The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.
Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Connective Tissue Growth Factor/*physiology ; Corpus Luteum/growth & development ; Ovarian Follicle/growth & development ; Ovary/*metabolism ; Rats, Wistar

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

OBJECTIVEEstrogen is closely associated with hypospadias. The present study was to explore the molecular mechanism of hypospadias caused by estradiol.

METHODSFibroblasts obtained from the prepuce of hypospadiac and normal children were cultured in vitro and treated with 17-beta ethinyl estradiol (17-EE) at the concentrations of 1 micromol/L to 0.1 nmol/L for 2 hours, or at 0.1 micromol/L for 0.5, 1, 2, 4, 8, 16 and 24 hours. MTT assay was used to evaluate the effect of 17-EE on the proliferation of the cells, and RT-PCR was employed to detect the expressions of the activating transcription factor-3 (ATF3) and connective tissue growth factor (CTGF) in the hypospadiac tissue. The results were compared with those obtained from the nonhypospadiac tissue.

RESULTSThe expressions of ATF3 and CTGF were significantly upregulated in the hypospadiac tissue as compared with the nonhypospadiac group. At the concentration of 1 micromol/L, 17-EE significantly inhibited the proliferation of the cells. ATF3 mRNA was elevated at 1-2 hours, while CTGF mRNA showed no significant changes in 24 hours.

CONCLUSIONATF3 and CTGF are two candidate genes involved in the etiology of hypospadias. And estradiol may induce hypospadias by upregulating the expressions of ATF3 and CTGF.

Activating Transcription Factor 3 ; genetics ; metabolism ; Cells, Cultured ; Child ; Connective Tissue Growth Factor ; genetics ; metabolism ; Estradiol ; pharmacology ; Estrogens ; pharmacology ; Fibroblasts ; metabolism ; Foreskin ; metabolism ; Humans ; Hypospadias ; genetics ; metabolism ; Male

OBJECTIVETo explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid.

METHODSCTGF antisense oligonucleotides (ASODN) conjugated with isothiocyanate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblast (HKF) culturing media. The intracellular distribution of CTGF ASODN was observed with fluorescence microscopy in the fixed HKF. The proliferation of HKF was measured by MIT test. The apoptosis of HKF was measured with a flow cytometer. The collagen synthesis of HKF was measured by using H3-proline incorporation method.

RESULTSThe CTGF ASODN inhibited the proliferation and collagen synthesis of the HKF, compared with the control, but it increased the apoptosis after the transfection (P < 0.01).

CONCLUSIONCTGF ASODN may has anti-fibrotic effects on human keloid in vitro, and the CTGF may play an important role in promoting the fibrosis of human keloid.

Apoptosis ; Cell Differentiation ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; Fibroblasts ; cytology ; Humans ; Keloid ; metabolism ; pathology ; Oligonucleotides, Antisense ; genetics ; Transfection

OBJECTIVETo explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells.

METHODSThe expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection.

RESULTSCTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells.

CONCLUSIONSCTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effect of chemically synthetic small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) on the synthesis and secretion of extracellular matrix (ECM) in hepatic stellate cells (HSC).

METHODSChemically synthetic siRNA targeting CTGF was transfected into HSC T6 (an active HSC line in rats) by oligofectamine package, and untreated HSC T6 were used as control. Total RNA and protein of the cells, after their incubation with siRNA for 24, 48 and 72 hours, were extracted, and the supernatants were collected. The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot. Contents of hyaluronic acid and type III pro-collagen in the supernatants were determined by radioimmunoassay.

RESULTSThe expression of CTGF at mRNA and protein level and type I and III collagen at mRNA levels were markedly down-regulated in siRNA-transfected HSCs. The contents of hyaluronic acid and type III pro-collagen in the supernatants decreased by 46%+/-7%, 52%+/-7%, 53%+/-7% and 29%+/-18%, 29%+/-7%, 27%+/-5%, compared with those of the blank control at 24, 48 and 72 hours.

CONCLUSIONSChemically synthetic anti-CTGF siRNA can significantly inhibit CTGF gene expression in HSC, and markedly reduce the synthesis and secretion of ECM including type I and III collagen and hyaluronic acid. The siRNA-directed suppression of CTGF gene in HSC was maintained for 72 hours. This suggests that chemically synthetic siRNA may be a potential in preventing and treating liver fibrosis and may have a promising future for development

Cell Line ; Connective Tissue Growth Factor ; Extracellular Matrix ; metabolism ; Gene Targeting ; Humans ; Immediate-Early Proteins ; genetics ; Intercellular Signaling Peptides and Proteins ; genetics ; Liver ; cytology ; metabolism ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the anti-fibrogenesis property of intraportal vein small interfering RNA (siRNA) injection targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl4) and its effect on hepatic stellate cell (HSC) activation.

METHODS24 male rats were randomly divided into four group. rats received CCl4 by subcutaneous injections every three days for 6 consecutive weeks, and meantime they also obtained either siRNA targeting CTGF (as CTGF siRNA group), saline (as model group) or a control siRNA (as control siRNA group) by intraportal vein injection to rats liver at the same approach. Other rats received saline intraportal vein injection for 6 weeks (as normal control group). The expressions of CTGF and a-SMA protein were detected by Western blot. Hepatic histology was evaluated by HE staining and Sirius red staining. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. The number of active HSC were evaluated by immunohistochemistry.

RESULTSSix weeks after CCl4 injection, prominent upregulations were observed in the expressions of CTGF and a-SMA protein in saline or control siRNA-treated rats livers. In rats with CTGF siRNA treatment, the protein expressions of CTGF and a-SMA in liver decreased by 95%+/-2% and 86%+/-11% (F=21.234 and 12.473, P<0.01) respectively, the number of active HSC in liver decreased by 76%+/-9% (F=9.179, P<0.01) as compared to the model group. The attenuation of liver fibrosis was also observed in rats with CTGF siRNA treatment.

CONCLUSIONIntraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuate liver fibrosis by decreasing the number of active HSCs.

Animals ; Connective Tissue Growth Factor ; genetics ; Gene Silencing ; Hepatic Stellate Cells ; metabolism ; Liver Cirrhosis ; genetics ; metabolism ; pathology ; therapy ; Male ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo design and synthesize small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) and to investigate its effect on liver fibrosis.

METHODSThe interference sequence of CTGF was designed and synthesized. Rat hepatic fibrosis model was induced by intraperitoneal injection of 40 % CCl4(3 ml/kg). Thirty male rats were randomly divided into 5 groups: in normal control and model groups rats received tail vein injection of normal saline every 3 days for 8 consecutive weeks; in preventive group rats received tail vein injection of CTGF siRNA (0.1 mg/kg) every 3 days for 8 weeks; in 2-w treatment group CTGF siRNA was given for 6 weeks starting from two weeks after CCl4 injection; in 4-w treatment group CTGF siRNA was given for 4 weeks starting 4 weeks after CCl4 injection. The serum and hepatic tissue samples were harvested 3 days after the last CCl4 injection. Hepatic fibrosis indices were measured. Expression of CTGF mRNA and protein in the liver was evaluated by RT-PCR and Western blot, respectively. Fibrosis in rat liver was analyzed by Masson staining.

RESULTSCompared with model group (0.544 0.019), the expression of CTGF mRNA and protein in liver of both preventive(0.105 ± 0.003) and 2-w treatment groups (0.190 ± 0.006) were markedly down-regulated (P<0.05). Inflammation, necrosis and fibrosis in hepatic tissue were significantly attenuated. In addition, the serum ration of liver fibrosis indices was greatly reduced(P<0.05). Compared with preventive and 2-w treatment groups, the expression of CTGF mRNA and protein in liver in 4 weeks of treatment group were up-regulated (P<0.05); inflammation, necrosis and fibrosis in hepatic tissue were relative increased; and the serum concentrations of liver fibrosis indices were relatively higher (P<0.05).

CONCLUSIONThe highly effective CTGF siRNA has been successfully synthesized, which can inhibit CTGF expression in liver, prevent hepatic fibrosis and its progress in rats.

Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; genetics ; metabolism ; Genetic Therapy ; Liver ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; therapy ; Male ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the expression of connective tissue growth factor (CTGF) in colorectal cancer(CRC) and its association with clinicopathologic parameters and overall survival rate.

METHODSFresh tumor tissues and matched distal normal colon tissues were collected from 92 patients diagnosed as CRC by surgical operation. The expression level of CTGF mRNA was quantified by quantitative reverse transcription PCR. Thirty out of 92 pairs of tissue specimens were selected randomly to detect CTGF protein by immunohistochemistry. All the cases were followed up to identify prognostic factors for survival.

RESULTSCTGF mRNA expression was up-regulated in CRC. The positive rate of CTGF protein expression tissues (73.3%) was significantly higher than that in the corresponding normal tissues (23.3%, P<0.01). CTGF expression was lower in patients with lymphatic metastasis or stage III/IIII disease (all P<0.05). A negative association was also observed between the CTGF protein positive rate and tumor infiltration depth (P<0.05). The relative expression of CTGF mRNA in tumor tissues was classified into high and low expression groups. The 5-year cumulative survival rate was lower in patients with low CTGF expression (29.3%) as compared to those with high CTGF expressions (68.3%) (P<0.01). Cox regression analysis revealed that the relative expression level of CTGF was independent factor of overall survival (RR=2.960, 95%CI:1.491-1.587, P<0.01). ROC curve analysis showed that sensitivity and specificity of CTGF mRNA expression for prediction of 5-year survival were 64.9% and 74.5%, respectively.

CONCLUSIONSThe aberrant expression of CTGF is associated with the malignant biological behaviors of CRC. Low expression of CTGF is associated with worse prognosis of CRC.

Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; diagnosis ; metabolism ; pathology ; Connective Tissue Growth Factor ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; RNA, Messenger ; genetics

Objective To investigate the effect of microRNA-133b(miR-133b)on cardiac fibrosis and its mechanism.Methods Human cardiac fibroblasts(CFs)were harvested.The proliferation of CFs was detected by CCK8 during the overexpression and knock-down of miR-133b.The expressions of connective tissue growth factor(CTGF),α-smooth muscle actin(α-SMA),collagen Ⅰ,and collagen Ⅲ were detected with qRT-PCR and Western blot analysis after miR-133b overexpression or downexpression.Target genes of miR-133b were predicted by bioinformatics software.Dual-luciferase activity assay were used to verify a target gene of miR-133b.Results qRT-PCR showed that the expression level of miR-133b in the miR-133b mimic group was significantly higher than that in the negative control group(=26.219,=0.000).The expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that in the negative control group(=6.738,=0.003).After 21,45,69,93,and 117 hours of transfection,the proliferation ability of CFs significantly decreased in the miR-133b mimic group but significantly increased in the miR-133b group(all <0.05,compared with the negative control group).After overexpression of miR-133b,the mRNA and protein levels of CTGF(=9.213,=0.001;=8.195,=0.001),α-SMA(=6.511, =0.003;=4.434,=0.011),collagenⅠ(=3.172,=0.034;=4.053,=0.015)and collagen Ⅲ(=6.404,=0.003;=5.319,=0.006)were significantly down-regulated.After the expression of miR-133b was knocked down,the mRNA and protein levels of CTGF(=9.439,=0.001;=14.100,=0.000),α-SMA(=4.519,=0.011;=4.377,=0.012),collagen Ⅰ(=5.966,=0.004;=5.514,=0.005)and collagen Ⅲ(=4.622,=0.010;=4.996,=0.008)were significantly increased.The relative luciferase activity of the cells co-transfected with miR-133b mimic and WT 3'UTR expression vector was significantly lower than that of the cells co-transfected with mimic control and WT 3'UTR expression vectors(=5.654,=0.005);however,there was no significant difference in relative luciferase activity between cells co-transfected with miR-133b mimic and MUT 3'UTR expression vectors and cells co-transfected with mimic control and MUT 3'UTR expression vectors(=0.380,=0.724).Conclusion miR-133b may affect the activation and proliferation of CFs by targeting CTGF and thus improve cardiac fibrosis.
Actins ; metabolism ; Cell Proliferation ; Cells, Cultured ; Collagen ; metabolism ; Connective Tissue Growth Factor ; metabolism ; Fibroblasts ; cytology ; Fibrosis ; Humans ; MicroRNAs ; genetics ; Myocardium ; pathology