發(fā)布時間：2018-04-19 09:24

  本文選題：弓形蟲 + 蛋白質(zhì)相互作用��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：第一部分弓形蟲棒狀體蛋白16的致病機制研究 剛地弓形蟲是一種專性細(xì)胞內(nèi)寄生原蟲,可感染幾乎所有的恒溫動物。近年來,隨著城市的發(fā)展、人口的增加,寵物飼養(yǎng)隊伍不斷地擴大,加上忽略飲食衛(wèi)生等因素,弓形蟲感染潛在危險性極度上升。孕婦感染弓形蟲后,通常能將這種感染傳給胎兒,如果感染發(fā)生在胚胎發(fā)育早期可導(dǎo)致智障、眼和腦疾病、其它多臟器疾病等嚴(yán)重后果,影響胎兒的發(fā)育,導(dǎo)致流產(chǎn)、畸胎、死胎、早產(chǎn)、出生缺陷等先天性弓形蟲病。調(diào)查顯示,我國先天性出生缺陷患兒當(dāng)中弓形蟲的感染率達(dá)到22%,弱智和精神病患者的發(fā)病與弓形蟲感染相關(guān)。 弓形蟲入侵人體細(xì)胞過程,棒狀體蛋白發(fā)揮了極其重要的作用。迄今僅發(fā)現(xiàn)兩種弓形蟲棒狀體蛋白可侵入人體細(xì)胞核內(nèi),分別是棒狀體蛋白16(ROP16)和PP2C-Hn。ROP16可快速侵入人體細(xì)胞核,業(yè)已證明ROP16可影響人體細(xì)胞的信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子(STAT)信號傳導(dǎo)途徑從而干擾人體細(xì)胞的增殖、分化、凋亡等功能。但該蛋白入核后發(fā)揮的具體功能并不知曉。 我們的假設(shè)是：1、既然ROP16可以進(jìn)入人體細(xì)胞核,對于異源性蛋白入侵,就可影響人體細(xì)胞的生理功能；2、既然弓形蟲對腦細(xì)胞有傾向性,那么侵入腦細(xì)胞的弓形蟲就可能影響這些細(xì)胞的增殖、分化、凋亡等生理功能,同樣ROP16入核后,可影響細(xì)胞核功能。3、既然弓形蟲感染胚胎腦細(xì)胞,那么就有可能影響早期大腦的發(fā)育。因此我們旨在從ROP16功能的基礎(chǔ)研究著手,探索它與人體相互作用的核蛋白,獲取下游靶基因的信息從中尋找與生長發(fā)育相關(guān)的因子,以及ROP16與相互作用的蛋白結(jié)合后對其功能的影響,揭示弓形蟲腦病的形成以及弓形蟲感染導(dǎo)致出生缺陷和智障的機制。 本研究工作主要集中于ROP16與人體相互作用核蛋白的篩選和鑒定及蛋白相互作用之后對功能的影響,主要內(nèi)容及結(jié)果如下： (1)成功構(gòu)建表達(dá)ROP16混合克隆細(xì)胞株：采用國際公認(rèn)的弓形蟲強毒株RH株感染C57小鼠,待其出現(xiàn)明顯的弓形蟲感染癥狀后從中抽取腹水提RNA,RT-PCR擴增目的基因ROP16,構(gòu)建真核表達(dá)載體PEXPR-IBA-105IP-ROP16,重組載體經(jīng)lip2000瞬時轉(zhuǎn)染入293T細(xì)胞,并用嘌呤霉素進(jìn)行篩選得到混合克隆細(xì)胞株,Western-Blot檢測目的蛋白成功表達(dá)。 (2)篩選出與ROP16相互作用的人體核蛋白：培養(yǎng)穩(wěn)定表達(dá)ROP16的293T細(xì)胞,以免疫沉淀技術(shù)獲得與ROP16存在相互作用的人體核蛋白,質(zhì)譜分析后得到7個可信的核蛋白,可能與ROP16存在相互作用。 (3)以免疫共沉淀技術(shù)分別證實ROP16與7個候選核蛋白中的5個(Histone H2B, Histone H4, lamin B1,hnRNP A2/B1,STMN1)之間的相互作用,證明lamin B1(LMNB1)和histone H2B (HIST1H2B)與ROP16存在相互作用。 (4)與ROP16相互作用蛋白LMNB1及HIST1H2B進(jìn)一步證實和定位：以免疫共沉淀雙向驗證ROP16與LMNB1和HIST1H2B之間相互結(jié)合,經(jīng)激光共聚焦證實ROP16與LMNB1共定位于細(xì)胞核膜,ROP16與HIST1H2B共定位于細(xì)胞核。 (5)ROP16對神經(jīng)細(xì)胞生長、分化的影響：將ROP16基因cDNA克隆入慢病毒載體PWPI,用293T細(xì)胞包裝病毒顆粒,將構(gòu)建好的慢病毒感染人腦星型膠質(zhì)母細(xì)胞瘤細(xì)胞(u-87MG)(?)口神經(jīng)母細(xì)胞瘤細(xì)胞(SH-SY5Y),real-time PCR方法檢測得到ROP16下調(diào)星型膠質(zhì)細(xì)胞和神經(jīng)元標(biāo)志基因。 (6)ROP16對神經(jīng)細(xì)胞凋亡影響：TUNEL和流式細(xì)胞術(shù)檢測ROP16對u-87MG和SH-SY5Y細(xì)胞凋亡的影響,證明ROP16促進(jìn)二者的凋亡,CCK-8實驗證實ROP16對二者增殖無顯著影響。 (7)ROP16與LMNB1相互作用后對其功能的影響：以全反式維甲酸(RA)誘導(dǎo)SH-SY5Y細(xì)胞向神經(jīng)元分化,電鏡檢測顯示少量神經(jīng)元細(xì)胞核仁增多、核膜空泡化、大量的微管成簇排列。 結(jié)論：ROP16分別與LMNB1和HIST1H2B存在相互作用。ROP16抑制u-87MG細(xì)胞的生長和SH-SY5Y細(xì)胞向神經(jīng)元的分化。ROP16促進(jìn)u-87MG和SH-SY5Y細(xì)胞的凋亡。ROP16與LMNB1相互作用后可能影響LMNB1維持核膜形態(tài)等功能,使神經(jīng)元細(xì)胞形態(tài)發(fā)生異常。 第二部分TFPI-2與PSAP相互作用的研究 組織因子途徑抑制物-2(tissue factor pathway inhibitor-2, TFPI-2)又稱胎盤蛋白—5(placenta protein-5, PP-5)和基質(zhì)相關(guān)絲氨酸蛋白酶抑制劑(matrix associated serine protease inhibitor, MSPI),是一種帶有Kunitz型結(jié)構(gòu)域的蛋白酶抑制劑,屬于絲氨酸蛋白酶抑制物超家族成員。成熟的人TFPI-2由富含酸性氨基酸的N端、三個串聯(lián)的Kuntiz (?)吉構(gòu)域和富含堿性氨基酸的C端組成。眾所周知,人TFPI-2能夠抑制胰蛋白酶、纖溶酶、糜蛋白酶、血漿緩激肽釋放酶、組織蛋白酶G和多種基質(zhì)金屬蛋白酶(MMPs)的活性,從而對抗多種惡性腫瘤的侵襲和轉(zhuǎn)移,如前列腺癌、肺癌、纖維肉瘤、黑色素瘤和神經(jīng)膠質(zhì)瘤等。此外,人TFPI-2還可以抑制動脈粥樣硬化斑塊的脫落。但迄今為止,對TFPI-2的生理功能尚不清楚,TFPI-2與其它蛋白質(zhì)相互作用的信息也知之甚少。 在前期研究中,我們從酵母雙雜交技術(shù)篩選出與TFPI-2相互作用的候選蛋白,其中之一為鞘脂激活蛋白原rosaposin (PSAP)。PSAP是一個具有多種神經(jīng)營養(yǎng)作用的多功能糖蛋白,最初被認(rèn)為是4種鞘脂激活蛋白A、B、C和、D的前體。鞘脂激活蛋白A、B、C和、D功能是作為鞘脂糖水解途徑中所涉及到溶酶體酶的激活劑。成熟的鞘脂激活蛋白由PSAP通過蛋白水解途徑產(chǎn)生,再運送至溶酶體。作為完整的分泌性蛋白,PSAP存在于多種生物體液中,如人乳汁、血清、腦脊液和精液漿等,也常見于神經(jīng)及其它質(zhì)膜中。 本項研究是對前期工作的進(jìn)一步延續(xù)和拓寬,主要集中于TFPI-2與PSAP內(nèi)源性相互作用的進(jìn)一步確認(rèn)、及二者蛋白相互作用后對各自功能的影響。通過酵母共轉(zhuǎn)化、GST-pulldown等實驗TFPI-2和PSAP在酵母及細(xì)胞內(nèi)外的相互作用；在通過細(xì)胞侵襲和遷移實驗驗證二者相互作用后在體外功能的影響,進(jìn)一步闡明TFPI-2的生理功能。主要內(nèi)容及結(jié)果如下： (1)確定了TFPI-2和PSAP內(nèi)源性相互作用。 (2)證明PSAP可以促進(jìn)人纖維肉瘤細(xì)胞的侵襲及遷移能力、TFPI-2可以抑制PSAP作用；PSAP可以增強HT1080細(xì)胞分泌的MMP-2的活性并呈劑量依賴性；同樣,TFPI-2也可以抑制PSAP。 (3) PSAP對MMP-2和MMP-9的表達(dá)量沒有影響。 結(jié)論：TFPI-2與PSAP存在內(nèi)源性相互作用,并對其促進(jìn)纖維肉瘤侵襲和遷移功能具有顯著的抑制作用。
[Abstract]:Study on the pathogenesis of the first part of the Toxoplasma gondii rod protein 16
Toxoplasma gondii is an obligate intracellular protozoan parasite can infect almost all warm blooded animal. In recent years, with the development of the city, the population increase, pets team continues to expand, and ignore the health diet and other factors, the potential risk of Toxoplasma infection was increased. Toxoplasma infection in pregnant women. Usually can be the infection to the fetus, if the infection occurs in the early embryo development can lead to mental retardation, eye and other brain diseases, multiple organ diseases and other serious consequences, affect fetal development, leading to miscarriage, stillbirth, premature birth, fetus, birth defects and congenital toxoplasmosis. The survey shows that China's congenital birth defects in children among the Toxoplasma infection rate reached 22%, the incidence of retarded and psychotic patients infected with Toxoplasma gondii.
The invasion of Toxoplasma gondii rhoptry protein in human cells, play a very important role. So far only found two kinds of Toxoplasma gondii rhoptry protein can invade the human body and the nucleus are rhoptry protein 16 (ROP16) and PP2C-Hn.ROP16 can quickly invade the human nucleus, proving that ROP16 can activate human cell signal transduction and transcription factor (STAT) signal transduction pathway which interfere with human cell proliferation, differentiation, apoptosis and other functions. But the specific function of protein into the nucleus after the play is not known.
Our hypothesis is: 1, since ROP16 can enter the cell nucleus of the human body, for heterologous protein invasion, can affect the physiological function of human cells; since 2, Toxoplasma on brain cells have the inclination, then invade the brain cells of Toxoplasma gondii may influence the cell proliferation, differentiation, apoptosis and other physiological functions, the same ROP16 into the nucleus, nucleus can affect the function of.3, the embryo brain cells of Toxoplasma infection, so it may affect early brain development. Therefore we aimed to research from the ROP16 function to explore the interaction between human and nuclear protein, obtaining information from the downstream target genes for growth and development of the and the ROP16 factor, and the interaction of protein binding on the function, reveal the formation of Toxoplasma encephalopathy and Toxoplasma infection mechanisms leading to birth defects and mental retardation.
This work focuses on the screening and identification of nucleoprotein interacting with human body and the effect of protein interaction on function after ROP16. The main contents and results are as follows:
(1) the successful construction of the expression of ROP16 hybrid cell clone: adopt internationally recognized Toxoplasma virulent strain RH. C57 mice were infected, the apparent Toxoplasma infection symptoms after extracted from ascites RNA, RT-PCR amplification of ROP16 gene and construct the eukaryotic expression vector PEXPR-IBA-105IP-ROP16, the recombinant vector was transiently transfected into 293T by lip2000 cells, and by puromycin screening hybrid cell clone, Western-Blot to detect protein expressed successfully.
(2) to screen human nucleoprotein interacting with ROP16: to cultivate 293T cells that stably express ROP16, to obtain human nucleoprotein interacting with ROP16 by immunoprecipitation technology, and to obtain 7 credible nucleoproteins by mass spectrometry, which may interact with ROP16.
(3) the interaction between ROP16 and 5 candidate nuclear proteins (Histone H2B, Histone H4, lamin B1, hnRNP A2/B1, STMN1) was confirmed by CO immunoprecipitation technology. It was proved that the interaction between lamin and H2B was existed.
(4) the interaction proteins LMNB1 and HIST1H2B with ROP16 were further confirmed and located: the combination of ROP16 and LMNB1 and HIST1H2B was confirmed by CO immunoprecipitation. Confocal laser scanning showed that ROP16 and LMNB1 were located in the cell nuclear membrane, and ROP16 and HIST1H2B were located in the nucleus.
(5) ROP16 on the growth of nerve cells, differentiation effect: ROP16 cDNA gene was cloned into the lentiviral vector PWPI with 293T cells to package virus particles, the constructed lentivirus infected human glioblastoma cells (u-87MG) (?) in neuroblastoma cells (SH-SY5Y), the detection of real-time method of PCR ROP16 down-regulation of astrocyte and neuron marker genes.
(6) the effect of ROP16 on neuronal apoptosis: TUNEL and flow cytometry were used to detect the effect of ROP16 on the apoptosis of u-87MG and SH-SY5Y cells. It was proved that ROP16 promoted the apoptosis of the two. CCK-8 test confirmed that ROP16 had no significant effect on the proliferation of the two cells.
(7) the interaction of ROP16 and LMNB1 on its function: the induction of SH-SY5Y cells into neurons by all trans retinoic acid (RA). Electron microscopy showed that a small number of neurons increased nucleolus, vacuolated the nuclear membrane, and a large number of microtubules were arranged in clusters.
Conclusion: ROP16 interaction between.ROP16 inhibition promotes neuronal apoptosis of.ROP16.ROP16 and LMNB1 u-87MG and SH-SY5Y cell interactions may influence the function of LMNB1 to maintain the nuclear morphology of cell growth and SH-SY5Y of u-87MG cells with LMNB1 and HIST1H2B, the neural cell morphology abnormalities.
The study of the interaction between second parts TFPI-2 and PSAP
Tissue factor pathway inhibitor -2 (tissue factor pathway inhibitor-2, TFPI-2) and placental protein 5 (placenta, protein-5, PP-5) and matrix associated serine protease inhibitor (matrix associated serine protease inhibitor, MSPI), is a Kunitz type domain with protease inhibitors belonging to the serine protease inhibitor superfamily. The mature man TFPI-2 is rich in acidic amino acids N terminal, three tandem Kuntiz (?) and Kyrgyzstan domains rich in basic amino acids C terminal. As everyone knows, TFPI-2 can inhibit plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G and a variety of matrix metalloproteinase (MMPs) activity against a variety of malignant in order to tumor invasion and metastasis, such as prostate cancer, lung cancer, fibrosarcoma, melanoma and glioma. In addition, people can also inhibit TFPI-2 The loss of atherosclerotic plaque. But to date, the physiological function of TFPI-2 is not clear, and the information of the interaction between TFPI-2 and other proteins is little known.
In our previous study, we screened candidate proteins interacting with TFPI-2 by yeast two hybrid technique, which is one of the prosaposin rosaposin (PSAP).PSAP is a variety of neurotrophic effect of multifunctional glycoprotein, was initially thought to be 4 kinds of sphingolipid activated protein A, B, and C, before the body of the D. The sphingolipid activator protein A, B, C and D, function as sphingolipid hydrolysis pathways involved in the activation of lysosomal enzymes. The mature protein produced by sphingolipid activator PSAP through proteolytic pathway, and then transported to the lysosomes. As a secretory protein complete, PSAP exists in many kinds of biological fluids such as, human milk, serum, cerebrospinal fluid and seminal plasma, are also common in other nerve and plasma membrane.
This research is a preliminary work on the further extension and widening, mainly concentrated in the TFPI-2 and PSAP further confirmed the endogenous interaction, and effects of the two protein interaction on their functions. Through yeast CO transformation, the interaction between GST-pulldown and PSAP in the TFPI-2 and yeast cells and in the cell invasion and; the migration experiments in vitro effects of functional interaction between the two, to further clarify the physiological function of TFPI-2. The main contents and results are as follows:
(1) endogenous interaction between TFPI-2 and PSAP was determined.
(2) it is proved that PSAP can promote the invasion and migration of human fibrosarcoma cells. TFPI-2 can inhibit the effect of PSAP. PSAP can enhance the activity of MMP-2 secreted by HT1080 cells in a dose-dependent manner. Similarly, TFPI-2 can also inhibit PSAP..
(3) PSAP has no effect on the expression of MMP-2 and MMP-9.
Conclusion: there is an endogenous interaction between TFPI-2 and PSAP, and it has a significant inhibitory effect on promoting the invasion and migration of fibrosarcoma.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R382.5

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