P_（16）基因、血管内皮生长因子和微血管密度与乳腺癌生物学行为的关系
摘要
目的：为研究多肿瘤抑制基因（p16）、血管内皮生长因子（VEGF）和肿瘤毛细血管密度（MVD）与乳腺癌生物学行为的关系。采用免疫组化染色技术对87例乳腺癌原发灶组织进行VEGF、p16蛋白及微血管密度（MVD）进行检测。研究VEGF、P16与乳腺癌间质微血管密度（MVD）及临床病理学参数之间的关系，探讨其在乳腺癌发生、发展以及血管生成中的作用。
材料与方法：选自收集吉林大学第一、二、三医院普外科2000～2003年间乳腺癌根治术或简化根治术切除标本共87例。患者术前未经抗癌治疗，经多位病理医师确诊为乳腺癌。组织类型按WHO标准分为浸润性导管癌（54例）、浸润性小叶癌（33例）,后者包括髓样癌、粘液腺癌、腺管样癌、分泌型癌。组织学分级参照Bloomand Richardson[1]标准。患者均为女性，年龄32-68岁，中位年龄49.8岁。TNM分期（1988年乳腺癌TNM分期标准）：Ⅰ期+Ⅱ期56例，Ⅲ期+Ⅳ期31例。肿瘤直径1-8cm，淋巴结有转移者46例，无转移者41例。全部标本按标准取材及处理后，备常规HE染色及免疫组织化学染色。免疫组织化学染色试：剂链霉菌抗生物素－过氧化酶免疫组化染色超敏试剂盒（Ultrasensitive S－P kit），CD34单克隆抗体，VEGF单克隆抗体及P16单克隆抗体均购自福州迈新生物技术开发公司，所有抗体均为既用型；抗原修复缓冲液（0.01M枸橼酸盐缓冲液PH值6.0）；工作液。免疫组织化学染色采用S－P法。免疫组织化学染色过程中，以PBS代替一抗作阴性对照，用已知CD34阳性、VEGF阳性和P16阳性
的乳腺癌组织切片作阳性对照。结果判定：分别以肿瘤细胞及血管内皮细胞胞浆、胞膜或胞核出现棕黄色颗粒，且其着色强度高于背景者判定为阳性。形态定量方法：1、微血管定量方法：参照Weidner报道的方法，在100倍光镜下挑选微血管分布最密区域，200倍视野下计数5个不重复视野中被CD34染成棕色的血管数目，取其均值作为微血管密度（Microvascular Density，MVD）。单位为个/200×，结果用均数±标准差（±s）表示。微血管的判定不以是否形成管腔或管腔内有无红细胞为记数单位，与邻近的微血管明显分离的染色阳性的单个内皮细胞或内皮细胞簇均作为一个血管计数，凡管腔大于8个红细胞，带较厚肌层的血管均不计数。2、VEGF计量方法：VEGF表达的判定以染色强度和阳性细胞百分率的得分之和进行评估。（a）染色强度：阴性为0，弱阳性为1，阳性为2，强阳性为3；（b）阳性细胞百分率：无阳性细胞为0，<25%为1，26-50%为2，>0%为3，a+b之和最高为6，0-2视为阴性，3-6视为阳性。3，P16计量方法：每张切片选取5个高倍镜视野，记数阳性细胞数后取其平均值。平均每视野阳性细胞数百分率大于10%为阳性，小于或等于10%为阴性。统计学处理：根据数据类型分别采用t检验、χ2检验、方差分析。
结果：1、87例乳腺癌中P16阳性表达率为55.17%（48/87），主要表达于肿瘤细胞的胞核，呈棕黄色颗粒，部分胞浆也有表达。P16表达与年龄、肿瘤大小、组织学分型、分化程度均无关（P>0.05），而与肿瘤部位、淋巴结转移和临床分期密切相关（P<0.05）。2、CD34在乳腺癌间质血管内皮细胞胞浆着色，乳腺癌中血管丰富，微血管走向紊乱，分布不均匀。微血管形态不规
则，管壁薄，仅有一层连续或不连续的内皮细胞，管腔大小差异也很大，部分血管无明显管腔。新生血管的内皮细胞形态不完全相同，有的内皮细胞增生肥大，有的内皮细胞变长变扁。87例乳腺癌MVD均值为39.81±15.12/200×，MVD值与临床分期、淋巴结转移密切相关（P<0.05），而与患者性别、年龄、肿瘤大小、组织学类型及分化程度无关（P>0.05）。3、在87例乳腺癌中，VEGF阳性表达率为64.37%（56/87），其阳性部位主要为肿瘤细胞胞浆或胞膜，癌巢边缘细胞染色强度较中心部强，间质炎细胞及邻近微血管内皮细胞也有不同程度的表达。在导管癌呈弥散分布，在腺癌则呈腺泡状分布，在癌细胞组成的岛状或腺状结构的基板上也存在强VEGF表达。VEGF表达与乳腺癌淋巴结转移、临床分期密切相关（P<0.05），而与患者年龄、肿瘤大小、组织学类型无关（P>.05）。4、乳腺癌组织中VEGF表达阳性组MVD值明显高于阴性组，两者之间差异有显著性（P<0.05），说明VEGF表达与间质微血管密度之间存在一致性。P16表达阳性组MVD值低于阴性组，但差异无统计学意义（P>0.05）。5、56例VEGF阳性病例中有32例P16阳性，31例VEGF阴性者中21例P16阳性，P16与VEGF表达呈负相关（P<0.05），即随着VEGF表达的上调，P16表达下调。
结论：1、乳腺癌P16主要表达于肿瘤细胞核内，其表达与乳腺癌淋巴结转移、肿瘤细胞及临床分期有关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。2、乳腺癌间质微血管丰富，MVD值与淋巴结转移、临床分期密切相关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。3、乳腺癌VEGF主要表达于
肿瘤细胞胞浆或胞膜，间质炎细胞及微血管内皮细胞也见少量表达；VEGF表达与乳腺癌淋巴结转移、临床分期有关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。4、乳腺癌VEGF表达与MVD值密切相关，P16表达与MVD值无关；乳腺癌P16与VEGF表达呈负相关。
Purpose: To study the association of P16, VEGF and MVD with the biological behavior of human breast cancers. 87cases of breast cancers were examined in terms of the original tissues of VEGF, P16 protein, and MVD by adopting the method of immunohistochemistry and pattern quantitative analysis in order to make clear the relationship between them and the clinical pathology parameters, and explore the function they play in breast cancer occurrence, development and tumor vascular forming.
Subjects and instruments: This was conducted in the General Surgery Department No 1, No2 and No3 Hospital of Jilin University. 87 removed samples of breast cancers from the year 2000 to 2003 were used. The patients were not treated in any way before the surgery and were diagnosed by several surgeons as flung cancers. According to the WHO, the tissues are classified into soked tube cancer (54cases), invasive lobular carcinoma (33cases), the latter one including marrowing carcinoma, pituita adenocarcinoma, glandulose ductal carcinoma and secrete cancer. The levels of the tissues are determined regarding the standard of Bloomand Richardson (1). All the patients are females, from 32 to 68, the average age is 49.8. TNM stages (the standard used in 1988): stage I + stage II, 56 cases; stage III + stage IV, 31 cases. The cross of the tumor: 1-8 cm, among whom 46 cases with metastasis in the lymph node, and 41 without. After all the samples are
elected and standardized, they are ready for the normal HE coloring and immunohistochemistry coloring. Immunohistochemistry coloring agents: Ultrasensirive S-P kit , single clone antibiotic CD34, VEGF and P16, all are instantly applied and all are purchased from Maixin Biological Technology Development Corporation, Fuzhou; Ag；liguid. S-P method is used in the process of immunohistochemistry coloring, and PBS is used instead of sample one to do the negative comparison, while the segment tissue of the breast cancer CD34 positive, VEGF positive, and P16 positive are used to do the positive comparison.
Results: Diagnose interior, exterior and nuclear of the tumor cells in the vascular endothelial cells with particles of brown and their colors are deeper than the background as positive ones.
Method of quantity-fixing in terms of the shapes:
1. Method of capillary quantity-fixing: Referring to the method reported by Weidner, choose the areas with the most microvasculars with the help of 100 times microscope, count the number of the microvasculars colored into brown by CD34 in 5 different areas with 200 times perspective, taking the average number as the MVD. The unit is /200 x, the result is expressed by (±s). The formation of microvascular is not based on whether there are vasculars or not, or if there are red cells in the vasculars. The isolated positive single endothelial cells or cell clusters are considered as one vascular, and the blood vessels with more than 8 red cells and with thicker muscles
are not counted.
2. Method of VEGF-counting: Evaluated by the amount of density of the color and the percentage of the positive cells. (a) Density of the color: the negative one is 0, the weak positive is 1, the positive is 2 and the strong positive is 3. (b) Percentage of the positive cells: the non-positive cells are 0, the one < 25% is 1, those between 26-50% are 2, the ones >50% are 3. The biggest number of a+b is 6, 0-2 are considered as negative and 3-6 positive.
3. Method of P16-counting: Choose 5 high-perspectives under the microscope for each section of the organic tissue, and take the average value after counting the number of the positive cells. Those more than 10% of the positive cells within each perspective are positive, and negative if they are less than 10%.
Method of statistics: Adopting the method of t-testing, x2 testing and analysis of variance respectively according to the types of the dada.
Results:
1. There are 55.17% (48-87) whose P16 is positive among the 87 breast cancer cases, the expression of P16 protein is main
材料与方法：选自收集吉林大学第一、二、三医院普外科2000～2003年间乳腺癌根治术或简化根治术切除标本共87例。患者术前未经抗癌治疗，经多位病理医师确诊为乳腺癌。组织类型按WHO标准分为浸润性导管癌（54例）、浸润性小叶癌（33例）,后者包括髓样癌、粘液腺癌、腺管样癌、分泌型癌。组织学分级参照Bloomand Richardson[1]标准。患者均为女性，年龄32-68岁，中位年龄49.8岁。TNM分期（1988年乳腺癌TNM分期标准）：Ⅰ期+Ⅱ期56例，Ⅲ期+Ⅳ期31例。肿瘤直径1-8cm，淋巴结有转移者46例，无转移者41例。全部标本按标准取材及处理后，备常规HE染色及免疫组织化学染色。免疫组织化学染色试：剂链霉菌抗生物素－过氧化酶免疫组化染色超敏试剂盒（Ultrasensitive S－P kit），CD34单克隆抗体，VEGF单克隆抗体及P16单克隆抗体均购自福州迈新生物技术开发公司，所有抗体均为既用型；抗原修复缓冲液（0.01M枸橼酸盐缓冲液PH值6.0）；工作液。免疫组织化学染色采用S－P法。免疫组织化学染色过程中，以PBS代替一抗作阴性对照，用已知CD34阳性、VEGF阳性和P16阳性
的乳腺癌组织切片作阳性对照。结果判定：分别以肿瘤细胞及血管内皮细胞胞浆、胞膜或胞核出现棕黄色颗粒，且其着色强度高于背景者判定为阳性。形态定量方法：1、微血管定量方法：参照Weidner报道的方法，在100倍光镜下挑选微血管分布最密区域，200倍视野下计数5个不重复视野中被CD34染成棕色的血管数目，取其均值作为微血管密度（Microvascular Density，MVD）。单位为个/200×，结果用均数±标准差（±s）表示。微血管的判定不以是否形成管腔或管腔内有无红细胞为记数单位，与邻近的微血管明显分离的染色阳性的单个内皮细胞或内皮细胞簇均作为一个血管计数，凡管腔大于8个红细胞，带较厚肌层的血管均不计数。2、VEGF计量方法：VEGF表达的判定以染色强度和阳性细胞百分率的得分之和进行评估。（a）染色强度：阴性为0，弱阳性为1，阳性为2，强阳性为3；（b）阳性细胞百分率：无阳性细胞为0，<25%为1，26-50%为2，>0%为3，a+b之和最高为6，0-2视为阴性，3-6视为阳性。3，P16计量方法：每张切片选取5个高倍镜视野，记数阳性细胞数后取其平均值。平均每视野阳性细胞数百分率大于10%为阳性，小于或等于10%为阴性。统计学处理：根据数据类型分别采用t检验、χ2检验、方差分析。
结果：1、87例乳腺癌中P16阳性表达率为55.17%（48/87），主要表达于肿瘤细胞的胞核，呈棕黄色颗粒，部分胞浆也有表达。P16表达与年龄、肿瘤大小、组织学分型、分化程度均无关（P>0.05），而与肿瘤部位、淋巴结转移和临床分期密切相关（P<0.05）。2、CD34在乳腺癌间质血管内皮细胞胞浆着色，乳腺癌中血管丰富，微血管走向紊乱，分布不均匀。微血管形态不规
则，管壁薄，仅有一层连续或不连续的内皮细胞，管腔大小差异也很大，部分血管无明显管腔。新生血管的内皮细胞形态不完全相同，有的内皮细胞增生肥大，有的内皮细胞变长变扁。87例乳腺癌MVD均值为39.81±15.12/200×，MVD值与临床分期、淋巴结转移密切相关（P<0.05），而与患者性别、年龄、肿瘤大小、组织学类型及分化程度无关（P>0.05）。3、在87例乳腺癌中，VEGF阳性表达率为64.37%（56/87），其阳性部位主要为肿瘤细胞胞浆或胞膜，癌巢边缘细胞染色强度较中心部强，间质炎细胞及邻近微血管内皮细胞也有不同程度的表达。在导管癌呈弥散分布，在腺癌则呈腺泡状分布，在癌细胞组成的岛状或腺状结构的基板上也存在强VEGF表达。VEGF表达与乳腺癌淋巴结转移、临床分期密切相关（P<0.05），而与患者年龄、肿瘤大小、组织学类型无关（P>.05）。4、乳腺癌组织中VEGF表达阳性组MVD值明显高于阴性组，两者之间差异有显著性（P<0.05），说明VEGF表达与间质微血管密度之间存在一致性。P16表达阳性组MVD值低于阴性组，但差异无统计学意义（P>0.05）。5、56例VEGF阳性病例中有32例P16阳性，31例VEGF阴性者中21例P16阳性，P16与VEGF表达呈负相关（P<0.05），即随着VEGF表达的上调，P16表达下调。
结论：1、乳腺癌P16主要表达于肿瘤细胞核内，其表达与乳腺癌淋巴结转移、肿瘤细胞及临床分期有关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。2、乳腺癌间质微血管丰富，MVD值与淋巴结转移、临床分期密切相关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。3、乳腺癌VEGF主要表达于
肿瘤细胞胞浆或胞膜，间质炎细胞及微血管内皮细胞也见少量表达；VEGF表达与乳腺癌淋巴结转移、临床分期有关，而与患者年龄、肿瘤大小、组织学类型、分化程度无关。4、乳腺癌VEGF表达与MVD值密切相关，P16表达与MVD值无关；乳腺癌P16与VEGF表达呈负相关。
Purpose: To study the association of P16, VEGF and MVD with the biological behavior of human breast cancers. 87cases of breast cancers were examined in terms of the original tissues of VEGF, P16 protein, and MVD by adopting the method of immunohistochemistry and pattern quantitative analysis in order to make clear the relationship between them and the clinical pathology parameters, and explore the function they play in breast cancer occurrence, development and tumor vascular forming.
Subjects and instruments: This was conducted in the General Surgery Department No 1, No2 and No3 Hospital of Jilin University. 87 removed samples of breast cancers from the year 2000 to 2003 were used. The patients were not treated in any way before the surgery and were diagnosed by several surgeons as flung cancers. According to the WHO, the tissues are classified into soked tube cancer (54cases), invasive lobular carcinoma (33cases), the latter one including marrowing carcinoma, pituita adenocarcinoma, glandulose ductal carcinoma and secrete cancer. The levels of the tissues are determined regarding the standard of Bloomand Richardson (1). All the patients are females, from 32 to 68, the average age is 49.8. TNM stages (the standard used in 1988): stage I + stage II, 56 cases; stage III + stage IV, 31 cases. The cross of the tumor: 1-8 cm, among whom 46 cases with metastasis in the lymph node, and 41 without. After all the samples are
elected and standardized, they are ready for the normal HE coloring and immunohistochemistry coloring. Immunohistochemistry coloring agents: Ultrasensirive S-P kit , single clone antibiotic CD34, VEGF and P16, all are instantly applied and all are purchased from Maixin Biological Technology Development Corporation, Fuzhou; Ag；liguid. S-P method is used in the process of immunohistochemistry coloring, and PBS is used instead of sample one to do the negative comparison, while the segment tissue of the breast cancer CD34 positive, VEGF positive, and P16 positive are used to do the positive comparison.
Results: Diagnose interior, exterior and nuclear of the tumor cells in the vascular endothelial cells with particles of brown and their colors are deeper than the background as positive ones.
Method of quantity-fixing in terms of the shapes:
1. Method of capillary quantity-fixing: Referring to the method reported by Weidner, choose the areas with the most microvasculars with the help of 100 times microscope, count the number of the microvasculars colored into brown by CD34 in 5 different areas with 200 times perspective, taking the average number as the MVD. The unit is /200 x, the result is expressed by (±s). The formation of microvascular is not based on whether there are vasculars or not, or if there are red cells in the vasculars. The isolated positive single endothelial cells or cell clusters are considered as one vascular, and the blood vessels with more than 8 red cells and with thicker muscles
are not counted.
2. Method of VEGF-counting: Evaluated by the amount of density of the color and the percentage of the positive cells. (a) Density of the color: the negative one is 0, the weak positive is 1, the positive is 2 and the strong positive is 3. (b) Percentage of the positive cells: the non-positive cells are 0, the one < 25% is 1, those between 26-50% are 2, the ones >50% are 3. The biggest number of a+b is 6, 0-2 are considered as negative and 3-6 positive.
3. Method of P16-counting: Choose 5 high-perspectives under the microscope for each section of the organic tissue, and take the average value after counting the number of the positive cells. Those more than 10% of the positive cells within each perspective are positive, and negative if they are less than 10%.
Method of statistics: Adopting the method of t-testing, x2 testing and analysis of variance respectively according to the types of the dada.
Results:
1. There are 55.17% (48-87) whose P16 is positive among the 87 breast cancer cases, the expression of P16 protein is main
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