磷酸化CB1R在大鼠骨骼肌挫伤愈合过程中表达及时间规律性研究

发布时间：2018-04-10 07:35

本文选题：法医病理　切入点：骨骼肌挫伤　出处：《中国医科大学》2009年硕士论文

【摘要】： 目的内源性大麻素系统是普遍存在的脂质类信号系统,在所有脊椎动物中起着重要的调节功能,主要由大麻素受体(cannabinoid receptor,CBR)、内源性大麻素(endocannabinoid,EC)及合成与降解内源性大麻素的酶组成。目前已经明确克隆出的CBR包括1型大麻素受体(cannabinoid 1 receptor,CB1R)和2型大麻素受体(cannabinoid 2 receptor,CB2R)两个亚型。CBR通过与G蛋白偶联后,调节某些信号传导通路,如抑制腺苷酸环化酶(adenylyl cyclase,AC),调节离子转运,激活粘着斑激酶、丝裂原活化蛋白激酶和胞浆型磷脂酶A_2,促进NO的产生等,并与细胞凋亡的发生密切相关。本实验在建立大鼠骨骼肌挫伤模型的基础上,应用免疫组织化学染色和Western blot方法,对大鼠骨骼肌挫伤愈合过程中磷酸化CB1R(phospho-cannabinoid 1 receptor,p-CB1R)的动态变化及其表达的时间变化规律在推断损伤时间中的可应用性进行了探讨。实验材料与方法健康成年雄性Sprague-Dawley(SD)大鼠50只,体重220～250g,适应性饲养1周后,将其随机分为10组,每组5只,其中9组为实验组,一组为正常对照组。乙醚吸入性麻醉后,将大鼠后肢置于伸膝、踝背屈90°位置,用自由落体式钝力打击器,以3m/s的速度和压深调控杆超出封闭螺扣7mm的长度(即形变量),一次性打击大鼠右后肢距跟骨2cm处右下肢部位,致骨骼肌挫伤。分别于伤后3h、6h、12h、1d、3d、5d、7d、10d和14d将大鼠脱颈椎致死,取右下肢腓肠肌。正常对照组大鼠未经打击,取相同部位同等大小腓肠肌,以备HE染色、免疫组织化学染色和Western blot检测应用。应用免疫组织化学和Western blot方法检测50例大鼠腓肠肌挫伤后各时间段p-CB1R变化情况,以非挫伤组大鼠腓肠肌作对照,显微镜×400倍下,在挫伤区及挫伤周边区随机选取10个视野,计数多核粒细胞、单个核细胞及成纤维细胞的细胞总数、阳性细胞数及阳性细胞率。应用Motic Images Advanced 3.2软件测各片段的平均灰度值,测得数据用均数±标准差((?)±s)表示。用SPSS13.0 forWindows进行单因素方差分析,以P＜0.05为差异有统计学意义。结果正常对照组大鼠骨骼肌未见p-CB1R表达。挫伤后各时间段大鼠骨骼肌未见p-CB1R表达,主要在单个核细胞和成纤维细胞中呈阳性反应。伤后3h～6h,骨骼肌间质内可见少量多核粒细胞和单个核细胞浸润,多核粒细胞未见p-CB1R表达,仅个别单个核细胞可见p-CB1R阳性;伤后12h,大量多核粒细胞的和少量单个核细胞浸润,p-CB1R在单个核细胞中呈阳性;伤后1d,随着单个核细胞的增多,p-CB1R阳性细胞数量也随之增加;伤后3d～5d,单个核细胞数量逐渐减少,成纤维细胞数量逐渐增加,p-CB1R阳性细胞主要以单个核细胞和成纤维细胞为主;伤后7d～10d,单个核细胞数量显著减少,p-CB1R阳性反应主要见于成纤维细胞,并于7d时达到高峰;伤后14d,成纤维细胞数量减少,p-CB1R表达量也随之减少。Western blot结果显示实验组各时间段p-CB1R表达强度有一定规律,伤后3h～12h,p-CB1R表达量较少;伤后1d～3d,p-CB1R表达逐渐增多;伤后5d,p-CB1R有所下降;伤后7d,p-CB1R达到高峰;伤后10d,随着时间的延长,p-CB1R表达逐渐下降;伤后14d,p-CB1R表达量进一步下降。各组间平均灰度值差异均具有统计学意义(P＜0.05)。结论 1、正常对照组大鼠骨骼肌未见p-CB1R表达。 2、挫伤后各时间段大鼠骨骼肌均未见p-CB1R表达。骨骼肌间质中浸润的多核粒细胞未见p-CB1R表达,单个核细胞和成纤维细胞可见p-CB1R表达,提示p-CB1R可能参与骨骼肌挫伤后的愈合过程。
[Abstract]:objective
The endocannabinoid system is lipid signaling system that exists in all vertebrates plays an important regulatory function, mainly by cannabinoid receptors (cannabinoid, receptor, CBR) endocannabinoids (endocannabinoid, EC) and the synthesis and degradation of endogenous enzymes. The large apocynin has been cloned CBR including type 1 cannabinoid receptor (cannabinoid 1 receptor, CB1R) and cannabinoid receptor type 2 (cannabinoid 2 receptor, CB2R) two subtypes of.CBR with G protein coupled after adjusting some signal transduction pathways, such as inhibition of adenylate cyclase (adenylyl cyclase, AC), the regulation of ion transport, activation of focal adhesion kinase, mitogen activated protein kinase and cytosolic phospholipase A_2, promote the production of NO, and have close relationship with cell apoptosis. In this experiment the establishment of rat skeletal muscle contusion model, immunohistochemical The dynamic changes of phosphorylated CB1R (phospho-cannabinoid 1 receptor, p-CB1R) in the process of skeletal muscle contusion healing in rats and the applicability of the time variation rule of their expression in the time of injury were studied by Western and blot staining.
Experimental materials and methods
Healthy adult male Sprague-Dawley (SD) 50 rats, weighing 220 ~ 250g, adaptive feeding for 1 weeks, they were randomly divided into 10 groups, 5 rats in each group, including 9 experimental groups, one group was the normal control group. Ether inhalation anesthesia, the rats in the hind knee, ankle 90 degrees of dorsiflexion position, striking device for freefall blunt force at a speed of 3m/s, and the pressure regulating screw rod deep closed beyond the length of the 7mm (i.e. shape variables), a single blow right hind limb of rats from the right calcaneus 2cm lower limbs, causing skeletal muscle contusion injury respectively. After 3h, 6h. 12h, 1D, 3D, 5D, 7d, 10d and 14d rats by cervical dislocation to death, take the right gastrocnemius muscle. The rats in the normal control group without a blow from the same location in the same size of gastrocnemius, for HE staining, immunohistochemical staining and Western blot detection.
Detection of immunohistochemistry and Western blot methods in 50 cases of rat gastrocnemius muscle contusion in different time after the changes of p-CB1R in rat gastrocnemius muscle contusion than controls, * 400 times under the microscope, randomly selected 10 fields in the surrounding area of contusion zone and contusion, count of polymorphonuclear cells, mononuclear cells and the total number of cells into cells, the number of positive cells and positive cell rate. The average gray Motic Images application software Advanced 3.2 measurement of each fragment value measured by standard deviation ((?) + s). Single factor analysis of variance with SPSS13.0 forWindows, P < 0.05 differences statistical significance.
Result
No p-CB1R normal control group rats skeletal muscle expression. The expression in different time after contusion of rat skeletal muscle was p-CB1R, mainly in the mononuclear cells and positive fiber cells. After injury 3H ~ 6h, skeletal muscle interstitial showed a few polymorphonuclear cells and infiltrating mononuclear cells, expression of nuclear granulocyte no p-CB1R, only a few mononuclear cells showed p-CB1R positive; 12h after injury, a large number of polymorphonuclear cells and a small amount of infiltrating mononuclear cells, p-CB1R in mononuclear cells were positive; 1D after injury, with the increase of mononuclear cells, p-CB1R positive cells were also increased after injury to 3D; 5D, the number of mononuclear cells gradually decreased, the number of fibroblasts increased, p-CB1R positive cells mainly mononuclear cells and fibroblasts; after injury 7d ~ 10d, the number of mononuclear cells was significantly reduced, p-CB1R positive reaction mainly in fibroblasts The cell, and reach the peak in the 7d; 14d after injury, the number of fibroblasts decreased, p-CB1R expression was also reduced.Western blot results showed that the experimental group while the expression of p-CB1R is regular strength 3h after injury to 12h, the expression of p-CB1R was less; after injury 1D ~ 3D, p-CB1R expression gradually increased; injury after 5D, p-CB1R decreased; 7d after injury, reached the peak at p-CB1R; 10d after injury, with the extension of time, the expression of p-CB1R decreased gradually; 14d after injury, the expression of p-CB1R decreased further. The average gray value differences between the groups were statistically significant (P < 0.05).
conclusion
1, no p-CB1R expression was found in skeletal muscle of normal control rats.
2, no expression of p-CB1R was found in skeletal muscle of rats at any time after contusion. No expression of p-CB1R was observed in multinucleated granulocytes from skeletal muscle stroma, and p-CB1R expression was observed in mononuclear cells and fibroblasts, suggesting that p-CB1R may participate in healing process after skeletal muscle contusion.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：D919

【参考文献】