【作者】 张先燎；

【导师】 施鑫；

【作者基本信息】 南方医科大学 ， 外科学， 2010， 硕士

【摘要】 肉瘤是最常见的原发性高度恶性肿瘤之一,起源于间叶组织(包括结缔组织和肌肉)的恶性肿瘤称为“肉瘤”。多发生于皮肤、皮下、骨膜及长骨两端。肉瘤生长迅速,肿瘤晚期常有坏死、出血、切面灰红色、质均匀细如生鱼肉状,早期即可发生血行转移。骨肉瘤是肉瘤中恶性程度最高,以青年人为多,以能够产生骨样组织的梭形基质细胞为特征,好发于生长活跃的干骺端,尤以股骨下端、胫骨上端及肱骨上端最多见,约占骨恶性肿瘤的1/3。骨肉瘤发展迅速,病程短,开始在皮质内生长,可逐渐向骨髓腔发展,有时向外突破骨膜,侵入周围软组织间叶组织的恶性肿瘤。目前已经证明,肿瘤的生长和转移必须依赖于血管的生成。体积在1-2mm3舳。以下的瘤体可通过渗透作用从周围组织中获得营养,以维持自身的生存。这时肿瘤生长极为缓慢。肿瘤的进一步生长必须依赖于新生血管生成,丰富的血管网为肿瘤细胞提供充足的氧气、营养成分和肿瘤生长因子等,也是肿瘤转移的通道。血管生成能力被认为是肿瘤生长、侵袭和转移的标志。肿瘤血管生成是从已存在的血管床形成新生毛细血管的过程,包括毛细血管基底膜降解、血管内皮细胞的活化、血管基底膜及细胞外基质蛋白酶降解、内皮细胞增生及迁移增殖、管腔样结构形成融合、血管壁的重建、血流贯通等步骤。这一过程既受机体神经内分泌因素等影响,又受肿瘤细胞和肿瘤基质细胞表达的生长因子调控。肿瘤血管生成过程中涉及到多种促血管生成因子与血管生成抑制因子之间的调节失衡。这一过程不仅涉及促血管生成因子分泌增加,而且内源性血管生成抑制因子产生相应减少。目前已分离和纯化了20多种血管生成因子,主要的血管生长促进因子有血管内皮细胞生长因子(VEGF)、酸性及碱性成纤维细胞生长因子(aFGF, bFGF)、肿瘤坏死因子α、转化生长因子α/β、胸苷磷酸化酶、血小板源性生长因子、肝细胞生长因子、表皮生长因子(EGF)、血管生成素(angiogenin)、胎盘生长因子(PIGF)、粒细胞集落刺激因子、血管素、血小板激活因子、增生素、P物质,乳酸酯、透明质酸片段和前列腺素等。内源性血管生成抑制因子主要包括血管抑素(angiostatin)、内皮抑素(endostatin)、凝血酶敏感蛋白(TSP-1)、金属蛋白酶抑制剂(TIMP)、血小板因子4(PF4)、干扰素α(IFN-α)、白介素-10(IL-10)、可溶性VEGF受体(sFlt-1)、可溶性酪氨酸激酶受体(sTie-2)等。血管内皮生长因子(vascular endothelial growth factor, VEGF)一种序列高度保守,高度特异性的促血管内皮细胞生长的因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等组织中,对内皮细胞具有强烈的细胞有丝分裂作用,刺激血管内皮细胞增殖和血管通透性增加,促进新生血管形成。VEGF通过与血管内皮细胞表面受体(vascular endothelial growth factor receptor, VEGFR)特异性结合发挥生物学效应。抑制VEGF及VEGFR的活性,可以减缓或阻滞骨肉瘤侵袭和转移,骨肉瘤侵袭和转移的分子调控机制是目前骨肉瘤分子生物学研究的热点,研究表明,VEGF及VEGFR对肿瘤血管及淋巴管的生成及肿瘤侵袭和转移起重要作用。基质金属蛋白酶(matrix metallopmteinase, MMPs)是一个蛋白水解酶家族,可降解细胞外基质(extracellular matrix, ECM)成分。近年来随着分子生物学研究的进展,学者们对MMP的结构、功能,调控及病理生理作用已进行了较为深入的研究,MMP在肿瘤侵袭、转移中的作用越来越受重视。金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinase, TIMP)是内源性MMP抑制剂,它通过抑制MMP活性形式及活化过程而施加双重影响。其C端功能区与MMP的其他部位结合,形成MMP-TIMP复合体,从而阻断MMP与底物结合,抑制MMP的活性,减缓或阻滞骨肉瘤侵袭和转移。骨肉瘤侵袭和转移的分子调控机制是目前骨肉瘤分子生物学研究的热点。研究表明,MMPs对肿瘤的侵袭和转移起重要作用。1971年Folkman首先提出了关于肿瘤血管形成以及通过抗血管形成抑制肿瘤生长的假设：肿瘤直径生长达到大于1-2mm3时,肿瘤细胞可以引起周围的血管内皮细胞开始增殖、迁移,在肿块内形成新的毛细血管,以保证肿瘤继续生长和转移；可以通过抑制肿瘤血管的形成来达到抑制肿瘤生长的目的,抑制肿瘤血管的形成不能消除所有的肿瘤细胞,但能阻止肿瘤细胞的生长。并在后来的实验中得时证实,从而为抑制肿瘤血管生成提供理论基础。1989年Ferrarra在牛垂体滤泡星状细胞培养液中分离纯化出来的一类糖蛋白,即血管内皮生长因子,它与肿瘤细胞分裂、生长、转移都与肿瘤血管形成有关(vascular endothelial growth factor, VEGF),1994年报道0’ Reilly博士发现了第一个内源性血管形成抑制剂Angiostatin后,又于1997年报道发现了Endostatin。2005年9月12日,我国自主研发的“重组人血管内皮抑制素”(商品名：恩度),为生物制品第一类抗肿瘤新药,它是世界上多靶点血管内皮抑制素抗癌新药。肉瘤新辅助化疗方案联合血管生长抑素在肉瘤的血管生成、侵袭和转移的目前肉瘤治疗—研究热点,为了观察肉瘤新辅助化疗方案联合血管生长抑制素对骨肉瘤中VEGF、MMPs及TIMP表达,进行动物模型研究,为临床的应用提供实验理论基础,提高肉瘤患者的无瘤生存时间。目的：1、观察S180鼠型肉瘤动物模型的肿瘤自然生长情况；2、观察顺铂联合恩度对S180鼠型肉瘤动物模型的肿瘤生长曲线及抑瘤率；3、免疫组化VEGF、MMPs及TIMP的表达与肿瘤血管生成之间的关系；方法：1、建立S180动物模型将南京大学生命科学院惠赠的液氮冻存S180细胞株连管一起迅速投入到37℃恒温水浴中,溶化1mim。室温下5mim内恢复至室温的20%胎牛血清培养基中稀释到原体积5倍,450rpm低速离心10min,去上清,转移到培养瓶中加入新鲜20%胎牛血清培养基,放入37℃,5%CO2培养箱中。复苏好的细胞接种于含10%FBS的D-MEM培养基中,于37℃,5%C02常规培养传代。隔日换液,细胞生长迅速,3-4d传1代,取对数生长期的细胞进行实验。S180细胞传代至足够数量后,收集培养好的S180骨肉瘤细胞,PBS液洗1次,调整细胞浓度,制成4×10^7/mL的细胞悬液,按4×10^6个细胞/只注入36只C57/BL6鼠后背皮下注射,每只小鼠后背皮下接种0.1ml。接种S180细胞后第6天C57/BL6鼠后背基本形成重约1g左右的实体瘤。2、对S180鼠肉瘤动物模型药物干预将36只C57BL/6小鼠模型随机分为4组,分别为：对照组、恩度组、顺铂组、顺铂+恩度联合组。每组9只,每组在接种第7天开始用药开始给药,注射部位于前肢腋窝皮下注射,给药药物剂量如下：对照组：生理盐水注射液10mg/Kg.d；恩度组：恩度10mg/Kg.d；顺铂组：顺铂5mg/Kg.d；顺铂+恩度联合组：恩度10mg/Kg·d.顺铂5mg/Kg.d,给药时间途径相同。按第1、4、7、10、13、16、19、22日共8次于前肢腋窝皮下注射药物,实验操作均遵循无菌原则。实验期间小鼠自由进食,末次给药后24h处死各组动物,剖取肿瘤标本。3、对剖取肿瘤标本进行免疫组化原理：酶标记的抗体与抗原形成抗原/一抗酶复合物,再用生物素抗体交联,其中生物素标记兔抗鼠IgM是标记有HRP(辣根过氧化物酶)和抗兔及抗鼠免疫球蛋白的多具体分子,相当于二抗,在生物素化的二抗结合一抗后,结合成抗原-抗体-生物素化二抗复合物,结合后的大分子即标记偶辣根过氧化物酶,再与DAB(3,3,-二氨基联苯胺)反应得显色。4观察指标：4.1测量移植瘤体积计算治疗后的抑瘤率自给药起第1、4、7、10、13、16、19、22天分别测量小鼠体重及肿瘤长短径,并据公式为：V=1/2 ab2(a为长径,b为短径),实验第28天处死实验小鼠,剥离肿瘤组织并测量肿瘤组织长径及短径,计算抑瘤率(tumorinhibition rate,TIR), TIR=(对照组肿瘤体积-实验组肿瘤体积)/对照组肿瘤体积×100%,绘制移计算肿瘤体积,得出各组的肿瘤生长曲线。4.2免疫组织化学检测移植瘤组织VEGF表达组织标本以10%甲醛溶液固定24h,常规石蜡包埋,制备5m厚切片,按免疫组化操作规程进行。用已知阳性标本作为阳性对照,以PBS代替抗作阴性对照。VEGF的阳性表达位于肿瘤细胞及血管内皮细胞,胞浆内出现黄染颗粒；对每一个切片随机选取5个高倍镜视野(×400)计数200个细胞,分别由两名病理科医师在双盲情况下对有标本进行观察,在光镜下判断其胞质的DAB染色强度,分别以0、1、2、3代表阴性、弱染色、中等染色及强染色等4个等级,H分值=[(弱染色强度细胞百分数×1)+(中度染色程度细胞百分数×2)+(强染色强度细胞百分数×3)]×100,再取两观察者的平均值作为最终H-评分,再取两观察者的平均值作为最终H-评分,H值得分介于0-300之间。4.3免疫组织化学检测移植瘤组织MMP-9表达MMP-9和TIMP阳性染色位于细胞胞浆内或胞膜出现黄染颗粒,呈棕黄色,结果采用半定量分析方法进行H-评分：对每一个切片随机选取5个高倍镜视野(×400)计数200个细胞,分别由两名病理科医师在双盲情况下对有标本进行观察,在光镜下判断其胞质的DAB染色强度,分别以0、1、2、3代表阴性、弱染色、中等染色及强染色等4个等级,H分值=[(弱染色强度细胞百分数×1)+(中度染色程度细胞百分数×2)+(强染色强度细胞百分数×3)]×100,再取两观察者的平均值作为最终H-评分,H值得分介于0-300之间。观察实验各组肉瘤动物模型中VEGF、MMPS及TIMP免疫的表达。4.4免疫组织化学检测移植瘤组织TIMP表达TIMP与MMP-9一样,阳性染色位于细胞胞浆内或胞膜出现黄染颗粒,呈棕黄色,结果与MMP-9同样的半定量分析方法进行H-评分。统计学分析所得数据采用SPSS13.0软件进行统计学分析。移植瘤体积定量结果用均数±标准差(X±s)表示,组间比较采用单因素的方差分析,两两组间比较采用LSD检验。各组移植瘤中的VEGF、MMP-9和TIMP的H值半定量资料均用中位数(最小值,最大值)表示,组间比较采用Kruskall-Wallis检验,两两组间比较采用Mann-Whitney U检验(检验水准采用Bonferroni法调整后的α=0.008)。以P<0.05认为有统计学意义。结果：1.移植瘤生长情况及抑瘤率瘤株肿瘤生长潜伏期均为3-5d,肿瘤呈膨胀性生长,质地略韧。对照组在治疗后第1、4、7、10、13、16、19、22天肿瘤体积平均为：395.70±34.92mm3、542.02±52.74mm3、637.48±51.69 mm3、713.88±61.78 mm3、795.39±68.88 mm3、864.14±77.63 mm3、932.244±83.27mm3、990.37±89.25mm3；恩度组在治疗后第1、4、7、10、13、16、19、22天肿瘤体积平均为：387.54±24.89mm3、506.70±28.07 mm、605.55±40.89 mm3、685.8±40.89mm3、764.48±43.98mm3、832.41±45.89 mm3、887.21±50.87mm3、930.78±52.96mm3；顺铂组在治疗后第1、4、7、10、13、16、19、22天肿瘤体积平均为：365.90±23.72 mm3、478.70±27.01mm3、553.73±34.65 mm3、580.60±40.66 mm3、551.95±37.81mm3、497.19±43.47mm3、431.43±38.81mm3、370.54±35.46mm3；联合组在治疗后第1、4、7、10、13、16、19、22天肿瘤体积平均为：392.78±42.81 mm3、477.82±47.74 mm3、524.79±38.14 mm3、534.89±22.05mm3、476.39±28.73mm3、392.15±28.73mm3、328.17±31.04mm3、275.83±32.50mm。用药后第4天对照组肿瘤依然迅速生长,恩度组较对照组略慢,仍持续性生长。顺铂组和联合组用药后第4天后肿瘤开始生长缓慢后,第7天后相对静止,而后逐渐缩小,并且随时间延长而迅速下降,与对照组及恩度组迅速增大形成鲜明对比如图所示(图3)。第22天联合组肿瘤缩小较快,顺铂组与联合组在肿瘤体积上比较有统计学意义(P<0.05)。2.免疫组化检测2.1 VRGF的免疫组化如图所示(图6-图9),对照组、恩度组的表达强度均为+++、++,而在顺铂组、联合组表达强度分别为++、+。H评分中位数(最小值,最大值)和平均秩次如表3所示。P<0.05,4组之间有统计学意义,可认为不同组合用药抑制肿瘤VRGF的表达有显著性差异。两两组间比较采用Mann-Whitney U检验,对照组与顺铂组有之间有统计学意义(P<0.008)；对照组与联合组有之间有统计学意义(P<0.008)；恩度组与联合组有之间有统计学意义(P<0.008)；顺铂组与联合组(P<0.008)；可推断对照组与顺铂组、对照组与联合组、恩度组与联合组、顺铂组与联合组VRGF的表达有差别。2.2 MMP-9的免疫组化H评分MMP-9的免疫组化如图所示(图10-图13),对照组、恩度组的表达强度均为+++、++,而在顺铂组、联合组表达强度分别为++、+。H评分中位数(最小值,最大值)和平均秩次如表3所示。P<0.05,4组之间比较有统计学意义,可认为不同组合用药抑制肿瘤VRGF的表达有差别。两两组间比较采用Mann-Whitney U检验,对照组与顺铂组、对照组与联合组、恩度组与顺铂组、顺铂组与联合组之间比较均有统计学意义(P<0.008),可以认为对照组与顺铂组、对照组与联合组、恩度组与顺铂组、顺铂组与联合组之间MMP-9的表达有差别。2.3 TIMP的免疫组化H评分TIMP的免疫组化如图所示(图14-图17),对照组、恩度组的表达强度均为+、++,而在顺铂组、联合组表达强度分别为++、+++。H评分中位数(最小值,最大值)和平均秩次如表3所示。P<0.05,4组之间有统计学意义,可认为不同组合用药抑制肿瘤TIMP的表达有显著性差异。两两组间比较采用Mann-WhitneyU检验,对照组与顺铂组、对照组与联合组、恩度组与联合组、顺铂组与联合组有之间比较均有统计学意义(P<0.008),可以认为对照组与顺铂组、对照组与联合组、恩度组与联合组、顺铂组与联合组有之间TIMP的表达有差别。结论：1、抗肿瘤药物联合恩度S180肉瘤抑制血管生成作用明显,显著抑制肿瘤体积增大,并且抑癌率高。2、肿瘤中VEGF与MMPs的表达基本平行,两者共同参与肿瘤血管的生成并协同表达,抗肿瘤药物联合恩度使瘤中VEGF与MMPs的显著降低。3、肿瘤中MMPs/TIMP比值降低可抑制肿瘤生长及侵润,恩度通过多种途经对VEGF及MMPs产生抑制作用,并增加TIMP的表达,减少肿瘤血管的生成。4、为抗肿瘤药物联合恩度治疗肉瘤临床上应用奠定实验基础,并为抗肿瘤药物联合恩度行抗血管治疗骨肉瘤动物模型,及骨肉瘤临床治疗方案开辟新治疗理念。更多还原

【Abstract】 The sarcoma is one of most common primary highly malignant tumors, it origins in the mesenchymic tissue (contracts organization and muscle including knot) the malignant tumor is called "the sarcoma". Occurs in the skin, hypodermic, the periosteum and the long bone both sides.The sarcoma growth is rapid, tumor later period common necrosis, the hemorrhage, cuts the face puttying red, the nature even thin like lives the fish flesh shape, the early time then has a blood line of shift. The osteosarcoma is in the sarcoma the malignant degree is highest, By youth artificial many, take can produce the osteoid organization’s spindle-shaped matrix cell as the characteristic, good sends in the growth active dry epiphysis end, especially by the thighbone lower extremity, the shinbone upper extreme and the humerus upper extreme most sees, approximately composes the bone malignant tumor 1/3.The osteosarcoma develops rapidly, the course of an illness is short, starts to grow in the cerebral cortex, may gradually to the medullary cavity of bones development, sometimes outward break through the periosteum, periphery the invasion the soft tissue mesenchymic tissue malignant tumor.At present already proved that, the tumor growth and the shift must rely on the blood vessel production.The volume may organize in the 1-2mm3 following lump body through the osmosis from periphery to obtain the nutrition, maintains own survival.By now the tumor grew extremely slowly, because the tumor further growth had to rely on the newborn blood vessel production, the rich network of bloodvessels provided the sufficient oxygen, the nutrition ingredient and the tumor growth factor for the tumor cell and so on, also was the tumor shift channel.Blood vessel production ability was considered is the tumor growth and the attack symbol.The tumor blood vessel production was from the blood vessel bed which existed has formed the newborn blood capillary the process, degraded, the blood vessel endothelial cell activation, the blood vessel basilar membrane and the extracellular matrix proteinase including the blood capillary basilar membrane degrades, the endothelial cell proliferation and the migration multiplies, the lumen type structure forms steps and so on fusion, blood vessel wall reconstruction, blood stream penetration.This process both influences and so on organism nerve internal secretion factor, and tumor cell and tumor matrix cell expression growth factor regulation.In the tumor blood vessel production process involves to many kinds of presses the blood vessel production factor and between the blood vessel production inhibiting factor adjustment is unbalanced.Not only this process involves presses the blood vessel production factor secretion to increase, moreover the endocardial blood vessel production inhibiting factor has the corresponding reduction.At present has separated and has purified 20 many kinds of blood vessel production factor, the main blood vessel growth promotion factor has blood vessel endothelial cell growth factor (VEGF), the acidity and the alkalinity becomes the ciliary cell growth factor (aFGF, bFGF), the tumor necrosis factor alpha, the transformation growth factor a/p, the chest glucoside phosphorylase, the blood platelet source growth factor, the liver cell growth factor, epidermis growth factor (EGF), blood vessel generator (angiogenin), placenta growth factor (PIGF), the granular cell colony stimulation factor, the blood vessel element, the blood platelet activation factor, the proliferation element, the P material, the lactate, the hyaluric acid fragment and the prostaglandin and so on.The endocardial blood vessel production inhibiting factor mainly includes blood vessel chalone (angiostatin), bast chalone (endostatin), the zymoplasm sensitive protein (TSP-1), metal proteinase inhibitor (TIMP), the blood platelet factor 4(PF4), the interferon alpha (IFN-a), Bai Jiesu-10(IL-10), the soluble VEGF acceptor (sFlt-1), the soluble tyrosine activating enzyme acceptor (sTie-2) and so on.The blood vessel bast growth factor (vascular endothelial growth factor, VEGF) one kind of sequence is highly conservative, highly specificity the factor which presses the blood vessel endothelial cell to grow, widely distributes in organizations and so on human and animal in vivo cerebrum, kidney, liver, spleen, pancreatic gland and skeleton, has the intense cell mitosis function to the endothelial cell, stimulates the blood vessel endothelial cell multiplication and the blood vessel permeability increases, promotion newborn vascularization. VEGF through and blood vessel endothelial cell surface acceptor (vascular endothelial growth factor receptor, VEGFR) specificity union display biology effect.Suppresses VEGF and the VEGFR activeness, may slow down or hinder the osteosarcoma attack and the shift, the osteosarcoma attack and the shift molecular regulation mechanism is the present osteosarcoma molecular biology research hot spot, the research indicated, VEGF and VEGFR make a sneak attack and shift the vital role to the tumor blood vessel and the lymph vessel production and the tumor.The matrix metal proteinase (matrix metallopmteinase, MMP) is a protein hydroltyic enzyme family, may degrade the extracellular matrix (extracellular matrix, ECM) the ingredient.In recent years along with the molecular biology research progress, the scholars to the MMP structure, the function, regulative and the pathology physiological action has conducted more thorough research, MMP in the tumor attack, the shift function has more and more received takes.The metal proteinase organization inhibitor (tissue inhibitor of metalloproteinase, TIMP) is the endocardial MMP inhibitor, it through suppresses the MMP active form and the reactivation process exerts the dual influences.Its C end function area and the MMP other spot union, forms the MMP-TIMP complex, thus blocks MMP and the substrate union, suppresses MMP the activeness, slows down or hinders the osteosarcoma attack and the shift. The osteosarcoma attack and the shift molecular regulation mechanism is the present osteosarcoma molecular biology research hot spot.The research indicated that, MMPs and shifts the vital role to the tumor attackIn 1971 Folkman first proposed about the tumor vascularization as well as the supposition which grows through the anti-vascularization suppression tumor:The tumor growth in thickness achieved when is bigger than 1-2mm3 periphery, the tumor cell may cause the blood vessel endothelial cell to start to multiply, the migration, forms the new blood capillary in the tumour, guaranteed the tumor continues to grow and the shift; May through the suppression tumor blood vessel formation achieve the suppression tumor grows the goal, the suppression tumor blood vessel formation cannot eliminate all tumor cell, but can prevent the tumor cell the growth.And when afterwards experiment confirmed, thus for suppresses the tumor blood vessel production to provide the rationale.In 1989 Ferrarra separated kind of glycoprotein in the cow pituitary gland follicle astral cell nutrient fluid which purified, namely blood vessel bast growth factor, it with tumor cell division, growth, shift all with tumor vascularization related (vascular endothelial growth factor, VEGF), after in 1994 reported Dr.O’Reilly has discovered first endocardial vascularization inhibitor Angiostatin, also in 1997 reported has discovered Endostatin. On September 12,2005, our country independent research and development "reorganizes the human blood vessel bast inhibin" (business commodity name:Graciousness), is the biological preparations first kind of anti-tumor new medicine, it is in the world the multi-target blood vessel bast inhibin anti-cancer new medicine.The sarcoma new assistance chemotherapy plan union blood vessel growth chalone in the sarcoma blood vessel production, the attack and the shift present sarcoma treats a research hot spot, in order to observe the sarcoma new assistance chemotherapy plan union blood vessel growth inhibin to the osteosarcoma in VEGF, MMPs and the TIMP expression, conducts the animal model study, and provides as the clinical application the animal experimentation rationale.And treats in the plan at the osteosarcoma to use the chemotherapy medicine union suppression tumor blood vessel production medicine to take one kind of new treatment idea.Objective:1、the observation and damps the lump rate along the platinum union graciousness to the S180 mouse sarcoma animal model tumor growth curve;2、immunity group VEGF, MMPs and the TIMP expression and the tumor blood vessel production, invades between Run’s relations;3、provides the zoology basic research for the anti-tumor medicine union graciousness line anti-blood vessel treatment sarcoma. Method:1、establish the S180 animal modelit is to put the S180 cell connecting pipe which is given by the Nanjing University Life Academy of science rapidly to invest together into 37℃the constant temperature water bath that liquid nitrogen frozen to save the S180 cell line connecting pipe, and to dissolve 1mim.Under the room temperature in 5mim restores to in the room temperature 20% embryo cow seroculture dilutes to original volume 5 times,450rpm low speed gentrifugalism lOmin, goes on clear, shifts joins the fresh 20% embryo cow seroculture to the culture bottle in, puts in 37℃,5%CO2 in the incubator.Recovers the cell vaccination in contains in the 10%FBS D-MEM culture medium, in 37℃,5%CO2 conventional raise transfer of generation.Trades the fluid every other day, the cell growth is rapid,3-4d passes on 1 generation, takes the logarithm vegetal period the cell to carry on the experiment.The S180 cell transfer of generation after the enough quantity, the S180 osteosarcoma cell which the collection raises, the PBS fluid washes 1 time, the adjustment cell density, makes 4x 10(?)7/mL the cell hangs the fluid/only pours into 36 C57/BL6 mouse back hypodermic injection according to 4×10(?)6 a cell, each mouse back hypodermic vaccinates 0.1ml.After vaccinates the S180 cell the 6th day C57/BL6 mouse back to form about heavy approximately 1g basically the entity lump.2、treated S180 mouse sarcoma animal model by drugsTo divides into stochastically to the S180 mouse sarcoma animal model medicine intervention 36 C57BL/6 mouse model 4 groups, respectively is:Control group, graciousness group, along platinum group, along platinum+graciousness union group.Each group of 9, each group is vaccinating 7 day to start to apply drugs starts for the medicine, the injection spot in the foreleg armpit hypodermic injection, is as follows for the medicine medicine dosage:Control group:Physiological saline inoculation fluid 1Omg/Kg·d; Graciousness group:Graciousness 10mg/Kg·d; Along platinum group:Along platinum 5mg/Kg·d; Along platinum+graciousness union group:Graciousness lOmg/Kg·d, along platinum 5mg/Kg·d, is same for the medicine time way.According to 1st,4,7,10,13,16,19, on 22nd altogether 8 times in the foreleg armpit hypodermic injection medicine, the experiment operates follows the asepsis principle.The experimental period mouse free feed, after end time 24h executes each group of animals for the medicine, splits takes the tumor specimen.3、take the tumor specimen to carry on the immunity groupThe principle enzyme mark immune body and the antigen form an antigen/antienzyme compound, again uses biological element immune body crossing linking, in which biology element mark rabbit anti-mouse IgM is the mark has HRP (horse radish peroxide enzyme) and the anti-rabbit and the anti-mouse immune globulin concrete member, is equal in two anti-, two anti-unifies after the biological element one anti-, combines to form the antigen-immune body-biology element two anti-compounds, after the union macro-molecule is the mark partner horse radish peroxide enzyme, again with DAB (3,3,-two aminobenzidines) responded results in the coloration.4 observation targets:4.1 survey transplant lump volume computation treatment damps the lump rate to be self-sufficientthe medicine to get up 1,4,7,10,13,16,19,22 days surveys the mouse body weight and the tumor long minor axis separately, and according to the formula is: V=1/2 ab2 (a is major axis, b is minor axis), tests the 28th day execution experiment mouse, the peeling tumor organizes and surveys the tumor organization major axis and the minor axis, the computation damps the lump rate (tumorinhibition rate, TIR), TIR= (control group tumor volume-experimental group tumor volume)/the control group tumor volume×100%, the plan moves the computation tumor volume, obtains each group the tumor growth curve.4.2 immunity group masculine result judgment standard:The slice carries on which according to the immunity group working instruction, under the microscope is located the tumor cell and the blood vessel endothelial cell with the VEGF masculine expression, in the cytoblastema appears yellow dyes the pellet; MMP-9 and the TIMP masculine gender dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, the dyeing intensity grading result uses the semi-quantitative analysis method to carry on H-to grade [2]: Selects 5 high power mirror fields of vision to each slice (×400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, according to the masculine cell percentage and the colored depth graduation, for details sees table 1.Above two grading adding together, no matter dyeing intensity, so long as the masculine cell< 10% all is a negative, by 0 represents the negative dyeing degree cell percentage; 3 divides into the weak masculine gender, by 1 represents weakness dyeing degree cell percentage masculine gender (+); 4-5 divides into the moderate masculine gender, by 2 represents the moderate dyeing degree cell percentage masculine gender (++); 6-7 divides into the strong masculine gender, by 3 represents the moderate dyeing degree cell percentage masculine gender (+++), by 0,1,2,3 on behalf of the negative, the weak dyeing, the medium dyeing and strong dyes 4 ranks.4.3 immunity histochemistry examination transplant lump organizes the VEGF expression organization specimen by 10% formaldehyde solution fixed 24h, the conventional paraffin wax embedding, prepares 5 m thick slices, carries on according to the immunity group working instruction. With the known masculine specimen took the masculine comparison, replaces by PBS anti-makes the negative comparison.The VEGF masculine expression is located the tumor cell and the blood vessel endothelial cell, in the cytoblastema appears yellow dyes the pellet; Selects 5 high power mirror fields of vision to each slice (×400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, respectively by 0,1,2,3 represents the negative, the weak dyeing, the medium dyeing and the strong dyeing and so on 4 ranks, H minute value= [(weak dyeing intensity cell percentage×1)+(moderate dyeing degree cell percentage×2)+(strong dyeing intensity cell percentage×3)] x 100, takes two viewer’s mean values to take again finally the H-grading, H is worth the minute being situated between 0-300Between.4.4 immunity histochemistry examination transplant lump organizes MMP-9 to express MMP-9 and the TIMP masculine gender dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, finally uses the semi-quantitative analysis method to carry on the H-grading:Selects 5 high power mirror fields of vision to each slice (x400) to count stochastically 200 cells, separately to has the specimen by two pathology department doctors in the double blind situation to carry on the observation, judges its butcher archery target DAB dyeing intensity under the light microscope, respectively by 0,1, 2,3 represents the negative, the weak dyeing, the medium dyeing and the strong dyeing and so on 4 ranks, H minute value= [(weak dyeing intensity cell percentage x1)+(moderate dyeing degree cell percentage x2)+(strong dyeing intensity cell percentage×3)] x 100, takes two viewer’s mean values to take again finally the H-grading, H is worth the minute being situated between 0-300Between.The observation tests in each group of sarcoma animal model VEGF, MMPs and the TIMP immunity expression.4.5 immunity histochemistry examination transplant lump organizes TIMP to express TIMP and MMP-9 is same, the masculine dyeing is located in the cell cytoblastema or the amnion appears yellow dyes the pellet, assumes the yellowish brown color, the result and the MMP-9 similar semi-quantitative analysis method carries on the H-grading.Statistics analysisThe obtained data uses the SPSS13.0 software to carry on statistics analysis.The transplant lump volume quota result (X±s) indicated with the mean value±standard deviation that, the group compares uses the single factor the variance analysis, during 22 groups compares uses the LSD examination. each group of transplant lump VEGF, MMP-9 and the TIMP H value semi-quantitative material uses the median (minimum value, maximum value) to indicate that, the group compares uses the Kruskall-Wallis examination, after during 22 groups compares uses Mann-Whitney the U-test (examination standard to use Bonferroni law adjustment alpha =0.008).By P< 0.05 thought has statistics significanceThe result:1. transplant lump growth situation and damps the lump rateThe lump tumor growth incubation period is 3-5d, the tumor assumes the expansibility to grow, the quality of material is slightly strong.The control group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:395.70±34.92 mm3,542.02±52.74 mm3,637.48±51.69 mm3,713.88±61.78 mm3,795.39±68.88 mm3,864.14±77.63 mm3,932.24±83.27mm3,990.37±89.25mm3; Graciousness group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally: 387.54±24.89mm3,506.70±28.07 mm3,605.55±40.89 mm3,685.8±40.89 mm3, 764.48±43.98 mm3,832.41±45.89 mm3,887.21±50.87 mm3,930.78±52.96 mm3; Along the platinum group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:365.90±23.72 mm3,478.70±27.01 mm3,553.73±34.65 mm3, 580.60±40.66 mm3,551.95±37.81mm3,497.19±43.47 mm3,431.43±38.81 mm3, 370.54±35.46mm3; The union group after the treatment 1st,4,7,10,13,16,19,22 day tumor volume is equally:392.78±42.81 mm3,477.82±47.74 mm3,524.79±38.14 mm3,534.89±22.05 mm3,476.39±28.73mm3,392.15±28.73 mm3, 328.17±31.04mm3,275.83±32.50mm3.After the medication the 4th day control group tumor still rapidly grows, graciousness group compares the control group to be slightly slow, still long-enduring growth.4th days the tumor starts slow-growing after the platinum group and the union group medication, after 7th days relatively static, after but reduces gradually, but and lengthens along with the time drops rapidly, increases rapidly with the control group and graciousness group forms distinctively shows (Figure to for instance the chart 3).The 22nd day union group tumor reduces quickly, (P 0.05) has statistics significance along the platinum group and the union< group in the tumor volume.2. immunity group examination2.1 VRGF immunity group H grades.VRGF immunity group like charts show (Figure 6-chart 9), the control group, graciousness group’s expression intensity is+++,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor VRGF the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Along platinum group and union group (P< 0.008); May infer union group VRGF the expression to be least, next is along the platinum group, is graciousness group once more, but the blank group are most.12 MMP-9 immunity group H grades.MMP-9 the immunity group like chart to show (Figure 10-chart 13), the control group, graciousness group’s expression intensity is+++,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor VRGF the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group with has along the platinum group has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); May infer union group MMP-9 the expression to be least, next is along the platinum group, is graciousness group once more, but the blank group are most。2..3TIMP immunity group H grading.The TIMP immunity group like chart shows (Figure 14-chart 17), control group, graciousness group’s expression intensity for+,++, but is being suitable the platinum group, the union group expresses the intensity respectively is++,+++.The H grading median (minimum value, maximum value) and average order as shown in Table 3.Between< the P 0.05,4 group has statistics significance, may think different combination medication suppression tumor TIMP the expression has the significance difference.Between 22 groups compares uses Mann-Whitney the U-test, the control group with has along the platinum group has statistics significance (P< 0.008); Between the control group and the union group has has statistics significance (P< 0.008); Between graciousness group and the union group have have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); Have between along the platinum group and the union group have statistics significance (P< 0.008); May infer blank group TIMP the expression to be least, next is graciousness group, but is once more along the platinum group, the union group most。Conclusion:1、the anti-tumor medicine union graciousness S180 sarcoma suppression blood vessel production function is obvious, obviously suppresses the tumor volume to increase, and damps the cancer rate to be high.2、in tumor VEGF and MMPs expression basic parallel, both participate in the tumor blood vessel together the production and coordinates the expression, anti-tumor medicine union graciousness causes in the lump VEGF and MMPs obviously reduces.3、in the tumor the MMPs/TIMP ratio reduces may suppress the tumor growth and invade Run, graciousness has the inhibitory action through many kinds of ways to VEGF and MMPs, and increases TIMP the expression, reduces the tumor blood vessel the production.4th, on clinical applies for the anti-tumor medicine union graciousness treatment sarcoma lays the experimental foundation, and treats the osteosarcoma animal model for the anti-tumor medicine union graciousness line anti-blood vessel, and the osteosarcoma clinical treatment plan opens the new treatment idea.更多还原