卵巢癌侧群细胞流式分选方法的建立及探讨

ESTABLISHMENT AND DISCUSSION OF A METHOD FOR SORTING OVARIAN CANCER SIDE POPULATION THOUGH FLOW CYTOMETRY

目的:探讨分选卵巢癌细胞株SKOV3中的侧群(side population,SP)细胞的荧光染料Hoechst33342的最佳染色浓度及SKOV3的最适细胞密度。方法:制备SKOV3细胞悬液,以荧光染料Hoechst33342和7-AAD染色,维拉帕米拮抗对照,选择不同浓度的Hoechst33342(2.5,3,4,5,6mg/L)孵育1×106个/mL SKOV3细胞90min,流式细胞仪检测SP细胞比例及活细胞比例;选取不同细胞密度的SKOV3细胞悬液(6×105,7×105,8×105,9×105,1×106个/mL),3mg/L的Hoechst33342孵育90min,流式细胞仪检测SP细胞比例及活细胞比例。结果:通过Hoechst33342蓝光和红光双参数图,SP细胞位于左下角两种荧光均很弱的区域。Hoechst33342浓度不变时,SP细胞比例随细胞密度的增加而升高;SKOV3细胞密度不变时,SP细胞比例及细胞活性随hoechst33342浓度升高而降低。卵巢癌SKOV3细胞株在细胞密度为8×105个/mL,加入终浓度为3mg/L的Hoechst33342,37℃水浴90min,为最佳染色条件,此时SP细胞比例为(1.12±0.104)%,且细胞保持良好的活性。结论:人卵巢癌细胞株SKOV3中存在SP细胞,Hoechst33342浓度及SKOV3细胞密度是影响SP细胞比例及细胞活性的重要因素。

Objective：To sorting side group （side population, SP） discusses the best dyeing concentration of Hoechst33342 and the cells in ovarian cancer cell line SKOV3, optimal cell density of SKOV3 suspen sion. Methods： Preparation SKOV3 single cell suspension, with fluorescent dye Hoechst33342 and 7-AAD dyeing, adding verapamil toantagonism group, incubation1 ×10^6/mL SKOV3 cell 90 min with different concentration of Hoechst33342 （2.5, 3, 4, 5, and 6 mg/L）, flow cytometry instrument detection propor- tion of SP cell and living cells choosing different cell density （6 ×10^ , 7 ×10^5 , 8 × 10^5 , 9 × 10^5 , 1× 10^6 cells/mL） of SKOV3 cell suspension, 3 mg/L Hoechst33342 incubation 90 minutes, flow cytometry in- strument detection proportion of SP cell and living cell. Results： Hoechst33342 blue and red double param- eter map showed that SP cells located in the lower left corner of the two fluorescences were weaker areas. For the same hoechst33342 concentration, with the increase of cell density, SP cell proportion increased; for the same SKOV3 cell suspension, with the increase of hoechst33342 concentration, proportion of SP cell and living cell decreased. The best dyeing conditions of ovarian cancer cell line SKOV3 were cell densi- ty of 8）〈 10s cells/mL, join Hoechst33342 final concentration was 3 mg/L, 37℃ water bath for 90 min. The SP cell ratio was （1.12 ± 0. 104）%, and cell can maintain good activity. Conclusion： SP cell subsetsexist in human ovarian cancer ce Hoechst33342 concentration and SK is an important factor affecting in SPine SKOV3, V3 cell density cell proportionand the activity of the cells.