Objective To study the effects of antisense oligodeoxynucleotides(ODN) of hEST2 (AODN) on telomerase activity and proliferation in ovarian cancer cell lines SKOV3 and COC1 Methods Antisense and sense human telomerse catalytic sub unit (hEST2) phosphorothioate (SODN)and random ODN were designed, synthesized and transfected into SKOV3 and COC1 cells by lipofectamine The expression of hEST2 mRNA and telomerase activity in SKOV3 and COC1 were tested by reverse transcription polymerase chain reaction and telomeric repeat amplification protocol before and after transfection The proliferation and growth in SKOV3 and COC1 were also investigated by methyl thiazolyl tetrazolium and growth curve before and after transfection Results AODN could down regulate the expression of hEST2 mRNA, inhibit telomerase activity and proliferation of ovarian cell lines The efficiency depends on dose and period of administration At 48 h, 30 ?mol/L AODN had the highest activity The expression of hEST2 mRNA were declined 54 6% and 44 6% in SKOV3 and COC1 respectively And also the inhibition of telomerase activity were 47 9% and 42 7% respectively in the two cell lines Conclusions AODN of hEST2 clearly inhibited the proliferation of ovarian cancer cell lines hEST2 may thus be a new target of gene therapy in ovarian carcinoma

Objective: To construct the packaging plasmid pSNAV-hEndostatin-CMV-EGFP of adeno-associated virus vector (AAV) encoding human Endostatin (hEndostatin) cDNA. Methods: The hEndostatin eDNA obtained from plasmid pCD-sEndostatin was subcloned into the packaging plasmid pSNAV of AAV by molecular clone ways. The recombinant plasmid pSNAV-hEndostatin-CMV-EGFP was identified by PCR analysis, restriction enzymes analysis and sequencing analysis.Results: The recombinant pSNAV-hEndostatin-CMV-EGFP was correctly constructed and confirmed by PCR analysis, restriction enzymes analysis and sequencing analysis. Conclusion: The constructed AAV-hEndostatin packaging plasmid pSNAV-hEndostatin-CMV-EGFP could be the packaging plasmid of rAAV-hEndostatin-EGFP.

Objective:To identify the relationship between expression of protein kinase R(PKR), phosphating PKR, EIF2α(p-PKR, p-EIF2α) in PKR→EIF2α signal transduction passage and the grades of cervical lesions, the role in generation and progression of cervical tumor and their effects to prognosis of cervical cancer patients. Methods:The expressions of PKR, p-PKR and p-EIF2α in human cervical cancer tissue of 63 cases, cervical intraepithelial neoplasia(CINⅠ-Ⅲ) of 114 cases and normal cervical epithelium of 15 cases were detected by immunohistochemical technique. Results:With the increase in grades of cervical lesions, the positive-expression rate of PKR increased and significantly correlated with the grades of cervical lesions(P < 0.05). With the increase in grades of cervical lesions, the positive-expression rates of p-PKR and p-EIF2α increased firstly, and then decreased. In cervical cancer group, the positive-expression rate of PKR was much higher than that of p-PKR(P < 0.01). The development and progression was quicker in later clinical stages of cervical cancer than that of earlier clinical stages of cervical cancer (P < 0.01). The development and progression of cervical cancer was quicker in patients with negative-expression of p-PKR and p-EIF2α than that in patients with positive-expression of p-PKR and p-EIF2α(P < 0.05). Conclusion:The positive-expression rate of PKR was correlated with the grades of cervical lesions. There are some factors which can impede PKR and EIF2α to be phosphorylated or make p-PKR and p-EIF2α dephosphorylate in high level cervical lesions, which promotes the development and progression of cervical lesions, worsens the prognosis of cervical cancer.

Objective To explore the relationship of signal transduction among human papillomavirus 18 E6 oncoprotein (HPV18E6), signal transducers and activators of transcription 1 (STAT1), protein kinase R( PKR )/α subunit of eukaryotic initiation factor 2 ( eIF2α ), nuclear factor-kappa Bp65 ( NF-κBp65 ), mitogen-activated protein kinase( MAPK)/c-Jun N-terminal kinase(JNK) ,and possible molecular mechanism. Methods Construct two lentiviral vectors which contain shRNA interfering sequence aiming at the targets of HPV18E6 oncogene and NC sequence( HPV18E6-RNAi-LV, NC-GFP-LV), based on the transduction with HPV18E6-RNAi-LV and NC-GFP-LV into HeLa cell to interfere the expression of HPV18E6 oncogene and NC sequence,the expressions of mRNA and protein( including phosphating patem)of HPV18E6, STATI, PKR, eIF2α, NF-κBp65, MAPK, JNK are measured with RT-PCR and Western blot, the difference of proliferation and sensitivity to carboplatin of HeLa cell are determined with Transwell cell methods and MTT among every groups. Results The expression of HPV18E6 oncogene can affect the expression level of mRNA and protein of NF-κBp65 and PKR genes, also affect phosphating levels of phosphating protein p-STAT1, p-PKR and p-eIF2α;the restraining rates of proliferation and sensitivity to carboplatin of HeLa cell are higher in HPV18E6-RNAi-LV group than the other groups( P＜0. 05 or P＜0.01 ). Conclusion HPV18E6 oncoprotein not only reduces the expression of PKR but dephosphorylates p-STAT1, pPKR and p-eIF2α to restrain activation of PKR/eIF2α signal transduction passage, maintain the proliferation and invading ability of HeLa cell and restrain apoptosis. The signal transduction among HPV18E6, MAPK/JNK are not clear.

Objective To investigate the roles of luteinizing hormone releasing hormone (LHRH), follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG) and ovary steroids in regulation of human endometrial stroma cell (ESC) decidualization Methods Stroma cells derived from human endometrium during the proliferative phase were cultured in vitro and treated with physiological doses of estradiol (E 2), testosterone (T),progesterone(P) or LHRH, FSH and hCG Their morphologic changes and prolactin (PRL) production in the media were examined and compared Results Addition of E 2 or T or P stimulated ESC proliferation, resulting in increase of the saturation density The fibroblast morphologic changes to polygonal shape and began to express PRL simutaneously after treatment of P or T P in presence of E 2 or T significantly enhanced PRL production( P

Objective To study the changes of DNA repair genes and enhanced anti-tumor effect of cisplatin induced by mifepristone in human ovarian cancer drug resistance cells.Methods The alterations of cisplatin concentration producing 50％inhibition(IC50)in the COC1/DDP cell lines were examined by methyl thiazolyl tetrazolium(MTT)assay.RT-PCR and flow cytometry were used to analyze the changes of the mRNA of ERCC1,BRCA1,hMLH1 genes and cell cycle and apoptosis.Subcutaneous implantation of COC1/DDP was established in nude mice and the enhanced anti-tumor effect of cisplatin by mifepristone was observed in vivo.ResultsCisplatin IC50 values of COC1/DDP cell were decreased from(3.71±0.38)μg/ml to(3.18±0.46),(1.95±0.14),(0.64±0,18)μg/ml respectively when treated with 2.5,5.0,10.0 μmol/L mifepristone.Mifepristone could down-regulate the mRNA levels of ERCC1,BRCA1,hMLH1 genes and enhance G0/G1 phase block effect pf cisplatin,and 2.5,5.0,10.0 μmol/L mifepristone combined with cisplatin increased rate of cell apoptosis from 0.08％to 5.11％,9.13％and 12.24％ respectively.The percentage of inhibition of xenograft tumor volume in combined treatment group was 70.1％,which was significantly different(P＜0.05).Conclusion By down-regulating ERCC1,BRCA1,hMLH1 genes,blocking G0/G1 phase,and increasing apoptosis rate,mifepristone could enhance anti-tumor effect of cisplatin.

Objective To Investigate the value of HPV genotyping in diagnosis of cervical intraepithelial neoplasia(CIN).Methods From July 2012 to February 2013,200 women from Tianjin Medical University General Hospital and 244 women from Affiliated Hospital of the Chinese People's Armed Forces Logistics College were selected to be HPV genotyping test and thin liqid-based cytology test.Consequently,132 samples were performed colposcopy test and cervical biopsy.Results HPV prevalence was 26.4％ (117/444) in this study.The infection of one type HPV was more common.The top 5 of HPV types were HPV16,58,33,18,and 52.The top 5 of the risk for CIN Ⅱ and above followed HPV16,33,39,52 and 18.There was no significant difference between age and HPV positive rate (x2 =0.948,P ＞ 0.05).Multiple infection and cervical lesions rank correlation analysis(r =0.132,P ＞0.05).For CIN Ⅱ and above disease,cytology positive rate was 90％ (44/49),and HPV positive rate was 96％ (47/49) cytology combine HPV positive rate was 98％ (48/49,x2 =0.063,P ＞ 0.05).Conclusions HPV infection should increasing trends with age.Cytology test and HPV genotyping test had good consistency.The combination of them can improve the sensitivity for high-grade lesions.

Objective:To investigate the effective method to avoid the ovarian hyperstimulation syndrome(OHSS)in infertile patients underwent in vitro fertilization-embryo transfer(IVF-ET)and whole embryo freezing and thawing 2-3 cycles after egg retrieval.Methods:The controlled ovarian hyperstimulation(COH),the number of retrieved oocytes,the number of embryo freezing,and clinical pregnancy rate were compared between 239 patients with the ET cycle(group A)and 164 patients with whole embryo freezing and thawing(group B).Results:The levels of initial dosage of gonadotropins(Gn)and total dosage of Gn were higher in group A compared with those of group B(P ＜ 0.01).The level of estradiol(E2)on day of human chorinonic gonadotrophin(hCG),the number of retrieved oocytes and the number of embryo freezing were lower in group A compared with those of group B(P＜ 0.01).There were no differences in patient age,COH,rate of OHSS and clinical pregnancy rate between group A and group B.Conclusion:Freezing the whole embryos and thawing ET 2-3 cycles after egg retrieval didn't influence the treatment results in infertile patients.

Objective: To analyze the influence of semen processing methods on the proportion of the aneuploid sperm by detecting the sperm's chromosome X, Y, 18 using fluorescence in situ hybridization. Methods: Ten patients with mild ohgoasthenosperia, who were received ICSI treatment, were included in this study. Five semen samples of the patients were randomly selected to detect using Swim-up method (A group) and 5 using sperm-grad double-density centrifugation method (B group).Another 5 patients with mild oligo-asthenosperia were as control (C group). The CEPX / Y and CEP18 probe was used to detect the sperm of these 15 patients by fluorescence in situ hybridization. The proportion of aneuploid sperm was compared in three groups. Results: The sex chromosome aneuploid rates were (4.21±2.49)%, (3.24～1.49) % and (2.62±0.89) % in control, A and B groups. The rates of aneuploid chromosome 18 were (3.00±1.22)%, (2.00～1.22)% and (2.00±1.22)% in control, A and B groups. There were no significant differences in three groups (P＞0.05). Conclusion: The results showed that the methods of Swim-up and Sperm-Grad double-density gradient centrifugation could select sperms in motility potential and teratospermia,but not in normal chromosome sperms.

Objective To study changes of cisplatin sensitivity by RNA interfering the excision repair cross-complementing (ERCC) 1 gene in ovarian cancer cell lines. Methods The small interference RNA (siRNA) targeting ERCC1 gene was designed and synthesized by transcription in vitro, and transfected to ovarian cancer cell line ES-2. The mRNA and protein of ERCC1 were evaluated by means of RT-PCR, western blot and immunocytochemistry. The changes of cisplatin sensitivity after interference were examined by methyl thiazolyl tetrazolium (MTT) assay. Results In ES-2 cell, the mRNA and protein levels of ERCC1 were dramatically decreased 24, 48 and 72 hours after transfection. The sensitivity to cisplatin of ES-2 cell line was increased by 53.88 times after disturbing the ERCC1 gene. Conclusion The sensitivity to cisplatin of ovarian cancer cell lines ES-2 could be enhanced by RNA interfering ERCC1 gene.