Objective To study the antitumor components from an actinomycete strain(N2010-37)of bottom mud in Zhanjiang Mangrove,South China Sea.Methods The components were isolated and purified by chromatographic techniques and recrystallization,and the structures were identified by spectral methods together with physicochemical analyses.The antitumor effects of these components were tested in vitro by MTT method.Results Three compounds were identified including two anthrones and one novel lactone.They are(3S,4R,7R,8R,9S)-3,8-dihydroxy-4,7,9-trimethyl-2,6-cyclononanediolacetone(1),2-hydroxy-l-methoxy-3-methylanthraquinone(2),and 1,6,8-thihydroxy-3-methyl-anthraquinone(3).Conclusion Compound 1 is a new compound,and compounds 1 and 3 show the favorablecytotoxic activities against human chronic granulocytic leukemia cell line K562 strain by MTT method in vitro.

Objective To investigate the expression of Rap1 GTPase-activating protein 1 (Rap1GAP),matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9),and their relation with clinical patterns in colorectal carcinoma.Methods Immunohistochemistry was used to detect the expression of Rap1GAP,MMP-2 and MMP-9 in colorectal carcinoma,villous adenoma,tubular adenoma and normal colorectal tissue,and their relationship with clinicopathological parameters was analyzed.Results The positive rate of Rap1GAP expression was 30.4 ％ (14/46),77.8 ％ (14/18),69.6 ％ (16/23) and 95.2 ％ (20/21) in colorectal carcinoma,villous adenoma,tubular adenoma,and normal colorectal tissue,respectively (x2 =30.659,P=0.000).The figures were 71.7 ％ (33/46),55.6 ％ (10/18),52.2 ％ (12/16) and 9.5 ％ (2/21) for the positive rate of MMP-2 expression (x2 =22.459,P =0.000),as well as for 76.1％ (35/46),61.1％ (11/18),56.5 ％ (13/23) and 14.3 ％ (3/21) for the positive rate of MMP-9 expression,respectively (x2 =22.643,P =0.000).In patients with colorectal carcinoma,the expression of Rap1GAP was correlated with tumor differentiation (x2 =5.275,P =0.022),but not sex,age,or lymphatic metastasis (all P ＞ 0.05).The expression of MMP-2 and MMP-9 were correlated with lymphatic metastasis (x2 =6.661,P =0.010;x2 =8.475,P =0.040),but not sex,age or tumor differentiation(all P ＞ 0.05).There was a negative correlation between expression of Rap1GAP and MMP-2,MMP-9 in colorectal carcinoma,respectively (r =-0.424,P =0.003;r =-0.294,P =0.048),but no correlation between the expression of MMP-2 and MMP-9 (r =0.101,P =0.505).Conclusions Rap1GAP,MMP-2 and MMP-9 play important roles in the malignant biological behavior of colorectal carcinoma,and the expression of Rap1GAP is negatively correlated with MMP-2 and MMP-9.The interactions among the three affect the occurrence and development of colorectal carcinoma.

Objective To investigate the expression of signal-induced proliferation-associated gene 1 (SIPA1), Ezrin and E-cadherin (E-cad), and their relationship with clinical patterns in epithelial ovarian carcinoma.Methods Immunohistochemistry was used to detect the expression of SIPA1, Ezrin and E-cad in normal ovarian tissue, benign epithelial ovarian tumor, borderline epithelial ovarian tumor and epithelial ovarian carcinoma,respectively. Results The positive rate of SIPA1 expression was 44.2 % (23/52), 64.5 %(20/31), 93.3 % (28/30) and 100.0 % (15/15) in epithelial ovarian carcinoma, borderline epithelial ovarian tumor, benign epithelial ovarian tumor, and normal ovarian tissue, respectively, and there was a statistical difference (χ2 = 29.159, P= 0.000). The corresponding rates were 57.7 % (30/52), 61.3 % (19/31), 90.0 %(27/30) and 93.3 % (14/15) for the positive rate of Ezrin expression (χ2= 14.555, P= 0.002), as well as for 23.1 % (12/52), 58.1 % (18/31), 86.7 % (26/30) and 0 (0/15) for the positive rate of E-cad expression, respectively (χ2= 45.731, P= 0.000). In patients with epithelial ovarian carcinoma, the expression of SIPA1 was correlated with tumor differentiation (χ2=3.895, P=0.048), but not with histological type and clinical stage (all P>0.05). The expression of Ezrin was not correlated with histological type, tumor differentiation and clinical stage (all P>0.05). There was a positive correlation between expression of E-cad and SIPA1, Ezrin in epithelial ovarian carcinoma, respectively (r= 0.339, P= 0.014; r= 0.284, P= 0.041), but no correlation between the expression of SIPA1 and Ezrin (r= 0.214, P= 0.128). Conclusions SIPA1, Ezrin and E-cad play important roles in the occurrence and development of epithelial ovarian carcinoma. They cooperate in the progression and their combined detection can better evaluate the prognosis of epithelial ovarian carcinoma.

The effects of continuous subcutaneous insulin injection (CSII, n=306) and multiple subcutaneous insulin injection (MSII, n=310)on hyperglycemia in patients with diabetes mellitus were compared. The results suggest that the effect of CSII is more prompt and efficient with lower incidence of hypoglycemia as compared with MSII.

Objective To develop a new method, capillary electrophoresis(CE) based on molecular beacon(MB),for rapid detection of polymerase chain reaction(PCR).To explor the roles of the IL-13 gene exon4 A2044G single nucleiotide polymorphism(SNP) in the pathogenesis of bronchial asthma.Methods The IL-13 exon 4 was amplified by PCR with genomic DNA used as templates from 20 healthy persons and 32 patients with dominantly allergic familial history living in the north of china.Then separating the amplification with capillary electrophoresis followed by hybridization of molecular beacons into the PCR product which were sequenced in the end. Results Detected with CE-MB method,there was significant difference in the distribution of A/G in IL-13exon 2044,A allele frequency was higher in asthma compared with normal controls,same as sequencing. Conclusion The method of capillary electrophoresis based on molecular beacon(MB-CE) is able to be developed as a clinical detecting method for genetic variation diseases.IL-13 A2044G SNP is important in the asthmatic mechanism.

AIM:To investigate the serum antibody of aquaporin 4 ( AQP4 - Ab ) in positive expression rate and the significance in patients with neuritis.
●METHODS: A total of 98 cases ( 128 eyes ) of patients with optic neuritis were studied to detect the patient′s serum AQP4-Ab positive rate of antinuclear antibodies ( ANAs) from Jan. 2012 to Dec. 2015 in ophthalmology center of our hospital. According to the expression of AQP4 - Ab group, the best corrected visual acuity between the two groups, peripapillary nerve fiber layer thickness (pRNFL), the volume of the macula, macular RNFL ( mRNFL ) , macular core layer volume ( mlNL ) measurement were compared.
●RESULTS:Ninety-eight patients (128 eyes) with optic neuritis cases diagnosed through examination revealed AQP4-Ab positive in 22 patients ( 22%) , negative in 76 patients ( 78%) , ANAs positive in 21 patients ( 21%) , negative 77 patients ( 79%) . Optic neuritis patients with serum AQP4 - Ab positive rate and ANAs positive significant correlation ( r = 0. 707, P < 0. 05 ). After examination revealed AQP4-Ab patients and negative-positive patients with best corrected visual acuity difference was not statistically significance (P>0. 05). After inspection found pRNFL, macular volume measured value AQP4 - Ab positive patients were significantly less than the negative patients, the differences were statistically significant (P<0. 05). AQP4-Ab positive patients and negative patients the mRNFL, mlNL measured values were not significantly different (P>0. 05).
●CONCLUSION:AQP4-Ab and ANAs expression in optic neuritis patients is a significant correlation. AQP4-Ab positive patients with optic neuritis pRNFL thinning of macular volume are decreased compared with negative patients.

Objective To detect the expression of MAGE-A9 in human hepatocellular carcinoma (HCC), and to investigate the association between expression of MAGE-A9 and the clinicopathological characteristics of HCC.Methods Reverse transcription-polymerase chain reaction (RT-PCR), one-step quantitative-PCR (qPCR) and immunohistochemistry (IHC) methods were performed to characterize the expression of MAGE-A9 in HCC cell lines and tissues.Kaplan-Meier survival analysis and Cox regression were employed to evaluate the prognosis of 100 HCC patients.Results The expression of MAGE-A9 mRNA in HCC (4.44±0.342) was significantly higher than that in non-cancerous cells and tissues (1.73±0.178) (P ＜ 0.05).The expression level of the MAGE-A9 protein in HCC was related to the pathological grade (P =0.003), portal vein invasion (P =0.001), distant metastasis (P =0.022) and TNM stage (P =0.005).Cox regression analysis revealed that MAGE-A9 expression is an independent prognostic factor for disease-free survival (P =0.006) and overall survival (P =0.022).Conclusion MAGE-A9 is a valuable prognostic biomarker for HCC patients, and its high expression suggests unfavorable survival outcomes in HCC patients.

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People′s Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43 % (50.35 %), 21.37 % (8.39 %) and 17.43 % (15.71 %) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P< 0.05). The methylation rates of Rap1GAP promoter in colonic adenocarcinoma were not correlation with age, sex, differentiation and the stage of TNM [ male vs. female: 42.74 % (70.44 %) vs. 21.98%(80.00%);≤60yearsoldvs.>60yearsold:36.26%(62.62%)and26.23%(76.42 %);well-differentiated vs. moderately/poorly-differentiated: 21.98 % (40.32 %) vs. 42.74 % (74.20 %); TNM Ⅰ-Ⅱ vsⅢ-Ⅳ: 25.31 % (48.27 %) vs. 36.26 % (75.55 %); all P> 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer.

Objective To confirm the main pathway of chemokine-chemokine receptor which mediates the accumulation of regulatory T cell ( Treg) in pancreatic cancer .Methods The concentrations of protein of FOXP3 and chemokines of CCL2, CCL3, CCL5, CCL17, CXCL8 in human and mouse pancreatic cancer and adjacent normal pancreatic tissue were measured by the method of enzyme-linked immunosorbent assay (ELISA).The receptor of chemokine CCL5 (CCR5) in human and mouse pancreatic cancer were determined by the immunofluorescent stain .Results The concentration of FOXP 3 protein in human pancreatic cancer and adjacent normal pancreatic tissue as (487.5 ±534.1) and (162.6 ±42.0) pg/mg, respectively, while they were (84.6 ±54.1) and (14.4 ±7.6) pg/mg, respectively in mouse.The concentration of FOXP3 protein were significantly higher in pancreatic cancer than those in adjacent normal pancreatic tissue .The concentration of CCL2 in human pancreatic cancer and adjacent normal pancreatic tissue as (76.9 ±37.5), (40.8 ±25.5) pg/mg, and the concentration of CCL3 as (38.0 ±22.6), (21.3 ±16.5) pg/mg, and the concentration of CCL5 were (390.2 ±158.5), (59.1 ±22.8) pg/mg, and the concentration of CCL17 as (7.2 ±2.0), (4.1 ±2.4)pg/mg, and the concentration of CXCL8 as (9.3 ±5.5), (6.3 ±5.2)pg/mg.The concentration of CCL2, CCL5, CCL17 in pancreatic cancer was significantly higher than those in adjacent normal pancreatic tissue (P<0.05).The concentration of CCL2 in mouse pancreatic cancer and adjacent normal pancreatic tissue as (77.9 ±30.5), (43.6 ±16.6) pg/mg, and the concentration of CCL3 was (27.4 ±18.2), (14.0 ±4.5)pg/mg, and the concentration of CCL5 was (302.2 ±55.8), (64.5 ±30.3) pg/mg; and the concentration of CCL17 was (4.4 ±1.4), (2.2 ±1.0)pg/mg;and the concentration of CXCL8 was (55.1 ± 55.1), ( 93.4 ±7.3 ) pg/mg.The concentration of CCL2, CCL5, CCL17 in pancreatic cancer were significantly higher than those in adjacent normal pancreatic tissue , and the difference between the two groups was statistically significant (P<0.05).The level of FOXP3 in pancreatic cancer was positively correlated with the concentration of chemokine CCL 5 both in human and mouse pancreatic cancer .Immunofluorescent staining indicated that the FOXP3 +cells also expressed CCR5.Conclusions The CCL5-CCR5 is the main chemokine-chemokine receptor pathway mediating the accumulation of Treg cells in pancreatic cancer .

BACKGROUND: Inhibiting the degradation of extracellular matrix in the intervertebral disc can delay the degenerative process of intervertebral disc. Matrix metalloproteinases-3 (MMP3) is considered as a key enzyme for degradation of extracelular matrix components such as type II collagen and aggrecan.
OBJECTIVE:To construct the short hairpin RNA lentiviral vector targeting human MMP3 gene and to detect its efficiency of gene silence by infecting human degenerative nucleus pulposus cells.
METHODS:According to the human MMP3 mRNA (NM_002422.4) sequence, four groups of the short hairpin RNA gene sequences targeting MMP3 were designed, synthesized and annealed to form double stranded DNA fragments, which were connected with the LV3 vectors digested by BamHI andEcoRI enzymes, and then transfected into the competent cels. The positive clones were identified by PCR, and analyzed by sequencing. The packaging and titer of lentivirus were determined after transfecting 293T cells. Human degenerative nucleus pulposus cels were infected with lentivirus vector, and the transfection efficiency of each group was observed under inverted fluorescence microscope. The interfering efficiency was detected by real time-PCR and western blot at 72 and 96 hours.
RESULTS AND CONCLUSION:The ds-oligo DNA was successfully inserted into the lentiviral vector as confirmed by electrophoresis and sequence analysis. The recombinant lentivirus was harvested from 293T cels with a viral titer of 1-5 ×108 TU/mL. RNA interference targeting the GCC AGG CTT TCC CAA GCA AAT sequences with the highest interfering efficiency in MMP3 gene at 72 and 96 hours resulted in suppression of MMP3 mRNA expression by 98% and 72%, respectively; and at 96 hours, the interfering efficiency of protein expression was 57.2%. The recombinant lentivirus vector containing RNA interference targeting MMP3 gene is successfuly constructed, which lays a foundation for further studies on the MMP3 function and gene therapy.