Objective:To study the change of tumor necrosis factor-?(TNF-?) , interlukin-1?(IL-1?) and interluldn-6(IL-6) in neonates with hypoxic-ischemic encephalopathy(HIE) ,and analyze the relation between their levels and the severity of HIE. Methods :TNF-?,IE-? and IL6 in the plasma and in the supernatant of cultured peripheral blood mononuclearcytes(PBMC) were mesured in 50 neonates with HIE and 30 normal term neonates by enzyme-linked immunosorbent assay. Results: TNF-?and IL-?plasma levels and the production by PBMC in neonates with FILE were markedly elevated than those in normal controls Levels of TNF-?,IL-?and IL-6 both in the plasma and the supernatant of PB-MC were much higher in acute stage of severe HIE compared with the control group and mild HIE, and plasma levels of TNF-a and IL-? were significantly decreased in convalescence stage, but IL-6 levels were still much higher than those in normal controls. Conclusion:TNF-? and IL-? plasma levels and the production by PBMC in neonates with I-LIE axe related to the severity of HIE, and IL-6 may participate in the repair of neurons after ischemic brain injuly. These cytokines can be indicators of the brain damage degree of IDE.

Objective To evaluate the value of homocysteine(Hcy) detection by different assays in the diagnosis of hypertension complicating cerebral infarction disease .Methods Serum concentration of Hcy in the patients with single hypertension ,hyperten‐sion complicating cerebral infarction and healthy controls were detected by the fluorescence quantification assay and the enzymatic cycling assay respectively ,the application value of the two assays in the diagnosis of hypertension complicating cerebral infarction disease was evaluated by the receiver operating characteristic (ROC) curve .Results Both the two assays showed that the serum Hcy level and incidence of high Hcy in the single hypertension group and the hypertension complicating cerebral infarction group were obviously higher than those in the healthy control group (P<0 .05) ,but there were no statistically significant differences be‐tween the single hypertension group and the hypertension complicating cerebral infarction group (P>0 .05) .The ROC curve analy‐sis showed that the sensitivity and the specificity in the enzymatic cycling assay were higher ,which were 67 .5% and 85 .3% respec‐tively ,but the fluorescence quantification assay had lower sensitivity (40 .0% ) and higher specificity (97 .1% ) .Compared to the sin‐gle index test ,the sensitivity of Hcy and triglyceride combination detection had higher sensitivity (72 .5% ) and higher specificity (94 .1% ) ,which were higher than the diagnostic performance of the single index .Conclusion The increase of serum Hcy level is closely related to hypertension ,but is not a direct causal relationship with hypertension complicating cerebral infarction .The enzy‐matic cycling assay is better than the fluorescence quantitative assay in the diagnostic performance of hypertension complicating cer‐ebral infarction .The combination detection of Hcy and triglyceride conduces to improve the diagnostic sensitivity and specificity .

Objective To explore the screening of peroxisome pathway reactive oxygen species (ROS) oxidative stress gene and its correlation with the antitumor sensitivity of artesunate against pancreatic cancer. Methods Based on microarray mRNA expressions of 55 tumor cell lines in the National Cancer Institute common database,peroxisome pathway-related key genes which were significant correlation with half-inhibitory concentration (IC50 )values of artesunate antitumor activity against human pancreatic cancer were selected by Kendall test.The candidate genes associated with artesunate sensitivity were identified and their mRNA expressions in pancreatic cancer cells were tested using fluorescent quantitative PCR.The contents of peroxi-dase in pancreatic cancer cells were detected through the DAB staining.Results Thirteen key genes mRNA expressions in peroxidase pathways were significantly correlated with IC50 values for artesunate antitumor activi-ty.Compared with normal liver cells HL-7702 (1.00),CRAT (2.89 ±0.06),PEX11B (1.90 ±0.07)and PEX16 (1.35 ±0.07)mRNA expression levels were significantly increased in pancreatic cancer Panc-1 cells which sensitive to artesunate (t =33.00,P <0.01;t =17.85,P <0.01;t =4.54,P <0.05).While CAT
(1.43 ±0.03),SOD1 (2.07 ±0.04)and SOD2 (1.15 ±0.01)mRNA expression levels were also signifi-cantly increased in Panc-1 cells which sensitive to artesunate (t =11.71,P <0.01;t =35.85,P <0.01;t =13.22,P <0.01).However,PEX12 (0.51 ±0.02),CAT (0.47 ±0.02),PRDX1 (0.43 ±0.01),and SOD1 (0.44 ±0.01)mRNA expression levels in pancreatic cancer BXPC-3 cells which resistant to artesunate were significantly lower than that of HL-7702 cells (t =37.53,P <0.01;t =16.52,P <0.01;t =84.20, P <0.01;t =48.24,P <0.01).DAB staining showed that the positive expression rate of peroxisomal content was apparently higher in Panc-1 cells (61.5%)than that of HL-7702 cells (43.8%),with a significant difference (χ2 =16.11,P <0.01).Conclusion Peroxisome and its related ROS antioxidant enzymes CAT, PRDX1,SOD gene expression may be the important factors that affect artesunate antitumor activity against human pancreatic cancer.

Objective To investigate the effect of dysadherin gene silencing on metastasis and invasion in pancreatic cancer cell line PANC1,BxPC3 in vitro.Methods PANC1 and BxPC3 cells were divided into dysa group,negative siRNA control group(HK),liposomes control group(control),dysa group and HK group were tranfected by dysadherin siRNA and Negative siRNA,respectively.The expression of dysadherin mRNA and protein were detected by RT-PCR and immunohistochemical method.Transwell test was used to evaluate the invasion ability of pancreatic cancer cells.Results After transfected by dysadherin siRNA,the dysadherin mRNA levels in PANC1 and Bxpc3 cells were decreased by 95.4% and 52.1%.The expression of dysadherin protein was also down-regulated by 91.2% and 83.6%,respectively,when compared with HK groups (P＜0.05 ).The numbers of invasive cells migrated in Transwell in PANC1 cells control group,HK group and dysa group were 163.2±15.5,154.4±17.3 and 53.6±7.9;the numbers of invasive cells in BxPC cells control group,HK group and dysa group were 30.7±3.2,27.5±2.8 and 4.7±2.4,respectively.The numbers in dysa group were significantly lower than those of HK group and control group (P＜0.01 ).Conclusions Silencing the dysadherin gene of PANC1,BxPC3 by RNA interference could significantly inhibit the invasive and migratory ability of canceroas cells.

Objective To investigate the effect of shRNA-mediated down-regulation of the receptor for activated C kinase 1 (RACK1) gene on the chemotherapeutic sensitivities in human lung adenocarcinoma cell line A549.Methods The shRNA recombinant plasmid targeting to human RACK1 gene was designed and transferred into A549 cells by lipofectin technique.The protein level of RACK1 was measured by Western blot to confirm the function of shRNA plasmid.Drug sensitivities of A549 cells to cisplatin,gemcitabine,pemetrexed and paclitaxel were analyzed by MTT assay.The protein expression of LRP and MRP were detected by Western blot.Results After 24 hours transfection,the relative expression quantity of RACK1 protein in RACK1-shRNA group was 0.267± 0.470,which was significantly lower than that in vector-shRNA group (0.821±0.109) and control group (0.842±0.060) (F =54.438,P ＜ 0.05).The results of MTT showed that the growth of A549 cells in the RACK1-shRNA group was markedly inhibited.The sensitivities of A549 cells to cisplatin and paclitaxel were significantly enhanced compared with that in the vector-shRNA group and control group (P ＜ 0.05).The relative expression quantity of LRP and MRP protein in RACK1-shRNA group were 0.163±0.056 and 0.246±0.050,which were lower than that in vector-shRNA group and control group (F LRP =19.430,F MRP =61.548,both P ＜ 0.05).Conclusion Targeted gene silencing of RACK1 improves the sensitivity of A549 cells to the ascisplatin and paclitaxel medicines,which might be achieved through down-regulation of the expression of LRP and MRP.

Objective:To investigate the expression of miR-92a in colorectal cancer (CRC) and its effect on the regulation mechanisms of tumor angiogenesis. Methods:The miRNA-92a expression in 25 CRC tissues and HT29, SW620, SW480, and HCT116 CRC cells was detected using quantitative real-time polymerase chain reaction. The CD31 positive expression of blood microvessels in CRC tissues was measured by immunohistochemistry. Pearson correlation analysis was used to analyze the relationship between miR-92a and tu-mor angiogenesis. The miR-92a mimic or inhibitor was transfected into HCT116 and SW620 cells in vitro to upregulate or downregu-late the miR-92a expression. The effect of miR-92a overexpression or suppression on the formation of human umbilical vein endotheli-al cell (HUVEC) tubules was detected by tube formation assay. Changes in phosphatase and tensin homolog (PTEN) protein expression were measured by Western blot. Results:The expression level of miR-92a in CRC tissues was significantly higher than that in matched adjacent tissues (P<0.01). The expression levels of miR-92a in HT29, SW480, SW620, and HCT116 colon cancer cell lines were signifi-cantly higher than that of the normal colorectal epithelium control (P<0.05). The number of CD31 positive expression of microvessel density (MVD) in CRC tissues was significantly higher than that in adjacent tissues (P<0.01), and the miR-92a expression level was posi-tively correlated with the MVD in CRC tissues (r=0.580, P=0.01). The cell culture supernatant of HCT116 with miR-92a overexpression could significantly promote the formation of HUVEC tubules (P<0.05). The upregulation of miR-92a expression could significantly inhib-it the expression of PTEN protein in CRC cells (P<0.01). Conclusion:The miR-92a was highly expressed in CRC cells and tissues, which was closely related to the formation of tumor angiogenesis in CRC. The miR-92a could promote tumor angiogenesis in CRC by inhibit-ing PTEN expression.

ObjectiveTo investigate the biological effects and its mechanisms of ascorbic acid on pancreatic cancer PANC1 cells. Methods PANC1 cells were treated by ascorbic acid of different concentrations (0 ～40 mmol/L) for 24,48,72 hours.The proliferation of PANC1 cells was analyzed by MTT method; cell cycle and apoptosis were assessed by flow cytometry (FCM); inverted microscopy and transmission electron microscopy were used to observe cell morphology. The membrane potential of mitochondria were mearured by with JC-1 staining and FCM.Meanwhile,the changes of cell morphology and mitochondrial membrane potential induced by ascorbic acid after pretreatment with hydrogen peroxidescavenging enzyme (catalase) and red blood cells were also detected. Results Ascorbic acid in pharmacologic concentrations selectively inhibited the proliferation of PANC1 cells in a dose and time dependent manner.PANC1 cells were arrested in G2/M phase after treatment with 5 mmol/L ascorbic acid [ (32.55 ± 7.14)％ vs (22.00 ±1.27)％,t =5.808,P＜0.05],but there was no changes on apoptosis rate [ (1.98 ± 1.80)％ vs (1.09 ±0.16)％ ].Inverted microscope and transmission electron microscopy showed that oncosislike cell death of PANC1 cells was induced after treatment with ≥5 mmol/L ascorbic acid.Mitochondrial membrane potential of PANC1 cells was significantly lower than that of the control group in a dose dependent manner.The descent of mitochondrial membrane potential was significantly inhibited by pretreatment with catalase and red blood cells,and the degree of cell oncosis was attenuated.ConclusionsAscorbic acid significantly inhibited the proliferation of pancreatic cancer PANC1 cells in vitro.Ascorbic acid induced PANC1 cell oncosis,but not apoptosis.The possible mechanisms of inducing oncosis may be related to the descent of mitochondrial membrane potential.

Objective To explore the expression characteristic of serum mir-16,mir-21,mir-29a, mir-92a and mir-143 in colorectal cancer , evaluate the clinical significance of candidate miRNAs in CRC diagnosis.Methods Case-control study was used.The expression levels of serum mir-16,mir-21,mir-29a, mir-92a and mir-143 from 50 CRC patients and 27 normal controls were detected by real-time PCR, the levels of miRNAs expression in serum of 8 CRC patients 1 day before and 7 days after radical surgery were analyzed.The sensitivity and specificity of serum miRNAs expression for the diagnosis of colorectal cancer were evaluated using the receiver operating characteristic ( ROC) curve.Results The expression level of mir-16, miR-21, mir-92a in serum of patients with CRC were (75.55 ±37.73), (35.96 ±23.81), (24.79 ±8.97) fmol/ml, significantly higher than that of the healthy control group (32.73 ±18.94), (24.36 ±13.27), (16.36 ±5.58) fmol/ml (tmir-16 =2.77, tmir-21 =2.34, tmir-92a =3.85,P <0.05).However, the expression levels of mir-29a and mir-143 showed no significant difference between two groups (tmir-29a=-0.17, tmir-143 =1.59,P>0.05).In addition, serum miR-16 and miR-92a levels of CRC patients 7 days after tumor resection were (36.02 ±19.95), (14.82 ±7.78) fmol/ml, significantly lower than that before operation (62.18 ±34.17), (24.06 ±12.99) fmol/ml (tmir-16 =3.59, tmir-92a =2.60,P<0.05).Area under the ROC curve ( AUC) of combined detection of mir-16, miR-21and mir-92a was 0.877, the sensitivity and specificity were 88% and 85%, respectively, which was higher than any of 3 miRNAs alone and the conventional tumor marker CEA.Conclusion Combination of mir-16, mir-21 and mir-92a in the diagnosis of CRC shows a better sensitivity and specificity , which would be expected as new tumor biomarkers for noninvasive diagnosis and monitoring progression of colorectal cancer.

Objective To evaluate the effect of microRNA-92a (miR-92a) on regulating cell proliferation by targeting Krüppel-like factor 4 (KLF4) in colon cancer.Methods The miR-92a expressions in 21 colon cancer tissues and matched normal tumor-adjacent tissues and 4 colon cancer cells (HT29,SW480,SW620,HCT116) were detected using quantitative real-time polymerase chain reaction (qRT-PCR).Models of over-expression and suppression of miR-92a were established by transient transfection of miR-92a-3p mimic to HCT116 and transient transfection of miR-92a-3p inhibitor to SW620,respectively.Cell proliferation activity was detected by the CCK-8 colorimetry method,cell cycles were detected by flow cytometry,KLF4 protein expression was detected by Western blotting,and cell luciferase activity was detected by the dual luciferase reporter gene experiment.Results The expression level of miR-92a in colon cancer tissues was (0.648 ± 0.489) fmol/μg total RNA,significantly higher than that in matched normal tumor-adjacent tissues [(0.064 ± 0.062) fmol/μg total RNA],with statistically significant difference (t =-5.420,P ＜ 0.001).In 4 colon cancer cell lines,the miR-92a expression level in HCT116 cells was the lowest,and highest in SW620 cell.When the expression of miR-92a was up-regulated,the cell proliferation activity of 72 h in HCT116 cells was higher than that in the negative control group (0.919 ±0.014 vs.0.765 ± 0.025),with statistically significant difference (t =-9.309,P =0.001),the proportion of S phase cells was also significantly increased [(41.670 ±0.461)％ vs.(38.703 ±0.554)％,t =-7.127,P =0.002),and KLF4 protein expression was decreased (0.460 ± 0.048 vs.0.758 ± 0.109,t =22.865,P =0.028).When the expression of miR-92a was down-regulated,the cell proliferation activity of 72 h in SW620 cells was lower than that in the negative control group (0.608 ± 0.011 vs.0.713 ± 0.005),with statistically significant difference (t =15.920,P ＜ 0.001),while the proportion of S phase cells was decreased [(31.935 ± 0.365) ％ vs.(34.955 ± 0.465) ％,t =8.849,P =0.001],and KLF4 protein expression was increased (0.694 ± 0.121 vs.0.479 ± 0.044,t =-5.246,P =0.034).KLF4 3'UTR wild-type dual luciferase report plasmids were co-transfected with miR-92a-3p mimic to HCT116 cell,and dual luciferase assay showed that miR-92a slightly repressed firefly luciferase actively,but the difference was not statistically significant (t =0.878,P =0.429).There was a negative correlation between the expression of miR-92a and the expression of KLF4 protein in colon cancer tissues,but with no statistical significance (r =-0.163,P =0.699).Conclusion miR-92a is highly expressed in colon cancer tissues.It can promote colon cancer cells proliferation via enhancement of the cell cycle transition of G0-G1 phase to S phase.Up-expression of miR-92a may play a role in down-regulating the expression of KLF4 protein in colon cancer cells.However,KLF4 is not a direct target gene of miR-92a.

Objective To analyze the infection characteristics and epidemic trend of 9 respiratory pathogens in Shenzhen popula-tion .Methods The 5918 patients with acute respiratory tract infection were collected ,indirect immunofluorescence was used to de-tect the IgM antibody of 9 respiratory pathogens ,including legionella pneumophila type 1 (LP1) ,mycoplasma pneumoniae (MP) , rickettsia Q (COX) ,rickettsia and chlamydia pneumoniae (CPn) ,adenovirus (ADV) ,respiratory syncytial virus (RSV) ,influenza A virus (INFA) ,influenza B virus (INFB) ,parainfluenza virus (PIVs) ,the results were analyzed statistically .Results A total of 1376 samples were detected at least one pathogen in 5918 serum samples ,the total positive rate was 23 .25% .The positive rate of MP was the highest (15 .19% ) ,followed by the INFB (8 .11% ) .The positive rates of other pathogens were relatively low .The mixed infection positive rate was 4 .29% .The positive rates of MP ,INFB and PIVs in women were significantly higher than those in men(P<0 .001) .The positive rates of MP ,INFB and PIVs were different in different seasons ,the differences were statistically sig-nificant(P<0 .001) .The positive rates of MP ,ADV and INFB in 0 to 14 years old group were significantly higher than those in >14 to 60 years old group and >60 years old group(P<0 .05) .The positive rates of MP ,CPn ,ADV ,RSV ,INFB and PIVs were closely related with age in infant and children (0 to 14 years old)(P<0 .05) .Conclusion The main respiratory pathogens of SARS in Shenzhen city were MP and INFB ,the 9 pathogens had their own infection characteristics and epidemic trend .