Objective To evaluate the effect of metformin on the proliferation of keratinocytes,and to investigate its possible mechanism.Methods HaCaT human keratinocytes were divided into several groups to remain untreated (control group) or be treated with different concentrations (25,50,75,100 mmol/L) of mefformin for 24 hours (intervention groups).Subsequently,CCK8 assay was conducted to evaluate the proliferation of HaCaT cells,real-time quantitative PCR to measure the mRNA expressions of miR-21-5p and its downstream target gene PDCD4,and Western blot to detect the expression of PDCD4 protein in HaCaT cells.Statistical analysis was done by using one-way analysis of variance for multiple group comparisons and SNK-q test for paired comparisons.Results After 24-hour treatment,the proliferation of HaCaT cells was inhibited by (5.43 ± 3.67)％,(19.61 ± 6.95)％,(45.93 ± 9.56)％ and (61.91 ± 6.93)％ by metformin of 25,50,75 and 100 mmol/L,respectively,with significant differences observed in cell proliferation inhibition rates among these intervention groups (F =246.90,P ＜ 0.05).Cellular proliferative activity was similar between the control cells (0.00 ± 3.00％) and those treated with 25 mmol/L metformin,but significantly higher in the control cells than in the other 3 metformin-treated groups (all P ＜ 0.05),and significantly different between the 4 metformin-treated groups (all P ＜ 0.05).The relative mRNA expression level (2-△△Q) of miR-21-5p was 0.90 ± 0.11,0.33 ± 0.05,0.21 ± 0.07 and 0.14 ± 0.04 (F =36.99,P ＜ 0.01),while that of PDCD4 was 2.11 ± 0.64,7.22 ± 1.13,11.16 ± 1.23 and 19.12 ± 3.16 (F =96.26,P ＜ 0.05),and the expression level of PDCD4 protein was 1.22 ± 0.08,2.09 ± 0.20,2.26 ± 0.1 1 and 2.37 ± 0.07 (F=75.37,P＜ 0.05),respectively,in HaCaT cells treated with metformin of 25,50,75 and 100 mmol/L.Similarly,no significant difference was observed between the control cells and those treated with 25 mmol/L metformin in the expression level of miR-21-5p mRNA,PDCD4 mRNA or protein,but decreased expression of miR-21-5p mRNA and increased expression of PDCD4 mRNA and protein were noted in cells treated with the other 3 concentrations of metformin compared with the control cells (all P＜ 0.05),and significant differences were also found in the expression levels of miR-21-5p mRNA as well as PDCD4 mRNA and protein among the 4 intervention groups (all P ＜ 0.05).Conclusion Metformin can markedly inhibit the proliferation of HaCaT cells in vitro,likely by downregulating miR-21-5p expression and upregulating PDCD4 expression.

Objective To explore the effects of inhibiting DDR2 expression by siRNA on hepatic stellate cells and evaluate the role of DDR2 gene in hepatic fibrogenesis. Methods (1) Three pairs of chemically synthesized siRNAs targeting DDR2 were respectively transfected into HSC-T6 cells for evaluation of silence efficacy, and the most effective siRNA was used. (2) HSC-T6 cells were divided into three groups, group A served as normal controls, group B served as negative control and group C was RNA interference DDR2 (siRNA-DDR2) expression of HSC. The most effective RNA interference sequences targeting DDR2 gene was chosen to transfect HSC-T6 cells by plasmid transfection. The tendency of DDR2, α-smooth muscle actin(α-SMA) and collagen-Ⅰ mRNA expression were estimated using RT-PCR, and the protein expression of DDR2 was evaluated by Western blot. Meanwhile, MTT assay was employed to analyze the proliferation of HSC. Results (1) DDR2 siRNA, which began at nt 868, inhibited DDR2 gone expression stronger than the other two siRNAs. (2) After transfection of siRNA-DDR2, the mRNA expression of DDR2 (P<0.01) and α-SMA (P<0.01) significantly decreased compared with the normal group, and the protein expression of DDR2 also significantly decreased (P<0.01). In addition, the proliferation of HSC was also markedly suppressed as compared with the normal group (P<0.01). However, compared with the negative control group, none of them was markedly suppressed. Conclusion SiRNA targeting DDR2 significantly suppresses the activation, proliferation of HSC, and thus attenuates hepatic fibrogonesis in vitro.

Objective To observe the clinical features, phenotypes and genotypes in a Chinese family with choroideremia (CHM). Methods A Chinese four-generation family (15 members) with CHM, including 5 patients (4 males/1 female), 2 female carriers and 8 healthy members, was enrolled in this study. Initially all family members underwent best corrected visual acuity (BCVA), indirect ophthalmoscopy, fundus fluorescein angiography, optical coherence tomography (OCT), visual field and full view electroretinogram (ERG). BCVA was followed up for 3 years. Venous blood samples were collected, and all of the 15 coding exons and flanking intron regions were amplified in the proband by polymerase chain reaction followed by direct sequencing. Protein structure was modeled based on the protein data bank and mutations in DeepView v4.0.1 to predict the effect of the mutations. A total of 180 healthy volunteers were enrolled as control group to matching CHM gene sequences. Results The visual acuity (VA) of 3/4 adult male patients began to decrease at less than 10, 10 and 30 years old, the average BCVA was 0.43. There were characteristic signs and symptoms of CHM including narrow visual field, extinguished rod and cone response in ERG, disappeared junction line and intermediate line of photoreceptor inner segment/outer segment on OCT. After 3 years, the mean BCVA decreased to 0.11. The BCVA of one young male patient was 1.0 in both eyes with minor changes fundus and visual field. The VA of the female patient began to decrease at 50 years old, her BCVA of two eyes were 0.5 and 0.25, respectively. The fundus changes were typical of CHM, with relative scotomas in the peripheral visual field of OD, and big scotomas in the OS. After 3 years, her mean BCVA decreased to 0.2. Of 2 female carriers, one had minor fundus changes (patches of pigmentary deposits, atrophy spots of retinal pigment epithelium cells), and the other was normal. A novel heterozygous c.1837G>A mutation in exon 15 of CHM was detected in the proband, which resulted in the substitution of serine by proline at codon 613 (p.D613N). Based on molecular modeling, the misfolded protein caused by the mutation might destabilize the structure of the helix that potentially could affect the global stability of the Rep-1 protein. Conclusions A novel c.1837G>A (p.D613N) mutation may be the causative mutation for CHM in this family. Female CHM carriers may have some signs and symptoms.

Objective To study normal flora components of conjunctiva, explore the relationship between normal flora and ocu- lar bacterial infections.Methods The data of 1 198 bacterial cultures of conjunctiva in non-infectious cataract patients were re- viewed and compared with the results of 985 specimens of conjunctivitis, keratitis and endophthalmitis from January 2000 to December 2004.Results Positive culture of conjunctiva in non-infectious cataract patients was 628 in 1 198 specimens (52.4%).These bacteria including gram-positive cocci and gram-neganve bacilli constituted the normal flora of conjunctiva. Staphylococcus epidermidis was the predominant bacteria (51.1%, 341/667) and its prevalence increased during this 5-year period.S.epidermidis and S.aureus were also the most frequently isolated pathogens in specimens of conjunctivitis, keratitis and endophthalmitis.The correlation was statistically significant between the positive rate in normal eye and conjunctivitis or keratitis in terms of these two species, but not statistically significant between normal eye and endophthalmitis.The resistance rate of normal flora of conjunctiva to ampicillin, amikacin, tobramycin, ciprofloxacin, and ofloxacin increased year by year. The resistance rate to gentamicin and the combination of two drugs decreased.The changing resistant pattern was similar in pa- tients with eye infection.Conclusions There is a close relationship between the normal flora S.aureus and S.epidermidis of conjunctiva and the bacterial infection of ocular surface, but no significant correlation between normal flora and the pathogen of endophthalmitis.

Objective To reveal the distribution of the TCM syndrome elements in patients with diabetic nephropathy ( DN) , by searching the literature of TCM diagnosis and treatment on DN. Methods Relevant lit-eratures on treating DN by syndrome differentiation of TCM were retrieved in CNKI, Wangfang Data and VIP from the beginning to August 9 th , 2014. Syndrome elements of disease nature or of disease location were analyzed from 159 eligible literature divided into two parts:syndrome differentiation only ( n=111 ) and syndrome differentiation combined with clinical stage (n=48). Results From the litrature of syn-drome differentiation only, kidney and spleen were the most common syndrome elements of disease loca-tion, while yin deficiency was the most common syndrome elements of disease nature. By the contrast, from the literature of syndrome differentiation combined with clinical stage, the main syndrome elements of location were kidney, spleen and liver and the most common syndrome element of nature was yin defi-ciency at early stage;at middle stage, the main elements of location were kidney and spleen and of nature was deficiency syndrome including yin deficiency, yang deficiency and qi deficiency; at end stage, the main elements of location were still kidney and spleen and of nature was yin-yang deficiency. Conclusion Study on syndrome elements of DN from literature is favorable for standardization of clinical syndrome differentiation and providing reference for therapy.

Objective To discuss the perioperative management of cirrhotic patients undergoing abdominal operation. Methods From Jan. 2000 to Jul. 2007, 93 patients with PHT underwent operation in our hospital. We retrospectively analyzed the data according to different therapy result. Results 21 cases developed postoperative ascites. The occurrence of ascites in patients dealt with PHT and coexistent other abdominal disease was 7% and 12% respectively for Child A and B grade patients. The occurrence of ascites in patients dealt with only abdominal disease was 13% and 17% respectively for Child A and B grade patients. The occurrence of ascites in patients dealt with PHT and abdominal disease was amazingly 83% for Child C cases. Patients suffered from tumors didn't have more risk of ascites than the other patients. Conclusion The proper perioperative management of patients can decrease the mortality. To deal with PHT together with abdominal operation is reasonable for patients in Child A or B liver function, and it would not increase the postoperative ascites. For the selective patients, we must try our best to decrease the ascites preoperatively to improve the condition.

Objective To assess the clinical value of high-frequency electrocautery in the treatment of massive hemorrhage of presacral venous plexus. Methods The clinical data of 8 patients with presacral venous hemorrhage treated with high-frequency electrocautery from February 2005 to March 2008 were analyzed. Once the presacral venous hemorrhage happened, the bleeding site was pressed with a gauze or finger and the accumulated blood was aspirated. Then, while simultaneously withdrawing the gauze or finger over the bleeding sites, high-frequency electrocautery was applied with a power of 80-100 W to coagulate all the bleeding sites. Results High-frequency electrocautery was used to stop bleeding in the 8 patients, and no electrocautery related complica-tions occurred postoperatively. Conclusions High-frequency electrocautery for massive hemorrhage of presacral venous plexus is simple, efficient and safe.

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Objective To explore the effects of silencing DDR2 expression by siRNA on CCl4-induced liver fibrosis and its mechanism in rats. Methods Liver fibrosis model was induced by intraperitoneal injection of CCl4 twice a week for 6 consecutive weeks. Some rats were administered siRNA targeting DDR2 (0. 3 mg/kg), saline or control siRNA every three days from the beginning of CCl4 injection via tail vein injection, while other rats were treated in the same pattern after 2-week CCl4 injection. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot were used to detect the mRNA and protein expressions of DDR2, MMP-2 and COL Ⅰ . Meanwhile, the pathological changes of liver tissues and the levels of liver function were also observed. Results QRT-PCR showed that the DDR2, MMP-2 and COL Ⅰ mRNA in the chemically synthetic cholesterol-modified siRNADDR2 group were significantly decreased as compared with those in the control group (P＜0.01) ,and the protein expressions of DDR2, MMP-2 and COL Ⅰ were also significantly decreased (P＜0. 01,4 wand 6w). In addition, in comparison with those in the control group, the pathological changes of liver tissues in the siRNA-DDR2 treated group were markedly attenuated, and the levels of ALT(1356.17 ±83.80 nkat/L vs 2532. 70±145.11 nkat/L,4w,1367. 60±321.76 nkat/L vs 2604.37±255.02 nkat/L,6w,P＜0. 01 ) and AST (2460. 80 ± 207. 58 nkat/L vs 3983. 70 ± 253. 08 nkat/L, 4w, P＜ 0. 01,2383.27±290.16 nkat/L vs 3227.70±353. 34 nkat/L,6w,P＜0. 05)were also significantly lowered,while the level of TBIL (7. 97 ± 1.60 μmol/L vs 3.80± 0.60 μmol/L, 4w, 10.40±1.61 μmol/L vs 6.10±0.79 μmol/L,6w,P＜0. 01)was markedly increased. Conclusion Systemic administration of cholesterol-modified siRNA targeting DDR2 could significantly suppress the expression of DDR2, decrease the contents of the extracellular matrix,and thus has a potential antifibrotic effect.

Background and purpose:Three-dimensional power Doppler angiography (3D-PDA) is a new technique to investigate the vessels in the organs, but the research in thyroid is limited. The purpose of this research was to investigate three-dimensional power Doppler angiography (3D-PDA) in differentiating malignant from benign thyroid nod-ules.Methods:This study prospectively evaluated 103 lesions in 94 patients who were scheduled for surgery. The patients underwent preoperative 3D-PDA scanning. Analysis of the 3D-PDA characteristics includes blood flow pattern, the num-ber of blood vessels, the shape of vessels, the spatial distribution of the vessels, the existence of rich local blood flow within nodules or in the parenchyma surrounding the nodules. This study also analyzed the difference between the benign lesions and the malignant lesions.Results:There were 50 benign lesions and 53 malignant lesions. The sensitivity and specificity of irregular vessels, the asymmetry spatial distribution, rich local blood flow within nodules or in the parenchyma surround-ing the nodules were 64.2%, 96.0%; 56.0%, 88.0%; 54.7%, 96.0%; 60.4% and 94.0%, respectively. The sensitivity, speci-ficity, positive predictive value, negative predictive value and accuracy of 3D-PDA were 83.0%, 94.0%, 93.6%, 83.9% and 90.3%, respectively.Conclusion:3D-PDA provides a useful tool to investigate vascularization of thyroid leisions.This technique is feasible for clinical application and plays an important role in diagnosis of thyroid nodules.