【作者】 李彬；

【导师】 张永莲；

【作者基本信息】 中国科学院研究生院（上海生命科学研究院） ， 生物化学与分子生物学， 2002， 博士

【摘要】 子宫肌瘤是一种受多基因控制的绝经前妇女生殖道最常见的良性肿瘤它是导致育龄期妇女行全子宫切除术的主要病因之一严重危害妇女的身心健康本实验室采用了两种技术来克隆肌瘤中有差异表达的基因并对所克隆到的部分基因进行了研究以期从分子水平对其发病机理有所了解在用mRNA 差异显示法DDPCR 筛选差异基因的过程中我们用了十二对引物将人的肌瘤和瘤旁组织进行了比较来获取差异片段并进而用反向Northern 的方法排除了假阳性用Northern 对真阳性克隆进行了验证结果我们从69 个DD 条带中找到了四个被Northern 所验证的cDNA 片段其中三个在肌瘤中高表达叫L1 L2 L3 一个在瘤旁组织中高表达叫M1 L1 经克隆并被证明是人核糖体蛋白S19 用13 种组织做Northern 分析发现它在所有组织中都有表达且有着不同的大小病理分布提示它在腮腺囊腺癌胰腺癌及乳腺癌中都有高表达在6 例病人中都未发现其有突变我们的结果表明它的过量表达是组织快速生长的指标在用抑制差减杂交法SSH 寻找肌瘤高表达的基因中我们建立了一个肌瘤高表达基因的cDNA 片段库这个库最终被证明含有88 个基因的cDNA 片段在用两轮反向Northern 筛过之后有二十个基因显示为在肌瘤中高表达我们选取了其中三个基因在Northern 分析中得到验证二十个基因中有17 个是已知基因3 个是未知功能基因它们都是首次显示出与肌瘤有关联其中一个叫磷酯酶A2 PLA2 的基因在6 例病人中的4 例都显示出被上调而在研究它的组织分布时发现它在前列腺睾丸心脏和骨骼肌中都有明显表达从Genbank 中的人EST 数据库中我们通过电子克隆得到了差减库中一个未知基因(061)接近全长的cDNA 序列(4.1kb, 包含整个编码框) 并经克隆测序和Northern 所证实利用人类基因组数据库我们将其定位于染色体12q24.11 并发现其由7 个外显子所组成序列分析表明它可能有两个不同的人子宫肌瘤相关基因的克隆和研究摘要<WP=3>剪接形式(4.1Kb, 2.7kb) Northern 结果证实了这个推测在16 种人的组织中的分布研究表明它的小mRNA 形式在人睾丸中的表达量显著高于其它组织在aa 水平上它与人的一个TNF 诱导蛋白的同源性为71 而与小鼠中一个未知基因则达到97更多还原

【Abstract】 As the most common reproductive tract neoplasm in premenopausal women,Uterine Leiomyoma (UL) is a multi-gene involved benign tumor. It is the main causeof hysterectomy for women at reproductive age, which affects their health greatly. Inour lab, we used two different techniques to clone differentially expressed genes inleiomyoma and studied part of them, which may help us to understand the mechanismof UL formation at molecular level.In searching of differentially expressed genes in human uterine leiomyomas,differential display was used with twelve pairs of primers to compare human uterineleiomyomas with matched myometrium. False positives were eliminated by reverseNorthern analysis. Positives were confirmed by Northern blot analysis. RESULTS: [1]Four of 69 cDNA fragments (3 up-regulated named L1, L2 & L3 and 1 downregulatednamed M1 in leiomyoma) were confirmed by Northern analysis. [2]Sequence comparison and Northern analysis proved that L1 is exactly the humanribosome protein S19. [3] It was present ubiquitously in 13 tissues tested but invarious levels and even in different size. [4] L1 was highly expressed in parotideancystadenocarcinoma, pancreatic cancer and breast cancer examined. [5] No mutationshave been found in human uterine leiomyomas (n=6). CONCLUSIONS: hRPS19overexpression might be a universal signal in rapid cell growth tissues.In searching for differentially expressed genes in human uterine leiomyomas(ULs), suppression subtractive hybridization was used to construct an UL up-<WP=5>regulated library, which turned out to represent 88 genes. After two rounds ofscreening by reverse Northern analysis, twenty genes were proved to be up-regulated,three of them were picked up and confirmed by Northern analysis. Among the twentygenes, seventeen of them are known genes and the other three are genes withunknown function, which were firstly associated with UL. One gene namedPhospholipase A2 (PLA2) showed up-regulation in 4/6 of the patients andinvestigation of tissue distribution indicated that it had obvious expression in prostate,testis, liver, heart and skeletal muscle.From human ESTs database at Genbank, we got the nearly full length cDNAsequence (4.1Kb, full coding sequence) of a unknown gene (061) derived fromsubtracted library, which is verified by sequencing and Northern results. Bycomparing its sequence with human genome database, we found it having sevenexons and located it on chromosome 12q24.11. From its sequence, we deduced it hadtwo forms of mRNAs because of alternative splicing and Northern results proved it.Distribution analysis among 16 human tissues indicated that its mRNA in small formwas especially abundant in testis. We found it had 71% homology with a TNF -induced protein in human at aa level, but 97% with a unknown gene in mouse.更多还原