BRS-3肺内生物学效应、基因表达调控研究及天然配体分离

发布时间：2018-01-02 13:07

本文关键词：BRS-3肺内生物学效应、基因表达调控研究及天然配体分离　出处：《中南大学》2007年博士论文　论文类型：学位论文

【摘要】： 目的：气道含有丰富的神经支配和神经内分泌细胞，，可感知内外环境变化，释放大量神经肽，调控气道的各项功能，维持气道微环境稳态。蛙皮素样肽(BLPs)在哺乳动物肺内广泛存在，在肺脏生理病理过程中发挥重要作用。相应的，哺乳动物体内BLPs受体家族目前已发现3个成员，分别是胃泌素释放肽受体(GRPR)、神经介素B受体(NMBR)、蛙皮素受体亚型-3(BRS-3)。BRS-3是一个含有399个氨基酸的蛋白质，在正常体内的绝大多数组织表达量很低，而在发育中的肺组织及肿瘤组织中表达增高，且BRS-3的cDNA就是首次从小细胞肺癌细胞株中分离出来的，提示BRS-3可能与细胞的生长或损伤修复有关。由于BRS-3的生物学效应、基因表达调控及天然配体等目前仍不清楚，所以本课题围绕BRS-3的肺内生物学效应，基因表达调控和BRS-3天然配体进行了一系列研究。方法与结果： 1)BRS-3在肺内的生物学效应研究我们用臭氧连续应激家兔8天，每天1小时建立气道高反应动物模型，用原位杂交观察了BRS-3在动物模型中的时空分布。结果显示BRS-3于臭氧应激的第2天开始缓慢增高，于第4天达到峰值，之后逐渐下降。BRS-3主要分布在细支气管的纤毛柱状上皮细胞、终末细支气管的单层柱状上皮、Ⅱ型肺泡细胞和一些间质细胞。为了研究BRS-3在AHR模型中表达上调的生物学意义，我们进一步观察了BRS-3激活对培养的人支气管上皮细胞损伤修复和增殖的影响。损伤修复测定采用机械损伤在融合的支气管上皮细胞单层上造成一小面积不规则缺损，用显微视频分析系统每隔4小时测量缺损面积一次，绘制时间与修复面积的直线回归方程，直线的斜率为损伤修复指数，斜率越大，损伤修复速度越快。应用这种方法，我们观察到BRS-3人工合成配体P3513可浓度依赖性促进BECs损伤修复，该效应可被PKA抑制剂H89所阻断，但似乎被钙调素抑制剂W7和酪氨酸激酶途径抑制剂PD98059所加强。MTT法也观察到P3513可浓度依赖性促BECs增殖，该效应可被H89、W7和PD98059所阻断。BRS-3的促损伤修复及促增殖效应可被BRS-3 ASO所阻断。我们还对BRS-3的抗损伤保护作用进行了研究，以~3H-UDR和LDH为损伤指标，Catalase为抗损伤指标，结果显示O_3应激使BECs ~3H-UDR释放率增加，LDH活性增高，导致细胞损伤。P3513可显著降低O_3应激的BECs ~3H-UDR释放率、LDH活性，增加Catalase活性。上述结果提示BRS-3在AHR肺组织的上调与活化可促进上皮的损伤修复，提高细胞抗氧化能力，发挥保护功能。 2)BRS-3基因表达调控研究为了进一步认识AHR中BRS-3基因表达上调机制，我们对臭氧应激条件下BRS-3的转录因子调节谱进行了研究。实验首先用TESS软件对BRS-3启动子区转录因子结合位点进行搜索，然后根据搜索结果，设计了10条探针，覆盖所有的转录因子结合位点，应用EMSA和ChIP的方法筛选了调控BRS-3表达的转录因子。结果显示探针1、4、8、10有探针与蛋白结合形成的滞后带，能被100倍未标记探针所竞争，为特异性结合。通过突变探针结合实验和抗体超迁移实验，证实这4条探针结合的转录因子分别为PPARα、MTF-1、AP-2α和HSF-1。ChIP实验显示，PPARα和AP-2α可特异性与BRS-3启动子结合。紧接着，我们用基因定点突变技术观察了四种转录因子对BRS-3启动子活性的影响，结果显示PPARα与AP-2α的结合位点突变后，BRS-3启动子活性下降，PPARα与AP-2α结合位点同时突变，可完全抑制臭氧应激条件下BRS-3的激活，这一结果在BECs和HLF中都得到验证。随后我们用反义寡核苷酸技术观察了四种转录因子对BRS-3表达的影响，Western及EMSA证实了四种ASOs的有效性，他们可分别阻断四种转录因子的蛋白表达和结合活性。应用四种ASOs后，我们用原位杂交观察到臭氧应激可促进BRS-3的表达，PPARα和AP-2αASO明显抑制BRS-3的表达，real-time PCR结果与上述结果一致。我们还用免疫荧光观察了PPARα的核转位，结果显示臭氧应激使PPARα核转位增加，PPARαASO可阻断臭氧应激条件下PPARα的核转位，这一结果在BECs和HLF中也都得到验证。通过进一步的研究，我们用EMSA观察到PPAR与AP-2α的活化在48小时的观察中具有双相性，real-time PCR检测显示BRS-3的表达与两种转录因子的激活一致，提示在臭氧应激条件下，PPARα与AP-2α特异性与BRS-3结合，共同调控BRS-3的表达。 3)BRS-3相互作用蛋白筛选 BRS-3表达质粒由美国Boston大学Weber教授惠赠，将该质粒酶切并克隆至PBT质粒，构建诱饵质粒。诱饵质粒经PCR、酶切和全长测序证实BRS-3插入正确，Western blot证实该质粒可在IPTG诱导下表达，自激活鉴定显示其没有自激活作用。人胎脑文库购于美国Stratagene公可。将诱饵质粒和文库质粒共转化并铺于含3-AT的选择性培养基进行初筛，随后将菌落移至含链霉素和3-AT的二重筛选平板进行二级筛选，然后提取单个文库质粒再分别与诱饵质粒共转化，并铺于选择性筛选培养基和非选择性筛选培养基上验证其相互作用，取双阳性克隆测序，及生物信息学分析，结果显示BRS-3可与13种蛋白相互作用，包括四种新蛋白。应甩PULL-DOWN实验对9种已知蛋白与BRS-3相互作用进行了验证，结果显示有7种蛋白可与BRS-3发生相互作用，分别为胃泌素释放肽、睫状神经营养因子、蛋白酶抑制子-5、丝氨酸蛋白酶抑制剂、酪氨酸蛋白激酶、蛋白激酶Cβ1、酪氨酸蛋白激酶2α1。应用生物信息学方法对四种新基因分析显示，除一种基因无完整ORF，且不能进行电子延伸外，其余3种均为在胎脑、胎肺、人胚干细胞及肿瘤细胞中高表达的蛋白，其中2种有跨膜区域，1种具有信号肽。 4)BRS-3天然配体分离实验首先建立BRS-3高表达细胞株，并用[Ca~(2+)]瞬变测定对可能含有BRS-3天然配体的组织进行了初筛，结果显示人胚肺组织匀浆25kD以下组分可能含有BRS-3天然配体。然后收集转染和未转染的COS-7细胞，加入人胚肺组织匀浆，于4℃结合3小时后，裂解细胞，进行免疫共沉淀，然后在免疫沉淀物中加入40μl PBS液，35℃孵育30min使配体解离，离心取上清。或在免疫沉淀物中直接加入上样缓冲液，进行Tricine-SDS-PAGE鉴定，结果显示转染细胞洗脱物可引起[Ca~(2+)]瞬变效应，并且含有一种1kD大小的多肽，该多肽在未经洗脱的免疫沉淀物中浓度较高，切胶后对其进行N端氨基酸序列测序。测序结果显示该多肽为含有10个氨基酸残基的多肽，在各种蛋白质数据库中未找到显著同源性的蛋白，与蛙皮素的同源性为36％。通过放射配基亲和实验和结合竞争抑制实验，结果显示受体数量为80 fmol／10~5cells，K_d值为0.17 nmol／L，未标记的配体可竞争抑制~(125)I-配体与受体的结合，并呈剂量效应关系，在浓度为反应浓度的100倍左右时可发生完全竞争抑制。结论： BRS-3的配体为一种具有10个氨基酸残基的多肽，BRS-3可与多种蛋白相互作用参与细胞信号转导及调控。在臭氧应激条件下，PPARα与AP-2α特异性与BRS-3启动子结合，促进BRS-3转录。BRS-3激活后，通过细胞内信号传递，促进BECs的损伤修复和增殖，提高细胞抗氧化能力，发挥保护功能。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R33

【引证文献】