氙气对3日龄新生大鼠脑白质损伤脑组织CLIC4 mRNA、Bcl-2蛋白表达的影响

发布时间：2018-05-13 23:27

本文选题：氙气 + CLIC4　；参考：《青岛大学》2017年硕士论文

【摘要】：目的腹腔内注射脂多糖联合缺氧缺血制作3日龄新生大鼠脑白质损伤模型,并对脑白质损伤新生大鼠给予氙气干预。通过检测其脑组织中CLIC4 m RNA、Bcl-2蛋白的表达,探讨氙气对新生大鼠脑白质损伤的神经保护作用机制。方法将3日龄SD新生大鼠120只随机分为空白对照组(n=24),脑损伤组(n=24)和氙气干预组(n=72)。空白对照组腹腔注射生理盐水(0.05mg/kg),仅游离右侧颈总动脉不做低氧处理;脑损伤组腹腔注射脂多糖(lipopolysaccharide,LPS,0.05mg/kg),游离并结扎右侧颈总动脉并进行低氧处理,但不进行氙气处理;氙气干预组腹腔注射LPS(0.05mg/kg),游离并结扎右侧颈总动脉并进行低氧处理及氙气吸入处理(50%氙气+30%氧气+20%氮气的充满20L恒温密闭箱,0.5L/min缓慢维持,持续处理3小时)。根据脑损伤后氙气延迟干预的时间(0小时、2小时和5小时),将氙气干预组分为C1、C2、C3三个亚组(n=24)。各组大鼠分别于0h、24h、48h、72h各取6只行多聚甲醛灌注后断头取脑组织,进行脑组织病理检测、蛋白质印迹法(Western Blot,WB)检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)蛋白含量、逆转录-聚合酶链反应(reverse transcriotion-polymerase chain reaction,RT-PCR)检测氯离子通道蛋白4(Chloride Intracellular Channel 4,CLIC4)m RNA的表达。采用独立样本t检验、单因素方差分析和Bonferroni法进行统计学分析。结果(1)实验新生鼠行为学监测:腹腔注射脂多糖及缺氧缺血处理后,脑损伤组和氙气干预组的新生鼠出现肢体抖动、烦躁、紫绀、呼吸急促等行为学异常;在氙气处理之后,C1组新生鼠的症状在约2小时后消失;而脑损伤组、C2组和C3组的症状约在5小时后消失;空白对照组新生鼠未出现明显异常反应。(2)HE染色观察脑组织病理变化:脑损伤组新生鼠可见脑白质苍白,结构疏松,细胞肿胀,核膜不清楚,细胞核固缩,可检测到细胞变性和坏死,神经纤维无序,胶质细胞数量增加;氙气干预组脑白质染色淡染,结构相对较完整,较少细胞变性坏死,各亚组之间无明显差异;而空白对照组脑组织结构正常,染色清晰,细胞排列整齐。(3)WB检测Bcl-2蛋白含量:在相同时间点,各组Bcl-2蛋白的表达量相比较,差异有统计学意义(P0.05);与脑损伤组相比,C1组和C2组中Bcl-2蛋白的表达在各时间点均显着增加,差异有统计学意义(P0.05),但在C3中组的Bcl-2蛋白的表达在48h和72h与脑损伤组比较差异无统计学意义(P0.05);与C1组相比,C2组和C3组Bcl-2蛋白表达在48h和72h显著降低,差异有统计学意义(P0.05)。(4)RT-PCR检测CLIC4 m RNA含量:与空白对照组相比较,在各时间点脑损伤组中新生鼠脑室周围白质CLIC4 m RNA的表达水平增加,差异有统计学意义(P0.05);与脑损伤组相比较,氙气干预各亚组中CLIC4 m RNA表达均明显减少,差异有统计学意义(P0.05)。结论(1)腹腔内注射LPS联合缺氧缺血可成功制作新生儿大鼠脑白质损伤模型;(2)氙气吸入干预可减轻新生儿大鼠脑白质损伤,减少细胞坏死;(3)新生大鼠脑白质损伤后Bcl-2蛋白表达水平升高,氙气可使其表达可下降,表明氙气可能通过抑制神经元凋亡进而减轻白质损伤作用;(4)氙气干预可下调CLIC4基因的表达,表明氙气可能通过抑制CLIC4基因的表达作用于Bcl-2蛋白相关途径抑制神经元凋亡而发挥神经保护作用;(5)氙气治疗脑白质损伤的时间窗至少有5小时,且越早干预效果越好。
[Abstract]:Objective to make intraperitoneal injection of lipopolysaccharide (lipopolysaccharide) and hypoxic ischemia to make a white matter injury model of 3 day old neonatal rats, and to give xenon intervention to neonatal rats with brain white matter injury. By detecting the expression of CLIC4 m RNA and Bcl-2 protein in the brain tissue, the mechanism of neuroprotective effect of xenon on brain white matter injury in newborn rats was investigated. The method was a new method of 3 days of age SD. 120 rats were randomly divided into blank control group (n=24), brain injury group (n=24) and xenon intervention group (n=72). The blank control group was intraperitoneally injected with normal saline (0.05mg/kg), and only the right common carotid artery was free from hypoxic treatment. The brain injury group was injected with lipopolysaccharide (lipopolysaccharide, LPS, 0.05mg/kg), free and ligated the right common carotid artery and carried out. Hypoxia treatment, but no xenon treatment; xenon intervention group intraperitoneal injection of LPS (0.05mg/kg), free and ligated right common carotid artery and conducting hypoxia treatment and xenon inhalation treatment (50% xenon +30% oxygen +20% nitrogen filled 20L constant temperature closed tank, 0.5L/min slow maintenance, sustained treatment for 3 hours). Xenon gas delayed intervention after brain injury, after the delayed intervention of xenon gas (0 hours, 2 hours and 5 hours), the xenon intervention group was divided into C1, C2, and C3 three subgroups (n=24). The rats in each group were respectively taken from 0h, 24h, 48h, and 72h to take the brain tissue after paraformaldehyde perfusion, and the brain tissue was detected, and the protein content was detected by the Western blot (Western Blot, WB), and the inverse of the protein content. Transcription polymerase chain reaction (reverse transcriotion-polymerase chain reaction, RT-PCR) was used to detect the expression of chloride channel protein 4 (Chloride Intracellular Channel 4, CLIC4) m RNA. Independent sample t test, single factor analysis of variance and statistical analysis were used. Results (1) behavioral monitoring of neonatal rats: intraperitoneal injection After the treatment of lipopolysaccharide and hypoxic ischemia, the newborn rats in the brain injury group and the xenon group appeared limb jitter, irritability, cyanosis, and respiratory shortness. After xenon treatment, the symptoms of newborn rats in group C1 disappeared after about 2 hours, while the symptoms of brain injury group, group C2 and C3 disappeared after 5 hours; the blank control group of newborn rats did not. There was an obvious abnormal reaction. (2) the pathological changes of brain tissue were observed by HE staining: the white matter was pale, the structure was loose, the cells were swollen, the nuclear membrane was not clear, the nuclear condensation was not clear, the cell degeneration and necrosis were detected, the nerve fiber disorder, the number of glial cells increased, the white matter in the xenon intervention group was pale staining, and the structure was relatively complete. Whole, less cell degeneration and necrosis, there was no significant difference between the subgroups, while the blank control group had normal brain structure, clear staining and orderly cells. (3) WB detection of Bcl-2 protein content: at the same time point, the expression of Bcl-2 protein in each group was statistically significant (P0.05); compared with the brain injury group, the C1 and C2 group Bcl-2 eggs The expression of white was increased at all time points, and the difference was statistically significant (P0.05), but there was no significant difference in the expression of Bcl-2 protein in C3 group in 48h and 72h with brain injury group (P0.05). Compared with the C1 group, the expression of Bcl-2 protein in C2 group and C3 group decreased significantly in 48h and 72h. (4) M RNA content: compared with the blank control group, the expression level of white matter CLIC4 m RNA around the ventricle of newborn rats in each time point brain injury group increased, the difference was statistically significant (P0.05). Compared with the brain injury group, the RNA expression of CLIC4 m in the xenon group was significantly decreased, the difference was statistically significant (P0.05). Conclusion (1) intraperitoneal injection (P0.05). LPS combined with hypoxia ischemia can successfully produce white matter injury model of neonatal rat brain; (2) xenon inhalation intervention can reduce brain white matter injury and decrease cell necrosis in neonatal rats. (3) the expression level of Bcl-2 protein in newborn rats after white matter injury increases, xenon can decrease its surface, indicating that xenon may inhibit neuronal apoptosis by inhibiting apoptosis. (4) xenon intervention can down regulate the expression of CLIC4 gene, indicating that xenon may play a neuroprotective role by inhibiting the expression of CLIC4 gene to inhibit neuronal apoptosis by Bcl-2 protein related pathway; (5) xenon treatment of brain white matter injury by xenon at at least 5 hours, and the earlier intervention the better.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742

【参考文献】