【作者】 黄若凡；

【导师】 林果为； 梁晓华； 许小平； 周鑫莉； 王小钦； 陈波斌；

【作者基本信息】 复旦大学 ， 内科学， 2009， 博士

【摘要】 目的1．了解内皮抑素对Namalwa淋巴瘤裸鼠移植瘤生长和肿瘤血管生成的抑制作用。2．初步探讨内皮抑素减少Namalwa淋巴瘤移植瘤肿瘤血管生成和调节血管内皮细胞叉头框蛋白O1表达的关系。方法1．体内实验：皮下注射Namalwa淋巴瘤细胞建立裸鼠移植瘤模型。在植瘤后第11天开始分组干预，每天分别于腹腔内注射内皮抑素2．5mg／kg、5mg／kg和10mg／kg；阳性对照组注射环磷酰胺75mg／kg，每2天1次，共3次；阴性对照组每天注射同容积生理盐水。每2天测量一次肿瘤的最大垂直径，按长×宽~2×0．52计算肿瘤体积，比较各组相对肿瘤体积(干预后肿瘤体积／干预前肿瘤体积)，并计算相对肿瘤增殖率。任一内皮抑素组出现抑瘤作用时停止干预，处死荷瘤裸鼠，分离肿瘤组织，进行血管内皮细胞CD31免疫组化染色和微血管密度计数。2．体外实验：小鼠微血管内皮细胞分别与0μg／ml、20μg／ml和80μg／ml内皮抑素在体外共培养16小时，用transwell小室观察细胞迁移能力，XTT法测定细胞增殖活性，RT-PCR和western blot测定细胞叉头框蛋白O1在mRNA和蛋白水平的表达。3．离体实验：建立Namalwa淋巴瘤裸鼠移植瘤模型后，分别注射有效剂量的内皮抑素(根据第一部分研究决定干预方案)和生理盐水，干预结束分离肿瘤组织，用磁珠分选CD31标记的血管内皮细胞，比较其叉头框蛋白O1表达水平。结果：1．移植瘤成瘤率100％，各组成瘤时间差异无统计学意义(P=0．523)，干预过程中各组荷瘤裸鼠未发生死亡。2．干预6天后，各组相对肿瘤体积差异无统计学意义(P=0．185)；干预8天后，环磷酰胺组相对肿瘤体积小于生理盐水组(P=0．003)；干预12天后，5mg／kg／d内皮抑素组相对肿瘤体积小于生理盐水组(P=0．027)，肿瘤相对增殖率为64．2％；而2．5mg／kg／d、10mg／kg／d组和生理盐水组差异无统计学意义，P值分别为0．693和0．604，肿瘤相对增殖率分别为87．4％和85．9％。3．干预12天后，5mg／kg／d内皮抑素组相对肿瘤体积大于环磷酰胺组(P=0．046)，后者肿瘤相对增殖率为38．4％。4．干预12天后，5mg／kg／d内皮抑素组肿瘤组织微血管密度计数少于生理盐水对照组，差异有统计学意义(P=0．006)，而2．5mg／kg／d和10mg／kg／d组与生理盐水组差异无统计学意义，P值分别为0．468和0．220。5．内皮抑素干预16小时后小鼠微血管内皮细胞迁移数减少，该作用随内皮抑素作用浓度提高而增加(P=0．000)。相应浓度干预后微血管内皮细胞增殖活性差异无统计学意义(P=0．484)6．与抑制迁移对应，内皮抑素干预16小时后上调小鼠微血管内皮细胞株叉头框蛋白O1的mRNA和总蛋白表达水平(P＜0．05)，磷酸化蛋白表达水平无变化(P=0．943)，以非磷酸化叉头框蛋白O1表达上调为主，该作用随内皮抑素浓度提高而增加。7．移植瘤组织的细胞悬液经磁珠阳性分选前后CD31阳性细胞分别为17．2％和90．9％，血管内皮细胞得到纯化。8．5mg/kg／d内皮抑素干预12天后的移植瘤组织CD3 1阳性细胞磷酸化叉头框蛋白O1表达水平和生理盐水对照组差异无统计学意义(P=0．896)，总叉头框O1蛋白表达高于生理盐水对照组(P=0．047)，以非磷酸化叉头框01蛋白表达升高为主；而未和磁珠结合的CD31阴性细胞无该蛋白表达。结论1．内皮抑素可抑制Namalwa淋巴瘤裸鼠移植瘤生长，即使肿瘤负荷较大，抑瘤作用仍显著。2．内皮抑素发挥抑瘤作用需要合适的剂量，过高或过低剂量的内皮抑素抑瘤作用反而减弱。3．内皮抑素抑制Namalwa淋巴瘤裸鼠移植瘤生长与减少肿瘤血管生成有关。4．内皮抑素抑制小鼠微血管内皮细胞迁移是内皮抑素减少肿瘤血管生成途径之一。5．内皮抑素抑制小鼠微血管内皮细胞迁移与上调非磷酸化Foxo1蛋白表达有关。6．内皮抑素上调肿瘤组织内血管内皮细胞非磷酸化Foxo1蛋白表达与减少Namalwa淋巴瘤移植瘤血管生成有关，非磷酸化Foxo1表达上调可能是内皮抑素的药理作用机制之一。更多还原

【Abstract】 Objects:1.To investigate the inhibitory effect of endostatin on the growth and angiogenesis ofxenografted Namalwa lymphoma in nude mice.2.To investigate the relationship between regulated Foxol protein expression anddecreased angiogenesis in xenografted Namalwa lymphoma by endostatin.Methods:1.Xenografted Namalwa lymphoma in nude mice was generated by subcutaneouslyimplanted Namalwa cells.And they were divided to five groups.Injections were givenintraperitoneally.Recombinant human endostatin was given at doses of2.5mg/kg/d,5mg/kg/d and 10mg/kg/d respectively.Negative control group was givensaline with same volume,and positive control group was given with cyclophosphamide(CTX) at the dose of 75mg/kg every two days for 3 times.Tumor volume wasmeasured with calipers,and the volume was calculated with the formula:width~2×lenth×0.52,as described previously.Relative tumor volume (RTV) and T/C%were also calculated.The endpoint was set when the RTV of any endostatin-treatedgroup was smaller than the saline-treated group significantly.2.Mouse microvessel endothelial celles were cocultured with 0μg/ml,20μg/ml and80μg/ml endostatin for 16 hours.The effect of endostatin on the migration ofendothelial cell was studied by transwell,and the proliferation was observed by XTT.Foxo 1 expression was studied by RT-PCR and western blot.3.Xenografted Namalwa lymphomas in nude mice were generated and given with salineor endostatin in protocol on the base of in vivo study.Tumor tissue was separated afterthe treatment,and was digested with collagenase I to single-cell suspension.Microbeads were used to seperate CD31 positive cells from the suspension.The Foxolprotein expression of CD31 positive cells was compared between endostatin- andsaline-treated groups.Results1.Xenografted Namalwa lymphoma occurred in 100% nude mice.No time difference wasfound when the tumor was palpable (P=0.523).And no mice died during theexperiment.2.No RTV difference was found among 5 groups after 6-days treatment (P=0.185).RTVof CTX-treated group was smaller than that of saline-treated group after 8-daystreatment (P=0.003).RTV of 5mg/kg/d endostatin-treated group was smaller than that of saline-treated group after 12-days treatment (P=0.027),and T/C% was 64.2%.Nodifference in RTV was found between the other two endostatin- and saline-treatedgroups,and T/C% was 87.4% and 85.9% respectively.3.The RTV of 5mg/kg/d group was larger than that of CTX group after 12-days treatment(P=0.046),and the T/C% of the latter is 38.4%.4.MVD in 5mg/kg/d group was lower than that in saline group after treatment for 12consecutive days,and the difference was significant(P=0.006),while MVD in2.5mg/kg/d and 10mg/kg/d groups were not significantly different from that in salinegroup。5.The number of endothelial cells passing the memberance decreased when treated withendostatin for 16 hours(P=0.000),and no effect was found on OD value of mousemicrovessel endothelial cell(P=0.484)6.The mRNA and non- phosphorylated Foxo 1 protein was upregulated by endostatin atthe same time(P＜0.05).7.CD31 positive cells of suspension from xenografted Namalwa lypmphoma accountedfor 17.2% before separation and 90.9% after separation.8.Endothelial Cells separated from endostatin-treated xenograted Namalwa lymphoma bymagnetic bead had more non-phosphorylated Foxol expression than those fromsaline-treated ones(P=0.047).But the CD31 negative cell had no Foxol expression(P=0.896)Conclusions:1.The growth of xenografted Namalwa lymphoma could be inhibited even if the tumorburden was prominent.2.The exact dose was necessary to inhibit the growth of xenograted Namalwa lymphoma.Higher or lower dose than the definite dose range was less effective.3.The inhibition on the growth of xenograted Namalwa lymphoma was related todecreased angiogenesis.4.Endostatin could inhibit the migration of mouse microvessel endothelial cell.Sincemigration was one of the most important steps in angiogenesis,it was one of theimportant ways in which endostatin decreased angiogenesis.5.There was common trend between inhibited migration and upregulated non-phosphorylated Foxol expression of endothelial cells treated by endostatin.And sothere may be relationship between them.6.Endostain raised non-phosphorylated Foxol expression,and at the same time endostatin decreased angiogenesis.Up-regulation of non-phosphorylated Foxolexpression was one of endostatin’s mechanisms.更多还原