Removal of mucus for ultrastructural observation of the surface of human gastric epithelium using pronase.
BACKGROUND
Helicobacter pylori adhering to the human gastric epithelium causes gastric diseases such as ulcer, carcinoma and lymphoma. It is thus important to observe in detail both the surface of the epithelial cells and the H. pylori that adhered to it for the elucidation of H. pylori-induced diseases by scanning electron microscopy (SEM). Since the thick mucus layer blocks the observation of the cell surface and the bacteria, it is generally eliminated during the processing for SEM by roughly mechanical methods, but these treatments also demolish the ultrastructure of the cells. We studied the nonmechanical method for removal of mucus layer of gastric epithelium using pronase.


METHODS
To determine the optimal concentration of pronase, mucin was used as a substrate for inhibition of the viscosity. Pronase was added in 2% mucin at the concentration of 10, 50, 100, 500, 1000, 2000 or 5000 unit/ml and the flowing time of the mixture was measured. Based on the digestion experiment, biopsied specimens from 24 patients with dyspepsic symptoms were fixed in glutaraldehyde and then washed in rolling with different concentration of pronase. After the pretreatment by pronase, the specimens were treated according to the standard process for SEM.


RESULTS
We succeeded in removing the mucus layer on the surface of epithelial cells from the biopsied specimens fixed in glutaraldehyde by rinsing with 2000 unit/ml pronase for 24 hours.


CONCLUSIONS
Using our digestive method without destroying the ultrastructure, the earliest stage which H. pylori has adhered onto the human gastric epithelium can be observed for the investigation of H. pylori-induced gastric disorders by SEM.
Saito
Nagahito
N
Department of Gastroenterology and Hematology, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan.
Sato
Fujio
F
Oda
Hisashi
H
Kato
Mototsugu
M
Takeda
Hiroshi
H
Sugiyama
Toshiro
T
Asaka
Masahiro
M
D001706:Biopsy; D004848:Epithelium / Q000382:microbiology / Q000473:pathology / Q000648:ultrastructure; D005753:Gastric Mucosa / Q000382:microbiology* / Q000473:pathology / Q000648:ultrastructure; D016481:Helicobacter Infections / Q000473:pathology*; D016480:Helicobacter pylori / Q000302:isolation & purification* / Q000648:ultrastructure; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009093:Mucus; D011402:Pronase; D011706:Pyloric Antrum / Q000382:microbiology / Q000473:pathology / Q000648:ultrastructure
D011402:Pronase