【作者】 许连军；

【导师】 高润霖；

【作者基本信息】 北京协和医学院 ， 内科学， 2010， 博士

【摘要】 药物洗脱支架(DES)是介入心脏病学的重要进展,DES使支架内再狭窄率由裸金属支架时代的20-30%下降到10%左右,然而DES轻度但有统计学意义地增加了极晚期血栓形成的发生率。其原因可能与不能降解的多聚物涂层引起的炎症或过敏反应有关；也可能与DES携载的抗细胞增殖药物有关,这些药物有效地抑制了支架置入术后血管平滑肌细胞过度增生,但同时也抑制了血管内皮细胞再生,延迟内皮愈合,从而增加支架内血栓的风险。人们期待研发出既能有效抑制平滑肌细胞增生,又对内皮细胞再生无明显影响的药物作为DES携载的药物。双环铂是继卡铂、顺铂后应用于临床抗肿瘤治疗的第三代铂类制剂,由北京兴大科学系统公司开发,是我国具有完全自主知识产权的化学Ⅰ类抗癌新药,双环铂的研究目前尚处于实验室研究和Ⅲ期临床研究阶段,现有资料表明,其抗癌疗效好,毒性低。我们拟研究其对平滑肌细胞及内皮细胞增生的影响,探讨作为支架携载药物的可能性。目前尚无双环铂对正常或良性增生的组织、细胞影响研究的文献报道。目的本研究通过体外实验,培养人主动脉平滑肌细胞(HASMC)和人主动脉内皮细胞(HAEC),MTS法检测不同浓度的双环铂对HASMC和HAEC增殖的影响,通过流式细胞仪检测不同浓度双环铂对HASMC和HAEC细胞周期的影响,探讨其可能的作用机制,旨在评价新药双环铂在防治支架置入术后再狭窄方面的潜在价值。在分子水平上,通过检测双环铂干预后HASMC和]HAEC细胞增殖核抗原PCNA,促凋亡蛋白Bax,抗凋亡蛋白Bcl-2,探讨双环铂在增殖和凋亡方面对HASMC和HAEC的影响。方法一、MTS法检测双环铂对HASMC和]HAEC增殖的影响通过体外扩增、培养HASMC和HAEC细胞株。10μg/ml,1μg/ml,100ng/ml双环铂干预培养HASMC,10μg/ml,1μg/ml双环铂干预培养HAEC,药物干预24小时、48小时、72小时,MTS法检测细胞增殖活性,计算细胞增殖抑制率,探讨双环铂的对HASMC和HAEC抑制增殖作用与时间、浓度的关系。双环铂100μg/ml起倍比稀释到0.39μg/ml共9个浓度组,干预培养HASMC72小时,双环铂200μg/ml起倍比稀释到1.56μg/ml共8个浓度组,干预培养HAEC72小时,MTS法检测细胞增殖活性,以log(双环铂浓度)与细胞生长率为变量作图,得出双环铂对HASMC和HAEC的半数抑制率IC50。双环铂浓度分别为10μg/ml,1μg/ml,100ng/ml,10ng/ml,1ng/ml,干预培养HASMC和HAEC72小时,使用雷帕霉素作为阳性对照药物,MTS法检测细胞增殖活性。使用SPSS10.0软件,单因素方差分析法进行统计分析,探求双环铂对HASMC和HAEC抑制增殖的最小有效浓度。二、流式细胞仪检测双环铂对HASMC和HAEC细胞周期的影响HASMC和HAEC接种24小时后,无血清培养24小时进行细胞周期同步化,HASMC分为6组,分别为正常对照组,雷帕霉素1ng/ml阳性对照组,双环铂1μg/ml组,100g/ml组,10ng/ml组,1ng/ml组。HAEC分为6组,分别为正常对照组,雷帕霉素10ng/ml组,双环铂10μg/ml组,1μg/ml组,100ng/ml组,10ng/ml组。药物干预培养48小时后,收集细胞,低温固定,碘化丙啶(PI)单染,通过流式细胞仪检测细胞周期变化。三、免疫印迹法(Western Blot)检测双环铂对细胞增殖蛋白PCNA和凋亡蛋白Bax, Bcl-2表达的影响HASMC和HAEC接种培养24小时后,改用无血清培养基培养24小时,HASMC分为6组,分别加入双环铂1μg/ml,100ng/ml,10ng/ml,1ng/ml,阳性对照组加入1ng/ml雷帕霉素,正常对照组予含2%血清的SMCM培养。HAEC分为6组,分别加入双环铂10μg/ml,1μg/ml,100ng/ml,10ng/ml,阳性对照组加入lOng/ml雷帕霉素,正常对照组予含有5%血清的ECM培养。药物干预48小时,提取细胞总蛋白。Western Blot法分别检测HASMC和HAEC的细胞增殖核抗原PCNA,促凋亡蛋白Bax和抗凋亡蛋白Bcl-2的表达情况。结果一、双环铂对HASMC和HAEC增殖的影响对于HASMC,双环铂10μg/ml组干预1天,2天,3天的增殖抑制率分别为16%,39%,75%,1μg/ml组增殖抑制率分别为13%,13%,27%,100ng/ml组增殖抑制率分别为11%,9%,20%。对于HAEC,双环铂10μng/ml干预1天,2天,3天的增殖抑制率分别为6.41%,11.05%,14.83%,1μg/ml组干预HAEC增殖抑制率分别为1.35%,0.60%,1.3%。双环铂对HASMC和HAEC的半数抑制浓度(IC50)分别为3.47μg/ml,72.44μg/ml,两者相差20.88倍。双环铂浓度分别为10μg/ml,1μg/ml,100ng/ml,10g/ml,1ng/ml时,干预培养HASMC和HAEC3天,10ng/ml以上浓度HASMC增殖受到明显抑制(P<0.05), lng/ml时双环铂对HASMC无明显增殖抑制(P=0.83)；在10μg/ml以上浓度时HAEC增殖受到明显抑制(P<0.001),双环铂浓度在1ng/ml-1μg/ml时,对HAEC无明显增殖抑制作用(P>0.05),双环铂对HAEC的最小有效抑制浓度较对HASMC的最小有效抑制浓度高1000倍。阳性对照药物雷帕霉素在1ng/ml时,即表现出对HASMC和HAEC的抑制增殖作用,相同浓度下,对HAEC抑制作用稍弱。二、双环铂对HASMC和HAEC细胞周期的影响双环铂干预HASMC生长48小时后以流式细胞仪检测细胞周期,试验重复4次,1μg/ml双环铂对HASMC作用48小时,G2/M期细胞百分比增加,细胞周期阻滞在G2/M期,10ng/ml-100ng/ml双环铂对HASMC作用48小时,S期细胞百分比增加,细胞周期阻滞于S期。阳性对照药物雷帕霉素对细胞周期的阻滞在Go/G1期。双环铂干预HAEC生长48小时后检测细胞周期,实验重复5次,在10μg/ml以上浓度时HAEC S期数目明显增加,双环铂对HAEC周期的阻滞处于S期。在10μg/ml以下浓度时双环铂对细胞周期的阻滞不显著。阳性对照药物雷帕霉素组,G0/G1期细胞数目增加,细胞周期阻滞在Go/G1期。在对HASMC和HAEC细胞周期检测中各药物组及正常对照组均未见凋亡峰(Sub-G1)。三、双环铂干预培养后HASMC和]HLAEC细胞增殖蛋白PCNA和凋亡蛋白Bax, Bcl-2表达的变化HASMC在不同浓度双环铂干预下,PCNA蛋白的表达量明显下降,其中双环铂100ng/mL组PCNA表达量达最低值。HAEC在不同浓度双环铂干预下,10μg/ml组和100ng/ml组PCNA表达量轻度减低,其他浓度组PCNA表达无明显变化。雷帕霉素在HASMC和HAEC中均抑制了PCNA的表达。在HASMC中,促凋亡蛋白Bax在各双环铂浓度组表达量均明显上升。抗凋亡蛋白Bcl-2表达在双环铂1μg/ml组升高,在100ng/ml组较正常组相比无明显差异,在10ng/ml、1ng/ml组Bcl-2表达稍减低,Bax/Bcl-2比值增高,即双环铂有促进凋亡作用。雷帕霉素组Bax/Bcl-2较正常对照组无明显变化。在HAEC中,Bax在各双环铂浓度组表达均升高,且随着药物浓度上升,Bax表达量有升高趋势。雷帕霉素组Bax表达较正常组降低。在同样的试验条件下,未检测到HAEC中Bcl-2蛋白表达。上述结果表明双环铂对HAEC也有促进凋亡的作用,低浓度的雷帕霉素无促凋亡作用。结论双环铂对HASMC和HAEC产生抑制增殖作用与药物作用时间、浓度呈正相关。随着药物作用时间的延长,药物浓度的增加,双环铂对细胞增殖的抑制作用逐渐加强。双环铂对HASMC的半数抑制浓度(IC50)为3.47μg/ml,对HAEC的半数抑制浓度为72.44μg/ml,两者相差20.88倍。双环铂对HAEC抑制增殖的最低有效浓度(10μg/ml)较对HASMC抑制增殖的最低有效浓度(10ng/ml)高1000倍。双环铂对HASMC和HAEC细胞周期的影响检测表明,双环铂为非特异性细胞周期阻滞药物。双环铂干预HASMC和HAEC后,HASMC表达PCNA显著下降,而HAEC表达PCNA正常或轻度下降,结果表明相同浓度下双环铂对HASMC的增殖抑制作用明显强于对HAEC的增殖抑制作用。双环铂干预HASMC和AEC后,促进HASMC凋亡蛋白的表达,表明双环铂在细胞凋亡的线粒体通路上,通过调节Bax和Bcl-2的表达,产生促进细胞凋亡的作用。但是这种促进凋亡的作用只体现在蛋白水平上,在细胞学实验中,流式细胞检测未见凋亡峰出现。综合上述实验结果,尽管在蛋白检测中发现细胞凋亡蛋白表达升高,但是细胞周期检测中在各个双环铂浓度组均未见凋亡峰,表明低浓度双环铂对细胞的抑制作用,主要是通过抑制细胞增殖来体现的。与雷帕霉素相比,双环铂抑制平滑肌作用尽管稍弱,但在纳克水平即可产生对平滑肌细胞的抑制作用；与雷帕霉素不同的是,双环铂对内皮细胞增殖的影响明显弱于对平滑肌细胞增殖的影响,在微克水平才产生对内皮细胞的抑制作用。双环铂这种对内皮细胞和平滑肌细胞抑制作用显著不同的特点,使其有希望成为药物洗脱支架的携带药物,值得进一步研究。更多还原

【Abstract】 BackgroundDrug-eluting stent (DES) has shown superior angiographic and clinical results compared with bare metal stent (BMS) in patient with coronary artery disease undergoing PCI. DES decreases late loss and restenosis by reduced neointimal proliferation following stenting, however it also inhibits re-endothelialization and vascular healing which can increase the incidence of in-stent thrombosis. We expect to approach a new drug which could inhibit the overgrowth of vascular smooth muscle cells (SMCs), and have no significant effect on re-endothelialization.Dicycloplatin is the third generation of platimum complex, developed by Xing Da Scientific Systems company, which own country has the independent intellectual property rights. At present the studies about dicycloplatin are in laboratory research and phrase Ⅲ clinical trial, which showed significant antitumor effects and safety characteristics, no studies about the effect on normal and benign proliferation cell and tissues is reported.ObjectiveThe objective of the study is to investigate the effects of dicycloplatin on proliferation of HASMC and HAEC in vitro and to approach the potential possibility of dicycloplatin to be used for DES. Human aortic smooth muscle cells (HASMC) and human aortic endothelial cells (HAEC) were cultured in vitro. In cytology level, MTS assay was used to evaluate the effect of dicycloplatin on the proliferation of HASMC and HAEC, flow cytometry was used to detect the effect of dicycloplatin on cell cycle. In protein molecular level, the proliferating cell nuclear antigen (PCNA), Bax, and Bcl-2protein expression were detected to evaluate the effects of dicycloplatin on proliferation and apoptosis in HASMC and HAEC. Methods1. MTS assay to evaluate the effects of dicycloplatin on proliferation of HASMC and HAEC.HASMC and HAEC were cultured in vitro. HASMC and HAEC were incubated with different concentration of dicycloplatin for24hours,48hours, and72hours, cell proliferation was measured by MTS assay to explore if the efficacy is time and dose dependent.Double diluted dicycloplatin from100μg/ml to0.39μg/ml was incubated with HASMC for72hours; double diluted dicycloplatin from200μg/ml to1.56μg/ml was incubated with HAEC for72hours, cell proliferation was measured by MTS assay, log(concentration) and growth rate were used to explore the50%inhibited concentration(IC50) for HASMC and HAEC.HASMC and HAEC were incubated with different concentration of dicycloplatin (10μg/ml,1μg/ml,100ng/ml,10ng/ml,1ng/ml) for72hour, rapamycin as positive control groups, cell proliferation was measured by MTS assay, SPSS software, One-Way ANOVA method were used to analyze the minimum effective concentration.2. Flow Cytometry to detect the influence of dicycloplatin and rapamycin on the HASMC and HAEC cell cycleHASMC and HAEC were cultured for24hours, then cultured with serum-free smooth muscle cell medium (SMCM) or endothelial cell medium (ECM) for24hours. HASMC were divided into6groups:negative control group, lng/ml of rapamycin group;1μg/ml,100ng/ml,10ng/ml,1ng/ml dicycloplatin groups. HAEC were divided into6groups:negative control groups,10ng/ml of rapamycin group,10μg/ml,1μg/ml,100ng/ml,10ng/ml of dicycloplatin groups. Cell samples were collected48hours after incubation, the samples were fixed in low temperature, PI dyeing for30min, the cell cycle was detected by flow cytometry.3. Western Blot to detect the effect of dicycloplatin on PCNA, Bax and Bcl-2protein expression in HASMC and HAEC.The divided group, treatment with different concentration of dicycloplatin and rapamycin were same as above. Protein samples were collected48hours after incubation. Protein expression of PCNA, Bax, Bcl-2were analyzed using Western blot.Result1. The effects of dicyclopaltin on proliferation of HASMC and HAEC.For HASMC incubated with lOμg/ml dicycloplatin for24hours,48hours,72hours, the proliferation inhibiting rate were16%,39%and75%, respectively; the inhibiting rate in1μg/ml group were13%,13%and27%, respectively; the inhibiting rate in100ng/ml group were11%,9%and20%. For HAEC incubated with lOμg/ml dicycloplatin for24hours,48hours,72hours,the proliferation inhibiting rate was6.41%,11.05%and14.83%, respectively; the inhibiting rate in1μg/ml group were1.35%,0.60%and1.3%, respectively. The inhibiting efficacy of dicycloplatin is in a dose and time dependent manner.The drug concentration at which maximal HASMC and HAEC proliferation were inhibited by50%(IC50) were3.47μg/ml and72.44μg/ml respectively. The IC50for HAEC is20.88times higher than that for HASMC.Dicycloplatin in lOng/ml-lOμg/ml remarkably inhibited proliferation of HASMC (P<0.05), compared with the negative control group. Dicycloplatin in lng/ml had no proliferation inhibiting effect on HASMC (P=0.83). Dicycloplatin in10μg/ml remarkably inhibited proliferation of HAEC (P<0.05), dicycloplatin in1ng/ml-1μg/ml had no proliferation inhibiting effect (P>0.05). The minimum effective inhibiting concentration for HAEC was103-times higher than it for HASMC. The positive control group, rapamycin in lng/ml inhibited proliferation both for HASMC and for HAEC, and the effect was weaker in HAEC than in HASMC.2. Cell cycle analysis demonstrated that:for HASMC, dicycloplatin in1μg/ml arrested cell cycle in G2/M phase; in lOng/ml-100ng/ml arrested in S phase. For HAEC, dicycloplatin in10μg/ml arrested cell cycle in S phase; the rest groups showed no significant difference in cell cycle. Dicycloplatin was a non-specific inhibiter on cell cycle. In all groups, there were no apoptosis peak (Sub-G1) detected.3. In HASMC, PCNA expression remarkably decreased in all dicycloplatin groups compared to negative control group, Bax expression was increased in all dicyclopaltin groups, Bcl-2expression was increased in dicyclopatin1μg/mL group, decreased in10ng/ml and lng/ml groups. The ratio of Bax/Bcl-2was increased in all dicycloplatin groups. In HAEC, PCNA expression was normal or decreased slightly compared with negative control group, Bax expression was increased in all dicycloplatin groups. In the same experiment condition, Bcl-2was not detected in HAEC.ConclusionThe inhibiting effect of dicycloplatin on proliferation of HASMC is in a dose and time dependent manner. The IC50for inhibiting HAEC proliferation is about20.88times higher than that for HASMC. The minimum effective concentration of dicycloplatin for inhibiting proliferation of HAEC is103-times higher than that for HASMC.Cell cycle analyze show that dicycloplatin is a non-specific inhibiter on cell cycle.At the same concentration, dicycloplatin inhibits PCNA expression more remarkably in HASMC than in HAEC. In the mitochondrion apoptosis pathway, dicycloplatin regulates the expression of Bax and Bcl-2, even in low concentratin, dicycloplatin can promote apoptosis in HAEC and HASMC. This effect is only detected in protein level, in cell cycle analysis there are no apoptosis peak detected.In summery, although in Western blot experiment apoptosis associate protein expression is up regulated, there are no apoptosis peak(Sub-G1) detected in cell cycle analysis. In low concentration, dicyclopatin inhibits cell proliferation rather than promotes apoptosis. Similar with rapamycin, dicyclopatin significantly inhibits proliferation of HASMC in nanogram level, but the effect is weaker than rapamycin. Different to rapamycin, dicycloplatin inhibiting proliferation of HAEC needs much higher concentration. This characteristic of dicycloplatin may have potential to be used for a drug carried by DES.更多还原