【作者】 杨幸子；

【导师】 唐步坚； 李力；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 卵巢癌死亡率在妇科肿瘤中居首位。尽管肿瘤减灭术及以铂类为基础的联合化疗在卵巢癌的治疗中起到了积极的作用,但因缺乏有效的早期诊断方法,70%的患者初诊时已为晚期患者。手术及化疗后复发率高、易出现耐药,因而患者的5年生存率长期停留在30%左右。基质酶类通过降解细胞外基质(ECM)在肿瘤的侵袭和转移中起了关键性的作用。因此,本课题探讨MMP-9, Hpa, CL与卵巢恶性肿瘤发生、发展、转移及其预后价值,构建了三种酶类联合诊断卵巢癌浸润转移诊断模型并对模型进行验证。最后,对已发表的基质酶类组织含量表达相关研究内容的文献进行META分析,用循证医学的观点对有争议的问题进行分析,从中找到解决问题的线索,以期更好、更快地解决上述卵巢巢癌酶学诊断中的主要问题。血清MMP-9、Hpa、CL的检测对卵巢癌浸润转移判断的临床价值目的：探讨卵巢肿瘤患者外周血基质酶类的表达及与其临床病理的相关性。方法：采用ELISA酶联免疫吸附法检测了217例卵巢恶性肿瘤、101例卵巢良性肿瘤及101例正常对照的血清中几种基质酶含量的表达,将结果与临床及病理资料进行统计学分析。结果：恶性卵巢肿瘤患者血中基质酶含量的表达均显著高于良性组及正常对照组,差异有显著性(P<0.05)；在恶性卵巢肿瘤患者血清中,基质酶类的表达与病理类型无关,与临床分期、组织分化程度有关；CL表达与腹腔转移有关,Hpa表达与远处转移有关,MMP-9表达与腹腔转移有关。相关分析显示血清CL与CA125的相关系数为0.051,P=0.434。P>0.05,二者无明显相关性。血清Hpa与CA125的相关系数为0.183,P=0.005。两者呈正相关,即Hpa含量越高患者,CA125含量也越高；血清mmp9与CA125的相关系数为0.131,P=0.044,两者呈正相关,即MMP-9含量越高患者,CA125含量也越高。结论：基质酶类的表达与卵巢恶性肿瘤的发生,发展有关,并与恶性肿瘤的浸润转移有关。认为几种基质酶类有望在临床上成为判断侵袭、转移的指标之一。血清基质酶类含量联合检测判断卵巢恶性肿瘤浸润转移诊断模型的建立及验证目的：构建了三种酶类联合诊断卵巢癌浸润转移诊断模型并对模型进行验证。方法：采用SAS8.0软件包进行统计学分析。模型建立方法采用logistic回归。以250例血清样本三种酶类含量来建立回归模型,168例血清样本三种酶类含量用于验证所得到模型,并与CA125检测结果进行对比。结果：最终所建诊断模型为：Logit(P)=14.90-43.24xCL-33.12xhl-43.80xml+71.08x(CLxhl)+55.83x(CLxml)1.模型对于肿瘤性质判断的灵敏度为86.4%,特异度为82.1%,ROC曲线下面积为0.935,P值为0.000,灵敏度及特异度均高于任一种标志物单一检测,故本模型用于判断卵巢恶性肿瘤有显著意义。2.模型对于肿瘤浸润转移判断的灵敏度为70.4%,特异度为63.9%,ROC曲线下面积为0.732, P值为0.000,灵敏度及特异度均高,故本模型用于判断卵巢恶性肿瘤浸润转移有显著意义。结论：本研究所建模型具有较好的诊断效能,在判断肿瘤性质及进展程度时的灵敏度特异度均较高。本实验尽可能收集多的血清样本,但样本量毕竟有限。如能更加大量增加样本量,对模型进行调试及完善,使其更加接近肿瘤实际演进情况,则以诊断模型协助诊断恶性肿瘤有望成为新的辅助诊断模式。液态芯片检测血清MMP-9、Hpa、CL的临床验证试验目的：探讨液态芯片卵巢肿瘤患者外周血基质酶类的表达及与其临床病理的相关性。方法：运用流式荧光法检测了96例卵巢恶性肿瘤、79例卵巢良性肿瘤及55例正常对照的血清中几种基质酶含量的表达,将结果与临床及病理资料进行统计学分析。结果：1.恶性卵巢肿瘤患者血中基质酶含量的表达均显著高于良性组及正常对照组,差异有显著性(P<0.05)；在恶性卵巢肿瘤患者血清中,基质酶类的表达与病理类型无关。2.液态芯片法检测基质酶对于判断卵巢肿瘤性质的判断灵敏度分别为：CL84.3%,Hpa 72.3%,MMP-973.5%；特异度分别为：CL76.0%,Hpa 84.0%,MMP-988.0%。3.液态芯片法检测基质酶对于判断卵巢肿瘤转移的判断灵敏度分别为：CL867.9%,Hpa 62.5%,MMP-975.0%；特异度分别为：CL66.7%,Hpa 63.0%,MMP-963.0%。4.液态芯片法检测基质酶含量,判断卵巢恶性肿瘤性质的检验效能总体上优于ELISA法,尤其在判断肿瘤良恶性时的特异度明显高于ELISA法。5.液态芯片法与ELISA法检测卵巢恶性肿瘤转移与否的检验效能相差不多。结论：液态芯片是可同时、快速、准确检测多种肿瘤标志物的综合技术,其灵敏度和特异度均高,有望用于肿瘤标志物检测。基质酶类在卵巢恶性肿瘤组织中表达的Meta分析目的：评价基质酶类在卵巢组织中的含量对肿瘤性质的判断。方法：检索CBMdisc光盘数据库、EMBASE数据库MEDLINE数据库,确定纳入标准,筛选符合纳入标准的2000年1月至2009年12月公开国内外发表的文献16篇。通过各研究间的异质性分析及固定效应、随机效应两种模型的综合分析得出生存结果,统计学方法采用Cochrane协作网提供的软件包RevMan 4.2.10。结果：在MMP-9表达的研究中,纳入的7个研究,OR=11.43,OR95%C1为6.78,19.27,P<0.00001。基质金属蛋白酶在恶性及非恶性卵巢肿瘤组织中含量有统计学差异；研究间无异质性。2.在Hpa表达的研究中,纳入的6个研究,OR=8.62,OR95%CI为4.57,15.28,P<0.00001。乙酰肝素酶在恶性及非恶性卵巢肿瘤组织中含量有统计学差异；研究间无异质性。3.在CL表达的研究中,纳入的3个研究,OR=2.70,OR95%CI为0.26,27.97,P=0.41。组织蛋白酶在恶性及非恶性卵巢肿瘤组织中含量无统计学差异；研究间存在异质性。结论：Meta分析的结果说明恶性组基质蛋白酶及乙酰肝素酶的表达显著高于非恶性组,组织蛋白酶未见差异。由于目前的循证医学证据均是一些分散的小样本病例,基质酶类在恶性卵巢中的表达作用,尚需要大规模多中心的研究。根据现有研究结果,基质酶类有望成为最新的卵巢肿瘤标志物。更多还原

【Abstract】 Mortality of ovarian cancer is the highest in gynecological malignancies. Despite the platinum-based chemotherapy and cytoreductive surgery has played a positive role in ovarian cancer management,70% of patients still displays at advanced stage in first diagnosis with newly method due to lacking of effective early diagnosis. The relapse and resistance after surgery and chemotherapy cause the 5-year survival rate has still remained about 30%. Matrix metalloproteinases is known to play an important role in cancer cell invasion by mediating the degradation of extracellular matrix (ECM). In this study, firstly, we sought to explore the relationship between MMP-9, Hpa, CL expressions and clinic-pathologic pattern in ovarian cancer. Secondly, It is also to explore the role of MMP-9, Hpa,CL in invasion and metastatic of ovarian cancer. Thirdly, we also construct and validate an ovarian cancer metastasis diagnosis model which is comprehensive judged by MMP-9, Hpa and CL. Finally, we have made a META analysis of the stroma enzymes expression of relevant researches which have been published in the tissue, and analysis the issues in dispute with evidence-based medicine method.The Detection of MMP-9, Hpa and CL on Tumor Invasion and Metastasis and Its Clinical SignificanceObjective:To explore the matrix enzyme on peripheral blood expression and its relationship with the clinic and pathological characteristics in ovarian carcinomas.Methods:Serum levels of CA125, MMP-9,Hpa and CL in patients with 217 cases of ovarian cancers, 101cases of benign tumors and 101 healthy women as controls were determined by enzyme linked immunosorbent assay(ELISA). Statistical method was used to analyze the clinical and pathological characterization by the analysis of the result.Results:The serum levels of stroma enzymes in ovarian cancers was significantly higher than that in the benign tumors and the control group(p<0.05); The serum expression of stroma enzymes was not associated with pathology types, but associated with pathological types, clinical stage and degree of differentiation. The expression of CL, MMP-9 is associated with peritoneal metastasis. The expression of Hpa is associated with distant metastasis. Correlation analysis showed the relationship between the serum levels of the CL and CA125 is 0.051 and P=0.434, P>0.05, no significant correlation between these two stroma enzymes. Correlation analysis showed the relationship between the serum levels of the Hpa and CA125 is direct correlation, and r=0.183, P=0.005. Correlation analysis showed the relationship between the serum levels of the MMP-9 and CA125 is direct correlation, and r=0.131, P=0.044.Conclusion:The results showed that serum expression levels of These stroma enzymes was correlated with carcinogenesis and progress of ovarian cancer and was expected to become as indicators for judging invasion and metastasis.The Establish and Validation of the combined Mathematical Diagnosis Model of serum levels of Stroma enzymes of Ovarian CancerObjective:To establish the combined diagnosis model of serum levels of stroma enzymes of ovarian cancer and validate the model’s effectiveness.Methods:The statistical analysis method (SAS 8.0 software) is used to analyze the mathematical model. The establish method of the diagnosis model is logistic Regresion by using 250 matrix enzyme cases.168 cases was used to validate the model’s effectiveness. The comparison result was made between the new model and CA125.Results:The established mathematical diagnosis model is: Logit(P)=14.90-43.24xCL-33.12xhl-43.80xml+71.08x(CLxhl)+55.83x(CLxml)1. Model to judge the nature of the sensitivity of the tumor is 86.4%, specificity is 82.1%, ROC area under the curve is 0.935, P= 0.000. Sensitivity and specificity are higher than any other markers which were detected, so this model is used to determine ovarian cancer are significant.2. Model to judge tumor invasion and metastasis’s sensitivity is 70.4%, specificity is 63.9%, ROC area under the curve is 0.732, P= 0.000, sensitivity and specificity are high, so this model is used to judge the invasion and metastasis of ovarian cancer is significant.Conclusion:The model in this study has good diagnostic performance. The sensitivity and specificity of progressing in determining the nature and extent of tumor are high. In this study, many of serum samples collected as much as possible, but the sample size is limited. If a more substantial increase in sample size, debuging and improving the model so that it closer to the actual tumor evolution. It is expected to become as indicators for judging invasion and metastasis.Clinical Value of Liquid Biochip in detection MMP-9, Hpa and CL’s value of serumObjective:To explore the matrix enzyme on peripheral blood liquid biochip expression and its relationship with the clinic and pathological characteristics in ovarian carcinomas.Methods:Serum levels of CA125, MMP-9,Hpa and CL in patients with 96 cases of ovarian cancers,79cases of benign tumors and 55 healthy women as controls were determined by flow fluorescence assay. Statistical method was used to analyze the clinical and pathological characterization by the analysis of the result.Results:1. Patients with malignant ovarian tumor expression levels of matrix enzymes were significantly higher than benign group and control group, the difference is significant (P<0.05); in serum of patients with malignant ovarian tumors, the expression of matrix enzymes and pathological types has no relationship.2. liquid silicon substrate enzyme assay for determining the quality of judging the sensitivity of ovarian cancer are:CL 84.3%, Hpa 72.3%, MMP-973.5%; specificity, respectively:CL 76.0%, Hpa 84.0%, MMP-988.0%.3. Liquid silicon substrate enzyme assay for the judgement to judge the sensitivity of ovarian tumor metastasis are:CL 867.9%, Hpa 62.5%, MMP-975.0%; specificity, respectively:CL 66.7%, Hpa 63.0%, MMP-963.0%.4. Detection of liquid silicon substrate enzyme levels to determine the nature of the test performance of ovarian cancer is generally better than ELISA, especially in benign and malignant tumors when judging the specificity was significantly higher than ELISA.5. Liquid chip technology and the ELISA assay test ovarian cancer metastasis have almost the same performance.Conclusions:Liquid Biochip can be used to detect multi tumor marker, and can increase the diagnosis sensitivity.The Meta Analysis of Relationship Between Several Stroma enzymes and Invasion and Metastasis in Ovarian CancerObjective:To explore the relationship of the value of stroma enzymes in ovarian and the ovarian cancer.Methods:We have searched CBMdisc database, EMBASE database and MEDLINE database, and found 16 papers between January 2005 and December 2009 complied with the standard. The survival results have been obtained through the study of the heterogeneity and fixed effects and random effects models. Statistical methods have been used by utilizing RevMan 4.2.10 software.Results:1. MMP-9 expression in the study, included 7 studies, OR= 11.43, Or 95CI is 6.78,19.27, P <0.00001. Matrix metalloproteinases in malignant and non-malignant ovarian tumor tissues and contents are significantly different; studies are homogeneous.2. Hpa expression in the study, included 6 studies, OR= 8.62, Or 95%CI is 4.57,15.28, P <0.00001. Heparanase in malignant and non-malignant ovarian tumor tissues and contents are significantly different; study are homogeneous.3. CL expression in the study, included 3 studies, OR= 2.70, Or 95CI is 0.26,27.97, P=0.41. Cathepsin in malignant and non-malignant ovarian tumor tissues and contents are significantly different; study are heterogeneity.Conclusions:The Meta analysis result has showed that the expression of stroma enzymes in much higher in ovarian cancer than that in normal ovarian tissue. Because the evidence-based medicine method has only several distribute examples, the effect of the expression of stroma enzymes in ovarian cancer is not very convincing. It needs more examples to research and study. Anyway, stroma enzymes have the possibility to be the new maker of ovarian cancer.更多还原