Abstract:Objective:To describe our novel modification in isolating pancreatic stellate cells (PSCs) from normal human pancreas and human pancreatic ductal adenocarcinoma (PDAC) tissue. Methods:Normal PSCs were isolated with enzyme digestion and ladder centrifuge. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrowth method. Isolated PSCs were cultured in DMEM/F12 containing 20% fetal bovine serum. Results:With our modifications,normal pancreas tissue from human yields an adequate number of PSCs (approximately 0.5-5 million/g pancreas) for in vitro studies,and the cell viability was about 90%. After new outgrowth method applied,tissue blocks were attached more tightly and cells grew out earlier compared to the previous method. Primary isolated PSCs were verified with appearance,auto-fluorescence,positive expression of α-SMA,Vimentin,Desmin,GFAP. Conclusion:Our modification for PSCs isolation significantly increase the isolating efficiency with shorter culture period,which can provide great convenience for future researches on PSCs.

Abstract:Objective:Vasohibin (VASH),one of new gene families,is believed to be involved in cancer-promoting. However,the exact function is still unknown. To analyze the expression level of nuclear VASH2 protein in human fetal tissues from different gestational ages. Methods:Immunofluorescent method (IF) was used to detect the expression of VASH2 protein in 293T and intracellular localization. Nuclear VASH2 polyclonal antibody was used to analyze the expression level of nuclear VASH2 protein in human fetal tissues from different gestational ages,and the change of embryonic cell mitotic counts and the expression of nuclear VASH2 was assessed by using immunohistochemistry (IHC). The cell growth was detected by methyl thiazolyl tetrazolium (MTT). Flow Cytometry (FCM) was used to determine the cell cycles. Results:We found that the protein expression of VASH2 (with 355 amino acid residues) was located in the cytoplasm while VASH2 (with 311 amino acid residues) was located in the nucleus. Nuclear VASH2 was generally lower in fully-developed embryos and markedly expression during undergoing several maturational processes. There is a significant positive correlation between nuclear VASH2 and mitotic counts (r = 0.826,P < 0.01). Nuclear VASH2 promoted proliferation of 293T cells. FCM analysis showed increasing proportion of S and G2 / M phase in nuclear VASH2 transfection. Conclusion:This is the first study to report differences in the intracellular localization of VASH2 protein. Nuclear VASH2 protein found in a variety of tissues is not a tissue-specific protein,and may play important roles in histogenesis and organogenesis.

Abstract:Objective:To study the influence and molecular mechanisms of low shear stress on local vascular endothelial injury and smooth muscle cell proliferation of BALB/c mice. Methods:Thirty BALB/c mice were randomly divided into three groups. Ten mice were taken as the sham-operated group and fed in a normal diet. The model one and model two were established by rapid perivascular collar placement at left common carotid artery(LCCA)and fed in a normal and a high-fat diet,respectively. The body weight was measured periodically. On the third day and eighth week post-operation,the alterations of peak blood flow,end-diastolic diameter of vessels and shear stress of carotid artery were assessed by carotid artery doppler ultrasound. The variations of lipid metabolism were detected by automatic biochemical analyzer during the eighth week post-operation. The pathomorphological and ultrastructural changes of carotid artery were evaluated by hematoxylin-eosin staining and electron microscope,respectively. The gene expression profile changes in the low shear stress blood vessels and in the self-controlled normal shear stress blood vessels were compared by microarray,and then verified by (quantificational real time- polymerase chain reaction,QRT-PCR). GO and KEGG signaling pathway were analyzed and focused on the changes of genes and signaling pathways related to cell migration and proliferation. Results:Compared with sham-operated group,the body weight and serum lipids levels had no significant differences(P > 0.05) in group model one fed with normal diet at the eighth week post-operation. However,the body weight and serum lipids(TC,TG,LDL-C and HDL-C)significantly increased(P < 0.05) in group model two. The shear stress of left common carotid artery-proximal (LCCA-P)was low in group model one and model two. Meanwhile,there were endothelial cell damages,media thickening and endothelial cell vacuolation in the LCCA-P of both group model one and group model two. The lesions in group model two were more serious than those in group model one. But no fatty streaks,plaque formations and lipid accumulations were observed by light microscope and electron microscope. Microarray results suggested that compared with the self-controlled blood vessels from normal shear stress,the blood vessels from low shear stress presented a remarkable miR-27a expression elevation(> 2.0-fold),PPAR signaling pathway down-regulation,MAPK,TGF-β,PI3K/Akt and NF-κB signaling pathway up-regulation,and the cell migration and / or proliferation-related genes up-regulation(TGFB2,END1,CTGF,etc.). The results of QRT-PCR were consistent with microarray. The expression of PPARG decreased to 0.14-fold(P < 0.05,n = 4),TGFB2 and EDN1 increased to 2.51-fold and 1.83-fold,respectively(P < 0.05,n = 4)in the blood vessels from low shear stress. Conclusion:Compared with the blood vessels from normal shear stress,the blood vessels from low shear stress showed the damage of local endothelial cells and media thickening after eight weeks post-operation in BALB/c mice. These changes may be associated with the down-regulation of local PPAR signaling pathways,thereby activating the genes expression and signaling pathways related to cell proliferation and migration.

Abstract:Objective:To investigate the expression level of PKM2 in cholangiocarcinoma (CCA)tissues,then study the effect of PKM2 down-regulation on migration,invasion and proliferation in cholangiocarcinoma cell lines. Methods:RNA and protein expressions of PKM2 in CCA tissues and paired adjacent tissues were detected by qRT-PCR and immunohistochemistry. PKM2 was down-regulated by a lentiviral vector expression system in cholangiocarcinoma cell lines HuCCT-1 and HCCC-9810. qRT-PCR and Western blot were performed to analyze the mRNA and protein expression of PKM2 in both cell lines. Cell migration, invasion and proliferation were assessed by wound-healing experiment, matrigel invasion and Cell Counting Kit-8(CCK-8). Results:The expression of PKM2 in CCA tissues had a higher level than that in paired adjacent tissues. The mRNA and protein expressions of PKM2 in the experimental group (PKM2-shRNA)were significantly lower than those in the two control groups,confirmed by qRT-PCR and Westen blot (P < 0.05). Compared to the empty vector group (PKM2-NC)and the normal control group (PKM2),the cell invasion,migration and proliferation were significantly decreased in the experimental group (PKM2-shRNA)(P < 0.05). Conclusion:Down-regulation of PKM2 by PKM2 shRNA can inhibit migration,invasion and proliferation of HuCCT-1 and HCCC-9810 cells.

Abstract:Objective:To explore whether polychlorinated biphenyl 118 (PCB118)could cause inflammation reaction in rat thyroid. Methods:①Forty male Wistar rats were randomly divided into 4 groups:solvent control group (corn oil),low dose,medium dose and high dose PCB118 groups,intraperitoneal injected with corn oil or different doses of PCB118 (10,100,1 000 μg/(kg-d),respectively)for 5 d per week,and weekly weighed. Thirteen weeks later,the thyroid tissues were stained using HE method and the infiltration of inflammatory cells were observed. ② The effects of PCB118 on FRTL-5 cells viability was assessed using a Cell Counting Kit-8 assay. ③Quantitative real-time PCR (qRT-PCR)was used to detect the mRNA expression of cytokines interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1 ). Results: ①In HE staining,there were a large number of inflammatory cells infiltration in PCB118-treated rat thyroid tissue; ②Relatively low concentrations of PCB118 had no significant effects on cells viability(P > 0.05); ③Compared to the control group,the mRNA levels of IL-6,TNF-α and ICAM-1 increased significantly(P < 0.05) after stimulating with different concentrations of PCB118. Conclusion:PCB118 could induce inflammation reaction in rat thyroid tissues.

Abstract:Objective:To investigate the effects of over-expressed mitogen-inducible gene 6 (MIG6)on apoptosis,proliferation and invasion of endometrial carcinoma cell. Methods:Ishikawa cells were transfected with MIG6 over-expressed plasmid by Lipofectamine 2000,and quantitative RT-PCR and Western Blotting were adopted to detect the expression level of MIG6 before and after transfection. Cell apoptosis and proliferation were measured by flow cytometry and BrdU incorporation assay,respectively. Expression of cleaved caspase 3 and Cyclin D1 was determined by Western Blotting. Invasive potential of Ishikawa cells was tested using Transwell assay. Results:Both mRNA and protein levels of MIG6 in Ishikawa cells transfected with MIG6 over-expression plasmid were significantly higher than those of negative control group. Fold changes were 24.2 and 2.8,respectively. Up-regulated MIG6 expression for 48 h,the expression of cleaved caspase 3 was significantly elevated(P < 0.05),and the early apoptosis rate and late apoptosis rate was 1.7 times and 2.7 times more than that of negative control group(P < 0.01,P < 0.05); the expressions of cyclin D1 and phosphorylation of ERK were both down-regulated significantly(P < 0.05),BrdU incorporation rate decreased from 43.4 ± 2.4% to 30.5 ± 4.3%(P < 0.05); meanwhile,the amount of trans-membrane cells decreased from 36.2 ± 6.9 to 26.2 ± 3.8(P < 0.05). Conclusion:Up-regulation of MIG6 inhibits proliferation and invasion,while promotes apoptosis of endometrial carcinoma Ishikawa cells in vitro.

Abstract:Objective:To investigate the expression of glypican-3 (GPC3)protein and mRNA in ovarian clear cell adenocarcinoma and its correlation with clinical pathologic parameters. Methods:The expression of GPC3 protein was detected by immunohistochemistry in 83 cases of ovarian cancer including 45 clear cell adenocarcinoma,20 serous cystadenocarcinoma,18 mucinous cystadenocarcinoma,20 cases of serous cystadenoma and 20 cases of normal ovary. The expression of GPC3 mRNA was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) in 20 clear cell adenocarcinoma,10 serous cystadenocarcinoma,8 mucinous cystadenocarcinoma, 5 serous cystadenoma and 5 normal ovary tissues. The correlation between GPC3 expression and relative clinical pathologic parameters was analyzed. Results:①In ovarian clear cell adenocarcinoma, the positive rate of GPC3 protein and the mRNA expression (82.2%;1.326 1 ± 0.493 6) were significantly higher than those in ovarian serous cystadenocarcinoma (20%;0.426 5 ± 0.029 4),mucinous cystadenocarcinoma (5%; 0.265 2 ± 0.103 9),serous cystadenoma (0; 0.141 3 ± 0.011 3) and normal ovarian tissue (0; 0.291 4 ± 0.048 1)(P < 0.01); ②Both the expression of GPC3 protein and the mRNA were relevant to the clinical stage of ovarian clear cell adenocarcinoma. GPC3 expression in stage Ⅲ or Ⅳ (100%;1.808 1 ± 0.265 7) was significantly higher than that in stage Ⅰ or Ⅱ (71.4%;1.045 8 ± 0.403 6) (P <0.01) ;③Both the expression of GPC3 protein and mRNA were relevant to the status of cisplatin resistance. GPC3 expression in cisplatin-resistant patients (100%;1.808 1 ± 0.265 7) was higher than that in cisplatin-sensitive patients (70.3%;0.991 8 ± 0.330 3) (P < 0.05);④GPC3 protein expression was associated with survival of clear cell adenocarcinoma,which was higher in patients with poor prognosis (P < 0.05). Kaplan-Meier analysis showed that GPC3 protein expression was a prognostic factor (P=0.048);⑤GPC3 protein expression in ovarian clear cell adenocarcinoma had no relavance with the lymph node metastasis or distant metastasis (P > 0.05). Conclusion:GPC3 has an important significance in the differential diagnosis. There was a significant relationship between GPC3 expression and clinical stage and cisplatin resistance in ovarian clear cell adenocarcinoma. GPC3 may play an important role in the development of ovarian clear cell adenocarcinoma and could be used as a helpful prognostic marker and potential target .

Abstract:Objective:To explore the effect on proliferation and apoptosis after treatment with afatinib and Mithramycin A(MIT)in hepatocellular carcinoma cell line HepG2,and detect the aberrant expression of related factors. Methods: HepG2 cells were treated by afatinib,MIT,and combination of two. MTT and flow cytometry(FCM)were used to observe cell viability and apoptosis. EGFR,Sp1,Sp3 and genes,which were related to proliferation and apoptosis like Cyclin-D1,Cyclin-E2,Bcl-2,Caspase3,Caspase9 and p53,were analyzed by qRT-PCR. Results:With the administration time increasing,the inhibition rate of HepG2 cells apparently raised,while the group treated by afatinib and MIT exhibited dramatic ascending (P < 0.05). In addition,FCM also confirmed that Combination of afatinib and MIT arreasted HepG2 cells in G0/G1 phase,and the highest apoptosis rate appeared in combined group (P < 0.05). The level of Cyclin-D1,Cyclin-E2 and Bcl-2 in HepG2 cells incubated with both afatinib and MIT were obviously down-regulated, while Caspase3 up-regulated. Besides,afatinib enhanced expression of Caspase9 and p53,while MIT down-regulated the level of EGFR (P < 0.05). Conclusion:Afatinib combined with MIT significantly suppressed proliferation and induced apoptosis in HepG2 cells by inhibiting of Cyclin-D1,Cyclin-E2 and Bcl-2 coupled with increase of Caspase3,Caspase9 and p53. Moreover,our resultes probably provide a novel EGFR-centered strategy for future combined targeting therapy in HCC.

Abstract:Objective:To evaluate the potential mitochondrial toxicity of A compound,one of DPP-Ⅳ inhibitor hypoglycemic drugs, and to explore the mechanisms of toxicity of A compound. Methods: HepG2 cells were cultured in troglitazone(TRO)50~300 μmol/L for 24 h or A compound 100~300 μmol/L for 24 h. The cell viability was assessed by CCK8 assay. HepG2 cells were cultured in TRO 100,200 and 225 μmol/L for 24 h or A compound 100,150 and 200 μmol/L for 24 h. The level of cellular ATP was detected by luciferase assay. The levels of intracellular free Ca2+,reactive oxygen species(ROS),mitochondrial membrane potential(-驻Ψm)and mitochondrial permeablity transition pore(MPTP)were determined using flow cytometer. Results: Compared with the vehicle control group,TRO at 50~300 μmol/L markedly inhibited cell viability(IC50=178 μmol/L),A compound at 100~300 μmol/L markedly inhibited cell viability(IC50=159 μmol/L). Compared with the vehicle control,levels of ATP and -驻Ψm were significantly decreased in the TRO treated group(≥200 μmol/L)(P < 0.01),levels of ROS were significantly decreased and intracellular free Ca2+ were significantly increased in the TRO treated group (≥100 μmol/L)(P < 0.05 or P < 0.01). After A compound treatment,compared with the vehicle control,levels of ATP and intracellular free Ca2+ were significantly decreased at 100 μmol/L(P < 0.01). A compound at 150 μmol/L increased ROS level significantly (P < 0.01),A compound at 200 μmol/L decreased -驻Ψm level significantly(P<0.05)and mitochondrial ultra-structural changes were observed. Conclusion: DPP-Ⅳ inhibitor A compound can induce mitochondrial toxicity by interfering with mitochondrial metabolism.

Abstract:Objective:To investigate the expression level of long noncoding RNA BANCR in non-small cell lung cancer (NSCLC) tissues and cell lines,and to study the effect of BANCR on NSCLC cell proliferation and invasion. Methods:Quantitative PCR was performed to detect the relative expression of BANCR in NSCLC cell lines and tissues. pCDNA-BANCR was transfected into SPC-A1 cells and A549 cells to up-regulate BANCR expression. Transwell assays were performed to detect the effect of BANCR on NSCLC proliferation and invasion. Western blotting was used to detect the expression level of E-cadherin and Vimentin,markers of epithelial-mesenchymal transition. Results:This study showed that BANCR was lowly expressed both in NSCLC samples and cell lines compared with their corresponding normal tissues and cell lines. MTT assays indicated that up-regulated BANCR inhibited the proliferation and invasion of SPC-A1 and A549 cells. BANCR overexpression elevated the expression of E-cadherin and suppresed the level of Vimentin,thus influencing epithelial-mesenchymal transition. Conclusion:BANCR promotes NSCLC cell proliferation and invasion by affecting epithelial-mesenchymal transition.

Abstract:Objective:To discuss the effects of RNA interference silencing MKK7 on SH SY5Y cells apoptosis. Methods:Three articles MKK7 siRNA(MKK7siRNA-1,MKK7siRNA-2 and MKK7siRNA-3)were designed and synthesized,the negative control siRNA with red fluorescence,which had no homology to the MKK7 gene,was transfected into SH SY5Y cells with the lipofectamine 2000 and the transfection effect was tested using fluorescence microscope,the MKK7 was detected by RT-PCR and Western blot, then were screened MKK7siRNAs. After the MKK7siRNAs were transfected into SH-SY5Y cells,the cell vitality was detested using CCK-8,and the apoptosis rate was tested by Hochest 33258 dyeing and flow cytometry instrument,the expression of p-JNK and the cleaved caspase-3 protein was detected by western blot. Results:①MKK7siRNA-3 has lowest MKK7 mRNA and protein expression;② compared with the control group,MKK7siRNA-3 group has no statistical difference in cell viability,lower p-JNK and cleaved caspase-3 expression and apoptosis rate. Conclusion: The RNA interference silencing MKK7 can down-regulate the JNK phosphorylation ,which forther inhibit the SH SY5Y cell apoptosis.

Abstract:Objective:To investigate the effects of erlotinib on proliferation and autophagy in HCC827,A549 and H1299 human non-small cell lung cancer cells. Methods:Three human non-small cell lung cancer cell lines HCC827,A549,H1299 were cultured in vitro,and were divided into control group,DMSO group,and groups treated by erlotinib in different concentrations. CCK-8 method was used for detecting the proliferation inhibition. The expression levels of autophagy-related proteins,microtubule-associated protein light chain (LC3)and Beclin-1 Were detected by Western blot analysis and cell immunofluorescence method. Results:Erlotinib inhibited the proliferation of HCC827,A549 and H1299 cells,proliferation inhibition rate showed that HCC827> A549 >H1299,IC50 of HCC827,A549 and H1299 cells were 8.72 μmol/L,32.09 μmol/L and 87.46 μmol/L,respectively. Compared with the control groups,erlotinib enhanced expression of LC3-II and Beclin-1 significantly(P < 0.05). Erlotinib induced autophagy in a concentration dependent manner in HCC827,A549 and H1299 cells(P < 0.05). Conclusion: Erlotinib inhibited proliferation selectively and activated autophagy in non-small cell lung cancer cells.

Abstract:Objective:To study the role of RNF123 in regulating protein stability of p27Kip1. Methods: Distribution of cell cycle was detected the by flow cytometer in SKOV3 cells,after treatment with serum starvation and release for synchronization purpose. Western blot was used to test the protein level of p27Kip1 of both sictrl cells and siRNF123 cells. SKOV3 cells were transfected with sictrl and siRNF123,western blot was used to analyze half-life of p27Kip1. Results:After serum deprivation for 48 h,SKOV3 cells were arrested in the G1/G0 phase,then serum releasing stimulated the proliferation of G1/G0 to S phase,and the expression of p27Kip1 was decreased during the progress. Moreover,the level of p27Kip1 was higher in siRNF123 cells than that in sictrl cells. After deceasing p27Kip1 expression,half-life of p27Kip1 was delayed. Conclusion:Our results suggest that down-regulated RNF123 can inhibit the degradation of p27Kip1.

Abstract:Objective:To study the association between polymorphisms in H19 gene,which transcribes a long non-coding RNA,and susceptibility to premature coronary artery disease (pCAD). Methods: Four polymorphisms(rs2067051,rs2251375,rs217727,rs4929984) of H19 gene were analyzed in 213 pCAD patients and 776 control subjects. Polymorphisms were genotyped by TaqMan technology. The statistical analysis was implemented in SPSS. Results: The frequencies of genotype CT,TT and allele T in pCAD group of rs217727 were higher than that in the control group. Multivariate logistic regression analysis showed that the rs217727 polymorhpism of H19 gene was independently associated with the occurrence of pCAD (P < 0.01). The risk of patients with T allele and TT homozygous genotype was 2.42 times(OR=2.42,95%CI=1.56~3.71)and 3.01 times(OR=3.01,95%CI=1.87~4.85)than that of patients with CC homozygous genotype. Conclusion: H19 gene rs217727 polymorphism is associated with the susceptibility of pCAD,and T allele may be a genetic susceptibility factor of pCAD.

Abstract:Objective:To analyse the clinical feature of atrial fibrillation (AF) with secundum atrial septal defect (ASD) and the outcomes of different treatment methods of AF. Methods:To calculate the incidence of AF in 641 patients with ASD before transcathter closure. And to find the risk factors in the patients with AF. To compare the outcomes between cardioversion of AF with catheter ablation and with antiarrhythmic drug therapy. Result:The incidence of AF was 4.8% in 641 patients with ASD,8.4% in patients above 40 years old and 25% in patients above 60 years old. Compared with the patients without AF,the patients suffering AF were characterized by much more males,higher pressure of right atrium,higher pressure of pulmonary,larger diameter of left atrium,larger left ventricular end diastolic diameter and lower left ventricular ejection fraction. Male,aged above 40 years old and larger diameter of left atrium were identified as risk factors for AF in ASD patients. The rate of cardioversion of AF with catheter ablation was higher than that of antiarrhythmic drug therapy. Conclusion:The incidence of AF was high in patients with ASD. Male,aged above 40 years old and larger diameter of left atrium were identified as risk factors for AF in ASD patients. Catheter ablation of AF should be a effective method for treatment of AF with ASD.

Abstract:Objective:To investigate the effects of Toll-like receptor 4 overexpression in human AEJ and the relationship between TLR4 overexpression and angiogenesis. Methods:Through retrospective analysis, a total of 43 surgically resected human AEJ specimens were investigated for TLR4 protein expression by immunohistochemical SP methods,CD34 antibody was used to mark microvascular endothelial cells and the microvessel density (MVD) was determined。The correlation among TLR4 protein expression and MVD and clinicopathologic features of AEJ was analyzed. Results:TLR4 protein positive rate was 69.8%, rate of 82.6% in group A which was metastasis and recurrence was significantly higher than that of the control group B(P < 0.05). TLR4 protein positive rate in the cases with lymph node metastasis (79.3%)was significantly higher than that in the cases without lymph node metastasis (50.0%)(P=0.05). TLR4 protein positive rate in the patients with stage Ⅲ and Ⅳ of TNM classification was 82.8%, which was significantly higher than that in the patients with stage ⅠandⅡ(42.9%,P < 0.05). MVD was closely related to TNM stage of AEJ (P=0.008). There was a strong positive correlation between TLR4 protein expression and MVD (P=0.012). There was no statistical significance among tumor grade,size,vascular metastasis,and neural invasion. Conclusions:TLR4 expression was closely related to the progression of tumor and prognosis of patients and might be related to the promotion of tumor angiogenesis.

Abstract:Objective:To Investigate the clinical efficiency and safety of ultrasound-guided sclerosing lauromacrogol injection therapy for thyroid cysts. Methods:Clinical data of 78 patients with thyroid cysts underwent ultrasound-guided aspiration and percutaneous lauromacrogol injections. All cysts were confirmed to be benign by fine-needle aspiration biopsy. Firstly, the complete aspiration of cystic contents was performed, and then lauromacrogol of approximately one third of the volume of thyroid cyst was injected into the cyst. We performed the therapy once a week, and the injection was repeated one to four times which depended on the reduction of the cysts. Results:1. One hundred and sixty-six times of injections were performed for 78 thyroid cysts. Volume of thyroid cysts decreased averagely by 71.6% ± 35.3% three months later, and there was a significant statistical difference before and after the therapy (P < 0.01). The completely cure rate was 52.6%(41/78)and the total effective rate was 84.6%(66/78); 2. The completely cure rates were different among diverse groups (P < 0.01). For the volume of cysts less than 5 mL, the completely cure rate was statistically lower than those above 5 mL, however, the statistical significance was not found in effective rate among different volume of thyroid cysts(P > 0.05);3. There was no obvious changes in thyroid function and titer of thyroid related antibodies after the therapy;4. The incidence of complications was low and no sever adverse effect was occured after therapy. Conclusion: Ultrasound-guided lauromacrogol injection is a feasible, effective and safe method, and can be chosen as the preferred therapy for thyroid cysts.

Abstract:Objective:To study the effects of endotoxin tolerance induced by subgingival plaques on the productions of inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 and the protein expressions of Toll-like receptor 2, 4 (TLR2, 4)in human macrophages. Methods:Macrophages were pretreated with subgingival plaques from moderate to serve chronic periodontitis patients or healthy controls (24h), washed (2h)and treated with the same plaques again (24 h). Levels of TNF-α and IL-10 in supernatant were detected by ELISA. Moreover, TLR2, 4 protein expressions in these cells were explored by flow cytometry. Results:The amounts of TNF-α and IL-10 secreted by macrophages stimulated with both kinds of subgingival plaques were increased significantly after 24h(P < 0.05). After repeated stimulation with the same plaques, secretions of TNF-α were decreased significantly compared with those following single challenge(P < 0.05), while only comparable levels of IL-10 were confirmed in both two kinds of plaques- tolerized cells(P > 0.05). However, no significant changes of TLR2 and 4 were detected in macrophages retreated with these plaques compared with those following single stimulations. Conclusion:Secretions of TNF-α could be suppressed by endotoxin tolerance induced by subgingival plaques, which might contribute to limiting periodontal inflammation. However, development of this tolerance might not be affected by TLR2 and 4.

Abstract:Objective:To evaluate the effects of micro-nano patterned titanium surface modification on Streptococcus mutans adhesion. Methods:According to different titanium surface modifications,there were four groups:Mechanical polishing group,Sandblasting group,Anodic oxidation group,and Sandblasting-anodic oxidation group. The specimen of each group were characterized by scanning electron microscopy. The contact angles of double distilled water on specimen of each group were calculated to evaluate the wettabilities. The specimens were incubated in Streptococcus mutans cell suspension,and the bacteria on specimens were numbered by colony-forming units and observed by scanning electron microscopy. Results:Scanning electron microscope demonstrated that the micro-nano patterned structure of nano-tube arrays within micro-pores grew on the titanium surface of Sandblasting-anodic oxidation group. The sequence of surface contact angles among four groups was as follows:Mechanical polishing group,Sandblasting group > Anodic oxidation group,Sandblasting-anodic oxidation group. The sequence of the bacterial adhesion quantities among four groups was as follows:Anodic oxidation group,Sandblasting-anodic oxidation group > Sandblasting group > Mechanical polishing group. Conclusion:In comparison with the nano-tube arrays,the micro-nano patterned surface modification did not increase the adhesion of Streptococcus mutans on the titanium surface.

Abstract:Objective:To investigate the effect of Er:YAG laser and/or acid-etching pretreatment on the bond strength of self-adhesive resin cement. Methods:Forty-four extracted human premolars were randomly divided into 4 groups according to the different pretreatment on the dentin surface:Control group,Er:YAG laser group,acid-etching group,and Er:YAG laser+acid-etching group. One tooth selected randomly from each group was cut into 3 mm dentin slices,then observed under Scanning electron microscopy (SEM) after pretreatment. The remaining ten teeth were prepared into the consistent standards dentin surface,then bonded with the self-adhesive resin cement RelyXTM Unicem and Everest ZS zirconia ceramic after pretreatment,tested shear bond strength after 24 h. Results: SEM showed the differences of dentin surface morphology pretreated by different methods. The results of statistical analysis showed that the shear bond strength in the order from high to low is Er:YAG laser group (7.18 ± 2.54MPa),Er:YAG laser with acid-etching group(5.50 ± 2.09MPa),Control goup(4.05 ± 1.04MPa),and Acid-etching group (3.61 ± 0.70MPa);there was a significant difference between Er:YAG laser group,Er:YAG laser with acid-etching group and Control goup (P < 0.05),however,there was no significant difference between Acid-etching group and Control goup(P > 0.05). Conclusion: The study shows that the influence of acid-etching on the shear bond strength of self-adhesive resin is not obvious,while Er:YAG laser pretreatment can obtain better dentin bonding surface,and improve the shear bond strength of the self-adhesive resin with dentin and zirconia ceramic。

Abstract:Objective:To build PBPK/PD models subcutaneous exposured to chlorpyrifos. Methods:①Animalt:Adult female SD rats were randomly divided into 3 treated groups and 1 control group. Subcutaneous injection was performed for once,and biological samples were collected at 3,6,12,24,48,72 hours. The indicators included serum CPF,TCP and urine,serum acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). ②The establishment of models:a:Determination the model structure. b:Set up of differential equation,and collection the parameters of model. c:Simulation. Optimize the model parameters. d:Validation of models. Results:The PBPK/PD model parameters was optimized according to experimental data of 139.5 mg/kg group. Changes of CPF,TCP and ChE were mocked in this model,and the simulation results were good. The experiments and model simulation results showed that CPF and TCP in serum increased initially and then decreased. ChE activity in serum decreased initially and then increased. Experimental data showed that serum CPF and TCP reached peak at 6-hour and 12-24-hour after exposure,respectively. Serum AChE and BuChE activity achived the minimum at 24-hour and 12-24-hour,respectively. Model simulation data showed that serum CPF and TCP reached peak at 6.7-hour and 24.7-hour,respectively. Serum AChE and BuChE activity achived the minimum at 32-35-hour and 15-28-hour,respectively. Conclusion:PBPK/PD models can simulate the disposal process of CPF,TCP and ChE.

Abstract:Objective:To investigate the prevalence of HCV infection and risk factors among paid blood donors in rural area of Jiangsu Province. Methods:Questionnaires and ultrasound examination were administered,and 5 mL blood sample of each subject was collected to detect HCV anti-body,HCV-RNA,and other biochemical indicators. EpiData and SPSS were used for statistical analysis of risk factors. Results:The prevalence of HCV infection among these paid blood donors was 65.3%(510/781),including 70.6% (360/510) of chronic infection and 29.4% (150/510) of spontaneous clearance. The dominant genotypes of HCV are 1b and 1b+3 mixed. The abnormal rate of ALT,AST,and GGT are highest in the group of chronic infection. Multivariate logistic regression analysis indicated that sex,history of donating blood,and history of donating plasma were the risk factors of HCV infection (OR=1.50-2.27,and 3.27,respectively). Age was a risk factor for chronicity of HCV infection with OR of 1.62. Conclusion:The prevalence of HCV infection among the paid blood donors was higher than that of general population due to illegal blood donation,especially donating plasma.