本文選題：法醫(yī)病理　切入點(diǎn)：骨骼肌挫傷　出處：《中國醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的 內(nèi)源性大麻素系統(tǒng)是普遍存在的脂質(zhì)類信號系統(tǒng),在所有脊椎動物中起著重要的調(diào)節(jié)功能,主要由大麻素受體(cannabinoid receptor,CBR)、內(nèi)源性大麻素(endocannabinoid,EC)及合成與降解內(nèi)源性大麻素的酶組成。目前已經(jīng)明確克隆出的CBR包括1型大麻素受體(cannabinoid 1 receptor,CB1R)和2型大麻素受體(cannabinoid 2 receptor,CB2R)兩個亞型。CBR通過與G蛋白偶聯(lián)后,調(diào)節(jié)某些信號傳導(dǎo)通路,如抑制腺苷酸環(huán)化酶(adenylyl cyclase,AC),調(diào)節(jié)離子轉(zhuǎn)運(yùn),激活粘著斑激酶、絲裂原活化蛋白激酶和胞漿型磷脂酶A_2,促進(jìn)NO的產(chǎn)生等,并與細(xì)胞凋亡的發(fā)生密切相關(guān)。本實驗在建立大鼠骨骼肌挫傷模型的基礎(chǔ)上,應(yīng)用免疫組織化學(xué)染色和Western blot方法,對大鼠骨骼肌挫傷愈合過程中磷酸化CB1R(phospho-cannabinoid 1 receptor,p-CB1R)的動態(tài)變化及其表達(dá)的時間變化規(guī)律在推斷損傷時間中的可應(yīng)用性進(jìn)行了探討。 實驗材料與方法 健康成年雄性Sprague-Dawley(SD)大鼠50只,體重220～250g,適應(yīng)性飼養(yǎng)1周后,將其隨機(jī)分為10組,每組5只,其中9組為實驗組,一組為正常對照組。乙醚吸入性麻醉后,將大鼠后肢置于伸膝、踝背屈90°位置,用自由落體式鈍力打擊器,以3m/s的速度和壓深調(diào)控桿超出封閉螺扣7mm的長度(即形變量),一次性打擊大鼠右后肢距跟骨2cm處右下肢部位,致骨骼肌挫傷。分別于傷后3h、6h、12h、1d、3d、5d、7d、10d和14d將大鼠脫頸椎致死,取右下肢腓腸肌。正常對照組大鼠未經(jīng)打擊,取相同部位同等大小腓腸肌,以備HE染色、免疫組織化學(xué)染色和Western blot檢測應(yīng)用。 應(yīng)用免疫組織化學(xué)和Western blot方法檢測50例大鼠腓腸肌挫傷后各時間段p-CB1R變化情況,以非挫傷組大鼠腓腸肌作對照,顯微鏡×400倍下,在挫傷區(qū)及挫傷周邊區(qū)隨機(jī)選取10個視野,計數(shù)多核粒細(xì)胞、單個核細(xì)胞及成纖維細(xì)胞的細(xì)胞總數(shù)、陽性細(xì)胞數(shù)及陽性細(xì)胞率。應(yīng)用Motic Images Advanced 3.2軟件測各片段的平均灰度值,測得數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示。用SPSS13.0 forWindows進(jìn)行單因素方差分析,以P＜0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果 正常對照組大鼠骨骼肌未見p-CB1R表達(dá)。挫傷后各時間段大鼠骨骼肌未見p-CB1R表達(dá),主要在單個核細(xì)胞和成纖維細(xì)胞中呈陽性反應(yīng)。傷后3h～6h,骨骼肌間質(zhì)內(nèi)可見少量多核粒細(xì)胞和單個核細(xì)胞浸潤,多核粒細(xì)胞未見p-CB1R表達(dá),僅個別單個核細(xì)胞可見p-CB1R陽性;傷后12h,大量多核粒細(xì)胞的和少量單個核細(xì)胞浸潤,p-CB1R在單個核細(xì)胞中呈陽性;傷后1d,隨著單個核細(xì)胞的增多,p-CB1R陽性細(xì)胞數(shù)量也隨之增加;傷后3d～5d,單個核細(xì)胞數(shù)量逐漸減少,成纖維細(xì)胞數(shù)量逐漸增加,p-CB1R陽性細(xì)胞主要以單個核細(xì)胞和成纖維細(xì)胞為主;傷后7d～10d,單個核細(xì)胞數(shù)量顯著減少,p-CB1R陽性反應(yīng)主要見于成纖維細(xì)胞,并于7d時達(dá)到高峰;傷后14d,成纖維細(xì)胞數(shù)量減少,p-CB1R表達(dá)量也隨之減少。Western blot結(jié)果顯示實驗組各時間段p-CB1R表達(dá)強(qiáng)度有一定規(guī)律,傷后3h～12h,p-CB1R表達(dá)量較少;傷后1d～3d,p-CB1R表達(dá)逐漸增多;傷后5d,p-CB1R有所下降;傷后7d,p-CB1R達(dá)到高峰;傷后10d,隨著時間的延長,p-CB1R表達(dá)逐漸下降;傷后14d,p-CB1R表達(dá)量進(jìn)一步下降。各組間平均灰度值差異均具有統(tǒng)計學(xué)意義(P＜0.05)。 結(jié)論 1、正常對照組大鼠骨骼肌未見p-CB1R表達(dá)。 2、挫傷后各時間段大鼠骨骼肌均未見p-CB1R表達(dá)。骨骼肌間質(zhì)中浸潤的多核粒細(xì)胞未見p-CB1R表達(dá),單個核細(xì)胞和成纖維細(xì)胞可見p-CB1R表達(dá),提示p-CB1R可能參與骨骼肌挫傷后的愈合過程。
[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.

【學(xué)位授予單位】：中國醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：D919

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