以Triton X-100和脱氧胆酸钠为萃取剂制备兔脱细胞神经基质:有最佳时间吗?

Preparation of acellular nerve matrix using Trito X-100 and sodium deoxycholate as extracting agent: Is there an optimal time?

背景：与其他脱细胞神经基质制备方法相比，化学萃取的去细胞同种异系（体）神经组织内细胞成分可以较完全地清除，进一步减少了发生免疫排斥反应的可能性，并能较好地保留神经支架的完整性，但制备过程中的相关问题有许多尚需讨论。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面神经，提出脱细胞神经基质制备的所需试剂及最佳时间。设计、时间及地点：随机分组，对比观察细胞学实验，于2009-02/06在滨州医学院附属医院口腔科学实验室完成。材料：3月龄新西兰大白兔15 只，体质量2.5~3.0 kg。Triton X-100、脱氧胆酸钠由美国Sigma 公司提供。方法：取兔双侧面神经，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12 h（为1个周期），室温下反复振荡1~5个周期。主要观察指标：常规苏木精-伊红染色，光镜下观察去细胞程度及纤维管道结构的完整性。结果：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，且许旺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，保留完整的许旺细胞基底膜，周边可见神经外膜和束膜。结论：大白兔面神经经 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。

BACKGROUND： Compared to other preparation method, chemical extraction can almost removed all cellular components, reduce the possibility of immunological rejection, and remain the integrality of nerve graft. However, there are still problems need to be explored. OBJECTIVE： To investigate the optimal condition of acellular nerve graft using Trito X-100 and sodium deoxycholate as extracting agent. DESIGN, TIME AND SETTING： Randomized grouping, controlled cytology observation. The experiment was performed at the Department of Stomatology, Affiliated Hospital of Binzhou Medical University, from February to June 2009. MATERIALS： Fifteen New Zealand white rabbits, aged 3-4 months, weighing 2.5-3.0 kg. Triton X-100 and sodium deoxycholate were provided by Sigma Company, USA. METHODS： The bilateral facial nerve were obtained from rabbits, and removed the adipose tissue and epineurium of the nerve surface under the surgery microscope, then divided these nerves into 66 segments, with each length of 10 mm. The 66 neurons were randomly divided into 11 groups, with 6 neurons in each group. Except the control group, all neurons were placed into Petri dish for 12 hours bathing using distilled water at room temperature, then 5 groups of which were cultured with Triton X-100 for 12, 24, 36, 48, and 60 hours, oscillation at room temperature; the remained 5 groups were cultured with 3% Triton X-100 for 12 hours, followed by 4% sodium deoxycholate for 12 hours, repeated for 1-5 cycles. MAIN OUTCOME MEASURES： Haematoxylin-eosin staining; degrees of decellularization and integrality of fiber pipe. RESULTS： Only use Triton X-100 to deal with the nerve of New Zealand white rabbits, even if 60 hours, could not to remove all the cellular components, and the basement membrane of Schwann cells were greatly destroyed. After 2 cycles treatment of Trito X-100 combined with sodium deoxycholate, cellular components and myelin sheath of nerve fibers and axons were removed effectively, and basement membrane of Schwann cell was remained, with epineurium and perineurium could be seen. CONCLUSION： Oscillation accompanied by 2 cycles treatment of Trito X-100 and sodium deoxycholate can obtain acellular nerve graft by removing cellular components completely, and reserving integrated basement membrane of Schwann cells.