【摘要】：實(shí)驗(yàn)?zāi)康? 應(yīng)用Triton X-100和脫氧膽酸鈉化學(xué)萃取劑處理新西蘭大白兔的坐骨神經(jīng),探索脫細(xì)胞神經(jīng)基質(zhì)制備的所需試劑劑最佳時(shí)間。用HE染色、固蘭染色、免疫組織化學(xué)方法、電鏡對(duì)其成分進(jìn)行分析;并對(duì)所得的數(shù)據(jù)進(jìn)行分析、整理,得出最佳作用時(shí)間;然后將按最佳作用時(shí)間制備好的ANG埋植于新西蘭大白兔的肌肉組織內(nèi)以檢驗(yàn)其組織相容性,為臨床篩選組織相容性良好的周?chē)窠?jīng)細(xì)胞外基質(zhì)支架,修復(fù)面神經(jīng)缺損提供實(shí)驗(yàn)基礎(chǔ)。 實(shí)驗(yàn)方法: 實(shí)驗(yàn)一:制備脫細(xì)胞神經(jīng)基質(zhì)。取15只健康成年新西蘭大白兔雙側(cè)坐骨神經(jīng),在手術(shù)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(zhǎng),共66條。將66條神經(jīng)隨機(jī)分成11組,每組6條。除正常對(duì)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(xì)胞和髓鞘在低滲液體中膨脹,使部分細(xì)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(gè)周期),室溫下反復(fù)振蕩1~5個(gè)周期。分別對(duì)每組脫細(xì)胞神經(jīng)基質(zhì)從脫細(xì)胞程度、纖維管道完整性、髓鞘染色程度三方面進(jìn)行評(píng)分,以方差分析進(jìn)行統(tǒng)計(jì)檢驗(yàn),設(shè)P0.05時(shí)結(jié)果有統(tǒng)計(jì)學(xué)意義。 實(shí)驗(yàn)二:組織相容性檢驗(yàn)。選用成年新西蘭大白兔6只,雌雄各半,沿雙側(cè)下肢外側(cè)各做4條縱行切口,深達(dá)肌層,將制備好的ANM分別包埋于切開(kāi)的肌肉中,每個(gè)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(xué)觀(guān)察,檢測(cè)其有無(wú)排異反應(yīng)、周?chē)荛L(zhǎng)入情況、和各種炎細(xì)胞浸潤(rùn)程度。 實(shí)驗(yàn)結(jié)果: 1.單獨(dú)應(yīng)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(xì)胞成分去除,且施萬(wàn)細(xì)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(gè)周期,可有效地去除神經(jīng)中的細(xì)胞成分及神經(jīng)纖維髓鞘和軸突,保留完整的施萬(wàn)細(xì)胞基底膜,周邊可見(jiàn)神經(jīng)外膜和束膜。 2.脫細(xì)胞神經(jīng)肌肉包埋后,傷口無(wú)紅腫、滲液等炎性反應(yīng)。術(shù)后1周取材時(shí)可見(jiàn)脫細(xì)胞神經(jīng)支架顏色稍紅,輕微炎性反應(yīng),炎細(xì)胞以中性粒細(xì)胞為主;術(shù)后2～3周可見(jiàn)脫細(xì)胞神經(jīng)支架與周?chē)M織黏附,不易分開(kāi),炎性細(xì)胞明顯減少,可見(jiàn)成纖維細(xì)胞及血管內(nèi)皮細(xì)胞生長(zhǎng),新生血管開(kāi)始形成;術(shù)后3～4周可見(jiàn)去細(xì)胞神經(jīng)支架與周?chē)M織黏附緊密,取出時(shí)周?chē)鲅黠@,未見(jiàn)炎細(xì)胞浸潤(rùn),新生血管較前增多。 實(shí)驗(yàn)結(jié)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(gè)周期,并不停地振蕩,可完全去除神經(jīng)組織中的所有細(xì)胞成分,保留完整的許旺細(xì)胞基底膜,得到脫細(xì)胞的神經(jīng)基質(zhì)。 2.其植入的臨近部位無(wú)急性炎癥反應(yīng),無(wú)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(xì)胞和血管內(nèi)皮細(xì)胞向ECM內(nèi)生長(zhǎng),形成較多富含紅細(xì)胞的血管;脫細(xì)胞神經(jīng)基質(zhì)在此過(guò)程中可引導(dǎo)再生細(xì)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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