【摘要】：實驗目的: 應用Triton X-100和脫氧膽酸鈉化學萃取劑處理新西蘭大白兔的坐骨神經(jīng),探索脫細胞神經(jīng)基質制備的所需試劑劑最佳時間。用HE染色、固蘭染色、免疫組織化學方法、電鏡對其成分進行分析;并對所得的數(shù)據(jù)進行分析、整理,得出最佳作用時間;然后將按最佳作用時間制備好的ANG埋植于新西蘭大白兔的肌肉組織內以檢驗其組織相容性,為臨床篩選組織相容性良好的周圍神經(jīng)細胞外基質支架,修復面神經(jīng)缺損提供實驗基礎。 實驗方法: 實驗一:制備脫細胞神經(jīng)基質。取15只健康成年新西蘭大白兔雙側坐骨神經(jīng),在手術顯微鏡下去除神經(jīng)表面的脂肪組織和神經(jīng)外膜,將其分為每段10 mm長,共66條。將66條神經(jīng)隨機分成11組,每組6條。除正常對照組外,其余10組均放入培養(yǎng)皿中,先用蒸餾水室溫下浸浴12 h ,以使細胞和髓鞘在低滲液體中膨脹,使部分細胞被脹破;然后將其中5組置于3% Triton X-100 12,24,36,48,60 h,室溫下振蕩,另外5組用3%Triton X-100和4%脫氧膽酸鈉先后作用12h(為1個周期),室溫下反復振蕩1~5個周期。分別對每組脫細胞神經(jīng)基質從脫細胞程度、纖維管道完整性、髓鞘染色程度三方面進行評分,以方差分析進行統(tǒng)計檢驗,設P0.05時結果有統(tǒng)計學意義。 實驗二:組織相容性檢驗。選用成年新西蘭大白兔6只,雌雄各半,沿雙側下肢外側各做4條縱行切口,深達肌層,將制備好的ANM分別包埋于切開的肌肉中,每個切口各置一條神經(jīng),分層縫合創(chuàng)口;分別在1、2、3、4周按自上而下的順序從每條創(chuàng)口取一條神經(jīng),HE染色后行組織學觀察,檢測其有無排異反應、周圍血管長入情況、和各種炎細胞浸潤程度。 實驗結果: 1.單獨應用Triton X 100處理神經(jīng)即使作用60 h ,仍不能將神經(jīng)中的所有細胞成分去除,且施萬細胞基底膜有較大破壞;Triton X 100配合脫氧膽酸鈉處理處理2個周期,可有效地去除神經(jīng)中的細胞成分及神經(jīng)纖維髓鞘和軸突,保留完整的施萬細胞基底膜,周邊可見神經(jīng)外膜和束膜。 2.脫細胞神經(jīng)肌肉包埋后,傷口無紅腫、滲液等炎性反應。術后1周取材時可見脫細胞神經(jīng)支架顏色稍紅,輕微炎性反應,炎細胞以中性粒細胞為主;術后2～3周可見脫細胞神經(jīng)支架與周圍組織黏附,不易分開,炎性細胞明顯減少,可見成纖維細胞及血管內皮細胞生長,新生血管開始形成;術后3～4周可見去細胞神經(jīng)支架與周圍組織黏附緊密,取出時周圍出血明顯,未見炎細胞浸潤,新生血管較前增多。 實驗結論: 1.大白兔坐骨神經(jīng)經(jīng)Triton X 100和脫氧膽酸鈉室溫處理2個周期,并不停地振蕩,可完全去除神經(jīng)組織中的所有細胞成分,保留完整的許旺細胞基底膜,得到脫細胞的神經(jīng)基質。 2.其植入的臨近部位無急性炎癥反應,無膿腫形成和組織壞死發(fā)生。 3.肌肉包埋后,宿主的成纖維細胞和血管內皮細胞向ECM內生長,形成較多富含紅細胞的血管;脫細胞神經(jīng)基質在此過程中可引導再生細胞定向排列。
[Abstract]:Objective of the experiment: The use of Triton X-100 and sodium deoxycholate chemical extractant to treat the sciatic nerve of New Zealand white rabbits and to explore the optimal reagent preparation for the preparation of acellular nerve matrix The components were analyzed by HE staining, solid-blue staining, immunohistochemical method and electron microscope, and the obtained data were analyzed, sorted, and the best effect was obtained. and then the ANG prepared according to the best action time is embedded in the muscle tissue of the New Zealand white rabbit to test the tissue compatibility of the rabbit, so as to provide an experimental basis for the clinical screening of the peripheral nerve extracellular matrix scaffold with good compatibility and the repair of the facial nerve defect. A. Real. Test method: Experiment 1: Preparation of Define Intracellular nerve matrix. 15 healthy adult New Zealand white rabbits with two-sided sciatic nerve were taken, and the adipose tissue and the outer membrane of the nerve surface were removed under the surgical microscope and divided into 10 mm in each segment. A total of 66. 66 nerves were randomly divided into 11 groups. 6. In addition to the normal control group, the remaining 10 groups were placed in a culture dish, the rest of the 10 groups were placed in a culture dish, and then the cells and the pulp were soaked in the low-permeability liquid at room temperature for 12 hours, so that the cells and the pulp fibers were expanded, and then the 5 groups were placed in 3% Triton X-100 12, 24, 36, 48, 60 h, The oscillation was carried out at room temperature. In the other 5 groups, 3% Triton X-100 and 4% sodium deoxycholate were used for 12h (1 cycle), and the oscillation was repeated at room temperature. The scores of the acellular nerve matrix in each group were measured from three aspects, such as the degree of decell, the integrity of the fiber and the degree of the staining of the pulp, and the statistical test was carried out by variance analysis, and the results were as follows: Statistical significance. Experiment 2: Tissue compatibility test. Six adult New Zealand white rabbits were selected. Six male and female half of the adult New Zealand white rabbits were divided into 4 longitudinal and deep muscle layers on the outer side of the lower limb of the double side. The prepared ANM was embedded in the cut muscle, each of which was set with a nerve. and carrying out histological observation after HE staining, detecting the presence or absence of a rejection reaction, a long-in condition of the surrounding blood vessels, and various inflammatory cell infiltration 紼嬪害. 瀹為獙緇撴灉: 1.鍗曠嫭搴旂敤Triton X 100澶勭悊紲炵粡鍗充嬌浣滅敤60 h ,浠嶄笉鑳藉皢紲炵粡涓殑鎵，

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