Objective To evaluate the effects of connective tissue growth factor (CTGF) on corneal neovascularization.Methods Twenty-five Wistar rats were divided into control and experimental groups.Corneal neovascularization (CNV) was induced by alkaline burn of the cornea with 1 mmol/L NaOH.On the 1st,4th,7th,and 14th day,CNV was observed,and the expression of CTGF was investigated with immunohistochemical method in rat cornea at the different time point.Results On the 4th day,7th day and 14th day after alkaline burn,the areas of CNV were (12.740 ±2.536) mm2,(26.068 ± 10.028) mm2,and (37.588 ± 8.066) mm2,respectively.CTGF was rarely expressed in the cornea of normal rats,and then CTFG expression was quickly increased after alkaline burn,reached the highest level on the 4th day (1.714 ± 0.185),and then declined remarkably on the 7th day (1.334 ± 0.198).Conclusions CTGF might be involved in the formation process of corneal neovascularization after corneal alkali burn.

Objective To construct RNAi combinant adenoviral expressive vectors specific to p65 subunit of NF-?B and to observe their gene silencing effect on p65 subunit.Methods Three pairs of complementary. single-strand DNA oligos targeting three various sites of p65 mRNA were designed and synthesized.Annealling was used to generate double-strand oligos(ds-oligos),and then the ds-oligos were cloned into pENTR~TM/u6 to generate the entry clone named pENTR.Recombination reaction in vitro with the pENTR and pAd/BLOCK-iT~TM- DEST was used to creat the adenovirus plasmid which contains the RNAi cassette.Then,the adenovirus plasmids digested with PacI were transfected into HEK293A cells to product adenovirus,and latter infected the HEK293A cells to amplify the adenoviral stock.Plaque forming assay was used to titer the adenoviral stock.The p65 gene silencing effect induced by the RNAi adenovirus was detected by Western blot and immunocytochemistry assay in ECV304 cells.Results The RNAi adenovirus specific to p65 subunit of NF-?B were produced with titer of 3.0 x 10~9pfu/ml to 2.5?10~10pfu/ml.The expression of p65 protein in ECV304 cells could be down-regulated efficiently by the RNAi adenovirus 48-72 h after infection,which would last for more than 6 days after infection.Conclusion RNAi adenovirus is an important tool inhibiting the expression of target gene efficiently.

Objective To elucidate the influence of fetal genotype in both non-diabetic gravidas and pregnant women on gestational diabetes mellitus (GDM) through analysis of the genotype discrepancy between maternal and fetal Pro12A1a single nucleotide polymorphism (SNP) of peroxisome proliferator-activated receptor gamma 2 (PPARG2) genes.Methods Pregnant women,who delivered in the Obstetrics and Gynecology Hospital of Fudan University from October 2005 to February 2007,and their newborn babies were selected,and were divided into GDM and control group.The GDM group consisted of 55 gravidas with GDM and 40 newborns born to the GDM mothers,and the control group consisted of 173 healthy gravidas and their 50 neonates.Polymerase chain reaction-denaturing high-performance liquid chromatography was applied to detect the distribution of PPARG2 Pro12Ala alleles in all subjects.The concentrations of plasma fasting blood sugar (FBS) and several bio-markers of lipids,including total cholesterol,triglyceride,apoprotein A,high-density lipoprotein and low-density lipoprotein,were also tested for the mothers.Results (1) No significant difference was found in the frequencies of Pro/Pro genotype between the GDM mothers and control mothers (94.6% vs 90.8%,P > 0.05),nor between the GDM offspring and control offspring (95.0% vs 94.0%,P >0.05) or between the GDM mothers and GDM offspring (P > 0.05).The same was shown in the frequencies of Pro/Ala genotype both between the GDM mothers and control mothers (5.5% vs 9.2%,P >0.05) and between the GDM offspring and control offspring (2.5% vs 3.0%,P > 0.05).(2) Within both GDM and control group,the maternal FBS and various lipids concentrations of Pro/ Pro genotype gravidas showed no significant difference compared to those of Pro/Ala genotype mothers (P > 0.05).(3) Based on the four possible PPARG2 genotype pairs between the mothers and fetuses,Pro/Pro mother and her Pro/Pro fetus,Pro/Ala mother and her Pro/Ala fetus,Pro/Ala mother and her Pro/Pro fetus,and Pro/Pro mother and her Pro/Ala fetus,less Pro/Pro pairs and more Pro/Ala pairs were found in the GDM group than in the control (72.5% vs 92.0%,P=0.014; 27.5% vs 6.0%,P< 0.05).Conclusions Neither the maternal nor the offspring's Pro/Ala genotypes is associated with the genesis of GDM.However,the discrepancy of PPARG2 Prol2Ala polymorphism between mother and her fetus implies a possible cause of GDM.

Background The pollution of agricultural products and the health risks caused by metals have become a hot spot of social concern. As China's main economic agricultural products, peppers are essential for health risk assessment. Objective By exploring the enrichment of common metals in different varieties of peppers in major growing areas of China, a bioavailability-based approach is used to assess dietary health risks of common metals in groups with different characteristics. Methods Through random sampling method, dried pepper samples from major pepper growing areas of China were purchased from the market, and were divided into Hippophae, Capsicum annuum, Magnoliopsida, Capsicum frutescens var, and Capsicum by morphological taxonomy, and a total of 667 batches of peppers were collected. Six common metals arsenic (As), cadmium (Cd), lead (Pb), nickel (Ni), copper (Cu), and zinc (Zn) were evaluated; physiologically based extraction test was designed to estimate the bioavailability of the metals in peppers and their associated dietary health risks were assessed. Results The concentrations of metals Cd and Ni in pepper exceeded the limits of China, and the disqualification rates were 6.1% and 22.7% respectively. The other metals were within the safe range; there were differences in the concentrations of As, Cd, Pb, Cu, and Zn among different pepper varieties (P<0.05). The order of bioavailability of the six metals in pepper from high to low was As (57.9%)>Cd (43.07%)>Zn (42.74%)>Pb (38.04%)>Ni (31.97%)>Cu (31.4%). Based on bioavailability, when the metal concentration in pepper was at the median level, the order of hazard quotient of metals in pepper was Cu>Cd>As>Ni>Zn>Pb, and at the 90th quantile level, the order was Cd>As>Cu>Ni>Zn>Pb; the hazard quotient of single metal element and the total target hazard quotient of combined metal elements were both less than 1, and these indicators of adults were higher than those of children. Conclusion In the collected pepper samples, the non-carcinogenic health risks of single metal elements and multiple metal elements are in the safe range. Based on gastrointestinal bioavailability, the dietary health risk of pepper is further reduced.

OBJECTIVETo explore the value of positron emission tomography (PET) in the Alzheimer's disease (AD) rat model verification and in monitoring the therapeutic effectiveness of cell transplantation.

METHODSA beta(1-40) hippocampus injected rat model was successfully established and neural stem cells were injected into hippocampus. Results of behavior tests and histological examinations were compared between model group and graft group, and then the N-methyl-[(11)C]2-(4 methylaminophenyl)-6-hydroxybenzothiazole ((11)C-PIB) and (18)F-fluorodeoxyglucose ((18)F-FDG) imaging were performed to observe whether the result of imaging was matched with behavior test and histological examination.

RESULTSThe Morris water maze showed that the latent period of the escape was significantly longer in model group than in control group (P<0.01). In histological examinations, the neuron loss and A beta deposition were found in hippocampus CA1 and dentate gyrus of rat model. (11)C-PIB imaging showed increased uptake in model rat hippocampus district (P<0.05), while (18)F-FDG imaging showed that the uptake in the injected side of hippocampus in model group was significantly lower than that in the same side in control group (P<0.001). After cell transplantation, the latent period of the escape was significantly shorter in graft group than in model group (P<0.01). Histological examinations showed that there was no obvious changes in A beta deposition; in addition, the neural stem cells differentiated and expressed neuronal nuclei-positive cells, and continuously expressed 5-bromodeoxyuridine-positive cells for six weeks. (11)C-PIB imaging and (18)F-FDG imaging showed the uptakes were not significantly different between between model group and transplantation group(P>0.05).

CONCLUSION(11)C-PIB imaging is useful in diagnosing AD and monitoring the pathological change of AD model in vivo, while (18)F-FDG imaging provides useful visual information for monitoring short-term therapeutic effectiveness of stem cell transplantation.

Alzheimer Disease ; diagnostic imaging ; pathology ; surgery ; Animals ; Disease Models, Animal ; Fluorodeoxyglucose F18 ; Male ; Positron-Emission Tomography ; methods ; Rats ; Rats, Sprague-Dawley ; Stem Cell Transplantation ; Thiazoles

OBJECTIVETo identify the expression characteristics of the 1700001022RIK (RIKEN cDNA 1700001022) gene in mice and explore its function by bioinformatic analysis.

METHODSUsing the expression profile of gene microarray, we detected the expression of a new testis-specific gene, 1700001022RIK, in mice. We analyzed its expression characteristics in the testis tissue and their changes in different developmental stages of the testis by RT-PCR, real-time RT-PCR, Western blot, and immunohistochemistry. We performed bioinformatic analysis using a bioinformatic software.

RESULTSThe 1700001022RIK gene was specifically expressed in the mouse testis in an age-dependent manner, most highly in the adult mice. The 1700001022RIK protein was mainly expressed in the spermatogonia, spermatocytes, and round spermatids of the adult mice. Bioinformatic analysis showed that the 1700001022RIK protein amino acid sequence had a high similarity in human and mice, which indicated that this gene was highly conserved in mammals.

CONCLUSION1700001022RIK is a testis-specific gene mainly expressed in the spermatogonia, spermatocytes, and round spermatids of seminiferous tubules, which might be involved in the regulation of spermatogenesis.

Age Factors ; Animals ; Blotting, Western ; Computational Biology ; DNA, Complementary ; Gene Expression ; Genomics ; Male ; Mice ; Molecular Chaperones ; genetics ; Seminiferous Tubules ; Spermatids ; Spermatocytes ; Spermatogenesis ; genetics ; Spermatogonia ; Testis

Background Endophthalmitis is a serious,sight-threatening condition.Identifying the causative microorganisms is very important for available treatment of endophthalmitis.Objective This survey was to analyze the spectrum of organisms causing culture-proven endophthalmitis and their sensitivities to commonly antimicrobial agents.Methods Medical data of patients with culture-proven endophthalmitis at Zhongshan Ophthalmic Center from January 2003 through December 2010 were respectively reviewed.The outcomes included intravitreal isolates and antibiotic sensitivities were analyzed.Results Four hundred and sixty-nine strains of organisms were isolated from 447 eyes of 447 patients with infective endophthalmitis,including 22 eyes of polymicrobial infection.In the organisms,gram-positive organisms were 241 (51.4％),fungi were 125 (26.7％) and gram-negative organisms were 103 (22.0％).The most common organisms were Staphylococcus epidermidis in 29.4％,Aspergillus in 7.7％ and Pseudomonas aeruginosa in 5.3％.In this group of infective patients,the most common clinic settings were posttraumatic endophthalmitis (72.7％),and then were postoperative endophthalmitis (10.5％),endogenous endophthalmitis (9.8％) and keratitis (6.9％).Most gram-positive organism and gram-negative organism were sensitive to levofloxacin and cefoperazone.The susceptibility rate of gram-positive organism to chloromycetin was increased in 2007-2010 years compared with 2003-2006 years (x2=5.398,P＜0.05).The susceptibility rate to ciprofloxacin of gram-negative organisms declined (x2 =5.398,P ＜ 0.05),but that to rifampicin increased in the duration of 2007-2010 compared with 2003-2006 (x2 =4.500,P ＜ 0.05).Conclusions Gram-positive organisms are the most commonly causative organisms of endophthalmitis.Most bacterial organisms are sensitive to levofloxacin and cefoperazone.Local data of culturing and susceptibility test offers a guideline for the treatment of infectious endophthalmitis.

Objective To investigate the effects of circDNMT1 on the proliferation,apoptosis and epithelial-mesenchymal transition(EMT)of triple-negative breast cancer(TNBC)cells by regulating miR-377-3p/PUM1 axis.Methods The TNBC tissues and adjacent normal tissues of 24 patients with TNBC treated in Danzhou People's Hospital from 2018 to 2021 were collected.qRT-PCR and Western blot were used to detect the expression of circDNMT1,miR-377-3p,and PUM1 protein in tissue and mouse normal breast epithelial cell line HC11 and TNBC cell lines 4T1,Eph41424,and JC.4T1 cells were divided into the 4T1 group(untransfected),the si-NC group(transfected with si-NC),the si-DNMT1 group(transfected with si-DNMT1),the si-DNMT1+anti-NC group(simultaneously transfected with si-DNMT1 and anti-NC),and the si-DNMT1+anti-miR-377-3p group(simultaneously transfected with si-DNMT1 and anti-miR-377-3p).qRT-PCR was used to detect the expression of circDNMT1 and miR-377-3p of 4T1 cells in each group;CCK-8 was used to detect the proliferation of 4T1 cells in each group;flow cytometry was used to detect the apoptosis of 4T1 cells in each group;Western blot was used to detect the expression of PUM1,EMT-related proteins and apoptosis-related proteins of 4T1 cells in each group;TargetScan website was used to predict the binding sites of miR-377-3p with circDNMT1 and PUM1;dual luciferase report was used to verify the targeting relationships of miR-377-3p with circDNMT1 and PUM1.After inoculation with 4T1 cells,BALB/c mice were randomly divided into the blank control group(injected with equal amount of normal saline),the negative control group(injected with si-NC via tail vein),the DNMT1 silencing group(injected with si-DNMT1 via tail vein),the combined control group(injected with si-DNMT1 and anti-NC via tail vein),and the combined silencing group(injected with si-DNMT1 and anti-miR-377-3p via tail vein),the tumor massess of mice were recorded and the morphological changes of tumors were observed by hematoxylin-eosin staining.Results circDNMT1 and PUM1 were up-regulated in TNBC tissues and cells,and miR-377-3p was down-regulated.The expression difference was most obvious in 4T1 cells,so 4T1 cells were selected for subsequent experiments.Compared with the 4T1 group and the si-NC group,the expression of miR-377-3p,the apoptosis rate of 4T1 cells,the expression levels of Bax,cleaved caspase-3 and E-cadherin protein of 4T1 cells in the si-DNMT1 group were significantly increased(P<0.05),the circ-DNMT1 level,the expression level of PUM1 protein,OD values at 24 hours and 48 hours,the expression level of Bcl-2,N-cadherin,Vimentin protein were significantly decreased(P<0.05).Compared with the si-DNMT1 group and the si-DNMT1+anti-NC group,the expression of miR-377-3p,the apoptosis rate of 4T1 cells,the expression levels of Bax,cleaved caspase-3 and E-cadherin proteins of 4T1 cells in the si-DNMT1+anti-miR-377-3p group were significantly decreased(P<0.05),the expression level of PUM1 protein,OD values at 24 hours and 48 hours,the expression levels of Bcl-2,N-cadherin,Vimentin proteins were significantly increased(P<0.05).Compared with the miR-NC+WT-circDNMT1 group,the cell luciferase activity in the miR-377-3p mimic+WT-circDNMT1 group was significantly decreased(P<0.05);compared with the miR-NC+WT-PUM1 group,the cell luciferase activity in the miR-377-3p mimic+WT-PUM1 group was significantly decreased(P<0.05).The tumor cells in the blank control group and the negative control group were densely arranged with clear boundary;the tumor cells in the DNMT1 silencing group and the combined control group were loosely arranged,the nuclei were pyknotic,and the cell fragments were increased;the tumor cells in the combined silencing group were densely arranged and the boundaries tended to be clear.Compared with the blank control group and the negative control group,the tumor mass of mice in the DNMT1 silencing group was significantly decreased(P<0.05).Compared with the DNMT1 silencing group and the combined control group,the tumor mass of mice in the combined silencing group was significantly increased(P<0.05).Conclusion Silencing circDNMT1 may inhibit the expression of PUM1 by up-regulating miR-377-3p,thereby inhibiting the proliferation and EMT of TNBC cells,and promoting cell apoptosis.

OBJECTIVESTo investigate the role of glycyrrhizin on TGFbeta1 stimulated signaling transduction in rat hepatic stellate cells (HSCs).

METHODSThe mice HSCs were isolated and cultured with or without glycyrrhizin (1 micromol/L-1000 micromol/L) in vitro after TGFbeta1 stimulation. The mRNA level of Smad2, 3, 7 were measured with RT-PCR; protein expression level of Smad2, 3, 7 and collagen I, III were analyzed with Western blot.

RESULTSTGFbeta1 increased the mRNA level and protein expression of Smad2, 3, 7 in HSC; it also increased protein expression of collagen I and III. 1 micromol/L-1000 micromol/L glycyrrhizin decreased the mRNA level and protein expression of Smad2, 3, 7; it also inhibited protein expression of collagen I and III gradually.

CONCLUSIONInterventing the TGFbeta signaling pathway and decreasing the synthesis of collagen, might be involved in the anti-fibrosis mechanism of glycyrrhizin.

Animals ; Glycyrrhizic Acid ; pharmacology ; Hepatocytes ; metabolism ; Male ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Transforming Growth Factor beta ; pharmacology

Cell Line, Tumor ; DNA Repair ; DNA Repair Enzymes ; Hep G2 Cells ; Humans