Objective To observe the biological activity of the recombinant immunotoxin hIL-2-LuffinP1 derived by gene engineering in vitro.Methods To observe the inhibitory effect on lymphocytes in vitro,the splenocytes isolated from C57 and BALB/c mice were used for mixed mouse lymphocyte response(MLR),and the lymphocyte proliferation was observed in vitro with BALB/c mice splenocytes stimulated by concanavalin A(ConA).Different concentrations of hIL-2-LuffinP1 was added into the reaction system,with LuffinP1 adopted as control.3H-TdR incorporation assay was used to detect the effects of hIL-2-LuffinP1 on lymphocyte proliferation,and its effects on CTLL-2 cells(IL-2 dependent cells) were also observed.The experiments consisted of six groups(PBS,IL-2,LuffinP1,LuffinP1+IL-2,hIL-2-LuffinP1 and hIL-2-LuffinP1+IL-2).CTLL-2 cells were cultured for 12h after being treated with different agents,and then the cells were stained by trypan blue to estimate the dead cells in every 100 cells.Results It was showed that CPM declined correspondingly to the gradually increased concentration of hIL-2-LuffinP1,and cell proliferation was inhibited obviously.The inhibition rate was up to 97% at the concentration of 10-6mmol/L of hIL-2-LuffinP1.The activity of inhibiting lymphocyte proliferation was proportional to the dosage.On the contrary,no obvious inhibition to lymphocyte proliferation was found in the control group treated with LuffinP1,the inhibition ratio only reached to 14% with the same concentration of hIL-2-LuffinP1.It was also found that,when hIL-2-LuffinP1 and LuffinP1 in different concentrations added to spleen cells stimulated by ConA,hIL-2-LuffinP1 still exhibited strong ability of inhibition on the splenocyte proliferation,while LuffinP1 has no obvious inhibiting effect on the lymphocyte proliferation in the two reaction systems mentioned above.MLR showed the same results.Furthermore,in the experiment of cytotoxic effects of IL-2-LuffinP1 on CTLL-2 cells,the inhibition rates of hIL-2-LuffinP1+IL-2 group and hIL-2-LuffinP1 group were 29.6% and 47.4% respectively,the difference was significant compared with IL-2 group(P

Objective To explore the mechanism that Staphylococcus aureus(S.aureus) α-toxin(Hla) regulate miR-142 expression and inhibit macrophage phagocytosis.Methods BALB/C mice and RAW264.7 cells were infected with wild type S.aureus(WT S.aureus),Hla deletion S.aureus(△HlaS.aureus) or phosphate buffered saline(PBS),and the expression level of miR-142 of skin tissues and cells were detected.miR-142 mimics and inhibitor were then applied in the RAW264.7 culture to examine the effect on phagocytosis.Direct targeting of miR-142 on protein kinase Cα(PKCα) was assessed by luciferase assay and western blotting.Results miR-142 expression in △HlaS.aureus group were significantly down-regulated than WT S.aureus group(P<0.05).MiR-142 mimics inhibited RAW264.7 phagocytosis ability and inhibitor enhanced RAW264.7 phagocytosis ability.PKCα was directly targeted by miR-142.MiR-142 mimics significantly decreased the mRNA expression levels of PKCα in RAW264.7 cells(P<0.05).Conclusion Hla could promote miR-142 expression in macrophages.MiR-142 could inhibit macrophage phagocytosis through down-regulated PKCα.

Objective To explore the occurance and risk factors of recurrence and metastasis of gastric cancer after gastrectomy.Methods From January 2001 to December 2004, the clinic pathological data of 141 patients with recurrence and metastasis after radical gastrectomy in Tianjin Medical University Cancer Institute and Hospital were analyzed retrospectively.The possible clinic pathological factors which may affect tumor recurrence were analyzed.Results After the surgery, the 1, 2, 3 and 5-year cumulative recurrence rates were 58.2%(82/141) 、80.1%(113/141)、89.4%(126/141) 、97.9 % (138/141)respectively.The results of multivariate analysis indicated that the tumor size,invasive depth, lymph node metastasis were independent factors which affected recurrence and metastasis after radical gastrectomy (P = 0.017, 0.003, 0.000).Invasive depth, lymph node metastasis and tumor differentiation degree were independent factors which affected early recurrence and metastasis after radical gastrectomy (P=0.042, 0.000, 0.039).Conclusions The tumor size,invasive depth, lymph node metastasis are the independent risk factors to predict the recurrence and metastasis after radical gastrectomy.Most of the recurrences and metastasis is found within 2 years after radical gastrectomy.Invasive depth, lymph node metastasis and tumor differentiation degree are the independent factors to predict early recurrence after radical gastrectomy.

Objective To investigate the clinical features and prognosis of recurrent gastric cancer. Methods The clinical data of 147 patients with recurrent gastric cancer was reviewed. Risk factors correlated with tumor recurrence and recurrent intervals were studied by logistic regression analysis. Survival analyses and comparisons were performed using Kaplan-Meier plots, the log rank test and the Cox proportional hazards model. Results Patients were divided into an early recurrence group consisting of 86 patients (recurred within one year after surgery) and a late recurrence group of 61 patients (recurred one year or more after surgery). There were significant difference in size of primary tumor, Borrmann stage, type of gastrectomy, T stage, N stage, TNM stage between the two groups(P ＜0.05). Multivariate analysis showed that the TNM stage and N stage independently influenced the recurrent time ( P ＜ 0. 05 ). In univariate survival analysis, post-gastrectomy chemotherapy(P ＜0. 05) , T stage (P ＜0. 05) , N stage(P ＜0.01) , TNM stage ( P ＜ 0. 01) , recurrence-free interval (P ＜ 0. 01) and reoperation (P ＜ 0.01) were significantly correlated with the prognosis. In multivariable analysis, TNM stage(P ＜0. 01) , recurrence-free interval ( P ＜ 0. 05 ) and reoperation ( P ＜ 0. 05 ) were independent factors predicting recurrence. Conclusions The TNM stage and N stage were the important factors predicting the time of recurrence after curative resection for gastric cancer. Patients with recurrent gastric cancer have poor prognosis and reoperation was associated with an improved survival in patients with recurrent gastric cancer.

Objective To investigate the clinical characteristics, treatment and prognostic factors of postoperative recurrent-metastatic gastrointestinal stromal tumors (GIST). Methods The clinical data of 56 patients with postoperative recurrent-metastatic GIST between January 1997 and December 2007 were analyzed retrospectively , and the prognostic factors were evalutated. Results Initial recurrence-metastasis was diagnosed after a median of 17.3 months from primary resection. Of the 56 cases, 19 cases received resection, 8 cases received imatinib targeted therapy,14 cases were treated by imatinib targeted therapy and surgery, the 3 year survival rates were 65% ,66% ,89% respectively. The rate of recurrence-metastasis after reoperation is 76%. Univariate analysis revealed that surgery, targeted therapy and age were related to the survival rates, multivariate analysis demonstrated that surgery and targeted therapy were the independent prognostic factors for survival. Conclusions For recurrent-metastatic GIST, a multimodal approach including surgery and targeted therapy improves survival.

Objective To investigate the treatment and prognosis of metastatic ovarian carcinoma from gastrointestinal tract. Methods The clinical data of 110 patients with metastatic ovarian carcinoma were reviewed. Results The median age of 110 patients was 45 years old.Metastatic ovarian carcinomas were from stomach (74.5%),and colon and rectum (25.5%).The median overall survival time of 110 patients was 16.3 months,and the median progression-free survival time of 110 patients was 8.2 months.Survival rates of 1-,3-and 5-year were 68.4% 、15.4% and 2.5%,respectively.In univariate survival analysis,different origins(P ＜ 0.01),extent of recurrent disease (P ＜ 0.01),cytoreductive surgery (P ＜0.01),intraperitoneal chemotherapy (P ＜ 0.05) and pathologic type of metastatic ovarian carcinoma (P ＜0.05) were correlated with the prognosis.Age,menstrual history,ascites,and chemotherapy have less effect on prognosis (P ＞ 0.05).In multivariable analysis,different origins (P ＜ 0.01),extent of recurrent disease(P ＜ 0.05) and cytoreductive surgery (P ＜ 0.01) were confirmed to be independent factor. Conclusion Patients with metastatic ovarian carcinomas have poor prognosis.Optimal cytoreduction significantly prolongs overall survival in patients with metastatic ovarian carcinoma from gastrointestinal tract.

Objective To investigate the correlation between thrombocytosis and the progress of gastric cancer. Methods The clinical data of 276 gastric cancer patients who had been admitted to Tianjin Cancer Hospital from August 2002 to January 2004 were retrospectively analyzed. Of all patients, 75 were with thrombocy-tosis and 201 were with normal level of platelet. Data were analyzed by chi-square test, and the prognostie factors for gastric cancer were analyzed by Cox regression model. The accumulative survival was determined by Kaplan-Meier method, and the difference in survival between patients with and without thrombocytosis was analyzed by Log-rank test. Results The 1-, 3-, 5-year survival rates of gastric cancer patients with normal level of platelet were 82.4%, 50.5% and 41.9%, and the 1-, 3-, 5-year survival rates of gastric cancer patients with thrombocy-tosis were 74.7%, 37.3% and 25.3%. There was a significant difference in 5-year survival rates between patients with or without thrombocytosis (χ~2= 7.358, P<0.05). Thrombocytosis was correlated with the depth of invasion, distal metastasis, TNM staging and the tumor size (χ~2=6.946, 5.625, 14. 925, 4.028, P<0.05). The result of multivariate analysis indicated that the content of platelet was not the independent factor of good prog-nosis in gastric cancer. Conclusions There is a close relationship between thromboeytosis and clinieopathological factors of gastric cancer. Thrombocytosis may be used as a reference indicator for the prognosis of gastric cancer.

Objective To explore the treatment methods and prognostic factors of metachronous liver metastases from gastric cancer.Methods The clinicopathological data of 102 patients with metachronous liver metastases from gastric cancer who were admitted to the Cancer Hospital of Tianjin Medical University from January 1996 to December 2008 were retrospectively analyzed.Sixty-four patients received systemic chemotherapy,19 received systemic chemotherapy + transcatheter arterial chemoembolization (TACE),and 19 received systemic chemotherapy + radical resection of the metachronous liver cancer.Patients were re-examined every 3 months within the first 3 years after operation,and every 6 months after postoperative year 3,and every 1 year after postoperative year 5.Physical examination,laboratory test and imaging examination were done during the follow-up.The followup was ended in October 2013.The cumulative survival rates of the patients were calculated and compared using the Kaplan-Meier method and the Log-rank test,respectively.The prognostic factors were analyzed using the COX regression model.Results The disease was alleviated in 15 patients,progressed in 27 patients,and the condition was stable in 22 patients after systemic chemotherapy.The disease was alleviated in 6 patients,progressed in 4 patients and the condition was stable in 9 patients after systemic chemotherapy + TACE.Of the 19 patients received systemic chemotherapy + radical resection of the metachronous liver cancer,1 was complicated with incisional infection,and no patient died perioperatively.Sixteen patients died of gastric cancer recurrence including 10 patients with local recurrence and 6 patients with multiple lesions recurrence.Eight patients missed the follow-up,the others were followed up for 9-149 months.The overall median survival time was 8 months (range,2-70 months),and the 1-,3-,5-year survival rates were 40.2％,17.7％ and 6.8％,respectively.The median survival time of the 64 patients who received systemic chemotherapy was 5 months (range,2-37 months),and the 1-,3-,5-year survival rates were 15.6％,3.5％ and 0,respectively.The median survival time of the 19 patients who received systemic chemotherapy +TACE was 6 months (range,3-36 months),and the 1-,3-,5-year survival rates were 26.1％,6.5％ and 0,respectively.The median survival time of the 19 patients who received systemic chemotherapy + radical resection of the metachronous liver cancer was 15 months (range,5-70 months),and the 1-,3-,5-year survival rates were 63.2％,31.6％ and 16.8％,respectively.The prognosis of patients who received systemic chemotherapy + radical resection of the metachronous liver cancer was superior to those who received systemic chemotherapy or systemic chemotherapy + TACE (x2=23.900,P ＜ 0.05).The results of univariate analysis showed that diameter and differentiation of primary tumor,extra-hepatic metastasis,type and number of liver metastases and treatment regimen were correlated with the prognosis of patients with metachronous liver metastases from gastric cancer (x2=6.307,7.908,4.375,45.188,18.234,23.900,P ＜ 0.05).The results of multivariate analysis showed that type and number of liver metastases were independent factors influencing the prognosis of patients with metachronous liver metastases from gastric cancer (OR=5.217,3.292,95％CI:1.428-2.882,1.054-2.514,P＜0.05).Conclusions Surgical resection of the metachronous liver cancer is important to improve the survival of patients.The type and number of liver metastases are important factors in deciding the treatment methods.

Objective To design and construct a controlled adenovirus vector in degradating by itself after induction for solving the problem of stimulating host immune and producing replication adenovirus and providing a secure exogenous gene vectors for clinical practice. Methods Based on the traditional adenovirus vector AdEasyTM system, we inserted the Cre gene which belongs to Cre-LoxP system into the downstream of Tet-On inducible expression system. Two LoxP sites were inserted into two sides of the shuttle plasmid′s right arm genome. Then, the full-length human insulin gene was inserted HindIII enzyme site. After the recombinant adenovirus infected the rat bone marrow-derived mesenchymal stem cells , fluorescent protein expression and insulin secretion were detected before or after induction by Dox. Results A new controlled recombinant adenovirus vector carrying human insulin gene was constructed successfully, and was named AdEasyN/INS. After the transfection of this new vector into QBI-293A cells and rat bone marrow mesenchymal stem cells , green fluorescent protein could be observed. After induction by Dox, both of the ratio of fluorescent cells/total cell and the levels of insulin significantly decreased. Conclusion Construction and preliminary validation of a controlledrecombinant adenovirus vector carrying human insulin gene is constructed successfully , it could infect rat bone marrow mesenchymal stem cells, and degradate by itself after Dox induction, realize the controllability of exogenous gene carrier.

Objective To analyze the risk factors of liver metastasis from gastric cancer. Methods The clinical data of 225 patients with liver metastasis from gastric cancer who had been admitted to our hospital from January 1996 to December 2001 were retrospectively analyzed. Synchronous liver metastasis was observed in 123 patients and metachronous liver metastasis in 102 patients. The risk factors of liver metastasis from gastric cancer were evaluated. Results The gender of patients, location and size of gastric cancer foci, differentiation and invasion depth of gastric cancer, Lanren classification, lymph node metastasis and lymph node metastasis in region Ⅷ, vascular invasion, extrahepatic metastasis, ascites and peritoneal metastasis were the significant factors associated with liver metastasis from gastric cancer (X2 = 43.560-263. 907, P<0.05). All the factors except the size of gastric cancer foci, extrahepatic metastasis and ascites were found to be the significant factors associated with different types of liver metastasis from gastric cancer (X2 = 6.673-26. 555, P < 0.05 ). Logistic regression analysis demonstrated that the gender of patients, lymph node metastasis and peritoneal metastasis were the factors that determined the types of liver metastasis from gastric cancer. Conclusion The gender of patients, lymph node metastasis and peritoneal metastasis are the important factors to evaluate the occurrence of different types of liver metastasis from gastric cancer.