Objective To investigate the effects of KAll gene transfection on proliferation,migration,invasion and VEGF expression of pancreatic cancer MiaPaCa-2 cells under hypoxic condition,and explore possible mechanism.Methods The pcMV-KAI1 vector which contained the full length of KAI1 cDNA was transfected into pancreatic cancer cells MiaPaCa-2,and KAI1,VEGF C,VEGF A protein expressions were determined by Western blot.The proliferation of pancreatic cancer cells was evaluated by MTT method.Wound-healing assay and cell invasion assay were used to detect the migration and invasion of pancreatic cancer cells.The expression of VEGF C,VEGF A in supernatant of culture was measured by ELISA method.Results The expression of KAI1 protein in MiaPaCa-2 K transfected with KAI1 was significantly higher than that in nontransfected cells [(0.549 ± 0.021) vs 0,P＜0.05].The proliferation under hypoxic condition was not significantly different,but the migration distance was significantly shorter and the number of transmembrane cell was significantly decreased [(14.0 ± 5.8) vs (43.0 ± 14.4),P ＜ 0.05].The expression of VEGF-C in cell was significantly decreased [(0.218 ± 0.043) vs (0.745 ± 0.069),P ＜0.05],but the expression of VEGF-A was not significantly different; the expression of VEGF-C in cell culture supernatants was significantly decreased [(1236 ± 247) vs (2045 ± 221) pg/ml,P ＜ 0.01].Conclusions The migration,invasion ability of pancreatic cancer MiaPaCa-2 cells with KAll transfection under hypoxic condition is decreased,and the possible mechanism of inhibiting lymphatic metastasis is down-regulation of VEGF-C expression.

Objective: Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in the TSC1 and TSC2 genes, but the molecular events contributing to TSC are not well understood.However, little is known about the role of microRNAs in TSC.To explore the microRNA differential expression profile between tuberous sclerosis complex cell line TSC2-/-MEFs and normal type cell line TSC2+/+ MEFs, and to provide new clues to study the mechanism of microRNA function in tuberous sclerosis complex.Methods: TSC2-/-MEFs and TSC2+/+ MEFs cell lines were cultured in vitro, each with three samples chosen as the experimental group and the control group respectively.Total RNA was isolated using TRizol and purified with RNeasy mini kit according to manufacturer''s instructions.RNA quality and quantity were measured by using nanodrop spectrophotometer and RNA integrity was determined by gel electrophoresis.Total RNAs were extracted by TRizol, followed by RNA quantification and quality control.MicroRNA profiles were analyzed by microarray and the threshold value used to screen up-regulated more than 2-fold change or down-regulated less than 0.5-fold change compared with controls.Real-time PCR was used to validate the reliability of microarray.Cell counting kit-8 (CCK-8) assay was performed to evaluate the proliferation.Results: Fourteen microRNAs, including miR-18a-5p, miR-376c-3p, miR-136-5p, miR-467c-5p, miR-467b-5p, miR-5104, miR-3098-3p, miR-30a-3p, miR-302b-3p, miR-18a-3p, miR-19b-1-5p, miR-19a-5p, miR-20a-5p, miR-155-5p, were up-regulated, while twenty-six microRNAs, including miR-200b-3p, miR-450a-1-3p, miR-542-5p, miR-199b-5p, miR-10a-5p, miR-466c-5p, miR-450a-5p, miR-450b-5p, miR-542-3p, miR-351-5p, miR-322-3p, miR-199a-3p, miR-335-5p, miR-10b-5p, miR-351-3p, miR-155-3p, miR-497a-5p, miR-503-5p, miR-148a-3p, miR-1843a-5p, miR-199a-5p, miR-490-5p, miR-450a-2-3p, miR-322-5p, miR-214-3p, miR-450b-3p, were down-regulated in tuberous sclerosis complex cell line TSC2-/-MEFs compared with normal type cell line TSC2+/+ MEFs (P<0.05).Real-time PCR confirmed the expressions of miR-136-5p, miR-30a-3p, miR-302b-3p, miR-10b-5p, miR-148a-3p, miR-199a-5p consistent with the microarray data (P<0.05).Furthermore, the overexpression of miR-199a-5p significantly inhibited cell proli-feration (P<0.05).Conclusion: There are differences in the expression of miRNA between the tube-rous sclerosis complex cell line TSC2-/-MEFs and normal cell line TSC2+/+ MEFs.MiRNA-199a-5p plays an important role in tuberous sclerosis complex, which may be developed as an important molecular target for the treatment of tuberous sclerosis complex.

Abstact:Aim To identify whether the petroleum e-ther fraction of Yueju pill (YJ-PE)could exhibit a po-tent rapid antidepressant effect and to investigate the underlying biological mechanism involved.Methods Fourty-four male KM mice were randomly assigned to five groups:the control group (saline group),low dosage of YJ-PE,middle dosage of YJ-PE,high dos-age of YJ-PE and ketamine.After a single drug admin-istration,we used tail suspension test (TST)to test the rapid antidepressant effect of YJ-PE in KMmice at 30 min.Another twenty-four male KM mice were ass-inged to three groups:control group,screened effective low dosage of YJ-PE group and ketamine group.We usd TST to assess these mice at 24 h post a single ad-
ministration.Western blot was performed to examine the expression of BDNF and TrkB in hippocampus of KMmice at 30 min and 24 h post a single administra-tion.Results An acute administration of YJ-PE de-creased the immobility time of KMmice in TST and in-creased the expression of BDNF and TrkB as soon as 30min post a single administration and lasted at least for 24 hours.Conclusion YJ-PE (low dosage)ex-hibits a rapid antidepressant-like potent,and the po-tent rapid antidepressant effects of YJ-PE are associat-ed with the up-regulation of BDNF and TrkB.

Objective To evaluate the results of thoracic endovascular aortic repair (TEVAR) for chronic type B aortic dissection.Methods From September 2005 to January 2013,30 patients with chronic type B aortic dissection received TEVAR.All patients were followed for 2-90 months [mean (33 ±25) months].Results All of the procedures finally achieved technical success.However,during TEVAR,there were transient endoleaks in 8 patients including type Ⅰ endoleaks in 3 patients,type Ⅱ endoleaks in 5 patients and persistent endoleaks in 3 patients which are type Ⅳ endoleaks.Type Ⅰ and type Ⅱ endoleaks were successfully managed during the procedures.There was no mortality or major complication during the perioperative period.Three patients died during follow-up:one patient died of carcinoma of the colon and two patients died of the complications of secondary interventions related to aortic dissection.Totally there were seven patients receiving secondary interventions.The Kaplan Meier actuarial survival curve showed a 5-year survival rate of 87.9％ and the 5-year survival rate without secondary intervention was 72.8％.Conclusions Early and midterm results showed that TEVAR was effective in treating chronic type B aortic dissection.

Objective To investigate the safety of laparoscopic common bile duct exploration and lithotomy with primary closure and without placing drainage tube postoperatively.Methods Forty patients who received laparoscopic common bile duct exploration and lithotomy at the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University from January 2011 to June 2013 were prospectively analyzed.All the patients were randomly divided into 2 groups according to the random number table.Twenty patients in the experimental group did not received drainage tube placement,and the other 20 patients in the control group had subhepatic drainage after operation.The operation time,duration of hospital stay and incidence of postoperative complications were compared between the 2 groups.Patients received computed tomography and B sonography at postoperative month 1 and 3,and then patients were reexamined every 6 months till postoperative year 3.The follow-up was ended on July 31,2013.The measurement data and the count data were analyzed using the independent sample t test and the Fisher exact probability,respectively.Results Patients in the 2 groups were cured after the operation.The operation time and duration of hospital stay were (117 ± 11) minutes and (5.6 ± 0.6) days in the experimental group,and (108 ± 12)minutes and (7.9 ± 0.7)days in the control group,with significant difference between the 2 groups (t =2.453,-ll.388,P ＜ 0.05).No complications including bile leakage,residual stones,obstructive jaundice,abdominal bleeding and subphrenic infection were detected after the operation.Thirty-one patients were followed up for 1 month to 2 years,no bile duct stone recurrence or biliary stricture were detected during the follow-up.Conclusion Laparoscopic common bile duct exploration and lithotomy with primary closure and without placing drainage tube postoperatively is safe and feasible.

Objective To investigate the ability of induction of specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cells (DCs) co-transfected with MUC1 and survivin mRNA of human pancreatic cancer,and to provide the experimental basis for the treatment of human pancreatic cancer with multi-epitope DC vaccine.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) of 6 patients with pancreatic cancer.Human pancreatic cancer cell line MiaPaCa-2 was routinely cultured,after being transcripted and amplified by RT-PCR,MUC1 and survivin mRNA were co-transfected or individually transfected into DCs by electroporation,and they were named as DC-MUC1,DC-survivin,DC-MUC1 + survivin.The expression of MUC1 and survivin mRNA in DCs were detected by real-time PCR.The survival rate of transfected DCs were determined by MTT method.The lymphocyte proliferation ability was evaluated by mixed cell culture method.The Th1 cytokine releasing of antigen-specific CTLs were measured by ELISA assay.Results Mature DCs were obtained,the positive expression rates of surface markers CD40,HLA-DR,CD83 and CD86 were 34.31％,50.21％,89.17％ and 73.62％,respectively.The expression amount of MUC1 mRNA of DC-MUC1 was 36.24 ± 5.17,and the expression amount of survivin mRNA of DC-survivin was 34.53 ± 4.02,while the expression amounts of MUC1,survivin mRNA of DC-MUC1 + surviving were 31.79 ±4.26 and 14.67 ± 2.96,which were significantly lower than that in individual transfection group (P ＜ 0.05).The survival rate of DC-MUC1 + surviving was decreased in a time dependent manner,which was significantly lower than that in individual transfection group (about 50.21％ vs 80％ at 24 h,P ＜0.05).When DC/T cells ratio was 1∶ 10,1∶ 20,the autologous T cell proliferation index of MUC1 and survivin mRNA in co-transfection DC group was significantly higher than that in individual transfection group (P ＜ 0.05) ;when DC/T cells ratio was 1∶ 40,1∶ 80,the difference of proliferation index was not statistically significant.When DC/T cells ratio was 1∶ 10,after 14 d culture,the expressions of IL-2 in DC-MUC1,DC-survivin,DC-MUC1 + surviving were (892.73 ± 32.9),(713.62 ± 56.37),(1884.37 ± 95.21) pg/ml,and the expressions of granzyme B were (501.62 ± 12.30),(203.84 ± 12.55),(1193.15 ± 86.04) pg/ml ; and the expressions of IFN-γ were (981.50 ± 47.82),(696.05 ± 41.66),(2237.94 ± 189.55) pg/mL.The corresponding values in DC-MUC1 + surviving group were significantly higher than those in individual transfection group (P ＜ 0.05) ; while the difference of IL-10 was not statistically significant.Conclusions DCs co-transfected with MUC1 and survivin mRNA have a stronger ability to stimulate specific CTL in vitro than individual antigen loaded DCs.

Objective To investigate expressions of CDev6 and p-ERK1/2 in AML patients and its clinical significance.Methods Expressions of CD44v6 and p-ERK1/2 on bone marrow blasts in 152 AML patients and 22 normal controls were determined by flow cytometry.Meanwhile,the effects of CD44v6 expression(CD+44v6 and CD-44v6) on relapse rate and survival time were analyzed by following up of 129 AML patients.Results Double positive expressions of CD44v6 and p-ERK1/2 were observed in 4.5%(1/22) of the normal bone marrow blasts while single positive expression of CD44v6 and p-ERK1/2 was observed in 36.2%(55/162) and 64.5%(97/162) of AML patients,respectively.The MFI of p-ERK1/2 expression on blast cells in AML patients was [14(7 -58)],which was higher than that in normal controls [8(6 - 10),U =4.2,P＜0.01].Furthermore,MFI of p-ERK1/2 expression on blast cells in CD+44v6 AML patients was [28(15-61)] ,which was significantly higher than that in CD-44v6 AML patients [18(6- 37),U =6.7,P＜0.01].A strong correlation was obtained between CD44v6 and p-ERK1/2 expression(rs = 0.7,P＜ 0.01).Among 129 patients followed up for 4 to 65 months,the data also revealed that the relapse rate of CD-44v6 AML patients was 32.1%(26/81) ,which was much lower than that in CD44v6 AML patients [81.3%(39/48),x2 = 29.13,P＜ 0.01).And the overall survival in CD44v6 AML patients was(28.5 ± 1.8)months,which was significantly worse than that of CD44v6 AML patients [(51.2±2.0) months,x2 =48.2,P＜ 0.01).Conclusion CD44v6 expression was associated with a poor survival in AML patients,and CD44v6 might promote the expansion of leukemic blast cells by up-regulating p-ERK1/2.

Objective To investigate the characteristics of immunophenotyping and clinical significance of MRD analysis in MM patients. Methods Multi-parameter flow cytometry was applied to analyze the immunophenotyping of malignant plasma cells from 172 MM patients, and normal plasma cells from 16 healthy individuals. MRD was analyzed in 32 MM patients with remission. Meanwhile, the effects of MRD status on the disease relapse and patient disease free survival ( DFS ) time was evaluated by following up patients. Results The immunophenotyping of normal plasma cells were CD38, CD138, CD19 and CD45 positive, while the predominant phenotype of MM cells were CD+38( 100.0% ), CD+138( 100.0% ), CD-19 ( 167/172, 97. 1% ), CD+56( 152/172, 88.4% ) and CD-45( 166/172, 96. 5% ). The characteristic markers for MM cells were CD+38, CD-138, CD-19, CD-45 and CD+56. MRD analysis revealed that, among 32 MM patients with remission, 14 patients were MRD negative and 18 patients were MRD positive. During follow-up of 2 to 16 months, the relapse rate in MRD negative patients was significantly lower ( 4/14, 28.6% ) than that of MRD positive patients ( 13/18, 72. 2% ;χ2 =6. 03, P ＜0. 05 ). Furthermore, the DFS time was significantly longer in MRD negative patients[ 16. 23( 10. 37-21.62 )months ] than that of the MRD positive patients [ 10. 07( 3. 79-16. 20 )months,χ2 =7. 53,P ＜0. 05 ]. Conclusions CD+38, CD+138, CD-19, CD-45 and CD+56 are the characteristic markers of MM cells compared to those of the normal plasma cells. MRD analysis is a valuable prognostic factor for MM patients.

Objective To investigate the effect of FK506 on the in vitro ostengenic potential of rat bone marrow-derived mesenchymal stem cells(MSCs)and the dose-response effect of FK506(5-5000 nmol/ L)on the ostengenic potential of MSCs in vitro.Methods MSCs derived from primary culture were sub- cultured for 16 days under four conditions:(1)?-MEM containing L-ascorbicacid-2-phosphate(AsAP)and?-glycemphosphate(?-GP)(basic culture medium)as a control;(2) AsAP and?-GP(basic culture medi- um) plus dexamethasone;(3)AsAP and?-GP(basic culture medium)plus FK506,(4)AsAP and?-GP (basic culture medium)plus FK506 and dexamethasone.Osteogenie potential was determined by testing os- teoblastic morphology,cell proliferation,alkaline phosphatase(APase)activity,bone nodule formation and the expression of osteocalcin mRNA.Results FK506 promoted the proliferation of MSCs,induced mineral- izing bone-like nodule formation,and increased APase activity and the expression of osteocalcin mRNA.The data also showed that the efficacy of FK506 was greater when used in combination with dexamethasone.Opti- mal ostengenesis was achieved with?-MEM containing 0.25mmol/L AsAP,10mmol/L?-GP,10nmol/L dexa- methasone and 50nmol/L FK506.Conclusion FK506 has powerful ostengenic ablility.It can be consid- ered as an osteogenic agent to repair bone defects.

Objective To investigate the relationship between PTEN mRNA expression and Akt phospholylation in acute leukemia(AL)and explore its clinical significance in the development of acute leukemia.Methods PTEN mRNA expression and Akt phospholylation level in the leukemia cell line K562, HL-60,Jurkat,21 healthy persons and 68 patients with AL were analyzed with RT-PCR and Flow cytometry, respectively.Results(1)The significantly different PTEN mRNA expression was found between the controls and AL patients.85.70% of the normal controls and 26.47% AL patients show positive respectively (?~2=23.38,P