Objective To explore the feasibility of hand-assisted laparoscopic splenectomy and azygos-portal disconnection. Methods Hand-assisted laparoscopy was performed in 12 patients with hypersplenia secondary to post-hepatitic hepatocirrhosis and a history of rupture and bleeding of esophago-gastric varicose vein.An ultrasound knife was used to dissect the ligaments of the spleen.The Endo-Cutter was used to cut off the pedicle of the spleen.Then the spleen was removed in a plastic bag.All of the varicose vessels around the fundus and the lower segment of the esophagus(6~8 cm in length) were dissected and disconnected according to the criteria of open surgery.Results The operation was successfully completed in 10 patients,while conversions to open surgery were required in 2 patients because of massive hemorrhage during the operation.The operating time was 2.5~5 h(mean,3.4 h) and the hemorrhagic volume was 100~500 ml(mean,250 ml).Postoperatively,1 patient experienced an intraperitoneal hemorrhage and received open surgery for hemostasis while the remaining patients had an uneventful recovery without complications.A total of 10 patients were followed for 0.5~2 years(mean,1.5 years).Four patients died of liver failure.Six patients presented small volumes of relapsed upper gastrointestinal bleeding around 1 year after operation.Gastroscopy showed portal hypertensive gastropathy in 3 patients,gastric ulcer in 1 patient,and ruptured varicose esophageal veins in 2 patients.All the 6 patients were cured by conservative medical treatment.Conclusions Hand-assisted laparoscopic splenectomy and azygos-portal disconnection is a feasible,effective,and safe surgical procedure.

Objective To explore the variations of regeneration hormones after hepatectomy for liver cancer,and evaluate the relationship between the liver regeneration hormones and cancer recurrence.Methods The clinical data of 129 patients with primary hepatic carcinoma in our hospital from Dec 2004 to Dec 2005 were collected.The patients were divided into three groups according to their recurrent times,which were one-month recurrence group,6 months recurrence group and one-year recurrence group.And at the same time,40 cases of liver cancer that received TAE treatment were as contrast group.Serum HGF value was detected before operation and 1,3,7,10 and 14 days after operation.c-met,which is the receptor of HGF,was also detected as c-met mRNA and protein expression in cancer tissue and near-carcinoma liver tissue by semi-quantitative RT-PCR and Western Blot.The differences between the level of expression and the time of recurrence were compared,and the results were also compared with pathological indexes.Results Serum HGF value elevated after hepatectomy,the crest time appeared at about 10 days after the operation,and decreased after 14 days.The elevated values of HGF in large HCC tumors were markedly higher than those in small HCC tumors.The change of c-met mRNA and protein levels,revealed that the earlier the recurrence in both large and small HCC,the higher the c-met levels,and the higher the rate of vascular cancer emboli.Conclusions There is marked elevation of HGF level after hepatectomy in patients with liver carcinoma,and the over expression of c-met of the tumor may be related to its early postoperative recurrence.

Objective To study the application of extracellular laccase from endophytic fungi to total flavonoid extraction. Methods The extracellular laccase vitality of an endophytic Fusarium sp.C-8 from Artemisa annua reached 36.1 U/mL when the fungus was cultivated in revised PDA medium with initial pH 7.0 at 20℃ on a rotary shaker incubator for 6 d. The crude laccase from the cultural medium was applied for extraction of flavonoid from the buds of Sophara japonica. The optimization on the ratio of dry buds to crude enzyme liquid,temperature,time,and pH value was carried out during the process of laccase-assistant treatment. Results After incubation with 40∶1 of crude enzyme (pH 7.0) at 20℃ to dry buds for 1 h,the extraction rate of total flavonoids was 11.4 %,a more increase of 28.7 % than that in regular extraction process. Conclusion The results present a practical method of laccase-assistant extraction process on total flavones in S. japonica buds.

Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.

Objective To investigate the effect of preoperative angiography and intraoperative Doppler blood flow detection in the surgery of colonic interposition for esophageal.Methods Thirty-seven patients who underwent the surgery of colonic interposition for esophageal were chosen,17 cases received colonic interposition for esophageal routine operation (routine group),and 20 cases received preoperative angiography of superior and inferior mesenteric arteries and intraoperative Doppler blood flow detection (improved group),the clinical efficacy was observed.Results Routine group occurred anastomotic leak in 4 cases on postoperative 6-15 d,operation time was (367.0 ± 53.3) min,and improved group occurred anastomotic leak in 1 case on postoperative 7 d,operation time was (302.0 ± 67.1) min,the operation time between two groups had significant difference (P ＜ 0.05).Conclusions The adoption of preoperative angiography and intraoperative Doppler blood flow detection can real-time directly display the vascular morphology of transplanted colon segment,observe the traveling direction and distribution of mesenteric arteries,provide accurate and rehable evaluation of blood vessel of transplanted colon,which can conduct to the optimum selection of transplanted colon and reduce the incidence of postoperative complications.

Objective To analyze the expression of miRNA-29a in U937 macrophages infected with Mycobacterium tuberculosis and its regulation of target genes.Methods The target genes of miRNA-29a were predicted with softwares miRnada,RNAhybrid and targetscan.The differentiation of U937 macrophages was induced by phorbol ester(PMA), the induced U937 cells were infected with bacilli calmette guerin (BCG).The expression levels of miRNA-29a and its target genes in U937 cells were detected with real-time fluorescence quantitative PCR(RT-fqPCR).The miRNA-29a over-and low-expression U937 macrophage cell lines were constructed and the levels of miRNA-29a and its garget genes were detected.The SPSS 18.0 software was used to analyze the data.Results As predicted by relevant softwares,the miRNA-29a regulate the expression of VEGFA,NFATC3 and TSC22D3 genes.After BCG infection,the expression of miRNA-29a increased to 1.33 fold(P <0.05), and the expression levels of VEGFA, NFATC3 and TSC22D3 were increased to 5.34,99.25 and 2.12 fold,respectively(P<0.01).In the miRNA-29a over-expressing U937 macrophages,the expression levels of VEGFA,NFATC3 and TSC22D3 were up-regulated to 1.35,1.29 and 3.38 fold,respectively(P<0.05 or <0.01).While in the U937 macrophages with miRNA-29a knock-down,the expression levels of VEGFA, NFATC3 and TSC22D3 were down-regulated to 0.07, 0.08 and 0.55 fold, respectively(P <0.01).Conclusion The results suggest that Mycobacterium tuberculosis infection can increase the expression of miRNA-29a in U937 macrophages,further targeting the regulation of VEGFA, NFATC3 and TSC22D3 gene expression, which may participate in the pathogenesis and development of tuberculosis.

The serious decrease in the number of functional β cells is one of the main features in the pathogenesis of diabetes mellitus. CDKN1B is a new kind of regulatory protein, which can bind and inactivate cyclin and cyclin-dependent kinase complex to control the process of cell cycle. It was suggested that down-regulation or deletion of CDKN1B in islet β cells could accelerate the proliferation of islet β cells, thus increasing the number of islet β cells, which is of great significance for treatments of diabetes.

Objective:To investigate the changes of T-lymphocyte subsets, T-cell immunoglobulin and mucin domain molecule-1 (TIM-1) and TIM-3 levels, and cytokines in the peripheral blood of patients with active tuberculosis.Methods:From December 2017 to December 2018, 50 tuberculosis patients and 50 cured tuberculosis patients in Zhejiang Hospital of Integrated Chinese and Western Medicine were selected as the tuberculosis group and cured tuberculosis group, respectively. Fifty healthy individuals in the same period were selected as the control group. Flow cytometry was used to detect the T-lymphocyte subsets in the peripheral blood. The mRNA levels of TIM-1, TIM-3, interferon(IFN)-γ and interleukin(IL)-4 in peripheral blood mononuclear cells (PBMC) were detected by quantitative real-time polymerase chain reacticn (PCR). T test was used for statistical analysis. Results:The ratio of CD4 + /CD8 + T lymphocytes in the tuberculosis group (1.21±0.50) decreased significantly, comparing with those in the cured tuberculosis group (1.88±0.62) and the control group (1.92±0.82). The differences were statistically significant ( t=2.148 and 2.207, respectively, both P<0.05). The mRNA levels of TIM-1, TIM-3 and IL-4 in PBMC in the tuberculosis group were 2.16±0.37, 1.59±0.36 and 1.52±0.69, respectively, which were all higher than those in the cured tuberculosis group (1.60±1.23, 1.01±0.52 and 0.91±0.36, respectively) and the healthy control group (1.40±0.27, 0.92±0.34 and 0.79±0.42, respectively). All of these differences were statistically significant ( t=14.120, 11.440, 17.130, 12.090, 12.050 and 17.030, respectively, all P<0.05). However, the IFN-γ mRNA level (0.43±0.11) was lower than that in the cured tuberculosis group (1.74±0.72) and the control group (1.82±1.17), and the differences were both statistically significant ( t=13.880 and 11.430, respectively, both P<0.05). Conclusion:The immune dysfunction in patients with active tuberculosis may be related to the low ratio of CD4 + /CD8 + T lymphocytes, the increased expressions of TIM-1 and TIM-3, and the imbalance of helper T lymphocyte (Th)1/Th2 cytokines.

Objective:To explore the prognostic risk factors of thymoma patients after resection, and establish a novel nomogram to predict progression free survival(PFS) of patients with thymoma.Methods:A retrospectively analysis was performed on clinicopathological datas of 267 cases of thymoma patients underwent thymoma resection in Beijing Tongren Hospital from January 2010 to December 2019. The univariate and multivariate Cox risk ratio models were used to analyze the related factors that might affect PFS, and the prediction nomogram of PFS after thymoma resection was established using the screened independent risk factors. Then the predictive ability of the model was evaluated. Results:The univariate analysis showed that age, type of surgery, completeness of resection, WHO histologic classification, TNM stage and postoperative adjuvant therapy were significantly correlated with PFS after thymoma resection( P<0.05). The multivariate analysis showed that only age and TNM stage were independent prognostic factors affecting PFS after thymoma resection( P<0.05). The concordance index( C- index) of the prediction model for the prognosis of thymoma patients established by this method was 0.866(95% CI: 0.809-0.923), which had remarkable predictive efficiency. Conclusion:The nomogram model is constructed and verified based on age and TNM stage, excluding the interference of other clinicopathological factors on prognosis assessment, and which is convenient for clinicians to quickly and individually evaluate the prognosis of patients after thymoma resection.

Objective: To investigate the effect of metformin on autophagy in muscle cells exposed to palmitic acid, and to explore its mechanism. Methods: L6 rat myoblasts were incubated with palmitic acid at various concentrations(0.1, 0.2, 0.4, 0.6 mmol/L) and metformin(0.5, 1, 2, 5, 10 mmol/L) for 24 h. CCK8 method was used to detect the survival rate of muscle cells. After muscle cells were treated with palmitic acid and metformin for 24 h, mRNA and protein expressions of microtubule-associated protein11ight chain3(LC3Ⅱ), Beclin 1, p62, and silent mating type information regulation2 homolog-3(SIRT3) were detected by RT-PCR and Western blot, respectively. AMP-activated protein kinase(AMPK) phosphorylation level was detected by Western blot. Results: Palmitic acid dose-dependently decreased the survival rate of muscle cells, which was attenuated by metformin at the concentration of 2 mmol/L. After muscle cells were incubated with 0.4 mmol/L palmitic acid and 2 mmol/L metformin for 24 h, palmitic acid significantly reduced the mRNA and protein expressions of LC3Ⅱ, Beclin1, and SIRT3 as well as phosphorylation level of AMPK(all P<0.05), and increased p62 mRNA and protein expressions(P<0.05). Those effects were all antagonized by metformin(all P<0.05). Conclusions Metformin treatment may promote the autophagy of muscle cells exposed to palmitic acid through AMPK/STRT3 pathway.