Abstract:Objective:To investigate the mechanism of aberrant Sonic hedgehog (Shh) signaling which contributes to acquired resistance to gemcitabine of pancreatic cancer cells SW1990. Methods:Gemcitabine-resistant pancreatic cancer cells SW1990-GEM were induced by increasing drug dosage intermittently. Quantitative real-time PCR and Western blot assay were used to evaluate the relative expression level of PIAS1 in SW1990 cells and SW1990-GEM cells. The ability of PIAS1 promoting acquired resistance to gemcitabine through up-regulating Shh signaling was evaluated by cell proliferation,soft agar colony formation assay and nude mice tumor transplantation assay. Results:SW1990-GEM (resistant strains) showed a high expression level of PIAS1. The overexpression of PIAS1 was involved in the increased cell proliferation and tumor formation ability of SW1990-GEM cells. Knockdown of PIAS1 in SW1990-GEM reduced the activity of Shh signaling thereby decreasing the drug resistance ability of this strain. Conclusion:PIAS1 is the key factor for acquired resistance to gemcitabine in pancreatice cancer cell SW1990 by up-regulating Shh signaling.

Abstract:Objective:To investigate the role of PUF family genes [Pumilio1 (Pum1) and Pumilio2 (Pum2)] in female fertility and meiotic maturation. Methods: We deleted the Pum genes by breeding mice harboring loxP sites in Pum1 (Pum1F/F Pum2-/-) with DEAD box polypeptide 4 (Vasa) promoter-mediated Cre recombinase mice (VDKO). Meanwhile. we analyzed the phenotype of VDKO mice. Results: Fertility test showed that female fertility of VDKO mice was reduced, but the mice could still produce offspring. However, in vitro meiotic maturation of VDKO oocytes was not affected. Conclusion: Pum proteins were not essential for mouse folliculogenesis or oocyte development.

Abstract:Objective:To validate fatty acid amide hydrolase (FAAH) expression in mouse spermatogonial stem cells (SSCs). Methods: We established a stable mouse SSCs line in vitro and verified FAAH expression in various tissues of mice and the cell line by Western blotting assay. Immunofluorescence was performed to detect FAAH location in the testis and SSCs. Results: Western blotting assay showed that FAAH was highly expressed in testicular tissues. Double-immunofluorescent staining showed that FAAH located on the membrane of mouse SSCs and co-localized with SSCs separation marker, GDNF family receptor alpha-1 (GFRα1). Conclusion: FAAH is a new candidate separation surface marker of SSCs, which provides a basis for the research of FAAH function in spermatogenesis.

Abstract:Objective:To clone the promoter sequences of stimulator of interferon gene (STING) and evaluate its activity, and to preliminarily investigate the transcriptional regulatory mechanisms. Methods: Promoter region was predicted by bioinformatics methods, and the 1 005 bp (-927~+77) fragment of 5′ upstream sequences of STING gene was amplified by PCR, and then cloned to pGL3-basic vector to construct the luciferase report gene recombinant plasmid. Three promoter fragments with different length were obtained by walking deletion and cloned into pGL3-basic vector. The vector expression activities were determined by transfection of the mouse NIH3T3 cells with the recombinant plasmids of STING gene promoter. Bioinformatics methods were performed to predict the potential transcriptional factor binding sequences. Resluts: The luciferase reporter gene recombinant vectors of mouse STING promoter were successfully constructed. Compared with the pGL3-basic plasmid, the relative luciferase activities of recombinant vectors of STING promoter were much higher (P < 0.05). In addition, the binding sequences of GATA, IK2, MZF1, SP1/SP3, and STAT may be included in the promoter region (-177~-48) of STING gene,which were predicted by the bioinformatics method. Conclusion: The luciferase report gene recombinant plasmids of STING gene promotor were constructed successfully and had strong transcriptional activity in NIH3T3 cells. By the activity comparison, it is speculated that the core promoter region of mouse STING is located in the -177~+77 region, which may contain a number of potential transcription factor binding sequences.

Abstract:Objective:To analyze lncRNA expression profiles in MIN6 cells,which belong to mouse pancreatic β cells, exposed to cytokines (IL-1β, TNF-α and IFN-γ) and to perform preliminary validation. Methods: MIN6 cells were incubated in the presence or absence of the cytokines mentioned aboved for 24 h. Cell apoptosis was detected by flow cytometry and cell viability was assessed using the Cell Counting kit-8. Affymetrix GeneChip Mouse Transcriptome Array 1.0 was performed to identify differently expressed lncRNA and mRNA in MIN6 cells exposed to cytokines compared with the normal control group. The interesting candidate lncRNAs were verified by Real-time quantitative PCR. Results: Cytokines promoted MIN6 cells apoptosis and inhibited the viability of MIN6 cells. A total of 723 differently expressed lncRNAs were identified in the cytokine-stimulated group compared with the control group with a set filter fold-change ≥ 2.0, of which 444 upregulated and 279 downregulated. Additionally, 111 differently expressed lncRNAs were identified with a set filter fold-change ≥ 5.0, of which 105 upregulated and 6 downregulated. And 2 180 differently expressed mRNAs were identified with a set filter fold-change ≥ 1.5. We also found, via quantitative PCR, that 10 lncRNAs were aberrantly expressed in the cytokine-stimulated group compared with the control group. The results of the qRT-PCR were consistent with the data from the microarray. Conclusion: The results revealed that there are differently expressed lncRNAs in MIN6 cells exposed to cytokines compared with the control group. These lncRNAs may play a key role in cytokine-induced apoptosis of pancreatic β cells.

Abstract:Objective:To investigate the effects and underlying mechanism of high mobility group protein box 1(HMGB1) on the proliferation and migration of human airway smooth muscle cells(HASMCs). Methods:Normal HASMC between passages 3 and 8 were randomly divided into the control group, HMGB1 group with different concentrations(125, 250, 500, and 1 000 ng/mL), and TGF-β(1 ng/mL) group as a positive control, respectively. The HMGB1-stimulated proliferation of HASMCs was evaluated by cell counting of CCK-8. The number of migrated cells was performed using Transwell migration assay. Western blotting assay was performed to detect the phosphorylation of Akt following HMGB1 stimulation. Result: The proliferation of HASMCs treated with HMGB1 was significantly increased in a dose-dependent manner than those of untreated control group (P < 0.05). Furthermore, the number of migrated cells was significantly increased after HMGB1 activation compared to the control group(P < 0.01). The phosphorylation level of Akt in the HMGB1 group(500 ng/mL) was significantly increased compared with the control group(P < 0.05). Conclusion:Our data suggest that HMGB1 contributes to airway remodeling by stimulating the proliferation and migration of HASMCs through regulating phosphorylation of PI3K/Akt pathway, which provides a promising target for future therapy of asthma.

Abstract:Objective:To observe the effects of chrysin on the expression of nuclear factor (NF)-κB in lung tissues in a murine model of asthma. Methods: Twenty-four female BALB/c mice were randomly divided into 4 groups, including control group, asthma group, chrysin group and budesonide group. BALB/c mice were sensitized by intraperitoneal injection and challenged via the airway with ovalbumin(OVA). Mice in the chrysin group were intragastrically administered with chrysin (50 mg/kg). Mice in the budesonide group were exposed to aerosolized budesonide. Pulmonary functions were measured to evaluate the resistance of expiration. The sections were stained with either hematoxylin & eosin to assess the inflammatory cell infiltrates. The levels of interleukin IL-4 and IL-13 in bronchoalveolar lavage fluid (BALF) and total immunoglobulin E (IgE) levels in serum were measured by ELISA. The protein expression of NF-κB in lung tissues was determined by Western blotting assay. Results: Airway inflammation and airway hyperresponsiveness (AHR) were increased in the asthma group. Chrysin significantly inhibited the airway inflammation and AHR, and decreased the levels of IL-4 and IL-13 in BLAF and total IgE levels in serum. In addition, chrysin significantly attenuated the increased protein expression of NF-κB in lung tissues of asthmatic mice. Conclusion: This study demonstrated that chrysin could suppress the progression of airway inflammation and AHR in a murine model of asthma. The effect may be due to inhibition of NF-κB expression.

Abstract:Objective:To investigate the effects of IL-11 on mouse liver warm ischemia/reperfusion (WI/Rp) injury. Methods: A total of 20 healthy male C57BL/6 mice, weighing (22 ± 3) g, were randomly divided into four main experimental groups (n=5 each), including normal group, sham group, ischemia/reperfusion (I/R) group, and IL-11 pretreatment group. We chose a nonfatal model of 70% liver WI/Rp (treated with 1 h ischemia ,and then 6 h reperfusion). The mice of I/R group were injected with PBS, and IL-11 pretreatment group with IL-11, at 2 h before operation. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) in serum were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of IL-6, IL-1β and TNF-α were examined by reverse transcription-polymerase chain reaction (RT-PCR). Histological haema (HE) stained sections were histopathologically examined using light microscopy. Results: The liver enzyme levels were significantly increased in the I/R and IL-11 pretreatment group, compared to those in the normal and sham group (P < 0.05). Meanwhile, the transaminase levels in the IL-11 pretreatment group were significantly reduced compared to those in the I/R group (ALT:P=0.003,and AST:P < 0.001, respectively). Liver cell hydropic and acidophilic degeneration were increased in the I/R and IL-11 pretreatment group, compared to those in the normal and sham group. Besides, the cell hydropic and acidophilic degeneration in the IL-11 pretreatment group was significantly reduced, compared to that in the I/R group. The expressions of IL-6, IL-1β and TNF-α of blood serum and liver tissue were reduced in the IL-11 pretreatment group, compared to those in the I/R group (P=0.0025, P=0.0159, P=0.0175, P=0.0076, P=0.0005, and P=0.0004, respectively). Conclusions: Pretreatment with IL-11 protects mouse livers from WI/Rp injury by suppressing the expressions of IL-6, IL-1β and TNF-α.

Abstract:Objective:To investigate the expression and function of CCN1 in hepatic ischemia-reperfusion (I/R)injury in mice. Methods:Partial warm ischemia was produced in the left and middle hepatic lobes of mice for 60 min,followed by reperfusion. Expression of CCN1 was detected by real-time PCR and Western blot at timed points. In indicated experiments,recombinant CCN1 or saline was injected via tail vein 1 h before hepatic ischemia. Levels of alanine aminotransferase (ALT)and aspartate aminotransferase(AST)in blood were examined at 6 h of reperfusion. Pathological analysis was performed with liver sections after hematoxylin and eosin staining. mRNA levels of cytokines were detected by real-time PCR. Results:CCN1 expression is up-regulated in hepatic I/R. Serum ALT and AST levels were significantly increased after CCN1 treatment. The pathological alterations were more severe compared with saline group. Gene transcripts for proinflammatory cytokines in liver tissue were elevated significantly in the CCN1 group. Conclusion:Induction of CCN1 in liver I/R aggravates hepatic injury,possibly by promoting the expression of inflammatory factors.

Abstract:Objective:To investigate the effects of 5 nm gold nanoparticles(GNPs) on newborn rat cortical neurons and the underlying mechanism. Methods:GNPs were prepared by the NaBH4 reduction method. Transmission electron microscope(TEM) and ultraviolet-visible(UV-Vis) spectra were obtained to characterize GNPs. Immunofluorescence staining was performed to indentify primary neurons and calculate the purity. After culture for 72 h, primary cortical neurons were incubated with neuron culture medium with(600, 1 200, 2 400 μg/L) or without GNPs for 48 h. TEM was used to investigate the celluar uptake and distribution of GNPs; Cell counting kit-8(CCK-8) was employed to evaluate cell viability; TUNEL assay was performed to analyze apoptosis; MDA and SOD were measured to assess oxidative stress. Results:TEM and UV-Vis showed that GNPs were homogeneous in size and shape and well dispersed in culture medium. The purity of primary neurons was(82.7 ± 2.3)%. GNPs were mainly distributed in cytoplasm, lysosomes and vesicles. The cell viability was found to decrease with increasing concentrations of GNPs(P < 0.05). In addition, GNPs were found to induce apoptosis in neurons. Particularly, higher concentrations(1 200 μg/L and 2 400 μg/L) markedly increased neural apoptosis(P < 0.05). Furthermore, our results suggested pro-oxidant ability of 5 nm GNPs,which is supported by escalating MDA and reducing SOD along with increasing concentrations of GNPs(P < 0.05). Conclusion:Five nm GNPs can significantly reduce cell viability and induce apoposis in newborn rat cortical neurons, which potentially links to oxidative stress.

Abstract:Objective:To observe the clinical significance of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and Cystatin C (Cys-C) in diagnosing elderly patients with chronic heart failure (CHF) and evaluating classification of cardiac function and prognosis. Methods: A total of 178 elderly patients with CHF were selected as the observation group, including 35 cases of cardiac function grade Ⅰ, 47 cases of gradeⅡ, 64 cases of grade Ⅲ, 32 cases of grade Ⅳ according to New York Heart Association Functional Classification (NYHA), and 87 healthy people taking health examination were selected as the healthy control group. NT-proBNP and Cys-C levels were detected, and left ventricular end-diastolic diameter (LVEDD), 1eft ventricular ejection fraction (LVEF) were measured. NT-proBNP and Cys-C levels were compared between the observation group and the healthy control group, and between patients with different cardiac function grades. Logistic regression analysis was performed to analyze the influencing factors of death end events in elderly patients with CHF, and the correlation among NT-proBNP, Cys-C and LVEDD, LVEF was analyzed by using Spearman correlation statistics. Results: NT-proBNP level, Cys-C level and LVEDD in the observation group were higher than those in the healthy control group, LVEF in the observation group was lower than that in the healthy control group (P < 0.05). NT-proBNP, Cys-C, and LVEDD were gradually increased from cardiac function grade Ⅰ to grade Ⅳ, while LVEF was gradually decreased (P < 0.05). Logistic regression analysis showed that NT-proBNP, Cys-C, and LVEF were risk factors of death of elderly CHF patients (P < 0.05). Correlation analysis showed that NT-proBNP was positively correlated with LVEDD (r=0.824, P < 0.05), while negatively correlated with LVEF (r=-0.723, P < 0.05), Cys-C was positively correlated with LVEDD (r=0.797, P < 0.05), while negatively correlated with LVEF (r=-0.714, P < 0.05). Conclusion: NT-proBNP and Cys-C level are useful in diagnosing elderly CHF patients and evaluating classification of cardiac function, and have certain predictive value for prognosis of elderly CHF patients.

Abstract:Objective:This study was designed to investigate prognosis differences among different negative induction test results in patients with paroxysmal atrial fibrillation underwent circumferential pulmonary vein isolation(CPVI). Methods:We retrospectively studied 133 patients (PTs)who underwent catheter ablation due to paroxysmal atrial fibrillation with the endpoint of non-inducibility(defined as atrial arrhythmias could not be induced or can be induced but lasted less than 3 minutes). The induction protocol was listed as follows:after successful CPVI,which was defined as completion of ablation set and bidirectional blockade of pulmonary vein(PV)-left atrium (LA)conduction,decremented burst stimulation (10 mA,2 ms pulse width)was attempted at coronary sinus orifice(CSo),and distal of coronary sinus (CSd)from 300 ms to loss of atrium capture (1∶1). If sustained (lasting >3 minutes)atrial arrhythmias were induced,the key site would be identified and further ablation performed. According to different negative induction test results at endpoint,patients were divided into group A (atrial arrhythmias could not be induced)and group B (atrial arrhythmias could be induced with duration< 3 minutes). All the patients were followed up regularly and receive free ECG/Holter. The blanking period was 3 months. Results:According to induction test results,74 PTs(55.6%) were detected in group A and 59 PTs(44.4%) in group B. After a mean follow-up of (21.3 ± 10.9)months,25 PTs in group A and 20 PTs in group B had AF relapse since the index ablation. Kaplan-Meier survival analysis showed no significant differences of AF relapse ratio between group A and B (P = 0.74). Conclusion:For patients with paroxysmal AF who underwent CPVI with the endpoint of non-inducibility,those who atrial arrhythmias could not be induced at the endpoint do not promise a better prognosis than those who have short(duration< 3 minutes)atrial arrhythmias could still be induced.

Abstract:Objective:To examine NJ001 recognized antigen expression in surgically resected human malignant tumor tissues,and to evaluate the relationship between its expression and tumor differentiation. Methods:NJ001 recognized antigen expression was immunohistochemically studied in lung adenocarcinoma,lung squamous carcinoma,pancreatic cancer,breast cancer,esophageal cancer,gastric cancer,liver cancer and matched non-neoplastic tissues,and all positive cases were compared on tumor differentiation. The results were scored by semiquantitative analysis,and statistical analysis was evaluated by chi-square test or Fisher exact test. Results:Expression of NJ001 recognized antigen was significantly high in lung adenocarcinoma (88.0%),lung squamous carcinoma(82.2%),pancreatic cancer(73.3%)and breast cancer(57.8%),and was significantly correlated with lower differentiation degree in all these tumors (P < 0.001 in lung adenocarcinoma;P=0.002 in lung squamous carcinoma;P=0.002 in pancreatic cancer;and P=0.024 in breast cancer). Conclusion:NJ001 recognized antigen is expected to be a tumor marker in lung adenocarcinoma,lung squamous carcinoma,breast cancer and pancreatic cancer,as well as a remarkable biomarker for identification of differentiation degree in multiple neoplasms.

Abstract:Objective:To analyze the relationship between epidermal growth factor receptor(EGFR) gene,EML4-ALK fusion gene and clinical features in patients with non-small cell lung cancer(NSCLC). Methods:Mutations of exons 19 and 21 of EGFR in 252 NSCLCs were detected by PCR amplification and gene sequencing, EML4-ALK fusion gene mutations were detected by Real-time PCR. The relationships between mutations and clinical features of NSCLCs were analyzed by SPSS software. Results:The total mutation rate of EGFR gene was 38.8%, including 15.8% of exon 19 and 23.0% of exon 21, respectively, and EGFR mutations usually occurred in the female, non-smokers and adenocarcinoma patients(P < 0.05). However, there was no relationship in age(P > 0.05). The total mutation rate of EML4-ALK fusion gene was 4.7%, EML4-ALK fusion gene mutations usually occurred in the female and younger age patients(P < 0.05). Mutations were not related to non-smoking and pathological type (P > 0.05). No mutation was detected to coexist in EGFR and EML4-ALK gene mutation. Conclusion:Due to high mutation frequency of EGFR in Chinese NSCLC patients, it is highly recommended to investigate EGFR gene mutations before treatment with tyrosine kinase inhibitors. EML4-ALK fusion gene defines another molecular subset of NSCLC with distinct characteristics, which provides a new option for the clinical treatment of patients with NSCLC. The coexistence phenomenon of EGFR mutation and EML4-ALK mutation is rare.

Abstract:Objective:To identify the factors influencing 24 h mean amplitude of glycemic excursion (MAGE) in patients newly diagnosed with type 2 diabetes. Methods:We analyzed the clinical data of 110 patients with type 2 diabetes without treatment. The 24 h MAGE was calculated using 3 d continuous glucose monitoring system (CGMS). Baseline data of patients were collected, and serum hemoglobin A1c(HbA1c), fasting blood glucose(FBG) and postprandial 2 h blood glucose(PBG), insulin and C peptide levels, blood lipid profiles, hepatic and renal functions were measured before treatment. Results:HbA1c was an independent index for 24 h MAGE in newly diagnosed type 2 diabetes mellitus patients (P < 0.05). MAGE in patients with HbA1c < 9.9% was lower than that with HbA1c ≥9.9%(5.97 mmol/L vs. 7.43 mmol/L, P < 0.05). HbA1c in patients with HbA1c < 8.5% was effected mainly by PBG, while FBG was an independent factor influencing HbA1c in newly diagnosed type 2 diabetes mellitus patients with HbA1c≥9.2%(P < 0.001). HbA1c in patients with FBG < 10.84 mmol/L was lower than that with FBG≥10.84 mmol/L (9.10% vs. 10.87%, P < 0.001). Conclusion:HbA1c is an independent factor of MAGE and closely related to FBG in patients newly diagnosed with type 2 diabetes mellitus.

Abstract:Objective:To investigate blood lipid profile and its changes of pregnant women,and to analyze if dyslipidemia occurred in those who suffered from gestational diabetes mellitus (GDM) or delivery of macrosomia. Methods: Based on the multi-center cohort study for reference intervals of gestational weight gain, total 516 singleton pregnant women were recruited from Danyang city, Jiangsu Province into the study for observation on serum lipid profiles during gestation. The general information of the subjects was collected and serum lipid [triglycerides(TG), total cholesterol(TC), low density lipoproten-cholesterol(LDL-C), and high density lipoprotein-cholesterol (HDL-C)] concentrations were tested in three trimesters of gestation. The complications during gestation and conditions of newborns were recorded as well. The lipid profiles during gestation were compared between women who delivered macrosomias and who did not, and between women with GDM and normal pregnancies. The lipid profiles were compared according to pregestational body mass index (BMI) levels in the first and third trimesters as well. Results: The serum lipid levels increased, the rate of HDL-C/TC decreased, and the rate of HDL-C/TC increased with progress of gestation in the investigated pregnant women (both P < 0.05). In same stage of pregnancy, there was little difference in lipid profiles between women who delivered macrosomias and who did not (both P > 0.05). The GDM women had higher serum TG level in first and second trimesters than normal pregnancies (both P < 0.05). Women with pregestational BMI≥24 kg/m2 were more likely to suffer from dyslipidemia in early pregnancy, and GDM morbidity of them was also higher than that of women with pregestational BMI<24 kg/m2. In our study, there was little difference in lipid profiles among different pregestational BMI levels in the third trimester. Conclusion: Hypertriglyceridemia in the first and second trimesters may be associated with higher risk of GDM. The women who are intended to conceive should keep a normal BMI to avoid dyslipidemia in early pregnancy.

Abstract:Objective:To identify the potential factors influencing the water quality of dental unit waterlines(DUWLs) by examining water samples collected from dental chair units(DCUs) from different departments, brands and service lives. Methods: Water samples were collected from 33 DCUs at the Affiliated Hospital of Stomatology, NJMU. Before patients arrived, a water sample of approximately 50 mL was collected from each DCU using a three-in-one air/water syringe. The water sample was diluted, and 0.1 mL of a prepared sample was spread on a sterilized R2A plate. After incubation, the number of microbial colony-forming units per millilitre was quantified with an automatic colony analyser. Statistical analyses were performed using SPSS 19.0 software. Results: According to the statistical analyses, no significant differences of the water quality were found in SIRONA C8+ with different service lives. However, the comparison of the water quality in different departments and brands showed significant difference(P≤0.05). SIRONA C8+DUWLs and DCUs of Endodontics Departments provided better quality water. Conclusion:Different service lives did not significantly influence the water quality of DUWLs. However, different DCU brands and different departments can influence the water quality. In this study, SIRONA C8+DUWLs and DCUs of Endodontics Departments provided better quality water.

Abstract:Objective:To establish a stable PNPLA7-expressed human hepatoma cell line (Huh7) and observe the effect of stable overexpression of PNPLA7 on the lipid contents in the cell line. Methods: pCMV6-PNPLA7-Flag plasmid was constructed and transfected into Huh7 cells with lipofectamine 2000 reagent. After G418 selection, mRNA and protein expression levels of PNPLA7 were examined by Western blot in these stable cell lines. Treated cells with oleic acid and the lipid contents in these cell lines were observed by oil red O staining. Results: Two lines stably expressing PNPLA7 were gained,and showed high expression level of PNPLA7 by Western blot analysis. The decreased lipid contents were observed in these cell lines after oleic acid treatment by oil red O staining. Conclusion: Huh7 cell lines stably expressing PNPLA7- Flag were established successfully. Stable overexpression of PNPLA7 may lead to the decreased lipid content in Huh7 cells after oleic acid treatment. It can be a reliable cell model for the study of functions and mechanisms of PNPLA7 in lipid metabolic pathways.

Abstract:Objective:To establish human embryonic stem cell (hESC)lines labeled with red fluorescent protein (RFP). Methods:The RFP expression vector pEF1α-DsRed-Express2 was transfected into hESCs by optimized nucleofection method. Alkaline phosphatase (AP) staining,RT-PCR,immunofluorescent staining and embryoid body (EB) formation were performed to compare the characteristics of hESCs before and after nucleofection. Results:AP staining of RFP-hESCs was positive. The expression of Oct4,Sox2,Klf4 and Nanog of hESCs were no difference before and after nucleofection (P > 0.05). RFP-hESCs could differentiate into the three germ layers and could stably passage over 30 generations. Conclusion:hESC cell lines stably expressing RFP has been successfully established in this study. RFP does not affect the pluripotency properties of hESCs. The cell lines can be used as seed cells for subsequent research.

Abstract:Objective:To genetically engineer an anti-H7N9 hemagglutinin Fab gene obtained by screening a full human Fab phage antibody library and to express it by antibody engineering, as well as verify its neutralizing activity to H7N9 avain influenza viruses. Methods: We screened a full human Fab phage antibody library using H7N9 avain influenza virus hemagglutinin and constructed IgG heavy and light chain expression vectors. Both vectors were transfected into 293 FreeStyle (293F) cells and the IgG antibody was expressed and purified. Then, the immunological activity was detected by ELISA assay and Western blotting assay, and the neutralizing activity was detected by microneutralization assay and prophylactic protection test in embryonated eggs. The hemagglutinin binding subunit was speculated by hemagglutination inhibition test. Results: A full human anti-H7N9 hemagglutinin Fab gene was successfully obtained and the IgG eukaryotic expression vectors were constructed. The sequences of both the heavy and the light chains of the antibody were correct and the antibody could bind to H7N9 hemagglutinin specifically. The antibody showed obvious neutralizing activity against H7N9 avain influenza viruses in microneutralization assay, and the neutralizing titer was 15.6 μg/mL. A dose of 200 μg IgG conferred a 100% survival rate in embryonated eggs. Hemagglutination inhibition test indicated that the IgG may bind the HA2 subunit of hemagglutinin. Conclusion: The reconstructed full human anti-H7N9 hemagglutinin IgG antibody could specifically recognize H7N9 avain influenza hemagglutinin and could obviously neutralize the virus, and laid a solid foundation for the further development of antibody drugs for the prophylaxis and treatment of avain influenza.

Abstract:Objective:To construct a new direct amplification PCR method to detect HBV rtA181T drug resistant mutation by nested PCR and specific primers. Methods:Nested PCR was performed to amplify the HBV polymerase rt domain fragment in the first round, and the base in the 3′end of the sense primer was designed same as the mutation one of HBV DNA rtA181T in the second round. Thus, the amplified fragment was the HBV DNA rtA181T mutation fragment. Using this method, we examined 43 specimens of various HBV DNA loading with HBV DNA rtA181T mutation and analyzed the sensitivity of this method; and compared it with the PCR-Sanger sequencing method in consistency under the level of low or high copy DNA HBV. Results:Confirmed by PCR products sequencing, this nested PCR based specific primers had successfully detected the HBV DNA rtA181T drug resistant mutation. This method had confirmed rtA181T drug resistant mutation even when the virus load was 24 U/-滋L and the mutation strain was less than 10% in the whole quasi-species pool. By detection of the 43 specimens, we found that the sensitivity of this method was 100%, significantly superior to Sanger sequencing, which was 74%(P < 0.05). Conclusion:Based on nested PCR and specific primers, our new method can successfully detect HBV DNA rtA181T mutation. Especially in low virus load circumstance, this new method is superior to Sanger sequencing in sensitivity, and also shows good specificity, thus is beneficial to early detection of HBV DNA drug resistant mutation and important for adjustment of the anti-HBV therapy in time.

Abstract:Objective:To evaluate the potential hazards of heavy metal in drinking water in primary and middle school students. Methods:We detected heavy metal elements in residential and school drinking water of primary and middle school students,and assessed risk of 8 kinds of heavy metals (As,Cr,Cd,Pb,Hg,Zn,Sb,Cu) using method recommended by the United States Environmental Protection Agency (EPA),and used Monte Carlo simulation method to analyze and evaluate the uncertainty. Results:The annual carcinogenic risks for carcinogenic elements,As,Cr,Cd were 1.6 × 10-6,1.5 × 10-7,1.4 × 10-7,respectively. For non carcinogens (Hg,Pb,Sb,Zn,Cu),the annual carcinogenic risks were 1.0 × 10-10,8.0 × 10-11,6.8 × 10-7,4.0 ×10-11,and 3.9 × 10-9 respectively. The total health risk was 2.5 × 10-6. Conclusion:The health risk of heavy metals caused by drinking water is more than the maximum acceptable risk level (1 × 10-6),and exerts potential adverse effects on health of primary and middle school students. The sensitivity analysis showed that the content of heavy metal elements in drinking water is the main factor affecting the health.