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Objective To study the effect of TGF?β1 gene therapy on the rat model of postpartum stress urinary incontinence and explore a novel non?operative treatment of this disease. Methods Two hundred and forty 6?month old SD female rats were used to prepare the model of postpartum stress urinary incontinence by vaginal dilation with a water sac. 148 rats from the 185 successfully prepared model rats were selected, and randomly divided into 5 groups: the TGF?β1 gene therapy, clentuterol treatment, electric stimulation therapy, injection of empty vector plasmid, and non?treated groups. In addition, 20 normal rats were selected as blank control group. Sneeze test and urodynamic test were conducted, the pelvic floor pubococcygeus muscle contractile force/muscle weight ratio was calculated, serum TGF?1 was detected by ELISA, and TGF?1 protein was detected by immunohistochemistry at 1, 21, 42 and 63 days after the treatment. Results At 21 days after treatment, all the maximum bladder capacity, leak point pressure, and urine or contractile force / muscle weight ratio of the TGF?β1 gene therapy group showed even better effects than those of the electrical stimulation group, but the differences were statistically not significant ( P>0?05 ) . Conclusions TGF?β1 gene therapy shows good therapeutic effect on the rat models of postpartum stress urinary incontinence, suggesting that TGF?β1 gene therapy may become a new type of non?surgical treatment for this disease.
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Objective To study the effect of IGF-1 gene therapy and electric stimulation therapy on the rat models of postpartum stress urinary incontinence, and explore the ideal treatment for this disease.Methods 240 SD female rats were used to establish the model of postpartum stress urinary incontinence by water sac vaginal dilation.148 model rats were randomly selected from 185 successful models and divided into 5 groups:IGF-1 gene therapy, clenbuterol treatment, electric stimulation therapy, injection of empty vector plasmid, and untreated groups.Besides, 20 non-modeled rats were used as blank control group.Urodynamic test was performed, pelvic floor pubococcygeus muscle/muscle weight ratio was calculated, and serum biochemical indices (LDH, CK) were detected, and the morphological changes of pubococcygeus muscle fibers were observed by light microscopy at 1, 21, 42 and 63 days after treatment.Results At 21 days after treat-ment, the maximum bladder capacity, leak point pressure, the contractile force/muscle weight ratio in the IGF-1 group and electric stimulation treatment group were significantly better (P>0.05), and the differences between the IGF-1 group and electric stimulation group were not significant ( P>0.05 ) .Conclusions The effect of IGF-1 gene therapy and electric stimulation on the rat models of postpartum stress urinary incontinence is better than that in the drug therapy group and oth-er groups.
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Objective To establish a satisfactory lung metastasis model of human choriocarcinoma using severe combined immunedeficient(SCID)mice and explore the appropriate cell concentration for the model.Methods Forty SCID mice aged between 5-6 weeks were randomly difided into four groups.1×107 cells/ml ×0.1 ml.5×106 cells/ml×0.2 ml and 1×106 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 weisht 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 witll naked eyes.then made pathological sections from the metastaticfoci 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 choriocareinoma 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 inoeutated 1×107 cells/ml×0.1 ml.all mice died when inoculating.In the group of 5×106 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 ram, which was choriocarcinoma confirmed by pathological sections.The special antigen β-hCG was detected by immunohistoehemical 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 ceils were variety from 19 to 128, and medal 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 × 106 cells/ml × 0. 2 ml is the suitable concentration and volume for the model.
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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 tranferred 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 trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, 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 transfeetion 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 μg/ml respectively.Inhibitory effect of GCV (10 μg/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.