過(guò)表達(dá)BRP-39對(duì)哮喘小鼠肺部炎癥的影響及其免疫機(jī)制研究
發(fā)布時(shí)間：2018-08-30 09:06
【摘要】：支氣管哮喘(asthma,簡(jiǎn)稱哮喘)作為一個(gè)世界范圍常見(jiàn)的呼吸道慢性炎癥性疾病,被認(rèn)識(shí)和熟知已超過(guò)二十余年。哮喘常被定義為一種以局部和全身的過(guò)敏性炎癥,可逆性的氣道結(jié)構(gòu)破壞,支氣管壁和管腔嗜酸粒細(xì)胞的浸潤(rùn)和氣道粘液的高分泌為特點(diǎn)的疾病。哮喘疾病的發(fā)生發(fā)展過(guò)程伴隨了一系列重疊和并行發(fā)生的炎癥反應(yīng),目前研究表明,這些炎癥反應(yīng)主要由CD4+的Th2型淋巴細(xì)胞介導(dǎo)。 BRP-39(breast regression protein39,在人類同系物中又叫做YKL-40)作為一個(gè)類殼質(zhì)酶蛋白,已經(jīng)被報(bào)道與許多以炎癥和組織重塑反應(yīng)為特點(diǎn)的疾病相關(guān)。人類的YKL-40和小鼠的BRP-39擁有共同的編碼基因殼質(zhì)酶3類似物1(CHI3L1),該基因定位在人的1號(hào)染色體和小鼠的2號(hào)染色體上,分別編碼了人40kDa和小鼠39kDa的蛋白分子。YKL-40和BRP-39均缺少殼質(zhì)酶的活性,被認(rèn)為是類殼質(zhì)酶蛋白質(zhì)在哺乳動(dòng)物中的一種表型。這兩種形式的蛋白都屬于18糖基水解酶家族,而YKL-40的晶體結(jié)構(gòu)已在文獻(xiàn)中得到了描述。最近,許多研究都聚焦在研究BRP-39對(duì)哮喘和其他過(guò)敏性疾病的調(diào)節(jié)功能上。Lee等發(fā)現(xiàn)BRP-39在小鼠Th2炎癥和組織重塑的起始和效應(yīng)階段發(fā)揮了重要的調(diào)節(jié)作用。另有報(bào)道稱,在Th2炎癥中,BRP-39/YKL-40參與了過(guò)敏性反應(yīng)的多個(gè)階段,通過(guò)刺激DCs的募集和活化調(diào)節(jié)了致敏過(guò)程和Th2型細(xì)胞因子的效應(yīng)功能。Lee等同時(shí)也發(fā)現(xiàn),在BRP-39基因敲除的哮喘小鼠肺組織中,髓系來(lái)源的DCs (dendritic cells,樹(shù)突狀細(xì)胞)表面CD86和CD40分子的表達(dá)水平顯著下降了,這提示了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發(fā)揮作用的具體機(jī)制目前仍不清楚。 為了研究BRP-39是否能影響哮喘小鼠的Th2炎癥及進(jìn)一步明確其是否通過(guò)調(diào)節(jié)DCs的功能來(lái)產(chǎn)生作用,我們構(gòu)建了攜帶CHI3L1基因的重組腺病毒載體(AdCHI3L1),然后把它轉(zhuǎn)入BMDCs (bone marrow-derived DCs,骨髓來(lái)源樹(shù)突狀細(xì)胞)。我們通過(guò)利用小鼠的哮喘模型來(lái)模擬人類哮喘的特征性表現(xiàn),隨后檢測(cè)了AHR (airway hyper-responsiveness,氣道高反應(yīng)性),氣道炎癥的相關(guān)指標(biāo),并觀察了肺組織在炎癥下的改變。 目的：BRP-39被廣泛地認(rèn)為是治療哮喘的一個(gè)潛在靶點(diǎn),然而其調(diào)節(jié)過(guò)敏性炎癥的具體機(jī)制仍然不清楚。本研究的目的是明確BRP-39在哮喘小鼠動(dòng)物模型中對(duì)樹(shù)突狀細(xì)胞的調(diào)節(jié)作用和對(duì)肺組織Th2炎癥的影響。 方法：本研究主要使用了卵白蛋白(OVA)誘導(dǎo)的哮喘小鼠模型。用過(guò)表達(dá)BRP-39的腺病毒和空白病毒分別感染骨髓來(lái)源的樹(shù)突狀細(xì)胞(BMDCs),然后通過(guò)過(guò)繼回輸?shù)姆绞綄⑵滢D(zhuǎn)入受體小鼠體內(nèi),繼而進(jìn)一步檢測(cè)氣道高反應(yīng)性(AHR),氣道的炎癥情況和肺組織病理學(xué)的改變。6-8周齡的Balb/c雌性小鼠按體重大小編號(hào),根據(jù)隨機(jī)數(shù)字表產(chǎn)生隨機(jī)數(shù)字,按照隨機(jī)數(shù)字大小分組,將實(shí)驗(yàn)小鼠平均分為Saline組、OVA-Control組、OVA-AdMock組和OVA-AdCHI3Ll組。Saline組給予normal saline致敏霧化,OVA組、OVA-AdMock組和OVA-AdCHI3L1組均予OVA致敏霧化,其中,OVA-AdMock組和OVA-AdCHI3L1組小鼠第一次霧化前通過(guò)尾靜脈分別回輸AdMock BMDCs和AdCHI3L1BMDCs,10×106個(gè)細(xì)胞/只。觀察各組小鼠支氣管肺泡灌洗液(BALF)中細(xì)胞總數(shù)、分類細(xì)胞計(jì)數(shù)和肺組織病理學(xué)檢測(cè)；采用熒光定量PCR和Western Blot檢測(cè)肺組織中BRP-39的表達(dá)水平；采用流式細(xì)胞術(shù)檢測(cè)BMDCs的純度、腺病毒轉(zhuǎn)染效率和過(guò)表達(dá)BRP-39的BMDCs表面分子標(biāo)記(CD80. MHCII)的表達(dá)水平；采用Buxco檢測(cè)小鼠氣道反應(yīng)性；采用ELISA的方法檢測(cè)血漿和培養(yǎng)上清BRP-39的表達(dá)水平及BALF中Th2型細(xì)胞因子的表達(dá)水平。 結(jié)果：本實(shí)驗(yàn)利用卵清蛋白致敏霧化構(gòu)建急性哮喘模型。通過(guò)對(duì)Saline組和OVA組的BALF細(xì)胞數(shù)、肺組織病理學(xué)以及炎癥因子表達(dá)等多項(xiàng)指標(biāo)的檢測(cè),符合哮喘模型的特征,提示造模成功。與生理鹽水組小鼠相比,BRP-39在哮喘小鼠的肺組織中表達(dá)量無(wú)論在mRNA水平(P0.01)還是蛋白水平均明顯升高。在霧化開(kāi)始前,我們將腺病毒感染過(guò)的BMDCs通過(guò)尾靜脈注射的方式回輸給小鼠。BMDCs感染了過(guò)表達(dá)BRP-39的腺病毒后與其成熟性和活性相關(guān)的表面分子標(biāo)記均較前升高。與回輸對(duì)照病毒感染的BMDCs小鼠相比,回輸過(guò)表達(dá)BRP-39病毒感染的BMDCs后進(jìn)一步加重了OVA誘導(dǎo)的AHR(P0.05)和嗜酸粒細(xì)胞(P0.05)在肺部的浸潤(rùn)。過(guò)表達(dá)BRP-39同時(shí)增加了OVA誘導(dǎo)的Th2型細(xì)胞因子在支氣管肺泡灌洗液(BALF)的表達(dá)水平(P0.05),如IL-4,IL-5和IL-13,但降低了IFN-γ的表達(dá)水平(P0.05)。 結(jié)論：我們證實(shí)了BRP-39在哮喘小鼠肺組織中的表達(dá)水平升高,并且促進(jìn)了樹(shù)突狀細(xì)胞在體外的成熟。我們的結(jié)果同時(shí)證實(shí)了BRP-39具有促進(jìn)哮喘小鼠Th2炎癥的應(yīng)答和AHR的作用,上述結(jié)果表明BRP-39是治療哮喘的一個(gè)潛在靶點(diǎn)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R562.25
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
1 王燕;韓偉忠;范春輝;程兆忠;;糖皮質(zhì)激素對(duì)哮喘大鼠氣道NGF和IGF-Ⅰ表達(dá)影響[J];青島大學(xué)醫(yī)學(xué)院學(xué)報(bào);2014年05期
2 胡祖權(quán);夏雪;楊曉芳;曾柱;;基于樹(shù)突狀細(xì)胞的抗腫瘤免疫治療[J];貴陽(yáng)醫(yī)學(xué)院學(xué)報(bào);2014年06期
3 張艷霞;高天e，
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