脑室周围白质软化大鼠脑组织TLR4表达变化及与细胞凋亡的关系

发布时间：2018-06-14 11:39

本文选题：缺氧缺血 + 脑室周围白质软化　；参考：《新乡医学院》2012年硕士论文

【摘要】：背景脑室周围白质软化(Periventricular Leukomalacia,PVL)是早产儿最常见的脑损伤形式,也是存活早产儿出现神经发育和行为障碍的主要原因。TLR4(Toll like receptor4)是介导脂多糖(Lipopolysaccharides.LPS)最主要的受体,在免疫应答和炎症反应中起重要作用。近年来研究发现在LPS介导的早产儿PVL的发病机制中TLR4参与其中发挥了重要作用,且最新研究显示TLR4在心、肺、肝、血管等缺血再灌注引起的炎症损伤中也起到了重要作用,但在缺氧缺血导致的PVL模型中,TLR4的变化规律及作用尚属少见,有待我们进一步研究。目的选择3日龄术成熟大鼠,通过缺氧缺血性损伤制作脑室周围白质软化模型,通过测定不同时间点脑组织细胞凋亡及TLR4在(?)mRNA和蛋白水平的表达变化情况,探讨TLR4在该模型中的表达变化规律以及与细胞凋亡的关系,进一步阐明TLR4在早产儿PVL发病机理中的作用,为该病的研究提供客观的实验依据。方法140只3日龄体重5.8-10.5g新生SD大鼠,性别不限,随机分为实验组(缺氧缺血组)75只和对照组(假手术组)65只。实验组通过结扎右侧预总动脉,置放于6%氧气十94%氮气混合气体中4小时,制作早产儿脑室周围白质软化动物模型。对照组新生大鼠用同样方法分离右侧颈总动脉,但仅将手术线从颈总动脉下穿过,不予结扎,亦不予缺氧处理。在模型制备后6小时、12小时、24小时、3天、7天将动物处死,取脑组织制作石蜡切片,或将脑组织置液氮罐中待用。实验过程中实验组死亡15只,对照组死亡5只,,故进入实验的两组均为60只。观察缺氧缺血前后大鼠行为改变并测量术后不同时点体重变化；常规观察脑组织的病理变化；Tunnel法测细胞凋亡情况；采用免疫组化及RT-PCR等实验方法检测该模型中不同时间点脑组织TLR4在mRNA和蛋白水平的表达情况。SPSS17.0进行统计分析,P0.05为差异有统计学意义。结果 1.实验组动物行为学及病理改变：缺氧缺血中大鼠主要表现为全身发绀,激惹、躁动不安,四肢强直抽动,大小便失禁,部分动物于缺氧缺血过程中死亡。缺氧缺血完成后,新生大鼠有吃奶减少,活动减少,动作不协调等异常表现。实验组新生大鼠于缺氧缺血后24h开始出现体重增长减慢,3d后开始出现体重追赶式生长,实验组缺氧缺血后24h、3d、7d体重与对照组相比,差异有统计学意义(P0.05)。实验组各组随缺氧缺血时间的延长,病理改变越来越重,缺氧缺血6小时可见脑组织白质内胶质细胞间隙增宽；12小时胶质细胞胞体肿胀,细胞走行出现紊乱；24小时细胞间隙增宽更明显,小胶质细胞增生,星形胶质细胞肥大；3天可见脑室周围白质广泛疏松,白质结构紊乱；7天右侧脑室旁白质可呈筛网状坏死。 2.细胞凋亡情况：实验组脑室周围白质及胼胝体区于缺氧缺血后6h即出现凋亡细胞百分率升高,3d达高峰,7d开始下降；对照组存在少量的凋亡细胞。实验组各时间点凋亡细胞百分率与对照组相比,差异有统计学意义(P0.05)。 3.TLR4在蛋白水平的表达状况：实验组脑室周围白质及胼胝体区于缺氧缺血后6h TLR4蛋白表达量开始增加,3d达高峰,7d开始下降；对照组各时间点TLR4蛋白的免疫阳性表达较少。实验组各时间点TLR4在蛋白水平表达与对照组相比,养异石统计学意义(P0.05)。 4. TLR4mRNA的变化：实验组脑组织于缺氧缺血后6hTLR4mRNA即开始增加,3d时达高峰,7d开始下降；对照组脑组织内可见少量的TLR4mRNA激活。实验组各时间点TLR4mRNA激活与对照组相比,差异有统计学意义(P0.05)。 5.未成熟大鼠缺氧缺血性脑室周围白质软化模型中,TLR4mRNA和蛋白水平的表达相关性分析显示二者呈正相关(r=0.675,P0.05)。 6.未成熟大鼠缺氧缺血性脑室周围白质软化模型中,TLR4mRNA和凋亡细胞百分比的相关性分析显示二者呈相关(r=0.575,P0.05)。 7.未成熟大鼠缺氧缺血性脑室周围白质软化模型中,TLR4蛋白表达量和凋亡细胞百分比的相关性分析显示二者呈正相关(r=0.774,P0.05) 结论1.新生3日龄大鼠单侧颈总动脉结扎加低氧(6%02)4h处理可以成功制作早产儿脑室周围白质软化损伤模型。 2.细胞凋亡在缺氧缺血导致的PVL的发病机制中有重要作用。 3.TLR4参与了缺氧缺血所致的早产儿脑室周围白质软化的发病机制。 4.TLR4有可能是通过促进细胞凋亡来发挥神经细胞的损伤作用。
[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.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.6

【参考文献】