發(fā)布時間：2018-06-03 20:56

  本文選題：PSD-95 + 單眼形覺剝奪��； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2012年碩士論文

【摘要】：研究背景 弱視是兒童時期常見的嚴(yán)重影響視功能發(fā)育的眼病,患病率約為1%-5%。弱視的發(fā)病機制與視覺神經(jīng)元突觸的發(fā)育和可塑性變化密切相關(guān),突觸可塑性的變化是形成弱視的中心環(huán)節(jié)。突觸后致密蛋白-95(PSD-95)在調(diào)節(jié)突觸發(fā)育和可塑性中起關(guān)鍵作用,在蛋白水平已證實PSD-95參與視覺早期的發(fā)育,而在基因水平的相關(guān)研究則相對較少。 目的 觀察幼年和成年單眼形覺剝奪性弱視大鼠和正常發(fā)育大鼠視皮質(zhì)中PSD-95的表達情況,從分子水平來探索弱視的發(fā)病機制,為臨床預(yù)防與治療弱視提供新的思路。 方法 1選取14日齡SD大鼠48只,雌雄不限,隨機分為單眼剝奪組(MD)24只和正常對照組(NC)24只。NC組隨機分為正常組Ⅰ組(NC Ⅰ)、正常組Ⅱ組(NCⅡ),每組各12只；MD組隨機分為剝奪組Ⅰ組(MD Ⅰ)、剝奪組Ⅱ組(MD Ⅱ),每組各12只。MD組所有大鼠在生后14天行單側(cè)眼瞼縫合術(shù)建立單眼剝奪模型。 2NC Ⅰ組和MD Ⅰ組大鼠共同飼養(yǎng)至生后45天(P45,幼年期)取材,所得標(biāo)本為NCP45、MDP45;NCⅡ組和MDⅡ組大鼠均飼養(yǎng)至生后90天(P90,成年期)取材,所得標(biāo)本為NCP90、MDP90, MD組取材前均經(jīng)圖形視覺誘發(fā)電位(P-VEP)檢測證實造模成功。 3應(yīng)用Nissl染色法觀察各組大鼠視皮質(zhì)神經(jīng)元的形態(tài)改變并定位分層,采用免疫組織化學(xué)方法和逆轉(zhuǎn)錄聚合酶鏈反應(yīng)技術(shù)(RT-PCR)檢測各組大鼠視皮質(zhì)中PSD-95蛋白表達和PSD-95mRNA轉(zhuǎn)錄水平的變化。 4通過攝像顯微鏡照相,圖像分析系統(tǒng)進行圖像分析得出PSD-95陽性反應(yīng)產(chǎn)物的平均光密度(AOD)值和PSD-95mRNA的相對表達量,數(shù)據(jù)用SPSS12.0統(tǒng)計軟件進行處理,以均數(shù)士標(biāo)準(zhǔn)差(x±s)表示,相同發(fā)育階段的組間比較采用成組設(shè)計的t檢驗,不同發(fā)育階段的組間比較采用方差分析。 結(jié)果 1P-VEP檢測結(jié)果：經(jīng)P-VEP檢測后可見,正常對照組大鼠P-VEP均有穩(wěn)定的波形、潛伏期和振幅。在幼年期(P45)和成年期(P90),MD組剝奪眼與NC組大鼠眼、MD組未剝奪眼比較,其P-VEP的P波潛伏期延長、波形不穩(wěn)定、波幅下降,差異均有顯著性意義(P0.05)；在幼年期和成年期,MD組未剝奪眼與NC組大鼠眼比較,差異均無統(tǒng)計學(xué)意義(P0.05)。 2Nissl染色結(jié)果：NC組大鼠視皮質(zhì)神經(jīng)元呈空泡狀,胞漿著色,核不著色。視皮質(zhì)的神經(jīng)元分布呈層狀,從皮質(zhì)淺層至深層依次為：分子層(Ⅰ)、外顆粒層(Ⅱ)、外錐體層(Ⅲ)、內(nèi)顆粒層(Ⅳ)、內(nèi)錐體層(Ⅴ)、多形細(xì)胞層(Ⅵ)。與NC組大鼠比較,MD組大鼠剝奪眼對側(cè)視皮質(zhì)Nissl染色結(jié)果未見明顯異常。 3免疫組織化學(xué)染色結(jié)果：光鏡下可見,PSD-95陽性反應(yīng)產(chǎn)物主要表達在神經(jīng)元的胞體中,呈棕色顆粒狀廣泛分布于視皮質(zhì)各層。PSD-95陽性反應(yīng)產(chǎn)物在NC組大鼠視皮質(zhì)中表達密集,其AOD值在NCP45組為0.397±0.008,在NCP90組為0.318±0.007,兩組間的差異有統(tǒng)計學(xué)意義(P0.05)；MD組大鼠視皮質(zhì)中PSD-95陽性表達產(chǎn)物的數(shù)量均減少,密度均減低,其AOD值在MDP45組為0.352±0.005(P4550.01),在MDP90組為0.259±0.011(P900.01)；MD組各發(fā)育階段間PSD-95陽性反應(yīng)產(chǎn)物的表達差異有顯著性(P0.05)。 4RT-PCR法檢測結(jié)果：PSD-95mRNA在各組大鼠的視皮質(zhì)中均有表達。PSD-95mRNA的相對表達量在NCP45組為0.667±0.009,在NCP90組為0.399±0.019,兩組間的差異有顯著性(P0.05)；不同發(fā)育階段MD組與NC組比較,PSD-95mRNA的表達均有明顯減少,其相對表達量在MDP45組為0.470±0.005(P450.01),在MDP90組為0.350±0.005(P900.01)；MD組各發(fā)育階段間PSD-95mRNA的相對表達量差異有顯著性(P0.05)。 結(jié)論 1在視覺發(fā)育的關(guān)鍵期內(nèi),視覺經(jīng)驗通過調(diào)節(jié)視覺神經(jīng)元間的突觸聯(lián)系來調(diào)控PSD-95的表達,PSD-95是弱視發(fā)病機制的重要分子生物學(xué)基礎(chǔ)之一； 2異常視覺經(jīng)驗?zāi)軌蛴绊懗赡甏笫笠暺べ|(zhì)內(nèi)PSD-95的表達,推測成年大鼠的視皮質(zhì)神經(jīng)元仍具有突觸可塑性； 3與幼年期正常發(fā)育大鼠比較,成年期正常發(fā)育大鼠視皮質(zhì)中PSD-95的表達減少,推測年齡因素是PSD-95表達的影響因素之一。
[Abstract]:Research background
Amblyopia is a common ocular disease affecting the development of visual function, which is about 1%-5%. amblyopia. The incidence of amblyopia is closely related to the development and plasticity of synapses in visual neurons. The change of synaptic plasticity is the central link in the formation of amblyopia. The postsynaptic dense protein -95 (PSD-95) plays a role in regulating synaptic development and plasticity. The key role of protein at the protein level has been confirmed that PSD-95 is involved in the early development of vision, while the relative research at the gene level is relatively small.
objective
To observe the expression of PSD-95 in the visual cortex of young and adult monocular deprived amblyopia and normal developing rats, and to explore the pathogenesis of amblyopia from the molecular level, and provide a new idea for the clinical prevention and treatment of amblyopia.
Method
1 14 day old SD rats were randomly divided into single eye deprivation group (MD) and normal control group (NC), and 24.NC groups were randomly divided into normal group I (NC I), normal group II Group (NC II) and each group 12. The MD group was randomly divided into group I (MD I) and group II (MD II) in deprivation group (MD II), and all rats in each group of 12.MD groups were 14 days after birth. Unilateral eyelid suture was performed to establish a monocular deprivation model.
Group 2NC I and group MD I were reared together to 45 days after birth (P45, young). The specimens were NCP45, MDP45, NC II and MD II rats were reared to 90 days after birth (P90, adulthood). The specimens were tested by the visual evoked potential (P-VEP) test before the samples were obtained from NCP90, MDP90, MD group.
3 the morphological changes of the cortical neurons of the rats were observed by Nissl staining and the stratification was located. The changes of the expression of PSD-95 protein and the level of PSD-95mRNA transcription in the visual cortex of each group were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).
4 the average light density (AOD) value of PSD-95 positive reaction products and the relative expression of PSD-95mRNA were obtained by image analysis system by image analysis system. The data were processed with SPSS12.0 statistical software, with the standard deviation of the number of men (x + s), and the group designed t test was used in the same development stage, and the difference between the groups was different. Variance analysis is used in the comparison between groups at the developmental stage.
Result
1P-VEP detection results: after P-VEP detection, the normal control group showed that P-VEP had stable waveform, latent period and amplitude. In juvenile (P45) and adult stage (P90), MD group deprived eye and NC group eyes, and MD group was not deprived of eye, the latency of P-VEP P wave extended, wave instability, amplitude decreased, the difference had significant significance (P0.05). In the juvenile and adulthood, there was no significant difference between the MD group and the NC group (P0.05).
2Nissl staining results: the neurons of the visual cortex in group NC were vacuolated, cytoplasm coloring and non coloring. The distribution of neurons in the visual cortex was stratified, from the superficial layer to the deep layer: the molecular layer (I), the outer layer (II), the outer pyramidal layer (III), the inner granular layer (IV), the inner pyramidal layer (V) and the multiform cell layer (VI). Compared with the NC group, the MD group was larger. There was no obvious abnormality in Nissl staining of lateral eye cortex in rat deprived eyes.
3 immunohistochemical staining results: the results showed that the PSD-95 positive reaction products were mainly expressed in the cell body of the neurons, and the.PSD-95 positive reaction products were widely distributed in the visual cortex of the NC group, and the AOD value was 0.397 + 0.008 in the NCP45 group and 0.318 + 0.007 in the NCP90 group and in the two groups. The difference was statistically significant (P0.05). The number of PSD-95 positive products in the visual cortex of group MD decreased and the density decreased. The AOD value was 0.352 + 0.005 (P4550.01) in the MDP45 group and 0.259 + 0.011 (P900.01) in the MDP90 group, and the expression difference between the PSD-95 positive reaction products of the MD group was significant (P0.05).
The results of 4RT-PCR assay: the relative expression of.PSD-95mRNA in the visual cortex of the rats of each group was 0.667 + 0.009 in the NCP45 group and 0.399 + 0.019 in the NCP90 group. The difference between the two groups was significant (P0.05). The expression of PSD-95mRNA in the MD group at different developmental stages was significantly reduced, and the relative expression was in MD. Group P45 was 0.470 + 0.005 (P450.01), which was 0.350 + 0.005 (P900.01) in group MDP90, and there was a significant difference in the relative expression of PSD-95mRNA between different stages of development in MD group (P0.05).
conclusion
1 in the critical period of visual development, visual experience regulates the expression of PSD-95 by regulating synaptic connections among visual neurons, and PSD-95 is one of the important molecular biological bases of the pathogenesis of amblyopia.
2 abnormal visual experience can influence the expression of PSD-95 in the visual cortex of adult rats. It is speculated that the neurons in the visual cortex of adult rats still have synaptic plasticity.
3 compared with the normal developmental rats, the expression of PSD-95 in the visual cortex of normal adult rats decreased, and the age factor was one of the factors affecting the expression of PSD-95.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R777.44

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本文編號：1974153