摘要
目的:研究缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在甲状腺乳头状癌(papillary thyroid carcinoma,PTC)中的表达,测定肿瘤微血管密度(microvessel density,MVD),探讨三者的相互关系及其与肿瘤生物学特征和肿瘤血管生成的关系。
方法:
1免疫组织化学方法检测43例甲状腺乳头状癌、20例甲状腺腺瘤和20例甲状腺正常组织中HIF-1α、VEGF蛋白的表达,用CD34标记新生血管,计算微血管密度,并结合甲状腺乳头状癌的临床病理资料进行分析;
2蛋白印迹法测定10例甲状腺乳头状癌、10例甲状腺腺瘤和10例甲状腺癌旁正常组织中HIF-1α蛋白表达情况。
结果:
1 HIF-1α蛋白的表达:HIF-1α蛋白主要表达于肿瘤细胞的胞质,肿瘤组织坏死边缘区域多见,正常甲状腺组织中仅见少量阳性着色。甲状腺乳头状癌组、甲状腺腺瘤组和正常组织组中HIF-1α蛋白阳性表达率分别是67.4%、20.0%和20.0%,组间比较差异有统计学意义(P＜0.01);乳头状癌组与腺瘤组和正常组织组比较,差异均有统计学意义(P＜0.01)。
2 VEGF蛋白的表达:VEGF蛋白主要表达于肿瘤细胞胞质,某些血管内皮细胞也可见阳性表达,极少数正常腺体上偶见微弱的棕黄色胞浆着色。甲状腺乳头状癌组、甲状腺腺瘤组和正常组织组中VEGF蛋白阳性表达率分别为72.1%(31/43)、25.0%(5/20)和15.0%(3/20),组间比较差异有统计学意义(P＜0.01);乳头状癌组与腺瘤组和正常组织组比较,差异均有统计学意义(P＜0.01)。
3 CD34的表达:CD34在肿瘤和正常组织的间质中均可见表达,定位于血管内皮细胞的胞质,微血管形态不规则,有的新生血管已呈管腔样结构,有的只见内皮细胞呈线状或点状分布。MVD值在甲状腺乳头状癌为114.11±6.23,甲状腺腺瘤为84.47±6.99,正常组织为49.83±6.12,组间比较差异有统计学意义(P＜0.01),且两两比较差异均有统计学意义(P＜0.01)。
4 HIF-1α与PTC临床病理资料的关系
HIF-1α蛋白表达与甲状腺乳头状癌患者性别、年龄、肿瘤大小、远处转移无关(P＞0.05),与淋巴结转移和UICC定义的肿瘤分期有关(P＜0.05)。存在淋巴结转移的甲状腺乳头状癌患者HIF-1α蛋白表达率(80.0%)明显高于无转移者(50.0%)(P＜0.05);HIF-1α蛋白表达率随着UICC定义的肿瘤分期的递增而增加(Ⅰ期36.4%,Ⅱ期60.0%,Ⅲ期75.0%,Ⅳ期92.9%)(P＜0.05);并且晚期患者UICCⅢ、Ⅳ期)HIF-1α蛋白表达率(86.4%)高于早期患者(Ⅰ、Ⅱ期)(47.6%)(P＜0.01)。
5 VEGF与PTC临床病理资料的关系
VEGF蛋白表达与甲状腺乳头状癌患者性别、年龄、肿瘤大小、远处转移无关(P＞0.05),与淋巴结转移和UICC定义的肿瘤分期有关(P＜0.05)。伴有淋巴结转移的PTC中VEGF蛋白表达率(84.0%)明显高于无转移者(55.6%)(P＜0.05);VEGF蛋白表达率随着UICC定义的肿瘤分期的递增而增加(Ⅰ期36.4%,Ⅱ期70.0%,Ⅲ期75.0%,Ⅳ期100.0%)(P＜0.01);其中Ⅳ期VEGF阳性表达率明显高于其他三期(P＜0.01);并且晚期患者(UICCⅢ、Ⅳ期)VEGF蛋白表达率(90.9%)高于早期患者(Ⅰ、Ⅱ期)(52.4%)(P＜0.01)。
6甲状腺乳头状癌中HIF-1α和VEGF的表达呈正相关关系(r_s=0.567,P＜0.01);HIF-1α阳性表达组的MVD值(117.59±5.72)高于阴性表达组(109.20±1.98)(P＜0.01);VEGF阳性表达组的MVD值(116.98±5.98)亦高于阴性表达组(109.31±2.21)(P＜0.01);HIF-1α和VEGF共同表达阳性组的MVD值(122.17±4.97)高于共同表达阴性组(108.21±0.98)(P＜0.01),随HIF-1α、VEGF表达强度的增加,MVD值逐渐增大。
7蛋白印迹法结果
HIF-1α蛋白在甲状腺乳头状癌中的表达明显高于腺瘤和癌旁正常组织组,各组的相对OD值为:乳头状癌组2.398±0.041;腺瘤组0.869±0.032;癌旁正常组织组0.199±0.027,组间比较差异有统计学意义(P＜0.01),两两比较差异均有统计学意义(P＜0.01),检测结果与免疫组织化学法结果相符。
结论:
1甲状腺乳头状癌与甲状腺腺瘤、正常组织相比,HIF-1α和VEGF蛋白表达增多,MVD值升高。提示HIF-1α、VEGF的表达和微血管的增殖与甲状腺乳头状癌的发生、发展紧密相关。
2 HIF-1α与VEGF蛋白表达均与甲状腺乳头状癌淋巴结转移和UICC定义的肿瘤分期密切相关,提示二者参与了甲状腺乳头状癌的恶性增殖和侵袭转移,HIF-1α和VEGF对临床判断甲状腺乳头状癌预后有一定价值。
3甲状腺乳头状癌中HIF-1α和VEGF蛋白呈正相关关系,并且与MVD值关系紧密,表明HIF-1α在缺氧时可能通过上调VEGF的表达诱导甲状腺乳头状癌微血管的生成,通过此途径为癌细胞生长及浸润转移提供物质基础。
Objective:By testing the expression of hypoxia-inducible factor-1α(HIF-1α), vascular endothelial growth factor(VEGF) proteins and assessing microvessel density(MVD) in papillary thyroid carcinoma(PTC),thyroid adenoma and normal thyroid tissue,we tried to reveal the relationship between the former factors and tumor biological characteristics,tumor angiogenesis.
Method:
1 We investigated the distribution and expression of HIF-1α,VEGF and assessed MVD in 43 PTCs,20 thyroid adenomas and 20 normal thyroid tissues by immunohistochemistry.And compared these former factors with clinical pathological datas of PTC.
2 Western blot was used to detect the expression of HIF-1αin 10 PTCs,10 thyroid adenomas and 10 adjacent tissues of thyroid tumor.
Result:
1 HIF-1αprotein was mainly expressed in the cytoplasm of tumor cells and a small quantity of normal tissues.Statistical analysis revealed that HIF-1α-positive stained rate was significant higher in PTCs(67.4%) than that in thyroid adenomas(20.0%) and normal thyroid tissues(20.0%)(P＜0.01). There were significant differences when thyroid adenoma group and normal thyroid tissue group compared with PTC group(P＜0.01).
2 VEGF protein was mainly expressed in the cytoplasm of tumor cells,some of the vascular endothelial cell could also be detected.Statistical analysis revealed that VEGF-positive stained rate was significant higher in PTCs (72.1%) than that in thyroid adenomas(25.0%) and normal thyroid tissues (15.0%)(P＜0.01).There were significant differences when thyroid adenoma group and normal thyroid tissue group compared with PTC group(P＜0.01).
3 CD34 protein was expressed in the cytoplasm of vascular endothelial cell that were in the mesenchyme of both tumor cells and normal cells.Statistical analysis revealed that MVD was significant higher in PTCs(114.11±6.23) than that in thyroid adenomas(84.47±6.99) and normal thyroid tissues (49.83±6.12)(P＜0.01).There were significant differences when the groups compared with each other(P＜0.01).
4 Statistical analysis revealed that the expression of HIF-1αprotein in PTCs had no significant relation with sex,age,tumor size or metastasis(P＞0.05), but it was correlated with lymph node metastasize,UICC stage(P＜0.05). Expression rate of HIF-1αprotein with lymph node metastasize(80.0%) was significant higher than that in without(50.0%)(P＜0.05).Accompany with the step up of UICC stage,the expression rates of HIF-1αprotein were rising(Ⅰ36.4%,Ⅱ60.0%,Ⅲ75.0%,Ⅳ92.9%)(P＜0.05).HIF-1α-positive stained rate was significant higher in advanced stage(Ⅲ,Ⅳ)(86.4%) than that in early stage(Ⅰ,Ⅱ)(47.6%)(P＜0.01).
5 Statistical analysis revealed that the expression of VEGF protein in PTC had no significant relation with sex,age,tumor size or metastasis(P＞0.05),but it was correlated with lymph node metastasize,UICC stage(P＜0.05).
Expression rate of VEGF protein with lymph node metastasize(84.0%) was significant higher than that in without(55.6%)(P＜0.05).Accompany with the step up of UICC stage,the expression rates of HIF-1αprotein were rising(Ⅰ36.4%,Ⅱ70.0%,Ⅲ75.0%,Ⅳ100.0%)(P＜0.01).There were significant differences when theⅣgroup compared with the others(P＜0.01).
VEGF-positive stained rate was significant higher in advanced stage(Ⅲ,Ⅳ) (90.9%) than that in early stage(Ⅰ,Ⅱ)(52.4%)(P＜0.01).
6 There was a positive relationship between HIF-1α-positive expression and VEGF-positive expression in PTC(r_s=0.567,P＜0.01).MVD in HIF-1α-positive stained PTC group(117.59±5.72) was significant higher in HIF-1α-negative stained group(109.20±1.98)(P＜0.01).MVD in VEGF-positive stained PTC group(116.98±5.98) was also significant higher in VEGF-negative stained group(109.31±2.21)(P＜0.01).In PTC with both HIF-1α-positive and VEGF-positive stained,the value of MVD was 122.17±4.97,but in both HIF-1α-negative and VEGF-negative stained tissues, the value was 108.21±0.98.The value of MVD was higher in the former than that in the latter(P＜0.01),accompany with the intensive expression of HIF-1αand VEGF protein.MVD was rising.
7 Western blot analysis of HIF-1αprotein expression in PTC was significant increased(2.398±0.041)than that in thyroid adenoma(0.869±0.032) and thyroid normal tissue(0.199±0.027)(P＜0.01).There were significant differences when the groups compared with each other(P＜0.01).
Conclusion:
1 Increased expression of HIF-1α,VEGF protein and MVD in PTC indicated that HIF-1αand VEGF were closely correlated with carcinogenesis and metastasis of PTC.
2 The expression rates of HIF-1αand VEGF protein were correlated with lymph node metastasize and UICC stage which manifest that both of them may have some effects in tumor malignancy and invasion.This may become an outcome indicator for PTC.
3 The close relationship of HIF-1α,VEGF protein and MVD in PTC indicated that HIF-1αmay mediated angiogenesis by activating transcription of VEGF, newly growth microvascular may have provided an essensial enviroment for tumor cell growth,malignancy and invasion.
引文
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