RNA interfering (RNAi) is a process of sequence-specific, posttranscriptional gene silencing in animals and plants. In mammalian cells, duplexes of 19～25 nts (nucleotides) RNAs efficiently inhibit gene expression. The pBS/H1SP vector expressing siRNA is used which inhibit specific HIV-1 gene expression. To assess the intracellular effect of this H1 promoter-driven siRNA, a reporter plasmid pEGFP-C1-HIV protein which expresses fusion protein of enhanced green fluorescent protein (EGFP) and HIV protein was used. The expression of the reporters can be easily visualized by fluorescence microscopy in living cells. siRNA-generating vectors targeted to several HIV-1 genomes were constructed and then co-transfected with respond reporter expression vectors into HEK293 cells. Cells transfected with pHIV-siRNA exhibited a significant inhibition of pEGFP-HIV expression compared with cells transfected with control vectors. By this way, it is successfully to select effective siRNA for silencing target HIV-1 genes. Then two or three siRNA transcripts targeted to different HIV genes were expressed by one plasmid, and a relative strong inhibition effect was observed.

BACKGROUND: It has been reported that Angiotensin Ⅱ (Ang Ⅱ) is related to occurrence and development of dermatofibrosis; however, less is explored about the expression and effect of AT1 and AT2 receptors in the fibroblasts of human hypertrophic scar.OBJECTIVE: To observe the expression of Ang Ⅱ type 1 (AT1) and type 2 (AT2) receptors in human hypertrophic scars, and explore their effects on collagen synthesis of fibroblasts.DESIGN, TIME AND SETTING: Randomized control experiment was performed at the Experimental Center, Guangzhou General Hospital of Guangzhou Military Area Command of Chinese PLA between August 2006 and November 2007. PARTICIPANTS: Samples of hypertrophic scare were taken from 18 patients (10 males and 8 females, 19-47 years Old). Seven specimens of normal skin served as control. All of the specimens collected were divided into two parts, one part for immunohistochemical staining after fixated by 4% paraformaldehyde, the other part for culturing fibroblasts.METHODS: The expression of both AT1 and AT2 receptors in fibroblasts of hypertrophic scare was detected with immunohistochemical staining and radioligand receptor binding assay. Collagen synthesis was examined in cultured fibroblasts of hypertrophic scars by measuring [3H]-proline incorporation into collagenous proteins.MAIN OUTCOME MEASURES: The expression of both AT1 and AT2 receptors in human hypertrophic scars; the [3H]-proline incorporation value in cultured fibroblasts.RESULTS: Positive staining signals of both AT1 and AT2 receptors were found in fibroblasts of hypertrophic scars. Similar results were also observed in cultured fibroblasts of hypertrophic scars, expression level of AT1 and AT2 receptors were (10.69±2.15) fmol/106 cells and (4.9±1.05) fmol/106cells, respectively. In cultured fibreblasts, Ang Ⅱ stimulation significantly increased collagen synthesis, which was inhibited by valsartan, an AT1 receptor blocker, but augmented by PD123319, an AT2 receptor antagonist.CONCLUSION: Both AT1 and AT2 receptors were expressee in the fibreblasts of hypertrophic scars, and Ang Ⅱ regulates collagen synthesis in hypertrophic scar fibroblasts through a negative cross-talk between AT1 and AT2 receptors, which might contribute, at least partly to formation and maturation of human hypertrophic scars.

OBJECTIVE To investigate the relationship between gastric polyps and Helicobacter pylori infection.METHODS The patients with gastric polyps were taken by gastroendoscopy in 2005.The tissues from their antrums were examined for presence of H.pylori.We collected and analyzed all of their general information and the data about their gastric polyps and H.pylori infection condition.RESULTS In the 95 gastric polyps patients,76 cases(80.0%) had inflammatory polyps and 19 cases(20.0%) had H.polyps.The total H.pylori infection rate was 33.7%.The H.pylori infection rate in the inflammatory polyps patients and H.pylori patients were 38.2% and 15.8%,respectively.CONCLUSIONS H.pylori infection promotes the formation of gastric inflammatory polyps.The examination and treatment for H.pylori is necessary for the gastric polyps patients.

Objective To knockout the cell division cycle 25 homolog C(Cdc25C) gene in HeLa human cervical cancer cells and to construct HeLa Cdc25C gene knockout stable strains using clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 gene-editing system.Methods The sequence of the small guide RNA(sgRNA),which could specifically recognize the first exon of Cdc25C,was designed according to the target-designing rules of CRISPR/Cas9 for construction of eukaryotic recombinant expressional plasmids.After sequencing,the plasmid was transfected into HeLa cells.The stable Cdc25C-knocking out strains were screened through the stress of puromycin,and the knockout effect was detected by Western blotting.The cell cycle was analyzed by flow cytometry.Results The stable Cdc25C-knocking out strains were obtained.Moreover,the gene′s knockout obviously delayed the progression of G2/M phase.Conclusion The HeLa Cdc25C gene knockout stable strain is successfully built using CRISPR/Cas9 system,facilitating studies on the function of Cdc25C and the mechanism of carcinogenesis.

Objective To separate human γ-tubulin ring complexes (γTuRC) .Methods Cell lysates prepared from 293FT cells were separated using gel filtration chromatography .Then, the eluate fractions containing γTuRC or γ-tubulin small complexes (γTuSC ) were determined by immunoblotting .Results As the constitutive components of γTuRC,γ-tubulin,γ-tubulin complex protein 2 (GCP2), GCP3 and GCP4 were eluted and enriched in the fourth fraction .The molecular mass of eluates in the fourth fraction was about 2000 ×10 3 .Following γTuRC, the constitutive components ofγTuSC including γ-tubulin, GCP2 and GCP3 were eluted and enriched in the fourteenth fraction .The molecular mass of eluates in the fourteenth fraction was about 310 ×10 3 .Unassembled free components were washed out in the eighteenth and subsequent fractions .γTuRC could be detected in the corresponding fractions by negative-PAGE separation .ConclusionγTuRC and γTuSC were successfully separated from the unassembled free components in the fourth ( 4#) and fourteenth (14#) eluted fraction, respectively.The eluates containing ofγTuRC orγTuSC can be used for microtubule assembly research.

Objective To discuss the efficacy of Compound Anisodine Injection combined with Iodized Lecithin Tablets in treatment of central serous chorioretinopathy.Methods Totally 60 patients with CSC were selected,and divided into two groups randomly.The patients in control group (29 cases) were given Iodized Lecithin Tablets.The patients in observation group (31 cases) were given Iodized Lecithin Tablets and Compound Anisodine Injection.The efficacy of Compound Anisodine Injection combined with Iodized Lecithin Tablets in treatment of central serous chorioretinopathy was evaluated by efficacy,visual acuity,light sensitivity,and adverse reaction during treatment.Results After treatment,the effective rates of observation and control groups were 93.5％ and 79.3％,and the observation group was significantly higher than control group (P ＜ 0.05).There were no statistical significance on visual acuity between two groups.After treatment,rhe visual acuity of two groups was increased and the visual acuity in the observation group was better (P ＜ 0.05).Before treatment,there were no statistical significance on light sensitivity between two groups.After 2 and 4 weeks treatment,the light sensitivity of two groups were increased and the light sensitivity in the observation group was higher (P ＜ 0.05).During treatment,there was no statistical significance on adverse reaction between two groups.Conclusion Compound Anisodine Injection combined with Iodized Lecithin Tablets has a curative effect on central serous chorioretinopathy.It could increase the visual acuity and improve the light sensitivity of eyes with good security.It is worthy of clinical use.

To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-β-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 μmol x L(-1).
Animals ; Biological Factors ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Cell Survival ; drug effects ; Macrophages ; cytology ; drug effects ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Mice ; Micrococcus ; chemistry ; genetics ; isolation & purification ; metabolism ; Molecular Structure ; Phylogeny ; RAW 264.7 Cells ; Seawater ; microbiology ; Secondary Metabolism

Objective To construct humanized monoclonal antibodies against CD 20 and check their affinity to CD 20 antigen and their anti-tumor activity.Methods Based on the computer model , human IgG1 candidates closest to rituximab in crystal structure were selected in the Protein Data Bank ( PDB) .With the selected human IgG 1 candidates as the frame , we modified and transplanted the complementarity determining region ( CDR) of rituximab .First,the target gene fragments were obtained by overlapping PCR.Then, the sequences of the light chains(L) and the heavy chains(H) were inserted in-to the pcDNA3.3 and pOptiVEC vectors.Next, the constructed clones were transfected into 293F cells through transient transfection.After a large-scale cell culture, the mAb was purified by affinity chromatography rProtein A column.The puri-ty and expression level of the humanized antibodies was tested by sodium dodecyl sulfate ( SDS)-polyacrylamide gelelectro-phoresis(PAGE).The affinity of the humanized antibodies to CD20 was assessed with Fortebio assay.Finally, the anti-tumor activity of the constructed antibodies was detected by checking the tumor growth inhibition of the nude mice transplan-ted with tumor .Results Three humanized monoclonal antibodies against CD 20 were expressed and purified successfully . In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 ×103 and 55 ×103 , respectively.The band size of the antibodies matched the expected value.Fortebio assay revealed that the humanized antibodies could bind to CD20 with high affinity (rituximab:6.48 ×10 -9mol/L, L4H7:1.91 ×10 -9mol/L, L5H5:7.35 ×10 -10mol/L,and L5H7:1.91 ×10 -9mol/L).The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7 mAb was better than that of rituximab .Conclusion Three humanized monoclonal antibodies against CD 20 have been successfully construc-ted and expressed.L5H7 mAb possesses high affinity for CD20 and a good ability to kill tumor cells.

Biopsy ; Histological Techniques ; instrumentation ; methods ; Humans ; Indicators and Reagents ; Paraffin Embedding ; Ultrasonics

Objective To investigate the effect of Pingfei Oral Liquid (POL) on the distribution of mast cells (MCs) and the expression of interleukin 6 (IL-6) in the lung tissue of radiation pneumonia rats. MethodsForty-five SD rats were randomized into 3 groups : normal control,model group and POL group. The rat model of radiation lung fibro-sis was set up by a single X-ray dose of 20Gy irradiation over the whole chest of the rats. POL (20 g·kg-1·d-1,once a day, five times a week) was given orally one week before irradiation and the treatment lasted 5 weeks. MCs in the lung tissue were stained with toluidine blue firstly and then were counted 2, 4 and 8 weeks after irradiation. IL-6 protein expression of lung tissue was measured by immunohistochemical assay 8 weeks after irradiation, and mRNA ex-pression was determined with RT-PCR 4 weeks after irradiation. ResultsIt's showed the aggregation of large amount of pulmonary mast cells and increase of IL-6 protein expression 8 weeks after irradiation (P < 0.01).IL-6 mRNA expression in the irradiated lung of rats increased 4 weeks after irradiation (P < 0. 01). POL could reduce the aggrega- tion of MCs (P < 0. 01) and the expression of IL-6 protein (P < 0. 01) and mRNA (P < 0. 05) in the lung tissue. ConclusionPOL can prevent radiation pneumonia in rats by reducing the aggregation of mast cells and inhibiting IL-6 expression in the lung tissue.