用于针刺穴位研究的结缔组织片模型

摘要

目的:先前研究发现,穴区功能与穴区结缔组织结构有密切关系,本文旨在建立基于穴区原位组织上的膜片钳研究方法.方法:急性分离出SD大鼠"后三里"穴皮下结缔组织,并安置在含有孵育液的浴槽内.组织片内肥大细胞可用甲苯胺蓝或中性红进行标记.在膜片钳实验中,通过记录电极对组织片内原位肥大细胞施加-30、-60和-90 cmH_2O的负压刺激,并记录细胞上机械激活性全细胞电流.结果:制备的皮下结缔组织片标本经甲苯胺蓝或中性红染色显示,肥大细胞散在分布于组织片中的基质中.肥大细胞脱颗粒现象也能通过染色清晰可见.膜片钳记录表明穴区组织片肥大细胞的全细胞电流随着机械负压的施加而增大.结论:大鼠穴区皮下结缔组织片原位肥大细胞全细胞电流能被机械刺激激活.结合显微技术和电生理技术,大鼠"后三里"穴区皮下结缔组织片可以做为研究针刺效应外周机制的一种模型,并提示穴位肥大细胞可能参与针刺过程中机械信号的转导.该模型也可为今后进一步在细胞水平上研究穴位中细胞、胶原纤维、蛋白多糖等各成分的特性及其之间的相互作用提供基础.%Objective To establish an acupoint-connective tissue model for studying the mechanism of acupuncture by using in vitro patch clamp technique in the rat skin slices. Methods The local connective tissue under the corium of "Housanli" (ST 36) area from SD rat was acutely and bluntly separated and fixed in a chamber filled with artificial incubation solution. Mast cells in the prepared connective tissue slice were labeled by toluidine blue (TB) or neutral red (NR). The whole-cell current of mast cells responding to pressure stimulation applied through a patch pipette was recorded in rat slices derived from acupoint ST 36 area by using in vitro patch-clamp technique. Results 1) After staining with TB and NR, the labeled mast cells were found to distribute in the extracellular matrix of the connective tissue samples, and their degranulation phenomenon could be seen clearly. 2) The whole-cell current of mast cells in response to mechanical stress stimulation was successfully recorded in the connective tissue slices of the rat acupoint ST 36 area. The cellular membrane currents increased evidently when pressure gradients of -30, -60 or -90 cmH_20 were applied to the recorded mast cells. Conclusion The connective tissue slice from the rat ST 36 area may be used as a model for investigating the peripheral mechanism of acupuncture by combining the microtechniques and electrophysiological techniques. The results obtained in this model prove for the first time by electrophysiology that the mast cells in the connective tissue are probably involved in the transduction process of the mechanical signal from acupuncture stimulation. This new model provides a base for investigating the characters of the cells, collagen fibers, proteoglycans, etc. and their interactions in the acupoint connective tissue in the future.