促血管生成因子及其部分相关因子在肾母细胞瘤中的表达及临床意义的研究
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摘要
前言与目的
肾母细胞瘤为儿童期较常见的恶性实体肿瘤,占小儿所有实体肿瘤的8%,在15岁以下儿童中,占泌尿生殖系恶性肿瘤的80%以上,据不完全统计,包括世界各国的任何种族,全世界每百万人口中,肾母细胞瘤的发生数约为7例,其在小儿外科疾病中占重要的地位。虽然肾母细胞瘤经手术及化疗等综合治疗,其预后得到了极大的改善,但肿瘤的复发和转移仍是最终造成患儿预后不良的重要原因,了解肾母细胞瘤生长过程中生物学特征及调控机制,对于研究针对性的治疗方法与药物有着重要的指导意义。
肿瘤中新生血管的形成是肿瘤生长的必需条件,血管生成在原发肿瘤的生长与侵入中至关重要,目前血管生成与实体肿瘤发展、转移等的关系为当前肿瘤学研究热点。实体肿瘤的发展必需有血管生成的参与。血管生成是实体肿瘤生长和转移的必要条件,目前已公认,血管内皮生长因子(vascular endothelial growthfactor,VEGF)是目前所知主要的血管内皮生长因子,VEGF在促进肿瘤血管内皮细胞增殖和增加血管通透性方面起着重要的作用,本研究以VEGF为中心,研究肾母细胞瘤组织中VEGF及与其密切相关的细胞因子的表达情况及临床意义,相对系统的研究肾母细胞瘤血管生成的调控机制。通过对肾母细胞瘤与血管内皮生长因子及血管生成素关系的研究可能为临床对肾母细胞瘤的治疗提供一条新的途径,成为治疗Wiims.瘤的新的突破点。
材料和方法
临床标本:取自卫生部小儿先天畸形实验室标本库,肾母细胞瘤标本52例,其中男27例,女25例,年龄在0.5~11岁,平均年龄4.2岁,均经病理证实;按NWTS-5标准根据间变细胞情况组织学分型:良好组织学类型35例,不良组织学类型17例。其中47例标本手术后立即在癌组织边缘以外的竖组织处取材,另有8例非肾脏疾病死亡尸检肾组织作为对照。标本均在手术中无菌取材后液氮处理,拟行RT-PCR的标本放入经DEPC水(diethyl pyrocarbonate,二乙基焦磷酰胺)处理并高压灭菌后的Eppendoff管中,入-80℃深低温冰箱保存。随访情况:所有病例随访2年以上者31例,7例因肿瘤复发转移而死亡。
免疫组化试剂:抗Ang1,Ang2及Tie2、Survivin、Stat3抗体为兔抗人多克隆抗体即用型(购自福建迈新生物技术公司),抗CD34、VEGF及Flt、HIF-1α抗体为兔抗人多克隆抗体即用型(武汉博士德生物技术公司)。RT-PCR试剂盒、Marker(DNA Marker DL2000)、Taq2酶、dNTP Mixture、琼脂糖均由TAKARA Biotechnolgy(大连)Co.提供:RNA裂解液购于TRIZOL Reagent(Total RNA isolation,美国)。
应用RT-PCR和免疫组化SABC方法检测WT标本中不同部位的血管内皮生长因子(VEGF)及其受体(FLT)和血管生成素(Ang1、Ang2)及其受体(Tie2)的表达,以免疫组织化学检测内皮细胞特异性的标志分子CD34,并且分析血管密度与促血管生成因子表达的关系。
采用免疫组化SABC法及RT-PCR方法研究Survivin及VEGF在52例肾母细胞瘤组织,47例瘤旁组织及8例肾组织在蛋白水平及mRNA水平的表达情况。结合临床及随访资料分析。
应用免疫组化SABC法辅以计算机图像分析的方法,应用Image-plus pro 5.1图像分析软件半定量研究Stat3、HIF-1α与VEGF的在52例WT组织,47例瘤旁组织及8例肾组织表达强度情况,结合临床资料加以分析。
结果
1、与正常肾组织比较,WT组织及瘤旁肾组织中VEGF表达明显增高(P＜0.05),Ang2在WT组织及瘤旁肾组织及正常肾组织比较,差异显著(P＜0.05),Ang1、Fk受体在各组织间表达虽未见明显差异(P＞0.2),但Ang1在WT组织及瘤旁肾组织中的表达存在增高的趋势。在肿瘤组织中,围绕着癌巢的新生血管数量明显增加,血管排列紊乱、密集、扭曲;而在瘤旁组织中,血管的形态呈向外扩展浸润性生长,密度高于其周围肾组织,与血管因子及其受体的表达高低是明显一致的。
2、肾母细胞瘤组织中Survivin免疫组化显示阳性表达率为61.5%(32/52),RT-PCR阳性率为67.3%(35/52);47例瘤旁组织中免疫组化显示阳性表达率为4.3%(2/47),RT-PCR阳性率为6.4%(3/47);肾组织中未见表达。52例肾母细胞瘤组织中VEGF免疫组化显示阳性表达率为75%(39/52),RT-PCR阳性率为71.2%(37/52);47例瘤旁组织中免疫组化显示阳性表达率为57.4%(28/47),RT-PCR阳性率为53.2%(25/47);肾组织中免疫组化显示阳性表达率为12.5%(1/8),RT-PCR阳性率为25%(2/8)。另外,Survivin及VEGF在预后不良型肾母细胞瘤组织及瘤旁组织中表达均明显增高。Survivin及VEGF阳性者2年存活率均低于其表达阴性者。
3、VEGF、Stat3及HIF-1α在肾母细胞瘤组织中表达强度较瘤旁组织及正常肾组织显著增强(P＜0.05)且瘤旁组织中VEGF表达强度高于正常肾组织。另外,临床分期Ⅲ～Ⅳ和预后不良病理类型的肾母细胞瘤组织中Star3及VEGF表达强度明显高于临床分期Ⅰ～Ⅱ的WT,预后不良病理类型及直径≥6cm的肾母细胞瘤组织中HIF-1α表达强度较预后良好型及直径＜6cm的肾母细胞瘤组织升高。
结论
1、在肾母细胞瘤的生长过程中,VEGF/FLT和Angiopietins/Tie2两条通路均有不同程度的活化,但不协调,使得肾母细胞瘤组织中的血管处于不成熟状态,这为进一步开发抑制血管生成的抗肿瘤生长药物提供了科学依据。
2、Survivin,VEGF表达与肾母细胞瘤的临床病理学特点有关,Survivin在血管形成的中间环节具有重要意义,Survivin和VEGF在肾母细胞瘤中表达具有一致性,二者在肾母细胞瘤组织的高表达与肿瘤的生物学行为密切相关,其与肿瘤血管的生成,血管内皮细胞的凋亡,稳定性密切相关,进而影响肿瘤的侵袭力。其检测有利于肾母细胞瘤的诊断及其预后情况的评价。
3、VEGF、Stat3及HIF-1α表达与肾母细胞瘤的发展预后有关,参与了肿瘤血管的生成及肿瘤的增殖侵袭,Stat3可能对HIF-1和VEGF的表达起着重要的调控作用,对于靶向治疗肾母细胞瘤具有一定意义。
INTRODUCTION AND OBJECTIVE
Wilms's tumor is a pediatric malignancy of the kidney and one of the most common solid tumors in children. It account to 8% of all the solid tumors in children and 80% of malignant tumor in urogenital system among the children younger than fifteen years old. There may be seven cases of Wilms' tumor among one million people in the world and it is an important disease in pediatric surgery. Despite the remarkable response to chemotherapy, 5-10% of tumours are fatal because of the occurrence of metastases and drug resistance. Therefore, it is important to find regulation mechanisms and biological characteristics of the process of growth of Wilms' tumor to guide the research of direct treatment and new drug.
Formation of the new vessels in tumor is essential for tumorous growth, angiogenesis is very important for the growth and invasion of the primartumor. It has been a hot spot of oncology research to study the relationship of angiogenesis and metastasis, development of solid tumor, it has been suggested that angiogenesis plays an important role in tumor progression and the spread of metastases through the bloodstream in solid tumors. It has been reported that several growth factors with angiogenic activity are produced by solid tumours. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is highly specific for endothelium, and also functions as a vascular permeability factor. This study focus on VEGF AND study the expressions and clinical significance of VEGF and related cell factors in Wilms' tumor, research the mechanisms of angiogenesis in Wilms' tumor. The study may be helpful for the treatment of wilm's tumor and provide a new way to cure the disease.
MATERIALS AND METHODS
1、Clinical specimens: All the specimens are from Congenital malformationslaboratory of the Ministry of Health. Wilms' tumor specimens of 52 patients, 27 male, 25 female, aged 0.5-11 years with a mean age of 4.2 years, were confirmed by pathology; According to NWTS-5 (National Wilms Tumor Study) standards by histological anaplastic cells, there are 35 cases of good histological types and 17 cases of adverse histologic types. Nephridial tissues close to the tumors were taken immediately after surgery in which 47 cases. Another eight cases of renal tissues were drawed as control group from autopsy. Aseptic surgical specimens were treated with liquid nitrogen and stored. The specimens for RT-PCR were placed in the Eppendoff tubes which managed by DEPC and autoclaving. They were preserved in -80℃refrigerator. There were 31 cases in all the patients to be followed up for more than two years and seven cases died due to tumor recurrence and metastasis.
2、Immunohistochemistry agents: Anti-ANG1, Anti-ANG1, Anti-TIE2,Anti-Survivin and Anti-Stat3 are polyclonal rabbit anti-human antibody (from the Fujian Maixin biotechnology companies). Anti-CD34, Anti-VEGF and Anti-FLT, Anti-HIF-1αare polyclonal rabbit anti-human antibody (Wuhan Boshide biotechnology companies). RT-PCR Kit, Marker (DNA Marker DL2000), Taq2 enzyme, dNTP Mixture and agarose are all from TAKARA Biotechnolgy Co.(Dalian) ; RNA lysates were purchased in TRIZOL Reagent (Total RNA isolation U.S).
3、RT-PCR and immunohistochemistry staining method were used to study theexpressions of vascular endothelial growth factor (VEGF) and its receptors (FLT), and angiopoietin (Ang1, Ang2) and its receptor(Tie2) in different parts of WT specimens, CD34(the endothelial cell-specific marker) was detected by immunohistochemical staining, then the relationship of vascular density and the expressions of angiogenesis factors was analyzed.
4、The expressions of Survivin and VEGF were detected by the immohistochemical staining and RT-PCR in 52 specimens from WT tissues, 47 from adjacent kidney tissues and 8 from normal kidney tissues in protein level and mRNA level. The data were analyzed combined with clinical and follow-up data.
5、The expression of Stat3, HIF-1αand VEGF were detected by theimmunohistochemical staining in 52 specimens from Wilms' tumor tissues, 47 from adjacent kidney tissues and 8 from normal kidney tissues, the expression intensity was analyzed by computer image processing of . Image-plus pro 5.1. The data were analyzed combined with clinical and follow-up data.
RESULTS
1、The expression levels of VEGF and Ang2 were significantly higher innephroblastoma group than the normal tissues(P<0.05), and so did the expression of CD34. The expressions of FLT and Ang1/Tie2 showed to be increased to various levels in tumor and tumor adjacent tissues as compared with normal kidney tissues. Compare with normal kidney tissue, expression of VEGF in the WT and adjacent tissues increased significantly (P <0.05). Expression of Ang2 was higher in the WT than in the adjacent tissues and kidney tissues, the difference was significant (P <0.05). Expressions of Ang1, Flt in various groups did not see significant differences (P> 0.2). But they had a trend of increase in the WT Ang1 and adjacent tissues compare to normal tissues. Increase in the number of angiogenesis, vascular disorder, intensive distorted could be seen in tumor tissues In adjacent tissues, vascular growth pattern was the expansion of invasive, much higher than the normal tissues, and it was consistency with the expressions of the vascular factors and its receptors.
2、By immunohistochemical staining, Survivin was expressed in 72.9% of WTtissue samples and in 4.3% of adjacent kidney tissues; by RT-PCR, Survivin mRNA was expressed in 67.3% of WT tissue samples and 6.4% in adjacent kidney tissues, while no Survivin and mRNA expression was detected in normal kidney tissues. Incontrast, VEGF was expressed in 75% of WT tissues and 19.1% of adjacent kidney samples(P<0.05) and 12.5% in normal kidney tissues by immunochemical staining; VEGF mRNA was expressed in 71.2% of Wilms' tumor tissues and 23.4% of adjacent kidney samples(P<0.05) and 25% in normal kidney tissues. The expression of Survivin and VEGF increased obviously in WT tissues with unfavourable prognosis and the cases with positive expressions of them were with higher mortality.
3、The expressions of Stat3, HIF-1 and VEGF were significantly up-regulated inWilms' tumor compare to that in adjacent tissues and normal kidney tissues(P<0.05). Stat3 and VEGF protein in Wilms' tumor of clinical stage III-IV and high risk histopathology was higher than that of clinical stage I - II and low risk histopathology. The higher expression of HIF-1 in Wilms' tumor was showed in tumors with high risk histopathology and with tumor size≥6cm.
CONCLUSIONS
1、In the process of growth of Wilms' tumor, the two pathways of VEGF/FLTAngiopietins/Tie2 have both different levels of activation, but they are disharmony and make vascular tissue of Wilms' tumor in the immature state, The results provide a scientific basis for the further development of drugs of anti-angiogenesis to inhibit growth of tumor.
2、Expressions of Survivin, VEGF in Wilms' tumor are associated with its clinicaland pathological features. Survivin plays an important part a in the middle stages of angiogenesis. Expressions of Survivin and VEGF in Wilms' tumor have consistency. High expressions of them in Wilms' tumor are closely related to the biological behavior, angiogenesis, apoptosis of vascular endothelial cells and stability of tumor vessels. They may affect the extent of tumor invasion, detection of them in Wilms' tumor is helpful in the diagnosis and evaluation of prognosis.
3、Increased expressions of Stat3, HIF-1 and VEGF were found in Wilms' tumor,and may be related to the development and angiogenesis of Wilms' tumor, may be associated with generation and invasion of the tumor. Stat3 may regulate expression of HIF-1 and VEGF, so it could be an effective target for inhibiting tumor VEGF expression and angiogenesis of Wilms' tumor.
肾母细胞瘤为儿童期较常见的恶性实体肿瘤,占小儿所有实体肿瘤的8%,在15岁以下儿童中,占泌尿生殖系恶性肿瘤的80%以上,据不完全统计,包括世界各国的任何种族,全世界每百万人口中,肾母细胞瘤的发生数约为7例,其在小儿外科疾病中占重要的地位。虽然肾母细胞瘤经手术及化疗等综合治疗,其预后得到了极大的改善,但肿瘤的复发和转移仍是最终造成患儿预后不良的重要原因,了解肾母细胞瘤生长过程中生物学特征及调控机制,对于研究针对性的治疗方法与药物有着重要的指导意义。
肿瘤中新生血管的形成是肿瘤生长的必需条件,血管生成在原发肿瘤的生长与侵入中至关重要,目前血管生成与实体肿瘤发展、转移等的关系为当前肿瘤学研究热点。实体肿瘤的发展必需有血管生成的参与。血管生成是实体肿瘤生长和转移的必要条件,目前已公认,血管内皮生长因子(vascular endothelial growthfactor,VEGF)是目前所知主要的血管内皮生长因子,VEGF在促进肿瘤血管内皮细胞增殖和增加血管通透性方面起着重要的作用,本研究以VEGF为中心,研究肾母细胞瘤组织中VEGF及与其密切相关的细胞因子的表达情况及临床意义,相对系统的研究肾母细胞瘤血管生成的调控机制。通过对肾母细胞瘤与血管内皮生长因子及血管生成素关系的研究可能为临床对肾母细胞瘤的治疗提供一条新的途径,成为治疗Wiims.瘤的新的突破点。
材料和方法
临床标本:取自卫生部小儿先天畸形实验室标本库,肾母细胞瘤标本52例,其中男27例,女25例,年龄在0.5~11岁,平均年龄4.2岁,均经病理证实;按NWTS-5标准根据间变细胞情况组织学分型:良好组织学类型35例,不良组织学类型17例。其中47例标本手术后立即在癌组织边缘以外的竖组织处取材,另有8例非肾脏疾病死亡尸检肾组织作为对照。标本均在手术中无菌取材后液氮处理,拟行RT-PCR的标本放入经DEPC水(diethyl pyrocarbonate,二乙基焦磷酰胺)处理并高压灭菌后的Eppendoff管中,入-80℃深低温冰箱保存。随访情况:所有病例随访2年以上者31例,7例因肿瘤复发转移而死亡。
免疫组化试剂:抗Ang1,Ang2及Tie2、Survivin、Stat3抗体为兔抗人多克隆抗体即用型(购自福建迈新生物技术公司),抗CD34、VEGF及Flt、HIF-1α抗体为兔抗人多克隆抗体即用型(武汉博士德生物技术公司)。RT-PCR试剂盒、Marker(DNA Marker DL2000)、Taq2酶、dNTP Mixture、琼脂糖均由TAKARA Biotechnolgy(大连)Co.提供:RNA裂解液购于TRIZOL Reagent(Total RNA isolation,美国)。
应用RT-PCR和免疫组化SABC方法检测WT标本中不同部位的血管内皮生长因子(VEGF)及其受体(FLT)和血管生成素(Ang1、Ang2)及其受体(Tie2)的表达,以免疫组织化学检测内皮细胞特异性的标志分子CD34,并且分析血管密度与促血管生成因子表达的关系。
采用免疫组化SABC法及RT-PCR方法研究Survivin及VEGF在52例肾母细胞瘤组织,47例瘤旁组织及8例肾组织在蛋白水平及mRNA水平的表达情况。结合临床及随访资料分析。
应用免疫组化SABC法辅以计算机图像分析的方法,应用Image-plus pro 5.1图像分析软件半定量研究Stat3、HIF-1α与VEGF的在52例WT组织,47例瘤旁组织及8例肾组织表达强度情况,结合临床资料加以分析。
结果
1、与正常肾组织比较,WT组织及瘤旁肾组织中VEGF表达明显增高(P＜0.05),Ang2在WT组织及瘤旁肾组织及正常肾组织比较,差异显著(P＜0.05),Ang1、Fk受体在各组织间表达虽未见明显差异(P＞0.2),但Ang1在WT组织及瘤旁肾组织中的表达存在增高的趋势。在肿瘤组织中,围绕着癌巢的新生血管数量明显增加,血管排列紊乱、密集、扭曲;而在瘤旁组织中,血管的形态呈向外扩展浸润性生长,密度高于其周围肾组织,与血管因子及其受体的表达高低是明显一致的。
2、肾母细胞瘤组织中Survivin免疫组化显示阳性表达率为61.5%(32/52),RT-PCR阳性率为67.3%(35/52);47例瘤旁组织中免疫组化显示阳性表达率为4.3%(2/47),RT-PCR阳性率为6.4%(3/47);肾组织中未见表达。52例肾母细胞瘤组织中VEGF免疫组化显示阳性表达率为75%(39/52),RT-PCR阳性率为71.2%(37/52);47例瘤旁组织中免疫组化显示阳性表达率为57.4%(28/47),RT-PCR阳性率为53.2%(25/47);肾组织中免疫组化显示阳性表达率为12.5%(1/8),RT-PCR阳性率为25%(2/8)。另外,Survivin及VEGF在预后不良型肾母细胞瘤组织及瘤旁组织中表达均明显增高。Survivin及VEGF阳性者2年存活率均低于其表达阴性者。
3、VEGF、Stat3及HIF-1α在肾母细胞瘤组织中表达强度较瘤旁组织及正常肾组织显著增强(P＜0.05)且瘤旁组织中VEGF表达强度高于正常肾组织。另外,临床分期Ⅲ～Ⅳ和预后不良病理类型的肾母细胞瘤组织中Star3及VEGF表达强度明显高于临床分期Ⅰ～Ⅱ的WT,预后不良病理类型及直径≥6cm的肾母细胞瘤组织中HIF-1α表达强度较预后良好型及直径＜6cm的肾母细胞瘤组织升高。
结论
1、在肾母细胞瘤的生长过程中,VEGF/FLT和Angiopietins/Tie2两条通路均有不同程度的活化,但不协调,使得肾母细胞瘤组织中的血管处于不成熟状态,这为进一步开发抑制血管生成的抗肿瘤生长药物提供了科学依据。
2、Survivin,VEGF表达与肾母细胞瘤的临床病理学特点有关,Survivin在血管形成的中间环节具有重要意义,Survivin和VEGF在肾母细胞瘤中表达具有一致性,二者在肾母细胞瘤组织的高表达与肿瘤的生物学行为密切相关,其与肿瘤血管的生成,血管内皮细胞的凋亡,稳定性密切相关,进而影响肿瘤的侵袭力。其检测有利于肾母细胞瘤的诊断及其预后情况的评价。
3、VEGF、Stat3及HIF-1α表达与肾母细胞瘤的发展预后有关,参与了肿瘤血管的生成及肿瘤的增殖侵袭,Stat3可能对HIF-1和VEGF的表达起着重要的调控作用,对于靶向治疗肾母细胞瘤具有一定意义。
INTRODUCTION AND OBJECTIVE
Wilms's tumor is a pediatric malignancy of the kidney and one of the most common solid tumors in children. It account to 8% of all the solid tumors in children and 80% of malignant tumor in urogenital system among the children younger than fifteen years old. There may be seven cases of Wilms' tumor among one million people in the world and it is an important disease in pediatric surgery. Despite the remarkable response to chemotherapy, 5-10% of tumours are fatal because of the occurrence of metastases and drug resistance. Therefore, it is important to find regulation mechanisms and biological characteristics of the process of growth of Wilms' tumor to guide the research of direct treatment and new drug.
Formation of the new vessels in tumor is essential for tumorous growth, angiogenesis is very important for the growth and invasion of the primartumor. It has been a hot spot of oncology research to study the relationship of angiogenesis and metastasis, development of solid tumor, it has been suggested that angiogenesis plays an important role in tumor progression and the spread of metastases through the bloodstream in solid tumors. It has been reported that several growth factors with angiogenic activity are produced by solid tumours. Vascular endothelial growth factor (VEGF) is an angiogenic factor that is highly specific for endothelium, and also functions as a vascular permeability factor. This study focus on VEGF AND study the expressions and clinical significance of VEGF and related cell factors in Wilms' tumor, research the mechanisms of angiogenesis in Wilms' tumor. The study may be helpful for the treatment of wilm's tumor and provide a new way to cure the disease.
MATERIALS AND METHODS
1、Clinical specimens: All the specimens are from Congenital malformationslaboratory of the Ministry of Health. Wilms' tumor specimens of 52 patients, 27 male, 25 female, aged 0.5-11 years with a mean age of 4.2 years, were confirmed by pathology; According to NWTS-5 (National Wilms Tumor Study) standards by histological anaplastic cells, there are 35 cases of good histological types and 17 cases of adverse histologic types. Nephridial tissues close to the tumors were taken immediately after surgery in which 47 cases. Another eight cases of renal tissues were drawed as control group from autopsy. Aseptic surgical specimens were treated with liquid nitrogen and stored. The specimens for RT-PCR were placed in the Eppendoff tubes which managed by DEPC and autoclaving. They were preserved in -80℃refrigerator. There were 31 cases in all the patients to be followed up for more than two years and seven cases died due to tumor recurrence and metastasis.
2、Immunohistochemistry agents: Anti-ANG1, Anti-ANG1, Anti-TIE2,Anti-Survivin and Anti-Stat3 are polyclonal rabbit anti-human antibody (from the Fujian Maixin biotechnology companies). Anti-CD34, Anti-VEGF and Anti-FLT, Anti-HIF-1αare polyclonal rabbit anti-human antibody (Wuhan Boshide biotechnology companies). RT-PCR Kit, Marker (DNA Marker DL2000), Taq2 enzyme, dNTP Mixture and agarose are all from TAKARA Biotechnolgy Co.(Dalian) ; RNA lysates were purchased in TRIZOL Reagent (Total RNA isolation U.S).
3、RT-PCR and immunohistochemistry staining method were used to study theexpressions of vascular endothelial growth factor (VEGF) and its receptors (FLT), and angiopoietin (Ang1, Ang2) and its receptor(Tie2) in different parts of WT specimens, CD34(the endothelial cell-specific marker) was detected by immunohistochemical staining, then the relationship of vascular density and the expressions of angiogenesis factors was analyzed.
4、The expressions of Survivin and VEGF were detected by the immohistochemical staining and RT-PCR in 52 specimens from WT tissues, 47 from adjacent kidney tissues and 8 from normal kidney tissues in protein level and mRNA level. The data were analyzed combined with clinical and follow-up data.
5、The expression of Stat3, HIF-1αand VEGF were detected by theimmunohistochemical staining in 52 specimens from Wilms' tumor tissues, 47 from adjacent kidney tissues and 8 from normal kidney tissues, the expression intensity was analyzed by computer image processing of . Image-plus pro 5.1. The data were analyzed combined with clinical and follow-up data.
RESULTS
1、The expression levels of VEGF and Ang2 were significantly higher innephroblastoma group than the normal tissues(P<0.05), and so did the expression of CD34. The expressions of FLT and Ang1/Tie2 showed to be increased to various levels in tumor and tumor adjacent tissues as compared with normal kidney tissues. Compare with normal kidney tissue, expression of VEGF in the WT and adjacent tissues increased significantly (P <0.05). Expression of Ang2 was higher in the WT than in the adjacent tissues and kidney tissues, the difference was significant (P <0.05). Expressions of Ang1, Flt in various groups did not see significant differences (P> 0.2). But they had a trend of increase in the WT Ang1 and adjacent tissues compare to normal tissues. Increase in the number of angiogenesis, vascular disorder, intensive distorted could be seen in tumor tissues In adjacent tissues, vascular growth pattern was the expansion of invasive, much higher than the normal tissues, and it was consistency with the expressions of the vascular factors and its receptors.
2、By immunohistochemical staining, Survivin was expressed in 72.9% of WTtissue samples and in 4.3% of adjacent kidney tissues; by RT-PCR, Survivin mRNA was expressed in 67.3% of WT tissue samples and 6.4% in adjacent kidney tissues, while no Survivin and mRNA expression was detected in normal kidney tissues. Incontrast, VEGF was expressed in 75% of WT tissues and 19.1% of adjacent kidney samples(P<0.05) and 12.5% in normal kidney tissues by immunochemical staining; VEGF mRNA was expressed in 71.2% of Wilms' tumor tissues and 23.4% of adjacent kidney samples(P<0.05) and 25% in normal kidney tissues. The expression of Survivin and VEGF increased obviously in WT tissues with unfavourable prognosis and the cases with positive expressions of them were with higher mortality.
3、The expressions of Stat3, HIF-1 and VEGF were significantly up-regulated inWilms' tumor compare to that in adjacent tissues and normal kidney tissues(P<0.05). Stat3 and VEGF protein in Wilms' tumor of clinical stage III-IV and high risk histopathology was higher than that of clinical stage I - II and low risk histopathology. The higher expression of HIF-1 in Wilms' tumor was showed in tumors with high risk histopathology and with tumor size≥6cm.
CONCLUSIONS
1、In the process of growth of Wilms' tumor, the two pathways of VEGF/FLTAngiopietins/Tie2 have both different levels of activation, but they are disharmony and make vascular tissue of Wilms' tumor in the immature state, The results provide a scientific basis for the further development of drugs of anti-angiogenesis to inhibit growth of tumor.
2、Expressions of Survivin, VEGF in Wilms' tumor are associated with its clinicaland pathological features. Survivin plays an important part a in the middle stages of angiogenesis. Expressions of Survivin and VEGF in Wilms' tumor have consistency. High expressions of them in Wilms' tumor are closely related to the biological behavior, angiogenesis, apoptosis of vascular endothelial cells and stability of tumor vessels. They may affect the extent of tumor invasion, detection of them in Wilms' tumor is helpful in the diagnosis and evaluation of prognosis.
3、Increased expressions of Stat3, HIF-1 and VEGF were found in Wilms' tumor,and may be related to the development and angiogenesis of Wilms' tumor, may be associated with generation and invasion of the tumor. Stat3 may regulate expression of HIF-1 and VEGF, so it could be an effective target for inhibiting tumor VEGF expression and angiogenesis of Wilms' tumor.
引文
1 Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature, 2000, 407(6801): 249-257.
2 Stratmann A, Acker T, Burger AM, et al. Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants. Int J Cancer, 2001, 91(3): 273-282.
3 Homer A, BordS, Kelsall AW, et al. Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone. Bone, 2001, 28(1): 65-71.
4 Weidner N, Semple JP, Welch WR, et al. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. _N Engl J Med, 1991, 324(1): 1-8.
5 Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996, 86(3):353-364.
6 Holmes DI, Zachary I. The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol, 2005, 6(2): 209.
7 Sato TN, TozawaY, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. _Nature, 1995, 376(6535): 70-74.
8. Lauren J, Gunji Y, Alitalo K. Is angiopoietin-2 necessary for the initiation of tumor angiogenesis?_Am J Pathol, 1998, 153(5): 1333-1339.
9 Helmlinger G, Yuan F, DellianM, et al. Interstitial pH and p02 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med, 1997, 3(2): 177-182.
10 von Marschall Z, Scholz A, Cramer T, et al. Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis. J Natl Cancer Inst, 2003, 95(6): 437-448
11 Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science,1997,277(5322):55-60.
12 赵万洲,韩锐.肿瘤血管生成抑制剂研究策略及进展.中华肿瘤杂志,2000,22(2):93-95.
13 Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human survivin is a direct inhibitor of caspase23 and 27.Biochemistry,2001,40(4):1117-1123.
14 Adida C,Crotty PL,McGrath J,et al.Developmentally regulated expression of the novel cancer anti-apoptosis gene surviving in human and mouse differentiation.Am J Pathol,1998,152(1):43-49.
15 Porter AG,Janicke RU.Emerging roles of caspase-3 in apoptosis.Cell Death Differ,1999,6(2):99-104.
16 Takamizawa S,Scott D,Wen J,et al.The survivin:fas ratio in pediatric renal tumors.J Pediatr Surg,2001,36(1):37-42.
17 Azuhata T,Scott D,Takamizawa S,et al.The inhibitor of apoptosis protein surviving is associated with high-risk behavior of neuroblastoma.J Pediatr Surg,2001,3(12):1785-1791.
18 林茂芳,孟小莉,蔡真,等.抗凋亡基因survivin在急性白血病细胞表达及其临床意义.中华血液学杂志,2002,23(5):251-253.
19 常青,秦仁义,裘法祖.Survivin在胆管癌中的表达.中华实验外科杂志,2003,20(5):398-399.
20 王贵红,夏荣龙,郑兰东.结肠癌组织survivin表达的意义.第四军医大学学报,2006,27(2):170-171.
21 高红,王常林,蔡炜嵩,等.Survivin Caspase23 mRNA在肾母细胞瘤中的表达及意义.中国当代儿科杂志,2004,6(5):381-384.
22 Nesbit M.Abrogation of tumor vasculature using gene therapy.Cancer Metastasis Rev,2000,19(1-2):45-49.
23 O'Connor DS,Schechner JS,Adida C,et al.Control of apoptosis during angiogenesis by survivin expression in endothelial cells.Am J Pathol,2000,156(2):393-398.
24 Tran J,Rak J,Sheehan C,et al.Marked induction of IAP famly antiapoptotic proteins survivin and XIAP by VEGF in vacular endothelial cells.Biochem Biophys Res Commum,1999,264(3):781-788.
25 Tran J,Master Z,Yu JL,et al.A role for Survivin in chemoresistance of endothelial cells mediated by VEGF.Proc Nat Acad Sci USA,2002,99(7):4349-4354.
26 Gray MJ,Zhang J,Ellis LM,et al.HIF-lalpha,STAT3,CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas.Oncogene,2005,24(19):3110-3120.
27 Xu Q,Briggs J,Park S,et a1.Targeting Star3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways.Oncogene,2005,24(36):5552-5560.
28 Kirkali Z,Tuzel E,MunganMU.Recent advance in kidney cancer and metastatic disease.BJU Int,2001,88(8):818-824.
29 Dankbar B,Padro T,Leo R,et al.Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma.Blood,2000,95(8):2630-2636.
30 张谦,单岩,陈清江.血管内皮生长因子在预测肾母细胞瘤转移中的作用.中华儿科杂志,2005,43(3):213-214.
31 Semenza GL,Wang GL.A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.Mol Cell Biol,1992,12(12):5447-5454.
32 Huang LE,Gu J,Schau M,et al.Regulation of hypoxia-inducible factor lalpha is mediated by an 02-dependent degradation domain via the ubiquitin-proteasome pathway.Proc Natl Acad Sci U S A,1998,95(14):7987-7992.
33 Semenza GL.HIF-1 and tumor progression:pathophysiology and therapeutics.Trends Mol Med,2002,8(4 Suppl):S62-67.
34 Carmeliet P, Dor Y, Herbert JM, et al. Role of HIF-1 α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature, 1998, 394 (6692): 485-490.
35 Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 α and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol, 2000, 157(2) :411-421.
36 Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an oncogene. Cell, 1999, 98(3): 295-303.
37 Ivanov VN, Bhoumik A, Krasilnikov M, et al. Cooperation between STAT3 and c-jun suppresses Fas transcription. Mol Cell, 2001, 7(3):517-528.
38 Bromberg J. Stat proteins and oncogenesis. J Clin Invest, 2002 ,109(9): 1139-1142.
1.Hill DA,Shear TD,Liu T,et al.Clinical and biologic signifance of nuclear unreast in Wilm's tumor.Cancer,2003,97(9):2318-2326.
2.Baish JW,Jain RK.Fractals and cancer.Cancer Res,2000,60(5):3683-3684.
3.Kirkali Z,Tuzel E,Mungan MU.Recent advance in kidney cancer and metastatic disease.BJU Int,2001,88(8):818-824.
4.Elizabeth AB,Wei D,Max RL,et al.Neurolastoma,apoptosis,and growth factors.The Am Surg,2003,69(1):28-31.
5.Kraizer Y,Mawasi N,Seagal J,et al.Vascular endothelial growth factor and angiopoietin inliver regeneration.Biochem Biophys Res Commun,2001,287(1):209-215.
6.Fong GH,Rossant J,Gertsenstein M,et al.Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.Nature,1995,376(6355):66-70.
7.Carmeliet P,Jain R_K,et al.Angiogenesis in cancer and other diseases.Nature,2000,407(1):249-257.
8.Risau W.Mechanisms ofangiogenesis.Nature,1997,386(6626):671-674.
9.Partanen J,Armstrong E,Makela TP,et al.A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.Mol Cell BIOL,1992,4(10):1698-1707.
10.Dales JP,Gacia S,Bonnier P,et al.Tie2/Tek expression in breast carcinoma:correlations of immunohistochemical assays and long-term follow-up in a series of 909 patients.Int J Oncol,2003,22(2):391-397.
11.Harris AL,Reusch P,Barleon B,et al.Soluble Tie2 and Fltl extracellular domains in serum of patients with renal cancer and response to antiangiogenic therapy.Clin Cancer RES,2001,7(7):1992-1997.
12.吴向华,覃文新,赵瑞胶,等.酪氨酸激酶受体Tie2在不同肿瘤中的表达及其意义.中国病理生理杂志,2004,20(5):836-839.
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14.Maisopierre PC,Suri C,Jones PF,et al.Angiopoietin-2,a natural antagonist for Tie2 that disrupt in vivo angiogenesis.Science,1997,277:55-60.
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21.徐敏,施诚仁,张文竹,等.儿童实体肿瘤中血管内皮生长因子的表达.中华小儿外科杂志,1999,20(5):280-282.
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2 Stratmann A, Acker T, Burger AM, et al. Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants. Int J Cancer, 2001, 91(3): 273-282.
3 Homer A, BordS, Kelsall AW, et al. Tie2 ligands angiopoietin-1 and angiopoietin-2 are coexpressed with vascular endothelial cell growth factor in growing human bone. Bone, 2001, 28(1): 65-71.
4 Weidner N, Semple JP, Welch WR, et al. Tumor angiogenesis and metastasis—correlation in invasive breast carcinoma. _N Engl J Med, 1991, 324(1): 1-8.
5 Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell, 1996, 86(3):353-364.
6 Holmes DI, Zachary I. The vascular endothelial growth factor (VEGF) family: angiogenic factors in health and disease. Genome Biol, 2005, 6(2): 209.
7 Sato TN, TozawaY, Deutsch U, et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. _Nature, 1995, 376(6535): 70-74.
8. Lauren J, Gunji Y, Alitalo K. Is angiopoietin-2 necessary for the initiation of tumor angiogenesis?_Am J Pathol, 1998, 153(5): 1333-1339.
9 Helmlinger G, Yuan F, DellianM, et al. Interstitial pH and p02 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat Med, 1997, 3(2): 177-182.
10 von Marschall Z, Scholz A, Cramer T, et al. Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis. J Natl Cancer Inst, 2003, 95(6): 437-448
11 Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science,1997,277(5322):55-60.
12 赵万洲,韩锐.肿瘤血管生成抑制剂研究策略及进展.中华肿瘤杂志,2000,22(2):93-95.
13 Shin S,Sung BJ,Cho YS,et al.An anti-apoptotic protein human survivin is a direct inhibitor of caspase23 and 27.Biochemistry,2001,40(4):1117-1123.
14 Adida C,Crotty PL,McGrath J,et al.Developmentally regulated expression of the novel cancer anti-apoptosis gene surviving in human and mouse differentiation.Am J Pathol,1998,152(1):43-49.
15 Porter AG,Janicke RU.Emerging roles of caspase-3 in apoptosis.Cell Death Differ,1999,6(2):99-104.
16 Takamizawa S,Scott D,Wen J,et al.The survivin:fas ratio in pediatric renal tumors.J Pediatr Surg,2001,36(1):37-42.
17 Azuhata T,Scott D,Takamizawa S,et al.The inhibitor of apoptosis protein surviving is associated with high-risk behavior of neuroblastoma.J Pediatr Surg,2001,3(12):1785-1791.
18 林茂芳,孟小莉,蔡真,等.抗凋亡基因survivin在急性白血病细胞表达及其临床意义.中华血液学杂志,2002,23(5):251-253.
19 常青,秦仁义,裘法祖.Survivin在胆管癌中的表达.中华实验外科杂志,2003,20(5):398-399.
20 王贵红,夏荣龙,郑兰东.结肠癌组织survivin表达的意义.第四军医大学学报,2006,27(2):170-171.
21 高红,王常林,蔡炜嵩,等.Survivin Caspase23 mRNA在肾母细胞瘤中的表达及意义.中国当代儿科杂志,2004,6(5):381-384.
22 Nesbit M.Abrogation of tumor vasculature using gene therapy.Cancer Metastasis Rev,2000,19(1-2):45-49.
23 O'Connor DS,Schechner JS,Adida C,et al.Control of apoptosis during angiogenesis by survivin expression in endothelial cells.Am J Pathol,2000,156(2):393-398.
24 Tran J,Rak J,Sheehan C,et al.Marked induction of IAP famly antiapoptotic proteins survivin and XIAP by VEGF in vacular endothelial cells.Biochem Biophys Res Commum,1999,264(3):781-788.
25 Tran J,Master Z,Yu JL,et al.A role for Survivin in chemoresistance of endothelial cells mediated by VEGF.Proc Nat Acad Sci USA,2002,99(7):4349-4354.
26 Gray MJ,Zhang J,Ellis LM,et al.HIF-lalpha,STAT3,CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas.Oncogene,2005,24(19):3110-3120.
27 Xu Q,Briggs J,Park S,et a1.Targeting Star3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways.Oncogene,2005,24(36):5552-5560.
28 Kirkali Z,Tuzel E,MunganMU.Recent advance in kidney cancer and metastatic disease.BJU Int,2001,88(8):818-824.
29 Dankbar B,Padro T,Leo R,et al.Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma.Blood,2000,95(8):2630-2636.
30 张谦,单岩,陈清江.血管内皮生长因子在预测肾母细胞瘤转移中的作用.中华儿科杂志,2005,43(3):213-214.
31 Semenza GL,Wang GL.A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation.Mol Cell Biol,1992,12(12):5447-5454.
32 Huang LE,Gu J,Schau M,et al.Regulation of hypoxia-inducible factor lalpha is mediated by an 02-dependent degradation domain via the ubiquitin-proteasome pathway.Proc Natl Acad Sci U S A,1998,95(14):7987-7992.
33 Semenza GL.HIF-1 and tumor progression:pathophysiology and therapeutics.Trends Mol Med,2002,8(4 Suppl):S62-67.
34 Carmeliet P, Dor Y, Herbert JM, et al. Role of HIF-1 α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature, 1998, 394 (6692): 485-490.
35 Talks KL, Turley H, Gatter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1 α and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Am J Pathol, 2000, 157(2) :411-421.
36 Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an oncogene. Cell, 1999, 98(3): 295-303.
37 Ivanov VN, Bhoumik A, Krasilnikov M, et al. Cooperation between STAT3 and c-jun suppresses Fas transcription. Mol Cell, 2001, 7(3):517-528.
38 Bromberg J. Stat proteins and oncogenesis. J Clin Invest, 2002 ,109(9): 1139-1142.
1.Hill DA,Shear TD,Liu T,et al.Clinical and biologic signifance of nuclear unreast in Wilm's tumor.Cancer,2003,97(9):2318-2326.
2.Baish JW,Jain RK.Fractals and cancer.Cancer Res,2000,60(5):3683-3684.
3.Kirkali Z,Tuzel E,Mungan MU.Recent advance in kidney cancer and metastatic disease.BJU Int,2001,88(8):818-824.
4.Elizabeth AB,Wei D,Max RL,et al.Neurolastoma,apoptosis,and growth factors.The Am Surg,2003,69(1):28-31.
5.Kraizer Y,Mawasi N,Seagal J,et al.Vascular endothelial growth factor and angiopoietin inliver regeneration.Biochem Biophys Res Commun,2001,287(1):209-215.
6.Fong GH,Rossant J,Gertsenstein M,et al.Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium.Nature,1995,376(6355):66-70.
7.Carmeliet P,Jain R_K,et al.Angiogenesis in cancer and other diseases.Nature,2000,407(1):249-257.
8.Risau W.Mechanisms ofangiogenesis.Nature,1997,386(6626):671-674.
9.Partanen J,Armstrong E,Makela TP,et al.A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains.Mol Cell BIOL,1992,4(10):1698-1707.
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