【作者】 尹凤玲；

【导师】 严春寅； 沈宗姬；

【作者基本信息】 苏州大学 ， 泌尿外科（专业学位）， 2013， 博士

【摘要】 第一部分4-HPR联合DDP对卵巢癌细胞株增殖抑制作用的体外实验研究目的：4-羟苯基维胺脂(N-4-hydroxyphenyl retinode,4-HPR)是一种全反式维甲酸的衍生物,实验研究和部分临床实验表明,它对各种肿瘤有化学预防和治疗双重作用；顺铂(cisi-platin,DDP)是卵巢癌常用的化疗药物,但因其毒副作用及其耐药等问题,临床应用受到限制。本研究探讨在体外环境下,4-羟苯基维胺酯(4-HPR)联合顺铂(DDP)对人卵巢癌细胞株SKOV-3增殖及凋亡的影响。方法：4-HPR单药,DDP单药及4-HPR+DDP联合用药方式多种剂量分别干预人卵巢癌细胞株SKOV-3; MTT法检钡SKOV-3细胞增殖抑制率变化；中效原理法判断联合用药的相互作用；HOCHST和流式细胞单染和双染分析凋亡细胞比例。实验结果计量资料以均数±标准差表示。应用SPSS13.0统计分析软件进行t检验,P<0.05被认为差异有统计学意义。结果：MTT检测方法结果：1、两种药物单独使用时均具有剂量依赖性和时间依赖性。2、DDP：药物作用48小时,各相邻药物浓度组之间,只有DDP20μM与DDP10μM浓度组之间有统计学差异(P<0.05)；药物作用72小时,从DDP2.5μM浓度组开始均与相邻药物浓度组之间有统计学差异。3、4-HPR;药物作用48小时,各相邻药物浓度组之间,只有4-HPR20μM与4-HPR10μM浓度组之间有统计学差异；药物作用72小时,从4-HPR10μM至4-HPR20μM浓度组均与相邻药物浓度组之间有统计学差异。4、两种药物联合应用后,根据中效浓度的计算,除4-HPR1μM+DDP5μM在48h的联合应用组外,在其余48h与72h的所有联合应用组,均CI<1,即两种药物联合应用产生了协同作用的效果。HOECHST检测方法：1)DDP与4-HPR在作用SKOV-3细胞株72h后,两种药物单独使用时,均具有剂量依赖性。2)DDP与4-HPR的联合应用,根据中效浓度的计算,4-HPR5μM+DDP组和4-HPR10μM+DDP5μM组均为CI<1,即两种药物联合应用产生了协同作用的效果。流式双染检测法结果：1)DDP与4-HPR在作用SKOV-3细胞株72h后,两种药物单独使用时均具有剂量依赖性。2)DDP与4-HPR的联合应用,根据中效浓度的计算,三组药物联合应用组均为CI<1,即两种药物联合应用产生了协同作用的效果。流式单染检测法：1)DDP与4-HPR联合应用组：DDP通过降低G1期细胞的比例,主要将细胞阻滞在G2期,抑制SKOV-3细胞的增殖,而4-HPR主要将细胞阻滞在S期和G2期。联合应用两种药物的效果综合了两种药物的单独作用效果。结论：SKOV-3细胞在4-HPR5μM+DDP5μM和4-HPR10μM+DDP5μM联合作用72h后,能够产生明显的抑制增殖的作用,且作用协同,其中4-HPR的使用浓度越高,抑制效应越明显。第二部分4-HPR联合DDP对卵巢癌移植瘤增殖抑制作用的体内实验研究目的：建立人卵巢癌皮下移植瘤裸鼠模型,探讨4-HPR和DDP单独应用及联合应用对移植瘤的作用,进一步探讨其作用机制。方法：先培养人卵巢癌细胞株SKVO-3,用细胞悬液接种法制备荷瘤鼠模型1只；再用套管针组织块接种法进行皮下接4-HPR组种,共接种30只裸鼠；观察成瘤情况；分组后腹腔分别给药：(1)对照组：生理盐水；(2)4-HPR组:8mg/kg;(3)顺铂组：3mg/kg;(4)联合用药组：4-HPR8mg/kg,4-HPR给药6小时后给顺铂3mg/kg,均为腹腔注射给药,一次注射剂量为0.2ml,每周两次,共三周。每周测量肿瘤长、短径,计算肿瘤体积,绘制肿瘤生长曲线,取瘤组织进行病理学检查；免疫组织化学S-P法检测瘤组织Caspase-3蛋白的表达；检测细胞凋亡率和细胞周期变化。结果：用2.5×1010个/只细胞悬液接种成功制备荷瘤鼠一只,套管针皮下接种SKVO-3瘤块后,2～3天皮丘消平,约4～5天可见微小的肿瘤结节,接种成功率为100%。治疗结束时测移植瘤体积分别为4937.72±563.28mm3、3726.34±462.32mm3、2687.78±631.91mm3、1532.69±723.84mm3,抑瘤率分别为0.00%、30.21%、42.78%、61.37%。移植瘤体积,各处理组均小于对照组,联合用药组小于单药组,差异均有统计学意义(p<0.05)；肿瘤组织病理：剖视肿瘤,对照组大部分呈类圆形,有的肿瘤可见表面血管,颜色灰白,质地较硬。光镜下见,对照组肿瘤具有恶性肿瘤细胞的一般特性,还具有特征性的腺腔样结构。实验组见不同程度点、片状坏死,并有炎性细胞的浸润,以联合用药组最重。免疫组织化学法检测Caspase-3的变化,对照组、4-HPR组、顺铂组、联合用药组,结果用免疫组化评分(HIS)分别为：2.78±1.33、4.15±0.76、5.22±0.62、6.37±1.25,各组与对照组相比,联合用药组与单药组相比,差异均有统计学意义(p<0.05)。流式细胞术结果显示：对照组、4-HPR组、顺铂组、联合用药组,细胞凋亡率逐渐升高,分别为6.43±2.31%、14.75±4.32%、21.54±1.47%、25.76±3.42%,细胞周期Go/G1期细胞逐渐减少,G2/M期细胞逐渐增多,S期细胞变化不明显。结论：4-HPR对SKVO-3细胞的增殖抑制作用呈时间-剂量依赖关系,4-HPR与顺铂联合应用可增强顺铂对SKVO-3细胞的增殖抑制作用。4-HPR可抑制SKVO-3细胞裸鼠移植瘤的生长,4-HPR与顺铂联合应用抑瘤效果增强。4-HPR可使SKVO-3细胞,细胞周期阻滞于G2/M期,诱导其凋亡,4-HPR与顺铂联合应用诱导SKVO-3细胞凋亡作用增强。4-HPR可通过增加Caspase-3蛋白的表达,增强SKVO-3细胞对顺铂的敏感性。更多还原

【Abstract】 SECTION ONE:THE SYNERGISTIC EFFECTS OF4-HPR COMBINED WITH CISPLATIN ON THE PROLIFERATION AND APOPTOSIS OF HUMAN OVARIAN CARCINOMA SKOV-3IN VITROobjective:N-4-hydroxyphenyl retinode (4-HPR) is a synthetic amide analogue of ATRA. It was reported that4-HPR could inhibit the growth of ovarian cancer cells in vitro. Cis-platinum (DDP) is a first choice chemical agent used in ovarian cancer chemo-therapy. To study the proliferation and apoptosis effects of combinative application of4-HPR and DDP on the human ovarian carcinoma SKOV-3Methods:Human ovarian carcinoma SKOV-3cell lines were interfered by4-HPR groups, DDP groups and combinative groups of4-HPR and DDP. The inhibitory effects of4-HPR and DDP on the proliferation SKOV-3were evaluated by MTT assay. The median-effect principle was used to analyze the mechanisms of the inhibitory effect after exposure to the combinative application of4-HPR and DDP. Cell apoptosis was detected by flow cytometry. Datas were shown as means±SEM by using SPSS software(version13.0; Chicago, USA). Significant differences were considered at P<0.05.Results:MTT:1. It showes time-dose dependent when the two drugs used alone.2. DDP:There are statistical differences only between DDP20μM and DDP10μM in each adjacent drug group after48hours. There are statistical differences from DDP2.5μM with their adjacent drug groups after72hours.3.4-HPR:There are statistical differences only between4-HPR20μM and4-HPR10μM in each adjacent drug group after48hours. There are statistical differences from4-HPR10μM to4-HPR20μM with their adjacent drug group after72hours.4. After the combination of two drugs, all the groups after48and72hours show CI<1except4-HPR1μM+DDP5μM after48hours.HOECHST:1. Both DDP and4-HPR have a dose-dependent manner when used72hours.2. DDP with4-HPR in combination, according to the calculation of the moderate effective concentration, Both4-HPR5μM+DDP5μM group and4-HPR10μM+DDP5μM group show CI<1, In other words, the combination of two drugs produces a synergistic effect.Streaming double staining method:1. Both DDP and4-HPR have a dose-dependent manner when used72hours.2. DDP with4-HPR in combination, according to the calculation of the moderate effective concentration, the three groups of drug combination group were CI<1, in other words, the combination of two drugs produces a synergistic effect.Streaming single dye detection method:DDP with the combination of4-HPR group:DDP by reducing the proportion of cells in G1phase, inhibit the proliferation of SKOV-3cells through the cell cycle arrest in the G2phase;4-HPR arrest the cell in the S and G2phase. Two drugs have additive effect.Conclusion:4-HPR5μM+DDP5μM and4-HPR10μM+DDP5μM significantly inhibited proliferation of ovarian cancer SKOV-3cells after72hours, and the effect is synergistic. The intensity increased with the concentration of4-HPR increased. SECTION TWO:THE SYNERGISTIC EFFECTS OF4-HPR COMBINED WITH CISPLATIN ON THE PROLIFERATION AND APOPTOSIS OF HUMAN OVARIAN CARCINOMA SKOV-3IN VIVOObjective. To establish the tumor-bearing nude mice models for human ovarian cancer, exam the effect of the application of4-HPR, DDP and combination of both4-HPR and DDP on transplanted tumor for further discussion of its mechanism of action.Methods. In preparation of the tumor-bearing mice model, the human epithelial ovary cancer cell line SKVO-3which we fostered in advance was vaccinated to the nude mice using the cell suspension method.30mice were subcutaneously vaccinated with4-HPR using the trocar tissue block inoculation method. They were subsequently divided into groups and treated with medicine separately according to the formation of the tumor:(1) Control group:physiological saline;(2)4-HPR group:8mg/kg;(3) Cisplatin group:3mg/kg;(4) Combined treatment group:3mg/kg of cisplatin6hours after8mg/kg of4-HPR, both treated with intraperitoneal injection,0.2ml each time, twice a week for3weeks, the size of the tumor was measured every week and the tumor formation curve was graphed, tumor was removed for histopathological analysis; the expression of caspase-3was detected by immunohistochemical S-P method; the apoptosis rate and the periodic change of the cells were also detected.Results. The mouse which was vaccinated with2.5×1010per mouse dosage cell suspension was successfully produced,2-3days after being subcutaneously vaccinated with SKVO-3tissue block, the ridge disappeared, tiny tumor nodules could be observed, the vaccination was successful. By the end of the treatment, the volumes of transplanted tumor were:4937.72±563.28mm3,3726.34±462.32mm3,2687.78±631.91mm3and1532.69±723.84mm3, the inhibition rates were:0.00%,30.21%,42.78%and61.37%. The sizes of the transplanted tumor in groups which were treated were significantly smaller than the size of the one in the control group; the transplanted tumor size in the combined treatment group was smaller than the one in single treatment groups, all differences have statistical significance(p<0.05); the histopathological analysis of the tumor tissue: according to the section view, the majority appeared to be round and tough grey surface vessels were observed. Through the light microscope, the tumor in control group demonstrated the general characters of malignancy and the distinctive structure of glandular cavity. Punctiform, flake necrosis emerged in in experimental groups with the infiltration of inflammatory cells, this phenomenon was most significant in the combined treatment group. Caspase-3which was examined by the immunohistochemical method for control group,4-HPR group, Cisplatin group and combined treatment group the IHS was:2.78±1.33,4.15±0.76,5.22±0.62and6.37±1.25. The differences among groups have statistical significance. The flow cytometry illustrated that the cell apoptosis rate in all groups gradually raised to6.43±2.31%,14.75±4.32%,21.54±1.47%and25.76±3.42%, G0/G1period cells reduced while the amount of G2/M period cells increased, and S period cells remained the same amount.conclusion.The4-HPR inhibition of SKVO-3cells proliferation illustrated its dependence on time and dosage, and the combined treatment of4-HPR and cisplatin demonstrated enhanced suppression on the formation of SKVO-3cells.4-HPR can block SKVO-3cells in the G2/M period and lead them to apoptosis and the effect is more efficient with cisplatin.4-HPR has the potential to improve the caspase-3expression and increase the sensitivity of SKVO-3cells to cisplatin.更多还原