Abstract:Objective：To investigate the effects of ETS1/2 on pancreas development of Xenopus laevis. Methods：The specific morpholino oligonueleitides(MOs) were designed to knockdown the ETS1/2 by microinjection. The expression of marker genes was tested during pancreas formation by whole mount in situ hybridization and quantitative RT-PCR. ETS1/2 downstream genes were screened by bioinformatics analysis. Results：Knockdown of ETS1 or ETS2 had no effect on embryo development of Xenopus laevis. Knockdown of both ETS1/2 promoted the expression of pancreas marker genes while inhibited the expression of intestine marker genes，and had no effects on the expression of liver and stomach marker genes. The binding sites of ETS1/2 were found in the promoter regions of pancreatic marker genes ngn3，igf1 and foxa1. Conclusion：Knockdown of ETS1/2 promoted the pancreas development of Xenopus laevis while inhibited the intestine development，and had no effects on the development of the liver and stomach.

Abstract:Objectives: To explore whether serine can protect myocardial fibrosis as a ligand of glycine receptor (GlyR). Methods: Serine or saline were injected intraperitoneally to mice a week before angiotensin Ⅱ(Ang Ⅱ) administrated by using an osmotic pump, and the myocardial fibrotic status in mice was detected 28 days later. The expression levels of inflammatory markers in cardiomyocytes response to angiotensin Ⅱ(Ang Ⅱ) were detected with or without serine in vitro. Primary cardiac fibroblast cells were isolated from neonatal rat and treated with Ang Ⅱ or serine，respectively，and then the expression levels of collagen Ⅰ and collagen Ⅲ were checked by quantitative PCR. The expression levels of collagen Ⅰ collagen Ⅲ were checked in both cardiac fibroblasts and cardiomyocytes co-cultured system. mRNA levels of collagen I and Ⅲ were checked after GlyR knocked down by siRNA transfection in cardiomyocyte. Results: In the presence of cardiomyocyte, production of collagen Ⅰ and collagen Ⅲ was enhanced in cardiac fibroblasts after Ang Ⅱ treatment, however，the enhancement is inhibited by serine. Importantly, the protective of serine on cardiac fibrosis was abolished by GlyR knockdown. Conclusion: Serine could blunt myocardial fibrosis through GlyR in cardiomyocyte.

Abstract:Objective：To investigate the therapeutics effect and the potential mechanism of paeoniflorin on the bleomycin-induced pulmonary fibrosis in mice. Methods：Sixty adult male SPF C57BL/6 mice were randomly divided into four groups：the control group (Sham operation，SH)，the control+paeoniflorin group (SH+PA)，the bleomycin group (BLM) and the bleomycin+paeoniflorin group (BLM+PA). From the day of operation，the mice of the SH+PA and BLM+PA groups were treated with paeoniflorin intragastrically (50 mg/kg，daily) while the mice of the SH and BLM groups were given the same volume of normal saline. The model of pulmonary fibrosis was established through intratracheally instillation with bleomycin (2 mg/kg) except for the control group，in which normal saline was used. All the samples were sacrificed on the day 14 after operation，and the change of body weight and mortality rates of each group were analyzed. The pathological section of lung tissues were harvested for hemotoxylin and eosin stain. Collagen deposition in the lung was determined by measuring the total hydroxyproline content. ELISA was performed to analyze the level of IL-18 and IL-1β in the lung homogenates. The levels of NLRP3 and TGF-β1 in the lung were detected by Western blotting. Results：Paeoniflorin significantly increased the survival rate and attenuated the collagen deposition in the bleomycin-induced pulmonary fibrosis mouse model compared with the control group (P＜0.05). The IL-18 and IL-1β in the lung homogenates were decreased notably after administration of paeoniflorin，when compared to the control group (P＜0.05). The expression of NLRP3 and TGF-β1 were down regulated significantly (P＜0.05). Conclusion：Paeoniflorin may attenuate bleomycin-induced pulmonary fibrosis in mice by suppressing NLRP3 inflammsome formation.

Abstract:Objective：To investigate the effect of ATP binding cassette transporter A6 (ABCA6) deletion on the metabolic status of mice fed on a ketogenic diet. Methods：ABCA6 knockout (KO) or wild type (WT) mice were placed on normal diet or ketogenic diet for 2 weeks，respectively. Expression of ABCA6 in the liver of mice fed on normal diet or ketogenic diet was determined by Western blot. Body weights were monitored. The blood samples and livers were harvested. Blood glucose and serum triglycerides (TG)，total cholesterol (TCH)，free fatty acids (FFA)，ALT，AST，and beta hydroxybutyric acid (β-HB) were tested. The contents of TG and TCH in the liver were examined. The hepatic expression levels of genes related to metabolism were detected by realtime-PCR. Results：Feeding of ketogenic diet induced expression of ABCA6 in the liver. No significant difference was observed in body weight，blood glucose and the levels of TG，TCH，FFA，ALT，AST，and β-HB in serum of ABCA6 KO or WT mice fed on normal diet. A 2-week ketogenic diet feeding significantly reduced the levels of blood glucose，serum TCH，and serum FFA in ABCA6 KO mice when compared with WT mice. Meanwhile，more TG and higher mRNA levels of FAS，DGAT2，PCK1，and HMGCS2 were detected in the liver of ABCA6 KO mice fed on ketogenic diet. Conclusion：Deletion of ABCA6 impaired the metabolism of mice fed on ketogenic diet. ABCA6 may be involved in the lipid metabolism of the liver.

Abstract:Objective：To investigate the impact of maternal high-protein diet on metabolism related hormones of rats offspring during pregnancy and lactation. Methods：Female Wistar rats (F0 generation) were mated and randomly assigned to three groups：ProⅠ，ProⅡ，ProⅢ，and were fed ad libitum with isocaloric purified diets：DietⅠ(protein，14%；carbohydrate，69.3%，E/E)，DietⅡ(protein，24%；carbohydrate，59.3%，E/E) and DietⅢ(protein，34%；carbohydrate，49.3%，E/E) respectively. On the 3rd day after birth (P3)，only 3 male rats (F1 generation) of each dam were kept. After 21 days of suckling period，rats of F1 generation from different groups were weaned onto the same normal diets till P77，when the research was over. Blood samples of offspring at P21/P49/P77 were kept and levels of hormone related to energy metabolism were detected. Results：Serum levels of IGF-1 of F1 generation were lower in ProⅠ than in ProⅡ(P＜0.01) at P21；IGF-1 levels were higher in both ProⅡ and ProⅢ than in ProⅠ(P＜0.01) at P49；IGF-1 levels of ProⅢ were higher than ProⅠ and ProⅡ(P＜0.01) at P77. Serum levels of Leptin of F1 generation were the highest in ProⅡ than in ProⅠ and ProⅢ(P＜0.01) at P21；Leptin levels of ProⅠ were lower than both ProⅡ and ProⅢ at P49 and P77(P＜0.05). Plasma levels of NPY of ProⅢ were lower than ProⅠ and ProⅡ(P＜0.01) at P49，with no differences between groups at P77(P＞0.05). Plasma levels of Ghrelin were higher in ProⅢ than in ProⅠ and ProⅡ (P＜0.01) at P77，with no differences between groups at P49(P＞0.05). Conclusion：Peripheral blood levels of leptin，ghrelin and IGF-1 were raised as a result of protein-rich diet during pregnancy and lactation in rats，with little influence on NPY levels.

Abstract:Objective：To investigate the effect of osthole on lipopolysaccharide (LPS)-induced inflammatory response in Caco2 cells and the underlying mechanism. Methods：Caco2 cells were treated with various concentrations of osthole prior to LPS treatment. The mRNA levels of interleukin (IL)-1β，IL-6 and TNF-α were detected by real-time PCR. Cells were treated with PKA inhibitors H89 or KT5720 prior to LPS at indicated doses to observe the effect of cAMP/PKA signaling pathway on osthole. The phosphorylations of p38，Erk and JNK were detected by Western blot. Results：The expressions of inflammatory factors IL-1β，IL-6 and TNF-α in Caco2 cells were significantly increased by LPS stimulation. Pretreatment of osthole significantly suppressed the up-regulation of inflammatory cytokines induced by LPS. PKA inhibitors failed to reverse the inhibitory effect of osthole. LPS induced significant phosphorylation of p38，Erk，and JNK in Caco2 cells，which were suppressed partly by osthole. Conclusion：Osthole attenuates LPS-induced nflammatory response in Caco2 cells. The inhibitory effect of osthole is independent on cAMP/PKA pathway. Osthole-induced reduction of phosphorylation of p38，Erk，and JNK may mediate its anti-inflammation action in Caco2 cells.

Abstract:Objective：To clone mouse Marf1 gene and let it ectopically express in HEK293T cells，for studying its expression and mechanism. Methods：Marf1 coding sequence was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and was cloned into the eukaryotic cell-expression vector pCMV6-AC-3DDK and pCMV6-AN-mKate using the advanced In-Fusion cloning system. After verification by restriction digestion followed by electrophoresis and sequencing，the correctly cloned plasmid DNA was transfected into HEK293T cells. The expression of MARF1-3DDK and MARF1-mKate fusion protein in the transfected HEK293T cells was assessed by both Western blot and immunofluorescence using the antibodies against DDK and mKate tag proteins. Results：The correct cloning of Marf1 was confirmed by restriction digestion and sequencing，and the expression of MARF1-3DDK and MARF1-mKate fusion protein was detected by Western blot. MARF1 fusion protein was detected to be localized either in the cytoplasm in a punctate manner or on the mitotic spindles. Conclusion：Mouse Marf1 gene was successfully cloned and expressed in HEK293T cells，which lays solid foundation for further studies on its functions and the related mechanisms.

Abstract:Objective：To analyze the differential expression profile of microRNAs in murine melanoma B16F0 and B16F10 cells，and to isolate the microRNAs related to the proliferation and metastasis of malignant melanoma. Methods：We verified the difference of B16F10 and B16F0 cell in vivo and in vitro. And then RiboArrayTM miDETECTTM Mouse Array was adopted to compare the microRNAs expressed in murine melanoma B16F0 and B16F10 cells. The differently expressed microRNAs were further confirmed by real time PCR. Results：B16F10 cells had stronger abilities of migration and proliferation in vivo and in vitro. Further more，compared with B16F0 cells，a total number of 38 microRNAs were statically differently expressed in B16F10 cells，with 15 upregulated and 23 downregulated. Consistent with RiboArray results，the expressions of miR-26a-1-3p，miR-28b，miR-29b-3p，and miR-24-2-5p were significantly upregulated and the expressions of miR-763，miR-431-5p，miR-205-5p，and let-7c-2-3p were markedly downregulated revealed by real time PCR. Conclusion：Our findings suggest that the differentially expressed microRNAs were related to the occurrence and development of malignant melanoma，and might serve as novel targets for early diagnosis and treatment of melanoma.

Abstract:Objectives：To reveal the relationship between microRNA-10b and gemcitabine (GEM) resistance of pancreatic cancer and its possible mechanism. Methods: The expression of microRNA-10b in PANC-1 cells (which are relatively resistant to GEM) and CFPAC-1 cells (which are relatively sensitive to GEM) were detected by RT-qPCR. PANC-1 and CFPAC-1 cells were treated with different doses of GEM，and then the cells were harvested. The expression of microRNA-10b was detected by RT-qPCR. CCK-8 assay and flow cytometry were performed to evaluate the drug sensitivity of CFPAC-1 cells，which were transfected with microRNA-10b mimics to GEM. The protein expressions of PI3K，p-Akt，Bcl-2 and Survivin in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were demonstrated using Western blotting assay. The gene expressions of PTEN in CFPAC-1 cells transfected with microRNA-10b mimics or negative control were detected by RT-qPCR. Results: PANC-1 cells showed significantly increased expression of microRNA-10b as compared to CFPAC-1 cells. The expression of microRNA-10b both in PANC-1 and CFPAC-1 cells increased with the rising of GEM concentration. The sensitivity of CFPAC-1 cells to GEM decreased when microRNA-10b was overexpressed and the expressions of PI3K，p-Akt，Bcl-2，and Survivin were much higher than their control groups. The expression of PTEN mRNA decreased as microRNA-10b increased. Conclusions: MicroRNA-10b might negatively regulate the gene expression of PTEN，which increases the protein expressions of PI3K，p-Akt，Bcl-2 and Survivin，thus reduces the sensitivity of CFPAC-1 cells to GEM，and as a result，pancreatic cancer cells become resistant to GEM.

Abstract:Objective：In the present study，we aimed to investigate the small-molecule B lymphoma Mo-MLV insertion region 1 (Bmi1) inhibitor，PTC-209，for its effects on endogenous Bmi1 expression and cell behaviors in tongue squamous cell carcinoma (TSCC) cell line in vitro. Methods：The TSCC cell line Cal27 was treated with PTC-209 in vitro，and the expression change of Bmi1 was detected by Western blotting and real-time RT-PCR assays. The changes of cell proliferation，invasion and migration were analyzed by MTT，transwell and wound-healing assay，respectivety. The cancer stem cell (CSC) subpopulations in Cal27 were further assayed by tumorsphere formation，colony-forming and flow cytometry assays. Results：Bmi1 mRNA and protein levels were significantly decreased in Cal27 cells treated with PTC-209 in both dose- and time-dependent manners (P＜0.05). PTC-209 inhibited cell proliferation，migration and invasion，and had synergic anti-cancer effects when combined with cisplatin and 5-FU. Moreover，colony formation，sphere formation，and ALDH+ subpopulation were significantly reduced upon PTC-209 exposure. Conclusion：PTC-209 treatment induces Bmi1 reduction in TSCC cell line in vitro，and shows potent anti-tumor activity with reduced cell proliferation，migration，invasion and CSC subpopulation.

Abstract:Objective：To study the anti-angiogenic effect of arsenic trioxide(As2O3) on breast cancer，and explore the possible molecular mechanisms. Methods：Human umbilical vein endothelial cells(HUVECs) were cultured in tumor conditioned medium derived from MCF-7 and treated with 0，4，8 μmol/L As2O3. Cell wound healing assay，Matrigel assay，CCK-8 assay，and Annexin V assay were carried out to detect cell migration rate，angiogenesis ability，cell proliferation and cell apoptosis，respectively. Western blotting assay was used for detecting protein level. Results：Compared to the control group，the As2O3 treated groups showed reduced ability of cell migration and tube formation. A decline in cell proliferation and a rise in apoptosis were also found in the As2O3 groups. The protein expression of FoxO3a and its downstream genes Fas-L and p27Kip1 were increased following As2O3 treatment. Conclusion：As2O3 could attenuate the angiogenic ability of HUVECs by inducing cell apoptosis and inhibiting cell proliferation through upregulation of FoxO3a and activating of its downstream pathways，which suggested that As2O3 may play an important role in anti-angiogensis in breast cancer.

Abstract:Objective：To investigate the expressions of LRPPRC and Bcl-2 in prostate cancer，and the effects of LRPPRC on apoptosis. Methods：Immunohistochemistry was performed to analyze the expressions of LRPPRC and Bcl-2 on 198 prostate cancer tissues. The correlation between LRPPRC and Bcl-2 expressions was analyzed. The relationship between LRPPRC and Bcl-2 expressions and prognosis was analyzed. siRNA LRPPRC was used to transfect prostate cancer cells to decrease the level of LRPPRC，and the effects on proliferation，apoptosis，the expressions of Bcl-2，Bax and caspase-3 protein were observed. Results：Prostate cancer and prostate hyperplasia tissues highly expressed LRPPRC in 61.6%(122/198) and 18.0%(18/100) of patients，respectively(P＜0.001). Prostate cancer and prostate hyperplasia tissues highly expressed Bcl-2 in 49.5%(98/198) and 45.0%(45/100) of patients，respectively(P=0.539). A significant association between the expressions of LRPPRC and Bcl-2 was observed(r=0.179；P=0.012). High expression of LRPPRC combined with Bcl-2 was related with shorter biochemical progress(BCP)-free survival and overall survival(P＜0.001). When LRPPRC level being down-regulated by siRNA，the survival rate of prostate cancer cells was decreased，the apoptosis rate was increased，Bcl-2 protein level was decreased，and cleaved caspase-3 protein level was increased. There were no significant effects on Bax expression. Conclusion：LRPPRC expression was increased in prostate cancer tissues. LRPPRC may inhibit endogenous apoptosis of prostate cancer cells. The effect may be related with regulating Bcl-2 expression.

Abstract:Objective：To investigate the expression of EZH2 in laryngocarcinoma tissues and cells，and its effect on proliferation，migration and invasion of laryngocarcinoma HEP-2 and SCC10A cells. Methods：Thirty laryngeal squamous cell carcinoma tissues and 10 normal para-carcinoma tissues were collected from Xuancheng Central Hospital，AnHui Province. The expressions of EZH2 were compared between laryngocarcinoma tissues and the para-carcinoma tissues，as well as laryngocarcinoma HEP-2 and SCC10A cells by qRT-PCR. EZH2 shRNA was transfected into HEP-2 cells by Lipofectamine2000 and the transfection efficiency was examined by qRT-PCR. Cell proliferation was evaluated by CCK8 assay. The cell migration and invasion abilities were detected by migration and invasion assay. The tumorigenesis assay in nude mice was performed to test the role of EZH2 in the tumor growth of HEP-2 cells in vivo. Results：The expressions of EZH2 in laryngocarcinoma tissues and cells were both significantly higher than those in para-carcinoma tissues(P＜0.01). EZH2 shRNA was successfully transfected into HEP-2 cells. The proliferation ability of HEP-2 cells was decreased significantly after transfection of EZH2 shRNA. The migration and invasion of HEP-2 cells were significantly inhibited. The EZH2 shRNA suppressed tumor growth of HEP-2 cells in vivo. Conclusion：EZH2 is overexpressed in laryngocarcinoma tissues and cells. The proliferation，migration and invasion of human laryngocarcinoma HEP-2 cells are inhibited after the transfection of EZH2 shRNA. EZH2 shRNA suppresses tumorigenesis of HEP-2 cells in vivo.

Abstract:Objective: To study the etiology, diagnosis and treatment of biliary complications after orthotopic liver transplantation (OLT) from donation after cardiac death (DCD). Methods：Eighty-seven recipients who had received liver transplantation from DCD in the First Affiliated Hospital of Nanjing Medical University between January 2015 and August 2016 were retrospectively reviewed. Fifty-eight cases received modified piggyback liver transplantation. The bile duct was reconstructed by end-to-end anastomosis of the common bile duct. No cases used the T tube. Results: Biliary complications was diagnosed cholangiographyically in 9 cases. Eight patients were cured，one patient was improved，and no patient died. The incidence for biliary complications was 10.1%(9/87). Conclusion: Long ischemic time of the graft, poor quality of donor’s liver graft, poor techniques for anastomosis and repair of the graft and other factors may contribute to the biliary complications after liver transplantation from DCD. Early cholangiography is helpful to diagnose biliary complications. Endoscopic and/or radiological interventions should be the main treatment for biliary complications.

Abstract:Objective：To investigate the clinical efficacy and safety of sidenafil and bosentan in the therapy of children with pulmonary artery hypertension(PAH) and congenital heart disease(CHD). Methods：Fifty cases of PAH combined with CHD were selected and randomly divided into the A group and B group. A group was given sildenafil therapy，and on this basis the B group was added with bosentan tablets. The two groups were followed up periodically to analyze the clinical outcome，adverse reaction and complication.Results：There were 17 cases males and 13 cases females in the A group，and 8 cases males and 12 cases females in the B.①World Health Organization functional class was improved significantly in both after therapy，and the B group was superior to the A；②The PAH after treatment in both groups was improved，moreover the effect in the B group；③There was no significant changes in the assay，Guardianship residence time and the duration of ventilatory support. No severe side effect and complication were observed. Conclusion：The results revealed the efficacy of bosentan in children with PAH and CHD，and bosentan is safety and validity.

Abstract:Objective：To evaluate the vitamin D status of pregnant women in different seasons from Jiangsu Province. Methods：A total of 18，478 healthy pregnant women were recruited from hospitals located in 8 areas from Jiangsu Province during January 2013 to December 2015，and fast blood samples were collected from all the subjects. Serum 25-hydroxyvitamin D3［25(OH)D3］ and 25-hydroxyvitamin D2［25(OH)D2］ concentrations were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). The information on characteristics of the pregnant women was collected by questionnaires. The serum concentration of 25-hydroxyvitamin D ［25(OH)D］ was the total concentration of 25(OH)D3 and 25(OH)D2. Results：The average serum concentration of 25(OH)D was (19.2±9.4) ng/mL among all the subjects. The rate of vitamin D deficiency ［25(OH)D＜20ng/mL］ in the subjects was 59.7%，the rate of serious vitamin D deficiency ［25(OH)D＜12ng/mL］ was 24.9%，and the rate of adequacy［25(OH)D＞30ng/mL］ was 13.7%. The average serum concentration of 25(OH)D was (17.3±8.5) ng/mL，(18.2±9.0)ng/mL，and (20.5±9.7)ng/mL in the year of 2013，2014，and 2015，respectively，and similarly the vitamin D deficiency rate was 69.4%，63.6%，and 54.4%，respectively in 2013，2014，and 2015. There were significant differences in the levels of serum 25(OH)D among the subjects in different years (P＜0.01). The average serum concentration of 25(OH)D was (19.2±9.5) ng/mL，(19.6±9.0) ng/mL，and (18.4±8.7) ng/mL in the southern，middle and northern part of Jiangsu Province，respectively，and the corresponding rate of vitamin D deficiency was 60.0%，57.9%，and 62.8%，respectively. There were significant differences in the serum concentrations of 25(OH)D among the subjects from the three regions (P＜0.01). The serum concentrations of 25(OH)D were positively correlated with ages of the subjects (r=0.8，P＜0.01). Seasonal difference was found in the serum concentrations of 25(OH)D. The average serum concentration of 25(OH)D in summer and autumn was higher than that in spring and winter，and the average serum concentration of 25(OH)D was (22.7±9.2) ng/mL，(22.2±9.3) ng/mL，(15.3±7.3) ng/mL，and (12.3±6.4) ng/mL in summer，autumn，spring，winter，respectively. Conclusion：It is vital for obstetrician to monitor the maternal vitamin D levels and guide the pregnant women with vitamin D deficiency to take vitamin D supplements timely.

Abstract:Objective：To analyze the genic characteristic of enterovirus 71(EV71) strains isolated from Jiangsu Province in 2014—2015. Methods：The EV71 RNA positive samples were inoculated into RD cells to separate the EV71 isolates，which were selected for sequencing and analysis of VP1 gene. The software DNA Star and MEGA 6.0 were used for comparison between EV71 and the various types of reference strains. Results：A total of 35 strains of EV71 were isolated and the positive rate was 29.17%. Sequence analysis of the 35 isolates showed that the homology of nucleotides and amino acids of VP1 gene were 93.3%～100.0% and 96.6%～100.0%，respectively. There were 95.6%～97.9% nucleotide acid identity and 97.9%～100% amino acid identity among these 35 strains and representative strain of C4a genotype isolated from Fuyang City. Phylogenetic analysis showed that the 35 EV71 strains gathered together with the representative strains of C4a genotype，and belonged to 2 viral transmission chains. Conclusion：The 35 EV71 strains from Jiangsu Province in 2014—2015 belong to C4a genotype. The major epidemic strain of EV71 in Jiangsu Province might have a same ancestor as domestic epidemic strains of EV71，and then evolve independently.

Abstract:Objective：To construct highly sensitive surface-enhanced Raman scattering (SERS) sensor and implement the rapid detection of trace formaldehyde from human urine. Methods：A novel SERS sensor was fabricated by modifying p-aminothiophenol (p-ATP) on the surface of Ag nanoparticles (Ag NPs). When the aqueous formaldehyde was dropped into Ag NPs colloid，the condensation reaction between p-ATP and formaldehyde resulted in the quenching of Raman signals of p-ATP. Thus，formaldehyde was indirectly detected by SERS. Results：Results showed that the method had a good linear relationship in the range of 1.0×10-8-1.0×10-7 mol/L with a correlation coefficient of 0.991 7. The limit of detection was 6.5×10-9 mol/L and the recoveries of formaldehyde were 97.5%~106.0% from the spiked urine samples，which were satisfactory. Conclusion：The method can be applied for the determination of trace formaldehyde in urine with the advantages of less time-consuming，non-preconcentrated procedure and high sensitivity.