| Objective:(1) To explore whether suspension tumor spheres exist in human gastric cell line BGC-823 or not, and investigate the expression of progastrin(Pro-GRP) in tumor spheres, then to detect ABCG2 (ATP-binding cassette superfamily G member 2),the former gastrin (progastrin, Pro-GRP),CD44,CD166 in the tumor spheres, and to probe the relationship of Pro-GRP and tumor spheres.(2) To study of the expression of the Pro-GRP in 32 cases of fresh gastric carcinoma, and the relationship of Pro-GRP and gastric carcinoma.(3) To detect whether the Pro-GRP was expressed in bone marrow mesenchymal stem cells (Mesenchymal Stem Cells, MSCs).Methods:(1) Adherent cells of BGC-823 were cultured by limit diluting assay and serum-free culture assay, and using soft agar colony formation to confirm the existence of suspended cells that possessed the characteristic of stem cells by colony formation capability assay in BGC-823. FCM has been applied to detected the expressions of ABCG2,CD4,CD166. Compare the different expressions of Pro-GRP between tumor spheres and adherent cells by ELISA, immunofluorescence and FCM.(2) Bone marrow mesenchymal stem cells (MSCs) were cultured by density gradient centrifugation and purification of adherent and passaged in serum-free DMEM/F12 medium, then amplified and viewed by inverted-phase-contrast microscope. The 1st generation,2nd generation,3rd generation of MSCs, were collected respectively to detected cell phenotype by FCM. Compare the different expressions of Pro-GRP between tumor spheres and MSCs through ELISA, FCM methods.(3) The fresh gastric carcinoma were collected to detected expressions of Pro-GRP by HE staining, immunohistochemical fluorescence, two-step immunohistochemical SP.Results:(1) The suspended tumor spheres were formed by limit diluting assay and serum-free culture assay. The difference of colony formation capability between suspended tumor spheres (37.63±5.99) and adherent cells(10.75±3.37) is significant(P=0.007). The expression of ABCG2 in suspended tumor spheres (57.17±3.49) was obviously higher than adherent cells(8.67±0.77)(P=0.000) by FCM. The expression of Pro-GRP in suspended tumor spheres (73.37±9.42) was markedly higher than adherent cells (5.11±1.03) (P=0.002).(2) The ELISA was used to detect the expressions of Pro-GRP in 1st generation,2nd generation,3rd generation of MSCs, and the results of FCM in MSCs was (52.31±4.07), tumor sphere (49.62±3.81) (P=0.64), significantly higher than that of adherent cells (7.54±1.56) (P=0.006), but no statistical significance in the MSCs and tumor sphere.(3) The widespread expression of Pro-GRP in fresh gastric by immunofluorescence staining, while not expressed in normal gastric tissues.Conclusion:(1) The suspended tumor spheres exist in human gastric cell line BGC-823 by method of combined limiting dilution and serum-free culture. The specific marker ABCG2 of SP cells and CD44 were expressed in suspended tumor spheres. They have the the characteristics of cancer stem cells.(2) The expression of Pro-GRP in suspended tumor spheres was significantly higher than that of adherent cells, suggesting that there is a high correlation between Pro-GRP and stem cells, meanwhile the Pro-GRP was expressed in gastric cancer in different levels, and the positive rate was increased with the increase of gastric cancer TNM classification, also suggesting that there is a closely correlation between Pro-GRP and the prognosis of patients with gastric carcinoma.(3) The Pro-GRP exists in Rat MSCs. suggesting that cancer stem cells of gastrin may be derived from bone marrow mesenchymal stem cells.
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