针刺“足三里”穴对端粒酶基因敲除小鼠海马区TRKB-ERK信号通路蛋白表达的影响

发布时间：2018-05-18 00:37

本文选题：Terc~(-/-)小鼠 + 行为活动　；参考：《福建中医药大学》2017年硕士论文

【摘要】：目的:观察不同基因型和不同月龄端粒酶基因敲除小鼠(telomerase-deficient,Terc-/-)行为学变化特点,并在此基础上研究针刺足三里穴对Terc-/-小鼠海马区TRKB-ERK信号通路蛋白表达的影响。方法:1.选用第3代Terc-/-小鼠,在小鼠3-4周龄时进行分笼、采血,经PCR法基因鉴定小鼠基因型。筛选出Terc-/-和野生型小鼠,随机选出2月龄、3月龄、5月龄、7月龄及9月龄小鼠各8只,共40只。对不同月龄Terc-/-和野生型小鼠进行行为学检测。检测完成后,对检测数据进行分析,观察Terc-/-和野生型小鼠随月龄增长其行为学变化特点。2.分别取24只7月龄Terc-/-和野生型小鼠,随机分为空白组、手针组和电针组,每组各8只,共48只。空白组在相同环境下饲养,仅模拟抓握;手针组取双侧足三里穴直刺约1.5mm,手针刺激30min,每5分钟行捻转手法一次,每天1次,连续干预7天;电针组取双侧足三里穴直刺约1.5mm,电针刺激30min,交替波,频率为2/50Hz,强度为2mA,每天1次,连续干预7天;干预结束后次日取脑组织,采用免疫组化法及免疫印迹法检验TRKB、ERK、P-ERK和NF-kB蛋白在海马区的表达。结果:1.Morris水迷宫:与野生型小鼠相比,Terc-/-小鼠随月龄增长其潜伏期逐渐延长,逃避潜伏期的目标象限时间比逐渐减小,两组间在2、3和5月龄时无显著差异(p0.05),但在7和9月龄时有显著差异(p0.05)。2.自主活动测试:与野生型小鼠相比,Terc-/-小鼠在2、3和5月龄时的自主活动均无显著差异(P0.05),但在7和9月龄时两组间出现显著差异(P0.05)。3.老化度评分:与野生型小鼠相比,Terc-/-小鼠的评分值在2、3月龄时无显著差异(p0.05),在5、7和9月龄时显著增高(P0.05)。4.免疫组化法结果:(1)干预后Terc-/-小鼠海马区TRKB蛋白阳性目标面密度:空白组(0.0233±0.007)、手针组(0.0312±0.005)、电针组(0.0363±0.007),电针组与空白组比较,差异具有显著性(p0.05);Terc-/-小鼠海马区ERK蛋白阳性目标面密度:空白组(0.0148±0.074)、手针组(0.0270±0.010)、电针组(0.0201 ±0.008),手针组与空白组比较,差异具有显著性(p0.05)。5.免疫印迹法结果:(1)干预后Terc-/小鼠海马区TRKB蛋白相对体积:空白组(0.6940±0.249)、手针组(0.9525±0.139)、电针组(0.9801±0.129),手针组和电针组与空白组比较,差异均具有显著性(P0.05)。Terc-/-小鼠海马区NF-kB蛋白相对体积:空白组(0.2891±0.070)、手针组(0.4066±0.126)、电针组(0.4856±0.066),手针组和电针组与空白组比较,差异均具有显著性(P0.05)。结论:1.与同龄野生型小鼠相比,Terc-/-小鼠随月龄增长其行为活动出现显著增龄性变化,Terc-/-小鼠的衰老特点与年龄密切相关,年龄越大,衰老速度及程度越大,尤其在7月龄以后更为明显。2.针刺"足三里"穴可促进Terc-/-小鼠海马区TRKB、ERK和NF-KB蛋白的表达,但对野生型小鼠影响不显著。
[Abstract]:Aim: to observe the behavioral characteristics of telomerase-deficient Terc-r-r in telomerase gene knockout mice with different genotypes and months, and to study the effect of acupuncture at Zusanli point on the expression of TRKB-ERK signaling pathway protein in the hippocampus of Terc-r-mice. Method 1: 1. The third generation of Terc-r-mice was divided into cages at 3-4 weeks of age and blood was collected. The genotypes of the mice were identified by PCR method. Terc-r- and wild-type mice were selected, and 40 mice were randomly selected, 8 mice were aged from 2 months to 3 months and 5 months from 7 months to 9 months old. The behavior of Terc-r-and wild-type mice were measured at different months of age. After the test was completed, the data were analyzed to observe the behavioral characteristics of Terc-r- and wild-type mice with increasing age. Twenty-four 7-month-old Terc-r- and wild-type mice were randomly divided into blank group, hand acupuncture group and electroacupuncture group with 8 rats in each group and 48 mice in each group. The control group was fed under the same environment, only simulated grip, the hand acupuncture group took bilateral Zusanli acupoint straight needling about 1.5 mm, hand acupuncture stimulation 30 minutes, twirling every 5 minutes, once a day, continuous intervention for 7 days; In the electroacupuncture group, the bilateral Zusanli acupoints were pricked directly for about 1.5 mm, and electroacupuncture was stimulated for 30 minutes, alternately, the frequency was 2 / 50 Hz, the intensity was 2 Ma, once a day for 7 days, and the brain tissue was taken the next day after the intervention. The expression of P-ERK and NF-kB in hippocampus was detected by immunohistochemistry and Western blotting. Results 1. Morris water maze: compared with wild-type mice, the latency of Terc-r-mice increased with the increase of age, and the target quadrant time ratio of escape latency decreased gradually. There was no significant difference between the two groups at the age of 2 and 5 months, but there was a significant difference between the two groups at the age of 7 and 9 months. Autonomous activity test: compared with wild type mice, there was no significant difference in autonomous activity between the two groups at the age of 2 and 5 months, but there was a significant difference between the two groups at the age of 7 and 9 months. Aging score: compared with wild-type mice, there was no significant difference in the scores of Terc-r-mice at the age of 2 to 3 months, but significantly increased at the age of 5 and 9 months. The results of immunohistochemical method showed that the positive surface density of TRKB protein in the hippocampal area of Terc-r-mice after intervention was 0.0233 卤0.007 in blank group, 0.0312 卤0.005 in hand acupuncture group, 0.0363 卤0.007 in electroacupuncture group and 0.0363 卤0.007 in electroacupuncture group. The positive target surface density of ERK protein in hippocampal area of mice was significantly higher than that in control group (0.0148 卤0.074), hand acupuncture group (0.0270 卤0.010), electroacupuncture group (0.0201 卤0.008). The difference between hand acupuncture group and blank group was significant (p 0.05. 5). The results of Western blotting showed that the relative volume of TRKB protein in hippocampal area of Terc-/ mice after intervention was 0.6940 卤0.249, 0.9525 卤0.139 and 0.9801 卤0.129 in the control group, 0.9801 卤0.129 in the electroacupuncture group, respectively. The comparison between the hand acupuncture group and the electroacupuncture group was compared with that in the blank group. The relative volume of NF-kB protein in hippocampal area of mice was significantly different from that in control group (0.2891 卤0.070), hand acupuncture group (0.4066 卤0.126) and electroacupuncture group (0.4856 卤0.066). Conclusion 1. Compared with the wild-type mice of the same age, the behavior of Terc-r-mice increased significantly with age. The aging characteristics of Terc-r-mice were closely related to their age. The older the age, the greater the speed and degree of senescence, especially after the age of 7 months. Acupuncture at "Zusanli" (Zusanli) could promote the expression of TRKBERK and NF-KB in the hippocampal area of Terc-r-mice, but had no significant effect on wild-type mice.
【学位授予单位】：福建中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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