【作者】 高坤；

【导师】 孙保国；

【作者基本信息】 郑州大学 ， 骨外科， 2003， 硕士

【摘要】 骨肉瘤(Osteosarcoma)是最常见的骨恶性肿瘤，占骨原发恶性肿瘤的20％，其生物学行为多呈高度恶性，早期即易发生血道转移。虽然大剂量化疗的临床应用使骨肉瘤的5年生存率达80％，但仍有相当病人死于肺转移。生长因子通过与特异性受体结合，激活受体后信号途径，对调节细胞的增殖、存活有重要作用。但大量产生正性生长因子或其受体可通过自(旁)分泌机制使细胞增殖失控，在多种肿瘤的发生中起重要作用。多数生长因子在细胞内的第二信号均为3，4，5-三磷酸磷脂酰肌醇(phosphatidylinositol3，4，5-triphosphate，PIP3)，PIP3可与其它激酶结合促使细胞进入分裂周期。抑癌基因与细胞骨架蛋白tensin同源的在肿瘤的10号染色体有缺失的磷脂酶(phosphatase and tensin homolog deleted on chromosome ten,PTEN)是一种蛋白酪氨酸激酶，可使PIP3去磷酸化成PIP2，逆转PIP3的作用，抑制多数生长因子介导的细胞增殖。细胞增殖核抗原(Proliferation cell nuclear antigen，PCNA)是DNA聚合酶δ的辅助蛋白，可评价细胞的增殖状态。表皮生长因子受体(epidermal growth factor receptor，EGFR)和PTEN在骨肉瘤中的表达研究国内外少见报道。 目的：探讨PTEN、EGFR及PCNA在人骨肉瘤中表达的生物学意义及其互相关系，为阐明骨肉瘤的发病分子机制和基因治疗提供一定依据。 材料与方法：选取30例人骨肉瘤标本(所有标本均根据细胞和组织分化程度，将骨肉瘤分为三级：Ⅰ级7例，Ⅱ级14例，Ⅲ级9例)，以10例骨软骨瘤为对照，采用免疫组化S-P法，检测PTEN、EGFR及PCNA在人骨肉瘤中表达。以P＜0.05作为差异有显著性的检验标准。 结果： 1．PCNA蛋白表达：1．1 骨软骨瘤组PCNA染色阳性率为20.0％(2/10)，其中阴2003年郑州大学硕士研究生毕业论文人骨肉瘤中PTEN、EGFR的表达及其与细胞增殖的关系 性(一)8例，弱阳性(+)2例;骨肉瘤组PCNA染色阳性率为100.0%(3。/30)， 其中弱阳性(+)13例，强阳性(++)17例。两组染色阳性率和染色强度均 有显著性差异(尸<0 .05)。1.2骨肉瘤I级组PCNA染色弱阳性(+)6例， 强阳性(++)1例;n、班级组PCNA染色弱阳性(+)7例，强阳性(++) 16例。两组差异有显著性(尸<0.05)。2.PTEN蛋白表达:2.1骨软骨瘤组阳性表达率为90.0%(9/1的，其中阴性(一) 1例，弱阳性(+)2例，中度阳性(++)2例，强阳性(+十+)5例;骨肉 瘤组阳性表达率为70.0%(21/30)，其中阴性(一)9例，弱阳性(+)7例，中 度阳性(++)12例，强阳性(+++)2例。骨软骨瘤，骨肉瘤的染色阳性率 无显著性差异(P>0 .05)，染色强度有显著性差异(P<0 .05)。2.2骨肉瘤中 PTEN蛋白的表达2.2.1骨肉瘤I级组阳性表达率为85.7%(6/7)，其中阴性 (一)1例，弱阳性(+)3例，中度阳性(++)3例;n、m级组阳性表达率 为65.2%(15/23)，其中阴性(一)8例，弱阳性(+)4例，中度阳性(++)9 例，强阳性(+++)2例。n、nI级组与I级组染色阳性率和染色强度相比， 无显著性差异(尸>0 .05)。2.2.2骨肉瘤PCNA弱阳性组PTEN的阳性率为 76.9%(10/13)，其中阴性(一)3例，弱阳性(+)3例，中度阳性(++)6 例，强阳性(+++)1例;PCNA强阳性组PTEN的阳性率为64.7%(n/17)， 其中阴性(一)6例，弱阳性(+)4例，中度阳性(++)6例，强阳性(+++) 1例。PcNA弱阳性组与强阳性组PTEN染色阳性率与染色强度无显著性差 异(P>0 .05)。3.EGFR蛋白表达:3.1骨软骨瘤组无阳性表达(0/10)，骨肉瘤组阳性表达率 为50.0%(15/30).骨软骨瘤、骨肉瘤染色阳性率有显著性差异(尸<0 .05)。3.2 骨肉瘤中EGFR蛋白的表达3.2.1骨肉瘤I级组阳性表达率为14.3%(1/7)， 11、m级组阳性表达率为60.9%(14/23).n、m级组与I级组染色阳性率相 比差异无显著性差异(P>0.05)。3.2.2骨肉瘤PCNA弱阳性组与强阳性组 EGFR阳性率分别为23.1%(3/13)和70.6%(12/17)，两组比较差异有显著性 (P<0 .05)。结论:1、PCNA在骨肉瘤与骨软骨瘤间，骨肉瘤各级间表达有显著性差异(P<0 .05)，提2003年郑州大学硕士研究生毕业论文人骨肉瘤中PTEN、EGFR的表达及其与细胞增殖的关系 示其可作为骨肉瘤定性、分级的指标;2、PTEN染色强弱在骨软骨瘤与骨肉瘤间存在显著性差异(尸<0.05)，提示其在 骨肉瘤的发生中起到重要的作用;P花N在骨肉瘤各级间，PCNA弱阳性组、 强阳性组间表达无显著性差异(尸>0.05)，提示其不可作为骨肉瘤的分级、恶 性增殖指标;3、EGFR在骨肉瘤与骨软骨瘤间，PCNA弱阳性组、强阳性组间表达有显著性 差异(尸<0.05)，提示其在骨肉瘤的发生及恶性增殖中起到一定的作用;EGFR 在骨肉瘤各级间表达无显著性差异(P>0.05)，提示其不可作为骨肉瘤的分级 指标。更多还原

【Abstract】 Osteosarcoma (OS) is the most common primary malignant tumor of bone, comprising 20% of all such malignancies. Its biological behavior is always highly malignant. Its hematogenous metastasis occurs more frequently and early. Though clinic apply of high-dose chemotherapy makes the 5-year survival rate of OS climb to 80%, a significant percentage of patients die from lung metastasis. Growth factors (GFs) activate post-receptor way by combining special receptors, which is important to cells survival. The second messenger of most GFs is phosphatidylinositol 3, 4, 5-triphosphate (PIPS). PIPS can promote proliferation of cells by combining other kinases. Tumor suppressor phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a kind of protein tyrosine phosphatase. It can dephosphate PIPS into PIP2, reverse the use of PIPS and refrain cells proliferation induced by most GFs. Proliferation cell nuclear antigen (PCNA) is affiliated protein of DNA ploymerase 6 . It can be used to evaluate the state of cell proliferation. So far, there are few reports on the expression of PTEN, epidermal growth factor receptor (EGFR) gene in human OS.Objective: In order to evaluate the role of PCNA, PTEN and EGFR in OS as well as their interaction, their expression in OS is investigated. It is expected that the study will help us to elucidate the molecular mechanism of OS and provide theoretical foundation for genie therapy.Materials and methods: Thirty specimens of human OS tissues (OST) were obtained. They were classified as three grades based on the grade of differentiation (I grade 7 cases, II grade 14 cases, III grade 9 cases). The control groups were 10 casesosteochondroma tissues (OCT). Immunohistochemical S-P method was used to exam the expression of PCNA, PTEN and EGFR in OS. There was a significant difference when P<0.05. Results:1. Expression of PCNA protein: 1.1 The positive immunostaining rate of PCNA in OCT was 20.0%(2/10), in which negative and strong stain were 8, 2, respectively; The positive immunostaining rate in OST was 100.0 %( 30/30), in which negative and strong stain were 13, 17, respectively. Comparing the staining rate and intensity of OCT and OST respectively, there were significant differences (P<0.05). 1.2 In grade I of OST, PCNA weak and strong positive staining were 6, 1, respectively. In grade II+III of OST, PCNA weak and strong positive staining were 7, 16, respectively. Comparing the staining intensity, there was significant difference between grade I and grade II+III of OST. (P<0.05).2. Expression of PTEN protein: 2.1 In OCT, the positive immunostaining rate of PTEN was 90.0%(9/10), in which negative, weak, moderate and strong immunostaining were 1, 2, 2, 5, respectively. In OST, the positive immunostaining rate of PTEN was 70.0 %( 21/30), in which negative, weak, moderate and strong immunostaining were 9, 7, 12, 2, respectively. Comparing the staining rate, there was no significant difference (P>0.05). Comparing the staining intensity, there was significant difference (P<0.05). 2.2 Expression of PTEN protein in OST: 2.2.1 In grade I of OST, the positive immunostaining rate of PTEN was 85.7%(6/7), in which negative, weak and moderate immunostaining were 1, 3, 3, respectively. In grade II+III of OST, the positive immunostaining rate of PTEN was 65.2%(15/23), in which negative, weak, moderate and strong immunostaining were 9, 4, 9, 2, respectively. Comparing the staining rate and intensity, there were no significant differences (P>0.05). 2.2.2 In PCNA weak positive staining group, the positive immunostaining rate of PTEN was 76.9%(10/13), in which negative, weak, moderate and strong immunostaining were 3, 3, 6, 1, respectively. In PCNA strong positive staining group, the positive immunostaining rate of PTEN was 64.7 %(11/17), in which negative, weak, moderate and strong immunostainingwere 6, 4, 6, 1, respectively. Comparing the staining rate and intensity of PTEN, there were no significant differences (P>0.05).3. Expression of EGFR prot更多还原