Laparoscopic surgery for upper-third gastric cancer has gradually been accepted by experienced surgeons as the mature of this technique.Different from the standardized and programmed D2 lymph node dissection in Laparoscopy-assisted Billroth Ⅰ gastrectomy,the indications and methods for laparoscopic splenic hilar lymphadectomy in the upper-third gastric cancer remains controversial.Unsolved problems include joint organs resection,appropriate surgical approach selection and variable vascular anatomy of the splenic hilum.Meanwhile,the long-term efficacy and safety of laparoscopic splenic hilar lymphadectomy for the upper-third gastric surgery need to be confirmed by evidence-based medical trials.With the advance of the theory and clinical practice,laparoscopic splenic hilar lymph node dissection will continue to progress.

Objective To observe the expression changes of matrix metalloproteinase 9(MMP-9),vascular endothelial growth factor(VEGF)in the esophageal squamous cell carcinomas,and to study the chinical significance. Methods The expression of MMP-9 and VEGF in 60 patients with esophageal carcinoma and 20 cases with adjacent normal mucosa were tested with immunohistochemical SP method. Results The positive rate of MMP-9,VEGF in the esophageal squamous cell carcinomas were 70%(42/60)and 80%(48/60),the positive rate of adjacent normal mucosa were 10%(2/20)and 20%(4/20).The positive rates of the two groups were compared,all the differences had statistical significance(P＜0.05); expressions of MMP-9,VEGF in the esophageal squamous cell carcinomas related to invasive depth of carcinoma and lymph node metastasis(P＜0.05).There were positive correlation(r=2.330,P＜0.05). Conclusion The higher expression of MMP-9 and VEGF in the esophageal squamous cell carcinomas played an important role in invasion and metastasis of esophagus squamous cancer.

Objective The aim of this study was to investigate the relationship between phosphotyrosine interac-tion domain containing 1 ( PID1 ) gene and variation in intramuscular fat ( IMF ) content and the possibility to generate transgenic animals by testicular injection .Methods Expression vector pIRES2-acGFP-PID1 carrying pig PID1 gene was incubated with transfection reagents and injected into the testes of male New Zealand rabbits .We examined the F1 genera-tion by fluorescence detection , PCR, Western blotting and measuring the IMF content .The F1 generation gave birth to the F2 generation.Then we examined the F2 generation through detecting the positive rate and the IMF content .Results The exogenous PID1 gene and fluorescent protein gene were expressed at different levels both in the F 1 generation and the F2 generation, and the positive rates were 35.88%and 34.33%, respectively.The IMF content was significantly elevated (P<0.05) in the transgenic positive individuals compared with the negative ones and the control group , and the PID1 protein expression was similarly higher .Conclusions The results of this study demonstrate that PID 1 gene affects intramuscular fat content significantly .Moreover, the results of our analysis provide further evidence that transgenic animals can be gener -ated by testicular injection , and the exogenous gene can be inherited steadily .

Objective To investigate the effect of miRNA-384 (miR-384)expression on hepatic steatosis in mice with nonalcoholic fatty liver disease (NAFLD)induced by high-fat diet (HFD). Methods A total of 30 male C57BL/6J mice were fed for 7 days to adapt to the environment and then randomly divided into 2 groups,with 15 mice in each group. The mice in the control group were given normal diet, and those in the model group were given HFD for 8 weeks and then the liver tissue was harvested. HE and Nile red staining were used to ob-serve the pathological changes of the liver. Microarray sequencing was performed to determine the whole-genome miRNA expression profile of liver tissue,and PCR was used to measure the relative expression of miR-384. The t-test was used for the comparison of continuous da-ta between groups. Results In the control group,the liver was red with sharp edges,the lobular structure was clear,and there was no he-patic steatosis;in the model group,the liver was yellow with blunt edges,and the hepatocytes were swollen with a large number of fat vacu-oles in the cytoplasm and nuclear deviation caused by the fusion of lipid droplets. Compared with the normal mice,the NAFLD mice had 12 upregulated miRNAs and 18 downregulated miRNAs in liver tissue. Some of the differentially expressed miRNAs between the control group and the model group were screened to obtain the same cluster diagram. Among the 8 miRNAs with significant changes,miR-384 showed a significant fold change. Conclusion The upregulation of miR -384 is closely associated with hepatic steatosis,but its mechanism still needs further study.

Pig copper zinc superoxide dismutase (CuZnSOD) is an important antioxidant enzyme. Some studies focused on the function of CuZnSOD gene, but the transcriptional regulation of the CuZnSOD gene is not yet fully elucidated. Therefore, the aims of the study were to determine the core promoter region and to explore its mechanism of transcriptional regulation. The 853 bp DNA sequence of 5'-flanking promoter was amplified by performing PCR. A series of CuZnSOD promoter fragments with gradually truncated 5'-end were produced by nested PCR and inserted into pGL3-Basic vector. The activities of the promoters were measured by the dual-luciferase assay system after transient transfection into the NIH/3T3 cells. The results demonstrated that there were 2 potential transcription start sites in the regions from initiation codon to -87 bp and -266 bp, respectively. The region from -383 bp to +67 bp in CuZnSOD gene promoter showed higher activity than other regions, and further deletion analysis demonstrated that the region from -75 bp to -32 bp contained an essential promoter sequence for pig CuZnSOD gene transcription. In addition, several potential transcription factor binding sites were predicted with bioinformatics method. These results suggest that these transcription factor binding sites may be involved in the transcriptional regulation of CuZnSOD gene.
Animals ; Cloning, Molecular ; Gene Expression Regulation ; Genetic Vectors ; Mice ; NIH 3T3 Cells ; Promoter Regions, Genetic ; Superoxide Dismutase ; genetics ; Swine ; Transfection

Objective:To explore the effect of adipose derived mesenchymal stem cells to regulate the differentiation of macrophage RAW264.7.Methods:First,we used RAW264.7 cells to simulate macrophage and induced them to M 1 macrophage with lipopolysaccharide ( LPS,1 μg/ml) .Then we cultured these RAW264.7 cells in culture mediums which were previously used to culture adipose derived mesenchymal stem cells to imitate the transplantation of ADMSC .Last,the mRNA relative expression of IL-10, IGF-1,Arg-1,TNF-α,FIZZ1,SPHK-1 was detected by real-time PCR.The protein expression of IL-12 p40,IL-27 Rα,IL-10 was detected by Western blot.Results:After been cultured in ADMSCCM and induced by LPS ,M1 markers (TNF-αmRNA,IL-12 p40;P<0.05) of the RAW264.7 cells declined while M2 markers (IGF-1 mRNA,IL-10 mRNA,IL-10;P<0.05) rose.Conclusion: ADMSC can secrete soluble cytokines to induce the RAW264.7 cell,which have been induced to the M1 macrophages,to differentiate towards M2 macrophages.

BACKGROUNDOncogenesis, development, invasion and metastasis of lung cancer are modulated by relative genes of lung cancer, and the expression, deletion or mutation of these genes are regulated by cell membrane signal transduction system and cell membrane ionic channels. The aim of this study is to explore the electrophysiological properties of human lung adenocarcinoma cell line A549 and cell proliferation affected by tetraethylammonium chloride (TEA), one potassium channel blocker, so as to know whether the voltage-gated potassium channels are required for the proliferation of A549 cell line.

METHODSIonic currents were recorded by whole-cell patch-clamp recording technique. The proliferation activity of A549 cells was measured by MTT assay.

RESULTSMembrane current was observed when cells were held at -70mV and test potentials ranged from -30 to +110mV. The current exhibited properties of voltage-dependent, outward rectification and no or little inactivation over the 500ms voltage pulse. Exposure of tumor cells to 10mmol/L TEA reduced the peak outward potassium current (evoked by depolarization to +110mV) from (1057.52±59.17)pA to (212.26±11.96)pA, the ratio of suppression was 79.92% (P < 0.01). Obvious inhibitive effect of TEA with different concentrations ranged from 20 to 60mmol/L on proliferation activity of the cells was found.

CONCLUSIONSThe voltage-gated potassium channels exist in human lung adenocarcinoma cell line A549 and play a great role in proliferation of A549 cells. TEA can inhibit proliferation of A549 cell through blocking the potassium channels and the inhibition is dose-dependent.

BACKGROUNDWith the development of patch clamp and molecular biology technique, and the application of them in the investigation of tumor cellular membrane ion channnel, the ion channel is becoming the hot spot of the tumor base research gradually. The aim of this study is to investigate the electrophysiological properties of human lung adenocarcinoma cell line A-549 and the role of K+ channel in inhibition of cell proliferation by the recombinant mutant human tumor necrosis factor (rmhTNF).

METHODSIonic currents were recorded using the whole-cell patch clamp recording technique. The proliferation activity of A-549 cells was measured by MTT assay. The cell cycle and apoptosis rates of the carcinoma cells were measured by flow cytometric analysis (FCM).

RESULTSWhole-cell patch clamp recording revealed a voltage-gated K+ current in A-549 cells, which could be blocked by the K+ channel blocker, TEA and CsCl. The amplitude of K+ current was markedly diminished in all cells incubated with different concentration of rmhTNF (P < 0.01). Obvious inhibitive effect of rmhTNF on proliferation of the cells was found in vitro in a dose-dependent manner (P < 0.01), the maximal inhibitory rate was 38.68% when the concentration was 400U/mL. The rmhTNF inhibited the cell cycle shifting from G1 phase to S phase and promoted apoptosis as determined by FCM analysis. The proportion of G1 cells increased from 53.02% to 72.93%, and the apoptosis rate increased from 2.08% to 8.68%. The difference were significant between the control and the high concentration groups ( 200U/mL and 400U/mL) (P < 0.01).

CONCLUSIONSrmhTNF exerts its cytotoxic effects on A-549 cells through inhibiting cell cycle shifting and inducing apoptosis. The K+ channels on the A-549 cell membrane can be blocked by rmhTNF partly, and the effect of inhibiting proliferation and activating apoptosis on A-549 cells is a result of depression of the K+ channel.

Objective:To observe any effect of repetitive low-frequency transcranial magnetic stimulation (rTMS) on sleep disorders and abnormal behaviors of children on the autism spectrum.Methods:Forty autistic children were randomly divided into an observation group and a control group, each of 20. Both groups were given sleep behavior training and individualized conventional rehabilitation training. Those in the observation group also received 30min of rTMS at 1Hz applied over the left dorsolateral prefrontal cortical area once a day, 5 days a week. Before and after 8 weeks of this treatment, both groups were evaluated using the Autism Behavior Checklist (ABC), the Child Autism Rating Scale (CARS) and the children′s Sleep Habit Questionnaire (CSHQ).Results:The average CARS and CSHQ scores, as well as the total ABC score of both groups increased significantly over the 8 weeks, but the average CARS and CSHQ scores, as well as the total ABC score of the observation group were then significantly better than in the control group. After the treatment, the average ABC scores for sensory ability, communication ability, motor ability, and language ability were significantly lower than before the treatment for both groups, but the observation group′s averages were then significantly better than those of the control group.Conclusions:Supplementing routine intervention with low-frequency rTMS can effectively improve the sleeping and correct the abnormal behavior patterns of autistic children.

Objective:To explore the effect of low-frequency repetitive transcranial magnetic stimulation (rTMS) on the executive functioning and core symptoms of preschool children on the autism spectrum.Methods:Forty-three preschool children showing signs of autism were randomly divided into an rTMS group of 21 and a control group of 22. In addition to routine rehabilitation and training in basic living skills, the rTMS group was additionally provided with 1Hz rTMS for 8 weeks. Before and after the treatment, both groups were evaluated using the preschool version of the Behavior Rating Inventory for Executive Function (BRIEF-P), the Social Responsiveness Scale (SRS), the revised version of the Repetitive Behavior Scale (RBS-R) and the Child Autism Rating Scale (CARS).Results:After the 8 weeks of treatment, the average BRIEF-P, SRS, RBS-R and CARS scores of both groups had improved significantly, but the rTMS group′s averages where then significantly better than those of the control group.Conclusions:Low-frequency rTMS in addition to conventional rehabilitation intervention can significantly improve the executive functioning and core symptoms of preschool children on the autism spectrum.