feration, DNA synthesis and apoptosis of three kinds of human pituitary adenoma (human prolactinoma, somatotropinoma and non-functional tumor) cells cultured in vitro. The results showed that (1) the positive staining of ChAT was obviously observed in the cells of the three kinds of human pituitary adenoma, however, it was lower than that in normal human pituitary gland; (2) ACh had a similar effect on the proliferation of the three kinds of human pituitary adenoma cells. ACh at 0.1～10 μmol/L decreased the ［3H］TdR incorporation and the MTT A value in a dose-dependent manner. At the same time, ACh decreased the ratio of S or G2 phase pituitary adenoma cells significantly, but increased the ratio of G1 phase pituitary tumour cells markedly; (3) the effect of acetylcholine on the proliferation of human pituitary adenoma cells was inhibited by atropine, but not by tubocurarine; (4) ACh had no effect on the apoptosis of human pituitary adenoma cells cultured in vitro. These data suggest that ACh may have a significant modulating effect on the proliferation of pituitary adenoma cells by means of paracrine or autocrine, and the effect is mediated by muscarinic receptor.
Key words: acetylcholine; pituitary gland; adenoma; proliferation; apoptosis
垂体腺瘤是临床内分泌系统和神经外科中的常见病变, 发病率约占颅内肿瘤的15％, 绝大多数为良性肿瘤, 但有三分之一为侵袭性的, 有一少部分(约1％)可转变为恶性肿瘤［1］。因此, 深入研究垂体腺瘤的发病机制及影响因素有着重要的意义。目前认为, 垂体腺瘤是由于垂体细胞首先发生突变(编码G蛋白α亚单位的基因存在突变), 然后在内外因素的促进下突变的细胞增生, 发展为垂体腺瘤。下丘脑的促激素和垂体内的旁分泌因子可能在垂体腺瘤形成的促进阶段起重要作用［2］。这些旁分泌因子包括乙酰胆碱(acetylcholine, ACh)、激活素、神经肽、生长因子、细胞因子、催乳素片段等。
研究表明, 多种动物的正常垂体前叶细胞及肿瘤细胞都可以合成并分泌ACh［3~5］。但是, 人垂体腺瘤细胞是否也可以合成、分泌ACh尚无直接的证据。而且, 虽然早在80年代初就有学者开始研究ACh与腺垂体功能的关系, 但到目前为止, 关于ACh作用的研究基本限于ACh对垂体和对垂体腺瘤细胞系的分泌功能的影响。由于实验方法的不同以及所使用的实验对象的不同, 众多学者对所观察到的结果尚未取得一致性的认识［6~8］。关于ACh对垂体前叶细胞增殖代谢的影响更无直接研究资料, 仅Fedotov观察了ACh对不同年龄组大鼠垂体细胞总蛋白及DNA合成的影响, 结果发现, ACh可减少青春期大鼠垂体细胞总蛋白合成, 但对新生鼠无影响, 而且ACh对新生鼠垂体细胞DNA合成亦无影响［9］。因此, 本实验通过免疫组化观察人的正常垂体前叶细胞及人无功能瘤、催乳素瘤和生长激素瘤等三种垂体腺瘤细胞的胞浆内是否存在合成ACh的限速酶胆碱乙酰转移酶(choline acetyl transferase, ChAT), 并利用多种手段探讨ACh对培养的人三种垂体腺瘤细胞增殖及凋亡的影响。
1材料和方法
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