【作者】 金泗虎；

【导师】 宫锋；

【作者基本信息】 中国人民解放军军事医学科学院 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 硫酸肝素蛋白多糖(heparan sulfate proteoglycan, HSPG)是基底膜和细胞外基质的重要构成成分,由一个核心蛋白和数个与之共价连接的硫酸乙酰肝素(heparan sulfate, HS)多糖侧链组成,其多糖侧链与多种生长因子和细胞因子相互结合并将这些因子固定在细胞外基质和基底膜中,参与并影响一系列的病理和生理过程。肝素酶(heparanase, HPSE)是一种β-葡萄糖苷内切酶,可特异性的识别和切割硫酸肝素蛋白多糖的硫酸肝素侧链,破坏细胞外基质和基底膜,并释放结合于硫酸乙酰肝素上的细胞因子及各种酶类分子,促进肿瘤的发生转移和血管生成,参与机体的炎症过程和自身免疫病。由于肝素酶与肿瘤发生转移的密切相关性,肝素酶已经成为肿瘤治疗的靶标。大多数真核生物基因都是断裂基因,包含了内含子(intron)与外显子(exon)。其中内含子在基因转录成mRNA前体(pre-mRNA)后会被RNA剪接复合体(spliceosome)移除,剩下的外显子连接在一起形成成熟的mRNA,约半数的基因其外显子与蛋白质结构域、亚结构域或基序有很好的对应关系。选择性剪接是指通过选择性地识别pre-mRNA中不同的剪接位点,由一条pre-mRNA生成多条成熟mRNA的过程,pre-mRNA的选择性剪接是增加蛋白质多样性与基因表达复杂程度的主要原因,是机体满足各种发育阶段、各种生理环境需要的重要调控机制,也是引起许多遗传病及肿瘤发生的重要原因。对这一领域的深入研究将有可能解开许多尚未明了的遗传性疾病的发病机制,找到某些肿瘤早期诊断的分子标记物,并有可能为遗传病以及肿瘤的治疗找到新的靶位,开辟新的途径。本课题的目的是克隆存在于人外周血白细胞及各种肿瘤细胞中的肝素酶可变剪接体,并研究其亚细胞定位和生物学功能,为进一步明确肝素酶在肿瘤发生转移中的作用和肿瘤的治疗提供新的思路。课题的研究内容主要包括三个方面:一、从人外周血白细胞和肿瘤细胞中筛选肝素酶可变剪接体并测序鉴定;二、构建肝素酶可变剪接体的亚定位研究载体,在哺乳动物细胞中表达各种肝素酶可变剪接体,研究其亚细胞定位;三、构建肝素酶可变剪接体的功能研究载体,在哺乳动物细胞中表达并筛选稳定表达细胞株,用于以后进行成瘤实验、克隆形成实验和酶活性研究。首先,根据人肝素酶的cDNA序列设计引物,用RT-PCR方法从正常人外周血白细胞、胃癌细胞(SGC-7901)、肝癌细胞(SMMC-7721)和前列腺癌细胞(Du145)中扩增肝素酶基因及其各种可变剪接体,随后构建至pGEM-T Easy载体中,转化大肠杆菌DH5α感受态细胞,筛选阳性克隆进行菌落PCR鉴定并测序。确定序列正确后,将肝素酶可变剪接体片段重组至pEGFP-N3质粒中(肝素酶位于融合蛋白的N端),实现肝素酶与绿色荧光蛋白的融合表达。将所构建的重组质粒分别转染HEK293FT细胞、B16F10细胞和CHO-K1细胞,通过激光共聚焦显微镜观察绿色荧光蛋白在细胞中存在的位置确定肝素酶可变剪接体的亚细胞定位。最后,将肝素酶及其可变剪接体片段重组至pcDNA3.1(+)质粒上,转染U87细胞和MCF-7细胞并筛选稳定表达细胞株,通过PCR和Western方法鉴定所得到的细胞株。研究结果:①在实验中共克隆得到5种肝素酶可变剪接体,分别命名为splice5、splice6、splice5&6、splice9&10、splice10,其中后四种为本课题组首次发现,我们在GenBank数据库中注册并获得序列号,分别为FJ517659、FJ517660、GQ337901、GQ337902。其中,在人白细胞中,我们筛选得到了所有的5种肝素酶可变剪接体形式;在人胃癌细胞(SGC-7901)中,筛选得到了splice5、splice9&10;在人肝癌细胞(SMMC-7721)中,筛选得到了splice5;在前列腺癌细胞(Du145)中,筛选得到了splice10。②构建了肝素酶的定位研究载体pEGFP-HPSE/s5/s9&10/s10,转染HEK293FT细胞、B16F10细胞和CHO-K1细胞,通过激光共聚焦显微镜观察发现全长肝素酶以颗粒状形式定位于细胞质中,但肝素酶的可变剪接体splice5、splice9&10、splice10与全长肝素酶不同,以弥散形式定位于细胞质中。③构建了肝素酶的功能研究载体pcDNA- HPSE/s5/s9&10/s10,转染U87和MCF-7细胞,并用G418筛选,但始终未筛选得到稳定表达细胞株。在本课题的研究中,首次克隆到了4个未报道的肝素酶可变剪接体splice6、splice5&6、splice9&10、splice10。其中splice6、splice5&6和Splice10在选择性剪接的过程中分别缺失了第6号外显子、5和6号外显子、10号外显子,并因此导致了移码突变,产生一个提前终止翻译的密码子(premature termination codon,PTC),但这三个剪接体并没有被细胞内的保护机制,即无意义密码子介导的mRNA降解(Nonsense-mediated decay,NMD)机制所降解,这说明splice6、splice5&6和splice10具有逃逸细胞内NMD监控的能力。其次,splice10与目前克隆到的其它所有的肝素酶可变剪接体不同,它没有因为提前终止翻译而缺失硫酸乙酰肝素(heparan sulfate, HS)的结合位点和活性位点,也就是说,splice10可能仍具有降解硫酸乙酰肝素的活性。因此,对缺失不同结构域的可变剪接体的功能研究,可能会为我们明确肝素酶不同结构域在肿瘤中的作用提供一些新的思路。肝素酶在内质网表面合成后转运至高尔基体,分泌到细胞外,与细胞表面的硫酸肝素蛋白多糖结合形成复合物,复合物通过内吞最终转运至溶酶体,溶酶体中的酸性环境以及其中的组织蛋白酶L(cathepsin L)促使肝素酶从65kD的前体形式转变为具有活性的异源二聚体形式。在肝素酶可变剪接体亚细胞定位的研究中,我们发现全长的肝素酶以颗粒状的形式定位于细胞质中,但肝素酶可变剪接体splice5、splice9&10、splice10的亚细胞定位与全长不同,它们是以弥散状的形式存在于细胞质中。这种定位的不同有两种可能:一是这些剪接体并没有转运至溶酶体中,因此也无法被组织蛋白酶L加工形成有活性形式的肝素酶异源二聚体;另外一种可能是在它们运送的过程中,经过了溶酶体并被加工形成了异源二聚体,然后又定位到细胞的其他部位行使功能。这种定位上的差异也预示着其功能上的不同,因此有必要进一步对这些可变剪接体的功能进行深入的研究。更多还原

【Abstract】 Heparan sulfate proteoglycans (HSPG) are macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) are connected. Heparan sulfate binds to and assembles ECM proteins, including growth factor and cytokine, is playing important roles in pathology and physiological processes. Heparanase (HPSE) is a mammalian endo-β-glucuronidase that can cleave heparin sulfate side chain of HSPG, facilitating structural alterations of the extracellular matrix and basement membrane underlying epithelial and endothelial cells, which makes it more susceptible to cellular invasion, and liberation of a multitude of biological mediators. Heparanase activity has been traditionally correlated with cancer metastasis, angiogenesis, inflammatory process and autoimmune disease.Almost all eukaryotic genes are interrupted gene which consists of exons and introns. RNA splicing is the process of excising the sequences in RNA that corresponds to introns, so that the sequence corresponding to exons are connected into a continuous messenger RNA. About half of the gene exons correlate with protein domains and motifs. Through recognizing different splicing sites, alternative splicing (AS) of precursor messenger RNA generates different isoforms from a single gene, which plays an important role in the generation of functional diversity of protein and the complexity of gene expression. Alternative splicing is also important for regulation of the levels and tissue specificity of gene expression and, if disrupted, can lead to disease. Along with profound study on this field will reveal the pathogenesis of hereditary disease which mechanism are not clear yet and find novel target of tumor therapy.The purpose of this research is to clone heparanase alternatively spliced variants from human peripheral blood leukocytes and tumor cells, then study their subcellular localization and biological function.The main contents of this research include three aspects as follows: 1). Screening and identification of heparanase alternatively spliced variants from human peripheral blood leukocytes and tumor cells. 2). Construction, expression of recombinant plasmids of heparanase alternatively spliced variants and studying their subcellular localization. 3). Construction eukaryotic expression plasmid of heparanase alternatively spliced variants and expression in mammalian cells for the purpose of enzymatic activity studying and tumorigenesis.First, the human heparanase alternatively spliced variants were amplified from peripheral blood leukocytes, SGC-7901, SMMC-7721 and Du145 by RT-PCR using specific primers based on the sequence of heparanase cDNA in GenBank, then cloned into pGEM-T Easy vector. The recombinant vector were further transformed into Escherichia coli strain DH5αand positive clones were screened and sequenced. Second, the human heparanase alternatively spliced variants were cloned into pEGFP-N3 vector. Transient transfection of HEK293FT and CHO-K1 were performed and heparanase alternatively spliced variants subcellular location were observed using laser scanning confocal fluorescence microscope. Third, the human heparanase alternatively spliced variants were inserted into the expression vector pcDNA3.1(+)and transferred into U87 and MCF-7. Stable transfected cells were selected and identified.Results: 1). We obtained five alternatively spliced variants of human heparanase, lacked exon 5, exon 6, exon 5 and exon 6, exon 9 and exon 10, exon 10 respectively, named splice5、splice6、slice5&6、splice9&10、splice10. The latter four were novel spliced form of human heparanase and were registered (GenBank :FJ517659、FJ517660、GQ337901、GQ337902). We obtainned all of the five heparanase alternatively spliced variants in peripheral blood leukocytes, however, only splice5 and splice9&10 in SMMC-7721 ,splice5 in SMMC-7721 and splice10 in Du145. 2). Plasmids pEGFP- HPSE/s5/s9&10/s10 were constructed and expressed in HEK293FT, B16F10 and CHO-K1. Results showed that wild-type heparanase exhibited predominantly granular pattern in cytoplasm, in contrast, splice5、splice9&10、splice10 all exhibited diffused pattern in cytoplasm. 3). Plasmid pcDNA- HPSE/s5/s9&10/s10 were constructed and transferred into U87 and MCF-7, but positive candidate cell line have not got yet.In this study, we cloned four novel splice variants of human heparanase splice6、splice5&6、splice9&10、splice10. It was noting that splice10, lack of exon 10, results in a premature termination codon (PTC), but it was not degradation by nonsense mediated decay (NMD). Furthermore, different from other heparanase alternatively spliced variants, splic10 have heparin sulfate binding sites and active sites. This means that splice10 may have potential heparanase enzymatic activity and its biological function may be different.Pre-pro-heparanase is first targeted to the endoplasmic reticulum via its own signal peptide. The 65 kD pro-heparanase is then shuttled to the Golgi apparatus, and is subsequently secreted via vesicles that bud from Golgi. Once secreted, heparanase rapidly interacts with cell membrane HSPGs, followed by a rapid endocytosis of heparanase-HSPG complex that appears to accumulated in endosomes. Conversion of endosomes to lysosome results in heparanase processing and activation. In this study, we found that wild-type heparanase exhibited predominantly granular pattern in cytoplasm. In contrast, splice5、splice9&10、splice10 exhibited diffused pattern in cytoplasm. These results suggested two possible explanations: On one hand, these alternatively spliced variants were not trafficked to lysosome and lack of heparanase enzymatic activity because they were not processed and activated. On the other hand, these alternatively spliced variants were trafficked through lysosome and then became heterodimer, an active form of heparanase, hereby localization in other place. The difference of subcellular localization implies different functions thus renders further study on these alternatively spliced variants necessary.更多还原