VP1 gene encoded by the newly identified caprine enterovirus CEV-JL 14 was amplified and cloned to prokaryotic expression vector pGEX-4T-1.Recombinant GST-VP1 fusion protein was expressed,purified,emulsified with Freund's complete adjuvant and used to immunize the BALB/c mice following a standard procedure.The spleen cells from immunized mice were collected and fused with myeloma cells after its antibody titer reached over 104 times detected by indirect ELISA.Hybridoma cell clones secreting monoclonal antibodies against VP1 were screened and their stability and specificity were further determined.The identified hybridoma cells were injected to mice intraperitoneally and ascites were collected at 7 DPI.Isotypes of the monoclonal antibodies against the recombinant VP1 protein were characterized to be either IgG1 or IgG2b,which showed a high specificity for detection of caprine enterovirus antigens by immunoperoxidase monolayer assay,thus laying a solid basis for future study related to viral pathogenesis,detection and diagnostics for caprine enterovirus infection.

With the development of laparoscopic instruments and surgery technology, the application of totally laparoscopic digestive tract reconstruction is more and more widely in the minimally invasive surgery of gastric cancer. Separation of gastric ligament tissue, dissection of lymph node, and digestive tract reconstruction under total laparoscopy can reduce surgical trauma and incisions. In particular for patients with rib arch narrow and obesity, totally laparoscopic radical gastrectomy for gastric cancer has more obvious advantages. However, due to difficult surgical operation and high technical requirements, totally laparoscopic gastrectomy for gastric cancer need strictly following the basic principles of digestive tract reconstruction, controlling the indications and contraindications, and evaluating intraoperative quality control, which is recommended to be carried out in medical centers with rich experience in laparoscopic gastric cancer surgery. The authors investigate technology and development of digestive tract recon-struction in totally laparoscopic radical gastrectomy for gastric cancer based on relevant literatures and their clinical practice.

OBJECTIVETo explore the feasibility and efficacy of laparoscopic D2 radical gastrectomy in patients with gastric neuroendocrine carcinoma (GNEC).

METHODSClinical data of 84 patients with GNEC undergoing laparoscopic D2 radical gastrectomy in Union Hospital from January 2006 to December 2012 were analyzed respectively. Among these patients, 44 cases underwent laparoscopic D2 gastrectomy (LAG group) and 40 cases underwent open gastrectomy (OG group). The short- and long-term outcomes, 3-year survival and recurrence-free survival were compared between two groups.

RESULTSThe LAG group and OG group did not differ significantly in terms of clinicopathologic characteristics. All the patients completed operations successfully and no patients in the LAG group ware converted to laparotomy. The operative time was similar (P>0.05). As compared to OG group, LAG group had less intra-operative blood loss [(85±21) ml vs. (192±89) ml, P=0.003], lower ratio of transfusion [2.3%(1/44) vs. 15.0%(6/40), P=0.048], shorter time to ambulation after surgery [(2.5±1.1) days vs. (3.5±1.1) days, P=0.001], faster postoperative gastrointestinal function recovery [(2.9±1.1) days vs. (5.1±1.0) days, P=0.001], shorter time to resume soft diet [(4.1±1.2) days vs. (5.7±1.3) days, P=0.001] and shorter postoperative hospital stay [(12.0±3.4) days vs. (15.0±5.5) days, P=0.002]. No significant difference was observed in average dissected lymph node number between LAG and OG group (35.0±16.4 vs. 31.6±12.1, P=0.204). Morbidity of postoperative complication of LAG group and OG group was 11.4%(5/44) and 17.5%(7/40) respectively (P=0.422). The overall 3-year survival rate was 54.0% for all the patients, while 3-year survival rate was 56.3% in LAG group and 51.4 % in OG group (P=0.478). In addition, there was no significant difference in recurrence-free survival between the two group (33.0 months vs. 31.5 months, P=0.703).

CONCLUSIONCompared with open gastrectomy, laparoscopic D2 radical gastrectomy has the advantages of faster recovery and less blood loss, and similar short-term and long-term outcomes in treatment of patients with GNEC, thus it is a safe and feasible treatment for GNEC.

Blood Loss, Surgical ; Carcinoma, Neuroendocrine ; surgery ; Female ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Laparotomy ; Length of Stay ; Lymph Node Excision ; Male ; Middle Aged ; Operative Time ; Postoperative Complications ; Postoperative Period ; Stomach Neoplasms ; surgery ; Survival Rate ; Treatment Outcome

Objective To explore the effect of unplanned reoperation (URO) on clinical efficacy after radical resection of gastric cancer (GC),and its causes and risk factors analysis affecting URO.Methods The retrospective case-control study was conducted.The clinicopathological data of 4 124 patients who underwent radical resection of GC in the Union Hospital of Fujian Medical University between January 2005 and December 2014 were collected.The initial operation was open or laparoscopic radical resection of GC.Observation indicators:(1) initial operation situations,results of pathologic examination and follow-up situations;(2) postoperative recovery situations;(3) causes and time interval of URO after radical resection of GC;(4) univariate analysis affecting URO after radical resection of GC;(5) multivariate analysis affecting URO after radical resection of GC.Follow-up using outpatient examination,telephone interview and Wechat was performed to detect postoperative 30-day recovery of patients.Measurement data with normal distribution were represented as (-x)±s,and comparisons between groups were done using the independent-sample t test.Count data and univariate analysis were done using the chi-square test or Fisher exact probability.Multivariate analysis was done using the logistic regression model.Results (1) Initial operation situations,results of pathologic examination and follow-up situations:① Initial operation situations of 4 124 patients,2 608 and 1 516 underwent respectively laparoscopic surgery and open surgery;2 259 and 1 865 underwent respectively total gastrectomy and distal gastrectomy.② Results of pathological examination of 4 124 patients:883,468,959 and 1 814 were respectively in T1,T2,T3 and T4a stages;1 414,571,683 and 1 456 were in N0,N1,N2 and N3 stages;1 073,825 and 2 226 were in Ⅰ,Ⅱ and Ⅲ stages.③) All the 4 124 patients were followed up within 30 days after initial operation,with a follow-up rate of 100.000％(4 124/4 124),including 52 with URO and 4 072 without URO,with a early URO rate of 1.261％ (52/4 124).(2) Postoperative recovery situations:of 4 072 patients without URO,575 had postoperative complications,with an incidence of 14.121％ (575/4 072);17 died after operation,with a mortality of 0.417％ (17/4 072),and duration of postoperative hospital stay was (14.0±9.0) days.Of 52 patients with URO,23 had complications after reoperation,with an incidence of 44.231％ (23/52);6 died after reoperation,with a mortality of 11.538％ (6/52),and duration of postoperative hospital stay was (28.0± 13.0) days.There were statistically significant differences in above indicators between groups (x2=37.550,t=10.900,P＜0.05).(3) Causes and time interval of URO after radical resection of GC:total time interval between initial operation and URO of 52 patients was (6.9±6.7)days.Causes of URO of 52 patients:23 (2 deaths),7,6 (2 deaths),5,5 (1 death),3,2 and 1 (death) patients were respectively due to intraperitoneal hemorrhage,anastomotic bleeding,anastomotic leakage,intra-abdominal infection,small bowel obstruction,dehiscence of abdominal incisions,enteric perforation and pancreatic fistula,and time intervals between initial operation and URO of them were respectively (3.9±3.8)days,(0.9±_0.5)days,(7.9±4.7) days,(14.9±4.6)days,(16.4±9.9) days,(10.0±6.O)days,(6.7±5.2) days and 12.0 days.(4) Univariate analysis affecting URO after radical resection of GC:results showed that age,body mass index (BMI) and volume of intraoperative blood loss were risk factors affecting URO after radical resection of GC (x2 =5.468,7.589,5.041,P＜0.05).(5) Multivariate analysis affecting URO after radical resection of GC:results showed that age ＞ 70 years old,BMI ＞ 25 kg/m2 and volume of intraoperative blood loss ＞ 100 mL were independent risk factors affecting occurrence of URO after radical resection of GC (odds ratio =1.950,2.288,1.867;95％ confidence interval:1.074-3.538,1.230-4.257,1.067-3.267,P＜0.05).Conclusions URO can increase postoperative complications and mortality,and extend duration of hospital stay after radical resection of GC.Intraabdominal bleeding,anastomotic bleeding and anastomotic leakage are the main causes affecting occurrence of URO after radical resection of GC,and age ＞70 years old,BMI ＞ 25 kg/m2 and volume of intraoperative blood loss ＞ 100 mL are independent risk factors affecting occurrence of URO after radical resection of GC.

Objective: To investigate the clinical efficacy of Da Vinci robotic total gastrectomy combined with spleen-preserving splenic hilar lymphadenectomy for gastric cancer. Methods: The retrospective cross-sectional study was conducted. The clinicopathological data of 47 patients with gastric cancer who were admitted to Fujian Medical University Union Hospital from September 2016 to June 2018 were collected. There were 37 males and 10 females, aged from 23 to 75 years, with an average age of 60 years. Patients underwent Da Vinci robotic total gastrectomy combined with spleen-preserving splenic hilar lymphadenectomy. Observation indicators: (1) intraoperative situations; (2) postoperative situations; (3) postoperative pathological examination; (4) follow-up. Follow-up was conducted by outpatient examination and telephone interview to detect postoperative survival of patients up to September 2018. Measurement data with normal distribution were represented as Mean±SD, and measurement data with skewed distribution were expressed by M (range). Count data were represented as absolute numbers. Results: (1) Intraoperative situations: 47 patients underwent Da Vinci robotic total gastrectomy combined with spleen-preserving splenic hilar lymphadenectomy for gastric cancer, without conversion to open surgery. The operation time, hilar lymph node dissection time, volume of intraoperative blood loss, number of lymph node dissected, number of splenic hilar lymph node dissected, number of metastatic lymph nodes, number of metastatic splenic hilar lymph node were (225±36) minutes, (20±6) minutes, (40±27) mL, 40 (range, 17-112), 2 (range, 0-10), 2 (range, 0-29), 0 (range, 0-3). (2) Postoperative situations: time to first out-of-bed activities, time to first anal flatus, time to gastric tube removal, time to initial liquid diet intake, time to initial semi-liquid diet intake, time to drainage tube removal, and duration of postoperative hospital stay were (2.0±0.3)days, (3.4±0.9)days, (3.4±1.1)days, (4.8±1.0)days, (6.7±1.5)days, (8.5±2.5)days, and (12.0±8.3)days, respectively. Of 47 patients, 3, 2, 1, 1, and 1 were detected pulmonary infection, abdominal bleeding, anastomotic fistula, postoperative intestinal obstruction, and abdominal infection. There were 1, 5, and 2 patients with complications in grade Ⅰ, Ⅱ, and Ⅲ of Clavien-Dindo classification. There was no death in the postoperative 30 days. Two patients with abdominal bleeding were cured by intervene therapy, and patients with other complications were cured by conservative treatment. (3) Postoperative pathological examination: tumor diameter, cases with nodal or vascular or neural invasion, cases with differentiated and undifferentiated tumor (histological differentiation), cases in T1, T2, T3, T4 stages (T staging), cases in N0, N1, N2, N3 stages (N staging), cases in Ⅰ, Ⅱ, Ⅲ stages (TNM staging) were (4.2±1.6)cm, 26, 31, 16, 5, 22, 17, 3, 18, 9, 8, 12, 13, 20, 14, respectively. (4) Follow-up: 47 patients were followed up for 3-25 months, with a median time of 9 months. During the follow-up, 3 of 47 patients died of tumor recurrence, 1 had tumor-bearing survival, and 43 had tumor-free survival. Conclusion Da Vinci robotic total gastrectomy combined with spleen-preserving splenic hilar lymphadenectomy for gastric cancer is safe and feasible.

OBJECTIVETo explore the prognostic assessment value of preoperative blood platelet-lymphocyte ratio (PLR) in patients with gastric mixed adenoneuroendocrine carcinoma (gMANEC) treated with radical surgery.

METHODSClinical and pathological data of 84 gMANEC patients who underwent radical resection from 2006 to 2016 in Department of Gastric Surgery, Fujian Medical University Union Hospital were analyzed retrospectively. Receiver operating characteristic (ROC) curve analysis was performed to determine the cutoff value of the PLR for predicting prognosis. The Cox proportional hazards regression model was used to identify prognostic factors of gMANEC.

RESULTSAll the patients underwent D2 lymph node dissection, including 26 cases of distal subtotal gastrectomy and 58 cases of total gastrectomy. The postoperative pathological TNM stage system(pTNM) demonstrated that the patients of stage I(, II(, and III( were 9(10.7%), 14(16.7%), and 61(72.6%) cases, respectively. The median follow-up time was 40(3 to 96) months. The recurrence rate was 41.7%(35/84). The median time to recurrence was 10 (1 to 40) months, and 82.9%(29/35) patients experienced recurrence within the first 2 years after operation. The median overall survival time was 27(3 to 39) month, and the median recurrence-free survival time was 21 (1 to 96) months. The 1-, 3-, and 5-year overall survival(OS) rates were 87.6%, 56.6%, and 47.4%, respectively, and the 1-, 3-, and 5-year recurrence-free survival (RFS) rates were 70.5%, 50.7%, and 44.9%, respectively. The best cutoff value of the PLR for predicting prognosis was 133 through ROC curve, which categorized all the patients into low PLR group (≤133) comprising 28 patients and high PLR group (>133) comprising 56 patients. The tumor recurrence rate was significantly higher in high PLR group (50.0%, 28/56) than that in low PLR group(25.0%, 7/28)(P=0.028). The live metastasis rate was significantly higher in high PLR group(35.7%, 20/56) than that in low PLR group(10.7%, 3/28)(P=0.015). Cox regression analysis showed that only pTNM stage (P=0.003) was independent prognostic factors of OS, while both pTNM stage (P=0.000) and blood PLR (P=0.015) were independent prognostic factors of RFS.

CONCLUSIONgMANEC patients with high preoperative PLR tend to present recurrence and metastasis, especially to present live metastasis, so they should be kept under surveillance more frequently after surgery.

Blood Platelets ; Carcinoma ; Female ; Gastrectomy ; Humans ; Lymph Node Excision ; Lymphocyte Subsets ; Lymphocytes ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neuroendocrine Tumors ; pathology ; therapy ; Prognosis ; Proportional Hazards Models ; ROC Curve ; Retrospective Studies ; Stomach Neoplasms ; pathology ; therapy

Objective To investigate the relationship between systemic immune-inflammation index(SII),red blood cell distribution width(RDW),albumin(ALB)ratio(RAR)and severity of disease and respiratory failure in patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD).Methods A total of 110 patients with AECOPD were divided into the severe group(n=37)and the non-severe group(n=73).They were also divided into the respiratory failure group(n=26)and the non-respiratory failure group(n=84)according to whether they had respiratory failure.Platelet count(PLT),neutrophil count(NEU),lymphocyte count(LYM),RDW,ALB,SII and RAR were compared between different groups.Multiple Logistic regression method was used to analyze influencing factors of severe and concurrent respiratory failure.Receiver operating characteristic(ROC)curves were plotted to analyze the predictive value of indicators for severe condition and respiratory failure.Results Compared with the non-severe group,there were increased NEU,SII,RDW and RAR,and decreased PLT and LYM in the severe group(P＜0.05).Compared with the non-respiratory failure group,there were increased NEU,SII,RDW and RAR,and decreased LYM and ALB in the respiratory failure group(P＜0.05).Multivariate Logistic regression analysis showed that increased SII and RAR were independent risk factors for severe condition or respiratory failure in patients with AECOPD.ROC curves indicated that the area under the curve(AUC)of SII combined with RAR for predicting severe condition and respiratory failure in patients with AECOPD were 0.882(0.806-0.935)and 0.908(0.837-0.954),both of which were higher than those of SII or RAR alone(P＜0.05).Conclusion Combination of SII and RAR can effectively help to evaluate the condition and the occurrence of respiratory failure in patients with AECOPD.

Objective To investigate the development trend,safety and clinical effects of laparoscopic radical gastrectomy (LRG) for gastric cancer.Methods The retrospective and descriptive study was conducted.The clinicopathological data of 4 435 patients with gastric cancer who underwent LRG in the Fujian Medical University Union Hospital between January 2008 and December 2017 were collected.There were 3 263 males and 1 172 females,aged (61±11)years,with a range of 12-93 years.According to the operation time,4 435 patients were divided into two periods,including 1 588 patients of the early period (2008-2012) and 2 847 patients of the later period (2013-2017).Observation indicators:(1) the clinicopathological data of patients;(2) intraoperative and postoperative situations;(3) postoperative complications;(4) follow-up and survival situations.Follow-up using outpatient examination,visit to home,mail and telephone interview was performed to detect survival of patients once every 3 months within 2 years postoperatively and once every 6 months after 2 years postoperatively up to June 2018.Survival time was from operation time to the last follow-up,death or deadline of follow-up database such as loss to follow-up or death of other diseases.Measurement data with normal distribution were represented as Mean±SD,and comparison between groups was analyzed by the t test.Measurement data with skewed distribution were represented as M (range).Count data were described as frequency or percentage,comparison between groups was analyzed using the chi-square test.Linear analysis was done using the unitary linear regression.The survival rate and survival curve were respectively calculated and drawn by the Kaplan-Meier method,and Log-rank test was used for survival analysis.Results (1) The clinicopathological data of patients:there were 3 263 males and 1 172 females of the 4 435 patients,accounting for 73.574％(3 263/4 435) and 26.426％ (1 172/4 435),respectively.TNM staging of the 4 435 patients:1 133 cases were detected early gastric cancer (T1 stage) and 3 302 cases were detected advanced gastric cancer including 518,1 431,1 353 in T2,T3 and T4a stages respectively.Linear regression analysis showed a linear correlation between the cases of LRG and operation year (R2 =0.911,P＜0.05) and a gradually increasing in cases of LRG.The sex (male),cases with tumor at upper stomach,middle stomach,lower stomach,＞ 2 regions (tumor location),tumor diameter,cases with undifferentiated and differentiated tumor (pathological types),cases in pT1,pT2,pT3,pT4a stages (pT staging),in pN0,pN1,pN2,pN3a,pN3b stages (pN staging),in Ⅰ A,Ⅰ B,Ⅱ A,Ⅱ B,ⅢA,ⅢB,ⅢC stages (pTNM staging) were 1 204,383,302,714,189,(4.8±2.7)cm,361,1 227,382,193,418,595,588,212,255,318,215,325,137,150,172,253,267,284 in patients of the early period,and 2 059,807,530,1 128,382,(4.3±2.6) cm,976,1 871,751,325,1 013,758,1 138,444,505,486,274,616,258,378,322,528,443,302 in patients of the later period,with statistically significant differences between patients of the two periods (x2 =6.411,15.699,t =10.946,x2 =57.801,90.437,26.502,98.773,P＜0.05).(2) Intraoperative and postoperative situations:the volume of intraoperative blood loss,cases with intraoperative blood transfusion,cases with Billroth Ⅰ,Billroth Ⅱ,residual stomach Roux-en-Y anastomosis,esophagogastric anastomosis,esophageal Roux-en-Y anastomosis of digestive tract reconstruction,number of lymph nodes dissected,time for initial fluid diet intake,time for initial semi-fluid diet intake,duration of postoperative hospital stay were (120±75)mL,38,599,122,0,32,835,32±13,(4.5±l.7)days,(8.6±2.5)days,(13.0± 7.3) days in patients of the early period,(104±68)mL,17,441,673,21,18,1 694,37±15,(4.1± 1.5)days,(7.9±2.8) days,(12.3±7.6) days in patients of the later period,showing statistically significant differences between patients of the two periods (t=2.169,x2 =26.843,397.185,t=-10.764,2.125,3.347,2.779,P＜0.05).Further linear regression analysis showed a linear correlation between the average number of lymph nodes dissected and operation year (R2=0.826,P＜0.05) and a gradually increasing in average number of lymph nodes dissected.(3) Postoperative complications:690 of 4 435 patients had postoperative complications,with an incidence rate of 15.558％ (690/4 435),including 242 patients of the early period and 448 of the later period,showing no statistically significant difference (x2 =0.191,P ＞ 0.05).Eight patients died of severe postoperative complications,with a death rate of 0.180％ (8/4 435),including 5 of the early period and 3 of the later period,showing no statistically significant difference (x2 =2.485,P＞0.05).Of 4 435 patients,561 had stage Ⅰ-Ⅱ complications,with an incidence rate of 12.649％ (561/4 435),129 had stage Ⅲ-Ⅳ complications,with an incidence rate of 2.909％(129/4 435).There were 196 and 46 patients of the early period with stage Ⅰ-Ⅱ complications and stage Ⅲ-Ⅳ complications,365 and 83 of the later period with stage Ⅰ-Ⅱ complications and stage Ⅲ-Ⅳ complications,showing no statistically significant difference between patients of the two periods (x2 =0.211,0.001,P＞0.05).(4) Follow-up and survival situations:4 250 of 4 435 patients including 1 465 of the early period and 2 785 of the later period were followed up for 1-123 months,with a median time of 37 months.The 5-year cumulative survival rate was 63.9％.The 5-year cumulative survival rate was 91.8％,80.2％ and 39.5％ in the stage Ⅰ,Ⅱ,Ⅲ patients,respectively,showing a statistically significant difference (x2 =810.146,P＜0.05).The 5-year cumulative survival rate was 60.8％ and 66.7％ in patients of the early and later period,respectively with a statistically significant difference (x2 =17.887,P＜0.05).Stratified analysis of TNM staging:the 5-year cumulative survival rates of stage Ⅰ A,Ⅰ B,Ⅱ A,Ⅱ B,Ⅲ A,Ⅲ B,Ⅲ C patients in the early period were 92.7％,85.6％,79.4％,74.5％,58.1％,37.6％,18.9％ and 95.6％,90.4％,87.6％,79.5％,52.7％,41.2％,19.5％ in patients of the later period,with no statistically significant difference (x2 =0.414,2.575,2.872,2.119,0.632,0.972,2.212,P＞0.05).Conclusions Surgical volume of the LRG has shown an increasing trend year by year,and the number of lymph nodes dissected and postoperative recovery of patients are improving.LRG is a safe procedure with acceptable clinical efficacy for gastric cancer.

Objective To investigate the application value of indocyanine green (ICG) fluorescence imaging in lymphadenectomy of laparoscopic radical gastrectomy for gastric cancer.Methods The retrospective cohort study was conducted.The clinicopathological data of 702 patients with primary gastric cancer who underwent laparoscopic radical gastrectomy and D2 lymphadenectomy in the Fujian Medical University Uuion Hospital between April and December 2017 were collected.There were 517 males and 185 females,aged from 22 to 91 years,with an average age of 61 years.Of the 702 patients,39 using ICG fluorescence imaging in the surgery and 663 not using ICG fluorescence imaging were allocated into ICG group and non-ICG group,respectively.Observation indicators:(1) surgical situations and postoperative recovery;(2) postoperative complications;(3) average number of lymph node dissected and positive lymph nodes;(4) follow-up situations.The number of lymph node dissected in the first station (No.1-7 group) and second station (No.8-12 group) were analyzed respectively.Follow-up using outpatient examination and telephone interview was performed to detect complications of patients up to June 2018.Measurement data with normal distribution were represented as Mean±SD,comparison between groups was analyzed using the t test.Measurement data with skewed distribution were described as M (range),comparison between groups was analyzed using the Mann-Whitney U test.Count data were represented as absolute number or percentage,comparison between groups was analyzed using the chi-square test.Comparisons of ordinal data were analyzed by the Mann-Whitney U test.Results (1) Surgical situations and postoperative recovery:702 patients underwent successfully laparoscopic radical gastrectomy and D2 lymphadenectomy,without injuries of important vessels and adjacent organs,without combined multiple organs resection or conversion to open surgery.Of 39 patients in the ICG group,cases undergoing total radical gastrectomy,distal subtotal gastrectomy and proximal subtotal gastrectomy,cases with Roux-en-Y esophagojejunostomy,Billroth Ⅰ anastomosis,Billroth Ⅱ anastomosis,Roux-en-Y gastrojejunostomy and esophagogastric anastomosis,operation time,volume of intraoperative blood loss,time for out-of-bed activities,time to initial anal exsufflation,time to first fluid diet intake and duration of postoperative hospital stay were 21,16,2,21,3,13,2,0,(173±28) minutes,40 mL (range,5-200 mL),(2.1±0.6)days,(3.5±1.4)days,(4.8± 1.3)days,(8.6±3.6) days.The above indexes were 363,299,1,363,27,267,1,5,(174±41)minutes,50 mL(range,0-1 750 mL),(2.2±0.8)days,(3.4± 1.1) days,(4.6± 1.5) days,(9.4± 5.0) days in the non-ICG group.There were statistically significant differences in the surgical type and digestive reconstruction method (x2 =9.550,11.388,P＜ 0.05) and no statistically significant difference in the operation time,volume of intraoperative blood loss,time for out-of-bed activities,time to initial anal exsufflation,time to first fluid diet intake and duration of postoperative hospital stay (t =0.221,Z =-0.651,t =0.492,-0.826,-0.842,0.995,P＞0.05).(2) Postoperative complications:92 out of the 702 patients had postoperative complications,without death of complications.The incidence of complication was 15.38％(6/39) and 12.97％(86/663) in the ICG group and non-ICG group,with no statistically significant difference between the two groups (x2=0.188,P＞0.05).Six patients with complications (1 of Clavien-Dindo Ⅳ,2 of Clavien-Dindo Ⅲa,3 of Clavien-Dindo Ⅰ) in the ICG group and 86 (6 of Clavien-Dindo Ⅳ,16 of Clavien-Dindo Ⅲ,61 of Clavien-Dindo Ⅱ,3 of Clavien-Dindo Ⅰ) in the non-ICG group were cured after symptomatic treatment.(3) Average number of lymph node dissected and positive lymph nodes:the average number of lymph node dissected and positive lymph nodes was 37 (range,3-112) and 1 (range,0-68) of 702 patients,38 (range,24-70) and 2 (range,0-42) in the ICG group,37 (range,3-112) and 1 (range,0-68) in the non-ICG group,with no statistically significant difference between the two groups (Z=-1.454,-0.514,P＞0.05).Stratified analysis:the average number of No.1-7 group lymph nodes dissected and positive lymph nodes was 34 (range,16-67) and 2 (0-38) in the ICG group,33 (range,3-91) and 1 (range,0-56) in the non-ICG group.The average number of No.8-12 group lymph nodes dissected and positive lymph nodes was 11 (range,4-22) and 0 (range,0-13) in the ICG group,9 (range,0-31) and 0 (range,0-25) in the non-ICG group.There was a statistically significant difference in the average number of No.8-12 group lymph nodes dissected between the two groups (Z=-1.984,P＜0.05).There was no statistically significant difference in the average number of No.1-7 group lymph nodes dissected,positive No.1-7 group lymph nodes and the average number of positive No.8-12 group lymph nodes between the two groups (Z =-1.302,-0.463,-0.758,P＞0.05).(4) Follow-up situations:702 patients were followed up for 6-14 months,with a median time of 10 months.There was no readmission caused by postoperative complications in the two groups.Conclusion ICG fluorescence imaging in lymphadenectomy of laparoscopic radical gastrectomy for gastric cancer is beneficial to dissection of perigastric lymph nodes and increase number of lymph nodes dissected,but cannot increase operation time and incidence of postoperative complications.

Objective:To investigate the mechanism of action and prognostic value of Dynamin 3 (DNM3) in gastric cancer.Methods:The bioinformatic analysis, experimental study and retrospective cohort study was conducted. The clinicopathological data, fresh gastric cancer tissues, paired normal tissues and the corresponding paraffin sections of 153 gastric cancer patients who underwent radical gastrectomy in Fujian Medical University Union Hospital from January 2013 to July 2018 were collected. Tissues and the corresponding paraffin sections were subjected to quanti-tative real-time polymerase chain reaction, immunoblotting assay, flow cytometric cell cycle assay and immunohistochemical staining, respectively, and clinicopathological data were used for prognostic analysis. The stomach adenocarcinoma (STAD) dataset from the Cancer Genome Atlas (TCGA) database was collected for bioinformatic analysis. Observation indicators: (1) DNM3 gene expression in TCGA-STAD in gastric cancer; (2) mutations and copy number alterations of DNM3 in gastric cancer; (3) methylation level of promoter of DNM3 in gastric cancer; (4) relative protein expression of DNM3 and p53 in gastric cancer; (5) DNM3 correlation and enrichment analysis; (6) ratio of G0/G1 phase, S phase and G2/M phase of cell cycle progression; (7) correlation between immune cell infiltration and DNM3 in gastric cancer; (8) correlation between results of immunohistochemical (IHC) staining and clinical features; (9) analysis of independent factors influencing 5-year overall survival rate of gastric cancer patients. Measurement data with normal distribution were represented as Mean±SD, and comparison among multiple groups was conducted using the ANOVA and further comparison between two groups was conducted using the LSD. Comparison between two groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers or percentages, and compari-son between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data was conducted using the rank sum test. The Pearson correlation coefficient or Spearman correlation coefficient was used to test the correlation between groups. Univariate and multivariate analyses were conducted using the COX proportional risk regression model. The Kaplan-Meier method was used to draw survival curves and calculate survival rates, and the Log-Rank test was used for survival analysis. The Benjamini-Hochberg false discovery rate correction was used for adjusting of the P-value. Results:(1) DNM3 gene expression in TCGA-STAD. The expression levels of DNM3 gene in the 27 tumor tissues and paired normal tissues of the TCGA-STAD database were 0.775(0.605,1.161) and 1.216(0.772,1.681), showing a significant difference between them ( Z=?2.64, P<0.05). The messenger RNA (mRNA) expression levels of DNM3 gene in 48 pairs of gastric cancer tissues and paired normal tissues of the author′s center were 4.370(2.870,6.040) and 2.520(0.850,4.170), showing a significant difference between them ( Z=?4.39, P<0.05). (2) Mutations and copy number alterations of DNM3 in gastric cancer. There were 16 gastric cancer patients in the TCGA-STAD database with DNM3 mutation or somatic copy number alterations, including 6 cases with missense mutations, 1 case with truncated mutation, 8 cases with copy number gain and 1 case with copy number loss. The mRNA expression levels of DNM3 gene before and after mutation in the 370 gastric cancer patients of the TCGA-STAD database were 6.13(5.40,7.08) and 5.02(3.98,5.46), showing a significant difference between them (Log 2FC=?1.11, Z=?2.59, P<0.05). (3) Methylation level of promoter of DNM3 in gastric cancer. There were 372 gastric cancer patients in the TCGA-STAD database undergoing DNM3 methylation and mRNA examinations, and the results showed that levels of methylation and mRNA expression of DNM3 was 0.198 (-0.458, 0.301) and 6.014 (5.141, 6.628), respectively. The levels of methylation in DNM3 was negatively correlated with its mRNA expression ( r=?0.38, P<0.05). Results of follow-up in 32 patients showed that the 3-year overall survival rate of 16 cases with high levels of methylation in DNM3 and 16 cases with low levels of methylation in DNM3 was 18.8% and 41.3%, respectively, showing a significant difference between them ( hazard ratio=1.40, P<0.05). Results of immunoblot-ting assay showed that the relative expression level of DNM3 protein in the AGS cells treated with 0, 0.5, and 1.0 μmol/L of 5-azacytidin was 0.270±0.020, 0.357±0.051 and 0.599±0.039, respectively, showing a significant difference among the three groups ( F=57.84, P<0.05). The relative expression level of DNM3 protein in the HGC-27 cells treated with 0, 0.5, and 1.0 μmol/L of 5-azacytidin was 0.316±0.038, 0.770±0.031 and 0.877±0.052, respectively, showing a significant difference among the three groups ( F=156.30, P<0.05). (4) Relative protein expression of DNM3 and p53 in gastric cancer. Results of immunoblotting assay showed that the relative expression of DNM3 and p53 protein was 0.688±0.047 and 0.872±0.041 in the AGS cells transfected with pCMV-DNM3 plasmid, versus 0.249±0.029 and 0.352±0.020 in the AGS cells transfected with control plasmid, showing significant differences in the above indicators between the two types of cells ( t=13.77,19.74, P<0.05). The relative expression of DNM3 and p53 protein was 0.969±0.069 and 1.464±0.081 in the HGC-27 cells transfected with pCMV-DNM3 plasmid, versus 0.456±0.048 and 0.794±0.052 in the HGC-27 cells transfected with control plasmid, showing significant differences in the above indicators between the two types of cells ( t=10.57, 12.06, P<0.05). (5) DNM3 correlation and enrichment analysis. Results of correlation analysis showed that DNM3 was positively correlated with genes such as RBMS3, CNTN4 and PDE1A ( r=0.52, 0.52, 0.50, P<0.05) and negatively correlated with genes such as SLC25A39, PAICS and GAPDH ( r=?0.41, ?0.40, ?0.40, P<0.05) in gastric cancer. Results of gene set enrichment analysis showed that the set of genes related to ribosome and oxidative phosphorylation were upregulated in gastric cancer patients with DNM3 low expression [normalized enrichment score (NES)=?3.30, ?2.16, P<0.05], while the set of genes related to immunomodulatory interactions between lymphocytes and non-lymphoid cells were upregulated in gastric cancer patients with DNM3 high expression (NES=1.67, P<0.05). Results of gene ontology analysis showed that the low expression of DNM3 was associated with the separation of mitotic sister chromatid (No.0000070), nonsense-mediation of nuclear transcriptional mRNA catabolic process, sister chromatid separation (No.0000819), nuclear transcriptional mRNA catabolic process and regulation of oxidative phos-phorylation (NES=?2.29, ?3.10, ?2.33, ?2.56, ?2.68, P<0.05). Results of Kyoto encycl opedia of genes and genomes analysis showed that metabolic pathway related to ribosome and oxidative phosphory-lation were upregulated and crosstalked in gastric cancer with low expression of DNM3 (NES=?3.34, ?2.21, P<0.05). (6) Ratio of G0/G1 phase, S phase and G2/M phase of cell cycle progression. Results of flow cytometric cell cycle experiments showed that the proportions of G0/G1 phase, S phase and G2/M phase in the cell cycle was 65.1%±3.0%, 17.3%±3.0% and 17.6%±1.0% in the AGS cells transfected with pCMV-DNM3 plasmid, versus 53.4%±4.0%, 26.3%±2.0% and 20.3%±3.0% in the AGS cells transfected with control plasmid, showing significant differences in the proportions of G0/G1 phase and S phase in the two types of cells ( t=4.05, 4.32, P<0.05). (7) Correlation between immune cell infiltration and DNM3 in gastric cancer. Results of immune cell infiltration examination showed that the expression level of DNM3 was positively associated with mast cells, NK cells, pDCs, B cells, follicular helper T cells, effector memory T cells, T cells, central memory T cells, CD8 T cells, DC cells, macrophages, γ-δ T cells (Tgd), iDCs and eosinophils infiltration (Spearman correlation coefficients as 0.41, 0.29, 0.26, 0.20, 0.22, 0.22, 0.13, 0.16, 0.15, 0.14, 0.14, 0.17, 0.18, 0.22, P<0.05) and negatively associated with Th17 cell, Th2 cells and NK CD56 dim cells infiltration ( r=?0.18, ?0.23, ?0.10, P<0.05). (8) Correlation between results of IHC staining and clinical features. Results of IHC staining analysis showed that the IHC score of DNM3 was 3(2,4) in the 105 gastric cancer tissues, versus 6(4,9) in the 105 paired normal tissues, showing a significant difference between them ( Z=-7.35, P<0.05). There were significant differences in gender, tumor location and N stating between the 70 patients with low expression of DNM3 and the 35 patients with high expression of DNM3 ( χ2=4.29, 7.67, 6.86, P<0.05). (9) Analysis of independent factors influencing 5-year overall survival rate of gastric cancer patients. Results of multivariate analysis showed that stage pT3?4 and low IHC score of DNM3 were independent risk factors for 5-year overall survival rate of gastric cancer patients ( hazard ratio=1.91, 0.51, 95% confidence interval as 1.06?3.43, 0.26?0.98, P<0.05). The 5-year overall survival rate was 44.3% in patients with low expression of DNM3, versus 65.7% in gastric cancer patients with high expression of DNM3, showing a significant difference between them ( χ2=5.02, P<0.05). Conclusion:DNM3 is a tumor suppressor and an independent predictor of poor prognosis for gastric cancer, which may regulate gastric cancer cell cycle and immunosuppression in the tumor microenvironment through methylation.