【作者】 黄延林；

【导师】 兰青；

【作者基本信息】 苏州大学 ， 神经外科学， 2010， 博士

【摘要】 目的采用传代的人脑多形性胶质母细胞瘤(glioblastoma multiforme,GBM)新鲜手术标本来建立呈侵袭性生长及表皮生长因子受体(epidermal growth factor receptor,EGFR)高表达的人脑GBM裸小鼠脑原位移植模型,然后在此已建立的能保持亲本肿瘤生物学特性的人脑恶性胶质瘤原位移植模型基础上,把EGFR抑制剂与常规化疗药物或与血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,VEGFR2)抑制剂联合应用来治疗人脑GBM移植瘤,以探讨不同作用机理的化学治疗药物联合应用后对GBM的疗效。方法将传代的新鲜人脑GBM手术标本行裸小鼠右尾状核接种后第3d用于分组实验。(1)40只鼠随机分为4组,10只/组,各组鼠分别应用靶向EGFR的蛋白酪氨酸激酶抑制剂AG1478(25mg/kg.次)、CDDP(3mg/kg.次)、AG1478+CDDP(25mg/kg.次+3mg/kg.次)及磷酸盐缓冲液(PBS,0.1ml/只.次)。PBS和药物均腹腔注射,每2d一次,共4次,用药后分别观察各组荷瘤鼠生存期。再取40只鼠,重复上述实验,肿瘤移植后14d处死所有组鼠取脑,HE染色后观察各组移植瘤形态及体积变化;EnVision和Western Blot法检测移植瘤中EGFR、磷酸化EGFR(P-EGFR)、增殖活性(Ki-67 labelling index,即Ki-67 LI)、血管内皮生长因子(vascular endothelial growth factor ,VEGF)、白细胞介素-8(IL-8)和微血管密度(microvessel density,MVD)变化;TUNEL法检测移植瘤细胞凋亡(apoptotic index即AI)变化。(2)40只荷瘤鼠随机分为4组,10只/组,分别应用靶向EGFR的单克隆抗体C225(50mg/kg.次)、靶向VEGFR-2的单克隆抗体DC101(40mg/kg.次)、C225+DC101 (50mg/kg.次+40 mg/kg.次)及PBS(0.1ml/只.次)。PBS组和药物均腹腔注射,每2d一次,共4次。肿瘤移植后14d,处死所有组鼠取脑,HE染色观察各组移植瘤形态及体积变化;EnVision法检测移植瘤中基质金属蛋白酶-1(matrix metalloproteinase,MMP1)、MVD及增值活性变化;TUNEL法检测肿瘤细胞凋亡变化。结果(1)与对照组生存期为19.4±0.6d比较,单用CDDP或AG1478后荷瘤鼠生存期分别为20.7±0.4d和20.8±0.6d(P>0.05),而CDDP+AG1478组荷瘤鼠生存期为33.6±0.9d(P<0.05);CDDP+AG1478能明显减小移植瘤体积、降低增殖活性及促进肿瘤细胞凋亡,而单独用药对此不明显。AG1478及CDDP+AG1478抑制了EGFR磷酸化,使P-EGFR表达下降,并降低VEGF、IL-8表达量及减少MVD。(2)与对照组比较,DC101组移植瘤体积下降了59.7%(P<0.05),DC101+C225组移植瘤体积下降了62.9%(P<0.05),而C225组对移植瘤体积几乎无影响(P>0.05);DC101组移植瘤侵袭性加强,移植瘤中MMP1表达为(++);C225组移植瘤侵袭性下降,移植瘤中MMP1表达阳性(+);C225+DC101组移植瘤侵袭性也下降,移植瘤中MMP1表达阳性(+);DC101组移植瘤中MVD减少了64(%P<0.05),C225+DC101组MVD减少了68(%P<0.05),C225组的MVD无明显变化(P>0.05);DC101组肿瘤细胞Ki-67 LI下降了53.2%(P<0.05),DC101+C225联合用药后Ki-67 LI下降了56.4%(P<0.05),而C225组下降不明显(P>0.05);DC101诱导细胞AI增加了66.7%(P<0.05),DC101+C225联合用药后AI增加了75%(P<0.05),而C225用药后增加不明显(P>0.05)。结论(1)AG1478与CDDP联合应用比单独用药有显著的抗肿瘤生长疗效,并在一定程度上抑制了肿瘤的血管生成,这为EGFR抑制剂和常规化疗药物联合治疗恶性胶质瘤提供了实验依据。(2)DC101与C225联合用药显著抑制GBM移植瘤生长,并降低了移植瘤侵袭和转移特性,两药合用疗效优于单用的疗效,这为抗肿瘤血管生成药物与抗肿瘤侵袭药物合用治疗恶性胶质瘤提供了实验依据。更多还原

【Abstract】 PartⅠExploring the effect on tumor growth and angiogenesis after AG1478 combined with chemotherapy for treating the orthotopic xenografts of human glioblastoma in nude miceObjective To evaluate the efficacy of antitumor activity and influence on angiogenesis after AG1478 combined with Cisplatin for treating the orthotopic xenografts of human gliobastoma multiforme.Methords Establishing orthotopic xenograft model of human gliobastoma multiforme with stable EGFR expression as described previously.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of AG1478(25mg/kg every time)、Cisplatin (3mg/kg every time)、AG1478+ Cisplatin(25mg/kg every time +3mg/kg every time)and PBS(0.1ml/20g every time)respectively with every 2 days for 4 times,then survival time were observed until mice bearing xenografts were natural death.The experiment was repeated,in which the same types of treatment were initiated on day 3.On day 14 after innnoculation ,every group of mice were sacrificed. Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin.Serial sections were stained with H&E,then tumor appearance and volume were observed and measured.The expression of EGFR,phosphorylated EGFR (P-EGFR) were detected by immunochemical or Western blot methods;VEGF,IL-8 or Ki-67 in the xenografts were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results The expression of P-EGFR were decreased because phosphorylation of EGFR were inhibited by AG1478,accordingly the expression of VEGF or IL-8 were decreased,and microvessel destiny was also declined.Compared with PBS group,the suvival were strikingly prolonged,and volume of xenografts were reduced by the combination of Cisplatin with AG1478;meanwhile, the combined application could significantly decrease proliferative activity and increase apoptotic rate,but no effect in drugs alone.Conclusion The combination of AG1478 with Cisplatin significantly increased the activities against tumor growth and inhibited the angiogenesis,which provided a rationale for its clinical evaluation in combination with both chemotherapy and other molecular target drugs for treating malignant gliomas.PartⅡAntitumor treatment efficiency by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma modelObjective: To explore the inhibitive effect on transplanted tumor of human glioblastoma multiforme in the brain of nude mice after t reatment of C225 and DC101 alone and combined application.Methods: Establishing the human GBM orthotopic models with high expression of EGFR and VEFR in the brain of nude according to the previous method.On day 3 postinnoculation,40 mice were divided into 4 groups randomly with 10 every group,then 4 groups of mice received i.p. injections of C225(50mg/kg)、DC101(40mg/kg)、CD225+DC101 (50 +40)mg/kg or PBS(0.1ml/20g)respectively with every 2 days for 4 times.On day 14 after innnoculation ,every group of mice were killed.Mouse brains were removed from the cranial cavity, fixed in formalin, bisected coronally, and embedded in paraffin. Serial sections were stained with H&E to observe tumor appearance and to measure the maximum or minimun diameter under light microscope so as to work out tumor volume.The expression of MMP1,MVD or Ki-67 were detected by immunochemical methods,and apoptosis were detected by TUNEL method.Results:When compared with controls(PBS) group,the xenograft volumes were decreased by 59.7% and 62.9% in the DC101 and the DC101+C225 group respectively(P<0.05),but no effect in the C225group(P>0.05);The invasion ability of xenograft was improved,and expression of MMP1 was increased to (++) in the DC101group. The invasion ability of xenograft was declined,and expression of MMP1 was decreased to (+) in the C225 group,which was also founded in the DC101+C225 group.MVD were reduced by 64% and 68% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225group(P>0.05);Ki-67 labelling index(LI) was decreased by 53.2% and 56.4% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05);Apoptotic index(AI) was increased by 66.7% and 75% in the DC101 and the DC101+C225 group respectively(P<0.05), but no effect in the C225 group(P>0.05).Results:The combined C225 and DC101 can inhibit the growth and invasion ability of transplanted tumor of human glioblastoma multiforme in nude mice. The effect of C225 + DC101 is better than that of C225 and DC101 alone,which provided a experimental foundation for treatmenting malignant glioma by the method of unifying drugs with different pharmacological features.更多还原