In situ hybridization technique was applied to identification of the cells producing structurally related hormones, gastrin and cholecystokinin (CCK). By this technique using biotinylated DNA probes and alkaline phosphatase histochemistry, we looked for the localization of each messenger RNA (mRNA) for the hormones on tissue sections of biopsy specimens of human gastric antrum and duodenum. As a result, gastrin mRNA positive cells were found in the pyloric glands, while CCK cells were not detectable in the gastric antral mucosa. In the proximal duodenum, CCK-and gastrin-cells were detected separately. In conclusion, detection of mRNA by in situ hybridization was considered to be a useful means for analyzing endocrine cells in the gut.Recent developments in gene technology have revealed the complete sequences of both human gastrin gene and human cholecystokinin (CCK) gene as well as their chromosomal locations (3, 5,12). As an application of the technology, in situ hybridization has allowed the localization of particular nucleic acid sequence on tissue sections (6, 9).Gastrin cells and CCK cells were demonstrated immunohistochemically using antisera raised against each polypeptide (1, 8), but because of the amino acid sequence homology between the two hormones, crossreactive immunostaining should be excluded. One of the solutions of this problem is the use of antisera against synthetic fragments containing no homologous region (10). We expected that the detection of each messenger RNA (mRNA) for the hormones could be another means to identify these cells separately, and searched for the localization of each transcript in human gastric antrum and duodenum by in situ hybridization. (Fig. 1). These probes were biotinylated with N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-l,3-propanediamine (photobiotin) (2) (Bresa, Adelaide). Tissue preparation Biopsy specimens of the gastric antrum and the proximal duodenum were obtained endoscopically from two normal volunteers. Tissues were fixed in 10% neutral buffered formalin for 24 hr at room temperature (RT), dehydrated through an ethanol gradient, and embedded in paraffin. Sections were cut at 4-6 ~Cm and placed on gelatin-coated slides. After passing through xylene and a graded series of ethanol, they were applied to in situ hybridization. Hybridization procedure After rinsing in 2 X SSC (1 X SSC: 0.15 M NaCI, 0.015 M Na citrate, pH 7.0) for 30 min, tissue sections were pretreated by sequential incubation in 0.2 M HCl for 20 min at RT; 2 X SSC for 30 min at 60°C; proteinase K solution at l pg/ml in 20 mM Tris-HC1, pH 7.4, 2 mM CaCl2 for 15 min at 37°C. Sections were rinsed in distilled water after each step. Slides were then dehydrated through an ethanol gradient, and incubated in a hybridization mixture for 24 hr at 37°C in a moist chamber. The hybridization mixture consisted of 2 X SSC, 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 0.02% (wt/vol) ficoll, 0.02% (wt/vol) polyvinyl pyrrolidone, 1 mg/ml bovine serum albumin (BSA), 100 ,ug/ml...
Ras gene family (c-Ha, Ki, N ras) and c-myc proto-oncogenes were analyzed in seven colonic and three gastric adenomatous polyps obtained from a patient with Turcot's syndrome. The rearrangement and amplification as well as overexpression of c-Ha ras gene in one colonic adenomatouspolyp were determined. The amplified c-Ha ras gene in this polyp revealed larger fragments of BamHI,PstI and Sad than those in the normal colonic mucosafrom the samepatient. But, such abnormalities were not observed in other polyps. Noabnormalities of c-Ki ras, c-N ras or c-mycgene were observed in any polyps. These results suggest that the alternations of c-Ha ras gene in this patient maynot be responsible for the adenomatous change, but may be related to the transition from adenomato carcinoma of the colon.
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