Effects of connective tissue growth factor gene-silencing on cell growth and cell cycle of hepatic stellate cells / 上海交通大学学报（医学版）

Objective • To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the expression of TGF-β1, Smad3 and Smad7 of rat hepatic stellate cell line HSCT6. Methods • The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructed by RNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The expression of green fluorescent protein (GFP) in the transfected HSCT6 cells was observed under fluorescence microscope. The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were tested by CCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzed by flow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the expression of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detected by real-time PCR and Western blotting, respectively. Results • The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfected by Ctgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P<0.05). The results of FCM revealed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was blocked in the S phase of cell cycle. The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene, down-regulated the expression of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the expression of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P<0.05). Conclusion • Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the expression of TGF-β1 and Smad3 and up-regulate the expression of Smad7. The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads (Smad3 and Smad7) signaling pathway.

Effects of recombinant human relaxin-2 on hepatic stellate cell proliferation, synthesis of Collagen Ⅰ,and mRNA expressions of TGF-β1 and CTGF in vitro / 西安交通大学学报(医学版)

ABSTRACT:Objective To validate that relaxin can resist hepatic fibrosis at the cellular level and explore its molecular mechanism in order to provide experimental basis for the treatment of liver cirrhosis.Methods Cultured HSC-T6s were treated with different concentrations (20,50 and 100 ng/mL)of recombinant human relaxin-2 (RLX-2).The proliferation of HSC-T6 was measured by MTT colorimetric assay.The content of type Ⅰcollagen in the cell culture supernatant of each group was detected by ELISA at 48 h of drug intervention;RT-PCR was used to detect the mRNA expressions of CTGF and TGF-β1 in HSC-T6 at 48 h of drug intervention.Results RLX-2 inhibited the proliferation of HSC and reduced type Ⅰ collagen content of HSC cells.It also inhibited the CTGF mRNA expression of HSC,but did not have a significant effect on the expression of TGF-β1 mRNA. Conclusion In the experiment of culturing HSC-T6 in vitro,RLX-2 may play a role in rat liver fibrosis by inhibiting cell proliferation and type Ⅰ collagen and CTGF mRNA expressions.

Effects of shRNA-CTGF on the expression of collagen-Ⅰ in keloid / 中华医学美学美容杂志

Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF (shRNA-CTGF) on the type Ⅰ collagen (COL-Ⅰ) protein expression in keloid,through RNA interference on connective tissue growth factor (CTGF) in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8％ and 54.1 ％,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8％ and 65.6％,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7％ and 48.0％,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar.

Renoprotective Effect of Yi-Shui Sheng-Xin Yin on Mechanism among Spontaneously Hypertensive Rats / 世界科学技术-中医药现代化

This study was aimed to observe renoprotective effect and possible mechanism on Y i-Shui Sheng-Xin Yin in spontaneously hypertensive rats. Sixty 12-week male SHR rats were randomly divided into six groups , which were the Y i-Shui She ng-X in Y in low-dose group , middle-dose group , high-dose group , Benazepril group , model group and blank control group , and ten rats for each group . The SHR rats were sacrificed after eight weeks . The urine microalbumin , blood urea nitrogen and cystatin were tested in each rat . The HE and Masson staining method were used to observe changes of renal pathology . Changes of expression of transforming growth factor-β1 ( TGF-β1 ) , connective tissue growth factor ( CTGF ) , FN were detected by immunohistochemistry . The results showed that compared with the blank control group , blood pressure in model group was associated with a significant rise after 8 weeks. Compared with the model group, blood pressure in the Yi-Shui Sheng-Xin Yin middle-dose group, high-dose group and Benazepril group significantly decreased. Compared with the blank control group , urine microalbumin , blood urea nitrogen and cystatin in model group were associated with a significant rise . Compared with the model group , urine microalbumin , blood urea nitrogen and cystatin in the Y i-Shui She ng-X in Y in middle-dose group , high-dose group and Benazepril group significantly decreased . Pathological examinations showed that pathological changes in model group were faster than all drug-groups , appeared pathological changes of glomerular hypertrophy , glomerular basement membrane thickening of heterogeneity and extensive vacuoles degeneration . Immunohistochemical staining showed that compared with the blank control group , expressions of TGF-β1 , CTGF and FN of rat kidney tissue in model group were obviously up-regulated ( P < 0 . 05 ) . Compared with the model group , expressions of TGF-β1 , CTGF and FN in the Y i-Shui She ng-X in Y in , middle-dose group , high-dose group and Benazepril group were down-regulated ( P < 0 . 05 ) . It was concluded that Y i-Shui She ng-X in Y in can reduce SHR rats' early renal glomerulosclerosis and renal interstitial fibrosis , which play roles of delaying the progress of hypertension and protecting kidney . Its mechanism of action may be related to TGF-β1 , CTGF , FN signal pathways .

The Effect of the Transcriptional Regulation of Sp1 for TGF-beta1 and CTGF Expression in Scar Formation

This study is to examine the relationship between TGF-b1 expression and CTGF expression, and to evaluate the effect of Sp1 blockade on the expression of TGF-b1, CTGF and extracellular genes, clones of fibroblasts stably transfected with Sp1 decoy ODN. R-Sp1 decoy ODN was highly resistant to degradation by nucleases or serum, compared to the linear or phosphorothioated-Sp1 decoy ODN. Skin wounds were created on the back of 36 anesthetized rats. They were divided into four groups-the rats with normal skin, with wounded skin without decoy, with wounded skin injected with R-Sp1 decoy, and with wounded skin injected with mismatched R-Sp1 decoy, respectively. Skins were collected at 3rd, 5th, 7th, 14th day after wounding. Cellular RNA was extracted by RT-PCR analysis. TGF-beta1 and CTGF were deeply related with skin fibrosis during scar formation and it appeared that TGF-beta1 may cause the induction of CTGF expression. R-Sp1 decoy ODN inhibited TGF-beta1 and CTGF expression both in cultured fibroblasts and in the skin of rats. These results indicate that targeting Sp1 with R-type decoy efficiently blocks extracellular matrix gene expression, and suggest an important new therapeutic approach to control the scarring in normal wound healing and fibrotic disorders.

Effects of simvastatin on myocardial fibrosis and connective tissue growth factor in renovascular hypertensive rats / 中国临床药理学与治疗学

AIM: To investigate the effects of simvastatin on myocardial fibrosis and connective tissue growth factor (CTGF) in renovascular hypertensive rats. METHODS: The experimental rats were randomly divided into three groups, such as sham operation group rats, hyperpiesia rats and hyperpiesia rats treated with simvastatin (5 mg㎏kg -1 ?d -1 ). Systolic blood pressure (SBP) was measured and collagen concentration (Coll) was also detected in terms of hydroxyproline concentration. VanGieson and immunohistochemistry staining combined with computed morphometry were used to evaluate the total collagen volume fraction (CVF) and CTGF expression in left ventricular tissue. RESULTS: Compared with sham operation group rats, the SBP,CVF, Coll and CTGF expression increased markedly in hyperpiesia rats (P

A Simple, Quantitative Method for Assessing Angiogenic Genes Using Skeletal Muscle by Electroporation-Mediated Naked DNA Delivery

BACKGROUND AND OBJECTIVES: For the development of an arteriogenic gene therapy in peripheral artery occlusive disease, we developed a novel angiogenesis assay, with electroporation-mediated naked DNA delivery to the skeletal muscle. MATERIALS AND METHODS: The levels of the expression CAT were compared between pJDK and pcDNA3.1, in HeLa and C2C12 cell lines, and skeletal muscle. The well known angiogenic gene, pJDK-hVEGF165, was injected, intramuscularly, into the tibialis anterior muscle of Balb/C mice, which was followed by electroporation. Two days later, the anterior tibialis muscles were divided into halves, embedded, and cultured in growth factor-reduced Matrigel. The capillary network area formed by the newly sprouting tube-like structures was calculated. For validation of this ex vivo assay, the connective tissue growth factor gene (pJDK-CTGF) was tested both by this new assay, and by the mice-hind limb ischemia model, with Laser Doppler imaging. RESULTS: The pJDK showed a significantly higher level of CAT expression than the pcDNA3.1. From the pJDK-hVEGF165 injected explants, endothelial cell migration and tube-like formation occurred on day 2, and the capillary network formation peaked on day 7. The capillary network formation in the pJDK-hVEGF165 group was markedly increased to that in the pJDK group. From the skeletal muscle assay, the pJDK-CTGF showed no angiogenic activity or attenuated VEGF-induced capillary network formation. The LDI flux ratio, on day 10 in the mice-hind limb ischemia model, for the mice treated with the pJDK-CTGF and pJDK-hVEGF165 was significantly lower than that of the mice treated with the pJDK-hVEGF165 alone. CONCLUSION: The skeletal muscle ex vivo assay, using an electroporation-mediated naked DNA delivery, is a simple, quantitative and reproducible method for assessing angiogenic genes. CTGF could be an anti-angiogenic factor due to its inhibition of VEGF.

Effects of Liuwei Dihuang Jiawei Capsula on renal protein kinase C activity and connective tissue growth factor of diabetic nephropathy rats / 中草药

Objective To investigate the protection of Liuwei Dihuang Jiawei Capsula(LDJ Capsula,Rehmanniae Capsula of Six Ingredients) on diabetic nephropathy(DN) rats′ kidney and effect on renal protein kinase C(PKC) activity and connective tissue growth factor(CTGF) of DN rats.Methods The DN rat models were induced by ip injection of streptozotocin(STZ).The rats were randomly divided into five groups: control group; DN model group;Lotensin group;LDJ Capsula group;and Lotensis and LDJ Capsula combination group.Drug intervention term was 12 weeks.Renal ultrastructure was observed by transmission electron microscope and Masson staining.Relative kidney weight,blood glucose level,serum and urine creatinine content,creatinine clearance,excretion rate of the 24 hour urine protein,renal PKC activity,and CTGF expression in renal cortex were measured by immunohistochemistry.Results Deposition of collagen in renal of DN rats was conspicuous.Relative kidney weight,blood glucose level,serum and urine creatinine content,creatinine clearance,excretion rate of 24 h urine protein,renal PKC activity and CTGF expression of DN rats increased obviously.All Lotensin,LDJ Capsula,and the combination of these two drugs could decrease renal PKC activity and CTGF expression and ameliorate proteinuria and renal function of DN rats.At the same time they all could abate the deposition of collagen in renal of DN rats.Combination of these two drugs could decrease renal PKC activity and CTGF expression more ob-viously and at the same time had more notable protective effect on kidney of DN rats.Conclusion All Lotensin,LDJ Capsula,and the combination of these two drugs could protect kidney of DN rats.The combination of these two drugs has more obviously protective effect than using Lotensin only.

Effect of electroacupuncture on learning and memory in diabetic rats and the expression of connective tissue growth factor in hippocampus / 西安交通大学学报(医学版)

Objective To observe the effect of electroacupuncture on improvement of learning and memory ability and the expression of connective tissue growth factor(CTGF)mRNA and protein in hippocampus in diabetic rats with cognitive impairment.Methods The rat diabetes model was induced by injecting streptozotocin(20 g/L),and then the rats were randomly divided into three groups:electro-acupuncture group(EA),diabetes-mellitus-untreated group(DM)and control group(CN).After four weeks of electroacupuncture treatment,blood glucose level was determined and the effect of electroacupuncture on learning and memory was examined with the device of Morris water maze.RT-PCR was used to detect CTGF mRNA level,and immunohistochemistry was used to detect CTGF protein expression.Results Blood glucose level and the latency period in DM group were increased compared with those in EA and CN groups(P