INFLUENCE OF SP AND CON A ON HISTAMINE RELEASE FROM CULTURED MAST CELLS OF RAT SKIN

The mast cells from enzymatically dispersed rat skin were incubated in DMEM mediumcontaining 10% fetal bovine serum. 1 day after culture, the various concentrations of substance P (SP),SP+Ca2+ or concanavalin A (Con A) were added into the media to stimulate mast cells for different periods of time. The media were rapidly seperated from the cultured tells using the micropore filter. The contents of histamine in the media were determined by spectrofluorimetry and the release rates of histaminewere caculated. The results showed that SP and Con A could stimulate in vitro mast cells tO release histamine in a time--and dose-dependent pattern, and that the effect of SP was significantly influenced by theconcentration of Ca2+.

Isolation of Side Population Cells and Detection of ABCG2 from SW480

Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation.from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods： side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results： SW480 contained 2.29% side population ceils. The fraction of side population ceils decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction （34.05%） of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. ConclusJon： our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.