【作者】 臧盛兵；

【导师】 黄爱民；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的:从mRNA和蛋白水平上,检测KiSS-1、KiSS-1R及MMP-9在肝细胞癌(hepatocellular carcinoma,HCC)中的表达情况,探讨KiSS-1、KiSS-1R及MMP-9表达与HCC侵袭转移的关系及其意义。方法:利用逆转录聚合酶链反应(RT-PCR)技术,检测33例HCC、2例静脉癌栓、26例癌旁肝组织、13例远癌肝组织中KiSS-1、KiSS-1R mRNA的表达情况,并分析其与各临床病理参数的关系及其意义;对手工制作组织芯片的方法进行改良,构建含有HCC、肝内转移灶、癌旁肝组织、远癌肝组织以及正常肝组织的组织芯片,应用免疫组化、原位杂交方法及彩色图像分析技术从蛋白质水平上检测了150例HCC、137例癌旁肝组织、98例远癌肝组织、37例肝内转移灶和16例正常肝组织及从mRNA水平上检测了137例HCC、119例癌旁肝组织、85例远癌肝组织、28例肝内转移灶和16例正常肝组织中KiSS-1、MMP-9的表达情况,并分析其与各临床病理参数的关系及其意义。结果:1、RT-PCR结果显示KiSS-1mRNA在HCC中表达明显低于癌旁、远癌肝组织(P<0.01);在HCC组织中,随病理学分级和临床分期的升高,KiSS-1mRNA的表达逐步降低(P<0.05);转移组HCC KiSS-1mRNA表达量显著低于未发生转移的HCC(P<0.05);2例静脉癌栓中均缺失表达。KiSS-1mRNA的表达与HCC年龄、性别、AFP水平、肿瘤大小、包膜侵犯、组织学类型均无明显相关性(P>0.05)。KiSS-1R的表达与HCC年龄、性别、AFP水平、肿瘤大小、包膜侵犯、组织学类型、分化程度、转移与否、病理学分级、临床分期等临床病理参数均无关(P>0.05)。KiSS-1和KiSS-1R mRNA的表达在HCC侵袭转移中存在负相关性( P < 0.05)。2、对手工制作组织芯片的方法进行了改良,成功构建8个35 mm×25 mm×10mm大小的含有HCC、癌旁肝组织、远癌肝组织以及肝内转移灶和正常肝组织共188个点的芯片蜡块。芯片经H-E、免疫组化、原位杂交染色后切片脱片率分别为1.40%,5.92%,9.76%;有效率分别为99.58%,96.25%和91.19%,芯片组织符合镜下组织学观察和进一步研究的需要。3、原位杂交及免疫组织化学染色结果显示无论是mRNA还是蛋白水平上,KiSS-1在癌旁、远癌肝组织及正常肝组织的表达明显高于HCC组(P<0.01);在HCC组织中,随HCC病理学分级和临床分期的升高,KiSS-1的表达逐步降低(P<0.05);转移组HCC KiSS-1表达量显著低于未发生转移的HCC(P<0.05);KiSS-1在肝内转移灶中的相对表达量显著低于原发灶(P<0.01);此外KiSS-1蛋白的表达与HCC性别、年龄、肿瘤大小、组织学类型均无明显相关性(P>0.05)。HCC组的MMP-9表达明显高于癌旁、远癌肝组织及正常肝组织组(P<0.01);在HCC组织中,HCC转移组MMP-9表达量显著高于未发生转移的HCC(P<0.01);包膜侵犯组中MMP-9 mRNA的表达明显高于包膜无侵犯组(P<0.05);肝内转移灶表达明显高于相应原发灶的表达(P<0.01);MMP-9的表达与HCC年龄、性别、肿瘤大体类型、组织学类型、分化程度及临床分期均无明显相关性(P>0.05)。在蛋白和mRNA水平上, KiSS-1和MMP-9的表达在HCC侵袭转移中存在负相关性。结论:1、对手工制作组织芯片的方法进行了改良,成功构建8个35 mm×25 mm×10 mm大小的含有HCC、癌旁肝组织、远癌肝组织以及肝内转移灶和正常肝组织的芯片蜡块。改良的手工制作组织芯片的方法省时省力,节约了实验费用,同时芯片脱片率低,而样本有效率高,芯片组织符合镜下组织学观察和后续进一步研究的需要;2、KiSS-1的缺失表达及MMP-9高表达与HCC的侵袭转移密切相关;3、KiSS-1R的表达与HCC年龄、性别、肿瘤大小、组织学类型、分化程度、转移与否、临床分期等临床病理参数均无关;4、在HCC组织中, KiSS-1R mRNA的表达随着KiSS-1 mRNA表达降低而升高,KiSS-1和KiSS-1R mRNA的表达存在负相关性,推测由于KiSS-1表达水平的下调反馈性的引起KiSS-1R表达水平的升高,两者表达可能存在反馈性的调控机制;5、在HCC组织中,无论是蛋白还是mRNA水平,KiSS-1和MMP-9的表达存在负相关性,提示KiSS-1、MMP-9在HCC进展的过程中彼此相互作用,共同调控着HCC的侵袭转移的生物学行为;6、KiSS-1的缺失表达及MMP-9高表达是促进HCC侵袭与转移的重要因素,可作为预测HCC转移潜能的有价值的参考指标。更多还原

【Abstract】 Objective: To detect the expression of KiSS-1, KiSS-1R and MMP-9 in hepatocellular carcinoma(HCC) and study the correlation of KiSS-1, KiSS-1R and MMP-9 expression to invasion and metastasis of HCC.Methods: The expression of KiSS-1, KiSS-1R mRNA in 33 cases of HCC samples, 2 portal vein tumor embolus samples, 26 non-neoplastic adjacent liver tissue samples and 13 non-neoplastic distant liver tissue samples were detected by RT-PCR, and the correlations of KiSS-1, KiSS-1R mRNA to clinicopathologic parameters of HCC were analyzed. The processing of tissue chip would be modified. Tissue chips were constructed, which contained HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. The expression of KiSS-1, MMP-9 protein were determined by tissue chips, immunohistochemistry(IHC) and semi-quantitative image analysis in 150 HCC samples, 137 non-neoplastic adjacent liver tissue samples, 98 non-neoplastic distant liver tissue samples, 16 normal liver tissue samples and 37 intrahepatic metastasis lesion samples . The expression of KiSS-1, MMP-9 mRNA were evaluated by tissue chips , in situ hybridization (ISH) and semi-quantitative image analysis in 137 HCC samples, 119 non-neoplastic adjacent liver tissue samples, 85 non-neoplastic distant liver tissue samples, 16 normal liver samples and 28 intrahepatic metastasis lesion samples. The correlations of KiSS-1, MMP-9 to clinicopathologic parameters of HCC were also analyzed.Results:1、The results of RT-PCR showed that compared with the non-neoplastic adjacent liver tissues,non-neoplastic distant liver tissues and normal liver tissues, the expression of KiSS-1 mRNA in HCC was significantly lower(P<0.01), which decreased with the advance of the pathologic grade and clinical stage in HCC. The expression of KiSS-1 mRNA in HCC with metastasis was lower than that in those without metastasis(P<0.05). KiSS-1 mRNA was not detected in 2 portal vein tumor embolus samples, but it was not associated with sex, age, AFP lever, tumor size, capsule invasion and histological type(P>0.05). The expression of KiSS-1R mRNA was not associated with all clinicopathologic parameters of HCC(P>0.05), however a negative correlation between KiSS-1and KiSS-1R mRNA expression was found in the proceeding of HCC invasion and metastasis (P < 0.05).2、The processing of tissue chips was manually modified to construct successfully 8 paraffin blocks of tissue chips in the size of 35mm×25mm×10mm . Every paraffin blocks contained 188 small tissues including HCC, non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues, normal liver tissues and intrahepatic metastasis lesions. After the tissue chips were stained by H-E, IHC and ISH, the average sheded ratio was 1.40%, 5.92% and 9.76% respectively, and effective rate was 99.58%, 96.25%and 91.19%respectively. Tissue chips were suitable for the histological observation by microscopy and further study.3、The results of ISH and IHC showed that on both mRNA and protein levels, the expression of KiSS-1 in the non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues or normal liver tissues was significantly higher than that in HCC(P<0.01). With the advance of the pathologic grade and clinical stage of HCC, the expression of KiSS-1 decreased(P<0.05). The expression of KiSS-1 in HCC with metastasis was lower than that in those without metastasis, but it was higher than that in the corresponding metastasis lesions. KiSS-1 expression is not associated with sex, age, tumor size, capsule invasion and histological types(P>0.05). Compared with the the non-neoplastic adjacent liver tissues, non-neoplastic distant liver tissues or normal liver tissues, the expression of MMP-9 in HCCs was significantly increased(P<0.01). The expression of MMP-9 in HCC with metastasis or capsule invasion was higher than that in those without metastasis or capsule invasion, but it was lower than that in the corresponding metastasis lesions. Negative correlation between KiSS-1 and MMP-9 expression was found in the proceeding of HCC invasion and metastasis.Conclusion:1、The modified processing of tissue chips manually was an easy and cost-effective method, decreased the sheded ratio, improved the effective rate and made the tissue chips suitable for the histological observation by microscopy and further study.2、Loss of KiSS-1 expression or overexpression of MMP-9 was closely associated with HCC invasion and metastasis.3、The expression of KiSS-1R mRNA had not associated with all clinicopathologic parameters of HCC.4、For a negative correlation between KiSS-1and KiSS-1R mRNA expression was found in the proceeding of HCC invasion and metastasis , it was suggested that up-regulation of KiSS-1R mRNA expression was a feedback of down-regulation of KiSS-1 mRNA expression. A possible feedback mechanism might exist between KiSS-1and KiSS-1R mRNA expression.5、KiSS-1 expression showed negative correlation with MMP-9 expression in the proceeding of HCC invasion and metastasis on both mRNA and protein levels, so KiSS-1 and MMP-9 expression might interactively co-regulate HCC invasion and metastasis.6、Loss of KiSS-1 expression and overexpression of MMP-9 played important roles in the proceeding of HCC invasion and metastasis and might predict an aggressive clinical behavior and associate with metastatic potential in HCC.更多还原