Abstract:Objective:To investigate the clinical significance of IMP3 expression, and to detect the relationship between IMP3 expression and genetic methylation. Methods:Paraffin sections of tumor tissues from 162 patients with HCC were examined for IMP3 expression by immunohistochemistry. Correlation analysis of clinical factors and IMP3 expression level was performed. One hundred and sixty-two patients with post-operation of HCC were follow-up for long-term survival analysis. P1 and P2 of IMP3 gene from normal and tumor tissues were detected by methylation-specific PCR and DNA sequencing. IMP3 protein was detected by Western Blot from cultured normal liver cells L02 and Huh7 hepatoma cells. DNA was extracted from two groups of cells,and the methylation status of promoter region CpG islands in IMP3 gene was detected. Results:IMP3 was expressed in 67.90% of HCC. IMP3 expression is related to age,tumor size, grading, staging,AFP,early relapse,but not gender and HBsAg. One hundred and sixty-two cases of postoperative follow-up found that postoperative long-term survival rate of IMP3 negative group was significantly higher than that of the IMP3-positive group(P < 0.01). Detection of P1 and P2 by methylation-specific PCR showed that CpG islands from P1 and P2 of tumor tissues showed unmethylated modification,however,those of normal tissues showed methylated modification. Normal liver cells were IMP3 negative,Huh-7 hepatoma cells showed strongly positive IMP3 expression. P1 and P2 of normal liver cells were highly methylated,and P1 and P2 of Huh-7 hepatoma cells showed unmethylated status. Conclusion:The level of IMP3 expression is high in HCC,and high IMP3 expression suggests poor prognosis;DNA methylated modification may participate in the processes,which regulates and controls IMP3 expression.

Abstract:Objective:To investigate the effects of miR-21 inhibition on proliferation and apoptosis of A549/DDP cells. Methods:MiR-21 expression was examined in A549 and A549/DDP cell lines. The inhibitor of miR-21(ASO-21) was transfected into A549/DDP. MTT and flow cytometry were performed to analyze the proliferation and apoptosis after transfection,respectively. Computational software and miR predicting database were used to predict the downstream effectors of miR-21 in the lung cancer cell line. Results:①miR-21 expression was approximately 3 times higher in A549/DDP compared with A549 cells. ②Apoptosis was induced and proliferation was suppressed in A549/DDP cells when miR-21 was inhibited. ③Computational softwares showed that multiple genes are possible downstream targets of miR-21. Some proteins could possibly regulate miR-21. Those genes form a regulating network via miR-21,resulting in regulating cisplatin chemoresistance. Conclusion:MiR-21 inhibition leads to apoptosis and proliferation suppression of A549/DDP cells, and it may be a therapeutic target to reverse cisplatin resistance in lung cancer treatment.

Abstract:Objective:To determine the expression of HOTAIR in common renal carcinoma and normal renal cell lines and establish a renal carcinoma cell line that highly expresses HOTAIR. Methods:①q-PCR was used to detect the expression of HOTAIR in four renal carcinoma cell lines (os-rc-2,kevt-3,achn and 769-p) and a normal renal cell line(HK-2). ②Infection with lentiviral (pLenti GFP Puro-HOTAIR) carrying HOTAIR gene in 769-p cell line and puromycin-resistance screening were performed to establish the cell line that highly expresses HOTAIR. Results:① HOTAIR was expressed in five cell lines (from high to low):kevt-3,os-rc-2,achn,769-p,and HK-2. ②HOTAIR was high expressed in 769-p after infected with lentiviral (pLenti GFP Puro-HOTAIR). Conclusion:①HOTAIR expression in high metastatic carcinoma cell lines(kevt-3,os-rc-2) was higher than that in low metastatic renal carcinoma cell lines (achn,769-p ) and normal renal cell line(HK-2).②The 769-p-HOTAIR cell line stably expressed HOTAIR.

Abstract:Objective:To explore VEGF expression induced by PGE2 in endometrial cancer cell and possibly involved signal transduction pathways. Methods:Ishikawa cells were treated with PGE2,EP1-4 receptor agonist,EP2 receptor antagonist AH6809,PKA inhibitor H89,and adenylate cyclase (AC) inhibitor SQ22536. Western blot was used to examine the expression of VEGF protein in Ishikawa cells cells. Results:PGE2 up-regulated the protein level of VEGF in Ishikawa cells. The level of VEGF protein was increased by 31.56%(P < 0.05)compared with the control group after treated with 10 μM PGE2 for 24 h. Treated with 10 μM four selective EP receptor agonists(17-phenyltrinor Prostaglandin E2,Butaprost,Sulprostone and Prostaglandin E1 Alcohol) respectivly,the protein expressions of VEGF in Ishikawa cells was increased compared with the control group. We found that EP2 receptor agonist increased the expression of VEGF protein to 87.8% (P < 0.05). The protein level of VEGF decreased by 45.66%(P < 0.05)after treated with 10 -滋M EP2 receptor antagonist compared with the group treated with PGE2. When treated with 25 -滋M AC inhibitor SQ22536 and 10 -滋M PKA antagonist H89 for 24 h,respectally,the protein levels of VEGF were decreased by 29%(P < 0.05)and 57.5%(P < 0.05)compared with the group treated with EP2 receptor agonist. Conclusion:PGE2 may up-regulate the protein expression of VEGF through EP2 receptor and Gαs-cAMP-PKA signaling pathway in Ishikawa cells.

Abstract:Objective:To explore the role of PI3K/Akt signal pathway in acetylation and aerobic glycolysis in Huh-7 cells. Methods:We treated Huh-7 cells with LY294002 (PI3K/Akt inhibitor) and rapamycin (mTOR inhibitor). DMSO treatment was set as negative control. Lactate concentrations were measured by lactate kits. The expression of ACL and acetylation was measured by Western blots. Cell viability was determined by WST assay. Results:①Under LY294002,lactate concentrations decreased by 66.15%. Expression levels of ACL declined by 20.84%. Acetylation levels decreased at several sites(P < 0.01,n = 3). ②Under rapamycin,lactate concentrations decreased significantly by 90.16%. Expression levels of ACL declined by 64.16%. Acetylation levels were down-regulated variously at different sites (P < 0.01,n =3 ). ③After 24 h culture with 20mmol/L LY294002 or 20 μg/L rapamycin,the cell growth inhibition ratio reached to 24.78% and 20.67%,respectively(P < 0.01,n = 3). Conclusion:PI3K/Akt/mTOR signal pathway promotes the aerobic glycolysis of cancer cells by up-regulating the expression of ACL genes and the levels of acetylation,which may contribute to the tumor growth.

Abstract:Objective:To study the effects of icotinib hydrochloride and 1α,25-dihydroxyxitamin D3 on proliferation and expression of EGFR of human lung adenocarcinoma cell line A549. Methods:The human lung adenocarcinoma A549 cell was cultured and treated by icotinib hydrochloride,1α,25-dihydroxyxitamin D3 and combination of these two medicines at different concentrations and time intervals. The CCK-8 assay was applied to analyze the proliferation inhibition rate. The expressions of EGFR total protein and EGFR mRNA were detected by western blotting and RT-PCR,respectively. Results:Compared with the single drug group,there was synergistic effect observed in the groups treated with icotinib hydrochloride and 1α,25-dihydroxyxitamin D3 along(P < 0.05). Compared with the control group and the group treated with icotinib hydrochloride alone,the expressions of EGFR total protein and EGFR mRNA of the group treated with 1α,25-dihydroxyxitamin D3 alone or the combination group were elevated dramatically(P < 0.05). Conclusion:There was synergistic effect in the cells treated with combination icotinib hydrochloride with 1α,25-dihydroxyxitamin D3. This may attribute to up-regulat of the expressions of EGFR mRNA and EGFR total protein by 1α,25-dihydroxyxitamin D3,and further to the anti-tumor effect of icotinib hydrochloride.

Abstract:Objective:To investigate the expression of insulin-like growth factor 2 mRNA binding protein 3 (IMP3) and insulin-like growth factor 2 (IGF2) in hepatitis B-virus associated hepatocelluar carcinoma (HBV-HCC) and study the correlation between IMP3 and IGF2 as well as their relationship with clinicopathological features of HBV-HCC. Methods:IMP3 and IGF2 were detected in 82 paraffin-embedded tumor tissues and corresponding adjacent benign tissues from HBV-HCC patients by immunohistochemical assay. IMP3 and IGF2 mRNA were detected in selected 32 freezing tissues by Real-time PCR. IMP3 and IGF2 proteins were detected in 4 strong positive and 4 negative immunohistochemical staining tissues by Western blot analysis. Results:IMP3 was expressed in 62.2% HBV-HCC tissues,but not detected in adjacent benign tissues. IGF2 was expressed in 59.8% HBV-HCC tissues and 14.6% adjacent benign tissues. Meanwhile,the expression of IMP3 was significantly correlated with IGF2 expression in HBV-HCC tissues (rn = 0.335,P = 0.002). Additionally,our results showed that IMP3 expression was significantly correlated with TNM 3-4 tumor stage (P = 0.001),Edmondson-Steiner 3-4 tumor grade (P = 0.001),Alpha Fetal Protein (AFP)> 200 ng/ml (P = 0.012) and portal vein tumor thrombus (P = 0.018). Similar to the expression of IMP3,IGF2 expression was significantly correlated with Edmondson-Steiner 3-4 tumor grade (P = 0.001),TNM 3-4 tumor stage (P < 0.001) and portal vein tumor thrombus (P < 0.001). Conclusion:Our results indicate that IMP3 was significantly correlated with IGF2 expression in HBV-HCC and detection of IMP3 and IGF2 was helpful to evaluate the differentiation and progression of HBV-HCC.

Abstract:Objective:To investigate the association between single nucleotide polymorphisms of IL-13 gene and susceptibility to laryngeal cancer in a Chinese population. Methods:A case-control study included 111 laryngeal squamous cell carcinoma (LSCC) patients and 340 cancer-free controls. Genotyping of the polymorphisms located at the 3’untranslated region (UTR) of IL-13 gene was conducted using MGB TaqMan Probe Assay. Logistic regression model was used to assess the contribution of genetic effects on the development of laryngeal cancer. Results:Individuals with IL-13 rs1295685 TT/CT genotypes had a increased risk of laryngeal cancer (adjusted OR = 2.00,95%CI:1.23~3.27,P = 0.005). Particularly among younger patients (OR = 2.72 and 95%CI:1.40~5.28),smokers (OR = 2.07 and 95%CI:1.19~3.58),drinkers (OR = 1.96 and 95%CI:1.03~3.73) and those without a family history of cancer (OR = 2.01 and 95%CI:1.18~3.41). Furthermore,compared with those,who were both nonsmokers and CC carriers,significantly increased risk was observed in those,who were smokers and TT/CT carriers (OR = 6.48 and 95%CI:2.77~15.14). The similar situation was found in drinkers(OR = 7.35 and 95%CI:3.15~17.12). No significant relationship was observed between rs1295685 polymorphism and progression of laryngeal cancer. Conclusion:The polymorphisms of IL-13 gene may play an important role in the etiology of laryngeal cancer.

Abstract:Objective:To investigate Th1 and Th2 cytokines production in the C57BL/6 mice vaccinated by recombinant ESAT-6 (6KD early secretory antigenic target) and FL (fms-like tyrosine kinase 3 ligand) plasmid DNA vaccine encapsulated with nano-chitosan. Methods:The chitosan nanoparticles were prepared,and recombinant plasmid DNA vaccines,namely pIRES-ESAT-6-FL,pIRES-ESAT-6,pIRES-FL and pIRES plasmids,were encapsulated by the nano-chitosan. Then,the size and morphology of nano-ESAT-6-FL plasmid were examined by transmission electron microscope and the expressions of corresponding protein were detected using Western blot. C57BL/6 mice were immunized with nano-ESAT-6-FL,nano-ESAT-6,nano-FL,and nano-pIRES, respectively. At the same time,the corresponding plasmids without nano-chitosan encapsultion,and PBS,nano-chitosan and BCG were also immunized with C57BL/6 mice. Nine weeks after vaccination,the splenocytes separated from the immunized mice were stimulated with PPD for 72 h in vitro,and the Th1 cytokines (IFN-γ and IL-12) and Th2 cytokines (IL-4 and IL-10) in splenocyte supernatant were measured using ELISA kit. Results:Nano-ESAT-6-FL and other chitosan-encapsulated plasmids were well prepared,and nano-ESAT-6-FL plasmid expressed the corresponding protein in glomerular mesangial cells (GMC). The mice immunized with nano-ESAT-6-FL plasmid group(nano-ESAT-6-FL) had significantly higher levels of Th1 cytokines (IFN-γ and IL-12) in splenocyte supernatants than those of the mice treated with control plasmids,but as for IL-4 and IL-10,the changes of the two cytokines in mice with mentioned-above plasmids did not show obvious differences. Conclusion:Recombinant ESAT-6-FL plasmid DNA vaccination encapsulated by nano-chitosan could markedly improve production of Th1 cytokines,suggesting that the mice vaccinated with nano-ESAT-6-FL plasmid DNA vaccine can enhance the cell immune responses.

Abstract:Objective:To investigate the major effects of environmental endocrine disruptors (EDCs) BPA on SD rat blood-brain barrier component expression,cell viability of astrocytes and explore the mechanism. Methods:The MTT assay was performed to evaluate the astrocyte viability,HPLC was performed to detect glutamate levels,and by real-time quantitative PCR the mRNA levels of Claudin-3,Claudin-4,Claudin-5,JAM-1,JAM-2,ZO-1 and E-cadherin were detected. Results:After astrocytes were exposed to BPA for 24 or 48 hours,the cell viability significantly decreased. The realtime-PCR data showed that mRNA levels of blood-brain barrier tight junctional protein,Claudin-3,Claudin-4,JAM-1,E-cadherin,were significantly down-regulated. Meanwhile,Claudin-5,JAM-2 and ZO-1 mRNA expressions were significantly up-regulated. Furthermore,HPLC showed that the concentration of glutamate was increased. Conclusion:Taken together,EDCs BPA demonstrated the potential toxic effects on the blood-brain barrier integrity.

Abstract:Objective:To measure the index of toxicokinetics and toxicodynamics of chlorpyifos in Sprague-Dawley rats after single oral-dose exposure to chlorpyrifos(CPF),and study the characteristic of toxicokinetics and toxicodynamics of CPF. Methods:The adult female SD rats were randomly divided into 5 groups (including 1 control group and 4 treated groups) according to the weight. Each group has fifteen rats. The dose of chlorpyrifos is respectively 12.5 mg/kg,25 mg/kg,50 mg/kg,100 mg/kg. Blood samples were collected through arteria cruralis after single dose gastric gavage exposure at 1 h,3 h,6 h,12 h,and 24 h,then rats were killed and cortexes were collected. The concentrations of CPF and TCP(3,5,6-trichloropyridinol)at every five time point in plasma were determined by HPLC(high performance liquid chromatography),and acetylcholinesterase(AChE) activity was estimated by the Pureauto CHE. Results:The peak concentration of CPF and TCP first peak were observed at 3 h and 6 h,respectively,then declined. The maximum inhibition of plasma AChE was observed between 3 to 6 h. Maximum inhibition of cortex AChE occurred between 6 to 12 h. Both the plasm concentration of CPF and TCP were linear negatively correlated with the plasm AChE activity. The plasm concentration of CPF and TCP showed a linear positive correlation. Conclusion:Through this study,we obtain several indexes of toxicokinetics and toxicodynamics of CPF,and represent the characteristics. These results will provide useful information to the physiologically based toxicokinetics and toxicodynamics(PBTK/TD)model.

Abstract:Objective:To evaluate the effect of minocycline on the long-term learning and memory ability for 30 days afte surgery in C57BL/6 aged mice,and to explore the role of astrocytes in the process. Methords:Forty-five male C57/BL6 aged mice were randomly divided into 3 groups with 15 in each group:sham operation group only received skin incision,operation group received 70% hepatectomy without drugs administration,minocycline group intraperitoneally injected with 45 mg/kg minocycline once a day for 30 days after surgery. Hippocampal-dependent spatial learning and memory ability was evaluated using the Morris water maze(MWM). Then mice hippocampal tissue was sampled. The hippocampal mRNA relative expression levels of tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) were tested using a real- time PCR. The expression levels of hippocampal glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1(Iba-1) were detected with Western blot. The remained hippocampal tissue was collected for immuno-histochemistry. Results:MWM tests showed that the escape latency and swimming distance in operation group was longer than that of sham and minocycline groups (P < 0.05). The relative expression of TNF-α mRNA of operation group was significantly higher sham and minocycline groups (P < 0.05),and the expression of TNF-α mRNA of minocycline group was less than operation group(P < 0.05). The expression of IL-6 mRNA in operation and minocycline group was significantly higher than that in sham group (P < 0.001),and the expression of IL-6 mRNA in minocycline groups was markedly less than that of operation group (P < 0.001). Compared with sham group,the expression of GFAP in operation and minocycline group was higher(P < 0.001),and the expression level of GFAP in minocycline group was less than that in operation groups(P < 0.001). There was no significant difference in Iba-1 expression among the three groups(P > 0.05). The changes of astrocytes in hippocampal tissue immunohistochemistry were consistent with the changes of GFAP. Conclusion:The activation of astrocytes in the hippocampus was likely to contribute to long-term decline of learning and memory ability in aged mice undergone partial hepatectomy. Minocycline can improve postoperative spatial learning and memory ability by decreasing the expression levels of TNF-a and IL-6 mRNA and inhibiting astrocyte activation.

Abstract:Objective:To preliminarily investigate whether fluvastatin has a therapeutic role on the peritoneum in a rat model of peritoneal dialysis. Methods:A rat model of peritoneal dialysis was induced by a daily intraperitoneal infusion of 4.25% peritoneal dialysate. Thirty SD rats were randomly divided into five groups:normal control group(n = 6),4.25% peritoneal dialysate group(n = 6),saline group(n = 6),Flu group(n = 6),4.25% peritoneal dialysate combined with Flu group(n = 6). Rats were sacrificed 4 weeks after the first treatment and the peritoneal tissues were gathered for further analysis. A portion of peritoneal tissues were used for histological examination. The expressions of TGF-β1 and FN of each group were examined by RT-PCR. Results:HE stain showed that compared with normal control group,the peritoneal tissue thickness of 4.25% peritoneal dialysate group and 4.25% peritoneal dialysate combined with Flu group increased significantly. Compared with 4.25% peritoneal dialysate group,the peritoneal tissue thickness of 4.25% peritoneal dialysate combined with Flu group decreased obviously. There was no obvious statistical significance among saline group,Flu group and normal control group. RT-PCR showed that compared with normal control group,the expressions of TGF-β1 and FN in 4.25% peritoneal dialysate group increased apparently. The expressions of TGF-β1 and FN in 4.25% peritoneal dialysate combined with Flu group were significantly lower than 4.25% peritoneal dialysate group(P < 0.05). There was no obvious statistical significance among saline group,Flu group and normal control group. Conclusion:Long-term treatment with high-glucose peritoneal dialysate could change the structure of peritoneum. Fluvastatin could be used for protecting peritoneum,however,the specific mechanism need tobefurtherstudied.

Abstract:Objective:To observe the effects of metformin on expressions of nuclear factor-κB and intercellular adhesion molecule-1 in cultured rat glomerular mesangial cells(MCs) and explore its reno-protective mechanisms. Methods:MCs were cultured in the medium with normal glucose(group NG), high glucose(group HG),and different concentrations of metformin (group M1, 0.5 mmol/L;group M2, 1.0 mmol/L;group M3, 2.0 mmol/L). After 48h exposure, MCs were collected for mRNA and protein expression of NF-κB, ICAM-1. The expression of NF-κB and ICAM-1 mRNA was analyzed by real time polymerase chain reaction. The expression of NF-κBp65 and ICAM-1 protein was visualized by western blotting . Results:①MCs expressed NF-κB and ICAM-1. ②After stimulated by high glucose, the levels of intracelluar NF-κB, ICAM-1 mRNA and protein expression were significantly increased compared with group NG(P < 0.05). ③Compared with group HG, the expression of intracellular NF-κB, ICAM-1 mRNA was significantly decreased in group M3(P < 0.05). ④Compared with group HG, the expression of intracellular NF-κBp65 and ICAM-1 protein was significantly decreased in group M1, M2 and M3 in a dose-dependent manner(P < 0.01). Conlusion:Metformin can suppress the expression of NF-κB and ICAM-1 in glomerular mesangial cells in a concentration-dependent mode, which may partly contribute to its reno-protection.

Abstract:Objective:To observe the three-dimensional (3D) structure and morphologic characteristics of domestic porous tantalum implanted in rabbit erector spinae and evaluate its histocompatibility,and to provide experimental evidence for repair and reconstruction of bone defects. Methods:The structure of the domestic porous tantalum was observed by scanning electron microscope (SEM). A total of 24 adult New Zealand rabbits were used to establish porous tantalum model. Two 1.5 cm × 1.5 cm × 0.2 cm porous tantalum materials were implanted in one side of rabbit erector spinae (the experimental group). Two 1.5 cm × 1.5 cm × 0.2 cm porous titanium materials (the control group) were implanted into the opposite side in the same rabbits. The rabbits were sacrificed successively at 2,4,8 and 12 weeks post implantation. Slices of hard tissue specimen were examined to observe fibrous capsule morphology and vascular formation. Muscle and fibrous tissue morphology around the material were observed by SEM. Results:The SEM showed the 3D structure of porous tantalum and pore diameter ranged from 400 to 600 μm. The muscle and fibrous tissue ingrowth was found in the surface and pores of porous tantalum in the early stage. In the late stage,collagen fibers and the pores of porous tantalum were filled with muscle tissue and connected to each other. Hard tissue slices observed that the loose and thick vascularized fibrous capsule formed in the porous tantalum-muscle interface at the beginning and became thin and compact in the end. The muscles,small vessels and fibrous tissues were found in the pores of materials. Conclusion:The Chinese porous tantalum has good histocompatibility,and its 3D porous structure is advantageous to ingrowth and extension on the surface and in the pores for the muscle tissue. The Chinese porous tantalum may has the potential to repair bone defect.

Abstract:Objective:To evaluate the protective effects of dopamine and norepinephrine on the actue lung injury(ALI) in a rabbit model of septic shock. Methods:A cecal ligation and puncture procedure combined with intravenous injection of lipopolysaccharides was used to make a septic shock model. Sixty three rabbits were randomly divided into 5 groups:sham operation group(group A,n =7),septic shock group(group B,n = 14),dopamine treatment group(group C,n = 14),norepinephrine treatment group (group D,n = 14),and dopamine combined with norepinephrine treatment group(group E,n = 14). Seven rabbits from each group were sacrificed 3 and 6 hours after septic shock,the concentration values of surfactant protein A and surfactant protein D(SP-A and SP-D)in plasma were tested by ELISA method and the morphological changes of lung tissue were performed at 3 and 6 h. Results:The concentrations of SP-A and SP-D in each group were significantly increased than those in group A(P < 0.05). No significant differences in the concentration values of SP-A and SP-D in plasma were found between group B and C at 3 and 6 h(P > 0.05);in group D and E,the concentration values of SP-A and SP-D in plasma were significantly lower than those in group B at 3 h and 6 h(P < 0.05). The morphological changes of lung tissue were lessened in group D and E than those in group B and C. Conclusion:The lung tissue was injured severely in septic shock model. Norepinephrine or norepinephrine combined with dopamine can relieve the lesion of lung tissue and play a protective role.

Abstract:Objective:To isolate and culture the normal and degenerative nucleus pulposus cells (NPCs) and bone mesenchymal stem cells (BMSCs) of SD rats,and then to compare the effects of normal and degenerative NPCs on BMSCs differentiatinto nucleus pulposus-like cells by a 3D noncontact coculture methord. Methods:The animal models with degenerative nucleus pulposus were established by acupuncture and aspiration. The BMSCs,normal and degeneratived NPCs of SD rats were isolated and cultured. The cells growth condition was observed by light microscope,and the second generation of NPCs and dNPCs and the third generation of BMSCs were selecte to combine alginate gel stents and culture in transwell plate. The BMSCs+NPCs or BMSCs+dNPCs were co-cultured in vitro as experiment group;the NPCs and BMSCs were cultured alone as positive and negative control,respectirely. 7 days later,the collagenⅡ and aggrecan of recycled BMSCs of each group were detected by western blot and RT-PCR. Results:The mRNA of collagenⅡand aggrecan showed a high level both in two co-culture groups,but the BMSCs/NPCs co-culture group is more similar to NPCs sample than BMSCs/dNPCs co-culture group. The results of the content of collagenⅡand aggrecan was the same with mRNA level. Conclusion:Under a 3D non-contact co-culture system,both the normal and degenerative NPCs can induce BMSCs differentiation into nucleus pulposus-like cells,but the induction effects of normal NPCs is more obviously than degenerative NPCs .

Abstract:Objective:To obtain the cDNA encoding Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)and construct the expression system of mAd-ASP-2 in E.coli. Methods:The cDNA encoding of the mature Ancylostoma-secreted protein 2 of Ancylostoma duodenale(mAd-ASP-2)was cloned by RT-PCR. The mAd-ASP-2 was inserted into the pET-22b(+)vector to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2. An optimized codon mAd-ASP-2 gene,designated mAd-ASP-2*,was designed and synthesized based on optimization of synonymous codon bias of E. coli,without modification of amino acid sequence,and inserted into pET-22b(+)to construct prokaryotic expression plasmid pET-22b-mAd-ASP-2*. The two expression plasmids were transformed into E. coli Rosetta-gami-2(DE3)cells. Results:SDS-PAGE analysis showed that the expected recombinant mAd-ASP-2 fusion protein was expressed in E. coli Rosetta-gami-2(DE3)cells induced by isopropyl-[3-thiogalactopyranoside(IPTG),and the mAd-ASP-2* with the optimized codon was more highly expressed than the original mAd-ASP-2 in the soluble fraction of E. coli cell lysates. The recombinant mAd-ASP-2* fusion protein was purified using His Trap HP affinity column. Western blot analysis showed that the recombinant mAd-ASP-2* protein was combined with mouse anti-His-Tag. So the expressed protein was definitely confirmed to be the target protein. Conclusion:The expression system of mAd-ASP-2 in E. coli was constructed successfully,which provided a fundamental basis for further studies on subunit vaccine for prevention of hookworm infections.

Abstract:Objective:To compare the biomechanical aspects among the three different fixation methods of Tossy type III acromioclavicular joint dislocation,and to guide the clinical treatment. Methods:Six adult cadaveric specimens were used in the study (12 shoulders). and made to the Tossy type Ⅲ acromioclavicular joint dislocation model. The 12 shoulder were randomly divided into 3 groups with 4 cases in each group,and the three groups received titanium cable fixation,anchor screw fixation and clavicular hook plate fixation respectively. After fixation,MTS biological material testing system was used to test the degree of excursion of three kinds of internal fixation. Results:All of the three internal fixation methods can be anatomical reduction. Compared with clavicular hook plate internal fixation group,the degree of activity in other two groups is larger(P < 0.05),and the degree of separation is smaller (P < 0.05). Conclusion:Congared with Tossy type Ⅲ acromioclavicular joint dislocation,the degree of separation of itanium cable fixation and anchor screw fixation is small,and the degree of activity is moderate,more closer to normal physiological state.

Abstract:Objective:To examine the discriminatory potential of the Cognitive-12 for moderate and severe Alzheimer＇s disease(AD). Methods:Patients with AD(61 moderate AD and 55 severe AD) were recruited. The COG-12,AD8,MMSE and CDR were tested by all AD subjects. Results:The total scores of COG-12,AD8,MMSE and CDR differed significantly between moderate and severe AD groups with higher scores in severe AD group. The results of multiple linear regression analysis showed insignificant influence of age or educational level on the COG-12 total score. COG-12 could classify moderate and severe AD efficiently with sensitivity of 90.9% and specificity of 70.5%. Conclusion:COG-12 is useful for staging of moderate and severe AD.

Abstract:Objective:To compare the serum levels of neuropeptide and cytokines in first episode depressive patients before and after citalopram treatment. Methods:The serum levels of neuropeptide[neuropeptide Y (NPY),β-endorphin (β-EP) and Motilin (MTL)]and cytokines[tumor necrosis factor α(TNF-α) and insulin-like growth factorsⅡ(IGF-Ⅱ)] were measured using radioimmunoassay in 50 first-episode drug-naive depressive patients before and after antidepressant treatment and 40 healthy controls. Severity of depression and treatment efficacy were assessed according to the Hamilton Depression Scale (HAMD-17) and Hamilton Anxiety Scale (HAMA),and analyzed with the SPSS17.0 statistical package. Results:The serum levels of β-EP (340.31 ± 94.97 pg/ml),TNF-α(14.64 ± 9.23 ng/ml) and IGF-II (0.80 ± 0.30 ng/ml) were significantly higher in first-episode drug-naive depression patients than controls (all P < 0.0001),and serum levels of NPY (96.41 ± 32.98 pg/ml) in depressive patients were significantly lower than control group (P < 0.0001). The serum levels of IGF-II reduced (P < 0.0001) after 6-week of therapy,while NPY increased (P < 0.05). Regression analysis showed that the serum NPY was the important risk factor of depressive (P = 0.012),and the change of MTL was the association factor influencing the therapeutic effect (P = 0.016). Conclusion:The change of serum neuropeptide and cytokines may be involved in pathophysiology of the first episode depressive patients. The low serum NPY may be a potential predictive index for the depressive episode,while the increase of MTL can predict the antidepressant efficacy.

Abstract:Objective:To observe the prognostic value of NT-proBNP testing in thrombolysis therapy in acute pulmonary thromboembolism. Methods:Sixty-two hemodynamically stable patients with acute pulmonary thromboembolism were randomly divided into thrombolysis therapy[TT(+)] group and non-thrombolysis therapy[TT(-)] group. The clinical aspects,vital sign,prognosis before and after thrombolysis treatment were compared between the two groups. And patients were divided into two groups based on serum NT-proBNP level. Clinical indicators of the two groups were compared. Resules:The difference of heart rate,systolic blood pressure,respiratory rate,D-dimer level,PaO2 level before and after treatment had no significant difference between TT (+) group and TT (-) group. Chest symptoms improvement rate and clinical adverse events rate had no significant difference. In patients with high NT-proBNP level,the improvement of heart rate,systolic blood pressure,respiratory rate,D-dimer level,PaO2 level,and chest symptoms improvement rate,clinical adverse events rate in TT (+) group were significantly better than those of TT (-) group. But in patients with normal NT-proBNP level,the clinical indicators had no significant difference. In patients without thrombolytic therapy,the improvement of heart rate,systolic blood pressure,PaO2 level in NT-proBNP normal group was significantly better than NT-proBNP increase group,while clinical adverse events rate was significantly lower than NT-proBNP increase group. But in patients undergo thrombolytic therapy,the difference between NT-proBNP normal group and NT-proBNP increase group was not significantly. Conclusion:Thrombolytic therapy can improve the prognosis of patients with high NT-proBNP level,but in patients with normal NT-proBNP level,the improvement is not obvious.

Abstract:Objective:To compare the outcomes of minimal invasive mitral surgery under minimal extracorporeal circulation with those under conventional middle sternotomy. Methods:Ninety patients with mitral stenosis and/or insufficiency,who need mitral repair or replacement,were randomized into minimal invasive (Group A,45 cases,minimal right thoracotomy under minimal extracorporeal circulation,MECC) or conventional middle sternotomy (Group B,45 cases). The following items are recorded and then compared:cardiopulmonary bypass time,aortic cross clamp time,the amount of blood lose during surgery,blood transfusion,peri-operative chest drainage,time of intubation and ICU stay post-operation. Results:There were no major complications and operative death in both groups. Both CPB time and cross clamp time in group A were longer then in Group B (P < 0.05). But the bloods lose during surgery,the amount of chest drainage and blood transfusion peri-operatively were significantly lower in Group A than in Group B (P < 0.05,P < 0.01,P < 0.05,respectively). The intubation time and ICU stay after surgery were obviously shorter in group A than in group B (P < 0.05),with lower hospital cost in group A. (P < 0.01). Conclusion:Mitral surgery through mini-thoracotomy under MECC improves the quality of surgery on following aspects:trauma,lower blood lose and transfusion,recovery,cost-effective,cosmetic effect and patients satisfaction even with relative longer CPB and cross clamp times.

Abstract:Objective:To evaluate the clinical value and safety of colorectal stenting as a bridge to primary anastomosis placed endoscopically using fluoroscopic guidance versus emergency surgical decompression on acute resectable malignant colorectal obstruction. Methods:From May 2001 to October 2012,94 patients were diagnosed with acute colorectal malignant obstruction. Thirty patients underwent metal stent placement as a bridge to an elective resection and primary anastomosis,while 64 patients underwent emergency surgery. The two group were compared for successful one-stage operation,operation time,postoperative ventilation time,hospital stay,hospital mortality and postoperative complications. And the clinical and technical success rate of stent placement,the rate of stent-related complication and after stenting accept laparotomy and laparoscopic surgery in the stent group was analyzed. Results:There was a significant difference in successful one-stage operation and morbidity between two groups. A resection and anastomosis stent group was significantly higher than emergency surgery group (96.67% vs 53.13%,P < 0.001). The postoperative morbidity in stent group was significantly lower than that in emergency surgery group (6.67% vs 25.0%,P < 0.05). There was no statistically significant difference in mortality rate in both groups. In stent group,operative time,postoperative ventilation time was (156.13 ± 49.79) min,(3.60 ± 1.40) d,which were significantly lower than those of the emergency surgery group. The stent group had no significant difference on hospital stay compared with emergency surgery group(P > 0.05). The stent insertion was 100% successful in attempted stent placements. The clinical success rate was 96.67% in the stent group. The stent-related complication was 6.67%. The mean interval between stenting and surgery was(8.9 ± 1.0)d. Patients in the sent group underwent significantly more laparoscopic surgery than in emergeney surgery group(P < 0.01). Surgery time stent group undergoing laparotomy is shorter than the stent group undergoing laparoscopic surgery (P < 0.05),laparotomy complications was significantly lesser than the minimally invasive laparoscopic surgery in the sent group (P < 0.05),but received laparotomy patient＇s hospital stay was significantly longer than patients undergoing laparoscopic surgery. Conclusion:Colorectal stenting placed endoscopically using fluoroscopic guidance as a bridge to a primary surgical procedure is effective. Elective surgery after stenting is more safer than emergency surgery. Elective surgery could increase the chance of primary anastomosis,and reduce postoperative complications,and can be used as an effective treatment for remission of malignant colorectal obstruction. Laparotomy is still the main choice after stenting for elective surgery. By stent implantation,patients can get the opportunity of minimally invasive surgery.

Abstract:Objective:To analyze the SLC39A4 gene mutation and mutating patterns in a sporadic Chinese patient with acrodermatitis enteropathica (AE) so as to provide a basis for gene diagnosis and genetic counseling of the disorder. Methods:Genomic DNA was extracted from whole blood by standard methods from one male AE patient and his parents. DNA samples were also extracted from 100 unrelated,normally individuals as controls. The whole coding region of SLC39A4 was amplified by PCR and products were analyzed by direct sequencing. Results:Molecular analysis of the SLC39A4 gene in this case of AE revealed a novel heterozygous mutation c.831G>A in exon 5 and c.1617delA in exon 10,which altered a methionine residue with isoleucine residue at position 277 of the protein sequence and caused a reading frame shift,respectively. The patient’s father was found to only carry heterozygous c.831G>A mutation,while his mother carried heterozygous c.1617delA mutation. The two novel mutations were not observed in 100 control individuals selected from a Chinese population. Conclusion:Our data suggest that c.831G>A and c.1617delA mutation of SLC39A4 gene is the genetic cause of AE.

Abstract:Objective:Non-invasive prenatal diagnosis of chromosome aneuploidies has been achieved by measuring the ratio of two alleles of a single nucleotide polymorphism (SNP) in circulating placental mRNA (the RNA–SNP allelic ratio approach) in maternal plasma. This research is conducted to develop a rapid,accurate and cost-effective assay for the non-invasive prenatal detection of fetal trisomy 21. Methods:Maternal plasma samples were collected from 50 eupliod pregnancies(gestational age range:15 to 21weeks)and 13 trisomy 21 pregnant cases(gestational age range:19 to 26 weeks). With the application of Multiple-SNaPshot assay in measuring five SNP allelic ratios and heterozygosis for the chromosome 21 transcribed placenta-specific-4(PLAC4) mRNA in maternal plasma,we successfully achieved noninvasive prenatal detection of trisomy 21 in cases with the heterozygous SNPs on PLAC4 mRNA. Also the genotypes of the SNPs located in the transcribed regions of the gene PLAC4 were detected in population samples collected from 200 voluteers in Southeast China by SNaPshot assay. Results:PLAC4 mRNA was detected in peripheral blood of all pregnant women. Of all 63 singleton pregnancies,17 gravidas were found at least one heterozygous SNP on PLAC4 mRNA in maternal plasma. Among them,3 pregnancies with a trisomy-21 fetus and 14 pregnancies with an euploid fetus were detected by the analysis of the RNA-SNP allelic ratio. The highest heterozygosity frequencies of five SNPs on gene PLAC4 were Rs8130833 and Rs4818219,which were estimated to be 0.278,0.343,respectively. Conclusion:The plasma placental RNA allelic ratio can be used for noninvasive prenatal detection of Downs Syndrome,if SNPs on PLAC4 mRNA in maternal plasma are heterozygous. The two SNPs with higher heterozygosity frequencies on gene PLAC4 were Rs8130833 and Rs4818219 in population of Southeast China.

Abstract:Objective:To develop an LC-MS/MS method for determination of sofalcone in urine samples,and to investigate its urinary excretion characteristics in healthy Chinese volunteers. Methods:The urine samples were treated with methanol and separated on a Poroshell 120 SB-C18 column,with a mobile phase of methanol and 2 mmol/L Ammonium acetate(85∶15,v/v). The rate was 0.20 mL/min and the temperature was 30℃. Electrospray ionization source was applied and operated in the negative ion mode using SRM. Two healthy Chinese volunteers were given single oral dose of sofalcone capsule(200 mg) for urinary excretion and characteristics. Results:The lower limits of quantitation for sofalcone were 1 ng/mL,and the linear range was 1~500 ng/mL. The data of intra and inter-run precision were all less than 6%. The matrix effects were between 93.1% and 114.8%. The extraction recovery was 84.7%~93.6%. The accumulated excretion rates of sofalcone in urine were lower than 0.01%. Conclusion:The developed method can be applied to evaluate the urinary excretion of the sofalcone with a very short running time.