本文選題：缺氧缺血 + 腦室周圍白質(zhì)軟化��； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2012年碩士論文

【摘要】：背景腦室周圍白質(zhì)軟化(Periventricular Leukomalacia,PVL)是早產(chǎn)兒最常見的腦損傷形式,也是存活早產(chǎn)兒出現(xiàn)神經(jīng)發(fā)育和行為障礙的主要原因。TLR4(Toll like receptor4)是介導(dǎo)脂多糖(Lipopolysaccharides.LPS)最主要的受體,在免疫應(yīng)答和炎癥反應(yīng)中起重要作用。近年來研究發(fā)現(xiàn)在LPS介導(dǎo)的早產(chǎn)兒PVL的發(fā)病機(jī)制中TLR4參與其中發(fā)揮了重要作用,且最新研究顯示TLR4在心、肺、肝、血管等缺血再灌注引起的炎癥損傷中也起到了重要作用,但在缺氧缺血導(dǎo)致的PVL模型中,TLR4的變化規(guī)律及作用尚屬少見,有待我們進(jìn)一步研究。 目的選擇3日齡術(shù)成熟大鼠,通過缺氧缺血性損傷制作腦室周圍白質(zhì)軟化模型,通過測(cè)定不同時(shí)間點(diǎn)腦組織細(xì)胞凋亡及TLR4在(?)mRNA和蛋白水平的表達(dá)變化情況,探討TLR4在該模型中的表達(dá)變化規(guī)律以及與細(xì)胞凋亡的關(guān)系,進(jìn)一步闡明TLR4在早產(chǎn)兒PVL發(fā)病機(jī)理中的作用,為該病的研究提供客觀的實(shí)驗(yàn)依據(jù)。 方法140只3日齡體重5.8-10.5g新生SD大鼠,性別不限,隨機(jī)分為實(shí)驗(yàn)組(缺氧缺血組)75只和對(duì)照組(假手術(shù)組)65只。實(shí)驗(yàn)組通過結(jié)扎右側(cè)預(yù)總動(dòng)脈,置放于6%氧氣十94%氮?dú)饣旌蠚怏w中4小時(shí),制作早產(chǎn)兒腦室周圍白質(zhì)軟化動(dòng)物模型。對(duì)照組新生大鼠用同樣方法分離右側(cè)頸總動(dòng)脈,但僅將手術(shù)線從頸總動(dòng)脈下穿過,不予結(jié)扎,亦不予缺氧處理。在模型制備后6小時(shí)、12小時(shí)、24小時(shí)、3天、7天將動(dòng)物處死,取腦組織制作石蠟切片,或?qū)⒛X組織置液氮罐中待用。實(shí)驗(yàn)過程中實(shí)驗(yàn)組死亡15只,對(duì)照組死亡5只,,故進(jìn)入實(shí)驗(yàn)的兩組均為60只。觀察缺氧缺血前后大鼠行為改變并測(cè)量術(shù)后不同時(shí)點(diǎn)體重變化；常規(guī)觀察腦組織的病理變化；Tunnel法測(cè)細(xì)胞凋亡情況；采用免疫組化及RT-PCR等實(shí)驗(yàn)方法檢測(cè)該模型中不同時(shí)間點(diǎn)腦組織TLR4在mRNA和蛋白水平的表達(dá)情況。SPSS17.0進(jìn)行統(tǒng)計(jì)分析,P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 1.實(shí)驗(yàn)組動(dòng)物行為學(xué)及病理改變：缺氧缺血中大鼠主要表現(xiàn)為全身發(fā)紺,激惹、躁動(dòng)不安,四肢強(qiáng)直抽動(dòng),大小便失禁,部分動(dòng)物于缺氧缺血過程中死亡。缺氧缺血完成后,新生大鼠有吃奶減少,活動(dòng)減少,動(dòng)作不協(xié)調(diào)等異常表現(xiàn)。實(shí)驗(yàn)組新生大鼠于缺氧缺血后24h開始出現(xiàn)體重增長(zhǎng)減慢,3d后開始出現(xiàn)體重追趕式生長(zhǎng),實(shí)驗(yàn)組缺氧缺血后24h、3d、7d體重與對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)組各組隨缺氧缺血時(shí)間的延長(zhǎng),病理改變?cè)絹碓街?缺氧缺血6小時(shí)可見腦組織白質(zhì)內(nèi)膠質(zhì)細(xì)胞間隙增寬；12小時(shí)膠質(zhì)細(xì)胞胞體腫脹,細(xì)胞走行出現(xiàn)紊亂；24小時(shí)細(xì)胞間隙增寬更明顯,小膠質(zhì)細(xì)胞增生,星形膠質(zhì)細(xì)胞肥大；3天可見腦室周圍白質(zhì)廣泛疏松,白質(zhì)結(jié)構(gòu)紊亂；7天右側(cè)腦室旁白質(zhì)可呈篩網(wǎng)狀壞死。 2.細(xì)胞凋亡情況：實(shí)驗(yàn)組腦室周圍白質(zhì)及胼胝體區(qū)于缺氧缺血后6h即出現(xiàn)凋亡細(xì)胞百分率升高,3d達(dá)高峰,7d開始下降；對(duì)照組存在少量的凋亡細(xì)胞。實(shí)驗(yàn)組各時(shí)間點(diǎn)凋亡細(xì)胞百分率與對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 3.TLR4在蛋白水平的表達(dá)狀況：實(shí)驗(yàn)組腦室周圍白質(zhì)及胼胝體區(qū)于缺氧缺血后6h TLR4蛋白表達(dá)量開始增加,3d達(dá)高峰,7d開始下降；對(duì)照組各時(shí)間點(diǎn)TLR4蛋白的免疫陽(yáng)性表達(dá)較少。實(shí)驗(yàn)組各時(shí)間點(diǎn)TLR4在蛋白水平表達(dá)與對(duì)照組相比,養(yǎng)異石統(tǒng)計(jì)學(xué)意義(P0.05)。 4. TLR4mRNA的變化：實(shí)驗(yàn)組腦組織于缺氧缺血后6hTLR4mRNA即開始增加,3d時(shí)達(dá)高峰,7d開始下降；對(duì)照組腦組織內(nèi)可見少量的TLR4mRNA激活。實(shí)驗(yàn)組各時(shí)間點(diǎn)TLR4mRNA激活與對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 5.未成熟大鼠缺氧缺血性腦室周圍白質(zhì)軟化模型中,TLR4mRNA和蛋白水平的表達(dá)相關(guān)性分析顯示二者呈正相關(guān)(r=0.675,P0.05)。 6.未成熟大鼠缺氧缺血性腦室周圍白質(zhì)軟化模型中,TLR4mRNA和凋亡細(xì)胞百分比的相關(guān)性分析顯示二者呈相關(guān)(r=0.575,P0.05)。 7.未成熟大鼠缺氧缺血性腦室周圍白質(zhì)軟化模型中,TLR4蛋白表達(dá)量和凋亡細(xì)胞百分比的相關(guān)性分析顯示二者呈正相關(guān)(r=0.774,P0.05) 結(jié)論1.新生3日齡大鼠單側(cè)頸總動(dòng)脈結(jié)扎加低氧(6%02)4h處理可以成功制作早產(chǎn)兒腦室周圍白質(zhì)軟化損傷模型。 2.細(xì)胞凋亡在缺氧缺血導(dǎo)致的PVL的發(fā)病機(jī)制中有重要作用。 3.TLR4參與了缺氧缺血所致的早產(chǎn)兒腦室周圍白質(zhì)軟化的發(fā)病機(jī)制。 4.TLR4有可能是通過促進(jìn)細(xì)胞凋亡來發(fā)揮神經(jīng)細(xì)胞的損傷作用。
[Abstract]:Background Periventricular Leukomalacia (PVL) is the most common form of brain injury in preterm infants. It is also the main cause of neurodevelopment and behavioral disorders in premature infants..TLR4 (Toll like receptor4) is the main receptor for mediating lipopolysaccharide (Lipopolysaccharides.LPS), which plays an important role in the immune response and inflammatory response. Recent studies have found that TLR4 plays an important role in the pathogenesis of LPS mediated preterm PVL, and the latest research shows that TLR4 plays an important role in the inflammatory injury caused by ischemia and reperfusion in the heart, lung, liver and blood vessels, but the changes and roles of TLR4 in the PVL model caused by hypoxia and blood deficiency. It is still rare and needs further study.
Objective to select the model of periventricular softening by hypoxic ischemic injury in 3 day old rats, and to explore the changes in the expression of TLR4 and the expression of mRNA and protein in the brain tissue at different time points, and to explore the relationship between the expression of TLR4 in the model and the relationship with the apoptosis, and further clarify the TLR4. The role of PVL in the pathogenesis of preterm infants provides an objective experimental basis for the study of this disease.
Methods 140 3 day old 5.8-10.5g newborn SD rats were divided into experimental group (hypoxic ischemia group) and control group (sham operation group), 65 rats were randomly divided into the experimental group and the control group (sham operation group). The experimental group was ligation of the right pretotal artery and placed in the 6% oxygen ten 94% nitrogen gas mixture for 4 hours, and the control group was new to the control group. The same method was used to separate the right common carotid artery, but only the operation line was passed under the common carotid artery, no ligation and no anoxia treatment. 6 hours, 12 hours, 24 hours, 3 days, 7 days after the preparation of the model, the animals were killed, the brain tissue was made of paraffin section, or the brain tissue was used in the liquid nitrogen tank. The experimental group died 15, right 5 rats were killed in the group, and 60 of the two groups entered the experiment. The behavior changes of rats before and after hypoxia ischemia were observed and the body weight changes were measured after the operation. The pathological changes of the brain tissue were observed, the apoptosis of the cells was measured by Tunnel method, and the brain tissue TLR4 at different time points in the model was detected by immunohistochemistry and RT-PCR. The expression level of mRNA and protein was analyzed by.SPSS17.0, and P0.05 was statistically significant.
Result
1. the animal behavior and pathological changes in the experimental group: the rats in the hypoxic and ischemic rats were mainly characterized by cyanosis, irritability, restlessness, ankylosis, incontinence of the limbs and incontinence, and some animals died in the process of hypoxia and ischemia. After the completion of hypoxia ischemia, the newborn rats had abnormality in the reduction of milk, reduced activity and disharmony of the movements. The experimental group was new. The body weight growth slowed down after hypoxic-ischemic 24h and body weight catching growth began to appear after 3D. The 24h, 3D, 7d body weight of the experimental group was statistically significant compared with the control group (P0.05). The experimental group was more and more serious with the prolongation of hypoxic and ischemic time, and the brain tissue was white after 6 hours of hypoxia ischemia. The interstitial glial cell gap widened; 12 hours glial cells were swollen and the cells were disorganized; 24 hours of cell gap widened more clearly, microglia proliferated, astrocytes hypertrophy; on the 3 day, the white matter around the ventricles was widely loose and the white matter structure was disorganized; the right lateral ventricles of the ventricles of the right lateral ventricle could be screened reticular necrosis on the 7 day.
2. cell apoptosis: the percentage of apoptotic cells in the white matter and the corpus callosum around the ventricle of the experimental group increased after hypoxic and ischemic 6h, the peak of 3D reached the peak, the 7d began to decline, and the control group had a small number of apoptotic cells. The percentage of apoptotic cells at each time point in the experimental group was significantly different from the control group (P0.05).
The expression of 3.TLR4 at the protein level: the expression of 6h TLR4 protein in the white matter and corpus callosum around the ventricle of the experimental group began to increase, the 3D reached the peak, and the 7d began to decline; the TLR4 protein expression in the control group at each time point was less. The expression of TLR4 at the protein level at each time point in the experimental group was compared with the control group, and the culture was compared with the control group. Study meaning (P0.05).
4. TLR4mRNA changes: the brain tissue in the experimental group began to increase after hypoxic and ischemic 6hTLR4mRNA, when 3D reached the peak, and the 7d began to decrease, and a small amount of TLR4mRNA activation was found in the control group. The difference of TLR4mRNA activation at each time point in the experimental group was statistically significant compared with the control group (P0.05).
The correlation analysis of the expression of TLR4mRNA and protein levels in the hypoxic-ischemic cerebral cortex periventricular softening model of 5. immature rats showed that there was a positive correlation between the two groups (r=0.675, P0.05).
The correlation analysis of the percentage of TLR4mRNA and apoptotic cells in the hypoxic-ischemic cerebral cortex periventricular softening model in 6. immature rats showed that the two were related (r=0.575, P0.05).
The correlation analysis of the expression of TLR4 protein and the percentage of apoptotic cells in the hypoxic-ischemic cerebral cortex periventricular softening model in 7. immature rats showed that the two were positively correlated (r=0.774, P0.05).
Conclusion 1. the neonatal rat's 3 day old rats with unilateral common carotid artery ligation and hypoxic (6%02) 4H treatment can successfully produce periventricular white matter softening injury model in premature infants.
2. apoptosis plays an important role in the pathogenesis of PVL induced by hypoxia ischemia.
3.TLR4 is involved in the pathogenesis of hypoxic-ischemic periventricular white matter softening in premature infants.
4.TLR4 may play a role in neuronal damage by promoting cell apoptosis.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R722.6

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本文編號(hào)：2017251