过表达BRP-39对哮喘小鼠肺部炎症的影响及其免疫机制研究
发布时间：2018-08-30 09:06
【摘要】：支气管哮喘(asthma,简称哮喘)作为一个世界范围常见的呼吸道慢性炎症性疾病,被认识和熟知已超过二十余年。哮喘常被定义为一种以局部和全身的过敏性炎症,可逆性的气道结构破坏,支气管壁和管腔嗜酸粒细胞的浸润和气道粘液的高分泌为特点的疾病。哮喘疾病的发生发展过程伴随了一系列重叠和并行发生的炎症反应,目前研究表明,这些炎症反应主要由CD4+的Th2型淋巴细胞介导。 BRP-39(breast regression protein39,在人类同系物中又叫做YKL-40)作为一个类壳质酶蛋白,已经被报道与许多以炎症和组织重塑反应为特点的疾病相关。人类的YKL-40和小鼠的BRP-39拥有共同的编码基因壳质酶3类似物1(CHI3L1),该基因定位在人的1号染色体和小鼠的2号染色体上,分别编码了人40kDa和小鼠39kDa的蛋白分子。YKL-40和BRP-39均缺少壳质酶的活性,被认为是类壳质酶蛋白质在哺乳动物中的一种表型。这两种形式的蛋白都属于18糖基水解酶家族,而YKL-40的晶体结构已在文献中得到了描述。最近,许多研究都聚焦在研究BRP-39对哮喘和其他过敏性疾病的调节功能上。Lee等发现BRP-39在小鼠Th2炎症和组织重塑的起始和效应阶段发挥了重要的调节作用。另有报道称,在Th2炎症中,BRP-39/YKL-40参与了过敏性反应的多个阶段,通过刺激DCs的募集和活化调节了致敏过程和Th2型细胞因子的效应功能。Lee等同时也发现,在BRP-39基因敲除的哮喘小鼠肺组织中,髓系来源的DCs (dendritic cells,树突状细胞)表面CD86和CD40分子的表达水平显著下降了,这提示了BRP-39在募集和活化肺部DCs中起了重要作用的可能性。然而,BRP-39/YKL-40发挥作用的具体机制目前仍不清楚。 为了研究BRP-39是否能影响哮喘小鼠的Th2炎症及进一步明确其是否通过调节DCs的功能来产生作用,我们构建了携带CHI3L1基因的重组腺病毒载体(AdCHI3L1),然后把它转入BMDCs (bone marrow-derived DCs,骨髓来源树突状细胞)。我们通过利用小鼠的哮喘模型来模拟人类哮喘的特征性表现,随后检测了AHR (airway hyper-responsiveness,气道高反应性),气道炎症的相关指标,并观察了肺组织在炎症下的改变。 目的：BRP-39被广泛地认为是治疗哮喘的一个潜在靶点,然而其调节过敏性炎症的具体机制仍然不清楚。本研究的目的是明确BRP-39在哮喘小鼠动物模型中对树突状细胞的调节作用和对肺组织Th2炎症的影响。 方法：本研究主要使用了卵白蛋白(OVA)诱导的哮喘小鼠模型。用过表达BRP-39的腺病毒和空白病毒分别感染骨髓来源的树突状细胞(BMDCs),然后通过过继回输的方式将其转入受体小鼠体内,继而进一步检测气道高反应性(AHR),气道的炎症情况和肺组织病理学的改变。6-8周龄的Balb/c雌性小鼠按体重大小编号,根据随机数字表产生随机数字,按照随机数字大小分组,将实验小鼠平均分为Saline组、OVA-Control组、OVA-AdMock组和OVA-AdCHI3Ll组。Saline组给予normal saline致敏雾化,OVA组、OVA-AdMock组和OVA-AdCHI3L1组均予OVA致敏雾化,其中,OVA-AdMock组和OVA-AdCHI3L1组小鼠第一次雾化前通过尾静脉分别回输AdMock BMDCs和AdCHI3L1BMDCs,10×106个细胞/只。观察各组小鼠支气管肺泡灌洗液(BALF)中细胞总数、分类细胞计数和肺组织病理学检测；采用荧光定量PCR和Western Blot检测肺组织中BRP-39的表达水平；采用流式细胞术检测BMDCs的纯度、腺病毒转染效率和过表达BRP-39的BMDCs表面分子标记(CD80. MHCII)的表达水平；采用Buxco检测小鼠气道反应性；采用ELISA的方法检测血浆和培养上清BRP-39的表达水平及BALF中Th2型细胞因子的表达水平。 结果：本实验利用卵清蛋白致敏雾化构建急性哮喘模型。通过对Saline组和OVA组的BALF细胞数、肺组织病理学以及炎症因子表达等多项指标的检测,符合哮喘模型的特征,提示造模成功。与生理盐水组小鼠相比,BRP-39在哮喘小鼠的肺组织中表达量无论在mRNA水平(P0.01)还是蛋白水平均明显升高。在雾化开始前,我们将腺病毒感染过的BMDCs通过尾静脉注射的方式回输给小鼠。BMDCs感染了过表达BRP-39的腺病毒后与其成熟性和活性相关的表面分子标记均较前升高。与回输对照病毒感染的BMDCs小鼠相比,回输过表达BRP-39病毒感染的BMDCs后进一步加重了OVA诱导的AHR(P0.05)和嗜酸粒细胞(P0.05)在肺部的浸润。过表达BRP-39同时增加了OVA诱导的Th2型细胞因子在支气管肺泡灌洗液(BALF)的表达水平(P0.05),如IL-4,IL-5和IL-13,但降低了IFN-γ的表达水平(P0.05)。 结论：我们证实了BRP-39在哮喘小鼠肺组织中的表达水平升高,并且促进了树突状细胞在体外的成熟。我们的结果同时证实了BRP-39具有促进哮喘小鼠Th2炎症的应答和AHR的作用,上述结果表明BRP-39是治疗哮喘的一个潜在靶点。
[Abstract]:Asthma (asthma) is a common chronic inflammatory disease of the respiratory tract in the world, which has been known for more than 20 years. asthma is often defined as a local and systemic allergic inflammation, reversible airway structural damage, eosinophil infiltration in the bronchial wall and lumen, and airway mucus. The development of asthma is accompanied by a series of overlapping and concurrent inflammatory reactions, which are mainly mediated by CD4+ Th2 lymphocytes.
BRP-39 (breast regression protein 39, also known as YKL-40 in human homology) has been reported to be associated with many diseases characterized by inflammation and tissue remodeling. Human YKL-40 and mouse BRP-39 share a common coding gene, chitinase 3 analogue 1 (CHI3L1), which is located in human No. 1 YKL-40 and BRP-39, which encode human 40kDa and mouse 39kDa proteins respectively on chromosome 2 and mouse chromosomes 2, lack the activity of chitinase and are considered to be a phenotype of chitinase-like proteins in mammals. Both proteins belong to the 18-glycosylhydrolase family, and the crystal structure of YKL-40 is in the literature. Recently, many studies have focused on the regulatory role of BRP-39 in asthma and other allergic diseases. Lee et al. found that BRP-39 plays an important role in the initiation and effect of Th2 inflammation and tissue remodeling in mice. Lee et al. also found that the expression levels of CD86 and CD40 molecules on the surface of myeloid derived DCs (dendritic cells, dendritic cells) decreased significantly in the lung tissues of BRP-39 knockout mice, suggesting that BRP-39 was involved in recruitment. However, the specific mechanism of BRP-39/YKL-40 is still unclear.
To investigate whether BRP-39 can affect Th2 inflammation in asthmatic mice and to further clarify whether BRP-39 plays a role by regulating the function of DCs, we constructed a recombinant adenovirus vector carrying CHI3L1 gene (AdCHI3L1) and then transferred it into BMDCs (bone marrow-derived DCs). Asthma models were used to simulate the characteristic manifestations of human asthma, and then AHR (airway hyper-responsiveness) and airway inflammation related indicators were detected, and lung tissue changes under inflammation were observed.
OBJECTIVE: BRP-39 is widely regarded as a potential target for asthma treatment, but the specific mechanism of its regulation on allergic inflammation remains unclear.
METHODS: OVA-induced asthmatic mice were used in this study. Bone marrow-derived dendritic cells (BMDCs) were infected with adenovirus over-expressing BRP-39 and blank virus respectively, and then transfused into recipient mice by adoptive reinfusion. Airway hyperresponsiveness (AHR) and airway inflammation were further detected. 6-8 weeks old female Balb/c mice were randomly divided into Saline group, OVA-Control group, OVA-AdMock group and OVA-AdCHI 3Ll group according to the size of the random number table. OVA sensitized nebulization was performed in both ock and OVA-AdCHI3L1 groups. AdMock BMDCs and AdCHI3L1 BMDCs were transfused into tail vein respectively before the first nebulization in OVA-AdMock and OVA-AdCHI3L1 groups, and 10 *106 cells / mouse were observed in each group. Fluorescence quantitative PCR and Western Blot were used to detect the expression of BRP-39 in lung tissue, flow cytometry was used to detect the purity of BMDCs, adenovirus transfection efficiency and the expression of CD80.MHCII on the surface of BMDCs overexpressing BRP-39, Buxco was used to detect the airway reactivity of mice, and ELISA was used to detect the plasma and culture. The expression level of BRP-39 and expression of Th2 cytokines in BALF were determined.
Results: The model of acute asthma was established by ovalbumin-sensitized nebulization. The number of BALF cells, lung histopathology and expression of inflammatory factors in Saline group and OVA group were detected, which accorded with the characteristics of asthma model, suggesting that the model was successful. Compared with normal saline group, BRP-39 was found in the lung tissue of asthmatic mice. BMDCs infected by adenovirus were injected back into mice via tail vein before atomization. BMDCs infected by adenovirus overexpressing BRP-39 showed higher surface molecular markers associated with maturity and activity than before. Overexpression of BRP-39 also increased the expression of Th2 cytokines in bronchoalveolar lavage fluid (BALF) induced by OVA (P 0.05), such as IL-4, IL-5 and IL-13. The expression level of IFN- gamma was decreased (P0.05).
CONCLUSION: We have demonstrated that BRP-39 is highly expressed in lung tissues of asthmatic mice and promotes the maturation of dendritic cells in vitro. Our results also confirm that BRP-39 can promote Th2 inflammatory response and AHR in asthmatic mice. These results suggest that BRP-39 is a potential target for asthma treatment.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25
[Abstract]:Asthma (asthma) is a common chronic inflammatory disease of the respiratory tract in the world, which has been known for more than 20 years. asthma is often defined as a local and systemic allergic inflammation, reversible airway structural damage, eosinophil infiltration in the bronchial wall and lumen, and airway mucus. The development of asthma is accompanied by a series of overlapping and concurrent inflammatory reactions, which are mainly mediated by CD4+ Th2 lymphocytes.
BRP-39 (breast regression protein 39, also known as YKL-40 in human homology) has been reported to be associated with many diseases characterized by inflammation and tissue remodeling. Human YKL-40 and mouse BRP-39 share a common coding gene, chitinase 3 analogue 1 (CHI3L1), which is located in human No. 1 YKL-40 and BRP-39, which encode human 40kDa and mouse 39kDa proteins respectively on chromosome 2 and mouse chromosomes 2, lack the activity of chitinase and are considered to be a phenotype of chitinase-like proteins in mammals. Both proteins belong to the 18-glycosylhydrolase family, and the crystal structure of YKL-40 is in the literature. Recently, many studies have focused on the regulatory role of BRP-39 in asthma and other allergic diseases. Lee et al. found that BRP-39 plays an important role in the initiation and effect of Th2 inflammation and tissue remodeling in mice. Lee et al. also found that the expression levels of CD86 and CD40 molecules on the surface of myeloid derived DCs (dendritic cells, dendritic cells) decreased significantly in the lung tissues of BRP-39 knockout mice, suggesting that BRP-39 was involved in recruitment. However, the specific mechanism of BRP-39/YKL-40 is still unclear.
To investigate whether BRP-39 can affect Th2 inflammation in asthmatic mice and to further clarify whether BRP-39 plays a role by regulating the function of DCs, we constructed a recombinant adenovirus vector carrying CHI3L1 gene (AdCHI3L1) and then transferred it into BMDCs (bone marrow-derived DCs). Asthma models were used to simulate the characteristic manifestations of human asthma, and then AHR (airway hyper-responsiveness) and airway inflammation related indicators were detected, and lung tissue changes under inflammation were observed.
OBJECTIVE: BRP-39 is widely regarded as a potential target for asthma treatment, but the specific mechanism of its regulation on allergic inflammation remains unclear.
METHODS: OVA-induced asthmatic mice were used in this study. Bone marrow-derived dendritic cells (BMDCs) were infected with adenovirus over-expressing BRP-39 and blank virus respectively, and then transfused into recipient mice by adoptive reinfusion. Airway hyperresponsiveness (AHR) and airway inflammation were further detected. 6-8 weeks old female Balb/c mice were randomly divided into Saline group, OVA-Control group, OVA-AdMock group and OVA-AdCHI 3Ll group according to the size of the random number table. OVA sensitized nebulization was performed in both ock and OVA-AdCHI3L1 groups. AdMock BMDCs and AdCHI3L1 BMDCs were transfused into tail vein respectively before the first nebulization in OVA-AdMock and OVA-AdCHI3L1 groups, and 10 *106 cells / mouse were observed in each group. Fluorescence quantitative PCR and Western Blot were used to detect the expression of BRP-39 in lung tissue, flow cytometry was used to detect the purity of BMDCs, adenovirus transfection efficiency and the expression of CD80.MHCII on the surface of BMDCs overexpressing BRP-39, Buxco was used to detect the airway reactivity of mice, and ELISA was used to detect the plasma and culture. The expression level of BRP-39 and expression of Th2 cytokines in BALF were determined.
Results: The model of acute asthma was established by ovalbumin-sensitized nebulization. The number of BALF cells, lung histopathology and expression of inflammatory factors in Saline group and OVA group were detected, which accorded with the characteristics of asthma model, suggesting that the model was successful. Compared with normal saline group, BRP-39 was found in the lung tissue of asthmatic mice. BMDCs infected by adenovirus were injected back into mice via tail vein before atomization. BMDCs infected by adenovirus overexpressing BRP-39 showed higher surface molecular markers associated with maturity and activity than before. Overexpression of BRP-39 also increased the expression of Th2 cytokines in bronchoalveolar lavage fluid (BALF) induced by OVA (P 0.05), such as IL-4, IL-5 and IL-13. The expression level of IFN- gamma was decreased (P0.05).
CONCLUSION: We have demonstrated that BRP-39 is highly expressed in lung tissues of asthmatic mice and promotes the maturation of dendritic cells in vitro. Our results also confirm that BRP-39 can promote Th2 inflammatory response and AHR in asthmatic mice. These results suggest that BRP-39 is a potential target for asthma treatment.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R562.25
【共引文献】
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1 王燕;韩伟忠;范春辉;程兆忠;;糖皮质激素对哮喘大鼠气道NGF和IGF-Ⅰ表达影响[J];青岛大学医学院学报;2014年05期
2 胡祖权;夏雪;杨晓芳;曾柱;;基于树突状细胞的抗肿瘤免疫治疗[J];贵阳医学院学报;2014年06期
3 张艳霞;高天e，
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