【作者】 李国利；

【导师】 孙东植；

【作者基本信息】 延边大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的:为了揭示精原细胞瘤发生发展的分子机制,探讨肿瘤分子病理改变与病理形态学和临床生物学行为之间的关系,进而为在临床上开辟以针对STAT3为靶点的治疗精原细胞瘤的新方法提供必要的理论依据。本课题通过检测信号传导与转录激活因子3(STAT3)蛋白的表达,评价信号传导在睾丸精原细胞瘤发生中所起的诱导和促进作用;检测VEGF和c-myc的表达,研究在精原细胞瘤发生过程中JAK/STAT3信号转导途径促进肿瘤细胞增殖作用;检测hiwimRNA和Bcl-xl蛋白表达,探究在肿瘤发展中STAT3/Bcl-xl抗凋亡作用。通过对Oct4、PLAP和p53基因表达的观察,探讨精原细胞瘤恶变过程中癌基因、抑癌基因的变化,为精原细胞瘤的早期鉴别诊断和治疗提供分子生物学依据。通过检测睾丸精原细胞瘤组织激素受体,如AR、ER-α和PR表达,研究激素受体与肿瘤发生的相关性,进而为精原细胞瘤的内分泌治疗积累实验证据。方法:应用免疫组织化学S-P法,检测STAT3、VEGF、C-myc、Bcl-xl、p53、AR、ER-α、PR、Oct4和PLAP基因蛋白在睾丸精原细胞瘤和正常睾丸组织中的表达;应用mRNA原位分子杂交技术,检测HIWI基因和AR基因在睾丸精原细胞瘤中的转录情况;采用饱和酚-氯仿抽提石蜡组织DNA,应用PCR-SSCP法,检测p53在睾丸精原细胞瘤中的突变及缺失情况。结果:在38例睾丸精原细胞瘤组织中STAT3蛋白表达于细胞核或细胞浆内。VEGF、C-myc和Bcl-xl蛋白表达于细胞浆内。四种蛋白阳性表达率分别为76.3%、71.1%、84.2%和71.1%,与10例正常睾丸组织中相应基因的表达均具有显著性统计学差异(p＜0.01),且上述基因与精原细胞瘤的TNM临床分期具有相关性。HIWI mRNA在睾丸精原细胞瘤中表达于肿瘤细胞核和部分细胞浆内。其阳性表达率为68.4%,与正常睾丸组织相比具有统计学差异(p＜0.05=,但与临床分期无关(p＞0.05)。STAT3、VEGF、C-myc、HIWI和Bcl-xl基因表达之间存在不同程度的相关性。STAT3与VEGF、HIWI和Bcl-xl分别呈正相关(p＜0.01);STAT3与C-myc呈正相关(p＜0.05);VEGF与C-myc呈正相关(p＜0.05)。HIWI与BCL-xl基因表达之间存在高度相关性(r=0.594,p＜0.01)。Oct4在睾丸精原细胞瘤中表达于肿瘤细胞核,阳性表达率为100%,在正常睾丸组织中不表达;PLAP阳性着色主要位于细胞膜,偶尔见于胞浆。阳性表达率为89.47%,在正常睾丸组织中同样不表达。同时根据镜下观察,Oct4表达的切片背景色非常低,而PLAP表达的许多切片上,背景色较重。VEGF在睾丸精原细胞瘤临床分期Ⅰ期的阳性表达率为33.33%,Ⅱ期的阳性表达率为90.91%,Ⅲ期的阳性表达率为100%。Ⅰ期与Ⅱ期、Ⅰ期与Ⅲ期各组间比较具有显著性统计学差异(p＜0.01),Ⅱ期与Ⅲ期之间比较无统计学差异(p＞0.05);在10例正常睾丸组织中,2例弱阳性,8例阴性。在睾丸精原细胞瘤中p53阳性物质见于肿瘤细胞核。临床Ⅰ期的阳性表达率为93.33%,Ⅱ期与Ⅲ期的表达率分别为54.55%和50%。Ⅰ期与Ⅱ期、Ⅰ期与Ⅲ期两组间比较具有统计学差异(p＜0.05),Ⅱ期与Ⅲ期之间比较无统计学差异(p＞0.05);10例正常睾丸组织均呈阴性表达。在抽取的11例睾丸精原细胞瘤样本中,p53基因突变的有5例,突变率为45.45%。其中第5外显子突变的有3例,第6外显子突变的有0例,第7外显子突变的有1例,第8外显子突变的有1例。p53和VEGF表达之间存在相关关系(p＜0.05);p53和STAT3表达之间存在高度相关性(p＜0.01)。在25例睾丸精原细胞瘤组织中,部分肿瘤细胞胞浆中存在AR mRNA与AR蛋白的表达。在AR mRNA阳性和AR蛋白阳性病例中,其阳性细胞面积与总细胞面积比值和OD值与正常睾丸组织相比均具有统计学差异(p＜0.05)。而ER-α、PR蛋白均呈阴性表达。提示生精组织恶变时,AR mRNA和AR蛋白的表达水平下降。结论:1.JAK/Stat3信号转导途径可激活C-myc的表达,引起生殖细胞的恶性增殖,诱导睾丸精原细胞瘤的发生与发展。2.JAK/Stat3信号转导途径可激活VEGF的表达,进而在睾丸精原细胞瘤的发展与转移中发挥重要作用。3.睾丸精原细胞瘤中,HIWI基因可激活STAT3/Bcl-xl信号通路,赋予肿瘤细胞自我更新和抗凋亡的能力。4.Oct4在睾丸精原细胞瘤中均呈阳性表达,是一个优秀的诊断精原细胞瘤的标记物基因,与PLAP相比更加灵敏。5.睾丸精原细胞瘤发生发展过程中,存在着p53基因表达异常,其调控作用可能是精原细胞瘤发生的早期事件。6.精原细胞瘤有AR蛋白阳性表达与阴性表达之分,ER-α和PR蛋白在睾丸精原细胞瘤组织中均无表达。更多还原

【Abstract】 OBJECTIVE:In order to reveal the molecular mechanism by which seminoma occurs,study the relationship of the changes in tumor molecular pathology to pathological morphology and clinical biological behave,and provide the academic evidence for the clinical therapy targeted to STAT3 in seminoma,the inducing and stimulating effects of the signal transduction was assessed by determining Stat3 protein expression in seminoma;the hyperplasia-stimulation effects of the JAK/STAT3 signal transducer pathway in this tumor was investigated by measuring VEGF and C-myc,and the anti-apoptosis effects of STAT3/Bcl-xl in development of seminoma were explored by detecting HIWI and Bcl-xl.In order to provide the molecular biologic evidence for the early identified diagnosis and treatment of seminoma,the alteration of oncogenes and anti-oncogenes in the seminoma canceration was studied by observing gene expression of Oct4, PLAP,VEGF,and p53.In order to accumulate experimental evidence for endocrine therapy for seminoma,the relationship between hormone receptor and tumor occurring was investigated by determining seminoma hormone receptors,such as AR,ER-αand PR.METHODS:Paraffin specimen of seminoma and normal human testis tissue were collected. IHC(immunohistochemistry) were used to detect the protein of Star3,VEGF,C-myc,Bcl-xl,p53, Oct4,PLAP,AR,ER-αand PR;ISH(in situ hybridization) were used to observe the mRNA transcription of HIWI and AR.Saturated hydroxybenzene chloroform were applied to gain the DNA from 11 paraffin tissue and PCR-SSCP(ploy chain reaction single-strand conformation polymorphism) were to examine the deletion and mutation of p53 in seminoma.RESULTS:The expression of STAT3 protein was in nucleus or cytoplasm,VEGF,C-myc and Bcl-xl protein was in cytoplasm in 38 seminoma.The positive expression rate of STAT3,VEGF, C-myc and Bcl-xl was 76.3%,71.1%,84.2%,71.1%,and comparing normal testis tissue they all had different significance in statistics(p＜0.01),and these genes all had relevance with the TNM clinical stage in seminoma.The expression of HIWI mRNA was in nucleus and partly in cytoplasm in 38 seminoma. The positive expression rate of HIWI mRNA was 68.4%,comparing normal testis tissue it had different significance in statistics(p＜0.05).But it was none of the business of the TNM clinical stage.There was positive relevance between STAT3 and VEGF(p＜0.01),STAT3 and C-myc (p＜0.05),STAT3 and Bcl-xl(p＜0.01),STAT3 and HIWI(p＜0.01),STAT3 and P53(p＜0.01), HIWI and BCL-xl(p＜0.01),C-myc and VEGF(p＜0.05).The expression of Oct4 protein was in nucleus and the positive expression rate in 38 seminoma was 100%,but in normal testicle tissue were zero.The expression of PLAP protein was at cell membrane.The positive expression rate of PLAP in seminoma were 89.47%,but in normal testicle tissue were zero too.However,on the base of the observation of microscope, there was little or no background staining in the all slices of Oct4,and there was a high level of background staining artifact in many slices of PLAP.The positive expression rate of VEGF in seminoma clinical stageⅠ,Ⅱ,Ⅲwere 33.33%, 90.91%,100%.There were different significance of VEGF between StageⅠand stageⅡ,stageⅠand stageⅢ(p＜0.01),there were no different significance of VEGF between StageⅡand stageⅢ(p＞0.05);10 normal testicle tissue samples,2 samples were weakly positive,8 samples were negative.The positive expression rate of p53 in seminoma clinical stageⅠ,Ⅱ,Ⅲwere 93.33%,54.55%,50%.There were different significance of p53 between StageⅠand stageⅡ, stageⅠand stageⅢ(p＜0.05),there were no different significance of p53 between StageⅡand stageⅢp＞0.05);10 normal testicle tissue samples were all negative.There were p53 mutations in seminoma,the mutation rate was 45.45%.By the research we confirm that there were expression of AR mRNA and AR protein in the cytoplasm of some seminoma.When the expression of AR mRNA is positive,comparing normal testis,it had different significance in statistics(p＜0.05),it suggest that when the primordial germ cell malignant,the expression of AR could be descend.CONCLUSIONS:1.The JAK/STAT3 signal transducer pathway can increase the expression of C-myc and cause the malignancy hyperplasy of germinal cell,which provocating seminoma. 2.The JAK/STAT3 signal transducer pathway can activate the expression of VEGF,and impulse the development of seminoma.3.In seminoma,the gene HIWI can activate the STAT3/Bcl-xl signal transducer pathway,give tumor cell the ability of self-renewal and anti-apoptosis.4.Oct4 is all positive in seminoma,it is an excellent marker gene of seminoma diagnostic.It is more sensitive than PLAP.5.p53 has the genes abnormality with the genesis and development of seminoma.6.Seminoma can be distinguish by the expression of AR protein to positive and negative.The ER-αand AR protein are both negative expression in seminoma.更多还原