Objective To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer.Methods The four pairs ITIH4 gene siRNA interference fragments(ITIH4-546,ITIH4-795,ITIH4-917 and ITIH4-1568) were designed respectively,and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently.Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment.The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells.The stably transfected cells-pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418).The experiment was divided into three groups,namely ITIH4-917 transfection group,the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group),and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group).Fluorescence quantitative reverse transcription(RT)PCR and western blot were used to detect the ITIH4 mRNA and protein expression.The cell proliferation,the cell cycle,colony formation of cells,cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT),flow cytometry,colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)],respectively.Results The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells,the relative copy number was only 0.26 ± 0.15.Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ±0.10,it significantly lower than that in empty vector group (1.87 ±0.12,P =0.008) and negative control group (1.58 ±0.21,P =0.032) ; Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells,empty vector group and negative control group were 0.51,1.64 and 1.74,respectively,there were statistically significant differences (0.51 vs.1.64,P =0.012; 0.51 vs.1.74,P =0.014).MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group,and there were statistically significant differences among them (P =0.001).The S ± G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2％,which was significantly higher than that in the empty vector group or negative control group (26.3％ and 31.3％,respectively,all P ＜ 0.05).The colony formation rate (55.7 ± O.7) ％ in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ±0.9) ％ (P =0.037) and negative control group (31.4 ± 0.3) ％ (P =0.043).Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18,whicht was significantly higher than that in the negative control group or empty vector (0.30 ±0.03,P =0.031 ;0.25 ±0.03,P =0.028,respectively).Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ±0.34) was higher than that in the control cells (1.05 ±0.68) and empty vector group (1.14 ±0.08),while there were not significant difference (P ＞ 0.05).Conclusion It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.

Objective To assess the suppression effect on WWOX gene in SKOV3/SB cell line by small interference RNA (siRNA). Methods Transfection of siRNA using lipofeetamine 2000 was conducted to silence WWOX gene expression, the expression levels of WWOX mRNA and protein were evaluated,and the effects on the cell cycles at 48 hours of transfection were assessed by RT-PCR, western blot and flow eytometry (FCM) respectively. The cisplatin resistance index was assayed after transfection of SKOV3/SB with siRNA by methyl thiazolyl tetrazolium(MTT) and the cisplatin concentration of SKOV3/SB cells transfected with siRNA of WWOX was measured by high performance liquid chromatography. Results(1) In SKOV3/SB cells transfected with WWOX interference fragment, whether at the mRNA or protein level, the expression of both of WWOX decreased. There was a significant difference (P<0.05) compared with SKOV3 cells and non-transfected cells. (2) After transfecfion of the WWOX interference fragment, the index of platinum resistance of SKOV3/SB decreased from 5.04 to 3.89. (3) The number of cell transfected with the WWOX interference fragment in G1 phase was increased, while that in S-phase was decreased. (4) The cisplatin concenla'ation of SKOV3/SB cells transfected with the WWOX interference fragment was increased from 9.43 ng/L to 23.45 ng/L compared with SKOV3/SB cells non-transfected with a significant difference (P<0.05). Conclusion WWOX gene may be involved in cisplatin resistance phenomenon in epithelial ovarian cancer.

Objective To clone cathepsin L (CTSL) gene and construct the eukaryotic expression plasmid pcDNA3. 1-CTSL and study the relationship between CTSL and invasion and metastasis in ovarian cancer cells in vitro. Methods The total RNA was extracted from the ovarian cancer tissue and the intact cDNA of CTSL was applied by reverse transcription (RT)-PCR. The product of RT-PCR was cloned to pMD18-T vector, and subcloned to pcDNA3. 1 vector. It was tested by the enzymation and DNA sequencing.The eukaryotic expression plasmid of CTSL was introduced into HO8910 cells by liposome transfection reagent. RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells. Western blot was used to confirm the CTSL protein expression in positive clones cells. The cell growth curves, clonogenicity efficiency were observed. The cell cycles were measured by flow cytometer.The ability of invasion, metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay, transwell migration assay and adhesion assay, respectively. Results The results from restrictive enzyme analysis and sequencing showed that the CTSL gene was successfully inserted into pcDNA3. 1.Result from RT-PCR and western blot showed that the ovarian cancer cells which transfected by recombinant plasmid could express CTSL gene and protein. There was no difference between HO8910-CTSL and HO8910-pcDNA3. 1 cells in proliferation and adhesion ability (0.16±0.04 versus 0. 19±0. 04) of the cells (P>0.05). There was difference between HO8910-CTSL and HO8910-peDNA3.1 cells in matrigel invasion ability (0.34±0.18 versus 0.17±0.04) and metastasis ability (1.252±0.114 versus 0.486±0.027) of cancer(all P<0.05). Conclusion CTSL maybe increase the ability of invasion and metastasis of ovarian cancer cells in vitro, which may be a molecular target of blocking invasion and metastasis of ovarian cancer.

Objective:The aim of this study was to identify fibrinogen ( FG ) on the development of intimal hyperplasia ( IH ), using an organ culture model. Methods：Segments(n=9 ) of human saphenous vein ( HSV ) wereharvested during coronary artery or infrainguinal vein bypass surgery. The culture medium supplemented with FG (from0 mg/ml to 5 mg/ml ). The proliferation of smooth muscle cell ( SMC ) quantified by 5′-Bromodeoxyuridine (5′-BrdU) uptake in the final four days of the culture period. Histologic analysis and computerized morphometric analysis were used to determine intimal and medial thickness and area，then the intima/media thickness ratio and intima/media area ratio were calculated. Monoclonal antibodies to 5′-BrdU were used as an immunohistochemical maker for proliferating SMC. Results：Addition of FG ( 2.5 mg/ml ) to the cultured medium caused a significant increase in median ( range ) of intima/media thickness ratio and intima/media area ratio of these segments when compared with the normal cultured vein segments ( Wilcoxon paired rank test )：0.387versus 0.215（P=0.017 ）and 0.396 versus 0.229（P=0.015 ），respectively. Addition of FG ( 5.0 mg/ml ) to the cultured medium also caused a significant increase in median ( range ) of intima/media thickness ratio and intima/media area ratio of these segments when compared with the normal cultured vein segments: 0.421 versus 0.215（P=0.008 ）and 0.382 versus 0.229 （P=0.011 ），respectively. However，there were no significant differences in the two vein segments which 2.5 mg/ml or 5.0 mg/ml FG in cultured medium (P>0.05 ).In addition, there was no significant difference in the median ( range ) of intima/media thickness ratio and intima/media area ratio of the segments which FG ( 0.5 mg/ml ) in cultured medium when compared with the normal cultured vein segments ( P>0.05 ). These were supported by SMC proliferation index using staining with 5′-BrdU. Conclusion：High concentration FG at local preianastimotic area may an important factor for IH and early postoprative vein graft restenosis or occlusion.

Objective To explore whether or not multi-drug resistance could be reversed by RNA interference the expression of Topo Ⅱα gene in epithelial ovarian cancer cell lines in vitro. Methods (1) The best silent small interference RNA (siRNA) of Topo Ⅱα gene was designed and chose and cloned into psilencer4, 1-CMV-neo vector. The psilencer4. 1-CMV-neo-Topo Ⅱα was transfected into SKOV3/DDP cell, then Topo Ⅱα siRNA (+) SKOV3/DDP cells was incubated. (2) The Tope Ⅱα mRNA and protein expression of the stability-transfecting cell lines were detected by RT-PCR and western blot method, respectively. The resistance index, the cell cycle and the cellular content of cisplatin were detected by methyl thiazolyl tetrazolium assay, the flow cytometry and high performance liquid chromatography method before and after Topo Ⅱα RNA interference in cells. Results (1) The Topo Ⅱα gene expression level in SKOV3/DDP cells could be inhibited after the plasmid DNA psilencer4, 1-CMV-neo-Topo Ⅱα transfeced. The expression level of Tope Ⅱα mRNA in Topo Ⅱα siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0 and 0.92±0.08; the expression level of Topo Ⅱα protein in Topo Ⅱα siRNA (+) SKOV3/DDP and SKOV3/DDP cells were 0.51±0. 04 and 1.95±0.09 (P<0.01). (2) The multi-drug resistance index of Topo Ⅱα siRNA (+) SKOV3/DDP cell was significantly lower compared with that in SKOV3/DDP cell (3.46 vs 5.05, P<0.05). (3) The percentage of G_0/G_1 and G_2/M phase cell in Topo Ⅱα siRNA(+) SKOV3/DDP cells were higher than that in SKOV3/DDP cells (P<0.05). (4) The content of cisplatin in Topo Ⅱα siRNA(+)SKOV3/DDP cells treated with cisplatin for 24 hours was significantly higher than that in SKOV3/DDP cell (157.20 vs 63.99 ng, P<0.05). Conclusion The results showed that the tolerance of cisplatin would be reversed by blocking the Topo Ⅱα gene expression in cisplatin-resistant epithelial ovarian cancer cells.

Aim To synthesize 8-bromo-ethoxy Rhein and investigate its mechanisms and inhibition effect on hepatitis B surface antigen ( HBsAg ) and e antigen ( HBeAg) in HepG2.2.15 cells.Methods 8-bromo-ethoxy Rhein was synthesized based on the chemical structure of Rhein , and its structure was identified by IR,1 H-NMR and 13 C-NMR spectra.MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on HepG2.2.15 cells.After the cells treatment by 8-bromo-ethoxy Rhein , the HBsAg and HBeAg in cell supernatant were detected by ELISA .The expres-sion of hepatitis B virus X gene ( HBx) was detected by Western blot .The cell cycles were examined with flow cytometry.The intracellular free calcium concentration was detected by laser scanning confocal microscopy . Results The structure of 8-bromo-ethoxy Rhein was confirmed by IR,1 H-NMR and 13 C-NMR.MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in HepG2.2.15 cells.After trea-ted with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%( IC50 ) was 14.29 mg? L-1 and 11.59 mg? L-1 , respectively .Using non-cytotoxic dose of 8-bromo-ethoxy Rhein , the inhibitory effect on HBsAg and HBeAg was gradually enhanced with increasing 8-bromo-ethoxy Rhein concentration . The inhibitory effect of synthetic product on hepatitis B virus was better than that of Rhein .8-bromo-ethoxy Rhein could down-regulate the expression of HBx , in-tracellular calcium ion concentration and block the hepatitis B virus ( HBV ) replication.Flow cytometry results showed 8-bromo-ethoxy Rhein didn′t affect the cell cycle .Conclusions Compare with Rhein , the synthesis of 8-bromo-ethoxy Rhein shows stronger inhi-bition on hepatitis B virus in HepG2.2.15, and its mechanisms may involve down-regulating the expres-sion of HBx and reducing calcium ion concentration .

Objective To investigate the role of BRAFV600E mutation in diagnosis of thyroid nodules when it is inconsonant with cytological results. Methods This study included 9837 patients who underwent US-FNA. We mainly analyzed 239 cases with benign or indeterminate cytology, but having a detection of BRAFV600E muta-tion. BRAFV600E mutation analysis was performed using a Amplification Refractory Mutation System Polymerase Chain Reaction. Results In 93 nodules with benign cytology results but positive BRAFV600E mutation, 84 nodules were malignant. Based on the results, US-FNA combined with BRAFV600E mutation analysis will improve sensitivity (Se=94.03％)and negative predictive value (NPV=2.69％) of the thyroid nodules diagnosis than using US-FNA alone(Se=71.03％, NPV=20.76％). Conclusion BRAFV600E mutation analysis is an important tool in the diagnosis of PTC with high sensitivity and NPV. When facing patients with benign or indeterminate cytology but positive BRAFV600E mutation, thyroidectomy should be considered.

The previous pharmacokinetic methods can be only limited to drug analysis in vitro, which provide less information on the distribution and metabolismof drugs, and limit the interpretation and assessment of pharmacokinetics, the determination of metabolic principles, and evaluation of treatment effect. The objective of the study was to investigate the pharmacokinetic characteristics of gene recombination angiogenesis inhibitor Kringle 5 in vivo. The SPECT/CT and specific 131I-Kringle 5 marked by Iodogen method were both applied to explore the pharmacokinetic characteristics of 131I-Kringle 5 in vivo, and to investigate the dynamic distributions of 131I-Kringle 5 in target organs. Labeling recombinant angio-genesis inhibitor Kringle 5 using 131I with longer half-life and imaging in vivo using SPECT instead of PET, could overcome the limitations of previous methods. When the doses of 131I-Kringle 5 were 10.0, 7.5 and 5.0 g/kg, respectively, the two-compartment open models can be determined within all the metabolic process in vivo. There were no significant differences in t1/2α, t1/2β, apparent volume of distribution and CL between those three levels. The ratio of AUC(0 ? 1) among three different groups of 10.0, 7.5 and 5.0 g/kg was 2.56:1.44:1.0, which was close to the ratio (2:1.5:1.0). It could be clear that in the range of 5.0–10.0 g/kg, Kringle 5 was characterized by the first-order pharmacokinetics. Approximately 30 min after 131I-Kringle 5 was injected, 131I-Kringle 5 could be observed to concentrate in the heart, kidneys, liver and other organs by means of planar imaging and tomography. After 1 h of being injected, more radionuclide retained in the bladder, but not in intestinal. It could be concluded that 131I-Kringle 5 is mainly excreted through the kidneys. About 2 h after the injection of 131I-Kringle 5, the radionuclide in the heart, kidneys, liver and other organs was gradually reduced, while more radionuclide was concentrated in the bladder. The radionuclide was completely metabolized within 24 h, and the distribution of radioactivity in rats was similar to normal levels. In our study, the specific marker 131I-Kringle 5 and SPECT/CT were suc-cessfully used to explore pharmacokinetic characteristics of Kringle 5 in rats. The study could provide a new evaluation platform of the specific, in vivo and real-time functional imaging and pharmacokinetics for the clinical application of 131I-Kringle 5.

Objective: To investigate the role of BRAF^V600E mutation in diagnosis of thyroid nodules when it is inconsonant with cytological results. Methods: This study included 9837 patients who underwent US-FNA. We mainly analyzed 239 cases with benign or indeterminate cytology, but having a detection of BRAF^V600E mutation. BRAF^V600E mutation analysis was performed using a Amplification Refractory Mutation System Polymerase Chain Reaction. Results: In 93 nodules with benign cytology results but positive BRAF^V600E mutation, 84 nodules were malignant. Based on the results, US-FNA combined with BRAF^V600E mutation analysis will improve sensitivity (Se=94.03%) and negative predictive value (NPV=2.69%) of the thyroid nodules diagnosis than using US-FNA alone (Se=71.03%, NPV=20.76%) . Conclusion BRAF^V600E mutation analysis is an important tool in the diagnosis of PTC with high sensitivity and NPV. When facing patients with benign or indeterminate cytology but positive BRAF^V600E mutation, thyroidectomy should be considered.