本文選題：鹽酸法舒地爾 + 視網(wǎng)膜缺血再灌注損傷　； 參考：《河北聯(lián)合大學(xué)》2014年碩士論文

【摘要】：目的建立一種能模擬人類RIRI特征的、可靠的動(dòng)物模型，經(jīng)兔耳緣靜脈注射鹽酸法舒地爾后，并分別通過HE染色法觀察視網(wǎng)膜形態(tài)學(xué)變化、TUNEL法檢測(cè)神經(jīng)細(xì)胞凋亡的變化及免疫組化法檢測(cè)凋亡基因Bcl-2、Caspase-3蛋白表達(dá)的變化及鹽酸法舒地爾對(duì)其的影響，從而進(jìn)一步探討鹽酸法舒地爾對(duì)RIRI的神經(jīng)保護(hù)及作用機(jī)制，為RIRI的臨床治療提供理論依據(jù)。 材料與方法1實(shí)驗(yàn)動(dòng)物分組：本研究選取54只健康雄性日本純種大耳白兔作為研究對(duì)象，按照平行對(duì)照原則隨機(jī)分為3組，其分別為：正常組（6只）、損傷組（24只）、治療組（24只），其中損傷組和治療組又按照不同的再灌注時(shí)間點(diǎn)（6h、12h、24h、48h、72h、120h）分為6組。2建立RIRI動(dòng)物模型：損傷組和治療組均采取前房灌注加壓法造成視網(wǎng)膜缺血，并在缺血1h后，將眼內(nèi)壓迅速降至正常水平，從而制作成RIRI動(dòng)物模型。3給藥途徑及方式：三組給藥途徑均為耳緣靜脈注射，治療組注射鹽酸法舒地爾劑量為10mg/kg，正常組和損傷組則注射等劑量的平衡鹽溶液。4標(biāo)本制作及方法：各組分別在再灌注后6h、12h、24h、48h、72h及120h處死動(dòng)物并迅速摘除眼球，對(duì)不同時(shí)間段的視網(wǎng)膜組織分別通過HE染色法觀察視網(wǎng)膜形態(tài)學(xué)變化，并通過TUNEL法檢測(cè)神經(jīng)細(xì)胞凋亡的變化和通過免疫組化法檢測(cè)凋亡基因Bcl-2、Caspase-3蛋白表達(dá)的變化。 結(jié)果1視網(wǎng)膜形態(tài)學(xué)的變化：治療組的視網(wǎng)膜損傷程度明顯輕于損傷組。2視網(wǎng)膜神經(jīng)細(xì)胞凋亡的變化：在6h、12h、24h、48h、72h時(shí)間點(diǎn)，三組間同一時(shí)間點(diǎn)凋亡細(xì)胞計(jì)數(shù)結(jié)果兩兩比較均具有顯著統(tǒng)計(jì)學(xué)意義（P＜0.01），而120h時(shí)各組間比較無明顯統(tǒng)計(jì)學(xué)意義（P＞0.05）。損傷組：各個(gè)時(shí)間點(diǎn)上凋亡細(xì)胞計(jì)數(shù)比較有顯著統(tǒng)計(jì)學(xué)意義（F值=13.06，P＜0.01）；治療組：各個(gè)時(shí)間點(diǎn)上凋亡細(xì)胞計(jì)數(shù)比較有顯著統(tǒng)計(jì)學(xué)意義（F值=22.54，P＜0.01）。3凋亡基因Bcl-2蛋白的表達(dá)：在6h、12h、24h、48h、72h時(shí)間點(diǎn)，三組間同一時(shí)間點(diǎn)Bcl-2蛋白表達(dá)兩兩比較均具有顯著統(tǒng)計(jì)學(xué)意義（P＜0.01），而120h時(shí)各組間比較無明顯統(tǒng)計(jì)學(xué)意義（P＞0.05）。損傷組：各個(gè)時(shí)間點(diǎn)Bcl-2蛋白表達(dá)比較有顯著統(tǒng)計(jì)學(xué)意義（F值=15.14，P＜0.01）；治療組：各個(gè)時(shí)間點(diǎn)之間Bcl-2蛋白表達(dá)比較有顯著統(tǒng)計(jì)學(xué)意義（F值=17.06，P＜0.01）。4凋亡基因Caspase-3蛋白的表達(dá)：在6h、12h、24h、48h、72h、120h時(shí)間點(diǎn)，三組間同一時(shí)間點(diǎn)Caspase-3蛋白表達(dá)兩兩比較均具有顯著統(tǒng)計(jì)學(xué)意義（P＜0.01）。損傷組：各個(gè)時(shí)間點(diǎn)之間陽性細(xì)胞計(jì)數(shù)差異有顯著統(tǒng)計(jì)學(xué)意義(F值=65.860，P＜0.01）；治療組：各個(gè)時(shí)間點(diǎn)之間陽性細(xì)胞計(jì)數(shù)差異有顯著統(tǒng)計(jì)學(xué)意義（F值=276.550，P＜0.01）。 結(jié)論1鹽酸法舒地爾能減輕視網(wǎng)膜缺血再灌注損傷，在維持視網(wǎng)膜的形態(tài)及功能中起著重要的作用；2鹽酸法舒地爾能抑制視網(wǎng)膜神經(jīng)細(xì)胞的凋亡，其作用機(jī)制可能與提高Bcl-2及降低Caspase-3表達(dá)有關(guān)，從而在視網(wǎng)膜缺血再灌注損傷中對(duì)視網(wǎng)膜神經(jīng)細(xì)胞起到明顯的保護(hù)作用。
[Abstract]:Objective to establish a reliable animal model which can mimic the characteristics of human RIRI. The morphological changes of retina were observed by HE staining and apoptosis of neurons was detected by Tunel method and the expression of apoptotic gene Bcl-2Caspase-3 was detected by immunohistochemical method and the effect of fasudil hydrochloride on it was also observed. To further explore the neuroprotection and mechanism of fasudil hydrochloride on RIRI and provide theoretical basis for the clinical treatment of RIRI. Materials and methods 1 Experimental animals were divided into three groups: 54 healthy male purebred Japanese white rabbits were randomly divided into 3 groups according to the principle of parallel control. They were: normal group (n = 6), injury group (n = 24) and treatment group (n = 24). According to different reperfusion time points, the injury group and the treatment group were divided into 6 groups to establish RIRI animal model: the injury group and the treatment group adopted anterior chamber. The retinal ischemia is caused by perfusion and compression. After 1 hour of ischemia, intraocular pressure was rapidly reduced to normal level, and then RIRI animal model was established. 3 administration route and method: all three groups were injected via ear vein. In the treatment group, the dose of fasudil hydrochloride was 10 mg / kg, the control group and the injury group were injected with the same dose of balanced salt solution of 4. 4 samples and methods: the animals were killed at 6 h, 12 h, 24 h, 48 h, 72 h and 120 h after reperfusion, and their eyeballs were removed quickly. The morphological changes of retina were observed by HE staining, the changes of neuronal apoptosis and the expression of apoptotic gene Bcl-2Caspase-3 were detected by TUNEL method and immunohistochemistry method respectively. Results 1 changes of retinal morphology: the degree of retinal injury in the treatment group was significantly less than that in the injured group (group .2): at the time point of 6h, 12h, 24h, 48h and 72h, the degree of retinal injury in the treatment group was significantly less than that in the injured group (group .2). The number of apoptotic cells in the three groups at the same time point was significantly different (P < 0.01), but there was no significant difference between the three groups at 120 h (P > 0.05). In the injury group, the number of apoptotic cells at each time point was significantly higher than that in the control group (P < 0.01), and in the treatment group, the number of apoptotic cells was significantly higher than that in the control group (P < 0.01), and the expression of apoptotic gene Bcl-2 protein was significantly higher in the treatment group than that in the treatment group at 12 h, 24 h, 48 h, 72 h, respectively. The expression of Bcl-2 protein in the three groups at the same time point was significantly higher than that in the control group (P < 0.01), but there was no significant difference between the three groups at 120h (P > 0.05). In the injury group, the expression of Bcl-2 protein was significantly higher than that in the control group (P < 0.01), and in the treatment group, the expression of Bcl-2 protein was significantly higher than that in the control group (P < 0.01). The expression of Caspase-3 protein of apoptotic gene was significantly higher in the treatment group than that in the control group at 12 h, 24 h, 48 h, 72 h and 120 h, respectively. The expression of Caspase-3 protein in the three groups was significantly different at the same time point (P < 0.01). In the injury group, there was significant difference in the number of positive cells between different time points (P < 0.01), and in the treatment group, there was significant difference in the number of positive cells between different time points (P < 0.01). Conclusion 1 Faxudil hydrochloride can attenuate retinal ischemia-reperfusion injury and play an important role in the maintenance of retinal morphology and function. Second, fasudil hydrochloride can inhibit the apoptosis of retinal nerve cells. The mechanism may be related to the increase of Bcl-2 and the decrease of Caspase-3 expression, which may play an important role in protecting retinal nerve cells from retinal ischemia reperfusion injury.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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本文編號(hào)：1812045