Lojda Z, Fric P
Laboratory of Histochemistry, 1st Faculty of Medicine, Charles University, Prague, Czech Republic.
Acta Histochem. 1996 Jul;98(3):285-93. doi: 10.1016/S0065-1281(96)80021-3.
Sucrase-isomaltase (SI), trehalase (T) and lactase-beta-glucosidase (LG) activities were assessed histochemically in samples of colorectal adenomas (11 tubular, 12 tubulovillous, 10 villous) and 30 adenocarcinomas obtained by biopsy during colonoscopy or from specimens removed by surgical intervention. Small samples of tumor tissue, tissue of the transitional zone and of macroscopically normal mucosa were quenched in heptan cooled in an acetone-dry ice mixture. Cryostat sections, transferred to non-precooled slides and in some cases to semipermeable membranes, were dried and subjected to the histochemical reactions for SI, T and LG. Sucrose, 2-naphthyl, 6-Br-2-naphthyl, and 5-Br-4-Cl-3-indoxyl alpha-D-glucosides, trehalose, and 5-Br-4-Cl-3-indoxyl-beta-D-fucoside were used as substrates. Sections of jejunal biopsies with normal activities of brush border glycosidases were used as controls. From samples of 5 adenomas, 5 adenocarcinomas and collected rests of jejunal biopsies with a normal finding 10% (w/vol) homogenates in 2% Triton X-100 were prepared. Homogenates were frozen and thawed 3 times and their supernatants subjected to isoelectric focusing on polyacrylamide gel plates. Zymograms were developed with the same methods as for the detection of alpha-glucosidases in sections. In no colorectal tumor LG was present. SI was found in 70% adenocarcinomas, 50% villous, 25% tubulovillous and 19% tubular adenomas when the method with sucrose, glucose oxidase-peroxidase and 3,3'-diaminobenzidine was used. Hardly discernible traces of activity were found in tumors with azo-coupling reactions applied at pH 5, 6 and 6.5. No reaction was detected with the indigogenic method applied at pH above 6.0. However, jejunal biopsies displayed very strong reactions confined to the brush border of enterocytes under the same conditions. A strongly positive reaction was seen in 7 of 12 tumors investigated recently when the indigogenic reaction was applied at pH below 6.0 (particularly at pH 5.0). In this case the deposition of indigo was due to membrane and lysosomal alpha-glucosidases of the tumor cells and lysosomal alpha-glucosidase of macrophages and leukocytes. These findings were corroborated by zymograms. T was detected in the same tumors as SI; its activity was lower, however. SI activity in colorectal tumors is a useful, but not general marker of these tumors.
采用组织化学方法评估了结肠镜检查活检获取的或手术切除标本中的11例管状、12例管状绒毛状和10例绒毛状大肠腺瘤以及30例腺癌样本中的蔗糖酶-异麦芽糖酶(SI)、海藻糖酶(T)和乳糖酶-β-葡萄糖苷酶(LG)活性。将肿瘤组织、过渡区组织和大体正常黏膜的小样本在丙酮-干冰混合物冷却的庚烷中淬灭。将低温恒温器切片转移至未预冷的载玻片上,在某些情况下转移至半透膜上,干燥后进行SI、T和LG的组织化学反应。使用蔗糖、2-萘基、6-溴-2-萘基和5-溴-4-氯-3-吲哚基α-D-葡萄糖苷、海藻糖和5-溴-4-氯-3-吲哚基-β-D-岩藻糖苷作为底物。具有正常刷状缘糖苷酶活性的空肠活检切片用作对照。从5例腺瘤、5例腺癌样本以及正常的空肠活检剩余组织中制备2% Triton X-100中的10%(w/vol)匀浆。匀浆冷冻和解冻3次,其上清液在聚丙烯酰胺凝胶板上进行等电聚焦。酶谱图采用与检测切片中α-葡萄糖苷酶相同的方法显影。在任何大肠肿瘤中均未检测到LG。当使用蔗糖、葡萄糖氧化酶-过氧化物酶和3,3'-二氨基联苯胺的方法时,在70%的腺癌、50%的绒毛状腺瘤、25%的管状绒毛状腺瘤和19%的管状腺瘤中发现了SI。在pH值为5、6和6.5时应用偶氮偶联反应,在肿瘤中几乎未发现可辨别的活性痕迹。在pH值高于6.0时应用产色原法未检测到反应。然而,在相同条件下,空肠活检显示出非常强烈的反应,局限于肠上皮细胞的刷状缘。当在pH值低于6.0(特别是pH值为5.0)时应用产色原反应,在最近研究的12例肿瘤中有7例出现强阳性反应。在这种情况下,靛蓝的沉积是由于肿瘤细胞的膜和溶酶体α-葡萄糖苷酶以及巨噬细胞和白细胞的溶酶体α-葡萄糖苷酶。这些发现通过酶谱图得到了证实。在与SI相同的肿瘤中检测到了T;然而,其活性较低。大肠肿瘤中的SI活性是这些肿瘤的一个有用但非普遍的标志物。