent microscope. The apoptosis of L-02 cells induced by oxidation was identified by TUNEL test, and the morphologic features of cells were also observed.</p><p><b>RESULTS</b>Agarohexaose at concentration of 1 mg x mL(-1) inhibited the oxidation of DCFH into DCF significantly. The fluorescence intensity and oxidized cell number decreased after the incubation with agarohexaose. The photomicrographs of antimycin A and agarohexaose treated cells revealed that agarohexaose could reduce the apoptotic morphologic features. The TUNEL results also indicated that the number of apoptotic cells decreased significantly after the treatment of agarohexaose.</p><p><b>CONCLUSION</b>Agarohexaose could inhibit the sudden increase reactive oxygen species (ROS) in cells significantly, and it also protected cells against oxidative stress injury in vitro.</p>


Subject(s)
Antimycin A , Pharmacology , Antioxidants , Pharmacology , Apoptosis , Cells, Cultured , DNA Breaks , Hepatocytes , Cell Biology , Metabolism , Humans , Oligosaccharides , Chemistry , Pharmacology , Oxidative Stress , Reactive Oxygen Species , Metabolism
16.
Chinese Journal of Oncology ; (12): 543-546, 2004.
Article in Chinese | WPRIM | ID: wpr-254305

ABSTRACT

<p><b>OBJECTIVE</b>To detect HPV 58, a common type of human papillomavirus (HPV), clone and express its E7 gene from biopsy specimens of cervical cancer.</p><p><b>METHODS</b>HPV 58 from 58 biopsy tissues of cervical cancer was detected by GP5+/GP6+ PCR followed by template-directed dye-terminator incorporation assay with fluorescence polarization detection (TDI-FP). HPV 58 E7 gene was amplified from one HPV 58-positive sample, and then cloned into pGEM-T Easy vector. The recombinant plasmid, HPV58E7-pGEM-T was confirmed by sequencing. Subsequently, E7 gene was cloned into prokaryotic expression vector pRSET-A. The constructed pRSET-58E7 plasmids were transfected into BL21(DE3) cells, and induced to express 58 E7 protein by IPTG.</p><p><b>RESULTS</b>Among the 58 biopsy tissues of cervical cancer, 10 were HPV 58-positive, accounting for 19.2% of 52 HPV-positive cases. HPV 58 E7 gene was amplified from one HPV 58-positive sample. The constructed plasmids were identified containing HPV58 E7 gene by restriction enzyme analysis and sequencing. SDS-PAGE analysis showed that HPV58 E7 His6 fusion protein of M(r) 16 x 10(3) was expressed by pRSET-58E7 after induction by IPTG. The fusion protein accounted for 30% of total bacterial proteins.</p><p><b>CONCLUSION</b>HPV 58 is not uncommon in Chinese women with cervical cancer in Shaanxi province. Constructed HPV58 E7 recombinant plasmids can be effectively expressed in E.coli, which may provide a tool in diagnosis and vaccine design for HPV of HPV58-associated tumors.</p>


Subject(s)
Adult , Cloning, Molecular , Escherichia coli , Metabolism , Female , Genes, Viral , Humans , Middle Aged , Papillomaviridae , Genetics , Papillomavirus E7 Proteins , Genetics , Papillomavirus Infections , Genetics , Plasmids , Recombinant Proteins , Genetics , Transformation, Genetic , Uterine Cervical Neoplasms , Genetics , Virology
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