Hammerhead ribozyme targeting connective tissue growth factor mRNA blocks transforming growth factor-beta mediated cell proliferation

PURPOSE: Excessive scarring following trauma or surgery of cornea, conjunctiva or retina can greatly impair visual outcome. At present, no agents are clinically available that selectively reduce activity of genes that regulate fibrosis. Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues, including cornea and conjunctiva. In this study, hammerhead ribozymes targeting CTGF mRNA were synthesized, kinetic parameters were measured, and the effect on TGF-beta-mediated cell proliferation was measured in cultured human fibroblasts. METHODS: The mRNA sequence of human CTGF was scanned for potential hammerhead ribozyme cleavage sites, and predicted secondary folding structures around the sites were calculated. Synthetic 12mer ribozymes and 33mer oligonucleotide mRNA targets corresponding to two sites were synthesized, and kinetic constants calculated from Hanes-Wolff plots of in vitro cleavage reactions. The ribozyme with higher percentage cleavage and kinetic rate was cloned into an expression plasmid (pTR-UF21) and stably transfected into cultured human fibroblasts. An inactive ribozyme plasmid served as a negative control. The effects of the ribozyme on expression of TGF-beta-induced CTGF mRNA and protein levels were measured using ELISA and real-time TaqMan quantitative RT-PCR. Finally, the effect of the CTGF ribozyme on TGF-beta-mediated proliferation of fibroblasts was measured using a non-radioactive cell proliferation microtiter assay. RESULTS: Of the eight potential hammerhead ribozyme cleavage sites in human CTGF mRNA, two sites (CHR 745, and CHR 859) were identified with optimal secondary folding. CHR 859 cleaved 94% of the target mRNA, compared to 46% cleavage for CHR 745 after 16 hr of reaction. CHR 859 had a K(m) of 1.56 microM and a K(cat) of 2.97 min(-1), while CHR 745 had a K(m) of 7.80 microM and a K(cat) of 5.7 min(-1). The turnover numbers (K(cat)/K(m)) of CHR 859 and CHR 745 were 1.9 x 10(6) M min(-1) and 7.4 x 10(5) M min(-1), respectively, indicating CHR 859 is 2.6 times more efficient than CHR 745 in destroying CTGF mRNA. Stable transfection of CHR 859 into human fibroblasts reduced CTGF mRNA levels 55% and protein levels 72% compared to the inactive ribozyme control. Furthermore, TGF-beta-induced cell proliferation was reduced 90% in fibroblasts stably transfected with CHR 859 compared to control cell groups. CONCLUSIONS: The CHR 859 hammerhead ribozyme cleaved human CTGF mRNA with high kinetic efficiency in vitro, effectively reduced levels of CTGF mRNA and protein in cultured human fibroblasts, and blocked TGF-beta-induced cell proliferation without nonspecific toxicity. These data support the concept that CTGF mediates TGF-beta-induced cell proliferation, and imply that regulating CTGF expression with ribozymes may be effective in reducing ocular scarring.     

体外反搏对减轻高脂饲养猪产生动脉粥样硬化的实验研究

目的探讨体外反搏对减轻高脂饲养猪产生动脉粥样硬化的效果及机制。方法选雄性乳猪16头,用高脂喂养建立动脉粥样硬化模型,对模型猪进行36h体外反搏治疗。对腹主动脉作苏丹Ⅲ染色,分析脂质浸润阳性率;用基因芯片分析猪的动脉血管内皮细胞基因的表达。结果腹主动脉苏丹Ⅲ染色图像定量分析阳性率,高脂组为6.49%±0.84%,高脂加反搏组为2.29%±0.46%,两组差异有统计学意义(P<0.05)。发现改变的基因:芯片1(反搏组Cy3/高脂组Cy5):整合素β10.06、CTGF0.28;芯片2(反搏组Cy3/高脂组Cy5):整合素β10.62、CTGF0.39。结论体外反搏可以下调内皮细胞整合素β1、CTGF基因的表达,降低血管內皮细胞对脂质的摄取,起到抗动脉粥样硬化的作用。     

VEGF及CTGF的表达与脑胶质瘤分级的相关性研究

目的通过检测脑胶质瘤患者肿瘤组织中血管内皮生长因子和结缔组织生长因子的表达水平,探讨其和肿瘤级别的关系及作用机制。方法应用免疫组化SP法检测49例胶质瘤手术标本中的VEGF和CTGF的表达水平,统计分析表达水平和肿瘤级别之间的关系。结果VEGF和CTGF在胶质瘤高级别组（Ⅲ-Ⅳ级）中的表达均明显高于低级别组（Ⅰ-Ⅱ级）,说明随着肿瘤级别的升高VEGF和CTGF表达也增强。结论VEGF和CTGF在胶质瘤组织中高表达,而且表达水平和恶性程度有密切联系。VEGF和CTGF的检测可作为胶质瘤恶性程度判断的参考,为从基因水平上探讨胶质瘤的生物学行为、预后及治疗提供新的思路。     

Effect of connective tissue growth factor delivered via porous sutures on the proliferative stage of intrasynovial tendon repair

Recent growth factor, cell, and scaffold‐based experimental interventions for intrasynovial flexor tendon repair have demonstrated therapeutic potential in rodent models. However, these approaches have not achieved consistent functional improvements in large animal trials due to deleterious inflammatory reactions to delivery materials and insufficient induction of targeted biological healing responses. In this study, we achieved porous suture‐based sustained delivery of connective tissue growth factor (CTGF) into flexor tendons in a clinically relevant canine model. Repairs with CTGF‐laden sutures were mechanically competent and did not show any evidence of adhesions or other negative inflammatory reactions based on histology, gene expression, or proteomics analyses at 14 days following repair. CTGF‐laden sutures induced local cellular infiltration and a significant biological response immediately adjacent to the suture, including histological signs of angiogenesis and collagen deposition. There were no evident widespread biological effects throughout the tendon substance. There were significant differences in gene expression of the macrophage marker CD163 and anti‐apoptotic factor BCL2L1; however, these differences were not corroborated by proteomics analysis. In summary, this study provided encouraging evidence of sustained delivery of biologically active CTGF from porous sutures without signs of a negative inflammatory reaction. With the development of a safe and effective method for generating a positive local biological response, future studies can explore additional methods for enhancing intrasynovial tendon repair. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2052–2063, 2018.

     

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin

BACKGROUND & AIMS: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS: When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.     

姜黄素对肝纤维化模型大鼠肝组织PDGF-BB、CTGF的影响

目的:研究姜黄素对肝脏血小板衍生因子-BB(PDGF-BB)、结缔组织生长因子(CTGF)表达的影响,探讨其抗肝纤维化的作用机制。方法:采用CCl4皮下注射复制肝纤维化大鼠模型。实验分为正常对照(等容生理盐水)、模型(等容生理盐水)、姜黄素(200mg·kg-1)和复方鳖甲软肝片(625mg·kg-1)组,以免疫组化法检测各组大鼠肝组织PDGF-BB、CTGF阳性表达情况。结果:与模型组比较,姜黄素组大鼠肝组织PDGF-BB、CTGF阳性表达显著降低(P<0.05)。结论:姜黄素能够抑制肝组织PDGF-BB、CTGF的生成,从而发挥其抗肝纤维化的作用。     

丹芍化纤对肺纤维化大鼠肺内结缔组织生长因子表达的影响

目的：观察辛伐他汀对博来霉素引起的肺纤维化大鼠肺内结缔组织生长因子（CTGF）表达的影响。方法：选取SD大鼠30只,随机分为正常对照组（对照组）、肺纤维化模型组（模型组）和丹芍化纤胶囊治疗组（治疗组）。模型组和治疗组气管内注射博来霉素（5 mg/kg）诱导肺纤维化,对照组气管内注射等量生理盐水。次日起治疗组大鼠给予丹芍化纤混悬液（0.8 g/kg/d）灌胃,其余两组给予等量生理盐水灌胃。治疗第28 d处死各组大鼠,计算肺系数,HE染色观察肺组织病理变化,碱水解法检测肺组织羟脯氨酸含量,免疫组化ABC法观察肺组织CTGF表达的变化。结果：与对照组比较,模型组大鼠肺系数明显增加,肺组织胶原沉积明显,羟脯氨酸含量、CTGF表达增加。与模型组比较,治疗组大鼠肺系数明显降低,肺组织胶原沉积有所减轻,肺组织羟脯氨酸含量、CTGF蛋白表达减少。结论：丹芍化纤具有较好的抗大鼠肺纤维化作用,其作用机制部分是通过下调肺组织内CTGF的表达,从而延缓甚至抑制纤维化的进展。     

结缔组织生长因子在非小细胞肺癌中的表达及相关性研究

目的：观察结缔组织生长因子（CTGF）在非小细胞肺癌（NSCLC）组织和正常肺组织中表达的差异性,并初步探讨CTGF表达与NSCLC恶性程度、侵袭和转移的关系。方法：采用免疫组织化学法（S-P法）检测50例NSCLC和10例正常肺组织中CTGF的表达情况,并分析CTGF与NSCLC患者临床病理特征之间的关系,以及CTGF在NSCLC组织中表达的相关性。结果：在50例NSCLC组织中有15例（30%）CTGF呈阳性表达,10例正常肺组织中有9例为阳性表达;统计结果显示,CTGF表达与NSCLC的临床分期（P=0.005）、淋巴结转移（P=0.04）有相关性。结论：CTGF在NSCLC组织中的表达较正常肺组织明显下调,与NSCLC的临床分期和淋巴结转移有相关性,提示CTG可能与NSCLC的侵润和转移有关。