超声弹性量化分析肝纤维化联合靶点治疗效果的实验研究

The essence of liver fibrosis is the imbalance of extracellular matrix (ECM) synthetic and degradation. It is known that transformating growth factor beta (TGF-β) and ET-1 play an important role in the liver fibrosis. Reseach shows that TGF-βl/Smads signaling pathway is one of most important signaling pathways to adjust the ECM synthetic and degradation. At present gene therapy may provide novel treatment strategies for a wide range of liver fibrosis.TGF-β1/Smads signaling pathway is the key target to prevent and control the ECM formation by gene therapy. However, there is not a convenient noninvasive diagnosis method to find accurately and timely at the coures of liver fibrosis pathological changes. So it is necessary to find a noninvasive method to reflect pathological changes of liver fibrosis. Thus, we design a study to observe the effect of treatment on liver fibrosis in rats by TβRI short hairpin RNA plasmid transfection combined ET-1 receptor blocks as following three parts of experiments. Firstly, we select TβRI as the target interfered with TβRI short hairpin RNA plasmid. Secondly, In vitro, hepatic stellate cell (HSC-T6) is induced by TβRI short hairpin RNA plasmid in order to silence TβRI mRNA and ET-1 receptor blocking to observe the effect of gene transfection and the relationship between TβRI silence and MMPs/TIMPs expression. The aim is to investigate the feasibility of interrupting the TGF-β1/Smads signaling pathway with TβRI silence combined ET-1 receptor blocking. Further, we want to investigate whether ET-1 is the key factor of ECM synthetic and degradation at the TGF-β1/Smads signaling pathway. Thirdly, at the hepatic structure and blood serum elements, immunohistochemical expression and RT-PCR mRNA expression levels we explore the feasibility of liver fibrosis targeted treatment induced by TβRI short hairpin RNA plasmid combined ET-1 receptor blocks. Collagen I、Ⅲ expression are detected by immunohistochemistry. TGF-β1、TβRI、Smad2、MMP-1、TIMP-1 expression in the liver are detected by western blotting. Ultrasound elastography quantitative analysis is used in the stage of liver fibrosis. Our purpose is to provide reliable experimental evidences for the ultrasound elastography quantitative analysis evaluating the liver fibrosis effects by combined targeted treatment, which could be a novel method in gene therapy of liver fibrosis.

肝纤维化的实质是细胞外基质（ECM）代谢失衡。TGF-βl／Smads信号传导通路是调节ECM的重要途径。研究表明TGF-βl以及ET-1在肝纤维化行程中扮演重要角色。目前如何及时判断肝纤维化是其关键，有必要寻找一种无创伤能够反映肝纤维化程度的检查方法。本项目利用RNA干扰高效沉默靶基因的原理, 构建TβRI 短发夹状RNA质粒；选取TGF-β1/Smad信号通路及ET-1受体两个作用靶点，通过体外探讨TβRΙ沉默与MMPs/TIMPs表达的关系，研究TGF-β1与ET-1在ECM形成中的相互作用机制；通过大鼠肝纤维化模型综合采用组织结构观察、免疫组化蛋白表达、RT-PCR基因mRNA表达三种层面来确认TβRI shRNA抑制TGF-β1联合ET-1受体拮抗对ECM形成的影响；利用超声弹性成像技术量化评估联合靶点治疗大鼠肝纤维化作用的效果，为肝纤维化的治疗寻找一个新的检测手段提供理论依据。

肝纤维化的实质是由肝星状细胞分泌的细胞外基质（ECM）代谢失衡。TGF-βl／Smads信号传导通路是调节ECM 的重要途径。研究表明TGF-βl以及ET-1在肝纤维化行程中扮演重要角色，如果阻断其表达，肝纤维化行程可能会停止甚至逆转，如何及时判断肝纤维化是其关键，超声弹性成像是无创伤反映肝纤维化程度的检查方法。本实验合成TGFβRI短发夹状RNA质粒并转染肝星状细胞，ET-1拮抗剂波生坦处理肝星状细胞，ELASA、RT-PCR、WB、细胞免疫荧光等技术检测Tβ通路下游表达蛋白和肝纤维化相关蛋白。分别于2/4/6/8/10/12W时进行超声检测， 12W时为观测截止点，超声测量结束后大鼠麻醉进行门静脉穿刺测量门静脉压力，然后心脏釆血，留取大鼠肝脏标本进行肝纤维化病理检测，病理诊断肝纤维化程度。结果表明：1.与模型组比较，干扰TβR1以及联合阻滞通过smad通路能够影响I、III、MMP、TIMP、smad3等变化，可以有效减少ECM的分泌，与模型组比较,治疗组和双靶点治疗组纤维化程度显著减轻；2.实验组中门静脉内径有逐渐增加的趋势，但血流速度无明显变化 ；3.门静脉内径各组之间无显著性差异，模型组的门静脉内径略高于其它各组。4.门静脉压力测定显示实验组与对照组之间有显著性差异（p＜0.01），抗纤维化治疗可以显著降低门静脉压力，而且早期、联合治疗明显优于4W干预治疗（p＜0.05）。门静脉压力与纤维化程度呈正相关，肝脏弹性硬度测值也与门静脉压力呈正相关。.另外，肝脏损伤时伴随有脂代谢异常，脂代谢异常与血管病变有关，而血管壁硬度的改变是一项重要指标，血管硬度分析（QAS）技术对于早期血管壁硬度变化可灵敏显示，本实验应用声辐射力脉冲成像（ARFI）技术检测慢性病毒性肝炎患者的肝脏硬度，应用射频超声血管定量技术检测慢性病毒性肝炎患者的颈总动脉弹性变化，评价其血管功能，结果表明： CHB患者和CHC患者的颈动脉IMT较对照组无明显升高，但功能弹性性参数已发生变化，可评估其肝纤维化的程度和动脉结构和功能变化情况，为临床对病毒性肝炎患者血管病变的关注提供参考依据。.