Objective: To construct the expression plasmid of a novel gene human NBEAL1 (neurobeachin like 1), and to study its relationship with the pathological grades of glioma. Methods: Total RNA of human glioma cell line U251 was extracted. NBEAL1 expression plasmid pGEX-KG/NBEAL1 was constructed and transferred into E. coli BL21. Recombinant NBEAL1 protein was induced by IPTG and further purified by GST affinity chromatographic column. The purity of recombinant NBEAL1 protein was examined by Western blotting analysis. A NBEAL1 protein specific monoclonal antibody was prepared and was used to study the relationship of NBEAL1 expression with pathological grades of glioma. Results: The NBEAL1 gene fragment was successfully cloned into pGEX-KG expression plasmid and verified by DNA sequencing. The recombinant NBEAL1 protein was expressed in inclusion bodies, with a yield of more than 30% of total bacterial proteins; the purity of purified NBEAL1 protein was above 95%. Western blotting analysis confirmed that the purified protein containing GST tag and NBEAL protein. NBEAL1 protein was lowly expressed in normal brain tissues and highly expressed in low grade glioma tissues; and the expression of NBEAL1 decreased with the increase of glioma malignancy. Conclusion: The NBEAL1 protein has been successfully cloned, expressed and purified. NBEAL1 protein expression in glioma tissues is negatively associated with the pathological grades of glioma.

Objective:To investigate the inhibitory effect of breast cancer-associated antigen 1(BRCAA1)gene silencing on gastric cancer MGC-803 cells and the related mechanism.Methods:Plasmid shRNA-BRCAA1 and shRNA-N were constructed and transfected with FuGene HD into gastric cancer cell line MGC-803.The transfection efficiency was examined using fluorescent microscope 24 h later.The total RNAs was extracted 48 h 'after transfection and the expression of BRCAA1 and GAPDH gene were analyzed by real-time PCR.The cell proliferation was assessed by MTT assay 24 h,48 h,and 72 h after transfection.The cell apoptosis was determined by Annexin V-PE/TAAD.The expression of Rb,Bax, Bcl-2 and BRCAA1 proteins was analyzed by Western blotting 48 h after transfection.Results:We found that the transfection efficiency of shRNA-BRCAA1 was(81.2?2.6)%24 h after transfection.Forty-eight hours after transfection with shRNA-BRCAA1 the expression of BRCAAI mRNA decreased by 61.4%;the inhibition rate of MGC-803 cells growth was 45.0%.The cell apoptosis rate of shRNA-BRCAA1 transfection group was significantly higher than those of untransfected group and mock plasmid transfected group([14.4?1.6]%vs[5.4?2.0]%,[4.4?2.5]%,P

Objective: To prepare a nanoprobe, anti-human melanoma ganglioside single chain variable fragment (GD/ScFvMEL) antibody conjugated with CdTe quantum dot, and to observe its ability to specifically bind human malignant melanoma cells. Methods: The GD/ScFvMEL gene was cloned into pET32a (+), and the plasmid was then transformed into E. coli BL21 (DE3) for GD/ScFvMEL protein antibody expression. The expressed GD/ScFvMEL antibody was purified by denaturing method and further refolded by modified dialysis method. The purified GD/ScFvMEL antibody was analyzed by SDS-PAGE. The GD/ScFvMEL-QDs nanoprobe was prepared by conjugating GD/ScFvMEL antibody with CdTe quantum dot, and its specificity was observed by incubating with MGC-803 cells and melanoma A375 cells. Results: The recombinant pET32a-GD/ScFvMEL was constructed and confirmed by PCR, restriction endonuclease analysis and DNA sequencing. The proportion of expressed GD/ScFvMEL antibody in total bacteria proteins was about 40% as detected by SDS-PAGE. The purified- and refolded-GD/ScFvMEL antibody was effectively conjugated with CdTe quantum dot, and the resulting GD/ScFvMEL-QDs nanoprobe was successfully prepared. The GD/ScFvMEL-QDs nanoprobe could specifically bind melanoma A375 cells, but could not bind stomach cancer MGC-803 cells. Conclusion: We have successfully prepared an anti-human melanoma ganglioside single-chain antibody-CdTe quantum dot nanoprobe, which can specifically bind melanoma cells.

Objective To observe the effect of a new cell delivery tool(MSC exo)on the proliferation of pancreatic cancer by transferring targeted genes.Methods Transmission Electron Microscope(TEM)and Nanoparticle Tracking Analysis(NTA)were used to identify human mesenchymal stem cell exosomes(MSC-exo)and transport miR-450a-5p into CFPAC-1,to explore the effect of miR-450a-5p targeting BZW2 on inhibiting the proliferation of pancreatic cancer cells.Results The expression of miR-450a-5p was low in pancreatic cancer tissue(P<0.05),and the expression of CD63 and TSG101 of MSC-exo-miR-450a-5p in CFPAC-1 cells was higher than that of MSC-exo by Western blot(P<0.05).CCK-8 and EdU results showed that MSC-exo-miR-450a-5p significantly inhibited the proliferation of CFPAC-1 cells(P<0.05).Cell scratch and Transwell experiments showed that MSC-exo-miR-450a-5p can inhibit the migration and invasion of CFPAC-1 cells(P<0.05).Through dual luciferase assay,it was confirmed that miR-450a-5p targets BZW2,and RT-qPCR and Western blotting showed a negative correlation(P<0.05)between miR-450a-5p and BZW2 expression.Overexpression of BZW2,CCK-8,EdU,cell scratch,and Transwell experiments confirmed that pc-BZW2 reversed the anti-cancer function of MSC-exo-miR-450a-5p on CFPAC-1.Western blot detected PCNA,Ki-67,MMP2,MMP9,and the results were consistent with the above experiments(P<0.05).Conclusion hMSC exo is a new delivery system,targeting BZW2 to transport miR-450a-5p to inhibit the biological malignancy of pancreatic cancer cells,which provides an important clue for the research of targeted treatment of pancreatic cancer.

OBJECTIVE：To e xplore the health economic burden of hypertension patients at county-level areas and its influential factors in China. METHODS ：A questionnaire survey was conducted on hypertension patients in 7 county-level public hospitals from 6 provinces as Hebei ，Shandong，Shanxi provinces by using a convenient sampling method. Catastrophic health expenditure was defined by the standard of “medical and health expenditure exceeding 10％ of household income ”. The incidence ， average gap and relative gap of catastrophic health expenditure were analyzed. A multi-factor Logistic regression model analysis was used to analyze the influential factors that lead to catastrophi c health expenditure. RESULTS ：A total of 1 378 questionnaires were sent out ，and 925 valid questionnaires were collected with effective rate of 67.13％ . The incidence of catastrophic health expenditure，average gap and relative gap among hypertension patients were 23.03％，19.37％ and 84.12％，respectively. At different income levels ，the incidence of catastrophic health expenditure，average gap and relative gap were 72.67％ ， 96.79％ and 133.18％ in the poorest household group ，and were 1.94％ ，0.47％ and 24.23％ in the richest household group. Among different types of medical insurance ，the incidence of catastrophic health expenditure in patients covered by “New Rural Cooperative Medical Scheme （NRCMS）”the highest （31.30％）. The household income ，complications and the type of health insurance had significant impacts on the incidence of catastrophic health expenditure in hypertension patients （P＜0.05）. CONCLUSIONS：The incidence of catastrophic health expenditure in hypertension patients with different income levels is different. As the income level raised ，the incidence of catastrophic health expenditures continued to decrease. But the protection of household health expenditure by NRCMS is weak. It is suggested that a certain policy preference should be given to families with low income and patients with chronic diseases ，so as to ensure the rights and interests of patients with hypertension .