本文選題：DMD + AAV9��； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:Duchenne型肌營養(yǎng)不良癥(Duchenne muscular dystrophy,DMD)是進行性肌營養(yǎng)不良癥中最常見的致死性X連鎖隱性遺傳肌病。該病是由于dystrophin基因發(fā)生了突變,使其編碼的抗肌萎縮蛋白的完全或部分缺失,導致肌纖維膜完整性、穩(wěn)定性破壞,最終導致肌肉的變性、壞死。DMD的主要臨床表現(xiàn)為進行性肌肉無力、肌萎縮和腓腸肌假性肥大,隨著疾病進展,DMD患者通常于12歲左右喪失行走功能,20歲左右因呼吸或心力衰竭死亡。DMD患者肌肉的主要病理特征為肌纖維直徑大小不等,可見肌纖維肥大、萎縮、變性和壞死,炎性細胞浸潤,肌纖維最終脂肪化和纖維化。然而dystrophin蛋白的缺失所致的肌細胞膜的不穩(wěn)定性及機械損傷并不能完全解釋DMD骨骼肌的持續(xù)性損害,在DMD的超早期階段甚至在肌纖維的壞死發(fā)生之前,就已經存在炎性信號通路的異常激活。近年來,越來越多研究表明炎性反應及炎性通路的活化在DMD的發(fā)生和疾病進展中發(fā)揮著極其關鍵的作用。對炎性反應這一重要靶點的調節(jié)是DMD治療極具前景的治療方法。C57BL/10ScSn-Dmdmdx/JNju鼠,簡稱mdx小鼠,是由于C57BL/10小鼠X染色體的23號外顯子發(fā)生了點突變,導致其編碼的dystrophin功能缺失而構建的DMD模型鼠,是目前研究DMD應用最廣泛的動物模型。基因治療是將正常的基因或有治療作用的基因通過一定方式導入靶細胞,以糾正基因的缺陷或者發(fā)揮治療作用。腺相關病毒載體(Adeno-associated virus,AAVs)是新近發(fā)展最為常用且具有臨床應用前景的基因載體之一。AAVs可以長期高效表達,具有對機體低毒性、低細胞免疫源性和低致病性等優(yōu)勢。系統(tǒng)性評估表明AAV9可以有效地轉染至骨骼肌和心肌。胰島素樣生長因子-l(insulin-like growth factor 1,IGF-1)是細胞增殖、分化和成熟過程中的重要營養(yǎng)因子。已有研究表明肌肉中特異性表達IGF-1基因可以增加mdx小鼠的肌纖維的再生,降低血清CK值且一定程度上改善肌力,這些效應是通過激活P13K/Akt信號通路實現(xiàn)的。IGF-1還可以從調節(jié)炎癥反應方面作用于骨骼肌,有研究指出IGF-1是通過抑制巨噬細胞移動抑制因子(macrophge migration inhibitory factor,MIF)、高遷移率族蛋白B(high mobility group protein-1,HMGB1)和核轉錄因子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對肌纖維再生的影響,很少關注IGF-1對肌肉炎癥的作用�？紤]到DMD是一種累及全身肌肉的肌病,尾靜脈注射的方式更適宜臨床應用。我們的研究是利用AAV9作為載體,攜帶hIGF-1基因一次性經尾靜脈注射給予6周齡的mdx小鼠治療。6周后通過常規(guī)組織病理染色,免疫熒光和蛋白免疫印跡(Western blot)技術評估m(xù)dx小鼠的炎癥反應,并進一步探究hIGF-1的作用機制。方法:選取周齡6周的mdx小鼠,隨機分為實驗組(AAV9-hIGF-1組)和實驗對照組(AAV9-GFP組),正常對照組為選取同等周齡的C57/10野生型小鼠,每組5只小鼠。給予實驗組mdx小鼠尾靜脈注射AAV9-hIGF-1病毒200μl(1×1012vg/ml),實驗對照組mdx小鼠尾靜脈注射AAV9-GFP病毒200μl(1×1012vg/ml),各組小鼠接受病毒尾靜脈注射后同等條件下觀察6周后取材。小鼠麻醉后分別剪取骨骼肌和心肌,進行包埋冰凍處理。熒光顯微鏡下觀察肌肉組織中AAV9在mdx小鼠的轉染情況后,觀察hIGF-1蛋白的表達。HE染色、ACP染色觀察炎癥反應的變化。檢測炎癥相關蛋白CD68,PP65的表達。結果應用spss13.0對數(shù)據進行分析。結果:1 AAV9在mdx小鼠體內的表達mdx小鼠不同組織中GFP均有所表達,其中脛骨前肌GFP表達最多,肱三頭肌表達次之,余組織僅可見較少量表達。2 AAV9-hIGF-1在mdx小鼠脛骨前肌的表達AAV9-hIGF-1組mdx小鼠脛骨前肌hIGF-1的表達效率較高,但仍有部分肌纖維未見表達。3 AAV9-hIGF-1對mdx小鼠肌肉的炎癥反應的影響HE染色和ACP染色觀察到AAV9-GFP組可見散在分布的吞噬現(xiàn)象,數(shù)個融合成片的炎細胞浸潤,AAV-9hIGF-1組炎細胞浸潤較分散,可見少量小灶狀炎細胞浸潤。CD68是巨噬細胞可靠的標記物,通過免疫熒光染色觀察到AAV9-hIGF-1組CD68+細胞較AAV-GFP組減少。對脛前肌炎癥區(qū)域所占百分比進行統(tǒng)計學分析,AAV-hIGF-1組為(1.78±0.47%),低于AAV-GFP組(3.4±1.22%),高于正常組(0%),差異均有統(tǒng)計學意義(p0.05)。4 NF-κB信號通路的變化運用Western-bloth觀察到AAV9-hIGF-1減少了PP65的表達,AAV-hIGF-1組為(0.45±0.07%),低于AAV-GFP組(0.76±0.13%),高于正常組(0.38±0.06%),差異有統(tǒng)計學意義。結論:1通過尾靜脈注射,AAV9能夠在mdx小鼠的多部位表達,其中脛前肌中表達最多。2 AAV9-hIGF-1尾靜脈注射治療能減輕了mdx小鼠肌肉的炎癥反應。3推測hIGF-1是通過下調NF-κB信號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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R746.2

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