本文關(guān)鍵詞：BRS-3肺內(nèi)生物學(xué)效應(yīng)、基因表達(dá)調(diào)控研究及天然配體分離　出處：《中南大學(xué)》2007年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 支氣管上皮細(xì)胞 氣道高反應(yīng)性 BRS-3 PPARα AP-2α細(xì)菌雙雜交

【摘要】： 目的： 氣道含有豐富的神經(jīng)支配和神經(jīng)內(nèi)分泌細(xì)胞，，可感知內(nèi)外環(huán)境變化，釋放大量神經(jīng)肽，調(diào)控氣道的各項功能，維持氣道微環(huán)境穩(wěn)態(tài)。蛙皮素樣肽(BLPs)在哺乳動物肺內(nèi)廣泛存在，在肺臟生理病理過程中發(fā)揮重要作用。相應(yīng)的，哺乳動物體內(nèi)BLPs受體家族目前已發(fā)現(xiàn)3個成員，分別是胃泌素釋放肽受體(GRPR)、神經(jīng)介素B受體(NMBR)、蛙皮素受體亞型-3(BRS-3)。BRS-3是一個含有399個氨基酸的蛋白質(zhì)，在正常體內(nèi)的絕大多數(shù)組織表達(dá)量很低，而在發(fā)育中的肺組織及腫瘤組織中表達(dá)增高，且BRS-3的cDNA就是首次從小細(xì)胞肺癌細(xì)胞株中分離出來的，提示BRS-3可能與細(xì)胞的生長或損傷修復(fù)有關(guān)。由于BRS-3的生物學(xué)效應(yīng)、基因表達(dá)調(diào)控及天然配體等目前仍不清楚，所以本課題圍繞BRS-3的肺內(nèi)生物學(xué)效應(yīng)，基因表達(dá)調(diào)控和BRS-3天然配體進(jìn)行了一系列研究。 方法與結(jié)果： 1)BRS-3在肺內(nèi)的生物學(xué)效應(yīng)研究 我們用臭氧連續(xù)應(yīng)激家兔8天，每天1小時建立氣道高反應(yīng)動物模型，用原位雜交觀察了BRS-3在動物模型中的時空分布。結(jié)果顯示BRS-3于臭氧應(yīng)激的第2天開始緩慢增高，于第4天達(dá)到峰值，之后逐漸下降。BRS-3主要分布在細(xì)支氣管的纖毛柱狀上皮細(xì)胞、終末細(xì)支氣管的單層柱狀上皮、Ⅱ型肺泡細(xì)胞和一些間質(zhì)細(xì)胞。 為了研究BRS-3在AHR模型中表達(dá)上調(diào)的生物學(xué)意義，我們進(jìn)一步觀察了BRS-3激活對培養(yǎng)的人支氣管上皮細(xì)胞損傷修復(fù)和增殖的影響。損傷修復(fù)測定采用機(jī)械損傷在融合的支氣管上皮細(xì)胞單層上造成一小面積不規(guī)則缺損，用顯微視頻分析系統(tǒng)每隔4小時測量缺損面積一次，繪制時間與修復(fù)面積的直線回歸方程，直線的斜率為損傷修復(fù)指數(shù)，斜率越大，損傷修復(fù)速度越快。應(yīng)用這種方法，我們觀察到BRS-3人工合成配體P3513可濃度依賴性促進(jìn)BECs損傷修復(fù)，該效應(yīng)可被PKA抑制劑H89所阻斷，但似乎被鈣調(diào)素抑制劑W7和酪氨酸激酶途徑抑制劑PD98059所加強(qiáng)。MTT法也觀察到P3513可濃度依賴性促BECs增殖，該效應(yīng)可被H89、W7和PD98059所阻斷。BRS-3的促損傷修復(fù)及促增殖效應(yīng)可被BRS-3 ASO所阻斷。 我們還對BRS-3的抗損傷保護(hù)作用進(jìn)行了研究，以~3H-UDR和LDH為損傷指標(biāo)，Catalase為抗損傷指標(biāo)，結(jié)果顯示O_3應(yīng)激使BECs ~3H-UDR釋放率增加，LDH活性增高，導(dǎo)致細(xì)胞損傷。P3513可顯著降低O_3應(yīng)激的BECs ~3H-UDR釋放率、LDH活性，增加Catalase活性。上述結(jié)果提示BRS-3在AHR肺組織的上調(diào)與活化可促進(jìn)上皮的損傷修復(fù)，提高細(xì)胞抗氧化能力，發(fā)揮保護(hù)功能。 2)BRS-3基因表達(dá)調(diào)控研究 為了進(jìn)一步認(rèn)識AHR中BRS-3基因表達(dá)上調(diào)機(jī)制，我們對臭氧應(yīng)激條件下BRS-3的轉(zhuǎn)錄因子調(diào)節(jié)譜進(jìn)行了研究。實驗首先用TESS軟件對BRS-3啟動子區(qū)轉(zhuǎn)錄因子結(jié)合位點進(jìn)行搜索，然后根據(jù)搜索結(jié)果，設(shè)計了10條探針，覆蓋所有的轉(zhuǎn)錄因子結(jié)合位點，應(yīng)用EMSA和ChIP的方法篩選了調(diào)控BRS-3表達(dá)的轉(zhuǎn)錄因子。結(jié)果顯示探針1、4、8、10有探針與蛋白結(jié)合形成的滯后帶，能被100倍未標(biāo)記探針?biāo)偁�，為特異性結(jié)合。通過突變探針結(jié)合實驗和抗體超遷移實驗，證實這4條探針結(jié)合的轉(zhuǎn)錄因子分別為PPARα、MTF-1、AP-2α和HSF-1。ChIP實驗顯示，PPARα和AP-2α可特異性與BRS-3啟動子結(jié)合。 緊接著，我們用基因定點突變技術(shù)觀察了四種轉(zhuǎn)錄因子對BRS-3啟動子活性的影響，結(jié)果顯示PPARα與AP-2α的結(jié)合位點突變后，BRS-3啟動子活性下降，PPARα與AP-2α結(jié)合位點同時突變，可完全抑制臭氧應(yīng)激條件下BRS-3的激活，這一結(jié)果在BECs和HLF中都得到驗證。隨后我們用反義寡核苷酸技術(shù)觀察了四種轉(zhuǎn)錄因子對BRS-3表達(dá)的影響，Western及EMSA證實了四種ASOs的有效性，他們可分別阻斷四種轉(zhuǎn)錄因子的蛋白表達(dá)和結(jié)合活性。應(yīng)用四種ASOs后，我們用原位雜交觀察到臭氧應(yīng)激可促進(jìn)BRS-3的表達(dá)，PPARα和AP-2αASO明顯抑制BRS-3的表達(dá)，real-time PCR結(jié)果與上述結(jié)果一致。 我們還用免疫熒光觀察了PPARα的核轉(zhuǎn)位，結(jié)果顯示臭氧應(yīng)激使PPARα核轉(zhuǎn)位增加，PPARαASO可阻斷臭氧應(yīng)激條件下PPARα的核轉(zhuǎn)位，這一結(jié)果在BECs和HLF中也都得到驗證。 通過進(jìn)一步的研究，我們用EMSA觀察到PPAR與AP-2α的活化在48小時的觀察中具有雙相性，real-time PCR檢測顯示BRS-3的表達(dá)與兩種轉(zhuǎn)錄因子的激活一致，提示在臭氧應(yīng)激條件下，PPARα與AP-2α特異性與BRS-3結(jié)合，共同調(diào)控BRS-3的表達(dá)。 3)BRS-3相互作用蛋白篩選 BRS-3表達(dá)質(zhì)粒由美國Boston大學(xué)Weber教授惠贈，將該質(zhì)粒酶切并克隆至PBT質(zhì)粒，構(gòu)建誘餌質(zhì)粒。誘餌質(zhì)粒經(jīng)PCR、酶切和全長測序證實BRS-3插入正確，Western blot證實該質(zhì)�？稍贗PTG誘導(dǎo)下表達(dá)，自激活鑒定顯示其沒有自激活作用。人胎腦文庫購于美國Stratagene公可。將誘餌質(zhì)粒和文庫質(zhì)粒共轉(zhuǎn)化并鋪于含3-AT的選擇性培養(yǎng)基進(jìn)行初篩，隨后將菌落移至含鏈霉素和3-AT的二重篩選平板進(jìn)行二級篩選，然后提取單個文庫質(zhì)粒再分別與誘餌質(zhì)粒共轉(zhuǎn)化，并鋪于選擇性篩選培養(yǎng)基和非選擇性篩選培養(yǎng)基上驗證其相互作用，取雙陽性克隆測序，及生物信息學(xué)分析，結(jié)果顯示BRS-3可與13種蛋白相互作用，包括四種新蛋白。應(yīng)甩PULL-DOWN實驗對9種已知蛋白與BRS-3相互作用進(jìn)行了驗證，結(jié)果顯示有7種蛋白可與BRS-3發(fā)生相互作用，分別為胃泌素釋放肽、睫狀神經(jīng)營養(yǎng)因子、蛋白酶抑制子-5、絲氨酸蛋白酶抑制劑、酪氨酸蛋白激酶、蛋白激酶Cβ1、酪氨酸蛋白激酶2α1。應(yīng)用生物信息學(xué)方法對四種新基因分析顯示，除一種基因無完整ORF，且不能進(jìn)行電子延伸外，其余3種均為在胎腦、胎肺、人胚干細(xì)胞及腫瘤細(xì)胞中高表達(dá)的蛋白，其中2種有跨膜區(qū)域，1種具有信號肽。 4)BRS-3天然配體分離 實驗首先建立BRS-3高表達(dá)細(xì)胞株，并用[Ca~(2+)]瞬變測定對可能含有BRS-3天然配體的組織進(jìn)行了初篩，結(jié)果顯示人胚肺組織勻漿25kD以下組分可能含有BRS-3天然配體。 然后收集轉(zhuǎn)染和未轉(zhuǎn)染的COS-7細(xì)胞，加入人胚肺組織勻漿，于4℃結(jié)合3小時后，裂解細(xì)胞，進(jìn)行免疫共沉淀，然后在免疫沉淀物中加入40μl PBS液，35℃孵育30min使配體解離，離心取上清�；蛟诿庖叱恋砦镏兄苯蛹尤肷蠘泳彌_液，進(jìn)行Tricine-SDS-PAGE鑒定，結(jié)果顯示轉(zhuǎn)染細(xì)胞洗脫物可引起[Ca~(2+)]瞬變效應(yīng)，并且含有一種1kD大小的多肽，該多肽在未經(jīng)洗脫的免疫沉淀物中濃度較高，切膠后對其進(jìn)行N端氨基酸序列測序。測序結(jié)果顯示該多肽為含有10個氨基酸殘基的多肽，在各種蛋白質(zhì)數(shù)據(jù)庫中未找到顯著同源性的蛋白，與蛙皮素的同源性為36％。通過放射配基親和實驗和結(jié)合競爭抑制實驗，結(jié)果顯示受體數(shù)量為80 fmol／10~5cells，K_d值為0.17 nmol／L，未標(biāo)記的配體可競爭抑制~(125)I-配體與受體的結(jié)合，并呈劑量效應(yīng)關(guān)系，在濃度為反應(yīng)濃度的100倍左右時可發(fā)生完全競爭抑制。 結(jié)論： BRS-3的配體為一種具有10個氨基酸殘基的多肽，BRS-3可與多種蛋白相互作用參與細(xì)胞信號轉(zhuǎn)導(dǎo)及調(diào)控。在臭氧應(yīng)激條件下，PPARα與AP-2α特異性與BRS-3啟動子結(jié)合，促進(jìn)BRS-3轉(zhuǎn)錄。BRS-3激活后，通過細(xì)胞內(nèi)信號傳遞，促進(jìn)BECs的損傷修復(fù)和增殖，提高細(xì)胞抗氧化能力，發(fā)揮保護(hù)功能。
[Abstract]:Objective:
The airway contains rich innervation and neuroendocrine cells, perceived changes in internal and external environment, the release of a large number of neuropeptides, various functions of regulating airway, airway homeostasis. Micro bombesin like peptides (BLPs) are widespread in mammalian lungs, play an important role in lung physiology and pathology process. Accordingly, mammalian BLPs receptor the family has found 3 members, respectively is the gastrin releasing peptide receptor (GRPR), neuromedin B receptor (NMBR), bombesin receptor subtype -3 (BRS-3).BRS-3 is a 399 amino acid protein, in the majority of normal tissues the expression is very low, and the lung tissue and in the development of tumor tissue in the expression levels of BRS-3 and cDNA is the first small cell lung cancer cell line isolated, suggesting that growth or repair of damaged BRS-3 cells and may have due to the biological effect of BRS-3. It is still unclear about gene expression regulation and natural ligands. Therefore, this study focused on the biological effects of BRS-3, gene expression regulation and BRS-3 natural ligands.
Methods and results:
1) study on the biological effects of BRS-3 in the lung
We use continuous ozone stress in rabbits for 8 days, 1 hours a day to establish animal model of airway hyperresponsiveness, temporal and spatial distribution of BRS-3 in animal models was observed by in situ hybridization. The results showed that BRS-3 in second days of ozone stress slowly increased, peaked on the fourth day, then gradually decreased.BRS-3 mainly distributed in the ciliated columnar epithelium cells in bronchioles, columnar epithelium terminal bronchioles, alveolar cells and some stromal cells.
In order to study the biological significance of expression of BRS-3 in the AHR model, we further examined the effects of BRS-3 activation on cultured human bronchial epithelial cell damage repair and proliferation. Repair was measured by mechanical damage caused by a small area of irregular defect in the confluent monolayer, by video microscopy measurement system every 4 hours the defect area of a linear regression equation, drawing time and repair area, the slope of the line for the repair index, the greater slope, repair faster. Using this method, we observed the BRS-3 synthetic ligand P3513 induced a dose-dependent BECs damage repair, the effect can be blocked by PKA inhibitor H89, but seems to be calmodulin inhibitor W7 and tyrosine kinase inhibitor PD98059 enhanced.MTT method was also observed in P3513 concentration dependent proliferation of BECs, the The effect can be blocked by BRS-3 ASO by the effects of H89, W7 and PD98059 on the repair and proliferation of.BRS-3.
We are also on the protective effects against injury of BRS-3 were investigated by ~3H-UDR and LDH as the damage index, Catalase damage index, the results showed that O_3 stress made BECs ~3H-UDR release rate increased, LDH activity increased, resulting in injury of.P3513 cells can significantly reduce the stress of O_3 BECs ~ 3H-UDR release rate, LDH activity, Catalase activity increased. These results suggest that BRS-3 AHR in the lung tissue increased and activation can promote the repair of epithelial cells, improve the antioxidant capacity, play a protective function.
2) Regulation of BRS-3 gene expression
In order to further understand the AHR BRS-3 gene expression mechanism, we on the ozone stress condition the BRS-3 transcription factor regulates spectrum are studied. The first use TESS software to start the search for transcription factor binding sites of BRS-3 promoter, and then according to the results, the design of the 10 probes, covering all transcription factor binding sites, methods the application of EMSA and ChIP were transcription factors regulate the expression of BRS-3. The results showed that 1,4,8,10 protein binding probe probe and hysteresis band formation, can be 100 times the unlabeled probe competition, combined with specific. The mutant probe binding assay and antibody supershift experiments confirmed that transcription factor 4 probes with respectively. PPAR MTF-1 AP-2, alpha, alpha and HSF-1.ChIP experiment, PPAR alpha and AP-2 alpha specifically binding to BRS-3 promoter.
Then, we used site directed mutagenesis to observe the effects of four kinds of transcription factors in BRS-3 promoter activity, the results showed that PPAR and alpha AP-2 alpha binding site mutation, BRS-3 promoter activity decreased, PPAR alpha and alpha AP-2 binding site mutation at the same time, can completely inhibit the activation of BRS-3 under ozone stress, the results are verified in BECs and HLF. Then we used antisense oligonucleotide technique to observe the effect of the four kinds of transcription factors on the expression of BRS-3, Western and EMSA confirmed the effectiveness of four ASOs, respectively, they can block the expression of four transcription factors and protein binding activity. The application of four kinds of ASOs, expression we used in situ hybridization to ozone stress can promote the expression of BRS-3, PPAR and AP-2 alpha alpha ASO significantly inhibited BRS-3, real-time and PCR results consistent with the above-mentioned results.
We also observed the nuclear translocation of PPAR alpha by immunofluorescence. The results showed that the translocation of PPAR alpha was increased by ozone stress. PPAR alpha ASO blocked the nuclear translocation of PPAR alpha under ozone stress, and this result was also verified in BECs and HLF.
Through further research, we observed by EMSA activation PPAR and AP-2 alpha is biphasic in 48 hour observation, real-time PCR showed consistent activation and BRS-3 expression of two transcription factors, suggesting that in the ozone stress conditions, PPAR and alpha AP-2 alpha specific binding with BRS-3, the expression of common control BRS-3.
3) screening of BRS-3 interacting protein
The expression plasmid BRS-3 by Hui professor Weber Boston University in the United States with the plasmid, and cloned into PBT plasmid, to construct the bait plasmid. The bait plasmid was identified by PCR, enzyme digestion and sequencing confirmed that the BRS-3 insert the correct length, Western blot confirmed that the expression can be induced by IPTG, the self identification showed that the live no self activation. Human fetal brain cDNA library was purchased from American Stratagene company. The bait plasmid and library plasmids were transformed into and spread in selective medium containing 3-AT were screened, then moved to the colony containing streptomycin and 3-AT double screening plate two level screening, and then extract a single plasmid respectively and bait plasmids. And shop on the selective screening medium and non selective screening medium to verify the interaction, the double positive clones were sequenced and bioinformatics analysis, the results show that BRS-3 can interact with 13 proteins, including four A new protein. PULL-DOWN should throw experiments for 9 kinds of known proteins interacting with BRS-3 were verified, results showed that 7 kinds of protein can interact with BRS-3, respectively, gastrin releasing peptide, ciliary neurotrophic factor, protease inhibitor -5, serine protease inhibitor, protein tyrosine kinase, protein kinase C beta 1 methods, protein tyrosine kinase 2 alpha 1. bioinformatics applications show the four new gene analysis, in addition to a complete ORF gene, and can not be extended electronic, the other 3 were in fetal brain, fetal lung, human embryonic stem cells and tumor cells with high expression of protein, 2 of them there are 1 transmembrane regions, with signal peptide.
4) separation of BRS-3 natural ligands
In the experiment, we first established a cell line with high expression of BRS-3, and used [Ca~ (2+)] transients to detect the tissues that might contain BRS-3 natural ligands. The results showed that the components of 25kD below human embryo lung homogenate may contain BRS-3 natural ligands.
Then collect the transfected and non transfected COS-7 cells into human embryonic lung tissue homogenate, with 4 DEG C to 3 hours after cell lysis and immunoprecipitation, then adding 40 L PBS solution in the immunoprecipitates, 35 C incubation 30min ligand dissociation, then centrifuged sample buffer or sink. Directly into the precipitate in the immune system, Tricine-SDS-PAGE identification, the results showed the transfected cells elution can induce [Ca~ (2+)] transient effect, polypeptide and contains an 1kD size, without a higher concentration of the peptide in the immunoprecipitates eluted in gel after N terminal amino acid sequencing of the sequencing. The results show that the peptide is a polypeptide composed of 10 amino acid residues, significant homology protein was not found in the protein database, and bombesin 36%. homology by radioligand binding affinity test and competitive inhibition experiment, results showed that The number of body was 80 fmol / 10~5cells, and K_d value was 0.17 nmol / L. The unlabeled ligand could competitively inhibit the binding of ~ (125) I- ligand to the receptor, and showed a dose effect relationship. When the concentration was 100 times of the reaction concentration, the complete competition inhibition could happen.
Conclusion:
BRS-3 ligand is a polypeptide with 10 amino acid residues, BRS-3 can interact with many proteins involved in cell signal transduction and regulation. In the ozone stress conditions, PPAR and alpha AP-2 alpha specific binding to BRS-3 promoter, promote BRS-3 transcription activation of.BRS-3, passed through the intracellular signaling, promote BECs damage repair and proliferation, improve the antioxidant ability of cells, play a protective function.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R33

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 喬莉娟;豬肺免疫防御相關(guān)基因—肺表面活性蛋白A的研究[D];中國農(nóng)業(yè)科學(xué)院;2008年

相關(guān)碩士學(xué)位論文 前1條

1 李孟蘭;BRAP對人支氣管上皮細(xì)胞抗原呈遞功能的影響[D];中南大學(xué);2011年

本文編號：1369344