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The isolation,culture and identification of CD133~+ OCSCPurposeTo isolate CD133~+ovarian cancer stem cell(OCSC)from human ovarian cancer cell line A2780 and to verify the cancer stem cell characteristics of self-renewal,proliferation and differentiation of CD133~+OCSC in vitro,which aims to lay the foundation for the treatment study of ovarian cancer targeting CD133~+OCSC.Methods1.CD133~+OCSC were sorted from A2780 cells which were incubated with CD133antibody labeled with allophycocyanin(APC-CD133)through flow cytometric assay(FCM).2.The CD133~+OCSC were cultivated in serum-free medium for sphere formation assay,biological characteristics and morphological of CD133~+OCSC were observed by optical microscope.3.The CD133 expression of A2780 cells,CD133~+OCSC and CD133~+OCSC after passage were examined via quantative real-time polymerase chain reaction(QPCR).Results1.The proportion of CD133~+OCSC sorted from A2780 cell line via FCM was merely0.1%.2.After cultivating in serum-free condition,CD133~+OCSC formed suspended spherical colonies,the amount and volume of sphere cells increased during cultivation.What's more,new colonies could formed again after passage.However,CD133~+OCSC were observed to be adherent to the bottom of the culture bottles.3.The CD133 expression in CD133~+OCSC before and after passage were compared via QPCR and detected a decrease of protein expression of CD133 after passage(P(27)0.05).ConclusionThere existed 0.1%CD133~+OCSC in ovarian cancer A2780 cells,which proved to have the cancer stem cell characteristics of self-renewal,proliferation and differentiation.And the CD133~+OCSC would be the targeting cells in the study of ovarian cancer treatment.Part ? Optimization of ultrasound combined Sono Vue transfecting CD133~+OCSCPurpose1.To investigate the effects of cell viability and transfection rate induced by various ultrasound and transfection parameters as well as culture conditions;2.To optimize the parameters suitable for enhancing the efficiency of transfecting CD133~+OCSC mediated by ultrasound combined Sono Vue in vitro so as to improve the transfection rate while reducing cell injury.Methods1.CD133~+OCSC were irradiated under various ultrasonic parameters[intensity and duty cycle(DC)].The cell viability and expression of mi R-let-7b of different parameters were observed and analyzed by inverted fluorescence microscopy and CCK-8 assay respectively.2.Transfect CD133~+OCSC with mi R-let-7b using different transfection parameters,inverted fluorescence microscopy was used to observe fluorescence expression of each combination 48h after transfection.The fluorescence intensity of each group was semiquantitatively analyzed by Image J software to evaluate the effects of each parameters on gene expression efficiency.Results1.After sonication of ultrasound combing Sono Vue,detachment cells were likely to be injured after sonication compared with adherent cells(P(27)0.05).What's more,cell viability was found to decrease while ultrasonic intensity and DC increased(P(27)0.05).2.The observation of inverted fluorescence microscopy and semi-quantitative analysis of fluorescent protein expression showed that the fluorescent protein expression of adherent cells was higher than that of suspended cells(P<0.05).While the ultrasonic intensity ranges from 0.6W/cm~2 to 1.0W/cm~2,the fluorescent protein expression increased as the increase of ultrasonic intensity(P<0.05).ConclusionThe cultivation method,ultrasonic and transfection parameter do have lots of influence on cell viability and transfection efficiency of CD133~+OCSC,cell injury could be decreased and transfection efficiency would be improved via optimization.After optimization,adherent cells,duty cycle 10%and ultrasonic intensity 1.0 W/cm~2 were preferred during transfection.Part ? Study of ultrasound combing Sono Vue mediated mi R-let-7b inhibiting the growth of CD133~+OCSCPurpose1.To design and construct the recombinant expression vector of mi R-let-7b and to investigate whether the growth of CD133~+OCSC could be inhibited by the expression of mi R-let-7b plasmid.2.To investigate the feasibility and application of UTMD-enhanced mi R-let-7b transfection with CD133~+OCSC,and to compare the effects of different transfection methods on transfection efficiency and growth inhibition of CD133~+OCSC.MethodsCD133~+OCSC were transfected by plasmid only,plasmid combing with ultrasound exposure,Sono Vue,UTMD and Lipofectamine 2000.The expression of enhanced green fluorescent protein(EGFP)of each group was observed by inverted fluorescence microscope.And the transfection efficiency and cell viability were analyzed by FCM and CCK-8assay.Finally,the apoptotic cells were observed by confocal microscopyResults1.The cell viability and growth of CD133~+OCSC were inhibited after transfection of mi R-let-7b mediated by UTMD.2.The observation of inverted fluorescence microscopy showed that the expression of EGFP in UTMD+P group was better than that in P,MB+P and US+P groups;the transfection rate of CD133~+OCSC detected by FCM showed the same result;the results of CCK-8 assay showed that the cell viability in the UTMD+P group(86.42±1.39%)was lower than that in the P group(93.44±1.84%),the US+P group(88.86±1.57%)and the MB+P(92.46±2.94%).And the observation of onfocal microscopy showed no obvious apoptosis of CD133~+OCSC after transfection.ConclusionIt would be a feasible way to transfect CD133~+OCSC with mi R-let-7b induced by UTMD,which could induce the cell viability and inhibit the growth of CD133~+OCSC. |
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