Mechanism for regulation of mucin secretion from intestinal goblet cells in parasitic infection

本研究旨在探讨肠上皮杯状细胞产生或分泌粘蛋白的调控机制。我们开发了一种方法来测量肠粘蛋白使用特异性凝集素结合粘蛋白。感染巴西新单胞菌（Nb）的C57 BL/6小鼠的肠粘蛋白量在感染后第7天达到峰值，然后在第10天降至正常水平。肠粘蛋白水平的变化与肠杯状细胞数的变化及排虫情况有较好的对应关系。Nb感染裸鼠的肠粘蛋白含量虽缓慢增加，但最大值与正常小鼠相近。在Nb感染的IL-4基因敲除小鼠中，肠粘蛋白水平与对照小鼠相比没有变化。腹腔注射Nb抗原、抗CD 4抗体或环磷酰胺均未改变Nb感染小鼠的肠粘蛋白。迄今为止，还没有发现能在体内改变Nb感染小鼠肠道粘蛋白水平的方法。从未感染的小鼠分离的肠细胞，当在体外培养与Con A，分泌更多的粘蛋白在培养基中。我们未能找到一个RT-PCR检测大鼠或小鼠肠道粘蛋白核心蛋白mRNA的条件。RT-PCR检测到人结肠癌细胞株LS 174 T中存在人粘蛋白核心蛋白（MUC 2）的mRNA表达，重组人TNF-α可促进LS 174 T细胞MUC 2 mRNA的表达。用重组人IL-13培养LS 174 T细胞刺激粘蛋白产生而不增加细胞数量。此外，用RT-PCR方法检测到LS 174 T细胞上存在IL- 13受体，为进一步研究LS 174 T细胞分泌粘蛋白的调控机制奠定了基础。

We examined methods of measuring mucin to study on the regulatory mechanism of mucin production or secretion from intestinal goblet cells. We developed a method to measure intestinal mucin using specific lectin binding to mucin. Amount of intestinal mucin in N.brasiliensis (Nb)-infected C57BL/6 mice was peaked on day 7 post-infection and then decreased to normal level on day 10. The change of intestinal mucin level corresponded well to change of intestinal goblet ceilnumber and worm expulsion. Although the intestinal mucin increased slowly in Nb infected-athymic nude mice, maximum amount of the mucin was almost similar to that of euthymic mice. In Nb-infected IL-4 gene knockout mice, intestinal mucin level was not changed from that in control mice. Intraperitoneal injection of Nb antigens, anti-CD4 antibodies or cyclophosphamide did not change intestinal mucin in Nb-infected mice. So far, no method have been found to change intestinal mucin level in Nb-infected mice in vivo. Separated intestinal cells from uninfected mice, when cultured in vitro with Con A, secreted more mucin in the culture medium. We failed to find a RT-PCR condition for detecting mRNA of mucin core protein in rat or mouse intestine. However, we could detected the mRNA of human mucin core protein (MUC2) by RT-PCR in the mucin producing human colon carcinoma cell line, LS174T.Addition of recombinant human TNF-alpha increased MUC2 mRNA expression in LS174T cells. Culture of LS174T cells with recombinant human IL-13 stimulated mucin production without increasing cell number. Furthermore, IL- 13 receptor was detected on LS 174T cells by RT-PCR.The experiment using LS 174T cells will facilitate the studies on regulation mechanism of mucin production.