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:Analysis of the clinical characteristics and genetic mutations of ten tuberous sclerosis complex(TSC) families to provide support for clinical diagnosis of TSC.Methods:Retrospective analysis of ten TSC family probands and their members were performed with detailed medical history collection and physical examination. The study consisted of 26 patients in 10 TSC families, including 12 males and 14 females, with male to female ratio of 1∶1.17, and aged 7-65 years. In terms of skin lesions, there were 24 cases of facial angiofibromas, 20 cases of hypomelanotic macules, 16 cases of ungual fibromas, and 9 cases of shagreen patch. There were 12 cases of renal angiomyolipoma, 4 cases of subependymal nodules based on craniocerebral CT, and 1 case of lymphangioleiomyomatosis. Four cases had a history of seizures. Peripheral blood was extracted for next generation DNA sequencing.Results:A total of 23 TSC patients were genetically tested, and a total of 14 patients in 6 families were TSC2 mutations(5 frameshift mutations, 5 heterozygous deletions, and 4 nonsense mutations), a total of 4 patients in 2 families were TSC1 nonsense mutations, and no mutations were seen in the 2 families.Conclusions:A total of 10 TSC families were summarized, and the patients mainly had skin manifestations and kidney lesions.There were 18/23 patients having TSC1/2 mutations, of which TSC2 mutations accounting for 14/18. In the two non-mutant families, patients mainly had facial angiofibroma and/or hypomelanotic macules.

Objective To observe the inhibitory effect on lymph node metastasis of pancreatic cancer and lymphangiogenesis in mice by injection of KAll gene within xenograft tumor.Methods Pancreatic cancer cell line MiaPaCa-2 wag used to construct the nude mice models bearing tumors,then the mice were divided into normal saline group,Ad group and Ad-KAI1 group.Since the successful model construction,normal saline,Ad,Ad-KAI1 was injected every week for 3 times,respectively in the three groups,then the tumor size was documented.50 d after model construction,the tumor and enlarged lymph nodes were collected and subjected to pathological exam,and the expression of LYVE-1 and the MLVD in xenograft tumor was detected by immunohistochemistry.Results Two weeks after MiaPaCa-2 implantation,the model was 100％ successfully constructed.The growth curve of subcutaneous tumor among 3 groups was not statistically significant (P＞0.05) ; the weights of subcutaneous tumor in the 3 groups were (2514.4 ±351.3),(2466.1 ± 295.5),(2294.4±255.4) mg after 50 d,and the difference among the 3 groups was not statistically significant (P ＞0.05).Enlarged lymph nodes metastasis was observed in 8 mice (80％) in normal saline group,and 20 lymph nodes were collected,with 2.0 lymph nodes per mice; and enlarged lymph nodes metastasis was observed in 7 mice (70％) in Ad group,and 15 lymph nodes were collected,with 1.5 lymph nodes per mice; while enlarged lymph nodes metastasis was observed in 4 mice (40％) in Ad-KAI1 group,and 6 lymph nodes were collected,with 0.6 lymph nodes per mice.All the lymph nodes were confirmed to be metastasis of the primary tumor after pathologic exam.The difference of lymph nodes metastasis,number of lymph nodes metastasis per mice among the 3 groups was statistically significant (F =3.14,3.35,P ＜ 0.05).The MLVD of subcutaneous tumor among the 3 groups was (18.70 ± 5.60),(19.40 ± 4.58),(9.80 ±4.10)/400 times magnification,the MLVD of Ad-KAI1 group was significantly lower than those in normal saline group and Ad group (F10.76,11.36,P ＜ 0.05),but the difference between normal saline group and Ad group was not statistically significant.Conclusions KAI1 can inhibit the lymph node metastasis of pancreatic cancer,and the mechanism may be related with decreased lymphangiogenesis and reduced lymphatic vessel density.

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 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

In order to know that Streptococcus exist in health swine herd,2 204 cases of nose swabs were collected in swine from different areas in Dongbei three province of northeast China.Seed swab to Streptococcus culture medium,24 hours after culturing,gram staining with becteria culture,gram positive Streptococcus were identified with PCR technology,then farther identified serotype with Streptococcus suis 1,2,7,9 serotype special primers.The results showed that Streptococcus exist rate in swine herd of Heilongjiang,Jinlin,Liaoning is 29 %,27 %,34 %,respectively.PCR typing assay indicated that 155 strains of Streptococcus suis were isolated including 7 strains of 1 serotype,39 strains of 2 serotype,4 strains of 7 serotype,11 strains of 9 serotype,94 strains of other serotype in three province of northeast China.

OBJECTIVETo understand the smoking-attributed mortality by inclusion of smoking information into all causes of death surveillance.

METHODSSince 2010, the information about smoking status, smoking history and the number of cigarettes smoked daily had been added in death surveillance system. The measures of training, supervision, check, sampling survey and telephone verifying were taken to increase death reporting rate and reduce data missing rate and underreporting rate. Multivariate logistic regression analysis was conducted to identify risk factors for smoking-attributed mortality.

RESULTSDuring the study period (2010-2014), the annual death reporting rates ranged from 6.5‰ to 7.0‰. The reporting rates of smoking status, smoking history and the number of cigarettes smoked daily were 95.53%, 98.63% and 98.58%, respectively. Compared with the nonsmokers, the RR of males was 1.38 (1.33-1.43) for all causes of death and 3.07 (2.91-3.24) for lung cancer due to smoking, the RR of females was 1.46 (1.39-1.54) for all causes of death and 4.07 (3.81-4.35) for lung cancer due to smoking, respectively.

CONCLUSIONThe study of smoking attributed mortality can be developed with less investment by using the stable and effective all causes of death surveillance system in Tianjin.

Cause of Death ; China ; epidemiology ; Female ; Humans ; Logistic Models ; Lung Neoplasms ; etiology ; mortality ; Male ; Multivariate Analysis ; Population Surveillance ; methods ; Risk Factors ; Smoking ; adverse effects ; mortality

OBJECTIVETo investigate the apoptosis inducing effects of ponicidin (PON) on leukemic K562 cells and its mechanisms of action.

METHODK562 cells in culture medium in vitro were given different concentrations of PON (10-50 micromol x L(-1)) for 24, 48 and 72 h. The inhibitory rate of the cells was measured by MTT assay, cell apoptotic rates were detected by flow cytometry (FCM) using Annexin V staining after K562 cells were treated with different concentrations of PON for 72 hours, and cell morphology was observed by Wright-Giemsa staining. Western blot was used to detect caspase-3 and poly(ADP-ribose) polymerase (PARP) expression, and the protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) as well as p-AKT and p-P85 in PI3K/AKT signaling pathways were also detected.

RESULTPON (over 30 micromol x L(-1)) could inhibit the growth of K562 cells in both time- and dose-dependent manner. FCM analysis revealed that apoptotic cells were gradually increased in a dose-dependent manner after treatment for 72 hours, and that marked morphological changes of cell apoptosis such as condensation of chromatin was clearly observed by Wright-Giemsa staining after treatment by 50 micromol x L(-1) PON. Western blot showed cleavage of the caspase-3 zymogen protein (32 kD), with the appearance of its 17 kD subunit, and a cleaved 89 kD fragment of 116 kD PARP was also found. Furthermore, Western blotting also showed that expression of p-AKT and p-P85 in PI3K/AKT signaling pathways was downregulated dramatically whereas the expression of p-P38 as well as p-ERK and p-JNK remained unchanged after the cells were treated by PON for 48 h.

CONCLUSIONThe results demonstrate that PON exhibits in vitro anti-leukemia effect by induction of apoptosis in K562 cells, and that PON induced apoptosis in K562 cells mainly related to activation of caspase-3 as well as inactivation of PI3K/AKT signaling pathway via down regulation of the expression of p-AKT and p-P85 protein levels. These results provide strong laboratory evidence for further anti-leukemia trials of PON.

Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Humans ; Poly(ADP-ribose) Polymerases ; metabolism ; Signal Transduction ; drug effects

Objective: To evaluate the expression of OCT4 and SALL4 in testicular diffuse large B-cell lymphoma (DLBCL), and the utility of an immunohistochemical (IHC) panel of OCT4, SALL4 and CD20 in the differential diagnosis of DLBCL and GCT of the testis. Methods: Eighteen cases of testicular DLBCL were selected.IHC method was used to detect the protein expression of CD20, CD3, CD5, CD10, bcl-6, MUM1, Ki-67, bcl-2, c-MYC, OCT4 and SALL4. Results: Among the 18 cases, CD20 and PAX5 were strongly and diffusely expressed in all cases, while CD21, CD3, cyclinD1, SALL4, CD117 and PLAP were all negative. CD5, bcl-2 and c-myc were expressed in 3, 16 and 8 cases, respectively. Ki-67 proliferation index ranged from 40%-95%. Bcl-2 and c-MYC were co-expressed in seven cases. Four cases were GCB-DLBCL and the remaining 14 cases were non-GCB-DLBCL, according to Hans algorithm. Nuclear OCT4 expression was present in two cases, which demonstrated moderate expression in >50% of neoplastic cells. Univariate analysis showed that clinical stage, CD5 and OCT4 expression were relevant to prognosis. Multivariate Cox regression analysis further confirmed that clinical stage, CD5 and OCT4 were independent prognostic factors in patients with testicular DLBCL. Conclusions Care should be exercised in using OCT4 as the sole marker of germ cell differentiation in the testis. The association of OCT4 and CD5, bcl-2 co-expression raises the question of whether OCT4 expression in DLBCL may reflect more aggressive biology.

Objective To evaluate the value of three-dimensional(3 D) printing technique in the diagnosis and treatment of complex congenital heart disease(CHD).Methods From March 2016 to February 2018,40 patients with complex CHD underwent heart CT scanning.The CT images were imported to Standard Template Library(STL) files after 3D reconstruction and then exported for 3D printing.The 3D printed models were then used for decision making and navigation during surgery.Results Thirty patients were indicated for surgical operation.Three patients underwent single ventricular repair,and biventricular repair were operated on 27 patients.The 3D printed models were quite in accordance with the actual anatomical findings in all the patients.And all the procedures carried on were exactly same as planned based on 3D printed model.Conclusion The 3D printing may help improve the diagnosis and treatment level in complex CHD.