蝎丝氨酸蛋白酶抑制剂Sj7170促神经胶质瘤生长和转移研究

发布时间：2018-06-18 20:13

  本文选题：sj7170 + 蝎毒素　； 参考：《武汉大学》2014年博士论文

【摘要】：神经胶质瘤是成人中最常见的原发性恶性脑瘤。目前被确诊为神经胶质瘤的患者,大约有一半是处于晚期阶段,神经胶质瘤患者平均预期的寿命约为14-18个月左右,因而神经胶质瘤是恶性程度最高的肿瘤之一。现有的科学研究表明神经胶质瘤侵袭行为的分子机制十分复杂且仍不清楚。因此,作为分子工具的神经胶质瘤调节剂(激活剂和抑制剂)的发现将有助于揭示神经胶质瘤发生的分子机制,也有利于研发治疗和诊断神经胶质瘤的药物和技术。 丝氨酸蛋白酶抑制剂(SPIs)广泛存在于各种生物体及其组织中,如寄生虫、吸血性无脊椎动物、两栖类动物的皮肤和有毒动物的毒腺分泌物,SPIs具有重要的生理/病理功能。近来研究表明Serpin和Kunitz家族的SPIs能导致肿瘤细胞恶性程度的增高。然而,所有报道的SPIs均是由内源性的人类细胞所分泌的,还没有任何关于外源性SPIs促肿瘤作用的报道,并且内源性SPIs促肿瘤的分子机制仍然不清楚,更为重要的是,SPIs促肿瘤研究还从未涉及神经胶质瘤。因此,发现新型SPIs并研究其促神经胶质瘤作用及其机制具有重要的科学意义与应用价值。 本研究从普洱真蝎cDNA文库中筛选鉴定到一条cDNA序列,命名为Sj7170。 Sj7170的全长cDNA序列为459个核苷酸,开放阅读框含有261个核苷酸共编码86个氨基酸残基,其中包含24个残基的信号肽和62个残基的成熟肽,形成5对二硫键。序列比对的结果表明Sj7170具有典型的Ascaris型抑制剂的特征,并且可能存在潜在的丝氨酸蛋白酶抑制活性。基于Sj7170的cDNA序列设计了特异性引物,通过PCR扩增成熟肽序列,构建重组表达质粒pET-28a-Sj7170,并将其转化到大肠杆菌中,得到了色谱纯的重组Sj7170多肽。在体内外检测了重组Sj7170多肽的丝氨酸蛋白酶抑制活性。结果表明,rSj7170可以特异性地抑制胰凝乳蛋白酶的活性,并且体外的抑制常数Ki值为3.9×10-7M,对胰蛋白酶和弹性蛋白酶的活性没有明显的影响。 进一步研究发现蝎丝氨酸蛋白酶抑制剂Sj7170能促神经胶质瘤细胞的增殖。细胞计数实验结果表明rSj7170对神经胶质瘤细胞U87和U251的增殖均有显著的促进作用。细胞克隆形成实验显示rSj7170同样能增强单个U87细胞形成克隆菌落的能力。细胞周期流式分析发现,rSj7170使U87细胞从G1期进入S期的时间缩短,从而加速了U87细胞周期的进程。Western blot研究结果表明,rSj7170促进了周期蛋白cyclinD1和周期转录激活子E2F1的表达,增强了Rb2/p130蛋白的磷酸化,抑制了周期转录抑制子E2F5的表达。通过构建下调周期蛋白cyclinD1细胞系进一步确证了cyclinD1在Sj7170促细胞增值过程中起到的重要作用。动物体内实验进一步证实,rSj7170促进了U87细胞在裸鼠体内肿瘤的形成和生长,同时上调了肿瘤组织中cyclinD1的表达。 研究还发现蝎丝氨酸蛋白酶抑制剂Sj7170能促进神经胶质瘤细胞的迁移和侵袭。Transwell实验表明,rSj7170能显著的促进神经胶质瘤U87和U251的迁移和侵袭,改变倍数均为10倍以上。EMT标记蛋白实验显示,rSj7170能上调神经胶质瘤细胞U87和U251中Vimentin和Snail蛋白的表达,抑制E-cadherin的表达,诱导神经胶质瘤细胞U87和U251上皮特征向间质特征转化。进一步构建下调转录因子Snail细胞系的结果表明,Snail下调后rSj7170的促迁移和侵袭作用消失。因此Snail蛋白的上调和EMT过程的发生对于Sj7170促神经胶质瘤细胞迁移和侵袭功能获得和发生起着不可或缺的作用。而cyclinD1下调后rSj7170仍然具有促进细胞迁移和侵袭的能力,因此cyclinD1并没有在此过程中发挥作用。免疫荧光实验结果显示,rSj7170通过Rho GTPase通路来使细胞骨架发生重排,从而使神经胶质瘤细胞U87的细胞球状生长形态发生改变,使之贴壁性更强。 最后进一步探索了Sj7170蛋白酶抑制活性和促神经胶质瘤生长和转移作用的关系。根据Sj7170的胰凝乳蛋白酶抑制活性关键位点,在分子水平上设计了10个Sj7170的突变体,采用overlapping PCR的方法获得了Sj7170的10个突变体基因序列,利用与野生型Sj7170相同的表达纯化系统,表达纯化了10个Sj7170突变体多肽,分别测定其蛋白酶抑制活性和促神经胶质瘤细胞增殖和转移能力。胰凝乳蛋白酶抑制活性测定实验发现,突变体Sj7170-G35A的二级结构和蛋白酶抑制活性与野生型Sj7170相比,均发生了显著的改变,其Ki值为2.1×10-9M,抑制活性较Sj7170提高了185.71倍。但神经胶质瘤细胞增殖实验结果显示与野生型Sj7170相比,突变体Sj7170-G35A并没有极大地促进神经胶质瘤细胞U87的生长和增殖。同时,其它9个突变体多肽的胰凝乳蛋白酶抑制活性较野生型Sj7170变化不大,并且也不具有促神经胶质瘤细胞增殖的能力。细胞迁移实验结果显示,Sj7170的10个突变体促进神经胶质瘤U87细胞迁移的能力与蛋白酶抑制活性之间并没有很好的相关性。这些结果均表明,Sj7170的这两种功能之间并没有直接的联系,其促神经胶质瘤细胞增殖和迁移功能并不依赖于其蛋白酶抑制活性功能,二者可能在生物体内发挥不同的生物学效应。 综上所述,本研究发现的Sj7170是首个分离鉴定的具有丝氨酸蛋白酶抑制活性和神经胶质瘤激活效应的双功能分子,不仅对深入了解蝎毒素的天然多肽库提供了新的视角,同时也为丝氨酸蛋白酶抑制剂作为肿瘤生长／转移调节剂提供了新的发现,加强了丝氨酸蛋白酶抑制和肿瘤生长转移激活剂之间的功能联系,这些都有助于进一步揭示肿瘤发生的分子机制,并提示其可能具有潜在价值。
[Abstract]:Neuroglioma is the most common primary malignant brain tumor in adults. About half of the patients diagnosed with glioma are in the advanced stage, and the average life expectancy of glioma patients is about 14-18 months, and neuroglioma is one of the most malignant tumors. The molecular mechanism of the invasion of glioma is very complex and not clear. Therefore, the discovery of neuroglioma regulators (activators and inhibitors) as a molecular tool will help to reveal the molecular mechanisms of glioma and the development of drugs and techniques for the treatment and diagnosis of neuroglioma.
Serine protease inhibitor (SPIs) is widely found in various organisms and their tissues, such as parasites, blood sucking invertebrates, the skin of amphibians and toxic animal glands of amphibians, and SPIs has important physiological / pathological functions. Recent studies have shown that SPIs in the family of Serpin and Kunitz can lead to the malignancy of tumor cells. However, all the reports of SPIs are secreted by endogenous human cells, and there are no reports about the effect of exogenous SPIs on tumor growth, and the molecular mechanism of endogenous SPIs promoting tumor is still unclear, and more importantly, the SPIs tumor research has never been involved in glioma. Therefore, the new SPIs is discovered and studied. The role and mechanism of glioma is of great scientific significance and application value.
In this study, a cDNA sequence was screened from the Puer true scorpion cDNA library. The full length cDNA sequence named Sj7170. Sj7170 was 459 nucleotides, and the open reading frame contained 261 nucleotides co encoding 86 amino acid residues, including 24 residues of signal peptide and 62 residues of mature peptide, forming 5 pairs of two sulfur bonds. Sequence alignment results It is shown that Sj7170 has the characteristics of typical Ascaris inhibitors and may have potential serine protease inhibitory activity. Specific primers are designed for the cDNA sequence based on Sj7170, and the recombinant expression plasmid pET-28a-Sj7170 is constructed by PCR amplification of the mature peptide sequence, and the recombinant plasmid pET-28a-Sj7170 is transformed into Escherichia coli and a purified recombinant Sj71 is obtained. 70 polypeptide. The serine protease inhibitory activity of recombinant Sj7170 polypeptide was detected in vitro and in vivo. The results showed that rSj7170 could specifically inhibit the activity of chymotrypsin, and the inhibitory constant Ki value was 3.9 x 10-7M in vitro, and had no significant effect on the activity of trypsin and elastase.
Further studies have found that the scorpion serine protease inhibitor Sj7170 can promote the proliferation of glioma cells. The results of cell count experiments show that rSj7170 has a significant effect on the proliferation of U87 and U251 in glioma cells. Cell clone formation experiments show that rSj7170 can also enhance the ability of single U87 cells to form cloned colonies. Cell cycle flow analysis showed that rSj7170 shortened the time of U87 cells from G1 to S phase and accelerated the process of U87 cell cycle.Western blot. The results showed that rSj7170 promoted the expression of periodic protein cyclinD1 and periodic transcriptional activator E2F1, enhanced the phosphorylation of Rb2/p130 protein and inhibited the periodic transcriptional suppressor E2F5. The important role of cyclinD1 was further confirmed by the construction of the cyclin cyclinD1 cell line. In vivo experiments in animals further confirmed that rSj7170 promoted the formation and growth of U87 cells in nude mice and increased the expression of cyclinD1 in the tumor tissues.
The study also found that the scorpion serine protease inhibitor Sj7170 can promote the migration and invasion of glioma cells and.Transwell experiments show that rSj7170 can significantly promote the migration and invasion of glioma U87 and U251, and the change multiplier is more than 10 times the.EMT labelled protein experiment, and rSj7170 can increase the U87 and U251 in glioma cells. The expression of Vimentin and Snail protein inhibits the expression of E-cadherin and induces the transformation of the U87 and U251 epithelial features to the interstitial characteristics of glioma cells. Further construction of the down-regulated transcription factor Snail cell line shows that the migration and invasion of rSj7170 after Snail downregulation is disappearing. Therefore, the up-regulation of Snail protein and the occurrence of EMT process Sj7170 promotes the migration and invasion of glioma cells and plays an indispensable role. But after the downregulation of cyclinD1, rSj7170 still has the ability to promote cell migration and invasion, so cyclinD1 does not play a role in this process. The results of immunofluorescence test show that rSj7170 makes the cell bone through the Rho GTPase pathway. The rearrangement of the scaffold results in a change in the morphology of spheroid growth of glioma cell U87, making it more adherent.
Finally, we further explored the relationship between the inhibitory activity of Sj7170 protease and the growth and metastasis of glioma. According to the key site of the inhibitory activity of Sj7170, 10 mutants of Sj7170 were designed at the molecular level, and 10 mutant gene sequences of Sj7170 were obtained by using overlapping PCR method. The same expression and purification system of Sj7170 was used to express and purify 10 Sj7170 mutant peptides. The inhibitory activity of the protease and the proliferation and metastasis of glioma cells were measured respectively. The assay of the inhibitory activity of chymotrypsin showed that the two stage structure and protease inhibitory activity of the mutant Sj7170-G35A were compared with the wild type Sj7170. The Ki value was 2.1 x 10-9M and the inhibitory activity was 185.71 times higher than that of Sj7170. However, the proliferation experiment of glioma cells showed that the mutant Sj7170-G35A did not greatly promote the growth and colonization of U87 in the glioma cells compared with the wild type Sj7170. At the same time, the other 9 mutant peptides of the pancreatic curd eggs The inhibitory activity of white enzyme was less than that of the wild type Sj7170 and did not have the ability to promote the proliferation of glioma cells. Cell migration experiments showed that there was no good correlation between the ability of the 10 Sj7170 mutants to promote the migration of glioma U87 cells with the protease inhibitory activity. These results all showed that the Sj7170 There is no direct connection between the two functions. The proliferation and migration function of glioma cells is not dependent on the function of its protease inhibitory activity. The two may play different biological effects in the organism.
To sum up, the Sj7170 is the first bifunctional molecule with the first isolation and identification of the serine protease inhibitory activity and the activation effect of glioma. It not only provides a new perspective for understanding the natural peptide library of scorpion toxin, but also provides a serine protease inhibitor as a tumor growth / transfer regulator. The new findings strengthen the functional association between serine protease inhibition and tumor growth transfer activator, which can help to further reveal the molecular mechanism of tumor occurrence and suggest that it may have potential value.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4

【相似文献】

相关期刊论文 前10条

1 陈赞,杨立庄,叶伟,王伊龙,赵鹏,李永利,康军,郑永日,曹阳;神经胶质瘤细胞热休克蛋白抗原肽复合物的研究[J];中华实验外科杂志;2003年05期

2 陈赞 ,杨立庄 ,叶伟;神经胶质瘤细胞热休克蛋白抗原肽复合物的研究[J];哈尔滨医科大学学报;2003年05期

3 张玲华;;神经胶质瘤的诊断与治疗现状[J];实用医技杂志;2007年24期

4 周文光;李南华;王廷芳;;鼻内型神经胶质瘤一例[J];临床耳鼻咽喉头颈外科杂志;1990年02期

5 ;颅面神经胶质瘤[J];国外医学.耳鼻咽喉科学分册;1997年02期

6 薛德麟,雷霆;要重视神经胶质瘤的治疗[J];中国临床神经外科杂志;2002年01期

7 肖敏 ,吴淼 ,曾冬竹 ,饶芸;树突状细胞瘤苗诱导的抗神经胶质瘤作用体外实验研究[J];现代医药卫生;2003年01期

8 戴捷,于\，

本文编号：2036726