發(fā)布時(shí)間：2018-05-23 08:45

  本文選題：華支睪吸蟲 + 分泌型磷脂酶A2��； 參考：《中山大學(xué)》2016年博士論文

【摘要】：華支睪吸蟲(Clonorchis sinensis,Cs)是一種致病性吸蟲,主要由食入感染華支睪吸蟲囊蚴的“魚生”感染,成蟲寄生于人及其他哺乳動物肝膽管系統(tǒng),蟲體的機(jī)械性刺激和分泌/排泄物可以刺激膽管上皮及結(jié)締組織增生,引起膽道炎癥、膽管阻塞、甚至膽管癌、肝硬化、肝癌,全世界均有患者發(fā)病和死亡,但主要流行于東亞地區(qū)包括中國、韓國和越南。目前中國有1300萬患者左右。近年來,華支睪吸蟲病發(fā)病呈上升趨勢,已成為我國流行最廣、危害最大的食源性寄生蟲病之一,2005年廣東省已將其列為重點(diǎn)防治的三大地方病之一,2006年衛(wèi)生部已將其列為重點(diǎn)防治的寄生蟲病。對華支睪吸蟲引起肝纖維化和肝硬化的分子機(jī)制進(jìn)行深入研究,對防治華支睪吸蟲病具有重要的意義。肝纖維化是由于肝細(xì)胞外基質(zhì)(ECM)合成和降解失衡,ECM過度沉積而發(fā)生的,肝星狀細(xì)胞(HSC)活化、增殖是肝纖維化的關(guān)鍵事件。已有實(shí)驗(yàn)證明華支睪吸蟲分泌排泄產(chǎn)物(Cs ESP)通過使肝星狀細(xì)胞活化增殖導(dǎo)致肝纖維化產(chǎn)生,還可使膽管上皮細(xì)胞(BEC)增生、癌變致膽管癌的發(fā)生,其中Cs ESP的成分之一分泌型磷脂酶A2(s PLA2)能引起HSC的活化增殖,這種作用不能被其酶活性的作用底物卵磷脂所阻斷,而只能被其特異性抗體所阻斷,顯示Css PLA2發(fā)揮使HSC活化增殖的作用不是通過酶活性而是通過結(jié)合相關(guān)受體實(shí)現(xiàn)的,但與其作用的受體蛋白及其活化HSC的具體分子機(jī)制仍不清楚。因此探究華支睪吸蟲分泌型磷脂酶A2(Css PLA2)相互作用的蛋白質(zhì)及其相關(guān)的信號通路,對進(jìn)一步了解華支睪吸蟲引起肝纖維化的有重要意義。研究目的和意義基于探究和華支睪吸蟲分泌型磷脂酶A2(Css PLA2)相互作用的蛋白質(zhì)及與其相互作用相關(guān)的信號通路,對進(jìn)一步了解該蛋白分子在華支睪吸蟲引起肝纖維化中的作用有重要意義。本課題以下列研究為目標(biāo),初步探討Css PLA2相互作用蛋白質(zhì)的篩選與其功能:(1)酵母雙雜交初步篩選出與Css PLA2相互作用蛋白質(zhì);(2)對篩選出與Css PLA2相互作用蛋白質(zhì)進(jìn)行免疫共沉淀、GST Pull-down等體外實(shí)驗(yàn)進(jìn)一步驗(yàn)證;(3)探究Css PLA2及其相互作用蛋白質(zhì)結(jié)合后對人肝星狀細(xì)胞系LX2細(xì)胞效應(yīng)及其可能涉及的相關(guān)細(xì)胞信號通路。研究方法1.利用酵母雙雜交篩選與Css PLA2相互作用的蛋白質(zhì)。構(gòu)建誘餌載體(p GBKT7-s PLA2),并轉(zhuǎn)化到酵母菌Y2HGold,誘餌蛋白自身激活測試、毒性測試及表達(dá)驗(yàn)證后,含誘餌載體的酵母Y2HGold與含人肝c DNA文庫載體(p GADT7)的酵母Y187進(jìn)行雜交(mating),雜交后的產(chǎn)物涂布于營養(yǎng)缺陷型培養(yǎng)基生長,選取生長出的酵母菌落,擴(kuò)大培養(yǎng)提取質(zhì)粒后PCR產(chǎn)物送測序。對測序結(jié)果進(jìn)行初步分析,篩選出和Css PLA2直接相互作用蛋白質(zhì)。2.免疫共沉淀(CO-IP)驗(yàn)證Css PLA2及篩選出的蛋白質(zhì)間相互作用將Css PLA2及相互作用候選蛋白質(zhì)TM7SF3分別克隆到真核表達(dá)載體p EGFP和pc DNA6,構(gòu)建好的重組載體利用Lipofectamine 2000瞬時(shí)共轉(zhuǎn)染轉(zhuǎn)染到293T細(xì)胞,以單獨(dú)轉(zhuǎn)染重組Css PLA2表達(dá)載體和重組相互作用蛋白質(zhì)表達(dá)載體作為對照組。轉(zhuǎn)染成功后293T細(xì)胞以RIPA液裂解,細(xì)胞裂解液與Protein A瓊脂糖珠在4°C共孵育過夜后離心沉淀后洗滌,再用抗c-Myc抗體及抗PLA2抗體分別進(jìn)行Western blotting分析。3.GST Pull-down驗(yàn)證Css PLA2及篩選出的蛋白質(zhì)間相互作用將Css PLA2及相互作用蛋白質(zhì)TM7SF3分別克隆到表達(dá)載體p MAL-c2x和p GEX-4T-1,在大腸桿菌中表達(dá),用親和層析法純化目的蛋白,并做SDS-PAGE分析。帶MBP標(biāo)簽的Css PLA2和帶GST標(biāo)簽的蛋白質(zhì)GST-TM7SF3混合物用Amylose resin進(jìn)行親和層析純化,以MBP蛋白、GST蛋白分別與MBP-Css PLA2混合后在Amylose resin中親和層析純化的產(chǎn)物作為對照組,純化后的產(chǎn)物均用抗GST抗體進(jìn)行Western blotting分析。4.對Css PLA2相互作用蛋白質(zhì)在LX2細(xì)胞上進(jìn)行免疫組化定位免疫組化法測定Css PLA2相互作用蛋白質(zhì)TM7SF3在LX2細(xì)胞上的細(xì)胞定位,LX2細(xì)胞與或不與抗TM7SF3抗體孵育后經(jīng)蘇木素染色,洗滌后觀察細(xì)胞染色情況及黃褐色顆粒分布位置。5.檢測Css PLA2及篩選出的蛋白質(zhì)對LX2細(xì)胞的作用及其涉及的相關(guān)信號通路LX2細(xì)胞分別用重組Css PLA2蛋白、重組Css PLA2及抗TM7SF3抗體混合物及PBS孵育24h后,CCK-8法分別測定LX2細(xì)胞增殖情況,實(shí)時(shí)熒光定量PCR檢測TGF-β、ERK、JNK and NF-κB等與肝纖維化相關(guān)的信號通路的表達(dá)變化。研究結(jié)果1.酵母雙雜交篩選與Cs PLA2相互作用的蛋白質(zhì)酵母雙雜交初步篩選出5個(gè)可能與Css PLA2相互作用的蛋白質(zhì),其中1個(gè)定位于細(xì)胞膜(TM7SF3)、1個(gè)為分泌蛋白質(zhì)(hemopexin,isoform CRA_a)、3個(gè)定位于線粒體(metallothionein-2、aminoacylase-1 isoform b、cytochrome c oxidase subunit I)。排除定位于線粒體不能與Css PLA2直接結(jié)合的蛋白質(zhì)以及分泌游離于血液中的蛋白質(zhì),選定有可能與Css PLA2直接作用的定位于細(xì)胞膜上的蛋白TM7SF3進(jìn)行進(jìn)一步驗(yàn)證。2.CO-IP驗(yàn)證Css PLA2及篩選出的蛋白質(zhì)間相互作用重組后Css PLA2和TM7SF3蛋白均能在293T細(xì)胞中表達(dá)。單獨(dú)轉(zhuǎn)染p EGFP-Css PLA2的293T細(xì)胞裂解液經(jīng)Protein A agarose進(jìn)行免疫沉淀再洗滌后只能被抗PLA2抗體識別,單獨(dú)轉(zhuǎn)染pc DNA6-TM7SF3細(xì)胞裂解液經(jīng)Protein A agarose進(jìn)行免疫沉淀再洗滌后既不能被抗c-Myc抗體識別,也不能被抗PLA2抗體識別,只有p EGFP-Css PLA2和pc DNA6-TM7SF3共轉(zhuǎn)染后的293T細(xì)胞裂解液經(jīng)Protein A agarose進(jìn)行免疫沉淀再洗滌后,能被抗c-Myc抗體和抗PLA2抗體同時(shí)識別,顯示Css PLA2和TM7SF3蛋白存在相互作用。3.GST Pull-down驗(yàn)證Css PLA2及篩選出的蛋白質(zhì)間相互作用MBP-Css PLA2和GST-TM7SF3在大腸桿菌中表達(dá),相對分子質(zhì)量分別約為42k Da和70k Da。只有MBP-Css PLA2和GST-TM7SF3蛋白質(zhì)混合物經(jīng)只能沉積含MBP標(biāo)簽蛋白Amylose resinpull down后的產(chǎn)物能被抗GST抗體識別,Css PLA2和TM7SF3蛋白存在相互作用。4.對Css PLA2相互作用蛋白質(zhì)在LX2細(xì)胞上進(jìn)行免疫組化定位免疫組化法測定Css PLA2相互作用蛋白質(zhì)TM7SF3在LX2細(xì)胞上的細(xì)胞定位,LX2細(xì)胞與或不與抗TM7SF3抗體孵育后經(jīng)蘇木素染色,洗滌后觀察細(xì)胞染色情況及黃褐色顆粒分布位置。只有與抗TM7SF3抗體孵育后LX2細(xì)胞顯現(xiàn)棕黃色,且黃褐色顆粒主要集中于細(xì)胞膜上。5.檢測Css PLA2及篩選出的蛋白質(zhì)對LX2細(xì)胞的增殖作用及有關(guān)的信號通路CCK-8檢測結(jié)果顯示Css PLA2孵育24h后相比PBS對照組OD450結(jié)果顯示PBS孵育組LX2細(xì)胞OD450約為1.2,Css PLA2孵育組LX2細(xì)胞OD450約為1.5,LX2細(xì)胞增殖率較PBS組升高了25%,而Css PLA2和抗TM7SF3抗體混合物孵育組LX2細(xì)胞OD450約為1.0,細(xì)胞增殖率較Css PLA2孵育組降低了33%。結(jié)果顯示Css PLA2能夠使LX2細(xì)胞增殖,抗體阻斷TM7SF3后Css PLA2對LX2細(xì)胞增殖作用減弱,間接驗(yàn)證了TM7SF3和Css PLA2共同作用使LX2細(xì)胞增殖�？筎M7SF3抗體阻斷TM7SF3后,Css PLA2對LX2細(xì)胞活化增殖作用受抑制。Css PLA2孵育LX2細(xì)胞24h后,相比空白對照組,肝纖維化相關(guān)信號通路,包括ERK、JNK、NF-k B、TGF-Smad等通路關(guān)鍵基因表達(dá)水平也上調(diào),抗TM7SF3抗體阻斷TM7SF3后,相比Css PLA2孵育LX2細(xì)胞上述通路關(guān)鍵基因表達(dá)水平下調(diào)。結(jié)論1.酵母雙雜交篩選出和Css PLA2相互作用的人蛋白質(zhì)TM7SF3等。2.免疫共沉淀、GSTPull-down進(jìn)一步驗(yàn)證Css PLA2與人TM7SF3的相互作用。3.免疫組化對TM7SF3在人肝星狀細(xì)胞系LX2細(xì)胞上定位,主要位于細(xì)胞膜。4.通過TM7SF3抗體阻斷等反向驗(yàn)證Css PLA2致肝纖維化所涉及的相關(guān)信號通路,包括ERK、JNK、NF-k B、TGF-Smad等通路。
[Abstract]:Clonorchis sinensis (Cs) is a kind of pathogenic fluke, which mainly infects the "fish students" infected with the cysts of Clonorchis sinensis, the adult parasites in human and other mammalian hepatobiliary system. The mechanical stimulation and secretion / excretion of the body can stimulate the hyperplasia of bile duct epithelium and connective tissue, cause biliary inflammation and bile duct. Obstruction, even cholangiocarcinoma, liver cirrhosis, liver cancer, and death in all the world, but mainly in East Asia, including China, Korea and Vietnam. At present, there are 13 million patients in China. In recent years, the incidence of Clonorchis sinensis has been on the rise, which has become one of the most widespread and hardest food borne parasites in China in 2005. Guangdong province has been listed as one of the three major endemic diseases in key prevention and control. In 2006, the Ministry of health has listed it as a major parasitic disease. The molecular mechanism of liver fibrosis and cirrhosis caused by Clonorchis sinensis is very important for the prevention and control of Clonorchis sinensis. The liver fibrosis is due to the liver extracellular matrix (ECM). It has been proved that the secretion and excretory product of Clonorchis sinensis (Cs ESP) is produced by the activation and proliferation of hepatic stellate cells (Cs ESP), and the proliferation of hepatic stellate cells (Cs ESP), and the proliferation of bile duct epithelial cells (BEC) and carcinogenesis of cholangiocarcinoma. One of the components of Cs ESP, secretory phospholipase A2 (s PLA2), can cause the activation and proliferation of HSC, which can not be blocked by its enzyme activity as a substrate for phosphatidylcholine, but can only be blocked by its specific antibody. It shows that Css PLA2 plays the role of activating HSC not by enzyme activity but by binding to related receptors. However, the specific molecular mechanism of its receptor protein and its activation of HSC is still unclear. Therefore, exploring the protein and related signaling pathway of the interaction of the secretory phospholipase A2 (Css PLA2) of Clonorchis sinensis is of great significance to the further study of the liver fibrosis caused by Clonorchis sinensis. The purpose and significance of this study are based on the exploration and China. The proteins interacting with the secretory phospholipase A2 (Css PLA2) of Clonorchis Clonorchis and the signaling pathway related to their interaction are of great significance for further understanding the role of this protein molecule in the liver fibrosis caused by Clonorchis sinensis. The following research aims at the preliminary study of the screening of Css PLA2 interaction protein and its work. (1) a preliminary screening of the interaction protein with Css PLA2 by yeast two hybrid; (2) further verification of the immune coprecipitation and GST Pull-down screening with Css PLA2 interaction proteins; (3) exploring the LX2 cell effect of Css PLA2 and its interacting protein binding to the human hepatic stellate cell line and its possible phase Cell signaling pathway. Study method 1. use yeast two hybrid to screen proteins interacting with Css PLA2. Construct bait carrier (P GBKT7-s PLA2), and convert to yeast Y2HGold, bait protein self activation test, toxicity test and expression validation, yeast Y2HGold containing bait carrier and human liver C DNA library carrier (P GADT7). Yeast Y187 was used to hybridize (mating). The products after hybridization were coated on the growth of nutrient deficient culture medium. The growing yeast colony was selected and the PCR products were sequenced. The sequencing results were preliminarily analyzed, and the direct interaction of Css PLA2 with.2. immunoprecipitation (CO-IP) was selected to verify Css PLA2 and to be screened out. Css PLA2 and the interaction candidate protein TM7SF3 were cloned to the eukaryotic expression vector p EGFP and PC DNA6, respectively. The recombinant vector was transfected to 293T cells by instantaneous co transfection by Lipofectamine 2000, and the recombinant Css PLA2 expression vector and recombinant protein expression vector were transfected as a pair. After the transfection, the 293T cells were lysed with RIPA solution, the cell lysate and Protein A agarose beads were incubated at 4 degree C for the night after the centrifuge precipitation. The Western blotting analysis of the Western blotting analysis.3.GST Pull-down was used to verify the.3.GST Pull-down and the interaction between the proteins and the screened proteins. Protein TM7SF3 was cloned to express vector p MAL-c2x and P GEX-4T-1, expressed in Escherichia coli, purified the target protein by affinity chromatography, and made SDS-PAGE analysis. Css PLA2 with MBP label and GST-TM7SF3 mixture with GST label were purified by affinity chromatography with Amylose. The products purified by affinity chromatography in Amylose resin were used as the control group, and the purified products were all Western blotting analysis with anti GST antibody and.4. on Css PLA2 interacting protein on LX2 cells, immunohistochemical localization method was used to determine the cell determination of Css PLA2. After incubation with or without anti TM7SF3 antibody, LX2 cells were stained with hematoxylin, and after washing, the staining of cells and the distribution of yellow brown particles were observed by.5.. The effects of Css PLA2 and the selected proteins on LX2 cells and related signaling pathways were used for LX2 cells, recombinant Css PLA2 protein, Css PLA2 and anti TM7SF3 antibodies. After the mixture and PBS were incubated for 24h, the proliferation of LX2 cells was measured by CCK-8 method, and the expression of TGF- beta, ERK, JNK and NF- kappa B and so on were detected by real-time fluorescent quantitative PCR. The results of 1. yeast two hybrid screening for protein yeast two hybrid screening with the interaction of Cs 1 of the interacting proteins are located in the cell membrane (TM7SF3), the 1 are secreting proteins (hemopexin, isoform CRA_a), and the 3 are located in the mitochondria (metallothionein-2, aminoacylase-1 isoform B, cytochrome c oxidase subunit). Protein in liquid, the protein TM7SF3, which is located directly on the membrane of Css PLA2, is selected for further verification by.2.CO-IP verification that the interaction of Css PLA2 and selected proteins can be expressed in 293T cells after the recombination of Css PLA2 and TM7SF3 protein. After immunoprecipitation and re washing, A agarose can only be identified by anti PLA2 antibody. PC DNA6-TM7SF3 cell lysate can not be identified by anti c-Myc antibody and can not be identified by anti PLA2 antibody after Protein A agarose. After immunoprecipitation and re washing, Protein A agarose can be identified by anti c-Myc and anti PLA2 antibodies. The interaction of Css PLA2 and TM7SF3 protein exists in the presence of.3.GST Pull-down to verify that Css PLA2 and selected proteins are interacted and expressed in Escherichia coli. And 70K Da. only MBP-Css PLA2 and GST-TM7SF3 protein mixture can be identified by anti GST antibody after only Amylose resinpull down containing MBP label protein, Css PLA2 and the interaction of the proteins exist. The interaction protein TM7SF3 was located on the cells of LX2 cells. LX2 cells were stained with or not incubated with anti TM7SF3 antibodies by hematoxylin. After washing, the staining of cells and the distribution position of the yellow brown granules were observed. Only after incubating with anti TM7SF3 antibody, the LX2 cells appeared brown yellow, and the yellow brown granules were mainly concentrated on the cell membrane.5. to detect Css. The effect of PLA2 and selected protein on the proliferation of LX2 cells and the related signal pathway CCK-8 detection results showed that Css PLA2 incubated 24h compared to the PBS control group OD450 results showed that LX2 cell OD450 in the PBS incubating group was about 1.2, and that the proliferation rate of Css cells was about 25%. The LX2 cell OD450 in the antibody mixture incubated group was about 1, and the cell proliferation rate was lower than that of Css PLA2 incubating group. The 33%. results showed that Css PLA2 could increase the proliferation of LX2 cells. After the antibody blocked TM7SF3, Css PLA2 could weaken the proliferation of LX2 cells. The effect of PLA2 on LX2 cell activation and proliferation was inhibited by.Css PLA2 incubating LX2 cells 24h. Compared with the blank control group, the key gene expression levels of liver fibrosis related signaling pathway, including ERK, JNK, NF-k B, TGF-Smad and so on, were also up-regulated. Conclusion 1. yeast two hybrids are used to screen the.2. immunoprecipitation of human protein TM7SF3, which interact with Css PLA2, and GSTPull-down further verifies the interaction of Css PLA2 and human TM7SF3 by.3. immunohistochemistry on TM7SF3 in the LX2 cells of human hepatic stellate cell line, which is mainly located in the reverse validation of cell membrane.4. through the blocking of antibodies. LA2 related signaling pathways involved in liver fibrosis include ERK, JNK, NF-k B, TGF-Smad and other pathways.
【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R383.2
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本文編號：1924054