AAV9-hIGF-1对mdx小鼠炎症反应的影响及机制研究

发布时间：2018-06-09 17:25

本文选题：DMD + AAV9　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:Duchenne型肌营养不良症(Duchenne muscular dystrophy,DMD)是进行性肌营养不良症中最常见的致死性X连锁隐性遗传肌病。该病是由于dystrophin基因发生了突变,使其编码的抗肌萎缩蛋白的完全或部分缺失,导致肌纤维膜完整性、稳定性破坏,最终导致肌肉的变性、坏死。DMD的主要临床表现为进行性肌肉无力、肌萎缩和腓肠肌假性肥大,随着疾病进展,DMD患者通常于12岁左右丧失行走功能,20岁左右因呼吸或心力衰竭死亡。DMD患者肌肉的主要病理特征为肌纤维直径大小不等,可见肌纤维肥大、萎缩、变性和坏死,炎性细胞浸润,肌纤维最终脂肪化和纤维化。然而dystrophin蛋白的缺失所致的肌细胞膜的不稳定性及机械损伤并不能完全解释DMD骨骼肌的持续性损害,在DMD的超早期阶段甚至在肌纤维的坏死发生之前,就已经存在炎性信号通路的异常激活。近年来,越来越多研究表明炎性反应及炎性通路的活化在DMD的发生和疾病进展中发挥着极其关键的作用。对炎性反应这一重要靶点的调节是DMD治疗极具前景的治疗方法。C57BL/10ScSn-Dmdmdx/JNju鼠,简称mdx小鼠,是由于C57BL/10小鼠X染色体的23号外显子发生了点突变,导致其编码的dystrophin功能缺失而构建的DMD模型鼠,是目前研究DMD应用最广泛的动物模型。基因治疗是将正常的基因或有治疗作用的基因通过一定方式导入靶细胞,以纠正基因的缺陷或者发挥治疗作用。腺相关病毒载体(Adeno-associated virus,AAVs)是新近发展最为常用且具有临床应用前景的基因载体之一。AAVs可以长期高效表达,具有对机体低毒性、低细胞免疫源性和低致病性等优势。系统性评估表明AAV9可以有效地转染至骨骼肌和心肌。胰岛素样生长因子-l(insulin-like growth factor 1,IGF-1)是细胞增殖、分化和成熟过程中的重要营养因子。已有研究表明肌肉中特异性表达IGF-1基因可以增加mdx小鼠的肌纤维的再生,降低血清CK值且一定程度上改善肌力,这些效应是通过激活P13K/Akt信号通路实现的。IGF-1还可以从调节炎症反应方面作用于骨骼肌,有研究指出IGF-1是通过抑制巨噬细胞移动抑制因子(macrophge migration inhibitory factor,MIF)、高迁移率族蛋白B(high mobility group protein-1,HMGB1)和核转录因子kappa B(nuclear factor-kappa B,NF-κB)的活性起到抗炎作用。之前的研究多集中在IGF-1对肌纤维再生的影响,很少关注IGF-1对肌肉炎症的作用。考虑到DMD是一种累及全身肌肉的肌病,尾静脉注射的方式更适宜临床应用。我们的研究是利用AAV9作为载体,携带hIGF-1基因一次性经尾静脉注射给予6周龄的mdx小鼠治疗。6周后通过常规组织病理染色,免疫荧光和蛋白免疫印迹(Western blot)技术评估mdx小鼠的炎症反应,并进一步探究hIGF-1的作用机制。方法:选取周龄6周的mdx小鼠,随机分为实验组(AAV9-hIGF-1组)和实验对照组(AAV9-GFP组),正常对照组为选取同等周龄的C57/10野生型小鼠,每组5只小鼠。给予实验组mdx小鼠尾静脉注射AAV9-hIGF-1病毒200μl(1×1012vg/ml),实验对照组mdx小鼠尾静脉注射AAV9-GFP病毒200μl(1×1012vg/ml),各组小鼠接受病毒尾静脉注射后同等条件下观察6周后取材。小鼠麻醉后分别剪取骨骼肌和心肌,进行包埋冰冻处理。荧光显微镜下观察肌肉组织中AAV9在mdx小鼠的转染情况后,观察hIGF-1蛋白的表达。HE染色、ACP染色观察炎症反应的变化。检测炎症相关蛋白CD68,PP65的表达。结果应用spss13.0对数据进行分析。结果:1 AAV9在mdx小鼠体内的表达mdx小鼠不同组织中GFP均有所表达,其中胫骨前肌GFP表达最多,肱三头肌表达次之,余组织仅可见较少量表达。2 AAV9-hIGF-1在mdx小鼠胫骨前肌的表达AAV9-hIGF-1组mdx小鼠胫骨前肌hIGF-1的表达效率较高,但仍有部分肌纤维未见表达。3 AAV9-hIGF-1对mdx小鼠肌肉的炎症反应的影响HE染色和ACP染色观察到AAV9-GFP组可见散在分布的吞噬现象,数个融合成片的炎细胞浸润,AAV-9hIGF-1组炎细胞浸润较分散,可见少量小灶状炎细胞浸润。CD68是巨噬细胞可靠的标记物,通过免疫荧光染色观察到AAV9-hIGF-1组CD68+细胞较AAV-GFP组减少。对胫前肌炎症区域所占百分比进行统计学分析,AAV-hIGF-1组为(1.78±0.47%),低于AAV-GFP组(3.4±1.22%),高于正常组(0%),差异均有统计学意义(p0.05)。4 NF-κB信号通路的变化运用Western-bloth观察到AAV9-hIGF-1减少了PP65的表达,AAV-hIGF-1组为(0.45±0.07%),低于AAV-GFP组(0.76±0.13%),高于正常组(0.38±0.06%),差异有统计学意义。结论:1通过尾静脉注射,AAV9能够在mdx小鼠的多部位表达,其中胫前肌中表达最多。2 AAV9-hIGF-1尾静脉注射治疗能减轻了mdx小鼠肌肉的炎症反应。3推测hIGF-1是通过下调NF-κB信号通路起到抗炎的作用。
[Abstract]:Objective: Duchenne muscular dystrophy (DMD) is the most common fatal X linked recessive myopathy in progressive muscular dystrophy. This disease is due to the mutation of the dystrophin gene, which encodes the complete or partial deletion of the amyotrophic protein, resulting in the integrity of the myofibrillar membrane, and the stability of the muscular dystrophy. The main clinical manifestations of.DMD are progressive muscle weakness, muscular atrophy and pseudohypertrophy of the gastrocnemius. As the disease progresses, the DMD patients usually lose the walking function around 12 years of age, and the main pathological features of the muscles of the patients with respiratory or heart failure at about 20 years old are the muscle fiber diameter. See muscle fiber hypertrophy, atrophy, degeneration and necrosis, inflammatory cell infiltration, muscle fiber final adipose and fibrosis. However, the instability of the myoblast and mechanical damage caused by the deletion of dystrophin protein can not fully explain the persistent damage of the DMD skeletal muscle, even before the necrosis of the muscle fibers in the ultra early stages of DMD. There has been an abnormal activation of inflammatory signaling pathways. In recent years, more and more studies have shown that inflammatory responses and inflammatory pathways play a crucial role in the development of DMD and the progression of the disease. The regulation of the important target of inflammatory response is a promising treatment for DMD,.C57BL/10ScSn-Dmdmdx/JNju rats, for short, mdx Mice, due to the point mutation of the exon 23 of the X chromosome of the C57BL/10 mouse, which lead to the deletion of the encoded dystrophin function, is the most widely used animal model in the study of the DMD application. Gene therapy is to import the normal gene or the therapeutic basis into the target cells in a certain way to correct the gene. Adeno-associated virus (AAVs) is one of the most commonly used and clinically promising gene vectors,.AAVs, which can be expressed in a long and efficient way. It has the advantages of low toxicity, low cellular immunity and low pathogenicity. Systematic assessment shows that AAV9 can be effective. Transfected into skeletal muscle and myocardium. Insulin like growth factor -l (insulin-like growth factor 1, IGF-1) is an important nutrient factor in cell proliferation, differentiation and maturation. Studies have shown that the specific expression of IGF-1 gene in muscle can increase the rebirth of muscle fibers in mdx mice, reduce the value of serum CK and improve muscle strength to a certain extent, These effects, which are achieved by activating the P13K/Akt signaling pathway, can also be used to regulate the inflammatory response in the skeletal muscle. There is a study that IGF-1 is through the inhibition of the macrophage migration inhibitory factor (macrophge migration inhibitory factor, MIF), the high mobility group of egg white B (high mobility group) and nuclear transcription. The activity of factor kappa B (nuclear factor-kappa B, NF- kappa B) plays an anti-inflammatory role. Previous studies focused on the effect of IGF-1 on muscle fiber regeneration, and little attention was paid to the effect of IGF-1 on muscle inflammation. Considering that DMD is a myopathy involving whole body muscles, the formula for the injection of the tail vein is more suitable for clinical application. Our study uses AAV9. As a carrier, the mdx mice of 6 weeks of age with hIGF-1 gene were injected into the tail vein for.6 weeks to be treated with routine histopathological staining, immunofluorescence and protein immunoblotting (Western blot) technique to evaluate the inflammatory response of mdx mice, and to further explore the mechanism of the action of hIGF-1. Methods: the mdx mice of 6 weeks of age were selected and randomly divided. In the experimental group (group AAV9-hIGF-1) and the experimental control group (group AAV9-GFP), the normal control group was selected as the same week old C57/10 wild type mice, with 5 mice in each group. The experimental group was given the tail vein of the mdx mice with AAV9-hIGF-1 virus 200 L (1 x 1012vg/ml), and the experimental control group was injected with AAV9-GFP virus 200 mu L (1 x 1012vg/ml) in the tail vein of the experimental control group. Each group was small. The mice were treated with the same condition after the tail vein injection of the virus for 6 weeks. The mice were harvested for 6 weeks. The mice were cut into the skeletal muscle and the myocardium to be frozen. The transfection of the mdx mice in the muscle tissue was observed under the fluorescence microscope. The expression of hIGF-1 protein was observed by.HE staining, and the changes of the inflammatory reaction were observed by ACP staining. Results of the expression of related protein CD68, PP65. Results the results were analyzed with SPSS13.0. Results: 1 AAV9 expressed GFP in different tissues of mdx mice expressed in mdx mice. The expression of GFP in the anterior tibial muscle was the most, the expression of triceps brachii was the second, and the remaining tissue was only a small amount of.2 AAV9-hIGF-1 in the tibial anterior muscle of mdx mice. The expression of hIGF-1 in the anterior tibial muscle of mdx mice was higher than that in -hIGF-1 group, but there were still some muscle fibers that did not express the effect of.3 AAV9-hIGF-1 on the inflammatory response of the mdx mice. HE staining and ACP staining showed that there were scattered phagocytosis in the AAV9-GFP group. Several inflammatory cells were infiltrated by fusion, and the infiltration of inflammatory cells in AAV-9hIGF-1 group was more than that of mdx mice. A small amount of small focal inflammatory cells infiltrated.CD68 was a reliable marker of macrophage. The decrease of CD68+ cells in group AAV9-hIGF-1 was observed by immunofluorescence staining. The percentage of inflammatory areas in the anterior tibial muscle was statistically analyzed, in group AAV-hIGF-1 (1.78 + 0.47%), lower than that in group AAV-GFP (3.4 + 1.22%), higher than that in the normal group (0%), The difference was statistically significant (P0.05).4 NF- kappa B signaling pathway changes using Western-bloth to reduce the expression of PP65, AAV-hIGF-1 group is (0.45 + 0.07%), lower than the AAV-GFP group (0.76 + 0.13%), higher than the normal group (0.38 + 0.06%), the difference has statistical significance. Conclusion: 1 via the tail vein, AAV9 can be more in mdx mice. The expression of site, in which the most.2 AAV9-hIGF-1 tail vein was expressed in the anterior tibial muscle, could reduce the inflammatory response to the muscle in mdx mice.3 speculates that hIGF-1 plays an anti-inflammatory role by downregulating the NF- kappa B signal pathway.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R746.2

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