The anti VEGF monoclonal antibody conjugated with nanometer particle of 5-FU

AIM:The characteristics of nanometer particles, which were prepared by the conjugation of anti-VEGF monoclonal antibodies and 5-fluorouracil-loaded polylactic acid nanometer particles (5-FU-NPs), were investigated for improving the anticancer activity of 5-FU. METHODS:The method of couple linkage of chemical bonds was used to prepare the 5-FU-NPs with VEGF antibody, then the appearance, distribution of particle diameter, releasing in vitro and the immunological activity were detected. RESULTS:The 5-FU-Ab-NPs appeared as regular globular, the average particle diameter was (202±23)nm. The 5-FU-Ab-NPs possessed the similar delayed release character of 5-FUs. More than 80% of the immmunogical activity were detected in conjugates-retained antibody by the immunological methods and electron microscopy. CONCLUSION:The 5-FU-Ab-NPs possess the similar delayed released activity to 5-FU-NPs and have double activity of immune targeting and delayed releasing, which may increase the local concentration of 5-FU.     

Relationship between genetic instability of nm23H1 gene and clinical pathological characteristic in hepatocellular carcinoma

AIM: The aim of this study was to examine the microstatellite instability (MSI) and loss of heterozygosity(LOH) of locus D17S396 on chromosome 17 and their influence on the expression of nm23H1 in hepatocellular carcinoma (HCC),which may provide experimental evidence for the mechanism of nm23H1 gene and tumor metastasis.METHODS: Techniques such as DNA extraction from formalin-fixed paraffin-embedded tissues,PCR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to assess the expression of nm23H1.RESULTS: ① The frequency of heredity instability of HCCs was 35.42%.The frequency of LOH in the cases with lymph node or distant organs metastasis or not and with intrahepatic metastasis or embolus of portal vein or not was significantly different (P<0.01),it was higher in stage TNM Ⅲ than that in stageⅠ and Ⅱ.Moreover,it was higher in high tendency to invasion or metastasis cases than that in the low tendency cases (P<0.01).② The expression of nm23H1 was 56.25%.It was significantly different in Edmondson grade,TNM stage and in lymph node or distant organ metastasis cases (P<0.01).The cases with high tendency of invasion or metastasis exhibited lower nm23H1 expression compared with low tendency cases (P<0.01).③ The positive rate of nm23H1 protein in LOH positive group was lower than that in LOH negative group (P<0.05).CONCLUSION: The results indicate that both MSI and LOH of nm23H1 gene control the development of HCC independently in different pathways.LOH inhibits the expression of nm23H1,which endows it with high aggressive and poor prognosis.Increase in the amount of nm23H1 protein expression effectively restrains the tendency to invasion or metastasis of HCCs and improves prognosis of patients.     

Effects of STK15 overexpression on growth of human esophageal carcinoma cell line KYSE150

AIM:To investigate the effects of serine/threonine kinase 15 (STK15) overexpression on the growth of human esophageal squamous-cell carcinoma (ESCC) cell line KYSE150. METHODS:Recombinant pEGFP-C1-STK15 expression vector was transfected into KYSE150 cells using LipofectamineTM 2000 and the expression of STK15 was detected by fluorescence microscopy and Western blotting. The proliferation of the cells in vitro was measured by MTT assay. The cell cycle distribution and apoptosis were detected by flow cytometry. The proliferation of the cells in vivo was measured by tumorigenicity experiment in nude mice. RESULTS:After recombinant pEGFP-C1-STK15 expression vector was stably transfected into KYSE150 cells, GFP-STK15 fusion protein localized to centrosome and spindle. The STK15-overexpressing colonies were further confirmed by Western blotting. MTT assay showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). Flow cytometry analysis showed that the percentage of the cells in G0/G1 phase and the cell apoptosis in STK15 overexpression group were decreased compared with control group (P<0.01). The tumorigenicity experiment in nude mice showed that the proliferation of the cells in STK15 overexpression group was increased compared with control group (P<0.01). CONCLUSION: Overexpression of STK15 in human ESCC KYSE150 cells promotes the cell growth in vitro and in vivo, indicating that STK15 may serve as a novel therapeutic target for esophageal carcinoma.     

6种大型海洋掠食性鱼类胃含物角质颚分类研究

为了研究大型海洋掠食性鱼类胃含物中角质颚的分类效果,于2017年10月,2018年3、5和11月,收集了南大西洋、中东太平洋和西印度洋海域的大型海洋掠食性鱼类的36个胃并分析其残留角质颚的形态。对采集的角质颚形态侧视图和顶视图进行种类鉴定,建立检索表,提取椭圆傅里叶描述子(EFDs)系数进行聚类分析。结果显示,通过对胃含物角质颚形态进行种类鉴定,共发现头足类2目10科17种。根据角质颚形态分类检索分析,17种头足类角质颚形态种间差异明显。枪形目(Teuthoidea)的13种头足类角质颚喙部形态分别为三角形和等腰三角形,侧壁为近似菱形,翼部发达;八腕目(Octopoda)的4种头足类角质颚有喙部发育,侧壁近似长四边形,有翼部发育。角质颚形态信息聚类分析在目级别区分效果显著。通过对胃含物角质颚形态进行分类研究,不仅可佐证胃含物头足类的识别,而且丰富了胃含物头足类的鉴定内容,也为大型海洋掠食性鱼类胃含物头足类分类鉴定体系奠定基础。     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

Up/down-regulation of miR-21 changes biological function of colon can-cer cells and sensitivity to cetuximab

AIM: To explore the effects of miR-21 on biological behavior of colon cancer cells and their sensitivity to epidermal growth factor receptor monoclonal antibody cetuximab. METHODS: Lentiviral vectors were constructed to generate up- and down-regulations of miR-21 lentiviruses (LV-miR-21 and LV-anti-miR-21, respectively), and the corresponding negative control viruses (LV-miR-21 NC and LV-anti-miR-21 NC, respectively) were also constructed. The viruses were used to infect human colon cancer RKO cells. The changes of the miR-21 expression level, the cell proliferation, the colony-forming ability, the cell apoptosis and the sensitivity of the cells to cetuximab were detected by real-time PCR, MTT assay, soft agar colony assay, flow cytometry and CCK-8 assay. RESULTS: The lentivirus titers of LV-miR-21, LV-miR-2 NC, LV-anti-miR-21 and LV-anti-miR-21 NC were 3.0×1012 TU/L, 6.0×1011 TU/L, 2.0×1012 TU/L and 8.0×1011 TU/L, respectively. The infection efficiency was over 80% by the observation of green fluorescence. The miR-21 expression level, the cell proliferation, and the colony-forming ability in LV-miR-21 group were significantly higher than those in LV-anti-miR-21 group. The early apoptotic rate and the inhibitory rate of cetuximab for the cells in LV-anti-miR-21 group were higher than those in LV-miR-21 group. CONCLUSION: miR-21 promotes the proliferation of colon cancer cells. Down-regulation of miR-21 enhances the sensitivity of the colon cancer cells to the targeted therapy drug cetuximab.     

Role of ACSL3 expression in suppressing prostate cancer cell metastasis

AIM:To analyze the difference of long-chain acyl-CoA synthetase 3 (ACSL3) expression between normal prostate epithelial cells and prostate cancer cells.METHODS:ACLS3 mRNA expression between normal prostate epithelial cells and prostate cancer cells was compared using RT-PCR. Meanwhile, ACSL3 gene was amplified from prostate cancer cells, and the eukaryotic expression plasmid pCDNA3.1(+)-Flag-ACLS3 and lentivirus Lenti-ACLS3 were constructed. After transfection of ACSL3-plasmid and lentivirus into the prostate cancer cells, ACSL3 expressive was detected by RT-PCR and Western blotting, and then Matrigel invasion assay was performed to investigate the alteration of the invasive ability of the prostate cancer cells with over-expression of ACSL3. RESULTS:A significant difference of ACSL3 mRNA level between normal prostate epithelial cells and prostate cancer cells was observed. ACSL3 was highly expressed in localized prostate cancer cells compared to metastatic prostate cancer cells, while ACSL3 expression was higher in androgen-dependent prostate cancer cells than that in androgen-independent prostate cancer cells. Furthermore, the eukaryotic expression plasmid and lentivirus containing ACLS3 gene were successfully constructed. The prostate cancer cell line which stably over-expressed ACLS3 was established. Up-regulation of ACSL3 inhibited the invasive ability of prostate cancer cells. CONCLUSION:ACSL3 plays an antagonistic role in invasiveness of prostate cancer.