Abstract:Objective:To clone mouse gene Gtf2h2 and have it stably expressed in HEK293 cells. Methods:Gtf2h2 was amplified by PCR using mouse oocyte cDNA as template,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 HEK 293 cells. The expression of GTF2H2-3DDK and GTF2H2-mKate fusion protein was detected by both immunofluorescence and Western blot using the antibodies of DDK and mKate tag proteins. Results:The correct cloning of Gtf2h2 was confirmed by restriction digestion and sequencing,and the expression of GTF2H2-3DDK and GTF2H2-mKate fusion protein was detected by Western blot. GTF2H2 fusion protein was detected in the nucleus in a punctate manner. Conclusion:Mouse Gtf2h2 gene was successfully cloned and expressed in HEK293 cells,which lays solid foundation for further studies of its functional roles and mechanisms.

Abstract:Objective:To establish a culture model for studying the interaction between oocytes and cumulus cells in the strain of ICR mice that are frequently used in biomedical research in China,and using this model to study the effect of oocytes and oocyte-derived paracrine factors growth differentiation factor 9(GDF9) and bone morphogenetic protein 15(BMP15) on cumulus expansion,which is one of the key ovulatory processes. Methods:Oocytectomized cumulus complexes (OOX)were constructed by microsurgical removal of the oocyte from the cumulus-oocyte complexes,and the effect of oocyte and paracrine factors on EGF-induced cumulus expansion was studied by co-culturing the OOX with oocytes and treating the OOX with recombinant GDF9 and BMP15. Results:The model of OOX culture was successfully established using ICR mice,and the oocyte and its paracrine factor GDF9 and BMP15 were found to be essential for EGF to induce cumulus expansion. Conclusion:Oocytes promote key ovulatory processes in ICR mice.

Abstract:Objective:To investigate the expression of miR-30e* and the role of miR-30e* in cisplatin induced mouse proximal tubular cells (mPTCs) apoptosis and mitochondrial injury. Methods:Expression of miR-30e* in mPTCs treated with cisplatin were determined by quantitative real-time PCR (qRT-PCR). To stably over expression or knock down miR-30e*, recombinant lentiviral expressing vectors were used for transfection of mPTCs. Flow cytometry was performed to analyze the apoptosis of mPTCs after transfection and treated with or without cisplatin. Mitochondrial DNA (mtDNA) copy numbers were determined by qRT-PCR and mitochondrial membrane potential was examined by JC-1 staining. Results:Cisplatin reduced the expression levels of miR-30e* in a dose and time-dependent manner. MPTCs transfected with over expression miR-30e* recombinant lentiviral expressing vector gained an increased expression of miR-30e* for seven fold. While knock down of miR-30e* decreased the expression of miR-30e* for 50% compared with vehicle. The apoptosis of mPTCs increased when treated with cisplatin,over expression miR-30e* reduced cisplatin induced apoptosis and knock down of miR-30e* facilitated cisplatin induced apoptosis. mtDNA significantly decreased after cisplatin treatment for 48 h,later than the decrease expression of miR-30e*. JC-1 staining revealed ectopic miR-30e* can protect mitochondrial membrane from cisplatin induced injury. Conclusion:Cisplatin reduced the expression levels of miR-30e* in vitro. Ectopic miR-30e* can protect mPTCs from apoptosis and mitochondrial membrane potential changes induced by cisplatin.

Abstract:Objective:To study the mechanisms of cardiomyocytes aging induced by advanced glycation end products. Methods:Neonatal rat cardiac myocytes were cultured with AGEs,anti-RAGE antibody for 48 h. Senescence-associated beta galactosidase (SA-β-Gal) activity was observed via SA-β-Gal assay in neonatal rat cardiomyocytes. p53,p16,LC3 and Beclin1 were measured by Western blot. We observed mitochondrial membrane potential and the generation of reactive oxygen species(ROS) by JC-1 and DCFH-DA methods. Results:After intervention for 48 h with AGEs,SA-β-Gal activity and the expression of p53(P < 0.01),p16(P < 0.01),LC3(P < 0.01) and Beclin1(P < 0.01) were significantly increased in the AGEs group compared to that of the control group,accompanied by the significantly decrease of mitochondrial membrane potential (P < 0.01) and remarkably increase of ROS(P < 0.01). After treated with anti-RAGE antibody,SA-β-Gal activity and the level of p53(P < 0.01),p16 (P < 0.01),LC3 (P < 0.05)and Beclin1(P < 0.01)were remarkably decreased compared with the AGEs group. However,inhibition of RAGE with anti-RAGE antibody remarkably increased mitochondrial membrane potential(P < 0.01) and significantly decreased ROS compared with AGEs group(P < 0.01).Conclusion:AGEs-RAGE may induce aging of cardiomyocytes by mitochondrial damage,the generation of ROS and autophagy.

Abstract:Objective:To investigate the effects of intra-myocardial transfer of bradykinin (BK)preconditioning(PC)endothelial progenitor cells (EPCs)in a mouse model of acute myocardial infarction. Methods:Cells were divided into three groups:human EPCs(hEPCs)without BK preconditioning group(EPCs group),BK PC hEPCs group(BK PC group),BK PC hEPCs + HOE140(icatibant)group (BK PC/HOE group). In vitro,proliferative ability was detected by MTT assay. Apoptosis induced by hypoxia was determined by annexin V-fluorescein isothiocyanate/propidium iodide staining and Hoechst 33342 staining. Cytokine concentrations in supernatant were measured. In vivo,hEPCs was labeled with carbocyanine near-infrared dye DiD prior to heart transplantation. Nude mice were allocated to the following treatment groups:sham group,MI + saline group (placebo group),MI + hEPCs group(EPCs group),MI + BK PC hEPCs group(BK PC group),MI + BK PC hEPCs + HOE140 group(BK PC/HOE group). Cardiac function was evaluated by echocardiography and postmortem analysis were assessed 10 days after transplantation. Results:In vitro,BK PC significanty promoted EPCs proliferation compared with the EPCs group(P < 0.01). Cell apoptosis rate post hypoxia was significantly reduced in the BK PC group compared with that in the EPCs group(P < 0.01),and was blocked by HOE140(P < 0.01). VEGF level was also significantly up-regulated in the BK PC group compared with that in the EPCs group(P < 0.01),and was also blocked by HOE140(P < 0.01). The BK PC group showed robust cell apoptosis inhibition,infarct size reduction,and cardiac function improvement in vivo and these effects were abrogated by the HOE140. Conclusion:Efficacy of BK PC hEPCs on improving cardiac function was greater than those by hEPCs in the mouse ischemia model. Its therapeutic effect could be achieved by promoting cell survival and resisting transplanted cells and host cardiomyocytes apoptosis.

Abstract:Objective:To investigate the effects and mechanisms of lipoxin A4(LXA4)in attenuating hypoxia/reoxygenation injury in human renal tubular epithelial cells(HK-2). Methods:HK-2 cells were exposed to hypoxia followed by reoxygenation with pretreatment of LXA4. Then the cell viability,γ-GT,NAG and LAP levels,SOD activity and MDA level were determined. The expressions of mRNA and protein of PPARγ and HO-1 were measured using real-time polymerase chain reaction (PCR) and Western blot,respectively. The expressions of HO-1 and Nrf2 were detected by using RNA interference technology through interference PPARγ expression. Results:Pretreatment with LXA4 increased the cell viability and SOD activity,and reduced the γ-GT,NAG,LAP and MDA levels. HO-1 inhibition by ZnPP and siRNA of PPARγ abolished the protective role of LXA4 on the cells undergoing hypoxia/reoxygenation injury. Furthermore,the expressions of HO-1,PPARγ and Nrf2 were increased in the cells pretreated with LXA4 significantly,whereas these overexpressions of HO-1 and Nrf2 were partly blocked by treatment with siRNA of PPARγ. Conclusion:This study reveals that LXA4 pretreatment serves a protective role against hypoxia/reoxygenation injury of human renal tubular epithelial cells via over-expression of HO-1 and activation of PPARγ/Nrf2.

Abstract:Objective:To detect the expression and study the role of nephroblastoma overexpressed gene (NOV CCN3)in hepatocelluar carcinoma(HCC). Methods:The expressions of CCN3 mRNA in HCC samples and cell lines were detected by real-time PCR,and the expressions of CCN3 protein in HCC tissues and adjacent non-cancer tissues were assessed by Western blot. In addition,invasion of HCC cells was observed after overexpressing CCN3 by Transwell invasion assay and wound healing assay. Results:In the total of 50 paired HCC specimens,compared with the adjacent non-cancer tissues,the expression of CCN3 mRNA was up-regulated in 39 cases,and the expression of CCN3 protein in HCC tissues was higher than that in adjacent non-cancer tissues. Overexpression of CCN3 enhanced HCC-derived MHCC-97H,SMMC-7721 cellular invasion in vitro. Conclusion:This study indicates CCN3 is frequently overexpressed in hepatocellular carcinoma,and contributes to tumor cell invasion. The study of CCN3 may provide a novel therapeutic idea for prevention and treatment of invasion in HCC.

Abstract:Objective:To investigate the effects and mechanism of BRD4 inhibitor JQ1 on the growth of human non-small cell lung cancer (NSCLC)cells including erlotinib resistant cells. Methods:The inhibitory effects of JQ1 on the growth of human NSCLC were observed at different concentrations for 72 hours by sulforhodamine B (SRB)assay. mRNA and protein levels of eIF4E were detected by quantitative real-time PCR assay and Western blot,respectively. Results:JQ1 inhibited the growth of NSCLC cells including erlotinib parental and resistant cells in a dose dependent manner. The mRNA and protein expression levels of eIF4E decreased significantly by JQ1 treatment. Conclusion:JQ1 inhibits the growth of NSCLC cells possibly through downregulating eIF4E expression.

Abstract:Objective:To investigate the effects of resveratrol on proliferation and apoptosis of human papillary thyroid carcinoma IHH4 cells and its possible mechanisms. Methods:The inhibition rate of resveratrol on IHH4 cells proliferation was detected by CCK-8 assay. Morphological changes of IHH4 cells were observed by inverted microscope,transmission electron microscope and DAPI staining,respectively. The expression of cleaved caspase-3 was detected by immunofluorescence cytochemistry and Western blot. The change of cell apoptosis and cell cycle distribution were analyzed by flow cytometry. Results:Resveratrol inhibited the growth of IHH4 cells in a time- and dose-dependent manner. Compared with normal control,after resveratrol treatment,IHH4 cells shrank,turned round and began to shed off,various sizes of vacuoles appeared in the cytoplasm,intracytoplasmic content increased under optical microscope. The transmission electron microscope examination showed pyknosis and margination of apoptotic cell nucleus chromatin. DAPI staining showed that the normal nucleichromatin was relatively dispersed with regular shape and smooth surface,while cells treated with resveratrol displayed condensed and fragmented nuclei,which were typical features of apoptosis. Immunofluorescence cytochemistry and Western blot revealed that the expression of cleaved caspase-3 increased significantly in a time- and dose-dependent manner. Resveratrol induced apoptosis of IHH4 cells in a dose-dependent manner with S phase cycle arrest. Conclusion:Resveratrol could inhibit proliferation and induce apoptosis of human IHH4 cells in vitro. One of the possible mechanisms is related to S phase cycle arrest.

Abstract:Objective:The aim of this study was to investigate in patients with type 1 diabetes (T1D):the prevalence of thyroid autoantibodies (T-Ab);the association between thyroid autoimmunity with either islet autoantibodies or thyroid function;and the differences of thyroid autoimmunity between adults and pediatrics. Methods:A total of 491 T1D patients were analyzed for thyroid autoimmunity in addition to islet autoantibodies and thyroid function. Results:Tweenty-two percentage T1D patients had thyroid autoantibodies (62% were female). Patients with T-Ab had higher prevalence of islet autoantibodies (all P <0.05). While thyroid peroxidase (TPOAb) was positively associated with all the 5 islet autoantibodies (all P < 0.05),thyroglobulin (TGAb) was only associated with glutamic acid decarboxylase antibody (GADA,P=0.005). In addition,the positive number of islet autoantibodies was positively associated with the prevalence of T-Ab (30.2%,P=0.000). Tweenty-two point six percentage adults with T1D had T-Ab,adults with double anti-thyroid autoantibodies positivity was more than that in the pediatric group (prevalence of double T-Abs:57.8% vs 36.4%,P=0.028). The presence of T-Ab was positively associated with serum thyroid-stimulating hormone (TSH) levels (P=0.008). The presence of both anti-thyroid antibodies was positively associated with thyroid dysfunction (prevalence of thyroid dysfunction:64.2% vs 30.9%,P=0.002). Conclusion:Thyroid autoimmunity was commonly happened in adults and pediatrics with T1D. Thyroid autoimmunity was associated with female gender,the persistence of GADA and multiple islet autoantibodies positivity. The risk of thyroid autoimmunity did not differ from adults to pediatrics;however,the thyroid autoimmunity was severe in the adults. Multiple anti-thyroid autoantibodies indicated thyroid dysfunction.

Abstract:Objective:To identify the positive rate of islet autoantibodies in patients with type 1 diabetes (T1D) according to age and disease duration. Methods: We collected the general information and laboratory tests of 537 patients diagnosed with T1D,and measured the levels of islet autoantibodies. The subjects were subdivided into different groups according to their age and disease duration. Then we investigated the association of islet autoantibodies with age and disease duration. Results:Among 537 patients,the average age and disease duration was 32.84 ± 17.98 year-old and 4.31 years,respectively. 75.4% of patients were positive for at least one islet autoanbotidy(55.7% within 5 year of the disease onset:44.1% after 5 year,P=0.032). 54.4% of the patients were positive for glutamic acid decarboxylase antibody (GADA),30.8% for protein tyrosine phosphatase antibody (IA-2A),28.2% for zinc transporter 8 antibody(ZnT8A) and 23.5% for islet cell antibody (ICA). The positive rate was significantly higher in patient whose age was less than 12-year-old than in those over 12 (GADA 76.9% vs 57.4%,IA-2A 65.4% vs 31.7%,ZnT8A 38.5 % vs 24.1%,at least one islet autoanbotidy 83.3% vs 75.7%). Conclusion:The rates of positive islet autoantibodies significantly decreased along with the disease duration. Younger patients had higher rate of positive islet autoantibodies. Therefore,more attention should be paid when type 1 diabetes is diagenozed in adults.

Abstract:Objective:To investigate the relationship of gall bladder diseases with insulin resistance (IR) and islet β cell function in people living in Gulou District,Nanjing. Methods:Questionnaire survey,physical examination,blood sample collection,OGTT and insulin release test were performed in 8 180 non-diabetic residents in Gulou district,Nanjing. We analyzed characteristics of different gall bladder-disease stages,compared IR and islet β cell function indexes and focus on the risk of diabetes in patients suffering from gall bladder diseases. Results:Gender,age,blood pressure,BMI,WHR,OGTT results,HbA1c,blood lipids,liver enzymes,life styles and incidence of diabetes showed differences in three groups. HOMA-IR,ISI and INSR120 differ in two gall bladder disease groups and the differences were statistical significant (P < 0.05). Post-cholecystectomy represents higher risk of diabetes as well as metabolic syndrome (MS). Conclusion:Gall bladder diseases are signs of IR. Post-cholecystectomy is a risk factor of diabetes in non-diabetic residents in Gulou District,Nanjing.

Abstract:Objective:To investigate the clinical characteristics of children’s white blood cells syndrome(HPS),diagnostic criteria,treatment process and related risk factors of prognosis. Methods:A retrospective analysis was conducted in 72 cases of children’s blood oncology of HPS treated in of 2009—2013 in Nanjing Children’s Hospital. Clinical manifestation,auxiliary examination,treatment and outcome,adopting Logistic methods were used to analyze the prognosis of children with risk factors. Results:In 72 cases,42 cases (58.3%) was HPS infection correlation,among which correlated with EB virus infection was the most conmon type. In the rest 30 cases,4 cases(5.56%) were related HPS infection,and 26 cases(36.1%)were unknown etiology. Clinical manifestatios included fever (91.7%),liver enlargement (83.3%),and spleen enlargement (66.7%). Peripheral blood routine test showed that the two of three lines decrease accounting to 72.2%,three lines decreased accounting to 27.8%,hemoglobin concentration reduction was 91.7%,the decrease of platelets was 77.8%. Elevated serum triglyceride was 38.9%. Coagulation routine inspection dysfunction was 41.7%. Elevated serum ferritin was 55.6%. Bone marrow cell morphology most found the white blood cells. Seventy-two cases of death in 10 cases (13.8%),improvement or basic recovery 24 cases (33.3%),14 cases (19.4%) lost to follow-up after discharge. Age < 3 years old, LDH > 2 500 U/L were the risk factos for adverse. Conclusions:Age and LDH leels are children that eats blood syndrome risk factors of poor prognosis,and should be treated early diagnosis and reduce the case fatality rate.

Abstract:Objective:To investigate the alteration of peripheral blood interleukin(IL)-4,IL-9,and IL-21 in patients with bronchial asthma. Methods:Forty-eight asthma patients in acute stage,41 patients in remission stage,and 48 sex and age paired healthy controls were enrolled in this study. The expression of IL-4,IL-9,and IL-21 from serum and peripheral blood mononuclear cells were analyzed by ELISA and real-time PCR. And the value of FEV1% and the expression level of serum IgE were detected by allergy method and ELISA respectively. Results:①The mRNA and protein expression levels of IL-4,IL-9,and IL-21 were significantly increased in acute asthma patients compared with patients in remission group (P < 0.05 or P < 0.01). Also the mRNA and protein expression levels of IL-4 and IL-9 were significantly higher in remission group than that of control group (P < 0.05 or P < 0.01). But there was no significant difference in the mRNA and protein expression levels of IL-21 between remission group and control group (P > 0.05). ②In asthma patients,no correlation was found among the serum concentration of IL-4,IL-9 and IL-21,except of correlation between IL-9 and IL-4. The serum levels of IL-4 and IL-9 from asthma patients were positively correlated with the serum level of total IgE (P < 0.05) but negatively correlated with FEV1% (P < 0.05). But no obvious correlation was found between the serum concentration of IL-21 and the serum level of total IgE or FEV1% (P > 0.05). Conclusion:IL-4,IL-9,and IL-21 may play important roles in the pathogenesis of bronchial asthma. IL-4 and IL-9 monitoring allows for an assessment of the asthma status which is helpful to guide clinical treatment.

Abstract:Objective:To detect the NJ001 antibody specific antigen expression in pancreatic cancer(PCa),and to investigate its relationship with clinicopathological features and prognosis value. Methods:The expression of NJ001 antibody specific antigen was evaluated through immunohistochemical staining in 89 PCa and 17 benign pancreatic disease. The results were scored by semiquantitative analysis. We analyzed the correlation of the results with clinicopathological parameters,and statistical analysis was evaluated by chi-square test or Fisher exact test. We also assessed the prognosis value using Kaplan-Meier survival curves. Results:The expression of this specific antigen was significantly higher in PCa tissues compared with benign pancreatic diseases(77.5% vs 17.6%,P < 0.001). We further found that the positive rate of NJ001 specific antigen in poor differentiation PCa patients was higher than that of well differentiation patients(83.0% vs 60.0%,P = 0.027). Meanwhile,the positive rate in patients with deep depth of tumor invasion or lymph nodes metastasis was higher than that of low invasion cases or no metastasis cases,respectively(82.2% vs 53.3%,P = 0.037;91.1% vs 62.8%,P = 0.002). Overall survival for patients with positive expression of the specific antigen was lower than that with negative expression(P= 0.036). Conclusion:The NJ001 antibody specific antigen expression in PCa was associated with differentiation,depth of invasion,lymph node metastasis and prognosis,suggesting the antigen may be a promising tumor marker for monitoring the development of Pca.

Abstract:Objective:To investigate whether the biomarker of uCTX-Ⅱ has a diagnostic and prognostic value in the early stage osteoarthritis or not. Methods:Forty patients with clinical diagnosis of early stage knee osteoarthritis and 35 patients with progression stage osteoarthritis were randomly assigned to receive 1 500 mg of glucosamine daily,100 mg of diacerein daily or both glucosamine and diacerein. Disease symptoms were assessed quarterly by WOMAC scoring. X-ray analysis was performed as baseline. uCTX-Ⅱ level in urinary were measured as a baseline,6 months,12 months after the initiation of the intervention. The parameters were also corrected for creatinine treatment. Results:A clinically detectable symptomatic benefit was observed as early as 3 months in all groups. In the combined group and the diacerein group,the WOMAC scores decreased singnificantly than that of the glucosamine goup(P < 0.05). The CTX-Ⅱ levels measured in early stage osteoarthritis was significantly higher in urine samples than that from the healthy controls(P < 0.05). A correlation was seen between the uCTX-Ⅱ levers and the WOMAC scores using bivariate analysis. Conclusion:The biomarker of uCTX-Ⅱhad diagnostic and prognostic value in the early stage oteoarthritis. Glucosamine and diacerein alone or combination could reduce pain effectively in the all groups of patients. The combination of glucosamine and diacerein may be more effective.

Abstract:Objective:To compare the clinical outcomes of Jack vertebral dilator-kyphoplasty (DKP)and balloon kyphoplasty(BKP)in treatment of osteoporotic vertebral compression fractures (OVCF). Methods:A retrospective study was conducted about 56 single OVCF cases from January 2010 to December 2012. Thirty-three cases were treated with DKP,while other 23 cases were treated with BKP procedures.The operating time,bleeding volume,cement injected volume were recorded during operation. The patients’ visual analogue scales (VAS)score and Oswestry disability index (ODI)score were also evaluated. The distribution of cement,the restoration of vertebral height and Cobb angle were also observed post-operation. Results:There were no significant differences in operating time,bleeding volume and cement injected volume between two groups (P > 0.05). The vertebral height,Cobb angle,VAS and ODI scores were significantly improved by those two operations (P < 0.05). The height of the anterior vertebrae and Cobb angle in DKP groups were restored more significantly than that of BKP group (P < 0.05). Cement leakages happened in one case (3.0%)in DKP group and in 5 cases (21.7%)in BKP group. Conclusion:DKP and BKP were both effective and safe for the treatment of OVCF. The height of anterior vertebrae and Cobb angle in DKP group were restored significantly than those in BKP groups,and there is lower cement leakage rate in DKP than that of BKP.

Abstract:Objective:To investigate the diagnostic value of real-time shear wave elastography(SWE)technology for the assessment of liver fibrosis in chronic hepatitis B (CHB)patients. Methods:Hepatic elastic modulus of 60 health control people and 68 patients with CHB were measured by SWE,and 68 patients had liver biopsy. The elastic modulus was compared with pathological stages of hepatic fibrosis. Results:The modulus of elasticity had a statistically significant difference in health control people and liver fibrosis patients. The elastic modulus of each pathological stages of hepatic fibrosis included S1 at (7.171 ± 1.819)kpa (n=14),S2 at(10.300 ± 3.121)kPa(n=18),S3 at(15.540 ± 4.341)kPa(n=20),and S4 at(23.919 ± 5.698)kPa(n=16). The elastic modulus of each pathological stages had statistically significant difference (F=50.807,P < 0.001). The elastic modulus was correlated with pathological stages in a correlation coefficient of 0.783 (P < 0.001). The ROC curve was set up by evaluating the pathological stages through elastic modulus. Areas under ROC curve of 0.940 for ≥S2 patients,of 0.947 for ≥S3 patients,of 0.949 for S4 patients. Conclusion:SWE has certain diagnostic efficiency in evaluating liver fibrosis stage in CHB patients.