【摘要】：豚鼠是研究人哮喘病、結(jié)核病等呼吸道疾病的理想動物模型之一。同時,豚鼠也是研究肺臟的先天免疫機制和呼吸道疾病的致病機理的常用模型。因此,本研究對豚鼠肺臟發(fā)育過程中上皮細胞的發(fā)育及其Toll樣受體(TLRs)信號表達進行了初步的研究。 試驗中,我們利用組織化學(xué)染色(HE)和免疫組織化學(xué)(IHC)染色方法對不同發(fā)育時期豚鼠呼吸道主要上皮細胞的類型與組成進行了形態(tài)學(xué)觀察,并對TLRs信號分子的發(fā)育表達利用定量反轉(zhuǎn)錄PCR(qRT-PCR)和免疫印跡(WB)法進行了分析。結(jié)果顯示,在胚胎(Embryonic)發(fā)育到45d和52d時,豚鼠肺臟分別處于假腺期和小管期；在胚胎發(fā)育到59d至出生前,肺臟呈現(xiàn)為肺泡增多的囊狀期；在出生后7d至成年期(3個月),豚鼠肺臟發(fā)育處于肺泡期。IHC結(jié)果表明,在胚胎發(fā)育的第45d,肺臟存在具有多能性的基底細胞和肺泡Ⅱ型細胞,當(dāng)胚胎發(fā)育到52d時,即肺臟發(fā)育的小管期,可以觀察到纖毛細胞和Clara細胞。 熒光定量PCR分析結(jié)果表明,在出生后3個月(成年)、28d、14d、7d利0d,以及胚胎期E45d、E52d和E59d的不同發(fā)育階段,豚鼠肺臟中TLR1、TLR2、TLR3、TLR4、TLR6、TLR7、 TLR8、TLR9、TLR10、MyD88和TRAF6,這11種TLR相關(guān)信號均有表達,但在出生前,各因子的表達量相對高于出生后。其中,在胚胎期E45d、E52d和E59d的豚鼠肺臟組織中,TLR1-10總體的表達水平為顯著遞增趨勢,E52d時,TLR4表達量最高,E45d表達量次之；在E52d和E59d時,TRAF6和MyD88相對表達量最高；而在出生后到成年(3個月)期,各因子的表達量顯著降低,且出生后的不同時期,同一因子間的表達差異不顯著。同時,利用蛋白免疫印跡Western Blotting技術(shù)在蛋白質(zhì)水平進一步對上述因子的表達進行了驗證。上述結(jié)果表明,豚鼠肺臟在發(fā)育過程中,隨著上皮細胞的增殖和分化,肺臟在形態(tài)學(xué)和功能上趨于成熟。TLRs在出生前的增高現(xiàn)象在分子水平提示豚鼠肺臟出生前的形態(tài)功能成熟性,并具備比較完善的免疫反應(yīng)能力；而出生后這些因子的表達顯著降低,將有利于避免機體誘導(dǎo)過度的免疫反應(yīng)對組織器官造成免疫損傷,并處于隨時識別免疫刺激源以激活先天免疫和獲得性免疫反應(yīng)的狀態(tài)。這些結(jié)果都為進一步探討豚鼠肺臟上皮細胞發(fā)育生物學(xué)的特征及其先天免疫機制的形成過程等提供了理論依據(jù)。
[Abstract]:Guinea pig is one of the ideal animal models to study respiratory diseases such as asthma, tuberculosis and so on. At the same time, guinea pig is also a common model to study the innate immune mechanism of lung and the pathogenic mechanism of respiratory diseases. Therefore, the development of epithelial cells and the expression of (TLRs) signal of Toll like receptor in guinea pig lung were studied in this study. In the experiment, the types and compositions of the main epithelial cells in the respiratory tract of guinea pigs at different developmental stages were observed by histochemical staining (HE) and immunohistochemical (IHC) staining. The developmental expression of TLRs signaling molecules was analyzed by quantitative reverse transcription PCR (qRT-PCR) and Western blot (WB). The results showed that the lungs of guinea pigs were in pseudoglandular phase and tubule stage at 45 days and 52 days of embryonic (Embryonic) development, and the lungs of guinea pigs showed a cystic phase of alveolar proliferation during the development of embryo from day 59 to pre-birth. From 7 days after birth to 3 months of adulthood, the lung development of guinea pigs was in alveolar phase. IHC results showed that there were pluripotent basal cells and alveolar type 鈪，

本文編號：2262415