大鼠肾脏5/6切除microRN-483-3p的表达变化及机制

【摘要】 目的 探讨慢性肾脏疾病中microRNA-483-3p及其相关靶基因表达情况,明确过表达miR-483-3p的治疗效果。方法 大鼠分为对照组（假手术组,12只）,模型组（5/6肾切除组,12只）,miR-483-3p过表达组（5/6肾切除+ miR 483 3p Agomir,12只）3组。miR-483-3p Agomir给予60mg/kg尾静脉注射,每两周一次。末次术后8周处死大鼠。测定血生化指标,使用ELISA方法测血清中hs-CRP及24小时尿蛋白含量;采用RT-PCR方法进一步筛查出miR-483-3p的下游靶基因;用RT-PCR和Western Blot方法测定三组大鼠肾组织中miR-483-3p及其靶基因表达变化。同时RT-PCR方法测定miR-483-3p宿主基因TGF-2的表达变化。结果 与对照组相比,模型组BUN、Cr明显升高, TC及LDL-C亦显著升高。干预组较模型组BUN、Cr明显降低,血脂水平治疗前后无明显变化。hs-CRP及24小时尿蛋白定量在模型组明显升高,干预组显著降低。RT-PCR结果显示与对照组相比,模型组肾脏组织中CTGF和EGFR/PTEN表达显著升高,而TGF-β、DPC4/Smad4及AANAT无明显变化。干预组较模型组表达显著降低。模型组较对照组肾脏组织中IGF-2的表达明显升高,干预组肾脏组织IGF-2的表达较模型组表达显著降低。结论 慢性肾脏疾病,miR-483-3p表达增加,通过抑制其靶基因CTGF、PTEN的表达,可延缓肾脏纤维化过程。     

Renal targeting of kinase inhibitors

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.     

PPAR-γ激活抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞Ets-1表达

目的研究过氧化物酶体增殖物激活受体-γ(PPAR-γ)激活对心肌纤维化的影响是否与血管紧张素Ⅱ(AngⅡ)-Ets-1通路有关。方法体外培养大鼠心脏成纤维细胞(CFs),分为对照组、AngⅡ组、AngⅡ+不同浓度rosiglitazone处理组、AngⅡ+不同PPAR-γ激动剂组、AngⅡ+不同PPAR-γ拮抗剂组,采用实时定量RT-q PCR、Western blot等检测Ets-1、CTGF mRNA及蛋白表达,并测定TGF-β1和Smad2/3的表达及磷酸化水平。结果在CFs中,AngⅡ诱导Ets-1 mRNA及蛋白表达(P0.05),上调Ets-1下游靶基因CTGF的蛋白的表达(P0.05),增加TGF-β1和Smad2/3的表达及磷酸化(P0.05)。PPAR-γ激动剂rosiglitazone,15d-PGJ2抑制AngⅡ诱导的Ets-1 mRNA及蛋白的表达(P0.05),下调AngⅡ诱导的CTGF蛋白表达(P0.05),部分阻断AngⅡ诱导的TGF-β1的表达、Smad2/3的表达及磷酸化(P0.05)。PPAR-γ拮抗剂GW9662及BADGE均可阻断rosiglitazone对Ets-1及CTGF表达的抑制作用(P0.05)。结论 PPAR-γ激活主要通过TGF-β1/Smad2/3通路介导抑制AngⅡ诱导的大鼠CFs转录因子Ets-1的过表达。     

Comparison of TGF-β, PDGF,and CTGF in hepatic fibrosis models using DMN,CCl4, and TAA

Three chemotoxins including dimethylnitrosamine (DMN), carbon tetrachloride (CCl₄), and thioacetamide (TAA) are commonly used in hepatofibrotic models. We aimed to draw characteristics of histopathology and pro-fibrogenic cytokines including TGF-β, PDGF and CTGF among three models. Rats were divided into six groups and intra-peritoneally injected with DMN (10?mg/kg, for three weeks, three consecutive days weekly), CCl₄ (1.6?g/kg, for 10 weeks, twice weekly), TAA (200?mg/kg, for 12 weeks, twice weekly) or their corresponded treatment for each control group. The liver weights were decreased in DMN model, but not other models. Ascites were occurred as 3-, 2-, and 7-rats in DMN, CCl₄, and TAA model, respectively. The lipid peroxidation was highest in CCl₄ model, serum levels of liver enzymes were increased as similar severity. The hepatofibrotic alterations were remarkable in DMN and TAA model, but not CCl₄ as evidenced by the Masson trichrome staining and hydroxyproline. The immunohistochemistry for α-SAM showed that the DMN model was most severely enhanced than other models. On the other hand, hepatic tissue levels of pro-fibrogenic cytokines including TGF-β, PDGF, and CTGF were generally increased in three models, but totally different among models or measurement resources. Especially, serum levels of three cytokines were remarkably increased by CCl₄ injection and CTGF levels in both hepatic tissue and serum were highest in CCl₄ group. Our results firstly demonstrated comparative study for features of morphological finding and pro-fibrogenic cytokines in serum and hepatic protein levels among three models. Above results would be a helpful reference for hepatofibrotic studies.     

清燥救肺汤联合黄芪精口服液对放射性肺损伤干预作用及对CTGF PDGF表达影响的研究

目的:观察清燥救肺汤联合黄芪精口服液对局部中晚期胸部肿瘤放射治疗的肺保护作用及对结缔组织生长因子(CTGF)、血小板源性生长因子(PDGF)表达水平的影响。方法:将局部中晚期胸部肿瘤放射治疗者随机分为治疗组和对照组。治疗组在放射治疗开始起服用清燥救肺汤200mL、黄芪精口服液10mL,2次/d,连续6个月。放射治疗前后测定血浆CTGF和PDGF,放射治疗开始15天起观察临床症状、高分辨率CT和肺弥散功能。结果:治疗组放射治疗前、后血浆CTGF分别为(118.32±81.31)pg/mL、(173.37±109.87)pg/mL,对照组放疗前、后分别为(121.01±82.34)pg/mL、(307.87±175.45)pg/mL,对照组放疗后明显升高(P<0.001)。治疗组与对照组放疗后血浆PDGF分别为(29.01±7.12)ng/mL和(43.31±7.08)ng/mL,有显著差异(P<0.001)。放射治疗开始后5个月和10个月治疗组CO弥散量下降情况较对照组差异有统计学意义(P<0.05)。治疗组和对照组急性放射性肺炎和肺纤维化的发生率比较差异有统计学意义(P<0.05)。结论:清燥救肺汤联合黄芪精口服液能有效抑制放射治疗后血浆CTGF和PDGF的高表达,降低放射治疗后弥散功能的恶化,对放射性肺损伤有预防作用。     

六味地黄加味胶囊对糖尿病肾病大鼠肾脏蛋白激酶C活性及结缔组织生长因子的影响

目的观察六味地黄加味胶囊对糖尿病肾病(DN)大鼠肾脏的保护作用,对肾脏蛋白激酶C(PKC)活性及结缔组织生长因子(CTGF)表达的影响。方法ip链脲佐菌素(STZ)建立大鼠DN模型,模型成功后随机分为5组:对照组、模型组、洛汀新组、六味地黄加味胶囊组、洛汀新与六味地黄加味胶囊合用组,药物干预12周后,透射电镜观察各组大鼠肾脏超微结构,免疫组织化学法检查肾皮质CTGF表达,检测相对肾质量、血糖、尿白蛋白排泄率(UPER)、血肌酐(SCr)、尿肌酐(UCr)、内生肌酐清除率(CCr)、肾脏PKC活性等。结果DN大鼠肾脏胶原沉积明显,相对肾质量、血糖、UPER、CCr、肾脏PKC活性、肾皮质CTGF表达等显著升高,洛汀新、六味地黄加味胶囊及两药合用均可降低肾脏PKC活性和肾皮质CTGF的表达,改善DN大鼠蛋白尿、肾功能,减轻肾脏胶原沉积,两药合用能更显著降低肾脏PKC活性、肾皮质CTGF的表达,降低蛋白尿和改善肾功能作用更强。结论洛汀新、六味地黄加味胶囊及两药合用均能保护DN大鼠肾脏,且两药合用较单用洛汀新作用更强。     

急性白血病治疗前后骨髓中VEGF、CTGFmRNA水平的研究

目的 探讨血管内皮生长因子-C（VEGF-C）、结缔组织生长因子（CTGF）mRNA在急性白血病患者骨髓表达及治疗前后变化情况,研究其在白血病发生发展、浸润转移中的相互作用和临床意义。方法 71例初发急性白血病患者,对照组30例缺铁性贫血患者。收集研究对象骨髓液单个核细胞,应用Real-time PCR方法对VEGF-C及CTGF mRNA表达水平进行检测。结果 初发急性白血病患者骨髓中VEGF-C、CTGF mRNA表达明显高于对照组,两者呈正相关（r=0.709,P<0.05）。经1周期化疗后,达完全缓解患者及部分缓解患者与未治疗时比较,VEGF-C、CTGF mRNA表达水平明显降低（P<0.05）,未缓解患者与初发时比较,差异无统计学意义（P>0.05）。VEGF-C、CTGF mRNA的表达在急性白血病骨髓增生不同程度各组及是否伴髓外浸润各组之间比较,差异有统计学意义（P<0.05）。结论 VEGF-C、CTGF在急性白血病发生发展中具有协同作用,共同参与了白血病的发生、浸润及转移,并且与白血病预后相关。     

抗纤灵冲剂对单侧输尿管梗阻大鼠CTGF基因调控的影响

目的 探讨抗纤灵冲剂防治肾间质纤维化可能的作用靶点及分子生物学机制.方法 将72只SPF级SD大鼠中54只行经典单侧输尿管结扎,制备UUO大鼠模型,造模成功后分为模型组、抗纤灵组、氯沙坦组.药物干预后分批于14d,21d,28d处死,留取左侧肾组织,运用PCR Array法,检测肾间质纤维化基因CTGF的阳性表达.结果 各时间点模型组CTGF基因水平分别较假手术组上调7.12倍、8.26倍及6.55倍,均＞2倍,具有非常显著差异.各时间点抗纤灵组CTGF基因表达分别比模型组下调1.79倍、1.63倍、1.63倍,下调均＞1.5倍,具有明显差异.结论 抗纤灵冲剂作用靶点及分子生物学机制之一是有效诱导CTGF基因表达下调而发挥抗肾间质纤维化效应.