【作者】 李月红；

【导师】 易建平；

【作者基本信息】 河北医科大学 ， 妇产科学， 2007， 硕士

【摘要】 目的:探讨氯丙嗪(Chlorpromazine,CPZ)对人宫颈癌Hela细胞体外生长的抑制作用及其相关机制,同时观察氯丙嗪联合抗癌药物顺铂(Cisplatin,DDP)对Hela细胞的体外作用。从而探讨其抗癌疗效,评价氯丙嗪治疗宫颈癌的安全性、可行性,以期对药物治疗宫颈癌提供新思路、新方法。方法:选取人宫颈癌Hela细胞为研究对象。实验分三组,即氯丙嗪、顺铂、氯丙嗪与顺铂联合组。1采用四甲基偶氮唑蓝(tetrzolium-based colorimetric assay, MTT)比色法观察不同浓度的氯丙嗪分别作用24h、48h、72h对Hela细胞增殖的影响,测定吸光度(OD)值。不同浓度的氯丙嗪作用Hela细胞48h后,流式细胞仪检测凋亡细胞百分率、细胞周期的改变以及凋亡基因Bax、Bcl-2表达量的改变; DNA凝胶电泳分析DNA片段的变化。2不同浓度各组药物作用48h后,采用MTT比色法检测对Hela细胞增殖的影响,测定OD值; HE染色分析细胞的形态学变化;链亲和素免疫组化法(S-P法)检测细胞p53的表达量变化。结果:1 MTT结果显示:一定浓度的氯丙嗪在一定范围内能明显抑制Hela细胞的增殖,且随氯丙嗪浓度的升高和作用时间的延长,抑制作用明显升高,呈现出明显的浓度依赖性和时间依赖性。分别用氯丙嗪、顺铂及联合用药处理细胞48h后,氯丙嗪在浓度为10-7mol /L、10-6mol /L、10-5mol/L、10-4mol /L时对Hela细胞的抑制率分别为22.74%、41.69%、51.39%、67.45%,与对照组(加入等量培养液)比较,差异有非常显著性(P<0.01);顺铂在浓度为0.2μmol/L、0.5μmol/L、1. 0μmol/L和2.0μmol/L时对Hela细胞的抑制率为43.88%、48.59%、61.49%和71.52%;两者合用可大大提高对Hela细胞的抑制作用,抑制率分别为49.70%、63.52%、70.42%、76.15%,明显高于单纯应用相同浓度的顺铂组。2流式细胞术结果分析:流式细胞仪检测Hela细胞周期分布和凋亡显示,10-7、10-6、10-5、10-4mol /L氯丙嗪处理Hela细胞48h后,随药物浓度的增加,G0/G1期细胞逐渐增多,S期和G2/M期细胞则逐渐减少,即氯丙嗪可阻滞Hela细胞周期于G1期,该作用呈剂量-效应依赖关系;10-7、10-6、10-5 mol /L氯丙嗪作用Hela细胞48h后,出现典型的亚二倍体凋亡峰,依氯丙嗪浓度增大而逐渐增高,凋亡百分率分别为:5.03%、9.35%、23.85%;流式细胞仪间接分析细胞Bax、Bcl-2的蛋白表达显示,10-7、10-6、10-5 mol /L氯丙嗪作用Hela细胞48h后,Bax的荧光指数FI值随处理浓度增大而逐渐增大,Bcl-2的荧光指数FI值随处理浓度增大而逐渐减小,对于每一种蛋白,其实验组与对照组的FI值,均具有显著性差异(P<0.01)。3 DNA凝胶电泳结果分析:10-7、10-6、10-5、10-4mol /L氯丙嗪处理Hela细胞48h后,提取的DNA表现为典型的阶梯状排列条带(DNA ladder),而对照组则没有出现。4 HE染色结果分析:细胞核染蓝紫,胞浆染粉红色。随药物浓度增大,Hela细胞由不规则形渐变圆,出现核固缩、浓聚、断裂后形成的凋亡细胞,呈致密紫蓝色大小不等的颗粒。顺铂组,当浓度为0.5μmol/L时,可见凋亡细胞;而两药联合组,当顺铂浓度为0.2μmol/L时,便出现上述表现。5免疫组化法(S-P)分析Hela细胞p53基因蛋白表达变化的结果:P53蛋白主要表达于细胞核内。氯丙嗪组,p53蛋白的表达呈升高趋势,当浓度为10-7mol/L时,p53蛋白染色细胞核的光密度值(OD)为0.1240±0.0551,当浓度升高至为10-5mol/L时,OD值为0.2020±0.0626,与对照组比较,差异具有显著性( P < 0.01)。顺铂组,p53蛋白的表达进一步上升,当浓度为0.5μmol/L时,光密度值为0.2260±0.0691,与对照组比较,具有显著性差异( P < 0.01)。两药联合组p53蛋白的表达明显增强,OD值升高明显,即最低浓度(10-7mol/L)氯丙嗪与最低浓度(0.2μmol/L)顺铂联合作用后,OD值达到0.2560±0.0305,与对照组相比,具有极其显著性差异( P < 0.01)。与对应浓度顺铂处理Hela细胞的p53蛋白染色阳性细胞光密度值比较,存在统计学差异( P < 0.01)。结论:1在一定剂量和时间范围内,氯丙嗪可以抑制人宫颈癌Hela细胞的增殖,并具有时间-剂量依赖关系;并可诱导该细胞凋亡。2氯丙嗪对顺铂有协同作用。氯丙嗪联合顺铂可明显增加顺铂对Hela细胞的增殖抑制作用,使p53蛋白的表达明显上升。更多还原

【Abstract】 Objective: By exploring the inhibition of cervical cancer Hela cells in vitro by Chlorpromazine(CPZ) and of the inhibiting mechanism, and by studying the effects of Chlorpromazine combined with anti-cancer agent Cisplatin on human cervical cancer cell Hela in vitro, this paper tries to obtain the anticancer therapeutic effect of Chlorpromazine, and to evaluate safety and the feasibility of Chlorpromazine treatment, so as to provide a new way in the medical treatment of cervical carcinoma.Methods: Human Hela cervical carcinoma cells are taken as study object and are divided into three groups: Chlorpromazine group, Cisplatin group and combination group. 1 Effect of CPZ of different concentrations on the proliferation of Hela cells with different functioning time(24h, 48h, 72h)and OD values were measured by MTT colorimetric method. Forty-eight hours after Chlorpromazine’s functioning, flow cytometer (FCM) was used to detect the changes of percentage and cell cycle of apoptosis cells,and to measure the amount of Bax and Bcl-2 protein and the analysis of changes of DNA-fragment were analyzed by DNA gelatum electrophoresis. 2 Forty-eight hours after combined functioning of Chlorpromazine and Cisplatin, the proliferation of Hela cells and OD values were measured by MTT colorimetric method; the HE staining was performed,and the morphological changes of Hela cells were observed by light microscopy; The immunohistochemical technique (S-P) was also used to identify the amount of P53 in Hela cells.Results: 1 Results of MTT indicate that CPZ, to certain extent, can inhibit the proliferation of Hela cells (compared with control group, P<0.01) and the inhibition enhances dramatically as concentration of CPZ is raised and functioning time is prolonged. The effect was presented in a dose- and time-dependent manner. When the concentration of CPZ was respectively 10-7mol/L, 10-6mol/L, 10-5mol/L, and 10-4mol/L the inhibition rate of Hela cells growth was 22.74%, 41.69%, 51.39% and 67.45% respectively, which was significantly different from the control group (same amount of culture solution was added) (P<0.01). When the concentration of Cisplatin was respectively 0.2μmol/L, 0.5μmol/L, 1.0μmol/L and 2.0μmol/L, the inhibition rate was 43.88%, 48.59%, 61.49% and 71.52% respectively. When Chlorpromazine and Cisplatin were used in combition, the inhibition rate increased to 49.70%, 63.52%, 70.42% and 76.15% respectively, much higher than those when Cisplatin only was used.2 Analysis of FCM results: the results of FCM test on cell cycle distribution and apoptosis of Hela celles indicate that the number of cells in G0/G1 phase increased gradually, while the number of cells in S phase and G2/M phase decreased gradually 48h after Chlorpromazine of 10-7, 10-6, 10-5 and 10-4mol /L CPZ were applied. That is, CPZ could hold cell cycle of Hela cells in G1 phase on a dose-dependent basis. In addition, 48h after Chlorpromazine of 10-7, 10-6, 10-5 mol /L was applied, a typical apoptotic peak was observed and the apoptosis increased gradually as Chlorpromazine concentration was raised, and the apoptotic percentage was 5.03%, 9.35% and 23.85% respectively. Analysis on expression of Bax and Bcl-2 by FCM showed that treating Hela cells with 10-7, 10-6, 10-5 mol /L CPZ respectively for 48h resulted in an increase of FI values of Bax and a decrease of FI values of Bcl-2 as Chlorpromazine concentration increased. To each kind of protein, its FI values differ dramatically between the Chlorpromazine-treated group and control group(P<0.01).3 The DNA gelaum electrophoresis showed that: forty-eight hours after treating Hela cells with Chlorpromazine of 10-7, 10-6, 10-5 and 10-4mol /L respectively, a typical DNA ladder was observed in every treatment group, while not in the control group.4 HE staining results showed that Hela cells gradually became round as Chlorpromazine concentration increased. lots of kyan-cellular nucleus, pink endochylema and apoptosis cells after karyopycnosis, thickness, collapse were observed . In Cisplatin group, the apoptotic cells were observed when the concentration was 0.5μmol/L, while the apoptotic cells were observed when the concentration was 0.2μmol/L in combination group.5 Analysis of P53 protein expression by immunohistochemical staining(S-P): P53 protein was expressed mainly in cell nucleus. In Chlorpromazine group, the expression of P53 protein showed a tendency of increasing; the optical density value of nuclear P53 protein was 0.1240±0.0551 when the concentration was 10-7mol/L; and when the concentration increased to 10-5mol/L, the OD value was 0.2020±0.0626. There was a significant difference compared with the control group (P < 0.01). In Cisplatin group, the expression of P53 protein went on increasing. When the concentration was 0.5μmol/L, the OD value was 0.2260±0.0691(P<0.01). There was a significant difference compared with the control group (P < 0.01). In combination group, the expression of P53 protein and the OD value increased markedly. That is when least concentration of Chlorpromazine(10-7mol/L)and Cisplatin(0.2μmol/L)are applied, the OD value reached 0.2560±0.0305. There was an exrtremely significant difference compared with the control group (P < 0.01) There existed a statistical significance compared with the Cisplatin group (P < 0.01) in which Hela cells were treated with Cisplatin of same concentration.Conclusions: 1 With certain dose and treating time, Chlorpromazine can inhibit proliferation of Hela cells in a dose- and time-dependent manner, and it can induce Hela cellular apoptosis. 2 The coordinative effect was observed through combining Chlorpromazine with Cisplatin. Cisplatin, if combined with Chlorpromazine, can further inhibit the proliferation of Hela cells in vitro and dramatically increase the expression of P53 protein.更多还原