發(fā)布時間：2018-06-22 03:33

  本文選題：顱內(nèi)動脈瘤 + 血管平滑肌細胞�。� 參考：《蘇州大學》2016年碩士論文

【摘要】：目的:顱內(nèi)動脈瘤(intracranial aneurysm,IA)具有自發(fā)性破裂出血的傾向,致殘率和致死率都很高。目前,在臨床上關(guān)于IA的治療僅僅局限于手術(shù)和介入治療,而對動脈瘤壁進行修復等非手術(shù)治療幾乎沒有。在對IA發(fā)病機制的研究中,我們發(fā)現(xiàn)動脈瘤壁血管平滑肌細胞(vascular smooth muscle cell,VSMC)具有增殖活性功能,結(jié)合目前生物治療和基因治療的發(fā)展,我們萌生了為未破裂顱內(nèi)動脈瘤壁進行修復治療的設(shè)想。VSMC是動脈壁組成的主要部分,在IA形成過程中,可表現(xiàn)出很高的增殖活性與可塑性,可見IA形成過程中血管壁VSMC的增殖將成為修復研究的主要方向。因此,本實驗通過建立大鼠顱內(nèi)動脈瘤模型對大鼠顱內(nèi)動脈瘤壁VSMC增殖活性進行探討,目的在于增強動脈瘤壁VSMC的增殖活性,增加平滑肌層,為今后人顱內(nèi)動脈瘤壁的修復治療提供理論基礎(chǔ)。方法:成年雄性自發(fā)性高血壓大鼠(spontaneously hypertensive rat,SHR)180只,隨機分為6組:對照組和實驗1、2、3、4、5月組(每組30只)。實驗組采用經(jīng)背部入路行雙側(cè)腎動脈后支結(jié)扎和左側(cè)頸總動脈結(jié)扎,對照組僅暴露血管,不結(jié)扎。各組術(shù)后1周開始喂養(yǎng)含1%氯化鈉的飲用水和0.12%β-氨基丙腈飼料。每組大鼠術(shù)前及處死前測量室溫安靜狀態(tài)下尾動脈血壓,處死前行7.0MRA檢查大鼠顱內(nèi)動脈。取下動脈瘤標本,用HE染色觀察動脈瘤的病理變化,免疫組化觀察瘤壁增殖細胞核抗原(proliferating cell nuclear antigen,PCNA)的表達和免疫熒光染色和蛋白質(zhì)印跡(Westernblot,WB)觀察瘤壁α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)、β-微管蛋白(β-Tubulin)和骨橋蛋白(osteopontin,OPN)的表達以及實時定量PCR觀測瘤壁SM22α與高血壓相關(guān)基因(hypertension-related gene,HRG-1)mRNA的表達來了解動脈瘤壁VSMC的增殖,末端標記法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)染色測定瘤壁VSMC的凋亡。結(jié)果:1.血壓變化:對照組平均血壓緩慢升高。實驗組平均血壓從1月組起至5月組逐漸升高,均明顯高于術(shù)前和對照組(P0.05)。2.MRA檢測情況:每組隨機抽取6只SHR行7.0 MRA檢測。對照組顱內(nèi)動脈左右對稱、粗細均勻,無明顯擴張增粗。實驗組所有檢測的右側(cè)顱內(nèi)動脈均有不同程度的增粗,血管病變率達100%,其中,4月組有3/6在增粗的血管上可見動脈瘤形成,5月組有5/6可見動脈瘤形成。3.動脈瘤病理變化:對照組顱內(nèi)動脈無明顯破壞,各層結(jié)構(gòu)保持完整。實驗組顱內(nèi)動脈從1月組起內(nèi)膜層開始破壞、VSMC退變、VSMC數(shù)目及層數(shù)減少、動脈壁逐漸變薄,至4、5月組時內(nèi)膜層已消失、彈性纖維斷裂,并有炎性細胞浸潤,其中,5月組最明顯,動脈壁最薄。4.免疫組化結(jié)果:對照組PCNA陽性細胞率低下。實驗組PCNA陽性細胞率從1月組起至5月組與對照組相比均明顯增高(P0.05);其中,4月組最高,5月組開始下降,與4月組相比有顯著差異(P0.05)。5.Western blot結(jié)果:對照組α-SMA和β-tubulin呈高表達,而OPN幾乎不表達。實驗組α-SMA和β-tubulin的表達從1月組起至5月組逐漸下降,均明顯低于對照組(P0.05);而OPN的表達從1月組起至5月組與對照組相比均明顯增高(P0.05);其中,4月組最高,5月組開始下降,與4月組相比差異顯著(P0.05)。6.免疫熒光結(jié)果:對照組α-SMA和β-tubulin呈高表達,而OPN幾乎不表達。實驗組α-SMA和β-Tubulin的相對熒光強度從1月組起至5月組逐漸降低,均明顯低于對照組(P0.05);而OPN的相對熒光強度從1月組起至5月組與對照組相比均明顯增高(P0.05),其中,4月組最高,5月組開始下降,與4月組相比差異顯著(P0.05)。7.實時定量PCR結(jié)果:對照組SM22α和HRG-1 mRNA相對表達較高。實驗組SM22α和HRG-1 mRNA相對表達從1月組起至5月組逐漸下降,均明顯低于對照組(P0.05)。8.TUNEL染色結(jié)果:對照組VSMC凋亡率低下。實驗組VSMC凋亡率從1月組起至5月組逐漸升高,均明顯高于對照組(P0.05)。結(jié)論:實驗性大鼠IA形成過程中動脈瘤壁VSMC具有增殖活性功能,且具有明顯的增殖活躍期;IA的形成與動脈瘤壁VSMC的增殖活性相對減弱密切相關(guān)。
[Abstract]:Objective: intracranial aneurysm (IA) has a tendency to spontaneous rupture and hemorrhage, and the rate of disability and lethality are high. Currently, the clinical treatment of IA is limited to surgery and interventional therapy, but nonsurgical treatment for the repair of the aneurysm wall is few. In the study of the pathogenesis of IA, we found the movement. Vascular smooth muscle cell (VSMC) has the function of proliferative activity. Combined with the development of biological therapy and gene therapy, we conceive that.VSMC is the main part of the arterial wall composition, which can show high proliferation in the process of IA formation. The proliferation of the vascular wall VSMC in the formation of IA will be the main direction of the repair research. Therefore, the rat intracranial aneurysm model was established to explore the VSMC proliferation activity of the intracranial aneurysm wall in rats. The aim of this experiment is to enhance the proliferation activity of the aneurysm wall VSMC and increase the smooth muscle layer for the future human intracranial. The repair of aneurysm wall provides a theoretical basis. Methods: 180 adult male spontaneously hypertensive rats (spontaneously hypertensive rat, SHR) were randomly divided into 6 groups: the control group and the experimental 1,2,3,4,5 month group (30 rats in each group). The experimental group was ligation of the posterior branches of the bilateral renal artery and the left common carotid artery by the back approach, and the control group was only violent. 1 weeks after operation, 1% sodium chloride containing drinking water and 0.12% beta aminonitrile feed were fed. The blood pressure of the caudal artery was measured at room temperature and quiet before and before the operation. Before and before the operation, the intracranial arteries of the rats were examined by 7.0MRA. The specimens of the aneurysm were taken and the pathological changes of the aneurysm were observed by HE staining and the immunohistochemical view was observed. The expression of proliferating cell nuclear antigen (PCNA) and immunofluorescence staining and Western blot (Westernblot, WB) observation of the tumor wall alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA), the expression of beta microtubulin (beta -Tubulin) and osteopontin and real-time quantitative observation The tumor wall SM22 alpha and hypertension-related gene (HRG-1) mRNA were expressed to understand the proliferation of the aneurysm wall VSMC. Terminal labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end) staining was used to determine the apoptosis of the tumor wall. Results: 1. blood pressure changes: the mean blood pressure in the control group increased slowly. The average blood pressure in the experimental group increased gradually from the January group to the May group, which was significantly higher than that in the pre operation and the control group (P0.05).2.MRA detection: each group was randomly selected 6 SHR for 7 MRA tests. The control group was symmetrical and thin, without obvious dilation and thickening. All of the right intracranial arteries in the experimental group were thickened in varying degrees. The rate of vascular lesions was 100%, of which, in the April group, the aneurysm was visible on the thickened vessels, and the pathological changes of the aneurysm formed in the group of 5/6 were seen in the May group. There was no obvious destruction of the intracranial artery in the control group, and the structure of each layer remained intact. The intracranial artery in the experimental group began to destroy from the inner membrane of the January group, the VSMC degeneration, the number of VSMC and the number of layers decreased in the experimental group. The arterial wall gradually thinned, the intima layer disappeared, the elastic fibers broke and the inflammatory cells infiltrated in the 4,5 month group. Among them, the May group was the most obvious, the thinnest.4. immunohistochemical results were found in the control group, and the rate of PCNA positive cells in the control group was low. The rate of PCNA positive cells in the experimental group increased significantly from the January group to the May group (P0.05), and 4 The month group was the highest, and the May group began to decline. Compared with the April group, there was a significant difference (P0.05).5.Western blot results: the control group of alpha -SMA and beta -tubulin were highly expressed, and OPN almost did not express. The expression of alpha -SMA and beta -tubulin in the experimental group decreased gradually from the January group to the May group, all significantly lower than the control group (P0.05), while the expression of OPN was from January to May group. Compared with the control group (P0.05), the April group was the highest and the May group began to decline. Compared with the April group, the difference was significant (P0.05).6. immunofluorescence results: the control group of alpha -SMA and beta -tubulin were highly expressed, and the OPN almost did not express. The relative fluorescence intensity of the alpha -SMA and beta -Tubulin in the experimental group decreased gradually from the January group to the May group, both significantly lower. Compared with the control group (P0.05), the relative fluorescence intensity of OPN increased significantly from the January group to the May group (P0.05), which was the highest in the April group, and the May group began to decline. Compared with the April group, the difference was significant (P0.05).7. real-time quantitative PCR results: the relative expression of SM22 alpha and HRG-1 mRNA in the control group was higher. The SM22 A and HRG-1 mRNA in the experimental group were relatively expressed. From the January group to the May group gradually decreased, both significantly lower than the control group (P0.05).8.TUNEL staining results: the control group VSMC apoptosis rate is low. The apoptosis rate of VSMC in the experimental group increased gradually from the January group to the May group, all obviously higher than the control group (P0.05). Conclusion: the arterial wall VSMC has the proliferative activity function in the experimental rat IA formation process, and it has a bright future. The proliferative phase was significantly correlated with the formation of IA and the relative attenuation of VSMC proliferation in aneurysm walls.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R743

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