Goang-Yao Yin, Wu-Ning Zhang, Xiao-Jing Shen, Yi Chen, Xue-Fen He

Goang-Yao Yin, Xiao-Jing Shen, Xue-Fen He, Wuxi No.3 peoples hospital, Wuxi 214041, Jiangsu Province, China
Wu-Ning Zhang, Yi Chen, Department of National Microanalysis Center, Fudan University, Shanghai 200433, Shanghai, China
Correspondence to: Dr. Goang-Yao Yin, Wuxi No.3 peoples hospital, 230 Eastern Tonghhui Road Wuxi 214041, Jiangsu Province,China. yinyao@pub.wx.jsinfo.net
Received: 2002-10-04 Accepted: 2002-12-03

Abstract
AIM: To carry out a comparative study on ultrastructure and molecular biological changes of chronic gastritis (CG), gastric cancer (GC) aand gastric precancerous lesions.

METHODS: By the use of histochemical staining, SEM with EDAX, TEM with EDAX, image analysis technique, RIA and chemiluminescence method, gastric mucosa of 168 patients were synchronously analyzed in morphology, trace elements, DNA, cAMP, SOD, 3H-TdR LCT and serum LPO were also done.

RESULTS: The incidence of epithelial nucleoplasmic ratio >1, lobulated nuclei, inter-chromatin aggregation of granules, nucleolar hypertrophy, and the content of DNA, Zn, Cu in nuclei and serum LPO of each group were showed as belows: normal control group (0.0, 0.0, 6.7, 0.0, 12.6±2.7, 7.6±0.4, 58.4±0.3, 2.6±0.6), CSG group (5.7, 2.9, 7.4, 2.9, 15.2±3.1, 8.1±0.5, 58.9±0.5, 4.2±0.7), CAG group (31.3, 29.7, 45.3, 42.2, 16.5±3.1, 8.6±0.4, 59.3±0.5, 4.5±0.6), CA group (100.0, 100.0, 72.2, 50.0, 30.7±8.2, 8.8±0.3, 59.5±0.4, 6.8±1.6), ATP⁺⁺group (61.5, 38.5, 23.1, 38.5, 23.5±8.9, 8.3±0.4, 59.1±0.4, 5.1±1.2), IM⁺⁺⁺ ATP⁺⁺group (77.8, 55.5, 33.3, 44.4, 25.1±7.2, 8.4±0.5, 59.5±0.4, 6.5±1.1), IM⁺⁺⁺⁺ATP⁺⁺ group (100.0, 100.0, 75.0, 62.5, 28.5±9.1, 8.9±0.5, 59.7±0.4, 7.6±0.7), IMII_b group (100.0, 62.5, 75.0, 50.0, 27.3±10.3, 8.6±0.3, 59.5±0.4, 6.1±0.9); whereas the content of Zn, Cu in mitochondria and cAMP, SOD in gastric mucosa, and ³H-TdR LCT of each group were showen as belows: normal control group (9.2±0.5, 58.3±0.3, 15.9±1.5, 170.5±6.1, 1079.7±227.4), CSG group (8.6±0.5, 57.8±0.3, 14.6±1.8, 163.3±5.6, 867.3±240.5), CAG group (8.3±0.4, 57.5±0.3, 13.4±1.8, 161.2±4.3, 800.9±221.8), CA group (8.9±0.4, 57.1±0.3, 10.2±3.9, 152.2±3.8, 325.7±186.8), ATP⁺⁺ group (9.1±0.4, 57.0±0.3, 12.4±1.8, 161.5±3.8, 642.9±174.3), IM⁺⁺⁺⁺ ATP⁺⁺group (8.6±0.4, 56.9±0.3, 12.0±2.3, 152.2±2.5, 326.3±160.3), IM⁺⁺⁺⁺ATP⁺⁺ group (8.5±0.3, 56.8±0.2, 10.4±0.9, 147.4±2.6, 316.1±170.7), IMII_b group (8.6±0.3, 56.9±0.3, 11.9±1.9, 150.0±2.8, 318.9±145.8), there were significant differences between groups (P<0.05-0.01).

CONCLUSION: There was a significant difference between CG and GC in their ultrastructure and molecular biology. Only on the condition of changes of internal environment in combination with the harmful effect of external environment, chronic atrophic gastritis can then develop into gastric cancer. Hence it might have similar epithelial cell ultrastructure and molecular biological changes in ATP⁺⁺, IMII_b and cancer, hence there were similar patterns of occurrence, development and transformation. Recognition of this trend might help to explore problems of prevention and cure.

Yin GY, Zhang WN, Shen XJ, Chen Y, He XF. Ultrastructure and molecular biological changes of chronic gastritis, gastric cancer and gastric precancerous lesions: a comparative study. World J Gastroenterol 2003; 9(4): 851-857
http://www.wjgnet.com/1007-9327/9/851.htm


INTRODUCTION
We conducted histochemical staining, detection of cAMP, SOD and DNA of gastric mucosa of 168 patients with chronic gastritis (CG) or gastric cancer (GC); SEM and TEM with EDAX were used to synchronously determine ultrastructures and trace elements and image analysis technique was used to determine nuclear DNA. ³H-TdR LCT and serum LPO were also cared out. Following was the comparative study.

MATERIALS AND METHODS
Materials
168 patients definitely diagnosed as suffering from CSG, CAG and GC were our subjects of study. According to "the standards for the classification of chronic gastritis, for gastrofiberscopic diagnosis and for histopathological diagnosis of atrophic gastritis" gastric mucosa tissue slices underwent HE staining and AB_PH2.5/PAS, Hid/AB_PH2.5 and HiD/AB_PH2.5 histochemical staining. ATP was classified into low-grade, middle-grade and heavy-grade and IM into IM I_a, IM I_b, IM II_a, IM II_b. 68 cases were definitely diagnosed as CSG, including 43 males, 25 females, average age 43 years, average course of disease 4a, 26 cases without IM or ATP; 31 cases with concomitant or solitary IM-19 cases low-grade, 10 cases middle-grade and 2 cases heavy-grade; 9 cases with solitary ATP-8 cases low-grade, 1 case middle-grade; 2 cases with IM and concomitant ATP, IM and ATP were both middle-grade. 64 cases were definitely diagnosed as CAG, including 37 males, 27 females, average age 47 years average course of disease 6a, 3 cases without IM or ATP; 39 cases with concomitant solitary IM (17 cases low-grade, 4 cases middle-grade and 18 cases heavy-grade); 8 cases with solitary ATP (2 cases low-grade, 6 cases middle-grade.) 14 cases with IM concomitant ATP (12 cases middle-grade IM, 2 cases heavy-grade IM, 8 cases low-grade ATP, 6 cases middle-grade ATP). 36 cases were definitely diagnosed as GC, including 22 males, 14 females, average age 52 years, average course of disease, 2a, all with IM or ATP. 12 cases with solitary IM (6 cases low-grade, 3 cases middle-grade, 3 cases heavy-grade); 7 cases with solitary ATP (1 case low-grade, 6 cases middle-grade); 17 cases with IM concomitant ATP (5 cases middle-grade IM, 12 cases heavy-grade IM; 7 cases low-grade ATP, 9 cases middle-grade ATP, 1 case heavy-grade ATP). Of the 45 healthy volunteers who underwent gastroscopy and biopsy, 15 cases had basically normal gastric mucosa tissues, including 6 males 9 females, average age 37, and they were referred to as normal control (NC) group.

Methods
Under gastroscopy, three pieces of gastric mucosa were taken from the focal and nonfocal areas in antral region and body of stomach as specimens for section preparation, SEM, TEM, detection of DNA, cAMP and SOD. Blood was taken for 3H-TdRLCT and LPO. For the observation of gastric mucosa ultrastracture and determination of its trace elements^[1-4], 501B SEM with 9100/60 EDAX was used to observe the three pieces of specimens of each patient, under the direct vision of SEM, the EDAX probe automatically detected the samples within 0.1-0.01 mm² range all the elements under 12 in atomic number and automatically calculated the weight percentage (WT%) of each element in element series. 15 points were fixed in the three pieces of mucosa of every patient to carry out 15 detections, and the average WT% of each element was taken as its actual WT%. In every detection, 21 elements such as Na, Mg, Al, Si, P, S, K, Ca, Fe, Cu, Zn, Ti, Cr etc were detected, and weight percentage (WT%) of each element between elements of gastric mucosa was calculated.
   By using EM430 TEM with 9100/60 EDAX, three pieces of mucosa specimens of every patient were magnified in unison by five magnifying powers (3 600, 7 200, 14 000, 19 000, 29 000) to randomly take the pictures of the panoramagram, local area and organelle and to detect the atomic number percentage (AT%) of each nuclear and mitochondrial element between trace elements^[5-10]. After gastric mucosa cell smear was stained by Feulgen staining, IBAS 2000 image analysis technique was adopted to detect IOD, which is taken as the relative content of nuclear DNA^[7,8]. RIA was adopted to detect the content of gastric mucosa cAMP (Pmol/g); chemiluminescence method was adopted to detect the activity of SOD (u/g)^[4]. ³H-TdRLCT. (Bq/L whole blood) was carried out; thiobarbituric acid development process was adopted to detect serum LPO (u mol/L)^[4].

Statistical method
x² and t test.

RESULTS
Histopathology of gastric mucosa
Between NC group. CSG group. CAG group and CA group, there were significant differences in the incidence rate and degree of different types of IM and ATP in glands propria of gastric mucosa (P<0.05-0.001, Table 1). The surface of normal gastric mucosa was isolated by crisscross small groves into many lesser gastric areas, assuming convolution shape (Figure 1). In these areas, there were many gastric pits (the mouths of gastric glands), which were shaped like craters. The concave walls had round or oval epithelial cells of almost the same size. In CSG gastric mucosa there were scattered denatured, diabrotic and necrotic exfoliated epithelical cells, on whose surface S-shape Helicobacter pylori (HP) were found (Figure 2). Massive epithelial cells became diabrotic, anabrotic and exfoliated forming micro ulcers. The ulcers spred out from their centers, with adjacent cells crushed, destructed in irregular shapes and arrangement. In CSG gastric mucosa, cyst was found accidentally (Figure 3), the epithelial cells of crater walls were atrophic and denatured, of different sizes and deranged; the cells became diabrotic and necrotic and had inflammatory cell infiltration and the glands propria of the serious cases were shaped like grid framework structure (Figure 4). The surfaces of the epithelial calls of IM gastric mucosa were thickly coated, villi were invisible and the intercellular boundaries were not clear (Figure 5). SEM was not able to definitely indentify gastric mucosa ATP, but only able to distinguish whether cells were hyperplastic or not. Degeneration, hyperplasia or cellular regeneration occured in gastric mucosa. The hyperplastic cells are of different sizes and states and the active memberanes were shaped like lesser tubercles (Figure 6, 7). The cells of gastric cancer were different sizes and shapes, deranged, characterized by obvious heteromorphic nature, conglobated and shaped like grapes or cauliflower. There were nearby or basal infiltration, destruction and ulceration of cancer cells, which have developed into ulcers.

The ultrastructure of gastric mucosa epithelial cells
The mucous cells on epithelial cell surface of relatively normal gastric mucosa were columna epithelial cells covering endogastric surfaces and inside wall of gastric pit. Their free surfaces had short micro villi; the mucous neck cells were distributed over the necks of gastric glands, and on the tops of these cells were a few short thick micro villi. Chief cells were distributed over the bodies and bottoms of gastric glands; parietal cells were big and conic and their conical tops turned towards gland cavities; the endocrine cells lied between chief cells and parietal cells and they were small, and their nuclei are round or oval, the nuclei envelopes are slightly bent, lobulated nuclei were few, the nucleocyotoplasmic ratio was less than 1.
   The nuclear chromatins were scattered, or associated with nucleoli or along nuclear perimeters. The light bright zones between heterochromatins in the nucleoli are euchromatins; the nucleoli had high electron density without capsule. Mitochondria were round or oval, scattering around nuclei. The mitochondria consisted of outer membrane, inner membrane, outer ventricle, inner ventricle and cristae. Crista, the inward folded inner membrane, was a hollow canal leading to outer ventricle. Some mitochondrial cristae directly led to cytoplasms. Cristae were generally in tabular arrangement, parallel to each other and vertical to mitochondrial long axes. In cytoplasms were many parallel rough surfaced endoplasmic reticuluas (RERs), secretory granules, well-developed Golgi's bodies and scattered mitochondria. The free surfaces of epithelial cells of CG gastric mucosa had dropped micro villi; the intercellular space expanded and cell conjuctions decreased; the karyoplasmic ratio of both ill-differentiated epithelial cells and IM cells was greater than 1, and lobulated nuclei increased; nucleolar granules became thick and nucleoli expanded with irregular margins (Figure 9).
   There occured nucleolar margination (Figure 10), multinucleoli (Figure 11) and nucleolar division (Figure 12). The obsolescent epithelial nuclei shrank and the karyoplasmic ratio was still less; the heterochromatins lied densely around nuclei; the electron density was low in the center of nuclei and nuclei were loop in shape. (referred to as chromatin margination); the shrunk nuclei were denticular, in which the electron density was moderately homogeneous and chromatins were not found (referred to as chromatin homogenization). There were mitochondrial swelling, hypertrophy, pyknosis, hyaline degeneration as well as vacuolar degeneration. The deformed mitochrondrias were in C-shape or U-shape. There were zigzag, longitudinal, sparse and pyknosis cristae, and deranged cristas. There was an decrease in the number of mitochondrias and their cristae. There were mitochondrial decreases in number, mitochondrial swelling, or crista fragmentation, vacuolar degeneration, and RERs in circular arrangement; the Golgi抯 bodies become atrophic and had lost their typical structures; the cytoplasmic incretory granules decreased. There were still greater epithelial cell changes in cancer cells of gastric cancer than in cells of gastric mucosa of chronic gastritis, intranuclear inclusion appeared (Figure 13).
   There was an increase in peri-chromatinic and interchromatinic granular densification (Figure 14). In euchromatins; there was a significant difference between CSG group and CAG group in nucleolar margination and nucleolar looping (P<0.05-0.001, Table 2, 3); between IM IIb group, ATP⁺⁺ group, IM⁺⁺⁺ATP⁺⁺ group and IM⁺⁺⁺⁺ATP⁺⁺ group, compared with NC group, CSG group and CAG group, the difference was also significant (P<0.05-0.001, Table 2, 3). There is no significant difference between IM⁺⁺⁺⁺ATP⁺⁺ group, IM II_b group and CA group (P>0.05, Table 2, 3).

The biochemical assay in gastric mucosa and serum
The content of nuclear Zn and Cu increased progressively in the sequence of NC group, CSG group, CAG group and CA group; the content of mitochondrial Zn and Cu decreased progressively in the same sequence. There was significant differences between these groups (P<0.05-0.001, Table 4). The content of Zn, Cu, cAMP and SOD in gastric mucosa decreased progressively in the same sequence; the content of DNA increased progressively in the above sequence. There were significant differences between these groups (P<0.05-0.001, Table 5). The content of ³H-TdRLCT decreased in the sequence of NC group, CSG group, CAG group and CA group; content of serum LPO increased in the same sequence; there was significant difference between these groups (P<0.05-0.001, Table 6). Between IMII_b group, ATP⁺⁺ group, IM⁺⁺⁺ATP⁺⁺ group and IM⁺⁺⁺⁺ATP⁺⁺ group, NC group, CSG group and CAG group, there was also significant difference (P<0.05-0.001, Table 4, 5, 6). Between IM⁺⁺⁺⁺ATP⁺⁺ group, IM II_b group and CA group, There was no significant difference (P>0.05, Table 4, 5, 6).

Table 1  Comparison between classification of IM and grading of ATP in CG and GC gastric mucosa n (%)

Either Solitary IM or IM with concomitant ATP in CSG, CAG or CA is included in IM classification statistically, compared with NC group. ^aP<0.05, ^bP<0.01; compared with CSG group. ^cP<0.05, ^dP<0.01; compared with CAG group. ^eP<0.05, ^fP<0.01. In table 2-6, the marks are the same as here.

Table 2  The ultrastructures of epithelial nuclei of gastric mucosa in CG and GC n (%)

Compared with CA group, ^gP<0.05, ^hP<0.01, Compared with IMII_b group, ⁱP<0.05, ^jP<0.01, Compared with ATP⁺⁺ group, ^kP<0.05, ^lP<0.01. Compared with IM⁺⁺⁺ATP⁺⁺ group, ^mP<0.05, ⁿP<0.01. In table 3-6, the marks are the same as here.

Table 3  Mitochondria utrastructures of gastric mucosa epithelial cells in CG and GC (x±s)

Table 4  Nuclear and mitochondriaL Zn, Cu of gastric mucosa in CG and GC (x±s)

Table 5  Gastric mucosa Zn, Cu, DNA, cAMP and SOD in CG and GC (x±s)