【作者】 张煜辉；

【导师】 刘建民； 岳志健；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 目的:评价替莫唑胺缓释微球(TM-MS)治疗C6脑胶质瘤大鼠的有效性及安全性,研究该药有效成分治疗作用的机制,进一步研究该药与抗肿瘤血管生成药物Vatalanib联合化疗的疗效和安全性。方法:一、SD大鼠C6脑胶质瘤模型的建立:细胞培养传代C6胶质瘤细胞,取其对数期细胞,在立体定向指引下,将2×10~6个C6细胞悬液注入大鼠左侧尾状核区,观察动物接种后表现,接种后第6天行头颅MRI检查,并随机抽样处死行HE染色、免疫组化GFAP和PCNA检查,并行透射电镜检查,根据MRI、HE染色、免疫组化和透射电镜结果判定大鼠C6脑胶质瘤模型制作成功。二、TM-MS治疗C6恶性胶质瘤:1、不同剂量替莫唑胺缓释微球治疗C6胶质瘤:将荷瘤大鼠分为5组:对照组、替莫唑胺原药(TM)组、TM-MSⅠ组、TM-MSⅡ组、TM-MSⅢ组。各组化疗均在模型植入C6细胞后第6天进行,对照组切除脑肿瘤表面脑组织;TM组予以口服替莫唑胺50mg/kg/day,连服5天,并切除脑肿瘤表面脑组织;TM-MSⅠ组切除脑肿瘤表面脑组织后植入TM-MS12mg;TM-MSⅡ组切除脑肿瘤表面脑组织后植入TM-MS 16mg;TM-MSⅢ组切除脑肿瘤表面脑组织后植入TM-MS 20mg。治疗14天后分别处死各组动物(各组大鼠中一半用以观察生存期,不予处死),统计平均生存期,计算肿瘤体积和抑瘤率,行PCNA免疫组化检查,评价TM-MS疗效,并寻找合适的TM-MS用药剂量。2、根据上部分获得的TM-MS合适剂量,将TM-MS分别与植入用5-FU和替尼泊甙(VM-26)作疗效对比:将荷瘤大鼠分为4组:对照组、TM-MS组、氟尿嘧啶(FU)组和VM-26组。TM-MS组予以切除脑肿瘤表面脑组织后植入TM-MS16mg,FU组切除脑肿瘤表面脑组织后植入FU 9mg,VM-26组予以VM-26静脉微泵(替尼泊甙3.6mg微泵每日1次,连用3天)。治疗后处死(各组大鼠中一半用以观察生存期,不予以处死),统计平均生存期,计算肿瘤体积和抑瘤率,行PCNA免疫组化检查,评价化疗疗效。3、评价TM-MS间质化疗的安全性:检查间质化疗后血常规、肝肾功能的改变,通过对TM-MS周围脑组织病理学检查评价TM-MS的体内生物相容性。三、TM-MS治疗C6脑胶质瘤大鼠的作用机制:将荷瘤大鼠分为对照组和TM-MS组。治疗方式同前,治疗14天后处死,行透射电镜检查和TUNEL法检测C6细胞凋亡情况,用Western Blot法检测P53蛋白和活化caspase-3蛋白在C6细胞凋亡信号转导途径中的表达水平。四、TM-MS与Vatalanib联合化疗治疗大鼠C6脑胶质瘤:将荷瘤大鼠分为4组:对照组、TM-MS组、Vatalanib组、联合化疗组。TM-MS组治疗方式同前;Vatalanib组从植入C6细胞后行Vatalanib治疗(Vatalanib 5mg口服1/日),治疗20天后处死;联合化疗组从植入C6细胞后开始行口服Vatalanib 5mg治疗,连续治疗20天,并在第6天时加用TM-MS行间质化疗,间质化疗14天后处死(各组大鼠中一半用以观察生存期,不予以处死)。处死大鼠前抽血查血常规、肝功、生化,评价联合化疗的安全性。统计平均生存期,计算测量肿瘤体积变化,行PCNA免疫组化检测,评价化疗对肿瘤细胞增殖能力的影响;检测VEGF和C31评价化疗对肿瘤血管生成的影响;检测ICAM-1和MMP-9的水平,评价化疗对肿瘤间质的影响。结果:一、SD大鼠C6脑胶质瘤模型的建立:本组实验10只SD大鼠有9只在肿瘤细胞种植后一周左右开始出现食欲减退,精神差、活动明显减少,皮毛粗糙,体重减轻。头颅MRI平扫和增强提示:左基底节占位,T1低信号,T2高信号,强化均匀明显,大鼠脑内占位形成。处死后标本GFAP和PCNA检查提示:GFAP在肿瘤细胞胞浆高表达提示肿瘤为神经胶质源性,PCNA在肿瘤细胞胞核高表达表示肿瘤细胞有较高的增殖能力。透射电镜检查显示:肿瘤细胞突触和微绒毛较多,是神经胶质源性肿瘤的特征之一,核内异染色质较多,表明细胞有较高的增殖能力。根据大鼠头颅MRI、免疫组化和电镜结果,说明大鼠C6脑胶质瘤模型成功建立,肿瘤种植成功率较高,达90%。二、替莫唑胺缓释微球治疗C6恶性脑胶质瘤大鼠:1、不同剂量替莫唑胺缓释微球治疗C6胶质瘤:不同对照组、TM组、TM-MSⅠ组、TM-MSⅡ组、TM-MSⅢ组的平均生存期依次为:22.2天、26.0天、26.4天、29.8天和32.4天。抑瘤率依次分别为:17.20%、35.28%、46.99%和48.94%。TM-MSⅡ、Ⅲ组与对照组相比均有明显差异(P＜0.01)。TM-MSⅡmg组与TM-MSⅢ组比较无明显差异提示:中浓度TM-MS和高浓度TM-MS相比疗效无明显差异。各组PCNA阳性率依次为63.2±5.9%、41.7±6.7%、37.5±5.8%、20.2±4.3%、18.8±3.4%。TM-MSⅠ、Ⅱ和Ⅲ组与对照组相比均有明显差异(P=0.0000),TM-MS间质化疗大鼠脑胶质瘤效果明显。TM-MSⅡ组与TM-MSⅢ组比较无明显差异(P＞0.05)提示:中浓度TM-MS和高浓度TM-MS相比疗效无明显差异,考虑高浓度TM-MS可能会对脑组织有更强的刺激,因此TM-MS间质化疗时采用16mg是较合适剂量。2、TM-MS分别与植入用5-FU和替尼泊甙(VM-26)作疗效对比结果:TM-MSⅡ组间质化疗较FU组和VM-26组疗效好,有明显差异(P＜0.01)。3、TM-MS间质化疗的安全性评价结果:TM-MSⅠ组、TM-MSⅡ组、TM-MSⅢ组的血常规、生化、肝功指标均正常,肝、肾病理切片显示未见明显坏死,炎症、水肿等病理改变。TM-MS的病理学结果提示TM-MS的生物相容性较好。三、TM-MS治疗C6脑胶质瘤大鼠的作用机制研究结果:透射电镜显示:C6细胞体积缩小,异染色质浓缩趋边提示该肿瘤细胞发生凋亡。TUNEL法对C6细胞染色显示:对照组肿瘤凋亡细胞阳性率为8.63±1.52%,TM-MS组肿瘤凋亡细胞阳性率为26.24±2.33%,两组间有明显差异(P=0.0000)。Western Blot方法测P53和活化caspase-3结果显示:TM-MS组P53蛋白表达上调,其表达量为对照组的2倍,两组间有明显差异(P=0.0000);TM-MS组活化caspase-3蛋白表达量较对照组高,两组间有明显差异(P=0.0000);四、TM-MS与Vatalanib联合化疗治疗C6胶质瘤:对照组、TM-MS组、Vatalanib组和联合化疗组的平均生存期依次为:21.7天、28.9天、26.4天、22.3天和31.6天。抑瘤率依次分别为:53.15%、16.39%和64.40%。PCNA细胞阳性率依次为:65.2±4.5%、30.1±6.2%、42.3±5.7%、和22.1±4.6%,四组之间存在差异(P=0.0000),TM-MS组与对照组之间有显著差异(P＜0.01),Vatalanib组与对照组之间有明显差异(P＜0.05),联合化疗组与对照组之间有显著差异(P＜0.01),联合化疗组与TM-MS组之间有明显差异(P＜0.05)。四个实验组的VEGF细胞阳性率依次为:70%、60%、50%、和40%,四组之间无明显差异(P=0.5682)。各组的微血管密度依次为:23.24±2.45、18.53±1.53、12.43±0.63、和5.48±0.87个/视野,四组之间存在差异(P=0.0000),TM-MS组与对照组相比有明显差异(P＜0.05),Vatalanib组与对照组之间有明显差异(P＜0.01),联合化疗组与对照组之间有明显差异(P＜0.01)。四个实验组的肿瘤细胞ICAM-1表达存在差异(P=0.0000),Vatalanib组与对照组相比有明显差异(P＜0.01),联合化疗组与对照组之间有明显差异(P＜0.01)。各组MMP-9表达程度存在差异(P=0.0000),TM-MS组与对照组相比有明显差异(P＜0.05),Vatalanib组与对照组之间有明显差异(P＜0.05),联合化疗组与对照组之间有明显差异(P＜0.05)。联合化疗中荷瘤鼠血常规、生化、肝功指标均正常。结论:1、采用立体定向法建立SD大鼠C6脑胶质瘤模型确实可靠,该模型能较好地模拟胶质瘤颅内生长情况,适于胶质瘤间质化疗和全身化疗的实验性研究。2、替莫唑胺缓释微球间质化疗能安全有效地治疗C6脑胶质瘤大鼠。3、替莫唑胺缓释微球通过诱导肿瘤细胞发生凋亡来抑制肿瘤生长。在促进肿瘤细胞凋亡的信号转导中,P53和caspase-3是参与其发生凋亡的重要蛋白质。4、替莫唑胺缓释微球间质化疗联合Vatalanib抗肿瘤血管治疗能安全、更有效地治疗大鼠C6胶质瘤。更多还原

【Abstract】 Objective To evaluate the effectivity and safety of Temozolomide/PLGA microspheres in interstitutial chemotherapy. Furthermore, to explore the mechanism of Temozolomide/PLGA microspheres in C6 rat glioma. Additionally, to investigate the changes in C6 rat glioma after the combined chemotherapy with temozolomide/PLGA microspheres and Vatalanib.Methods Fifty SD male rats were injected with 2×10~6 C6 cells into the left caudate nucleus area using steoreotaxis techniques. The animals were scanned with MRI at the 4th day after implantation to monitor tumor growth. The rats were divided into five groups. The control group (n=10) received sham operation. The temozolomide group were treated with temozolomide 50mg/kg/day p.o.for 5 days from the 6th postoperation day. The Temozolomide/PLGA microspheres (TM-MS) 12mg group (n=10), TM-MS 16mg group (n=10) and TM-MS 20mg group (n=10) were treated with interstitial TM-MS chemotherapy. The biocompatibility of TM-MS was investigated through histology on different days after the operation. Then, forty SD rats were divided into 4 groups . The control group (n=10) received sham operation. The TM-MS 16mg group (n=10) were treated with interstitial TM-MS chemotherypy. The 5-FU group were treated with interstitial 5-FU chemotherapy, and the VM group were treated with VM-26 via intraveins approach. The technical aspects, therapeutic effectivity, and complications of chemotherapy were analyzed. The therapeutic efficacy was evaluated by histology and transmission electron microscopy investigation. In order to investigate the antitumor mechanism of the Temozolomide/PLGA microspheres, C6 cells apoptosis were measured by transmission electron microscopy investigation and TUNEL method. P53 and caspase-3 were measured by Western Blot method. Forty rats were divided into the four groups. The control group (n=10) received sham operation. The TM-MS 16mg group (n=10) were treated with interstitial TM-MS chemotherapy. The Vatalanib group were treated with Vatalanib with 5ml/kg p.o. for 20 days and the combined chemotherapy group were treated with both TM-MS and Vatalanib. All the animals were undertaken autopsy and pathology examinations. All statistical analyses were performed with computer software SPSS 11.0. For all statistical analyses, P <0.05 was considered statistically significant. Results The tumorigenic rate was 90%. Rat’s head enhanced MRI shows the glioma in the left caudate putamen area. Pathology shows the GFAP and PCNA were positive in tumor cells. Transmission electron microscopy investigation discovered the C6 tumor cells. All rats tolerated the chemotherapy well. TM-MS group acquired effective outcome (P=0.0000), the TM-MS 16mg group didn’t show any statistic difference with TM-MS 20mg group (P>0.05) ,and 16mg is the appropriate dose. TM-MS showed good biocompatitility. The TM-MS 16mg group got better outcome than FU group and VM-26 group. The TUNEL data discovered the apoptosis cells in TM-MS group were more than those in sham operation group (P=0.0000). P53 was up-regulated in the TM-MS group (P=0.0000).Caspase-3 show obvious difference between the TM-MS group and the sham operation group (P=0.0000). All rats well tolerated with the combined chemotherapy. Pathology showed PCNA in combined chemotherapy group was down-regulated (P<0.01), microvessel density (MVD) was reduced in combined chemotherapy group, VEGF was also decreased in combined chemotherapy group(P<0.01). ICAM-1 and MMP-9 were reduced in combined chemotherapy group (P<0.01).Conclusions (1) the models of C6 glioma rat were suitable for the study of interstitial chemotherapy and combined chemotherrpy. (2) Temozolomide/PLGA interstitial chemotherapy was a safe and effective therapeutic option for C6 glioma. (3) Temozolomide/PLGA induced tumor cells apoptosis. P53 and caspase-3 were associated with tumor cell apoptosis. (4) Combined chemotherapy with Temozolomide/PLGA and Vatalanib was safe, and more effective than single drug chemotherapy.更多还原