本文選題：sj7170 + 蝎毒素　； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：神經(jīng)膠質(zhì)瘤是成人中最常見的原發(fā)性惡性腦瘤。目前被確診為神經(jīng)膠質(zhì)瘤的患者,大約有一半是處于晚期階段,神經(jīng)膠質(zhì)瘤患者平均預(yù)期的壽命約為14-18個月左右,因而神經(jīng)膠質(zhì)瘤是惡性程度最高的腫瘤之一�，F(xiàn)有的科學(xué)研究表明神經(jīng)膠質(zhì)瘤侵襲行為的分子機(jī)制十分復(fù)雜且仍不清楚。因此,作為分子工具的神經(jīng)膠質(zhì)瘤調(diào)節(jié)劑(激活劑和抑制劑)的發(fā)現(xiàn)將有助于揭示神經(jīng)膠質(zhì)瘤發(fā)生的分子機(jī)制,也有利于研發(fā)治療和診斷神經(jīng)膠質(zhì)瘤的藥物和技術(shù)。 絲氨酸蛋白酶抑制劑(SPIs)廣泛存在于各種生物體及其組織中,如寄生蟲、吸血性無脊椎動物、兩棲類動物的皮膚和有毒動物的毒腺分泌物,SPIs具有重要的生理/病理功能。近來研究表明Serpin和Kunitz家族的SPIs能導(dǎo)致腫瘤細(xì)胞惡性程度的增高。然而,所有報道的SPIs均是由內(nèi)源性的人類細(xì)胞所分泌的,還沒有任何關(guān)于外源性SPIs促腫瘤作用的報道,并且內(nèi)源性SPIs促腫瘤的分子機(jī)制仍然不清楚,更為重要的是,SPIs促腫瘤研究還從未涉及神經(jīng)膠質(zhì)瘤。因此,發(fā)現(xiàn)新型SPIs并研究其促神經(jīng)膠質(zhì)瘤作用及其機(jī)制具有重要的科學(xué)意義與應(yīng)用價值。 本研究從普洱真蝎cDNA文庫中篩選鑒定到一條cDNA序列,命名為Sj7170。 Sj7170的全長cDNA序列為459個核苷酸,開放閱讀框含有261個核苷酸共編碼86個氨基酸殘基,其中包含24個殘基的信號肽和62個殘基的成熟肽,形成5對二硫鍵。序列比對的結(jié)果表明Sj7170具有典型的Ascaris型抑制劑的特征,并且可能存在潛在的絲氨酸蛋白酶抑制活性�；赟j7170的cDNA序列設(shè)計了特異性引物,通過PCR擴(kuò)增成熟肽序列,構(gòu)建重組表達(dá)質(zhì)粒pET-28a-Sj7170,并將其轉(zhuǎn)化到大腸桿菌中,得到了色譜純的重組Sj7170多肽。在體內(nèi)外檢測了重組Sj7170多肽的絲氨酸蛋白酶抑制活性。結(jié)果表明,rSj7170可以特異性地抑制胰凝乳蛋白酶的活性,并且體外的抑制常數(shù)Ki值為3.9×10-7M,對胰蛋白酶和彈性蛋白酶的活性沒有明顯的影響。 進(jìn)一步研究發(fā)現(xiàn)蝎絲氨酸蛋白酶抑制劑Sj7170能促神經(jīng)膠質(zhì)瘤細(xì)胞的增殖。細(xì)胞計數(shù)實驗結(jié)果表明rSj7170對神經(jīng)膠質(zhì)瘤細(xì)胞U87和U251的增殖均有顯著的促進(jìn)作用。細(xì)胞克隆形成實驗顯示rSj7170同樣能增強(qiáng)單個U87細(xì)胞形成克隆菌落的能力。細(xì)胞周期流式分析發(fā)現(xiàn),rSj7170使U87細(xì)胞從G1期進(jìn)入S期的時間縮短,從而加速了U87細(xì)胞周期的進(jìn)程。Western blot研究結(jié)果表明,rSj7170促進(jìn)了周期蛋白cyclinD1和周期轉(zhuǎn)錄激活子E2F1的表達(dá),增強(qiáng)了Rb2/p130蛋白的磷酸化,抑制了周期轉(zhuǎn)錄抑制子E2F5的表達(dá)。通過構(gòu)建下調(diào)周期蛋白cyclinD1細(xì)胞系進(jìn)一步確證了cyclinD1在Sj7170促細(xì)胞增值過程中起到的重要作用。動物體內(nèi)實驗進(jìn)一步證實,rSj7170促進(jìn)了U87細(xì)胞在裸鼠體內(nèi)腫瘤的形成和生長,同時上調(diào)了腫瘤組織中cyclinD1的表達(dá)。 研究還發(fā)現(xiàn)蝎絲氨酸蛋白酶抑制劑Sj7170能促進(jìn)神經(jīng)膠質(zhì)瘤細(xì)胞的遷移和侵襲。Transwell實驗表明,rSj7170能顯著的促進(jìn)神經(jīng)膠質(zhì)瘤U87和U251的遷移和侵襲,改變倍數(shù)均為10倍以上。EMT標(biāo)記蛋白實驗顯示,rSj7170能上調(diào)神經(jīng)膠質(zhì)瘤細(xì)胞U87和U251中Vimentin和Snail蛋白的表達(dá),抑制E-cadherin的表達(dá),誘導(dǎo)神經(jīng)膠質(zhì)瘤細(xì)胞U87和U251上皮特征向間質(zhì)特征轉(zhuǎn)化。進(jìn)一步構(gòu)建下調(diào)轉(zhuǎn)錄因子Snail細(xì)胞系的結(jié)果表明,Snail下調(diào)后rSj7170的促遷移和侵襲作用消失。因此Snail蛋白的上調(diào)和EMT過程的發(fā)生對于Sj7170促神經(jīng)膠質(zhì)瘤細(xì)胞遷移和侵襲功能獲得和發(fā)生起著不可或缺的作用。而cyclinD1下調(diào)后rSj7170仍然具有促進(jìn)細(xì)胞遷移和侵襲的能力,因此cyclinD1并沒有在此過程中發(fā)揮作用。免疫熒光實驗結(jié)果顯示,rSj7170通過Rho GTPase通路來使細(xì)胞骨架發(fā)生重排,從而使神經(jīng)膠質(zhì)瘤細(xì)胞U87的細(xì)胞球狀生長形態(tài)發(fā)生改變,使之貼壁性更強(qiáng)。 最后進(jìn)一步探索了Sj7170蛋白酶抑制活性和促神經(jīng)膠質(zhì)瘤生長和轉(zhuǎn)移作用的關(guān)系。根據(jù)Sj7170的胰凝乳蛋白酶抑制活性關(guān)鍵位點,在分子水平上設(shè)計了10個Sj7170的突變體,采用overlapping PCR的方法獲得了Sj7170的10個突變體基因序列,利用與野生型Sj7170相同的表達(dá)純化系統(tǒng),表達(dá)純化了10個Sj7170突變體多肽,分別測定其蛋白酶抑制活性和促神經(jīng)膠質(zhì)瘤細(xì)胞增殖和轉(zhuǎn)移能力。胰凝乳蛋白酶抑制活性測定實驗發(fā)現(xiàn),突變體Sj7170-G35A的二級結(jié)構(gòu)和蛋白酶抑制活性與野生型Sj7170相比,均發(fā)生了顯著的改變,其Ki值為2.1×10-9M,抑制活性較Sj7170提高了185.71倍。但神經(jīng)膠質(zhì)瘤細(xì)胞增殖實驗結(jié)果顯示與野生型Sj7170相比,突變體Sj7170-G35A并沒有極大地促進(jìn)神經(jīng)膠質(zhì)瘤細(xì)胞U87的生長和增殖。同時,其它9個突變體多肽的胰凝乳蛋白酶抑制活性較野生型Sj7170變化不大,并且也不具有促神經(jīng)膠質(zhì)瘤細(xì)胞增殖的能力。細(xì)胞遷移實驗結(jié)果顯示,Sj7170的10個突變體促進(jìn)神經(jīng)膠質(zhì)瘤U87細(xì)胞遷移的能力與蛋白酶抑制活性之間并沒有很好的相關(guān)性。這些結(jié)果均表明,Sj7170的這兩種功能之間并沒有直接的聯(lián)系,其促神經(jīng)膠質(zhì)瘤細(xì)胞增殖和遷移功能并不依賴于其蛋白酶抑制活性功能,二者可能在生物體內(nèi)發(fā)揮不同的生物學(xué)效應(yīng)。 綜上所述,本研究發(fā)現(xiàn)的Sj7170是首個分離鑒定的具有絲氨酸蛋白酶抑制活性和神經(jīng)膠質(zhì)瘤激活效應(yīng)的雙功能分子,不僅對深入了解蝎毒素的天然多肽庫提供了新的視角,同時也為絲氨酸蛋白酶抑制劑作為腫瘤生長／轉(zhuǎn)移調(diào)節(jié)劑提供了新的發(fā)現(xiàn),加強(qiáng)了絲氨酸蛋白酶抑制和腫瘤生長轉(zhuǎn)移激活劑之間的功能聯(lián)系,這些都有助于進(jìn)一步揭示腫瘤發(fā)生的分子機(jī)制,并提示其可能具有潛在價值。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.4

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