卵巢癌组织中KLK11基因的表达意义
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摘要
卵巢癌是女性生殖系统最常见的三大恶性肿瘤之一，死亡率占女性生殖系统恶性肿瘤的首位，至今发病机制尚未阐明，缺乏有效的早期诊断和治疗方法。人激肽释放酶Kallikrein(KLK)基因家族包括KLK1～KLK15基因，定位于19q13～14染色体上，编码相应的人激肽释放酶HK1～HK15，为一组丝氨酸蛋白酶，广泛存在于各种组织和体液中，参与多种生理和病理过程。研究发现70％的卵巢癌患者血清中HK11水平是升高的，提示HK11可能是卵巢癌潜在的生化标记物，但KLK11的表达与临床分期、分化程度、及组织学分型的关系尚未见报道。
本研究采用逆转录—聚合酶链式反应(RT-PCR)和免疫组化技术检测卵巢癌KLK11 mRNA及蛋白的表达，并结合其临床病理特征进行分析，旨在探讨KLK11基因的表达与卵巢癌发病的关系，以期为临床卵巢癌的诊治提供实验依据，并初步探讨其发生的分子机制。
一、方法
1．RT-PCR：取48例卵巢癌新鲜手术标本，提取总RNA逆转录成cDNA。通过PCR扩增KLK11基因，PCR反应产物经电泳检测。
2．免疫组织化学染色：采用SP法，按常规方法操作。一抗为鼠抗人KLK11单克隆抗体，DAB显色。
3．图像分析：采用Leica RX 250型图像分析系统，对免疫组化阳性染色进行定量分析。测量视野中阳性染色的相对面积(即阳性细胞面积／镜下细胞及间质总面积×100％)和阳性染色积分灰度值。
4．统计学分析：应用Spss11.0软件对不同临床病理分级及组织学类型的卵巢癌样本数据采用x~2检验、t检验和方差分析等方法进行统计学分析。
二、结果
1．RT-PCR：KLK11基因在卵巢癌中高表达，KLK11 mRNA表达与卵巢癌临床病理特征关系密切。随临床分期增加，KLK11基因的mRNA表达水平增高，晚期卵巢癌(Ⅲ／Ⅳ期)KLK11基因mRNA表达水平明显高于早期卵巢癌(Ⅰ／Ⅱ期)；中低分化卵巢癌KLK11 mRNA表达明显高于高分化卵巢癌；KLK11 mRNA表达水平与卵巢癌组织学分型无关。
2．免疫组化染色：HK11蛋白阳性染色定位于细胞质中，在晚期卵巢癌中的HK11阳性表达程度高于早期的卵巢癌；分化差卵巢癌中HK11阳性表达程度高于分化好的卵巢癌；不同的组织学分型间的表达程度无明显差异。
3．图像分析结果
晚期卵巢癌和分化差卵巢癌中阳性染色的相对面积和积分灰度值均高于早期卵巢癌和分化好的卵巢癌，其差异有统计意义。
三、结论
1．KLK11在卵巢癌中高表达，HK11蛋白表达定位于细胞质。KLK11 mRNA及蛋白的表达与临床分期相关，晚期卵巢癌中KLK11 mRNA及蛋白的表达水平明显高于早期卵巢癌，中低分化卵巢癌中KLK11 mRNA及蛋白的表达明显高于高分化卵巢癌，但KLK11 mRNA及蛋白的表达与组织学类型无关。
2．KLK11基因表达可能与卵巢癌的恶性程度、临床分期、肿瘤侵袭进展等有关，在卵巢癌的发生发展过程中起一定的作用。
Ovarian cancer is one of the three most common malignancies Offemale reproductive system, mortality of ovarian cancer is the firstfemale reproductive system cancer mortality. The mechanisms of theovarian cancer is not clear. The Effective methods for early diagnosisand treatment are lack. The Human Kallikrein gene family contains15 genes. All genes share important similarities, including mapping at thesame chromosomal locus(19q 13.4). All members of the human kallikreinmultigene family encode for serine proteases that are found in diversetissues and biological fluids and involved in a variety of physiologicaland pathological processes. Borqono CA demonstrate that elevated serumlevels of HK11 were found in 70% of women with ovarian cancer,whichsuggest KLK11 expression may abnormal in the ovarian cancer.
In this research, KLK11 mRNA expression was examined byRT-PCR. The expression and distribution of KLK11 in ovarian cancerwere detected by immunohistochemistry and image analytical technique.We analyzed the results combination with clinic pathological conference. The purpose of this study was to examine expression level ofthe human tissue kallikrein gene (KLK11) in ovarian cancer. Somolecule regulation mechanisms and experiment proof of ovarian cancercan be obtained. The aim of present study was to determine the potentialrole of KLK11 mRNA expression in ovarian cancer development andprogression. The result were analyzed in term of tumor type, clinicalstage and histological grade.
Methods
1.RT-PCR
For RT-PCR analysis, fresh surgical ovarian cancer tissue samplewere collected from 48 patients treated surgically, Extraction ofmRNA from ovarian tissue and cDNA synthesis were carried out.cDNA was synthesized by random hexamer priming. KLK11 mRNAexpression levels were determined by RT-PCR.Reaction product waspanned successfully using electrophoresis technique.
2. Immunohistochemistry stain
It was operated by SP method, the monoclonal antibody of rat anti-KLK11 were used as first antibody with DAB colouration.
3. Image analysis
Positive staining of immunohistochemistry to ovarian cancer wastaken with quantitative analysis by Germany Leica RX 250 image analytical system. Relative areas and integral gray scale of positivestaining of KLK11 expression were measured in campus visualis.
4. statistics analysis
For statistical analysis, spss11.0 software package was used toassess expression of KLK11 gene. Associations between clinicopathologicparameters, such as stage, grade, histotype, and residual tumor, andKLK11 expression were analyzed by the x~2 test,F test, and t test.
Results
1. RT-PCR
High KLK11 mRNA expression remained significantly associatedwith clinicopathological features. The expression level of KLK11 genemRNA upload with the development of the clinical stage. Expression ofKLK11 mRNA levels showed significantly higher in patients with theadvanced stages (ⅢorⅣ) compared to the early stages (ⅠorⅡ)Expression of KLK11 mRNA levels showed significantly higher inpatients with the poor and moderate histological type than the goodhistological type. On the other hand, no significant association was foundin KLK11 expression grade of ovarian carcinoma
2. Immunohistochemistry stain
KLK11 protein was detected in the cytoplasm. The positive rate ofKLK11 protein showed significantly higher in patients with the advancedstages (ⅢorⅣ) compared to the early stages (ⅠorⅡ) The positive rate of KLK11 protein showed significantly higher in patients with thepoor and moderate histological type than the good histological type.There is no difference in Serous cystadenocarcinoma, Mucinouscystadenocarcinoma, Endometrial adenocarcinoma and Clear CellCarcinoma
3. Image analytic result
Relative areas and integral gray scale of positive staining of HK11expression of the advanced stage and the poor or moderate cancerdifferentiation groups were higher than those of the early stage and thehigh differentiation groups, the difference has statistical significance.(P＜0.05)
Conclusions
1. KLK11 protein was detected in the cytoplasm. Expression of KLK11both on mRNA and protein levels showed significantly higher in patientswith the advanced stages (ⅢorⅣ) compared to the early stages (ⅠorⅡ).Expression of KLK11 both on mRNA and protein levels showedsignificantly higher in patients with the poor and moderate histologicaltype than the good histological type. On the other hand, no significantassociation was found in KLK11 expression grade of ovarian carcinoma
2. The different expression of KLK11 might be related with themalignancy of ovarian carcinoma.KLK11 gene could play critical role as a prognostic marker in the future.
本研究采用逆转录—聚合酶链式反应(RT-PCR)和免疫组化技术检测卵巢癌KLK11 mRNA及蛋白的表达，并结合其临床病理特征进行分析，旨在探讨KLK11基因的表达与卵巢癌发病的关系，以期为临床卵巢癌的诊治提供实验依据，并初步探讨其发生的分子机制。
一、方法
1．RT-PCR：取48例卵巢癌新鲜手术标本，提取总RNA逆转录成cDNA。通过PCR扩增KLK11基因，PCR反应产物经电泳检测。
2．免疫组织化学染色：采用SP法，按常规方法操作。一抗为鼠抗人KLK11单克隆抗体，DAB显色。
3．图像分析：采用Leica RX 250型图像分析系统，对免疫组化阳性染色进行定量分析。测量视野中阳性染色的相对面积(即阳性细胞面积／镜下细胞及间质总面积×100％)和阳性染色积分灰度值。
4．统计学分析：应用Spss11.0软件对不同临床病理分级及组织学类型的卵巢癌样本数据采用x~2检验、t检验和方差分析等方法进行统计学分析。
二、结果
1．RT-PCR：KLK11基因在卵巢癌中高表达，KLK11 mRNA表达与卵巢癌临床病理特征关系密切。随临床分期增加，KLK11基因的mRNA表达水平增高，晚期卵巢癌(Ⅲ／Ⅳ期)KLK11基因mRNA表达水平明显高于早期卵巢癌(Ⅰ／Ⅱ期)；中低分化卵巢癌KLK11 mRNA表达明显高于高分化卵巢癌；KLK11 mRNA表达水平与卵巢癌组织学分型无关。
2．免疫组化染色：HK11蛋白阳性染色定位于细胞质中，在晚期卵巢癌中的HK11阳性表达程度高于早期的卵巢癌；分化差卵巢癌中HK11阳性表达程度高于分化好的卵巢癌；不同的组织学分型间的表达程度无明显差异。
3．图像分析结果
晚期卵巢癌和分化差卵巢癌中阳性染色的相对面积和积分灰度值均高于早期卵巢癌和分化好的卵巢癌，其差异有统计意义。
三、结论
1．KLK11在卵巢癌中高表达，HK11蛋白表达定位于细胞质。KLK11 mRNA及蛋白的表达与临床分期相关，晚期卵巢癌中KLK11 mRNA及蛋白的表达水平明显高于早期卵巢癌，中低分化卵巢癌中KLK11 mRNA及蛋白的表达明显高于高分化卵巢癌，但KLK11 mRNA及蛋白的表达与组织学类型无关。
2．KLK11基因表达可能与卵巢癌的恶性程度、临床分期、肿瘤侵袭进展等有关，在卵巢癌的发生发展过程中起一定的作用。
Ovarian cancer is one of the three most common malignancies Offemale reproductive system, mortality of ovarian cancer is the firstfemale reproductive system cancer mortality. The mechanisms of theovarian cancer is not clear. The Effective methods for early diagnosisand treatment are lack. The Human Kallikrein gene family contains15 genes. All genes share important similarities, including mapping at thesame chromosomal locus(19q 13.4). All members of the human kallikreinmultigene family encode for serine proteases that are found in diversetissues and biological fluids and involved in a variety of physiologicaland pathological processes. Borqono CA demonstrate that elevated serumlevels of HK11 were found in 70% of women with ovarian cancer,whichsuggest KLK11 expression may abnormal in the ovarian cancer.
In this research, KLK11 mRNA expression was examined byRT-PCR. The expression and distribution of KLK11 in ovarian cancerwere detected by immunohistochemistry and image analytical technique.We analyzed the results combination with clinic pathological conference. The purpose of this study was to examine expression level ofthe human tissue kallikrein gene (KLK11) in ovarian cancer. Somolecule regulation mechanisms and experiment proof of ovarian cancercan be obtained. The aim of present study was to determine the potentialrole of KLK11 mRNA expression in ovarian cancer development andprogression. The result were analyzed in term of tumor type, clinicalstage and histological grade.
Methods
1.RT-PCR
For RT-PCR analysis, fresh surgical ovarian cancer tissue samplewere collected from 48 patients treated surgically, Extraction ofmRNA from ovarian tissue and cDNA synthesis were carried out.cDNA was synthesized by random hexamer priming. KLK11 mRNAexpression levels were determined by RT-PCR.Reaction product waspanned successfully using electrophoresis technique.
2. Immunohistochemistry stain
It was operated by SP method, the monoclonal antibody of rat anti-KLK11 were used as first antibody with DAB colouration.
3. Image analysis
Positive staining of immunohistochemistry to ovarian cancer wastaken with quantitative analysis by Germany Leica RX 250 image analytical system. Relative areas and integral gray scale of positivestaining of KLK11 expression were measured in campus visualis.
4. statistics analysis
For statistical analysis, spss11.0 software package was used toassess expression of KLK11 gene. Associations between clinicopathologicparameters, such as stage, grade, histotype, and residual tumor, andKLK11 expression were analyzed by the x~2 test,F test, and t test.
Results
1. RT-PCR
High KLK11 mRNA expression remained significantly associatedwith clinicopathological features. The expression level of KLK11 genemRNA upload with the development of the clinical stage. Expression ofKLK11 mRNA levels showed significantly higher in patients with theadvanced stages (ⅢorⅣ) compared to the early stages (ⅠorⅡ)Expression of KLK11 mRNA levels showed significantly higher inpatients with the poor and moderate histological type than the goodhistological type. On the other hand, no significant association was foundin KLK11 expression grade of ovarian carcinoma
2. Immunohistochemistry stain
KLK11 protein was detected in the cytoplasm. The positive rate ofKLK11 protein showed significantly higher in patients with the advancedstages (ⅢorⅣ) compared to the early stages (ⅠorⅡ) The positive rate of KLK11 protein showed significantly higher in patients with thepoor and moderate histological type than the good histological type.There is no difference in Serous cystadenocarcinoma, Mucinouscystadenocarcinoma, Endometrial adenocarcinoma and Clear CellCarcinoma
3. Image analytic result
Relative areas and integral gray scale of positive staining of HK11expression of the advanced stage and the poor or moderate cancerdifferentiation groups were higher than those of the early stage and thehigh differentiation groups, the difference has statistical significance.(P＜0.05)
Conclusions
1. KLK11 protein was detected in the cytoplasm. Expression of KLK11both on mRNA and protein levels showed significantly higher in patientswith the advanced stages (ⅢorⅣ) compared to the early stages (ⅠorⅡ).Expression of KLK11 both on mRNA and protein levels showedsignificantly higher in patients with the poor and moderate histologicaltype than the good histological type. On the other hand, no significantassociation was found in KLK11 expression grade of ovarian carcinoma
2. The different expression of KLK11 might be related with themalignancy of ovarian carcinoma.KLK11 gene could play critical role as a prognostic marker in the future.
引文
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