| IntroductionStem cells are a group of undifferentiated cells, and scholars propose tumor stem cell theory based on deep research into stem cells and the pathogenesy of tumours. The theory considers stem cells as the origin of tumour, which is a kind of diseases of stem cells. The Musashi family is an evolutionarily conserved group of neural RNA-binding proteins, Musashi-1 (Msi-1) is selectively expressed in neural stem/progenitor cells. Notablely, numerous researchs indicated that Msi-1 may be a marker for stem/progenitor cells of gastrointestinal tract. Msi-1 keeps stem cells or immature cells in an undifferentiated status during post-transcriptional gene regulation, and possess a significant role in maintenance of the stem cell status and tumorigenesis.Gastric cancer pathogenesis refers to a multifactorial and multistep pathological process. Correa has been propose a theoretical model of gastric cancer pathogenesis: by multifactorial long term effectiveness, gastric mucosa is injuryed with varying degree, through the chronic superficial gastritis, the chronic atrophic gastritis, intestinal metaplasia and dysplasia finally towards gastric cancer. Now the relationship between stem cells and disease process and the role of Msi-1 are not identified clearly.ObjectiveIn the present study, immunohistochemistry was employed to detect expressive intensity of Msi- 1 in the different stage of the progression of gastric carcinogenesis, the correlations of Msi-1 expression and gastric carcinogenesis was explored. Furtherfore, using Msi-1~+/PCNA~- as potential marker for gastric stem cells, the characteristic and variation of location of Msi-1~+/PCNA~- cells at the lesions mentioned previously and the relationship between gastric stem cells and gastric cancer stem cells were investigated.Materials and methodsClinical material: surgically resected early gastric cancer specimens (35), the chronic superficial gastritis (31), the chronic atrophic gastritis(7), intestinal metaplasia(32) and dysplasia(14) were collected from the oncosurgery of No.1 Hospital of China Medical University from August 2006 to October 2007. Additional 112 cases of advance gastric cancer from 6 archived blocks of tissue microarrray were collected from 4th Laboratory, Cancer Institute of No.1 Hospital of China Medical University.Immunohistochemistry and result assessment: the ABC and Envision immunohistochemical methods were used to detect expression of Msi-1 and PCNA correspondly in this study. Rat anti-Msi-1 antibody (kindly presented by Professor Okano H, Keio University School of Medicine, Tokyo, Japan. Working dilution 1:150), mouse anti-PCNA monoclonal antibody (Maixin biology company, ready to use). In addition, to some of the cases immunohistochemical double-labelling stainning method was used to determine the distribution of Msi-1~+/PCNA~- cell in different lesions of gastric mucosa, immunohistochemical double-labelling stainning kid was from Zhongshan biology company (ready to use). Known positive tissue sections were used for positive control, and PBS instead of the primary antibodies for negative control. Result assessment: Clearly red fine granules in cytoplasm was defined as Msi-1 positive cell. Firstly, intensity of immunostaining was assessed into four grades: 0, achromatic color; 1, plum;2, red; 3, crimson. Secondly, according to percentage of positive cells to divide into four grades: 1,5%～20%;2, 20%～50%; 3,>50%. Finally, according to the product of staining intensity and percentage of positive cells to divide: weakly positive(+), 1～2; moderately positive(++), 3～6; strongly positive(+++), 7～9. Clearly brown fine granules in nuclei was defined as PCNA positive cell. According to percentage of positive cells to divide into four grades: negative, 0; weakly positive(1), 5～20%; moderately positive(2), 20%～50%; strongly positive(3), >50%. Immunohistochemical double-labelling stainning: Clearly blue fine granules in cytoplasm and unstained nuclei within one cell were defined as Msi-1~+/PCNA~- cells.Statistical analysis: All data were analyzed by SPSS11.5 statistical software to evaluate the differences via Nonparametric Tests and Kendall's tau-b correlation coefficient, with significance defined as P< 0. 05.ResultsExpression of Msi-1 and PCNA in the chronic superficial gastritis, the chronic atrophic gastritis, intestinal metaplasia, dysplasia and cancer: From the chronic superficial gastritis, the chronic atrophic gastritis, intestinal metaplasia to dysplasia, expressive intensity of Msi-1 gradually increased along with the serious degree of gastric mucosal lesions, which showed a positive correlation (P<0.001); while from dysplasia to gastric cancer, expressive intensity of Msi-1 gradually decreased, which showed a negative correlation (P<0.001). Expression of Msi-1 is different between early gastric cancer and advanced gastric cancer, and expression intensity of Msi-1 gradually decreased along with the progression of gastric mucosal lesions (P<0.001). The expression intensity of Msi-1 in gastric cancers was generally related with histological type and lymph node metastasis, but irrelevant with age and sex of gastric cancer patients and the Borrmann's type of gastric cancer. From the chronic superficial gastritis to gastric cancer, PCNA expressed with a gradually increased trend. Expression of PCNA in gastric cancers was generally related with histological type and lymph node metastasis, but irrelevant with age and sex of gastric cancer patients.Distribution of Msi-1~+/PCNA~- cells in the lesions mentioned previously: in the gastric mucosa of the chronic superficial and gastritis chronic atrophic gastritis, Msi-1~+/PCNA~- cells mainly located in the isthmus/neck region of the gastric glands; in the intestinal metaplasia, Msi-1~+/PCNA~- cells expressed specifically within the intestinal metaplasia's crypts; in the dysplasia, Msi-1~+/PCNA~- cells scattered among the epithelial cells of atypia glandular tube; in the gastric cancer, Msi-1~+/PCNA~- cells scattered in the tumor tissue or located along the leading edge of tumor invasion.Relationship between the expression of Msi-1 and PCNA: The cells with both Msi-1~+ and PCNA~+ were commonly in the proliferative zone(such as isthmus/neck region of the gastric glands, intestinal metaplasia's crypts and the leading edge of tumor invasion). There was a trend of negative correlation between Msi-1 and PCNA expression in gastric cancers.ConclusionsOur study shown that distribution feature of Msi-1~+/PCNA~- cell in the chronic superficial gastritis, the chronic atrophic gastritis, intestinal metaplasia, dysplasia and cancer was consistent with the region of stem/progenitor cells of gastrointestinal tract, agree with that Msi-1 may be a objective marker for stem/progenitor cells of gastrointestinal tract. Presence of Msi-1~+/PCNA~- cells in the gastric cancer tissues suggeste that mutant of the gastrointestinal stem/progenitor cells may be as a source of gastric cancer stem cells.In the different stage the progression of gastric carcinogenesis, the variation of Msi-1 expression indicated close correlation between expression of Msi-1 and the model of gastric cancer pathogenesis. |
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