OBJECTIVE: The study aimed to investigate the efficacy of intrauterine instillation of granulocyte colony-stimulating factor (G-CSF) on the day of ovulation triggering or oocyte retrieval in infertile women with a thin endometrium. METHODS: Fifty women whose endometrial thickness (EMT) was ≤8 mm at the time of triggering during at least one previous in vitro fertilization (IVF) cycle and an index IVF cycle were selected. On the day of triggering (n=12) or oocyte retrieval (n=38), 300 µg of G-CSF was instilled into the uterine cavity. RESULTS: In the 50 index IVF cycles, the mean EMT was 7.2±0.6 mm on the triggering day and increased to 8.5±1.5 mm on the embryo transfer day (p<0.001). The overall clinical pregnancy rate was 22.0%, the implantation rate was 15.9%, and the ongoing pregnancy rate was 20%. The clinical pregnancy rate (41.7% vs. 15.8%), the implantation rate (26.7% vs. 11.7%), and the ongoing pregnancy rate (41.7% vs. 13.2%) were higher when G-CSF was instilled on the triggering day than when it was instilled on the retrieval day, although this tendency was likewise not statistically significant. Aspects of the stimulation process and mean changes in EMT were similar in women who became pregnant and women who did not. CONCLUSION: Intrauterine instillation of G-CSF enhanced endometrial development and resulted in an acceptable pregnancy rate. Instillation of G-CSF on the triggering day showed better outcomes. G-CSF instillation should be considered as a strategy for inducing endometrial growth and good pregnancy results in infertile women with a thin endometrium.
Embryo Transfer ; Endometrium* ; Female ; Fertilization in Vitro ; Granulocyte Colony-Stimulating Factor* ; Granulocytes* ; Humans ; Oocyte Retrieval ; Ovulation ; Pilot Projects* ; Pregnancy ; Pregnancy Rate

Bovine embryos (day 5) were cultured to day 10 with or without 100 ng/mL PGF2α in medium supplemented with control; 100 nM Dex; 1,000 U/mL recombinant human leukemia inhibitory factor (rhLIF); or Dex+rhLIF. Although the rates to development to the blastocyst were not significantly different among groups, the hatching rate after additional culture with Dex +/or rhLIF was significantly higher in all supplemented groups than the control (p < 0.05). In the presence of PGF2α, the hatching rate was significantly restored in all supplemented groups relative to the group treated with only PGF2α and the control (p < 0.05). Embryo transfer (ET) was performed with blastocysts (day 7). PGF2α levels of control recipient cows were significantly higher in the circulatory blood samples collected 60 min after ET than in samples collected 60 min before ET (p < 0.005), and were decreased in cows injected with loading medium supplemented with Dex+rhLIF (p < 0.005). Pregnancy rate was significantly higher in the ET group that received supplemented embryo-loading medium than in the non-supplemented control (p < 0.05). The intrauterine administration of Dex and rhLIF at ET prevented increased PGF2α in circulatory blood and resulted in enhanced pregnancy rate.
Animals ; Blastocyst ; Cattle* ; Dexamethasone* ; Embryo Transfer* ; Embryonic Structures* ; Fertilization in Vitro ; Humans* ; Leukemia Inhibitory Factor* ; Leukemia* ; Pregnancy Rate* ; Pregnancy* ; Prostaglandins F

OBJECTIVETo evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer.

METHODSA total of 12 female New Zealand rabbits were used in this study. Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear. Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique. After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad-HGF group 1, Ad-HGF group 2, Ad-HGF group 3, Ad-GFP (a reporter gene) group and the solvent group. Immediately after surgery, 6 x 10(7) pfu Ad-HGF, 6 x 10(8) pfu Ad-HGF, 6 x 10(9) pfu of Ad-HGF, 6 x 10(9) pfu of Ad-GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively. One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad-GFP (6 x 10(9) pfu) into its wounds. Ice slides of wounds from this animal were observed under fluorescence microscopy. Another additional rabbit was used to evaluate the expression of HGF and TGFbeta1 after transferring Ad-HGF (6 x 10(9) pfu) into each of its wound. Immunohistochemistry was used for detection.

RESULTSThe effect of HGF on reducing excessive dermal scarring was observed by adenovirus-mediated gene transfer. Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over-expression of TGFbeta1, which plays an essential role in the progression of dermal fibrogenesis. Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring.

CONCLUSIONHGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing.

Animals ; Cicatrix ; prevention & control ; Female ; Gene Transfer Techniques ; Hepatocyte Growth Factor ; pharmacology ; Rabbits ; Random Allocation ; Wound Healing ; drug effects ; physiology

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNA^Gln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.
Humans ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Eukaryotic Initiation Factor-4A/genetics* ; Cell Line ; RNA, Transfer/metabolism* ; RNA ; Cell Proliferation

To investigate therapeutic efficiency of Ad/CMV- hTGF-beta1 gene for rabbit intervertebral disc degeneration model. 60 Japanese white rabbits were selected to form the 1.5-L6 Anterior-Lateral-Anulus-Fibrosus-Incision-Induced model in order to simulate human intervertebral disc degeneration. 36 rabbits, whose corresponding intervertebral discs were injected with 20 microl (10 x 10(6) pfu) of Ad/CMV- hTGF-beta1 gene, constituted the therapy group, 12 were injected with 20 microl (10 x 10(6) pfu)of Ad/CMV-LacZ gene as comparison group, while 12 were only injected with equivalent capacity of saline for empty comparison group, 3 weeks after injection, examples were taken for investigation of HE staining, MRI, Western Blotting and immunohistochemical research TGF-beta1. Wide distribution of TGF-beta1 was detected by immunohistochemical research in the degenerated annulus fibrosus after injection. Western Blotting research showed significant increase of TGF-beta1 content in intervertebral discs treated with TGF-beta1 gene than comparison groups. MRI signal transformed from low to comparatively high and that intervertebral disc pathological degree improved. Ad/CMV- hTGF-beta1 gene transfection is a potential method to increase TGF-beta1 content and reverse intervertebral disc degeneration.
Adenoviridae/genetics ; Gene Therapy ; Gene Transfer Techniques ; Genetic Vectors ; *Intervertebral Disk/pathology ; Lumbar Vertebrae ; Spinal Diseases/*drug therapy ; Transfection ; Transforming Growth Factor beta/*genetics ; Transforming Growth Factor beta1

OBJECTIVETo test the possibility of modification of human degenerative lumbar disc cells by the exogenous growth factor gene, transforming growth factor beta(1) (TGF-beta(1)) cDNA, and the expression of the encoded protein.

METHODSNucleus pulposus samples were surgically obtained from 8 patients with degenerative lumbar disc disease. The cells were cultured and directly infected by two adenoviral constructs, Ad/CMV-EGFP containing the enhanced green fluorecence protein (EGFP) gene (marker gene) and Ad/CMV-TGF-beta(1) containing the potentially therapeutic TGF-beta(1) gene. Transgene expression was analyzed by fluorescence production and immunohistochemical staining (Ad/CMV-TGF-beta(1)).

RESULTSCulture cells transduced by Ad/CMV-EGFP showed specific green fluorescence under the fluoroscope and expression sustained for at least 4 weeks. When infected by Ad/CMV-TGF-beta(1), approximately 30% of cultured cells were stained brown (+) with TGF-beta(1) staining.

CONCLUSIONThis study established the strategy of delivering a potentially therapeutic gene, TGF-beta(1), by using an adenoviral vector to human degenerative lumbar intervertebral disc cells.

Adenoviridae ; genetics ; Adult ; Female ; Gene Transfer Techniques ; Humans ; Intervertebral Disc ; cytology ; Lumbar Vertebrae ; Male ; Middle Aged ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

OBJECTIVE: To evaluate the clinical efficacy of serum insulin-like growth factor-I (IGF-I), IGF-II, and IGF binding protein-3 (IGFBP-3) levels in predicting the prognosis of in vitro fertilization and embryo transfer (IVF-ET). MATERIALS AND METHODS: In 84 patients undergoing IVF-ET, serum levels of IGF-I , IGF-II, and IGFBP-3 were measured using immunoradiometric assay (IRMA) before the gonadotropin administration and on the hCG day of controlled ovarian hyperstimulation (COH). Serum levels of IGFs and IGFBP-3, and the outcomes of IVF-ET were retrospectively analyzed and compared between the pregnant (n=18) and nonpregnant (n=66) groups. RESULTS: There were no significant differences in the outcomes of COH such as total dosage of gonadotropins used, duration of COH, serum estradiol (E2) level on the hCG day, numbers of oocytes retrieved and fertilized, and number of embryos transferred between the pregnant and nonpregnant groups. No differences were found in serum levels of IGF- I , IGF-II, and IGFBP-3, and their ratios before the gonadotropin administration and on the hCG day of COH. Basal serum level of IGF-II was lower with the borderline significance in the pregnant group (796.9+/-159.6 vs. 908.9+/-338.9 ng/ml, p=0.056). The ratio of change in IGF-I to that of IGF-II was significantly higher in the pregnant group (0.066+/-0.489 vs. -0.582+/-2.091, p=0.045). CONCLUSION: Even though basal serum level of IGF-II was lower and the ratio of changes in IGF-I to IGF-II was higher in the pregnant group, serum levels of IGF-I , IGF-II, and IGFBP-3 do not seem to predict the prognosis of IVF-ET. Further investigations are necessary in a larger group of patients to elucidate the clinical efficacy of serum IGFs and IGFBPs levels in predicting the prognosis of IVF-ET.
Embryo Transfer* ; Embryonic Structures* ; Estradiol ; Fertilization in Vitro* ; Gonadotropins ; Humans ; Immunoradiometric Assay ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor Binding Proteins ; Insulin-Like Growth Factor I ; Insulin-Like Growth Factor II* ; Oocytes ; Prognosis* ; Retrospective Studies

OBJECTIVE: To investigate the correlation between the concentrations of insulin-like growth factor-II (IGF-II), insulin-like growth factor binding protein-1, 3 (IGFBP-1, 3) in the follicular fluid and the cumulative embryo score (CES) in the patient who underwent in vitro fertilization and embryo transfer (IVF-ET). MATERIALS AND METHODS: A total of 21 cycles of 18 patients which underwent IVF-ET cycle after controlled ovarian hyperstimulation (COH) were included in this study. Using immunoradiometric assay (IRMA), we measured the concentrations of IGF-II, IGFBP-1, 3 in the follicular fluid. The patients were grouped into the pregnant and non-pregnant group. The concentrations of IGF-II, IGFBP-1, 3 in the follicular fluid were compared between the two groups and the correlations of the follicular concentrations of IGF-II, IGFBP-1, 3 and cumulative embryo score were evaluated. Results were analyzed with Mann-Whitney U test and Spearman's rank correlation coefficient and statistical significance was defined as p<0.05. RESULTS: There were no statistical significance in the follicular concentrations of IGF-II, IGFBP-1, 3 between the pregnant group and non-pregnant group. There were signifiant correlation between the follicular concentration of IGF-II and cumulative embryo score (p=0.001). There might be correlations between the follicular concentration of IGFBP-3, and free IGF-II and cumulative embryo score (p=0.053, p=0.056, respectively). CONCLUSION: The follicular IGF-II and free IGF-II might have an influence to development of good- quality embryos in patients undergoing IVF-ET.
Embryo Transfer ; Embryonic Structures* ; Female ; Fertilization in Vitro ; Follicular Fluid ; Humans ; Immunoradiometric Assay ; Insulin-Like Growth Factor Binding Protein 1* ; Insulin-Like Growth Factor Binding Protein 3 ; Insulin-Like Growth Factor II*

OBJECTIVEThe aim of this study was to investigate suppression effects of the transfection of human tumor necrosis factor-alpha (hTNF-alpha) gene on tongue carcinoma cells.

METHODSThe shuttle plasmid containing hTNF-alpha gene was extracted and purified, then it was transferred into Tca8113 tongue carcinoma cells with cationic liposome DOSPER. The control group was only given equivalent liposomes, except the plasmid. After culturing for 24, 48, 72 and 96 hours, the expression of hTNF-alpha gene in Tca8113 cells was analysed by ELISA and, the survival rate of transferred cells was assayed by MTT enzymatic labeling technique.

RESULTSThe transferred Tca8113 cells displayed significantly overexpression of hTNF-alpha (P < 0.05). The survival rate of the transferred Tca8113 cells was decreased significantly (P < 0.05).

CONCLUSIONTransfection of hTNF-alpha gene in vitro mediated by cationic liposomes can induce the overexpression of hTNF-alpha and inhibit the growth of tongue carcinoma cells.

Carcinoma, Squamous Cell ; genetics ; pathology ; Cell Division ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Plasmids ; genetics ; Tongue Neoplasms ; genetics ; pathology ; Transfection ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; genetics

To investigate the improving effect of inter-chain disulfide formation on protein trans-splicing, we introduce a Cys point mutation at Tyr(664) in heavy chain and at Thr(1826) in light chain of B-domain-deleted FVIII (BDD-FVIII). By co-transfection of COS-7 cell with the two Cys mutated chain genes, the intracellular protein splicing, inter-chain disulfide formation, secreted BDD-FVIII and bioactivity in culture supernatant were observed. The data showed that a strengthened spliced BDD-FVIII with an inter-chain disulfide detected by Western blotting and an elevated secretion of spliced BDD-FVIII (128 +/- 24 ng mL(-1)) compared to control (89 +/- 15 ng mL(-1)), assayed by a sandwich ELISA. A Coatest was performed to assay the secretion of bioactivity in culture supernatant and shown a much higher value (0.94 +/- 0.08 u mL(-1)) compared to that of control (0.62 +/- 0.15 u mL(-1)). It suggests that inter-chain disulfide formation could improve protein trans-splicing based dual-vector delivery of BDD-FVIII gene providing experimental evidence for ongoing in vivo study.
Animals ; COS Cells ; Cercopithecus aethiops ; Cysteine ; genetics ; metabolism ; Disulfides ; metabolism ; Factor VIII ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; Mutation ; Peptide Fragments ; genetics ; metabolism ; Protein Splicing ; Transfection