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目的研究UW液低温保存方法对大鼠坐骨神经组织结构、细胞活性及免疫原性的影响。方法 SD大鼠坐骨神经4℃条件下用UW液保存(UW组),分别保存1、4、6周,以新鲜坐骨神经作对照(对照组)。HE染色和透射电镜扫描观察组织结构改变;LIVE/DEAD Viability/Cytotoxicity试剂盒和TUNEL法分别评估细胞活性和凋亡程度;Western blot法检测细胞间黏附分子-1(ICAM-1)和主要组织相容性复合体Ⅱ(MHCⅡ)的表达以了解免疫原性改变。结果各保存时相点UW组神经外膜和雪旺细胞基底膜结构均较完整,与对照组无明显差别。各UW组细胞活性程度降低,TUNEL阳性细胞数增多,ICAM-1和MHCⅡ表达降低,与对照组相比均有明显统计学差异(P<0.05);且4、6周组TUNEL阳性细胞数均高于1周组(P<0.05),ICAM-1和MHCⅡ表达均低于1周组(P<0.05),但4、6周组间均未见明显差异。结论 UW液低温保存方法能有效维持周围神经组织神经外膜和雪旺细胞基底膜结构完整性,并降低保存神经的免疫原性,但有效维持神经组织细胞活性的时限不超过1周。
Objective To investigate the effect of cryopreservation of UW solution on the structure, cell viability and immunogenicity of sciatic nerve in rats. Methods SD rat sciatic nerve was stored in UW solution (UW group) at 4 ℃ for 1, 4 and 6 weeks, respectively. Fresh sciatic nerve was used as a control (control group). The changes of tissue structure were observed by HE staining and transmission electron microscopy. The cell viability and apoptosis were evaluated by LIVE / DEAD Viability / Cytotoxicity kit and TUNEL assay respectively. The expressions of ICAM-1 and major histological phases Capacitive Complex II (MHC II) Expression to Understand Immunogenicity. Results At the time of preservation, the structures of basilar membrane of Schwann cells and epineurium in UW group were all intact, and no significant difference with control group. The level of TUNEL positive cells and the expression of ICAM-1 and MHCⅡ in UW group were significantly lower than those in control group (P <0.05), and the number of TUNEL positive cells in 4 and 6 weeks groups The expression of ICAM-1 and MHCⅡ were lower than those of the 1-week group (P <0.05), but no significant difference was found between the 4 and 6-week groups. Conclusion The cryopreservation of UW solution can effectively maintain the structural integrity of the peripheral nerve tissue and Schwann cell basilar membrane and reduce the immunogenicity of nerve preservation, but the effective time to maintain the activity of nerve tissue cells is less than one week.