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Objective: To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system., Methods: A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis., Results: The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]., Conclusion: HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.

Objective: To investigate the effects and possible mechanisms of platelet-activating factor (PAF) on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy., Methods: (1)Serous type ovarian cancer cell line OVCA429 with platelet-activating factor receptor (PAFR) positive and mucinous type cell line RMUG-L (PAFR negative) were treated with 100 nmol/L of the PAF, cell invasion ability was determined by transwell cell migration assay. (2) For determination of the optimal PAF concentration, ovarian cancer cell OVCA429 was treated by 0, 0.1, 1, 10, 100, and 1000 nmol/L of PAF for 10 minutes or 24 hour, respectively. To observe the different time point of protein changes, OVCA429 were treated by 100 nmol/L of PAF for 0, 5 minutes, 10 minutes, 30 minutes, 1 hour or 12 hours, respectively. The total proteins of treated cells were extracted according to standard protocol. The expression of p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), transcription factor response element-binding protein (CREB), phosphorylated CREB (p-CREB) and matrix metalloproteinase-2 (MMP-2) were detected by western blot. (3) To verify the pathway involved in the PAF induction of the cancer cell invasion, we repeated the experiments by adding the inhibitors when treating cells with PAF. The inhibitors used were as follows, PAFR inhibitor-WEB2076 (50 µmol/L), p-p38 MAPK inhibitor-SB203580 (10 µmol/L), CREB binding protein (CBP)-CREB interaction inhibitor-217505(25 µmol/L). All treated cells were divided into 6 groups: control group, PAF group, PAF + WEB2076 group, PAF + SB203580 group, PAF + 217505 group and PAF + SB203580 + 217505 group., Results: (1) By transwell assay, 100 nmol/L of PAF could improve the invasion ability of OVCA429 cell significantly [PAF: (118 ± 23) cells vs. control: (36 ± 8) cells, P < 0.01], while the same treatment had no effect on RMUG-L cells [PAF: (45 ± 13) cells vs. control: (53 ± 9) cells, P > 0.05]. (2) Even a very low concentration of PAF (0.1 nmol/L) could increase the expression of p-CREB and MMP-2, while the most effective concentration of PAF was 100 nmol/L. The highest p-CREB protein expression was detected at 10 minutes after administration of 100 nmol/L PAF, as well as the expression of p-p38 MAPK protein. Even 12 hours after treatment the p-p38 MAPK protein could be detected, while there was no significant difference in the expression of CREB (P > 0.05). (3) As compared with PAF group, both in PAF + WEB2076 group and PAF + SB203580 group, the expressions of p-p38 MAPK, p-CREB and MMP-2 protein were decreased significantly; in PAF + 217505 group, although the expression of p-p38 MAPK and p-CREB protein was significantly higher than the control group, the expression of MMP-2 protein was significantly lower; in PAF + SB203580 + 217505 group, the expression of these three proteins were also significantly lower, but there was no significant difference as compared with that in the PAF + WEB2076 or PAF + SB203580 group., Conclusion: PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.

Objective: To observe the gene and protein expression of herpes simplex virus type I-thymidine kinase (HSV(1)-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV(1)-tk gene mediated by GE7 delivery system., Methods: GE7-polylysine/pCMV-HSV(1)-tk/polylysine-HA20 complexes were constructed. Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein, respectively. The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection. RT-PCR and Western Blot were preceeded to determine the expression of HSV(1)-tk gene and protein in the tumor tissues and other organs., Results: In the group of arterial injection with 4-element complexes, the HSV(1)-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs. In the group of arterial injection with saline buffer, no HSV(1)-tk gene and protein was detected in both tumor tissues and other organs. In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys., Conclusion: High target and gene transfer rates can be obtained when HSV(1)-tk gene is transferred via the artery route mediated by GE7 delivery system. HSV(1)-tk protein can be expressed after the gene transfer. The results may provide a new strategy for target killing of HSV(1)-tk/GCV system in ovarian tumors.

Objective: To establish a satisfactory lung metastasis model of human choriocarcinoma using severe combined immunodeficient (SCID) mice and explore the appropriate cell concentration for the model., Methods: Forty SCID mice aged between 5 - 6 weeks were randomly divided into four groups. 1 × 10(7) cells/ml × 0.1 ml, 5 × 10(6) cells/ml × 0.2 ml and 1 × 10(6) cells/ml × 0.1 ml of human choriocarcinoma cells JEG-3 were respectively injected in SCID mice of experimental groups by lateral tail vein, the remain group was assigned to the control group. The status and weight of mice were observed every three days. When these mice were being dying, the size and the number of the lesions of lung metastasis in every mouse were inspected with Micro CT. After Micro CT inspection, the SCID mice were executed dissected to note whether there were tumors on all organ surfaces with naked eyes, then made pathological sections from the metastatic foci of fresh lung tissues, and cultured primarily cells and purified cells and passaged cells isolated from the same metastastic foci. The pathological sections were observed under the microscope. The special antigen human chorionic gonadotropin-beta subunit (β-hCG) of the choriocarcinoma cells was immunohistochemically detected in the pathological sections and the cells out of cultured primarily cells. The chromosomes of the cells out of cultured primarily cells were analysed., Results: Of the group inoculated 1 × 10(7) cells/ml × 0.1 ml, all mice died when inoculating. In the group of 5 × 10(6) cells/ml × 0.2 ml, when inoculating, 3 mice died; the remain 7 mice were being dying on (18.0 ± 2.0) days after injection. 5 of them, there were 1 - 3 lesions of lung metastasis after Micro CT inspection in each mice, and the diameter of the tumors lesions reached 1.5 - 3.5 mm, which was choriocarcinoma confirmed by pathological sections. The special antigen β-hCG was detected by immunohistochemical method in the pathological sections of pulmonary tissue with tumor and in the cells, which were purified and passaged from being cultured primarily cells isolated from metastastic foci of fresh lung tissues from the SCID mice. The chromosome numbers of these cells out of cultured primarily cells were variety from 19 to 128, and modal numbers were variety from 70 to 79., Conclusions: We successfully established the lung metastatic model of human choriocarcinoma in SCID mice by injecting JEG-3 cells into lateral tail vein, of which 5 × 10(6) cells/ml × 0.2 ml is the suitable concentration and volume for the model.

Objective: To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer., Methods: Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK). By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B. The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency. The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR). The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay. The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line. The insertion sites of foreign gene transferred by PB transposon in genome were analyzed by inverse PCR., Results: (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully. (2) Using three different transfective reagents, PB transposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7+/-9.2)%] was higher than that of lipofectamine 2000 [(54.1+/-11.4)%] and jetPEI [(46.5+/-7.4)%, all P<0.05]; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfection efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7+/-9.2)%, (74.4+/-8.9)% and (83.2+/-9.7)% respectively, which all were higher than that on HEC-1B [(39.5+/-8.7)%, P<0.05]. (3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines. (4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 microg/ml respectively. Inhibitory effect of GCV (10 microg/ml) on SKOV3 transfected with pPB/TK was (86+/-9)%, which was superior to that transfected with pORF-HSVtk alone [(52+/-12)%, P<0.05]. (5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites. mRFP1 expression still could be detected in three months after transfected., Conclusions: PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression. It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

Objective: To establish the high-performance liquid chromatography (HPLC) method for measuring concentrations of methotrexate (MTX) in rat lung and some other tissues through internal iliac artery infusion., Methods: Fifty female Sprague-Dawley rats were included in this study. The rats were randomly assigned to two groups. Methotrexate was injected to group one through internal iliac artery, and was injected to group two through femoral vein. Blood and tissues were collected in each group at 15, 30, 60, 90 and 120 minutes for detection of the drug concentrations with HPLC., Results: The area under the concentration time curve (AUC) in rat lung, ovary and uterus in the artery group were separately (3.77 +/- 0.28), (4.40 +/- 0.40), (9.97 +/- 0.89) microgxh(-1)xg(-1), which were significantly different from those of the vein group [(2.31 +/- 0.25), (3.91 +/- 0.19), (7.65 +/- 1.54) microgxh(-1)xg(-1); P < 0.05]. The AUC in the rat plasma, heart, kidney, liver and spleen in the artery group were separately (6.13 +/- 0.53), (1.90 +/- 0.11), (5.32 +/- 0.89), (14.16 +/- 1.96), (0.76 +/- 0.20) microgxh(-1)xg(-1). There were no significant differences from the vein group [(5.79 +/- 0.71), (1.64 +/- 0.29), (5.15 +/- 1.69), (14.29 +/- 3.47), (0.76 +/- 0.13) microgxh(-1)xg(-1); P > 0.05]., Conclusions: Through internal iliac artery infusion, there are higher drug concentrations in lung, uterus and ovarian compared to venous injection. The internal-arterial chemotherapy may be used to treat pulmonary metastasis of gynecological tumor.

Objective: To prepare follicle stimulating hormone (FSH) polypeptide modified nanoparticles (NP) in order to achieve specific ovarian tumor targeting., Methods: Expression of FSH receptor protein in human liver cancer and ovarian cancer cell lines BEL-7402, SKOV-3 and Caov-3 was detected by immunocytochemistry. The polypeptide fragment of FSH beta 81-95 amino acids (FSHL81-95) was synthesized and covalently coupled to NP. The specific binding of FSHL81-95 and FSHL81-95-NP was examined by fluorescence microscopy and flow cytometry., Results: BEL-7402 and SKOV-3 cells were negative for FSH receptor staining, while Caov-3 cells were positive. The diameters of NP were about 100 nm, with a Zeta potential of -25 mV or so. Caov-3 cells showed a more specific interaction with FSHL81-95-NP than SKOV-3 cells (4.17 +/- 0.86 and 2.30 +/- 0.21; P < 0.05). The uptake of FSHL81-95-NP was more than NP in Caov-3 cells (4.17 +/- 0.86 and 0.41 +/- 0.32; P < 0.05). FSHL81-95-NP showed a selective targeting at Caov-3 cells compared with control NP., Conclusion: FSH polypeptide modified NP could selectively target ovarian cancer cells expressing FSH receptor, which might contribute to specific endocytosis mediated by FSH receptor.

Objective: To investigate the impact of danazol alginate microspheres used for uterine arterial embolization (UAE) on ovarian function and subsequent pregnancy using rabbit as a model., Methods: A total of 32 female rabbits were divided into 3 groups: a control group, danazol alginate microspheres (DKMG) group and alginate microspheres (KMG) group. Basal serum estradiol (E(2)), follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) levels before UAE and 1 - 3 months after UAE were compared for all rabbits. In breeding field all rabbits mated after UAE. Estrus, and pregnancy rate were observed by veterinary., Results: There were no significant changes from baseline FSH, LH, E(2), T levels measured at 1, 2 and 3 months after UAE (P > 0.05). The total pregnancy rate of DKMG or KMG group was 0 within 2 - 4 months after UAE. Compared to the control group (4/8), the difference was statistically significant (P < 0.05); the total pregnancy rate of DKMG, KMG and control groups within 5 - 7 months after UAE, respectively 17% (2/12), 25% (3/12) and 5/8 (P > 0.05); the total pregnancy rate was 42% (5/12), 50% (6/12) and 6/8 respectively within 8 - 10 months after UAE, there were also no significant differences between the three groups (P > 0.05)., Conclusions: There is no obvious effect of danazol alginate microspheres used for uterine arterial embolization on ovarian function in rabbits. After UAE some animals are able to achieve pregnancies, while harmful effects are observed on short term pregnant rate.

Objective: To study the anti-tumor immunotherapeutic effect induced by the suicidalcancer vaccine FC/TK, and to evaluate the safety of this vaccine., Methods: The suicidal cancer vaccine, named FC/TK, was prepared by fusion of suicide gene (HSVI,-TK gene) -modified ovarian carcinoma NuTu-19 cells with rat bone marrow-derived dendritic cells (DCs). The morphology of FC/TK was evaluated by scanning electron microscopy. The stimulatory effect of FC/TK on T cells was determined by T cell proliferation assay. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of FC/TK on days 7 and 14, compared to controls treated with irradiated FC/TK, FC or PBS, respectively. Tumor incidence and volume were measured in 90 days after challenge. To determine the killing effect of FC/TK in vivo, TUNEL assays were applied to detect apoptotic cell death in spleen of vaccinated rats with prodrug ganciclovir administration., Results: FC/TK cells were of irregular shape with surface membrane processes. Compared to the control groups, FC/TK significantly promoted T cell proliferation (P <0.01). The rats vaccinated with FC/TK and FC significantly inhibited the tumor growth compared to rats vaccinated with irradiated FC/TK (P <0.05) or with PBS ( P <0.01). The immunotherapeutic effect induced by FC/TK was similar to that using FC. Fluorescence microscopy showed that fluorescein-stained FC/TK cells migrated into spleen also showed to be TUNEL-positive, suggesting that the FC/TK cells were killed by ganciclovir in vivo., Conclusion: Our data indicate that suicidal cancer vaccine is an effective and safe therapy for ovarian carcinoma and may serve as a broadly applicable approach for other cancer vaccines in the future.

Objective: To explore possibility of vasculogenic mimicry induced by hypoxia and the inhibitive effect by sirolimus in epithelial ovarian carcinoma in vitro., Methods: Based on three-dimensional cell culture system developed by Matrigel, ovarian cell lines SKOV3 and ES2 were induced under conditions of hypoxia, hypoxia added with sirolimus and no-hypoxia, respectively. Potential formation of tumor channels and their characterization of network were observed by light microscopy and scanning electronic microscopy. Relative hypoxia-inducible factor (HIF) 1alpha mRNA expression was detected by RT-PCR simultaneously., Results: The micrograph showed both SKOV3 and ES2 cells appeared expanded and re-shaped, then formed blood vessel-like structures such as cavity, channel, branch and network. These capabilities of cells were inhibited by sirolimus and no-hypoxia. The levels of HIF-1alpha mRNA expression of SKOV3 and ES2 were 0.801 +/- 0.034 and 0.736 +/- 0.059 under hypoxia, which were significantly higher than under hypoxia added with sirolimus (0.025 +/- 0.007, 0.231 +/- 0.035; P < 0.01, P < 0.05), and those under no-hypoxia (0.010 +/- 0.004, 0.011 +/- 0.002; both P < 0.01)., Conclusions: Hypoxia plays a key role in development of vasculogenic mimicry in epithelial ovarian carcinoma. Sirolimus can inhibit vasculogenic mimicry effectively by blocking HIF-1alpha at transcription level.

Objective: To investigate the efficiency of antitumor immune responses induced by a controlled live dendritic cell (DC) vaccine., Methods: DC precursors were isolated from Fischer 344 rat bone marrow and cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4. The rat ovarian tumor cell line NuTu-19 was genetically modified by retroviral-mediated suicide gene (HSV(1)-TK), and the positive clones were selected using G418. Live DC vaccine was then fused with DC and NuTu-19/TK cell by polyethylene glycol. The characteristics of live DC vaccine were assayed with flow cytometry and confocal laser scanning microscopy. The specific expression of HSV(1)-TK gene in live DC vaccine was evaluated by RT-PCR and western blot. The sensitivity of live DC vaccine to ganciclovir (GCV) was evaluated by methylthiazoletetrazolium assay. In vivo, rats vaccinated twice with live DC vaccine were compared to those vaccinated with killed DC vaccine, unfused DC and NuTu-19/TK cell or phosphate buffered saline. Seven days following the last immunization, the rats were sacrificed to test the specific cytotoxic T lymphocyte (CTL) activity by lactate dehydrogenase release assay, or challenged with NuTu-19 and tumor incidence was observed., Results: The fusion efficiency was approximately (23 +/- 14). Live DC vaccine displayed an up-regulated expression of major histocompatibility complex (MHC)-IIOX6 (87.6 +/- 3.4)%, costimulatory molecule B(1 - 2) (71.1 +/- 9.3)%, integrin OX 62 (68.0 +/- 7.4)%, and adhesion ICAM-1 (77.1 +/- 2.0)%, and specifically expressed HSV(1)-TK gene. Our data showed that spleen T lymphocytes from rats vaccinated with live vaccine displayed enhanced CTL activity (61.8 +/- 8.3)% contrast to that of rats vaccinated with killed vaccines (26.0 +/- 3.8)% (P < 0.05). Compared to the control groups, rats immunized with live DC vaccine demonstrated a significant delay in tumor development [(39 +/- 8) d vs (70 +/- 16) d], reduced tumor incidence (100% vs 80%) and decreased tumor volume [(806 +/- 553) mm(3) vs (89 +/- 53) mm(3), P < 0.05]. Seventy-three percent of TK-transduced live DC vaccine was killed after 7 of GCV treatment by a functional assay., Conclusion: The results demonstrate that DC live vaccine modified by HSV(1)-TK gene can induce efficient protective immunity and be killed by GCV.

Objective: To investigate the efficiency and targeting of the GE7 system mediated gene transfer in the chemically induced ovarian tumor by intraarterial route., Methods: Animal models were chemically induced by 7,12-dimethylbenz[a]anthracene (DMBA). GE7-polylysine/beta-galactosidase (beta-gal)/polylysine-HA20 complexes were constructed. Fifteen rats with induced ovarian tumor were divided into three groups, the complexes were injected through ovarian artery and tail vein, respectively. The tumor, heart, liver, spleen, lung and kidney were obtained at 72 h. X-gal staining was used to check expression of beta-gal., Results: beta-gal was expressed strongly and well-distributed in tumor in the first group, and stained blue in the liver, spleen, lung and kidney, but much weaker than that of tumor. In the second group, beta-gal was expressed in the tumor, and weakly expressed in the liver, none was detected in the other organs. In the third group beta-gal was detected in the lung slightly, none was detected in the tumor and other organs., Conclusions: When GE7 complexes were administrated through ovarian artery, the introduced gene expressed preferentially in ovary. This result was the basis of intraarterial administration of therapeutic gene to treat ovarian cancer.

Objective: To investigate gene transfer efficiency of a novel target non-viral vector GE7 and effects of herpes simplex virus thymidine kinase (HSV(1)-tk)/ganciclovir (GCV) mediated by it in vitro., Methods: The epidermal growth factor receptor (EGF-R) target gene delivery system GE7 was constructed. Human ovarian cancer cell line CAOV3 was transfected in vitro with beta-galactosidase (beta-gal) as reporter gene and HSV(1)-tk gene as therapeutic gene using this gene delivery system. By means of the assay of X-gal staining, Northern blotting, cell growth-inhibiting curve and so on, the transferring efficiency of exogenous genes and killing effects are observed., Results: It showed that gene transfer efficiency is over 80%. When 10 mg/L GCV was put into ovarian cells transfected with HSV(1)-tk gene, 95% of cells were killed, and the apoptosis ratio reached up to 30., Conclusions: The GE7 gene delivery system is an effective and safe delivery system. GE7/HSV(1)-tk/GCV therapeutic gene system is appraising for ovarian cancer.