肿瘤干细胞在喉癌Hep-2细胞化疗抵抗中的作用及机制研究
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摘要
第一部分免疫磁珠分选与化疗药物富集喉癌Hep-2细胞中肿瘤干细胞的比较
目的:比较免疫磁珠分选(MACS)及化疗药物顺铂(DDP)筛选富集喉癌Hep-2细胞系肿瘤干细胞的效果。方法:以CD133免疫磁珠分选、DDP作用两种方法来富集喉癌Hep-2细胞系中的肿瘤干细胞,并以流式细胞仪(FCM)检测处理后CD133~+细胞百分率。同时观察细胞形态学的改变,判断两种方法分选后细胞对后续实验的影响。结果:经FCM检测,MACS分选喉癌Hep-2,CD133~+细胞得率为64.33%,不同浓度DDP作用于喉癌Hep-2细胞48 h,FCM检测CD133~+细胞有不同的得率,其中浓度为4μg/ml时,所得CD133~+细胞百分率最高,为50.7%。MACS与DDP各组比较差异均有显著性(P＜0.01);两种方法处理后细胞的存活状态MACS组要好于DDP组。结论:MACS分选纯度较高,对细胞损伤小,适合后续培养;但分选前耗时长,每次分选仅能用于一种marker。DDP筛选简单易行,符合临床TSC抵抗化疗的模式。但阳性细胞得率与细胞毒性成正比。MACS和DDP筛选肿瘤干细胞各有其优势及适用范围,实验中可根据不同目的来确定所用筛选方法。
第二部分喉癌Hep-2细胞中肿瘤干细胞化疗抵抗及其机制
目的:探讨喉癌Hep-2细胞中肿瘤干细胞对化疗药物DDP的抵抗作用。
方法:培养细胞分别接种于96孔板和6孔板,经浓度分别为1μg/ml、2μg/ml、3μg/ml、4μg/ml、5μg/ml的DDP作用于喉癌Hep-2细胞,作用时间为24h、48h、72h,收集96孔板内细胞,酶标仪检测各组细胞存活率,收集6孔板细胞行FCM检测细胞中CD133~+细胞百分率。
结果:在所研究剂量及时间范围内,细胞存活率与DDP剂量及作用时间不呈线性关系;CD133~+细胞比例在2μg/ml、24h检测CD133~+比例小于空白组,其余各浓度及各时间点CD133~+比例就有不同程度地富集;48h CD133~+细胞富集率最显著,浓度为2μg/ml在各时间点上的检测值均低于其它浓度,浓度为4μg/ml的DDP在各时间点富集率都高于其它浓度。
结论:DDP作用后,CD133~+细胞比例上升,喉癌Hep-2细胞中CD133~+细胞可以抵抗化疗药物DDP的作用。特定浓度下可以诱导Hep-2-CD133~+细胞分化。
第三部分Oct-4维持喉癌Hep-2中肿瘤干细胞干性并调节其分化
目的:检测Oct-4在喉癌Hep-2细胞中及经DDP作用后的表达情况。
方法:培养的人喉癌Hep-2细胞,经浓度分别为1μg/ml、2μg/ml、3μg/ml、4μg/ml、5μg/ml的DDP作用48h后,免疫细胞化学法检测细胞中Oct-4的表达情况,并与CD133表达情况相比较。
结果:ICC结果:喉癌Hep-2细胞中Oct-4微量表达,经DDP作用后,细胞中Oct-4表达位于细胞膜及细胞浆,表达情况与CD133表达情况相一致。
结论:Oot-4可能与CD133同为喉癌Hep-2细胞中的标志物,CD133~+细胞可能通过Oct-4信号传导通路维持Hep-2-TSC的干性,并调节其分化。
PartⅠEffect of magnetic activated cell sorting and chemotherapy agent DDP in enriching tumor stem cells of laryngeal carcinoma Hep-2 cells
Objective:To compare effect of chemotherapy agent DDP to MACS in sorting TSC of laryngeal carcinoma Hep-2 cells.
Method:CD133 magnetic beads were applied to sort Hep-2 cells. Different concentrations of DDP were used to treat Hep-2 cells for 48 hours. Enrichment rate of CD133~+ cells by MACS and after DDP treatment was detected by FCM.Morphologic change was observed under inverse-phase microscope.
Result:FCM showed that the sorting rate of CD133~+ cells through MACS was 64.33%,while after DDP treatment for 48 hours,CD133~+ cells was enriched significantly in each group of different DDP concentrations,with the maximal rate of 50.7%,at the concentration of 4μg/ml.There is a significant difference between MACS and each group of DDP(P＜0.01).Cells treated with DDP were abnormal in morphology.
Conclusion:MACS and DDP sorting has respective advantages in enriching TSC in Hep-2 cells.
PartⅡTumor stem cell and chemotherapy resistance in laryngeal carcinoma Hep-2 cells
Objective:To observe the effect of laryngeal carcinoma Hep-2-TSC in chemical drug DDP resistance.
Method:The cell survival rate was detected through MTT and the expression rate of CD133~+ cells was detected through FCM after each group had been treated with different concentrations of DDP for 24 hours,48 hours and 72 hours.
Result:Cell survival rate did not have the relation with concentrations of DDP in the range of experiment.Percentage of CD133~+ cells at concentration of 1μg/ml,at 24 hours was less than that in control group.Percentage of CD133~+ cells was higher than that in other groups at concentration of 4μg/ml at each check point.At the point of 48 hours,percentage of CD133~+cells was the highest.
Conclusion:Rate of CD133~+ cells in laryngeal carcinoma Hep-2 cells changed after different DDP treatment of different concentrations for different time intervals.CD133~+ cells could resist the effect of chemical drug DDP.At the concentration of 2μg/ml,DDP could induce differentiation of Hep-2-TSC.
PartⅢOct-4 maintains stemness of Hep-2-TSC and regulates its differentiation
Objective:To detect expression of Oct-4 in laryngeal carcinoma Hep-2 cells and after different concentrations of DDP treatment for 48 hours.
Method:Human laryngeal carcinoma Hep-2 cells were cultured and different concentrations of DDP were added.Expression of Oct-4 of the cells was detected through ICC after 48 hours.
Result:ICC showed Oct-4 was expressed in Hep-2 cells.Expression of Oct-4 after different concentrations of DDP treatment has positive correlation with that of CD133.
Conclusion:Oct-4 may be a marker of laryngeal carcinoma Hep-2 cells as CD133.Oct-4 maintains stemness of Hep-2-TSC and regulates its differentiation.
目的:比较免疫磁珠分选(MACS)及化疗药物顺铂(DDP)筛选富集喉癌Hep-2细胞系肿瘤干细胞的效果。方法:以CD133免疫磁珠分选、DDP作用两种方法来富集喉癌Hep-2细胞系中的肿瘤干细胞,并以流式细胞仪(FCM)检测处理后CD133~+细胞百分率。同时观察细胞形态学的改变,判断两种方法分选后细胞对后续实验的影响。结果:经FCM检测,MACS分选喉癌Hep-2,CD133~+细胞得率为64.33%,不同浓度DDP作用于喉癌Hep-2细胞48 h,FCM检测CD133~+细胞有不同的得率,其中浓度为4μg/ml时,所得CD133~+细胞百分率最高,为50.7%。MACS与DDP各组比较差异均有显著性(P＜0.01);两种方法处理后细胞的存活状态MACS组要好于DDP组。结论:MACS分选纯度较高,对细胞损伤小,适合后续培养;但分选前耗时长,每次分选仅能用于一种marker。DDP筛选简单易行,符合临床TSC抵抗化疗的模式。但阳性细胞得率与细胞毒性成正比。MACS和DDP筛选肿瘤干细胞各有其优势及适用范围,实验中可根据不同目的来确定所用筛选方法。
第二部分喉癌Hep-2细胞中肿瘤干细胞化疗抵抗及其机制
目的:探讨喉癌Hep-2细胞中肿瘤干细胞对化疗药物DDP的抵抗作用。
方法:培养细胞分别接种于96孔板和6孔板,经浓度分别为1μg/ml、2μg/ml、3μg/ml、4μg/ml、5μg/ml的DDP作用于喉癌Hep-2细胞,作用时间为24h、48h、72h,收集96孔板内细胞,酶标仪检测各组细胞存活率,收集6孔板细胞行FCM检测细胞中CD133~+细胞百分率。
结果:在所研究剂量及时间范围内,细胞存活率与DDP剂量及作用时间不呈线性关系;CD133~+细胞比例在2μg/ml、24h检测CD133~+比例小于空白组,其余各浓度及各时间点CD133~+比例就有不同程度地富集;48h CD133~+细胞富集率最显著,浓度为2μg/ml在各时间点上的检测值均低于其它浓度,浓度为4μg/ml的DDP在各时间点富集率都高于其它浓度。
结论:DDP作用后,CD133~+细胞比例上升,喉癌Hep-2细胞中CD133~+细胞可以抵抗化疗药物DDP的作用。特定浓度下可以诱导Hep-2-CD133~+细胞分化。
第三部分Oct-4维持喉癌Hep-2中肿瘤干细胞干性并调节其分化
目的:检测Oct-4在喉癌Hep-2细胞中及经DDP作用后的表达情况。
方法:培养的人喉癌Hep-2细胞,经浓度分别为1μg/ml、2μg/ml、3μg/ml、4μg/ml、5μg/ml的DDP作用48h后,免疫细胞化学法检测细胞中Oct-4的表达情况,并与CD133表达情况相比较。
结果:ICC结果:喉癌Hep-2细胞中Oct-4微量表达,经DDP作用后,细胞中Oct-4表达位于细胞膜及细胞浆,表达情况与CD133表达情况相一致。
结论:Oot-4可能与CD133同为喉癌Hep-2细胞中的标志物,CD133~+细胞可能通过Oct-4信号传导通路维持Hep-2-TSC的干性,并调节其分化。
PartⅠEffect of magnetic activated cell sorting and chemotherapy agent DDP in enriching tumor stem cells of laryngeal carcinoma Hep-2 cells
Objective:To compare effect of chemotherapy agent DDP to MACS in sorting TSC of laryngeal carcinoma Hep-2 cells.
Method:CD133 magnetic beads were applied to sort Hep-2 cells. Different concentrations of DDP were used to treat Hep-2 cells for 48 hours. Enrichment rate of CD133~+ cells by MACS and after DDP treatment was detected by FCM.Morphologic change was observed under inverse-phase microscope.
Result:FCM showed that the sorting rate of CD133~+ cells through MACS was 64.33%,while after DDP treatment for 48 hours,CD133~+ cells was enriched significantly in each group of different DDP concentrations,with the maximal rate of 50.7%,at the concentration of 4μg/ml.There is a significant difference between MACS and each group of DDP(P＜0.01).Cells treated with DDP were abnormal in morphology.
Conclusion:MACS and DDP sorting has respective advantages in enriching TSC in Hep-2 cells.
PartⅡTumor stem cell and chemotherapy resistance in laryngeal carcinoma Hep-2 cells
Objective:To observe the effect of laryngeal carcinoma Hep-2-TSC in chemical drug DDP resistance.
Method:The cell survival rate was detected through MTT and the expression rate of CD133~+ cells was detected through FCM after each group had been treated with different concentrations of DDP for 24 hours,48 hours and 72 hours.
Result:Cell survival rate did not have the relation with concentrations of DDP in the range of experiment.Percentage of CD133~+ cells at concentration of 1μg/ml,at 24 hours was less than that in control group.Percentage of CD133~+ cells was higher than that in other groups at concentration of 4μg/ml at each check point.At the point of 48 hours,percentage of CD133~+cells was the highest.
Conclusion:Rate of CD133~+ cells in laryngeal carcinoma Hep-2 cells changed after different DDP treatment of different concentrations for different time intervals.CD133~+ cells could resist the effect of chemical drug DDP.At the concentration of 2μg/ml,DDP could induce differentiation of Hep-2-TSC.
PartⅢOct-4 maintains stemness of Hep-2-TSC and regulates its differentiation
Objective:To detect expression of Oct-4 in laryngeal carcinoma Hep-2 cells and after different concentrations of DDP treatment for 48 hours.
Method:Human laryngeal carcinoma Hep-2 cells were cultured and different concentrations of DDP were added.Expression of Oct-4 of the cells was detected through ICC after 48 hours.
Result:ICC showed Oct-4 was expressed in Hep-2 cells.Expression of Oct-4 after different concentrations of DDP treatment has positive correlation with that of CD133.
Conclusion:Oct-4 may be a marker of laryngeal carcinoma Hep-2 cells as CD133.Oct-4 maintains stemness of Hep-2-TSC and regulates its differentiation.
引文
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