脱细胞神经基质的制备及组织相容性研究
发布时间：2018-12-15 03:28
【摘要】：实验目的: 应用Triton X-100和脱氧胆酸钠化学萃取剂处理新西兰大白兔的坐骨神经,探索脱细胞神经基质制备的所需试剂剂最佳时间。用HE染色、固兰染色、免疫组织化学方法、电镜对其成分进行分析;并对所得的数据进行分析、整理,得出最佳作用时间;然后将按最佳作用时间制备好的ANG埋植于新西兰大白兔的肌肉组织内以检验其组织相容性,为临床筛选组织相容性良好的周围神经细胞外基质支架,修复面神经缺损提供实验基础。 实验方法: 实验一:制备脱细胞神经基质。取15只健康成年新西兰大白兔双侧坐骨神经,在手术显微镜下去除神经表面的脂肪组织和神经外膜,将其分为每段10 mm长,共66条。将66条神经随机分成11组,每组6条。除正常对照组外,其余10组均放入培养皿中,先用蒸馏水室温下浸浴12 h ,以使细胞和髓鞘在低渗液体中膨胀,使部分细胞被胀破;然后将其中5组置于3% Triton X-100 12,24,36,48,60 h,室温下振荡,另外5组用3%Triton X-100和4%脱氧胆酸钠先后作用12h(为1个周期),室温下反复振荡1~5个周期。分别对每组脱细胞神经基质从脱细胞程度、纤维管道完整性、髓鞘染色程度三方面进行评分,以方差分析进行统计检验,设P0.05时结果有统计学意义。 实验二:组织相容性检验。选用成年新西兰大白兔6只,雌雄各半,沿双侧下肢外侧各做4条纵行切口,深达肌层,将制备好的ANM分别包埋于切开的肌肉中,每个切口各置一条神经,分层缝合创口;分别在1、2、3、4周按自上而下的顺序从每条创口取一条神经,HE染色后行组织学观察,检测其有无排异反应、周围血管长入情况、和各种炎细胞浸润程度。 实验结果: 1.单独应用Triton X 100处理神经即使作用60 h ,仍不能将神经中的所有细胞成分去除,且施万细胞基底膜有较大破坏;Triton X 100配合脱氧胆酸钠处理处理2个周期,可有效地去除神经中的细胞成分及神经纤维髓鞘和轴突,保留完整的施万细胞基底膜,周边可见神经外膜和束膜。 2.脱细胞神经肌肉包埋后,伤口无红肿、渗液等炎性反应。术后1周取材时可见脱细胞神经支架颜色稍红,轻微炎性反应,炎细胞以中性粒细胞为主;术后2～3周可见脱细胞神经支架与周围组织黏附,不易分开,炎性细胞明显减少,可见成纤维细胞及血管内皮细胞生长,新生血管开始形成;术后3～4周可见去细胞神经支架与周围组织黏附紧密,取出时周围出血明显,未见炎细胞浸润,新生血管较前增多。 实验结论: 1.大白兔坐骨神经经Triton X 100和脱氧胆酸钠室温处理2个周期,并不停地振荡,可完全去除神经组织中的所有细胞成分,保留完整的许旺细胞基底膜,得到脱细胞的神经基质。 2.其植入的临近部位无急性炎症反应,无脓肿形成和组织坏死发生。 3.肌肉包埋后,宿主的成纤维细胞和血管内皮细胞向ECM内生长,形成较多富含红细胞的血管;脱细胞神经基质在此过程中可引导再生细胞定向排列。
[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 绋嬪害. 瀹為獙缁撴灉: 1.鍗曠嫭搴旂敤Triton X 100澶勭悊绁炵粡鍗充娇浣滅敤60 h ,浠嶄笉鑳藉皢绁炵粡涓殑鎵，
本文编号：2379883
[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 绋嬪害. 瀹為獙缁撴灉: 1.鍗曠嫭搴旂敤Triton X 100澶勭悊绁炵粡鍗充娇浣滅敤60 h ,浠嶄笉鑳藉皢绁炵粡涓殑鎵，
本文编号：2379883
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