细胞周期蛋白D1和细胞周期蛋白依赖激酶4在宫颈鳞癌中的表达

                作者：姬宏宇　成日青　薄爱华 舒丽莎

  【摘要】目的：探讨Cyclin D1和CDK4在正常宫颈上皮、CIN和宫颈鳞癌中表达状况。方法：收集子宫颈活检及手术标本118例，其中正常鳞状上皮14例，CIN25例,宫颈鳞癌79例。采用SP免疫组织化学方法，检测CyclinD1和CDK4蛋白的表达情况。结果：Cyclin D1和CDK4在正常宫颈上皮、CIN和宫颈鳞癌的中表达水平存在显著性差异（P<005），在不同分化程度的宫颈鳞癌间无显著性差异（P>005）。结论：在宫颈鳞癌发生过程中伴有Cyclin D1和CDK4 表达水平的增高，检测其含量改变有助于宫颈鳞癌诊断。

    【关键词】宫颈鳞癌，细胞周期蛋白D1；细胞周期蛋白依赖激酶4

    1MATERIALS AND METHODS

    11Samples collection

    Specimens were taken from uterine cervix of 118 cases The surgical material was from hysterectomy or biopsy All samples were confirmed by pathological diagnosis There were normal squamous epithelium in 14 cases and CIN in 25 cases Normal cervices from hysterectomy specimens were removed for leiomyomas served as controls 79 cases were cervical squamous cell carcinoma, among which 8 cases were well differentiated squamous cell carcinoma, 58 cases were moderately differentiated squamous carcinoma and poorly differentiated squamous cell carcinoma were 13 cases The above mentioned specimens were fixed in 10% formalin, embedded in paraffin and sliced into the thickness of 5μm The cellular protein expressions of CyclinD1 and CDK4 were studied with streptavidin peroxidase immunohistochemical method which has high sensitivity and strong specificity

    12Immunohistochemistry reagents

    2006年2月Ji Hongyu,et al:Expression of CyclinD1 and CDK4 Protein in Cervical Squamous Cell Carcinoma第1期2006年2月河北北方学院学报（医学版）第1期Mouse monoclonal AntiCyclinD1 protein, Mouse monoclonal Anti CDK4 protein, immunohistochemical staining SP kit and DAB were purchased from Maixin LTD

    13Methods

    Immunohistochemical SP staining method was used in the experiment The conventional staining procedures were carried out The main procedures were as follows:

    The tissue sections were routinely dewaxed and hydrated, then treated with 3 % peroxide for 10 minutes Antigen restoration was carried out by heating in citrate buffer, blocked with normal goat serum, incubated overnight with CyclinD1 monoclonal antibody or CDK4 monoclonal antibody at 4℃, washed three times with PBS, treated with antibody II for 30 minutes at 37℃ and then with antibody III for 30 minutes at 37℃ Color was displayed with DAB Negative control was designed with PBS instead of antibody I The known positive tissue sections were used as positive control

    14Statistic analysis

    SPSS100 software was used for statistical analysis

    2RESULTS

    Under lightmicroscope the CyclinD1 and CDK4 immunoreactive products showed as granules with brown color Granules were located in nucleis in all the CDK4 positive cells and most CyclinD1 positive cells, while it could be found in cytoplasm of a few cyclinD1 positive cell Statistical analysis was performed using SPSS for windows(version 10) A probability of <005 was considered satistically significant Detailed expressions of CyclinD1 and CDK4 in tissues of normal cervical squamous epithelium, cervical intraepithelial neoplasia and cervical squamous cell carcinoma are shown in Table 1,2 and 3

    The mean age of all 79 cases with cervical squamous cell carcinoma patients is 5087 The median age is 5000 The positive rates of CyclinD1 and CDK4 are similar in younger or elder patients (P>005)

    Table 1The expressions of CyclinD1, CDK4 in normal squamous epithelium, CIN and CSCC

    Pathologictypesn CyclinD1 （+） n% CDK4 （+）n%Normal

    epithelia141714%1714%CIN251248%114400%CSCC79698734%637976%χ2〖4〗χ2=44488〖6〗χ2=32306P〖4〗P=0000〖6〗P=0000Table 2The Relation between CyclinD1 or CDK4 expression and different degree CSCC

    differentdegreenCyclinD1 （+）n % CDK4 （+）n%  Well differentiated88100%78750%Moderately

    differentiated58508620%447886%Poorly

    differentiated13118462%129231%Total79698734%637976%χ2〖4〗χ2=1314〖6〗χ2=2110P〖4〗P=0518〖6〗P=0348Table 3correlation between the expression of CyclinD1 and that of CDK4 in CSCC

    CyclinD1CDK4+-n+61869-2810n631679Significant coexpression was seen for CyclinD1 and CDK4 r=056596,P=00001

    3DISCUSSION

    Cell growth is directed by complex molecular cascades that involve a number of genes, all acting in concert to achieve the biochemical and structural changes required for correct cell duplication In particular, cell cycle progression is controlled by the products of timely expressed genes[2] CyclinD1 gene is located in the 11q13 region CyclinD1 protein consists of 295 amino acids, which express in early G1 phase and go to its peak in middle G1 phase and associated with cyclin dependent kinase 4(CDK4) The major targets of Cyclin D1CDK4 complexes are the retinoblastoma family of protein Rb[3] Phosphorylation of Rb in midG1 leads to the release of active forms of the E2F family of transcription factors Free E2F mediates transcription of E2fdependent genes, including DNA polymerase, thymidine kinase[4] In normal human tissues, the expression of CyclinD1 protein was rather low and negative by immunohistochemistry[5] The expression of cell cycle regulators could be used as prognostic markers in some malignant tumors[6] In our study, The positive rates of CyclinD1 protein were 714% (1/14)in normal squamous epithelium However, in 12/25 (48%) cases of CIN and in 69/79 (8734%) cases of CSCC,the expression of CyclinD1 was positive In the course of invasive cervical squamous carcinomas change (normal epithelium→ CIN epithelium→ change of carcinoma epithelium), The protein expression of CyclinD1 and CDK4 shows gradual uptrend The differentiation between three groups was statistically significant (P<005) The over expression of CyclinD1 and CDK4 are correlation (P<00001, r=056596), which are accompanying factor in cervical squamous cell carcinomaThe detection of CyclinD1 and CDK4 protein by immunohistochemistry is helpful in the diagnosis of CIN or early stage CSCC Some studies showed that the positive expression of CyclinD1 was statistically associated with welldifferentiated tumors[7] In our study, the expression of CyclinD1 and CDK4 protein had no correlation with CSCCs pathologic differentiation and patients age (P>005)

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