OBJECTIVE： To investigate the correlation of ADPRT rs1136410 polymorphism with the occurrence of non-small cell lung cancer （NSCLC） in Han nationality from northern Jiangsu. METHODS： A total of 283 patients with primary NSCLC of Han nationality in Northern Jiangsu were selected from the Affiliated Hospital of Xuzhou Medical University during Nov. 2015-Dec. 2018 as NSCLC group. A total of 210 healthy subjects underwent physical examination were included in control group. PCR-RFLP was utilized to determine the genotypes at ADPRT rs1136410 locus. Logistic regression model was used to evaluate the effect of polymorphism and its interaction with smoking on the occurrence of NSCLC. RESULTS： There was no statistical significance in age and gender between 2 groups （P＞0.05）. The proportion of smoker in NSCLC group was significantly higher than control group （P＜0.05）. TT， TC and CC genotypes were detected at rs1136410 locus of ADPRT gene. The frequency of TT， TC and CC genotype were 41.9％，44.8％ and 13.3％， and those of allele T and C were 64.3％ and 35.7％ in control group. The frequency of TT， TC and CC genotype were 21.6％， 50.2％ and 28.2％， and those of allele T and C were 46.6％ and 53.4％ in NSCLC group， respectively. The frequencies of genotypes in 2 groups were in accordance with Hardy-Weinberg equilibrium （P＞0.05）， while there was significant difference in genotype and allele frequencies between 2 groups （P＜0.05）. Compared with TT genotype， the risk of NSCLC in individuals carrying TC and CC genotypes raised by 1.179， 3.122 folds [ORTC＝2.179， 95％CI （1.435， 3.309）， P＜0.05； ORCC＝4.122，95％CI（2.401，7.075），P＜0.05]. Compared with individuals carrying TT genotype， the risk of NSCLC occurrence in non-smokers carrying TC and CC genotypes increased by 0.371， 1.328 fold [ORTC＝1.371，95％CI （0.927，3.428），P＜0.05； ORCC＝2.328，95％CI （1.249，4.622），P＜0.05]； and the risk of NSCLC occurrence in smokers carrying TC and CC genotypes increased by 0.928， 2.182 folds [ORTC＝1.928，95％CI （1.257，2.957）， P＜0.05；ORCC＝3.182，95％CI （1.760，5.754）， P＜0.05]. CONCLUSIONS： The rs1136410 locus mutant genotype of ADPRT gene is the risk factor of NSCLC in Han nationality from Northern Jiangsu， and smoking raises this risk of NSCLC occurrence in individuals with mutation genotypes of ADPRT rs1136410.

Objective: To observe morphological change and diversity of periodontium and alveolar bone after tooth transplantation into artificial tooth socket. Methods: 6 dogs were divided randomly into 2 groups: 2 dogs were used as the controls and 4 used for the experiment. In the control group 4 teeth were autotransplanted into the inherent sockets. In the experiment group 4 teeth were autotransplanted into the artificial sockets. The dogs were sacrificed at the 16th week after operation. The healing condition of periodontal tissue and the remodeling of alveolar bone were examined. Results: None of the transplanted teeth in both groups was loosen or dropped. Mircro-CT examination showed that cancellous bone and bone trabecula around the transplanted teeth lined tightly,no significant difference of bone trabecula thickness was observed between the 2 groups. Hard tissue slice examination revealed that parodontium of both groups grew and adhered to the teeth,and the quantity of new-born bone between the top of alveolar ridge and the neck of transplanted teeth was fundamentally the same in the 2 groups. Conclusion: Autotransplantation of teeth into the artificial socket is similar to that into inherent socket.

Aim To investigate the effects of PLCε on the invasion and migration of human osteosarcoma cancer cells U2OS. Methods RNA interference ( RNAi) was used to inhibit PLCεexpression, and the proliferation of cancer cells was measured by CCK-8 assay. The migration of the cells was measured by scratch wound healing assay and migration chamber as-say. Gelatin zymography was performed to measure the MMP2 activities in U2OS cells. Results PLCε ex-pression was suppressed by siRNA. CCK-8 assay showed that PLCε had no effect on the proliferation of cancer cells. PLCε knockdown inhibited cell invasion and activities of MMP2 . Conclusion PLCε knock-down can inhibit the migration and invasion of human osteosarcoma cancer cells U2OS.

Background and objective Rap2a, a member of the small GTPase superfamily, plays a critical role in regulating the function of integrin and cell adhesion, thereby controlling cell motility and cell/matrix interactions. However, the function of Rap2a in carcinogenesis is still poorly understood. To clone Rap2a cDNA, which belongs to human Ras-related small G protein superfamily, we constructed its eukaryotic expression vector and determined its expression in lung cancer cells. hTe aim of this study is to explore the role of Rap2a in carcinogenesis. Methods hTe levels of endogenous Rap2a protein in lung cancer cells were measured by Western blot. Total RNA of human osteosarcoma cells U2OS was extracted and reverse-transcribed into cDNA by RT-PCR. hTen, Rap2a gene was ampliifed by PCR and inserted into pcDNA3.1(+). hTe re-constructed plasmid was identiifed by restricted enzyme digestion and sequencing. pcDNA3.1(+)-Rap2a was transfected into H1299 and A549 cells, the expression of Rap2a was detected by Western blot. In addition, the migratory abilities of lung cancer cells were evaluated by Transwell assay. Matrix metalloproteinase (MMP)2 enzyme activity was evaluated by gelatin zymogra-phy. Results Rap2a is signiifcantly upregulated in lung cancer cells. hTe results of enzyme digestion and sequencing showed that the coding sequence of pcDNA3.1(+)-Rap2a was right and was inserted into the vector correctly. hTe results of Western blot showed that H1299 and A549 cells were transfected successfully. Transwell assay indicated that the ectopic expression of Rap2a promotes lung cancer cells migration. Correspondly, enzyme activity of MMP2 also increased. Conclusion Eukaryotic expression plasmid pcDNA3.1(+)-Rap2a was constructed successfully. Rap2a could be expressed in lung cancer cells effciently and promotes lung cancer cell migration.

Objective To investigate the effect of acetyl-L-carnitine (ALC) preconditioning on the PC12 cell apoptosis induced by oxygen-glucose deprivation.Methods PC12 cells were seeded in 96-well plates and randomly divided into 5 groups ( n =6 each):control group (group C),cell injury group (group Ⅰ) and preconditioning with different concentrations of ALC groups (groups A1-3 ).In group C,the cells were incubated with DMEM liquid culture medium containing glucose 0.5 g/L for 3 h.In groups Ⅰ and A1-3 the cells were incubated with DMEM liquid culture medium containing sodium hydrosulfite (Na2S2O4) 3 mmol/L and glucose 0.5 g/L for 3 h,and in addition the cells were pre-incubated with ALC 0.2,0.4 and 0.6 mmol/L for 24 h in groups A1-3 respectively.Cell viability was evaluated by MTF assay,while the apoptosis in cells was detected using TUNEL.The activities of ATPase and SOD and MDA content were also detected.Results Oxygen-glucose deprivation significantly increased the number of apoptotic cells and the content of MDA,and decreased the cell viability and activities of SOD and ATPase in group Ⅰ compared with group C ( P ＜ 0.05).Preconditioning with ALC significantly increased the cell viability and the activities of SOD and ATPaes,and decreased the number of apoptotic cells and the content of MDA in groups A1-3 compared with group Ⅰ ( P ＜ 0.05).Conclusion ALC preconditioning can attenuate PC12 cell injury induced by oxygen-glucose deprivation through inhibition of apoptosis in cells.

Objective To study the molecular mechanism of glutamate receptor-6 (GluR6) in the pathogenesis of epilepsy. Methods Seizure model of SD rats was induced by intraperitoneal injection of kainate (KA). Immunoprecipitation and immunoblotting were performed to examine the interactions of GluR6 and MLK3 with PSD95 at various time points after KA injection. The effect of Tat-GluR6-9c on the MLK3 phospharylation induced by kainate was observed with immunoblotting and immunohistochemistry. Results The assembly of GluR6 and MLK3 with PSD95 was induced after KA hippocampal CA3 region, and bagan to decrease one day later. Pretreatment after KA injection in CA3 region (P<0.05). Conclusion KA induces the assembly of the GluR6-PSD95-MLK3 signaling module and subsequently activates MLK3, which ultimately results in brain injury.

Objective To study the effects of oncolytic adenoviruses ZD55 harboring IL-24 gene (ZD55-IL-24) on the apoptosis of human melanoma cell line A375. Methods The oncolytie adenoviruses ZD55-IL-24 were verified by PCR. Then, the viruses were propagated, purified, and titrated by HEK293 cell plaque assay. A375 cells were cultured, divided into three groups transfected with ZD55-1L-24, ZD55 fused with enhanced green fluorescent protein (ZD55-EGFP), and replication-deficient adenovirus ZD55 carrying IL-24 gene (AD-IL-24), respectively. The multiplicity of infection was 0.1, 1, 10 and 100, respectively.Subsequently, the eytotoxity of these viruses and proliferation of A375 cells were determined by crystal violet staining and methyl thiazolyl tetrazolium (MTT) assay, respectively. The expressions of EIA and IL-24 protein were detected by Western blot in A375 cells. Results PCR verified that the adenoviruses ZD55-IL-24 contained IL-24 gene without wild adenovirns contamination. Crystal violet staining revealed that ZD55-IL-24 had an obvious eytotoxic effect on A375 cells, and MTT assay indicated that ZD55-IL-24 inhibited the proliferation of A375 cells in a time-and concentration-dependent manner. As shown by Western blot analysis, ZD55-1L-24 expressed IL-24 and E1A protein in A375 cells with a high efficiency. Conclusions The oncolytic adenoviruses ZD55-IL-24 can efficiently express IL-24 gene, inhibit the proliferation of, and induce the apoptosis in A375 cells.

Objective To evaluate the antitumor effect of oncolytic adenovirus expressing human IL-18 gene on malignant melanoma implanted in nude mice.Methods BALB/c nude mice were subcutaneously inoculated with A375 cells to establish a model of malignant melanoma.When the volume of implanted tumor reached 100-150 mm3,murine models were randomly divided into three groups to receive a 3-day intratumoral injection of IL-18 gene-expressing human oncolytic adenovirus named ZD55-IL-18,IL-18 gene-expressing adenovirus named Ad-IL-18,phosphate buffer saline(PBS),respectively.The tumor size was measured at an interval of 4 days for 9 weeks.Hematoxylin and eosin (HE)staining was performed to observe the morphological changes of tumor cells.The protein expression of IL-18 and E1A.microvessel density in tumor tissue,and apoptosis of tumor xenografts were detected by immuno fluorescence assay,immunohistochemistry and in situ end labeling technique (TUNEL).respectively.Results The treatment with ZD55-IL-18 significantly inhibited the growth of tumor.Forty-four days after the treatment,the mean tumor volume was 1039.378±29.67 mm3 in ZD55-IL-18-treated mice.significantly smaller than that in Ad-IL-18 treated mice(2900.46±62.65 mm3)and PBS-treated mice(3980.24±63.78 mm3).HE staining showed that the nuclei of tumor cells were heavily stained with few nucleoli in ZD55-IL-18-treated mice.Increased positivity rate of IL-18 was noticed in ZD55-IL-18-treated mice vs.AD55-IL-18-treated mice(83.4%±3.2%vs 24.4%±2.1%.P＜0.01).Moreover,immunofluorescence assay revealed the presence of E1A protein in tumor tissue.A decrease was found in the microvessel density in ZD55-IL-18-treated mice compared with the PBS-treated mice(P＜0.01).The apoptosis rate in tumor cells from high to low was 86.28%±3.25%in ZD55-IL-18-treated mice,43.67%±3.46%in Ad-IL-18-treated mice,and 10.73%±2.48%in PBS-treated mice;there was a significant difference between the three groups(all P＜0.05).Conclusion The oncolytic adenovirus expressing human IL-18 gene,ZD55-IL-18,has a significant inhibitory effect on the growth and metastasis of malignant melanoma implanted in nude mice.

AIM: To explore the mutual effect of co-culture of wild-type astrocytes (ASC) and motor neurons VSC4.1 (VSC) on the respective ability to produce reactive oxygen species (ROS). METHODS: The inhibition rates of cell growth in ASC and VSC in co-culture or independent culture was detected after exposed to excitatory stimulus by MTT method. Real-time observation of ROS production by ASC and VSC labeled with Hoechst 33342 was detected by confocal microscopy under the conditions of co-culture or independent culture. RESULTS: Higher concentration of glutamate induced a higher inhibition rate in mixed cell growth than that in ASC alone, while lower concentration of glutamate induced a higher inhibition rate in mixed cell growth than that in VSC only. Real-time observation by confocal microscopy showed that ROS production by VSC under the condition of co-culture, which showed a notable increase at 15 min, was significant less than that in independent culture, which peaked at 5 min and was gradually decreased. ROS production by ASC in co-culture began to increase significantly at 10 min. CONCLUSION: Compared to independent culture, ASC reduces the resting ROS production by co-cultured with VSC, while ASC prolongs the duration of ROS production by VSC after exposed to excitatory stimulus.

Objective:to investigate the clinical feature and dynamic changes of the cervical dural sac and spinal cord during neck flexion in Hirayama disease(juvenile muscular atrophy of distal upper extremity).Methods:Clinical data were taken and MRI in neutral neck position and a fully flexed neck position were performed on 27 cases of Hirayama disease.Results:(1)All patients were consistent with the diagnostic criteria of Hirayama disease who had asymmetric muscular atrophy and weakness of the hand and forearm.All patients were young males and right handed of whom 77.8% had initial symptoms before they were 19 years old.More patients(20 cases,74%)had muscular atrophy in the right hand than in the left at onset.The duration after disease onset was from 2-72 months[(26.48?15.57)months].(2)In neutral neck position by MIR examination,16 patients showed abnormal cervical curvature,14 showed atrophy of the lower cervical cord and 2 patients had intramedullary abnormal high signal.(3)In a fully flexed position of the neck,all patients showed forward displacement and flattening of the lower cervical cord,and a crescent-shaped high signal area behind the cord.(4)The crescent-shaped area was enhanced on T1-weighed imaging and disappeared after the patient returned to a neural position in one case.Conclusion:Hirayama disease occurs mainly in young males.There are obviously dynamic changes of the cervical cord during neck flexion in Hirayama disease by MRI examination,which can help the doctor make diagnosis in the early stage.