Abstract:Objective: To detect the expression of high mobility group protein 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) in the tissues of patients suffering from cholangiocarcinoma, and to study the possible mechanism of HMGB1 and RAGE on the growth and epithelial-mesenchymal transition (EMT) of human cholangiocarcinoma cells. Methods:Immunhistochemical study was performed to analyze the expression of HMGB1 and RAGE in cholangiocarcinoma tissues with or without metastasis; ELISA method was used to detect the level of HMGB1 in the bile of patients with hepatolithiasis and cholangio-carcinoma;The effect of HMGB1 and RAGE on cell proliferation and invasion was detected by CCK8 and Transwell assay; the expression of HMGB1 and p-ERK in tissues of cholangiocarcinoma was detected by Western blot. Results: HMGB1 and RAGE were significantly higher in cholangiocarcinoma tissues with metastasis than in those without metastasis. HMGB1/RAGE can obviously promote the growth of cholangiocarcinoma cell line in vitro and promote the expression of EMT relative cytokines. Conclusion: HMGB1 and RAGE can promote the EMT and proliferation of cholangiocarcinoma cell line

Abstract:Objective:To study the effects and possible mechanism of action of KL1 protein in human non-small cell lung cancer cells A549. Methods:Western blot was used to detect the overexpression of KL or KL1 in A549 cells transfected with pcDNA3.1-MYC-KL1 or pcDNA3.1-MYC-KL. MTT assay,colony-forming assay and flow-cytometry analysis were designed to determine the change of cell growth,proliferation and apoptosis of A549 cells mediated by KL or KL1, respectively. Effects on the phosphorylation of ERK1/2 in the middle and lower target site of the basic fibroblast growth factor(bFGF) signaling pathway were detected by Western blot after transfection. Results:After transfected 48 h,exogenous KL or KL1 was overexpressed significantly in A549 cells (both P < 0.01). Compared with the control group,the cell growth was inhibited in A549 cells after transfected with KL1 (P < 0.01),the proliferation was remarkably inhibited (P < 0.01)and the apoptosis was promoted significantly(P < 0.01). KL1 overexpression in A549 cells was associated with reduced bFGF-induced phosphorylation of ERK1/2 (P < 0.01). There was no signifcant difference between overexpressed KL and KL1 in cell growth,proliferation and activation of bFGF signal pathway (P > 0.05). Conclusion:The KL and KL1 have a similar signifcant ability to inhibit the growth,proliferation and promote the apoptosis of A549 cells. This may be due to the same ability to inhibit bFGF pathway. Thus,KL1 may be the main functional fragment of the KL protein.

Abstract:Objective:To research whether G-protein coupled receptor kinase-interacting protein 1(GIT1) regulates the differentiation of bone marrow cells into osteoclasts through Notch signaling pathway. Methods:Bone marrow cells, which obtained from eight GIT1+/+ and eight GIT1-/- mice of 8-week-old, were isolated and cultured,then were induced to osteoclast differentiation. The tartrate-resistant acid phosphatase (TRAP) activity was detected to evaluate the osteoclast differentiation at day 4 and 7. Real-time RT-PCR were performed to detect the expression of cathepsin K (CTSK),calcitionin receptor(CTR),matrix metalloproteinase 9 (MMP9) and Notch signaling pathway at day 4 and 7. Results: The size,number and density of osteoclasts were lower in the GIT1 gene knockout group compared with the wild type group. The expression of CTSK,CTR and MMP9 in GIT1 gene knockout group was significantly lower than that of the wild type group (P < 0.05). However,The expression of Jagged1,Notch2 and Hes1 in the GIT1 gene knockout group was significantly higher than that of the wild type group (P < 0.05). Conclusion:The deficiency of GIT1 inhibited the differentiation of bone marrow cells into osteoclasts through upregulating Notch signaling pathway.

Abstract:Objective:To investigate the effect of ransforming growth factor-β (TGF-β) on regulating autophagic activity in osteoblasts cell line. Methods:Osteoblasts cell line (hFOB1.19) was treated with TGF-β to induce autophagy. Then,RT-PCR and Western blotting assay were performed to examine the expression of mRNA and protein of Beclin1 and microtubule-associated protein light chain 3(LC3),respectively. Transmission electron microscope(TEM) was performed to identify autophagosomes. Data was analyzed by SPSS 19.0 statistical software. Results:Compared with the control group,after 3 h of TGF-β treatment in hFOB1.19 cells,the mRNA expression of Beclin 1 was initially decreased (P < 0.05). Meanwhile,the mRNA expression of LC3 was increased and reached the highest level at 6 h (P < 0.05),and then decreased with time. At the same time,Beclin 1 was initially increased at protein level after 3 h,and then decreased at 6 h,12 h,and 24 h(P < 0.05),and LC3-Ⅱ was increased after 6 h,reached its peak at 12 h(P < 0.01). Autophagosomes were observed in hFOB1.19 cell line by transmission electron microscope TEM at 12 h. Conclusion:TGF-β can induce autophagic activity in osteoblasts cell line hFOB1.19.

Abstract:Objective: To detect whether suppression of endoplasmic reticulum (ER) stress maintains hepatocyte growth factor (HGF) expression in hepatic stellate cells (HSCs) and its potential mechanism. Methods: Rat hepatic stellate cell line HSC-T6 was treated with the ER stress agonists 5.0 -滋g/mL tunicamycin and 0.2 -滋mol/L thapsigargin, and ER stress inhibitors sodium 4-phenylbutyrate (4-PBA) 4.0 mmol/L and salubrinal 200.0 -滋mol/L were used as pretreatment. Recombinant lentivirus LV-eIf2α-shRNA-GFP was produced to block eIf2α activated by ER stress. Levels of HGF, glucose-regulated protein 78 (GRP78), eukaryotic translation initiation factor 2α (eIf2α), phospho-eIF2α, activating transcription factor 4(ATF4) and C/EBP (CCAAT/enhancer binding protein) homologous protein (CHOP) in vitro were measured by quantitative RT-PCR and Western blot. Results: Our results demonstrated that tunicamycin or thapsigargin stimulated GRP78 expression, and activation of ER stress inhibited HGF expression in HSC-T6 cells. The inhibition of HGF could be partly prevented in the presence of 4-PBA or salubrinal, but their effects on ATF4 and CHOP expression were different. Interfering eIf2α mRNA proportionately down-regulated HGF expression. Conclusion: The activation of ER stress inhibits HGF expression of HSCs through decreasing eIF2α expression.

Abstract:Objective: To explore the regulation function of transforming growth factor β (TGF-β1)-induced cardiac fibroblast differentiation by DNA methylation. Methods: Cardiac fibroblasts isolated from neonatal Sprague-Dawley rats were cultured and characterized using immunocytochemistry. First-passage cardiac fibroblasts were used throughout the experiment and stimulated with TGF-β1, DNA methyltransferases (DNMT), 5-aza-2′-deoxycytidine (5-aza-dC) and TGF-β-neutralizing antibody, respectively. The protein level of α-smooth muscle actin (α-SMA) was determined by Western blot assay and immunofluorescence method. The mRNA levels of α-SMA,DNMT1,DNMT3a,and DNMT3b were determined by quantitative polymerase chain reaction,and the global DNMT activity was measured. Results: TGF-β1 and 5-aza-dC both significantly upregulated the expression of α-SMA in cardiac fibroblasts. DNMT1 and DNMT3a expressions were significantly down-regulated and the global DNMT activity was inhibited when treated with TGF-β1. Conclusion: Cardiac fibroblasts differentiation may be associated with the DNA methylation. Our research provides new insights in cardiac fibrosis from the perspective of epigenetics.

Abstract:Objective:To investigate the association and potential mechanism between single nucleotide polymorphism (SNP)in 3′untranslated region(3′-UTR) of COL6A2 and the risk of congenital atrial septal defect(ASD). Methods:SNPs in 3′-UTR of COL6A2 were searched in PubMed and Hapmap database for minimum allele frequency (MAF)>0.05 in Han Chinese population. Then the potential functional SNPs were predicted in website of miRNA-SNP. Meanwhile,the association between SNPs and the risk of ASD was checked in our previous Genome-wide association study (GWAS) database of congenital septation defects. Results:Rs1044598 mutant genotype AA decreased the risk of ASD by 36% compared with wild genotype TT. The luciferase reporter assay further confirmed that the significant differences were detected in cotransfected plasmid (rs1044598) with miR-4252 in HEK293T,H9C2 and cardiac cells from newborn SD rats cell lines(all P < 0.05). Conclusion:Rs1044598 may be a new independent susceptibility locus of ASD. MiR-4252 down-regulates COL6A2 expression by binding to 3′-UTR of COL6A2,and rs1044598 could influence the procedure and decrease the risk of ASD.

Abstract:Objective:To investigate the effect and mechanism of different macrophage subtypes on the induction process of regulatory T cells(Tregs). Methods: The cells was divided into the normal group(noM group),the primary macrophages effect group (M0 group),the M1 effect group (M1 group) and the M2 effect group (M2 group) according to different induction conditions. Foxp3 expression of Tregs and proliferation of effector T cells in co-culture and Transwell experiments were tested by flow cytometry. Serum of ALT was analyzed by automatic biochemistry analyzer and the injury of liver in ischemia reperfusion(IR) was detected by H&E staining. Results: Compared with the noM group, the expression of Foxp3 was reduced in the M0 group and the M2 group(both P < 0.01), while no significant difference was observed in the M1 group. What’s more,the suppressive ability of Tregs from the M0 and M2 group were less than those from the M0 and M2 group both in vitro and in vivo (both P < 0.01). Mechanism study proved that the regulatory effect of macrophage to Tregs was depended on cell-cell contact(P < 0.05). Conclusion: Our study suggests that peritoneal macrophages play an important role in regulating the induction of Tregs in vitro,which present key therapeutic potential in maintaining immune balance.

Abstract:Objective: The proteomic analysis of isobaric tags for relative and absolute quantitation(iTRAQ) technique was performed to analyze the differential plasma membrane proteins of superior olivary complex (SOC) of SD rats. Methods: The cochlear nuclear complex(CN),SOC and inferior colliculus(IC) were isolated from P60 healthy male Sprague Dawley(SD) rats,the rest of the brain was used as control. Then the iTRAQ technique was used to detected and identified the differential plasma membrane protein expression in these regions. Results: A total of 1 937 plasma membrane proteins showed significantly different expression,between two regions. We found 14 region-typical proteins in SOC. Gene ontology (GO) functional analysis showed that alanyl-tRNA synthetase (Aars),glutamyl-prolyl-tRNA synthetase(Eprs),α-internexin (Ina) and solute carrier 44A1 (Slc44A1) mignt be involved in neural development, neurotransmission,synaptic remolding, and affect the neurotransmission of auditory neurons. Conclusion: The quantitative comparison of protein screened reveals that several interesting candidate proteins, including Aars,Eprs,Ina,Slc44A1, are related to central auditory processing, which provides a theoretical basis for further study of auditory processing disorder.

Abstract:Objective:To apply CRISPR/Cas9 technology to construct stable OSBPL2 gene knockout HeLa strain. Method:Three single-guide RNA(sgRNA) targeting respectively to exon 2,3,5 of OSBPL2 gene were designed, then 3 recombinant eukaryotic expressional plasmids by the carrier of PGK1.1 were constructed. After transfection to HeLa cell respectively, puromycin was performed to screen positive cells and then cruiserTM gene knockout detection in combination with the sequencing were used to analyze targeting effect of the three plasmids. HeLa cells with the plasmid of the optimal targeting effect continued to screen monoclonal cell. The knockout effect was measured by Western blot. Results: SgRNAs were correctly inserted into the recombinant plasmids, OSBPL2 protein was undetected in HeLa cell after transfection with plasmid targeting to exon 2 and screening of monoclonal cell. Conclusion:Stable OSBPL2 gene knock-out HeLa strain can be successfully built.

Abstract:Objective:To study the effects and the mechanism of Faecalibacterium prausnitzii(F.prausnitzii)combined with Kuikeling on colitis of rats with inflammatory bowel disease(IBD). Methods:A total of 50 rats was randomly divided into 5 groups,which named the normal group,the model group,the Kuikeling group,the F.prausnitzii group and the F.prausnitzii combined Kuikeling group,respectively. Each group was given medicine by gavage for a week before model establishment (the normal and model group received saline,the other groups received corresponding F.prausnitzii and Kuikeling). Colitis model was established by 2,4,6-trinitrobenzene sulfonic acid (TNBS) method(80 mg/kg TNBS combined with equad volume ethanol). All rat were killed after 3 days,and mucosal lesion area and colon mucosal inflammation were assessed by the colorectal histological damage scores (HDS). Levels of interleukin-10 (IL-10),interleukin-12 (IL-12),tumor necrosis factor-a (TNF-α)and interferon-γ were detected by ELISA in the plasma,and then we calculated the ratio of IL-10/IL-12. The expressions of IL-10 and TNF-α in the colon mucosal tissues were detected by immunohistochemical(IHC) method. Results:Compared with the model group,mucosal lesion area and the colon HDS were significantly decreased (P < 0.05),the levels of IL-12 and IFN-γ in the plasma(P < 0.05,P < 0.001)and the expression of TNF-α in the colon mucosal tissues (P < 0.05) were significantly decreased in F.prausnitzii combined Kuikeling group,and the level of TNF-α in the plasma was decreased without statistical significance,the levels of IL-10 in the plasma and the colon mucosal tissues(P < 0.05,P < 0.01)and the ratio of IL-10/IL-12(P < 0.05)were significantly increased in the F.prausnitzii combined Kuikeling group. Compared with the Kuikeling group and the F.prausnitzii group,mucosal lesion area and the colon HDS were decreased,the levels of IL-12,IFN-γ,and TNF-α in the plasma and the expression of TNF-α in the colon mucosal tissues were decreased,the levels of IL-10 in the plasma and the colon mucosal tissues,the ratio of IL-10/IL-12 were increased in the F.prausnitzii combined Kuikeling group,but those differences had no statistical significance. Conclusion:The preventive and therapeutic effects ofF.prausnitzii combined Kuikeling is better than single treatment. The immune regulation mechanism may be related to promoting secretion of IL-10 in the plasma and the colon mucosal tissues and decreasing IL-12,TNF-α,IFN-γ levels in the plasma and TNF-α level in the colon mucosal tissues.

Abstract:Objective:To investigate the clinical effectiveness and safety of double dosage of clopidogrel and ticagrelor in patients with clopidogrel resistance after percutaneous coronary intervention(PCI). Methods:A total of 188 patients with clopidogrel resistance(platelet inhibition rate<30%)after PCI were randomly divided into the conventional clopidogrel group (group A,n=64),the double dosage of clopidogrel group (group B,n=61)and the ticagrelor group (group C,n=63). The platelet inhibition rate of the adenosine diphosphate (ADP) pathway was tested at the seventh day after PCI,high sensitivity C reactive protein (hsCRP)was measured at the first and the seventh day after PCI,the incidence rates of major adverse cardiovascular events (MACE)and slight bleeding were compared among the three groups at the first month and the sixth month follow-up. Results:At the seventh day after PCI,the platelet inhibition rates in group B and C were significantly higher than that in group A (both P < 0.05),and the rate in group C was higher than that in group B(P < 0.05). At the first day after PCI,no significant difference in hsCRP was observed among the three groups (P > 0.05). However,at the seventh day after PCI,hsCRP in group B and C was significantly reduced compared with the group A,respectively(both P < 0.05),and hsCRP in group C was lower than that in group B(P < 0.05). At the first month follow-up,there were no significant differences in the incidence rates of MACE and the slight bleeding among the three groups(P > 0.05). At the sixth month follow-up,the incidence rates of MACE in group B and C were significantly reduced compared with group A(both P < 0.05),and the rate in group C was lower than that in group B (P < 0.05),but there was still no significant difference in the incidence rate of slight bleeding among the three groups (P > 0.05). Conclusion:In patients with clopidogrel resistance after PCI,ticagrelor is more effective without increasing adverse reactions.

Abstract:Objective:To explore the expressions of inhibitors of DNA binding 1 (Id-1)and matrix metalloproteinase-9(MMP-9)in human colorectal adenocarcinoma tissues and study their correlation with the progression of colorectal adenocarcinoma. Methods:The expressions of Id-1 and MMP-9 in 50 specimens of coloreetal adenocarcinoma tissues and 50 specimens of normal eolorectal tissues were detected by immunohistochemistry,reverse transcription polymerase chain reaction (RT-PCR),and Western blotting assay. Correlations between the expressions and clinicopathological features were analyzed. Results:The positive rates of Id-1 and MMP-9 in colorectal adenoeareinoma tissues were 72.00% and 78.00%,respectively,while in normal colorectal tissues were 24.00% and 22.00%,respectively. The differences were significant (all P < 0.01). Expression of Id-1 was positively related with expression of MMP-9 in colorectal adenocarcinoma tissues (r=0.469,P=0.000). Expression levels of Id-1 and MMP-9 were correlated with the depth of tumor invasion,TNM stage,lymph node metastasis,vessel invasion,and liver metastasis (all P < 0.05),but not with age,gender,tumor size,and differentiation degrees(all P > 0.05). The results of RT-PCR and Western blotting assay were consistent with those of immunohistochemistry. Cox multi-factor analysis showed that Id-1 and MMP-9 expressions were independent prognosis factors for colorectal adenocarcinoma. Conclusion:The expression levels of Id-1 and MMP-9 have a high correlation with the invasion and metastasis of colorectal adenocarcinoma,and joint detection of the above indicators to judge the invasion,metastasis and prognosis of colorectal cancer has important guiding significance.

Abstract:Objective: To examine the expression of crystallin-alpha B (CRYAB) in human laryngeal squamous cell carcinoma(LSCC) and investigate the relationship between its expression and clinical characteristics of LSCC. Methods: One-step quantitative reverse transcription-polymerase chain reaction (qRT-PCR) test in 10 fresh LSCCs as well as non-cancerous tissue samples and immunohistochemistry analysis by tissue microarrays (80 LSCC samples and 20 tumor-adjacent normal samples) were performed to characterize CRYAB expression in LSCC. Cox regression and Kaplan-Meier survival analyses were carried out to evaluate the prognosis of LSCC. Results: The results of qPCR test and immunohistochemistry analysis showed that the expression of CRYAB in LSCC was significantly higher than that in tumor-adjacent normal tissues(P = 0.0005 and P = 0.021, respectively). Moreover, the expression level of CRYAB protein in LSCC was significantly related to tumor differentiation(P = 0.022), TNM stage(P = 0.013),lymph node metastasis (P = 0.026) and overall survival (P = 0.001). COX multi-factor analysis showed that CRYAB expression (P = 0.036) was recognized as an independent prognosis factors for LSCC. Conclusion: The data suggest that CRYAB expression is correlated with malignant phenotypes of LSCC and it may serve as a novel prognostic factor for LSCC.

Abstract:Objective: To explore the early-stage clinical results of ROI-C and MC+ fusion cage for patients treated by anterior cervical discectomy and fusion. Methods: Forty seven patients suffered from cervical spondylosis were treated by the same orthopedist with anterior cervical surgery (23 treated by ROT-C+ and 24 treated by MC(+) from May 2013 to Jan 2015). The evaluation indexes included Japanese Orthopaedic Association (JOA) scores,cervical physiological curvature,post-operative dysphagia,rate of bone fusion,intraoperative blood loss,duration of operation and costs of operation,duration of hospitalization were detected before suragury,postoperation 3 d,postoperation 3 month and the last follow-up respectively. Results: JOA scores of patients used ROI-C and MC+ at three-month post operation and the last follow-up were better than JOA scores of pre-opertaion,and the differences were statistically significant. Cervical physiological curvature of patients used ROI-C and MC+ at each follow-up time point was better than that of pre-operation. Average vertebral height at each follow-up time point was higher than that of pre-operation, the differences were statistically significant, but there were no statistical significances observed between ROI-C and MC+. Only 4 patients with dysphagia in 3 day post-operation, and dysphagia was disappeared in 7-day post-operation. Anterior vertebral soft tissue at 3-day was thicker than that at pre-operation (P < 0.05), there were no statistical significances between pre-operation and last follow-up. Conclusion: The ROI-C and MC+ fusion cage is a new kind of cage with less intraoperative blood loss, duration of operation and easy to operate.

Abstract:Objective:To investigate the changes in cervical curvature relative to the gravity line in cervical kyphosis and to provide better reference for orthopedic treatment. Methods:Measurements were performed on 60 patients with cervical kyphosis in the sagittal view in the standard,hyper flexion,and hyper extension position including∠A-∠B-∠C,and A,B and C were calculated to ascertain the value of the changes during cervical flexion and extension. The apex of the cervical curve was determined to compare changes in the distribution characteristics with 60 normal people as well as the D value. Results:In standard position,∠A was positive and increased from C2 to C7. In hyper extension position,∠A was decreased and the reduction in amplitude decreased from C2 to C7,while in hyper flexion position,∠A increased and the increase in amplitude decreased from C2 to C7. ∠B followed similar patterns of change with a larger mean value. During cervical flexion and extension,change in ∠A of the upper vertebral body(∠D) was almost equal to change in ∠A of the lower vertebral body and change in ∠C between the two vertebral bodies(∠E). The curve apex was mainly distributed between C4 and C5. Conclusion:During cervical flexion and extension movement,the dynamic changes and regularities of cervical kyphosis patients is consistent with normal people. Cervical vertebral body movement is a rotation and displacement based on a central apex in the sagittal plane during dynamic flexion and extension in both cervical kyphosis patients and normal people. The correction of the cervical kyphosis can be carried out from the apex of the cervical spine,which lays a solid theoretical foundation for the correction of the cervical kyphosis.

Abstract:Objective:We sought to determine the accuracy of digital models generated from impressions and casts respectively by an ortho insight 3D laser scanner (Motion View Software,Chattanooga,Tenn)and compare them with surface area analysis method. Methods:Two sets of maxillary and mandibular digital models of 20 subjects were obtained. The models were made from impressions and casts were scanned by ortho insight 3D digital scanner,respectively. Each patient’s matched pairs of maxillary and mandibular models were superimposed by using a software program and a best-fitalgorithm; surface-to-surface analysis was then performed. The average linear differences between the 2 files at all points on the surfaces were measured,and tolerance levels of 0.05,0.1,0.25,0.5,0.75,and 1.0 mm were set to determine the surface correlation amounts between the 2 files. Additionally,6 linear measurements from predetermined landmarks were also measured and analyzed. Results:The average maxillary model linear difference was 0.097 to 0.191 mm,whereas the average mandibular model linear difference ranged between 0.092 and 0.257 mm. Greater than a 94% surface correlation was obtained on average at 0.5 mm tolerance level. The mean differences obtained from the linear measurements indicated strong agreement (0.972≤single measure ICCs≤0.998) between the maxillary and mandibular pairs. Conclusion:Surface-to-surface analysis of digital models generated from impressions and casts by ortho insight 3D digital scanner pointed to a fair overlap between the protocols. Therefore,the digital model generated directly from impressions is an alternative for that from casts.

Abstract:Objective:To investigate the effects of low-dose of long-and short-acting gonadotrophin releasing hormone analogue(GnRH-a) agonist on patients with diminished ovarian reserve. Methods:Retrospective analysis was performed on 565 in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI)cycles,which are consistent with diminished ovarian reserve and using the low-dose long down-deregulation protocol. According to the type of GnRH agonist,they were divided into the long-acting group(group A)and the short-acting group (group B). Then,we compared basic situation,ovulation induction process and outcome of ET and FET cycle of the two groups. Results:There were no significant differences between groups A and B in the general situation and assisted reproductive method. Compared with group B,the egg number and the number of effective embryos of group A had no statistical difference. However,both gonadotripin(Gn)stimulation days and Gn doses were significantly higher in group A compared with group B. In the ET cycle,there was no significant difference in clinical pregnancy rate,abortion rate and delivery rate between group A and B. However,in the FET cycle,the clinical pregnancy rate,implantation rate and delivery rate of group B were significantly higher than those of group A. The cumulative clinical pregnancy rate and delivery rate of group B were also significantly higher than that of group A. Conclusion:Patient with diminished ovarian reserve can still use low-dose long down-deregulation protocol to obtain more effective embryos and fresh embryo transplant opportunity. The clinical pregnancy rates were also high. Long-acting GnRH-a increased the amount of Gn,and the clinical pregnancy rate of this group in FET cycle was lower. This study suggests that these patients use low-dose short-acting GnRH agonist protocol.