局麻药布比卡因诱导卵巢癌和前列腺癌细胞凋亡及其机制研究

发布时间：2018-04-09 16:29

本文选题：布比卡因　切入点：卵巢癌细胞　出处：《华中科技大学》2015年博士论文

【摘要】：第一部分布比卡因对肿瘤细胞活性及耐药性的影响目的：明确布比卡因是否有抑制肿瘤细胞活性的作用,并观察布比卡因处理后是否能增加肿瘤细胞对抗肿瘤药物的敏感性。验证布比卡因的促肿瘤细胞凋亡作用是否通过调控凋亡蛋白酶的活性。方法：体外培养人卵巢癌细胞(SKOV-3)、前列腺癌细胞(PC-3)和正常肾小管上皮细胞(HK-2),实验前将肿瘤细胞种植在24孔细胞培养板中,24小时后细胞达到70%-80%融合度时开始试验。实验组肿瘤细胞培养基中加入不同容积布比卡因以达到1μM、10μM、100μM、1mM四种不同的终浓度,对照组加入相等容积的生理盐水,继续培养24h或72小时后用MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)比色法检测肿瘤细胞的活性。为比较布比卡因对肿瘤细胞和正常细胞作用的差异性,将正常的人肾小管上皮细胞(HK-2)与1mM布比卡因共同培养24小时后,用MTT比色法测定细胞的活性。在观察布比卡因与抗肿瘤药物共同作用的实验组中,加入l00μM、1mM布比卡因的同时给予治疗浓度的抗肿瘤药物紫杉醇(taxol),共同培养24小时后同样以MTT比色法测定肿瘤细胞的活性。凋亡蛋白酶检测组加入1mM布比卡因,24小时后免疫荧光染色法测定细胞中活性凋亡蛋白酶caspase3、 caspase8和caspase9的表达量。之后使用相应蛋白酶抑制剂和FAS配体中和抗体处理,观察凋亡相关蛋白酶和细胞膜表明死亡受体在布比卡因诱导的肿瘤细胞凋亡中的作用。结果：MTT法检测细胞活性的结果显示,只有最高实验浓度(1mM)的布比卡因在24和72小时的培养后均显著抑制了SKOV-3和PC-3肿瘤细胞的活性。100μM组肿瘤细胞活性虽可见轻微下降,但未及显著性差异。SKOV-3和PC-3肿瘤细胞在1mM布比卡因存在下培养72小时后细胞活性下降的程度明显大于24小时,表明布比卡因对肿瘤细胞活性的抑制随时间的延续逐步增加。SKOV-3细胞活性下降的程度较PC-3更大,体现局麻药布比卡因对不同癌细胞的差异性杀伤作用。HK-2细胞在1mM布比卡因处理24小时的情况下细胞活性轻微下降,而无显著性差异,说明了局麻药有效的杀伤作用更多的体现在肿瘤细胞上,提示我们其可能干扰的是肿瘤细胞特异的生存条件。将100μM、1mM布比卡因与紫杉醇混合培养肿瘤细胞时,表现出的是布比卡因与抗肿瘤药物对肿瘤细胞的叠加杀伤效应,而非协同效应。免疫荧光分析显示,1mM布比卡因处理24h后,SKOV-3细胞中活性caspase3,8和9的表达均明显上升,Caspase8和9抑制剂部分逆转了SKOV-3的凋亡。PC-3细胞中只有活性caspase3和9的表达上调,而caspase8未见改变,同时只有caspase9抑制剂部分逆转了布比卡因诱导的肿瘤细胞凋亡,caspase8抑制剂无效。最后,FAS配体中和抗体处理后并未改变布比卡因引起的肿瘤细胞凋亡。结论：布比卡因能够显著抑制肿瘤细胞活性,且具有剂量和时间依赖性。其诱导肿瘤细胞凋亡的作用与凋亡蛋白酶的激活有关,与细胞膜表明死亡受体无关。第二部分布比卡因对肿瘤细胞增殖与迁移能力的影响目的：明确布比卡因是否有抑制肿瘤细胞增殖和迁移力的作用。方法：体外培养卵巢癌(SKOV-3)和前列腺癌(PC-3)细胞,增殖实验前将肿瘤细胞种植在置有玻片的24孔板上,在24孔板中培养24小时后细胞达到70%-80%融合度时开始试验。细胞增殖实验中肿瘤细胞SKOV-3和PC-3的培养基中加入1mM布比卡因后培养24h,之后免疫荧光染色法测定细胞增殖重要标记物ki67蛋白的表达量。肿瘤细胞迁移实验中使用划痕实验。迁移实验前将肿瘤细胞终于细胞培养皿中,24小时后当其铺满皿底形成单细胞层后开始实验。用一毫升枪头尖端在皿底做“十”字形划痕并在皿底座标记,以保证位置固定,显微镜下拍照记录划痕造成的间隙形态。之后肿瘤细胞培养液中加入浓度1mM的布比卡因,培养24小时后再次显微镜下拍照记录划痕形态,用Image-Pro Plus软件比对前后缺损面积的变化,根据公式(初始缺损面积-最终缺损面积)/初始缺损面积,计算出创伤愈合率,由此估算细胞整体迁移率。结果：荧光强度分析结果显示,培养基中加入1mM布比卡因培养24小时后,SKOV-3和PC-3细胞中Ki67的表达均显著下降,表明1mM布比卡因有效抑制了这两种肿瘤细胞的增殖能力。细胞迁移实验中,SKOV-3和PC-3在1mM布比卡因的影响下,较对照组的创伤愈合率显著下降,表明其细胞迁移能力因布比卡因的存在而下降。结论：布比卡因能有效抑制SKOV-3和PC-3肿瘤细胞的增殖能力和迁移率。第三部分GSK-3β在布比卡因诱导的肿瘤细胞凋亡中的影响目的：探索GSK-3β的活性对布比卡因诱导的SKOV-3和PC-3肿瘤细胞凋亡有何种影响。方法：体外培养卵巢癌(SKOV-3)和前列腺癌(PC-3)细胞,实验前将肿瘤细胞种植玻片上,在24孔板中培养24小时后细胞达到70%-80%融合度时开始试验。在lmM布比卡因处理24小时后,免疫荧光法测定磷酸化的总GSK-3β、活化型GSK-3β(GSK-3βtyr216)以及抑制型GSK-3β (GSK-3βser9)的量。其它实验在1mM布比卡因加入培养液的基础上,用GSK-3抑制剂SB-216763抑制其活性,观察肿瘤细胞存活情况。再应用基因沉默技术用siRNA sc-35527沉默GSK-3β基因,在此基础上加入1mM布比卡因,培养24小时后用免疫荧光技术观察凋亡相关蛋白酶Caspase3, Caspase8, Caspase9的表达情况,以及最终肿瘤细胞的生存情况。结果：实验结果显示1mM布比卡因作用24小时后,SKOV-3磷酸化的总GSK-3β、GSK-3βryr216和GSK-3βser9均有显著增加,其中代表GSK-3β活性的磷酸化GSK-3β tyr216升高最为明显,有将近一倍的提升。而PC-3中磷酸化的总GSK-3β、 GSK-3βtyr216和GSK-3βser9均无显著改变。抑制剂和SiRNA的运用都部分减轻了布比卡因诱导的SKOV-3细胞凋亡,但这些作用并未在PC-3细胞上发现。在沉默GSK-3ββ基因后,我们发现GSK-3β仅有微弱表达,免疫荧光分析显示,凋亡相关蛋白酶Caspase3, Caspase8,Caspase9的表达随着GSK-3β基因的沉默也显著下降。结论：1mM布比卡因的处理增加了SKOV-3磷酸化的总GSK-3β、 GSK-3β tyr216和GSK-3β ser9,其中活化型GSK-3β升高最为显著。但是对PC-3无影响。GSK-3β活性的存在是布比卡因诱导SKOV-3肿瘤细胞凋亡的必要条件,而其对布比卡因诱导的PC-3肿瘤细胞的凋亡无显著影响。
[Abstract]:Part 1 Effect of bupivacaine on the activity of tumor cells and drug resistance
Objective: to determine whether bupivacaine inhibit tumor cell activity, and observe whether bupivacaine after treatment can increase the sensitivity of tumor cells to anticancer agents. Promote tumor cell apoptosis by regulating the apoptosis of bupivacaine is verified protease activity.
Methods: human ovarian cancer cells in vitro (SKOV-3), prostate cancer cells (PC-3) and normal renal tubular epithelial cells (HK-2), before the experiment to tumor cells grown in 24 well cell culture plate, began to test 24 hours after cells reached 70%-80% fusion degree. Adding different volume of bupivacaine medium to reach 1 mu the experimental group M of tumor cell culture, 10 M, 100 M, 1mM four different final concentration, the control group normal saline with the same volume, cultured for 72 hours with MTT or 24h (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) than tumor cell assay activity. The difference of bupivacaine on tumor the role of cells and normal cells, normal human renal tubular epithelial cells (HK-2) and 1mM bupivacaine co cultured for 24 hours, the activity of cells was measured by MTT colorimetry. In the observation of antitumor drug and total bupivacaine The experimental group with the effect of adding l00, M, 1mM and bupivacaine anticancer drug paclitaxel in the treatment of concentration (Taxol), co culture activity also using the MTT colorimetric assay of tumor cells after 24 hours. 1mM bupivacaine join apoptosis protease testing group, 24 hours after immunofluorescence staining was used to determine the activity of apoptosis protease Caspase3 cells, expression of caspase8 and caspase9. After using the corresponding protease inhibitors and FAS ligand neutralizing antibody treatment, observation of apoptotic proteases and cell membrane showed that the role of death receptor in tumor cell apoptosis induced by bupivacaine.
Results: MTT cells viability assay results showed that only the highest concentration (1mM) of bupivacaine after cultured 24 and 72 hours after significantly inhibited the activity of.100 M group, the activity of tumor cells SKOV-3 and PC-3 tumor cells showed a slight decline, but no significant difference between.SKOV-3 and PC-3 tumor cells in 1mM bupivacaine the presence of decreased cell activity after cultured for 72 hours was significantly greater than 24 hours, and that the continuation of bupivacaine on the activity of tumor cells inhibition with time gradually decreased with the increase of.SKOV-3 cell activity degree is more serious than PC-3, reflect the local anesthetics bupivacaine on different cancer cell killing effect of.HK-2 cells in 1mM cells treated with bupivacaine activity for 24 hours under the condition of slight decrease, but no significant difference, that local anesthetic effective killing effect is more reflected in the tumor cells, suggesting that I have The possible interference is a tumor specific survival condition. 100 M, 1mM bupivacaine and paclitaxel mixed culture of tumor cells, showing the killing effect of bupivacaine is superimposed with anti tumor drugs on tumor cells, and a synergistic effect.
Immunofluorescence analysis showed that 1mM of bupivacaine after 24h treatment, the activity of caspase3,8 in SKOV-3 cells and 9 expression were significantly increased, and Caspase8 9 inhibitors partially reversed the SKOV-3 apoptosis in.PC-3 cells only active Caspase3 and 9 expression, while caspase8 was not changed, with only caspase9 inhibitor partially reversed the apoptosis of tumor cells induced by bupivacaine the caspase8 inhibitor is invalid. Finally, did not change the apoptosis of tumor cells induced by bupivacaine neutralizing antibody FAS ligand.
Conclusion: bupivacaine can significantly inhibit the activity of tumor cells, and has a dose and time dependent manner. The apoptosis of tumor cells and apoptosis protease activation, and cell membrane showed that death receptor independent.
The second part of bupivacaine on tumor cell proliferation and migration
Objective: to determine whether bupivacaine inhibit the proliferation and migration of tumor cells.
Methods: in vitro cultured ovarian cancer (SKOV-3) and prostate cancer (PC-3) cells, the proliferation of tumor cells before the experiment will be planted in the 24 hole plate glass on the train began to test 24 hours after cells reached 70%-80% fusion degree in 24 well plates. 24h culture medium added 1mM bupivacaine SKOV-3 tumor cells and PC-3 cells in the experiment of proliferation after expression after immunofluorescence staining was used to determine the cell proliferation important marker Ki67 protein. The migration of tumor cells using scratch test in the experiment. Before the experiment the migration of tumor cells and finally cell culture dish, after 24 hours when the covered dish bottom form a single cell layer after the start of a test. Ml gun head tip in the dish bottom "ten" shape and on the base plate scratch marks, in order to ensure the fixed location, space form under the microscope photograph scratches caused. After adding concentration of 1mM of tumor cell culture solution Bupivacaine, training again after 24 hours under the microscope and photographed by Image-Pro Plus scratch morphology, software defect area ratio before and after the change, according to the formula (initial defect area - Final defect area) / initial defect area, calculate the wound healing rate, which estimates the overall cell migration rate.
Results: the fluorescence intensity showed that cultured 24 hours after adding 1mM bupivacaine medium, the expression of Ki67 and SKOV-3 in PC-3 cells were significantly decreased, 1mM showed that the two bupivacaine can effectively inhibit the proliferation of tumor cells. The ability of cell migration experiment, SKOV-3 and PC-3 in 1mM bupivacaine, compared with the control group the wound healing rate was significantly decreased, showed that the cell migration ability because of the existence of bupivacaine decreased. Conclusion: the proliferation and migration of bupivacaine can effectively inhibit SKOV-3 and PC-3 tumor cells.
The third part of GSK-3 beta in tumor cell apoptosis induced by bupivacaine
Objective: To explore the GSK-3 beta activity has any impact on SKOV-3 and PC-3 bupivacaine induced apoptosis of tumor cells.
Methods: in vitro cultured ovarian cancer (SKOV-3) and prostate cancer (PC-3) cells, before the experiment to tumor cell seeding slides, culture began to test 24 hours after cells reached 70%-80% fusion degree in 24 well plate. The lmM of bupivacaine after 24 hours of treatment, the total determination of phosphorylation of GSK-3 beta immunofluorescence, activation GSK-3 beta (GSK-3 beta tyr216) and inhibition of GSK-3 beta (GSK-3 beta ser9). The amount of other experiments in 1mM bupivacaine culture medium was added on the basis of inhibiting the activity of GSK-3 inhibitor SB-216763, tumor cell survival. Using gene silencing technology with siRNA sc-35527 silencing GSK-3 gene, on the basis of joining the 1mM bupivacaine, after 24 hours of incubation with immunofluorescence technique to observe the apoptosis protease Caspase3, Caspase8, Caspase9 expression, and the final survival of tumor cells.
Results: the results showed that 1mM of bupivacaine after 24 hours, the total SKOV-3 phosphorylation of GSK-3 beta, GSK-3 beta ryr216 and GSK-3 beta ser9 increased significantly, the phosphorylation of GSK-3 beta tyr216 GSK-3 beta activity increased most obviously, there are nearly one fold increase in PC-3 phosphorylation. The total GSK-3 beta. There were no significant changes in GSK-3 tyr216 and GSK-3 ser9 beta beta. The use of inhibitors and SiRNA are partially alleviate the apoptosis of SKOV-3 cells induced by bupivacaine, but these effects are not found in PC-3 cells. The silencing of GSK-3 beta gene, we found only weak expression of GSK-3 beta, immunofluorescence analysis showed that apoptosis related protein Caspase3 the expression of Caspase9, Caspase8, GSK-3 with beta gene silencing also decreased significantly.
Conclusion: 1mM bupivacaine increased phosphorylation of SKOV-3 GSK-3 beta, GSK-3 beta tyr216 and GSK-3 beta ser9, the activation of GSK-3 beta increased most significantly. But no influence on PC-3.GSK-3 beta activity is a necessary condition for bupivacaine induced SKOV-3 cell apoptosis, and the PC-3 of tumor cells induced by bupivacaine apoptosis has no significant effect.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614;R737.31;R737.25

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