雌或孕激素联合紫杉醇对人卵巢癌细胞生长调控及对Drosha表达的影响

发布时间：2018-03-18 15:17

本文选题：卵巢癌　切入点：雌激索　出处：《南京中医药大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：研究雌孕激素联合紫杉醇对人卵巢癌细胞体外生长的调控作用,并且探索改变细胞生长状态的可能机制,为其在临床中用于卵巢癌的辅助治疗提供前期的理论依据。方法：梯度浓度紫杉醇体外作用人卵巢癌细胞,四甲基偶氮唑兰(methyl thiazolyl tetrazolium, MTT)比色法检测药物作用24h、48h、72h后细胞的抑制率；根据IC50确定下一阶段实验药物的浓度。根据前期实验结果,10－4nol/L雌孕激素抑制卵巢癌细胞生长。组别设置为对照组(control)、雌激素组(E2)、孕激素组(P4)、紫杉醇组(Taxol)、雌激素联合紫杉醇组(E2+Taxol)、孕激素联合紫杉醇组(P4+Taxol),各组药物作用于卵巢癌细胞48h。 MTT检测各组卵巢癌细胞的抑制率；流式细胞仪检测各组卵巢癌细胞的凋亡率和细胞周期；实时荧光定量PCR (qRT-PCR)法和蛋白质印迹(Western blot)法检测各组卵巢癌细胞drosha基因和蛋白的表达。结果：①MTT显示：E2(10-4mol/L)、P4(10-4mol/L)、Taxol (10ug/ml)、E2+Taxool、 P4+Taxol均能抑制人卵巢癌细胞生长,诱导细胞凋亡：雌激素对ER(-)的卵巢癌细胞生长抑制作用强于ER(+)的卵巢癌细胞(P0.05),并且与紫杉醇具有协同作用；孕激索对卵巢癌细胞也具有生长抑制作用(P0.05)。②流式细胞技术检测细胞凋亡显示：紫杉醇可以提高卵巢癌细胞的凋亡率,与对照组比较具有显著性差异(P0.05)：雌激素作用卵巢癌细胞后凋亡率增加,且.ER(-)的卵巢癌细胞凋亡率高于ER(+)的卵巢癌细胞,雌激素联合紫杉醇凋亡率也增加；孕激素单用或联合紫杉醇,卵巢癌细胞凋亡率也有所提高(P0.001)。③流式细胞技术检测细胞周期显示：雌激素作用后大量细胞阻滞在S期,孕激素作用后细胞周期无明显改变,紫杉醇作用后大量细胞阻滞在G2/M期。④qRT-PCR和Western blot结果显示：紫杉醇能上调drosha基因及蛋白表达(P0.001)：雌激素对drosha基因及蛋白的影响与卵巢癌细胞ER表达相关：ER(-)者,drosha基因及蛋白表达上调(P0.001)：ER(+)者,drosha基因及蛋白表达差异无统计学意义。两药联合作用后,drosha基因及蛋白的表达较对照组上调(P0.001)；孕激素、孕激素联合紫杉醇均能上调drosha基因及蛋白表达(P0.001),但两药联合较紫杉醇组差异无统计学意义。结论：雌、孕激素联合紫杉醇能够抑制体外培养的人卵巢癌细胞的生长,这三种药物均能改变细胞的凋亡率,雌激素和紫杉醇能够改变细胞周期。雌孕激素联合紫杉醇通过上调drosha表达,起到抑癌或者增敏作用,并且与雌激素受体的表达相关。
[Abstract]:Objective: to study the effects of estrogen and paclitaxel on the growth of human ovarian cancer cells in vitro, and to explore the possible mechanism of changing the growth state of human ovarian cancer cells, and to provide a theoretical basis for the clinical application of estrogen and paclitaxel in the adjuvant treatment of ovarian cancer. Methods: human ovarian cancer cells were treated with gradient concentration of paclitaxel in vitro. The inhibition rate of the cells was determined by MTT colorimetry after 24 h, 48 h and 72 h exposure to methylazolam methyl tetrazolium (MTT). Determine the concentration of the next phase of the experimental drug based on IC50. According to the previous experimental results, 10-4 nol-1 / L estradiol inhibits the growth of ovarian cancer cells. The group is divided into three groups: control group, estrogen group, estradiol group, progesterone group, taxolol group, estrogen combined with purple. Estradiol group, progesterone combined with paclitaxel group, P4 Taxolol group. The inhibition rate of ovarian cancer cells in each group was detected by MTT for 48 h. The apoptosis rate and cell cycle were detected by flow cytometry, and the expression of drosha gene and protein were detected by real-time quantitative PCR qRT-PCR and Western blotting. Results: the results showed that 10 ~ (-4) mol / L ~ (-1) Taxol of E _ (2) N _ (10 ~ (-4)) P _ (10 ~ (-4)) P _ (4) Taxol could inhibit the growth of human ovarian cancer cells and induce apoptosis: estrogen had a stronger inhibitory effect on the growth of human ovarian cancer cells than that on ER () ovarian cancer cells, and had synergistic effect with paclitaxel. The results of flow cytometry showed that paclitaxel could increase the apoptosis rate of ovarian cancer cells. Compared with the control group, there was a significant difference (P 0.05): the apoptosis rate of ovarian cancer cells treated with estrogen was increased, and the apoptosis rate of ovarian cancer cells with. ER-) was higher than that of ovarian cancer cells with ER- (). The apoptosis rate of estrogen combined with paclitaxel was also increased. The apoptosis rate of ovarian cancer cells was also increased by progesterone alone or in combination with paclitaxel. The results of flow cytometry showed that a large number of cells were blocked in S phase after estrogen treatment, but the cell cycle did not change after progesterone treatment. After paclitaxel treatment, a large number of cells were blocked in G _ 2 / M phase .4qRT-PCR and Western blot. The results showed that paclitaxel could up-regulate the expression of drosha gene and protein (P0.001). The effect of estrogen on drosha gene and protein was related to ER expression of ovarian cancer cells. There was no significant difference in the expression of drosha gene and protein between the two groups. The expression of drosha gene and protein was higher than that of the control group. Progesterone combined with paclitaxel could up-regulate the expression of drosha gene and protein, but there was no significant difference between the two groups. Conclusion: estrogen and progesterone combined with paclitaxel can inhibit the growth of human ovarian cancer cells in vitro. Estrogen and paclitaxel can change cell cycle. Estrogen and progesterone combined with paclitaxel can inhibit cancer or increase sensitivity by upregulating the expression of drosha and is related to the expression of estrogen receptor.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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