本文選題：菩人丹超微粉　切入點：OLETF大鼠　出處：《承德醫(yī)學院》2016年碩士論文

【摘要】：隨著人均壽命的延長和人們生活方式的改變,糖尿病視網(wǎng)膜病變(diabetic retinopathy,DR)的患病率、致盲率正逐年升高,成為人類不可逆性盲的重要原因。因此,DR的防御及治療成為糖尿病及其并發(fā)癥研究領(lǐng)域的一個重要課題。近年來研究表明,DR不僅是一種微血管病變更是一種視網(wǎng)膜神經(jīng)組織損傷的退行性變,視網(wǎng)膜神經(jīng)細胞凋亡是DR早期最主要的病理表現(xiàn)形式。JNK信號通路作為介導(dǎo)細胞凋亡的一條重要通路在DR的發(fā)生發(fā)展過程中發(fā)揮著重要作用,c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)和半胱氨酸天冬氨酸激酶-3(cysteine aspartic acid specific protease-3,Caspase-3)作為JNK信號通路中的兩個重要因子與DR關(guān)系密切。中藥復(fù)方在防治DR方面具有多靶位、多途徑的獨特優(yōu)勢。菩人丹超微粉(Purendan superfine powder,PRD)是針對糖尿病(diabetes mellitus,DM)病機關(guān)鍵“熱、虛、瘀”而設(shè)的中藥組方,具有益氣清熱、活血通絡(luò)之功效[1]。本研究采用自發(fā)性2型糖尿病大鼠模型OLETF大鼠為研究對象,探討PRD對2型糖尿病大鼠模型視網(wǎng)膜病變的保護作用,為PRD治療DR提供理論基礎(chǔ)和研究依據(jù)。目的:探討PRD對糖尿病大鼠視網(wǎng)膜p-JNK和Caspase-3表達的影響。方法:1以自發(fā)性雄性2型糖尿病大鼠模型OLETF大鼠和其同系非糖尿病對照鼠LETO雄性大鼠為實驗對象,空腹血糖(fasting blood-glucose,FBG)峰值16.7 mmol/L和負荷后120 min血糖11.1 mmol/L作為造模成功標準,將24只雄性成模OLETF大鼠按隨機數(shù)字表法分為DR模型組、PRD治療組,每組12只,同時選取12只同周齡雄性leto大鼠為空白對照組(正常組)。成功建立模型后,prd治療組大鼠給予prd(1.8g/kg/d)灌胃2個月。2采用快速血糖儀法監(jiān)測大鼠血糖,觀察大鼠生存狀況。3he染色觀察視網(wǎng)膜形態(tài)結(jié)構(gòu)的變化。4采用脫氧核糖核苷酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標記法(tunel)檢測大鼠視網(wǎng)膜神經(jīng)細胞的凋亡情況。5sp免疫組織化學染色法和westernblotting法檢測視網(wǎng)膜磷酸化jnk(p-jnk)、caspase-3蛋白的表達。6采用逆轉(zhuǎn)錄聚合酶鏈法(reversereanscription-polymerasechainereaction,rt-pcr)檢測大鼠視網(wǎng)膜jnkmrna、caspase-3mrna的表達。結(jié)果:1造模前各組大鼠fbg無明顯差距,成模時除正常組外,dr模型組、prd治療組fbg顯著升高,差異有統(tǒng)計學意義(p0.01)。用藥后,與dr模型組比較,prd治療組fbg水平顯著降低,有統(tǒng)計學意義(p0.01)。2各組大鼠視網(wǎng)膜病理學改變:正常組大鼠視網(wǎng)膜組織結(jié)構(gòu)完整、內(nèi)界膜光滑、分層清晰;節(jié)細胞圓形、橢圓形,染色淺,排列整齊;內(nèi)網(wǎng)狀層較厚、疏松;內(nèi)核層染色稍深,由3~5層細胞構(gòu)成;外網(wǎng)狀層較內(nèi)網(wǎng)狀層明顯變薄;外核層染色深,由8~10層細胞組成,排列較緊密;外界膜邊界清楚、整齊。dr模型組可見視網(wǎng)膜內(nèi)界膜明顯腫脹、增厚,部分內(nèi)界膜破裂,內(nèi)界膜界線不清晰,部分細胞空泡樣變、細胞核固縮、溶解。prd治療組視網(wǎng)膜分層較為清晰,內(nèi)界膜輕度腫脹,可見少量空泡樣變細胞。3各組大鼠視網(wǎng)膜神經(jīng)細胞的凋亡情況:正常組大鼠視網(wǎng)膜切片未見明顯tunel染色陽性細胞;dr模型組及prd治療組陽性細胞分布范圍擴大,tunel染色陽性產(chǎn)物表現(xiàn)為褐色、顆粒狀,位于胞核中,主要分布部位為視網(wǎng)膜內(nèi)核層和神經(jīng)節(jié)細胞(retinalganglioncells,rgcs)層。3組大鼠視網(wǎng)膜中ai的總體比較差異有統(tǒng)計學意義,與正常組相比,dm模型組大鼠視網(wǎng)膜神經(jīng)細胞ai明顯升高(p0.01);與dm模型組相比,prd治療組大鼠視網(wǎng)膜神經(jīng)細胞ai明顯降低(p0.01)。4各組大鼠視網(wǎng)膜p-JNK的表達:免疫組織化學染色結(jié)果顯示,p-JNK免疫陽性產(chǎn)物為棕黃色、細顆粒狀物質(zhì),位于胞核和胞質(zhì)中,陽性細胞主要分布部位為RGCs層及內(nèi)核層。Western blotting法結(jié)果顯示,p-JNK蛋白條帶位于42 kDa處,β-actin條帶位于43 kDa處。RT-PCR結(jié)果顯示,JNK mRNA條帶位于422 bp處,β-actin條帶位于425 bp處。糖尿病模型組大鼠與正常組大鼠相比,糖尿病模型組大鼠視網(wǎng)膜組織p-JNK蛋白和mRNA表達明顯升高(P0.01);PRD治療組大鼠與糖尿病模型組大鼠相比,PRD治療組大鼠視網(wǎng)膜組織p-JNK蛋白和mRNA表達明顯降低(P0.01)。5大鼠視網(wǎng)膜Caspase-3的表達:免疫組織化學染色結(jié)果顯示,Caspase-3免疫陽性產(chǎn)物為棕黃色、細顆粒狀物質(zhì),位于胞核中,陽性細胞主要分布部位為內(nèi)核層和RGCs層。Western blotting法結(jié)果顯示,Caspase-3蛋白條帶位于34 kDa處,β-actin條帶位于43 kDa處。RT-PCR結(jié)果顯示,Caspase-3 mRNA條帶位于329 bp處:β-actin條帶位于425 bp處。糖尿病模型組大鼠與正常組大鼠相比,糖尿病模型組大鼠視網(wǎng)膜組織Caspase-3蛋白和mRNA表達明顯升高(P0.01);PRD治療組大鼠與糖尿病模型組大鼠相比,PRD治療組大鼠視網(wǎng)膜組織Caspase-3蛋白和mRNA表達明顯降低(P0.01)。結(jié)論:PRD可以改善糖尿病視網(wǎng)膜病變中神經(jīng)組織的損傷,表現(xiàn)為神經(jīng)細胞凋亡的減少,這可能與下調(diào)p-JNK、Caspase-3的表達相關(guān)。
[Abstract]:With the extension of life expectancy and the change of people's lifestyle, diabetic retinopathy (diabetic, retinopathy, DR) the prevalence of blindness rate is increasing year by year, has become an important cause of human irreversible blindness. Therefore, the prevention and treatment of DR become an important research topic in the field of diabetes and complications in recent years. Research shows that DR is not only a change of microvascular disease is a degenerative retinal nerve tissue injury and apoptosis of retinal nerve cells is an important pathway in early DR, the main pathological manifestation of the.JNK signal pathway is mediated by apoptosis plays an important role in the development of DR, c-Jun terminal kinase (c-Jun N-terminal, kinase, JNK) and cysteine aspartate kinase -3 (cysteine aspartic acid specific protease-3, Caspase-3) as the two important in the JNK signaling pathway The factor is closely related with DR. Traditional Chinese medicine compound with multi targets in the prevention and treatment of DR, the unique advantages in many ways. Purendan Supermicropowder (Purendan superfine powder PRD (diabetes) for diabetes mellitus, DM) the key pathogenesis of heat, deficiency, blood stasis and prescription of traditional Chinese medicine ", has the effect of heat. Effect of Huoxue Tongluo [1]. the spontaneous type 2 diabetes OLETF rats as the research object, to investigate the protective effect of PRD on retinal lesions in type 2 diabetic rat model, and provides a theoretical basis and research basis for the treatment of PRD DR. Objective: to discuss the impact of PRD on the expression of p-JNK and Caspase-3 in retina of diabetic rats methods: 1 OLETF male spontaneous type 2 diabetic rat model and rat syngeneic non-diabetic control rats LETO rats as experimental subjects, fasting blood glucose (fasting, blood-glucose, FBG) 16.7 mmol/L and peak load of 120 min Blood glucose of 11.1 mmol/L as the successful model of the standard, 24 male model OLETF rats were randomly divided into DR model group, PRD treatment group, 12 rats in each group at the same time, a total of 12 week old male Leto rats into normal control group (normal group). After the model, PRD treatment rats were given PRD (1.8g/kg/d) by gavage for 2 months.2 glucose by fast blood glucose meter monitoring rats,.3he rats survival staining nick end labeling changes of.4 retinal morphology using terminal deoxynucleotidyl transferase mediated (TUNEL) detection of rat retinal cell apoptosis.5sp immunohistochemical staining of retinal phosphorylated JNK method and westernblotting method (p-JNK), the expression of.6 caspase-3 protein by reverse transcriptase polymerase chain reaction (reversereanscription-polymerasechainereaction, RT-PCR) detection of jnkmr in retina of rats Na, the expression of Caspase-3mRNA. Results: 1 of the rats in FBG no obvious difference between model except the normal group, Dr model group, PRD treatment group, FBG was significantly increased, the difference was statistically significant (P0.01). After treatment, compared with Dr model group, PRD treatment group and FBG level were significantly decreased. There was statistical significance (P0.01) pathological changes of retina of rats in.2 groups: normal rat retinal tissue structural integrity, membrane smooth, clear stratification; ganglion cell round, oval, stained and neatly arranged; the inner plexiform layer is thick and loose; the core layer was slightly darker, composed of 3~5 layers of cells; the outer plexiform layer than the inner reticular layer was thinner; the outer nuclear layer was deep, composed of 8~10 layers of cells, arranged more closely; outside the membrane boundary clear, neat.Dr model group showed retinal membrane swelling, thickening, part of the internal limiting membrane rupture, internal limiting membrane boundary is not clear, cell vacuolar degeneration fine. Pyknosis, dissolved in.Prd treatment group layered retina is clear, the internal limiting membrane swelling, apoptosis of a small amount of vacuolar degeneration of cells in.3 group rat retinal neural cells: the positive cells of normal rats retinal slices without obvious TUNEL staining; Dr model group and PRD treatment group distribution of positive cells to expand, tunel positive product is brown, granular, is located in the nucleus, the main distribution of retinal inner nuclear layer and ganglion cell layer (retinalganglioncells, RGCs) general.3 rats retina in AI had a significant difference, compared with the normal group, DM model group of rat retinal nerve cells significantly increased AI (P0.01); compared with the DM model group, PRD treatment significantly decreased the AI of retinal nerve cells in rats (P0.01) expression of p-JNK in retina of rats in.4 groups: immunohistochemical staining showed that p-JNK immune The positive product was brown yellow, fine granular material, is located in the nucleus and cytoplasm, the positive cells mainly distributed position display for the RGCs layer and core layer.Western blotting results, p-JNK protein bands at 42 kDa and beta -actin bands located at 43 kDa.RT-PCR results showed that the JNK mRNA band at 422 BP -actin, beta band at 425 BP. The diabetic model rats compared with normal rats, the expression of diabetic retinal tissue of rats in the model group of p-JNK protein and mRNA increased significantly (P0.01); PRD rats in the treatment group compared with the diabetic rats in the model group, PRD treatment group significantly decreased expression of rat retina p-JNK protein and mRNA (P0.01) expression in Caspase-3 rat retina.5: immunohistochemical staining showed that Caspase-3 immunoreactive product was brown yellow, fine granular material, is located in the nucleus. The positive cells mainly distributed to the kernel layer and parts RGCs.Western blotting assay showed that Caspase-3 protein band at 34 kDa, beta -actin bands located at 43 kDa.RT-PCR results showed that the Caspase-3 mRNA band at 329 BP beta -actin band at 425 BP. The diabetic model rats compared with normal rats, the expression of diabetes model the retinal tissue of rats Caspase-3 protein and mRNA increased significantly (P0.01); PRD rats in the treatment group compared with the diabetic rats in the model group, PRD treatment group significantly decreased expression of rat retinal tissue Caspase-3 protein and mRNA (P0.01). Conclusion: PRD can improve the nerve tissue of the diabetic retinopathy is to reduce nerve damage, cell apoptosis, which may be related to the down-regulation of p-JNK, Caspase-3 expression.

【學位授予單位】：承德醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R276.7

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