本文關(guān)鍵詞： 炎癥 腹膜炎 巨噬細胞 Na~+依賴性核苷轉(zhuǎn)運載體 剪接異構(gòu)體 CNT2b炎癥因子 表達調(diào)控作用 RNA干擾　出處：《第三軍醫(yī)大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】： 炎癥和免疫反應(yīng)是一種常見的病理過程,大多數(shù)的人類疾病均與此有關(guān)。除了人們所熟知的感染性疾病外,風(fēng)濕性關(guān)節(jié)炎、類風(fēng)濕疾病、系統(tǒng)性紅斑狼瘡、肝纖維化、動脈粥樣硬化、心肌梗塞、阿爾茨海默病以及惡性腫瘤等多種疾病也與炎癥密切相關(guān)。炎癥免疫因素參與了這些疾病的發(fā)生、發(fā)展及轉(zhuǎn)歸整個過程。巨噬細胞是機體免疫系統(tǒng)的一種重要細胞,不僅具有很強的吞噬功能,而且是主要的抗原提呈細胞,在多種炎癥和免疫反應(yīng)中起關(guān)鍵作用�；罨木奘杉毎煞置诙喾N生物活性物質(zhì),如一氧化氮、白介素-1、腫瘤壞死因子α、活性氧以及前列腺素、白三烯等花生四烯酸代謝產(chǎn)物。誘生型環(huán)氧酶(cyclooxygenase,COX)—COX-2可大量表達于巨噬細胞中。種種研究跡象表明,COX-2并非專一性地參加炎癥反應(yīng),還在保護胃粘膜、維護腎臟功能、學(xué)習(xí)記憶等方面發(fā)揮重要作用,可能還存在其它亞型。眾多的炎癥因子在炎癥反應(yīng)的不同時期組成復(fù)雜的網(wǎng)絡(luò),相互作用,共同調(diào)控炎癥反應(yīng)的的進程和預(yù)后。巨噬細胞在炎癥免疫反應(yīng)中所起的重要作用,且其在誘導(dǎo)狀態(tài)下能大量表達COXs,因此我們擬針對COX的保守序列設(shè)計簡并引物,選擇急性炎癥誘導(dǎo)下的巨噬細胞cDNA作為模板克隆新型COX亞型或炎癥相關(guān)性基因,并對其表達特征和功能進行初步研究。 方法 1.分析不同物種、不同亞型的COX的保守序列,設(shè)計簡并引物。 2.采用腹腔注射酵母多糖A的方法建立SD大鼠急性腹膜炎模型,采集腹腔巨噬細胞,提取總RNA,進行RT-PCR,擴增產(chǎn)物純化后連接T載體,轉(zhuǎn)化DH5α細菌,抽提質(zhì)粒,測序。 3.選取新型序列,進行3’,5’快速末端反應(yīng),獲取全長基因序列。 4.采用Realtime RT-PCR法分別檢測新基因CNT2 mRNA、CNT2b mRNA在大鼠不同臟器組織中的表達情況,以及在酵母多糖誘導(dǎo)的急性腹膜炎大鼠腹腔巨噬細胞中表達的時相特征。 5.應(yīng)用Realtime RT-PCR法觀察抗炎藥物阿司匹林(12mg/kg)、塞來昔布(0.13mg/kg)和潑尼松(0.4mg/kg)對大鼠腹腔巨噬細胞中CNT2b mRNA和CNT2 mRNA表達的影響。 6.針對CNT2b序列設(shè)計siRNA干擾序列,采用脂質(zhì)體轉(zhuǎn)染體外培養(yǎng)NR8383大鼠巨噬細胞;Realtime RT-PCR法檢測干擾后細胞中炎癥相關(guān)因子IL-1α、IL-1β、IL-6、TNF-α、COX-2、IL-10以及CNT2、CNT2b等分子的mRNA表達情況。 結(jié)果 1.不同物種的COX-1、COX-2和COX-3序列中存在兩段氨基酸保守序列。 2.從大鼠腹腔巨噬細胞中發(fā)現(xiàn)并克隆了一個新基因,全長13330nt,為一mRNA序列,對應(yīng)DNA位于大鼠第三號染色體3q35區(qū)。經(jīng)BLAST分析與序列比對,該基因與Na+依賴性核苷轉(zhuǎn)運載體(Na+/nucleoside cotransporter, CNT)2有較高同源性,保留了CNT2的第1、7~14、16、17內(nèi)含子;并且,在CNT2的最后一個(第18個)外顯子后,又附加了一段3169nt長的序列。由于該基因和CNT2均包含加尾信號和polyA尾巴,均為完整的轉(zhuǎn)錄產(chǎn)物,故確定為CNT2的剪接異構(gòu)體,已提交Genbank數(shù)據(jù)庫并獲得登錄號:EU032627.2,重新命名為CNT2b。 3. CNT2 mRNA在全身各器官均有表達,其中在心臟、肝臟、平滑肌、脾臟表達較高;CNT2b mRNA在正常器官中表達量遠遠少于CNT2 mRNA,其中心臟、平滑肌、肝臟表達稍高。 4. CNT2b mRNA在腹膜炎大鼠腹腔巨噬細胞中的表達具有顯著時相特征。注射酵母多糖后,其表達顯著升高,至2h時達到最高點;隨后,表達量逐漸降低,至24h時基本恢復(fù)至0.5h時的水平;致炎后36h,表達量再次明顯升高,至第48h時又降至36h時的47.52%。CNT2 mRNA在腹膜炎大鼠腹腔巨噬細胞中的表達也具有類似特征,其時相變化幅度遠少于CNT2b mRNA。 5.三種抗炎藥物阿司匹林、潑尼松、塞來昔布均能明顯降低CNT2b mRNA表達量。在臨床使用劑量下,潑尼松作用最強,阿司匹林次之,塞來昔布最弱。 6.抑制CNT2b基因表達后NR8383細胞中IL-1βmRNA,IL-6 mRNA,TNF-αmRNA,COX-2 mRNA的表達均顯著升高,IL-10 mRNA的表達明顯降低。 結(jié)論 1.在大鼠腹腔巨噬細胞中發(fā)現(xiàn)并克隆了一個新基因CNT2b。 2. CNT2b屬于炎癥誘導(dǎo)性高表達基因,在正常組織中很少表達,而在急性炎癥刺激的巨噬細胞中表達顯著增加。在大鼠急性腹膜炎過程中,腹腔巨噬細胞CNT2b mRNA的表達具有明顯時相性,呈雙峰特征�？寡姿幬锬苊黠@抑制CNT2b mRNA的表達。 3.抑制CNT2b的表達能促進多種促炎因子的表達,降低抗炎因子IL-10的表達。 4. CNT2b參與了炎癥反應(yīng)的調(diào)控過程,其功能與抑制炎癥反應(yīng)有關(guān)。
[Abstract]:Inflammation and immune response is a common pathological process, most human diseases are related to this. In addition to well-known infectious diseases, rheumatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, hepatic fibrosis, atherosclerosis, myocardial infarction, Alzheimer's disease and a variety of malignant diseases are closely related inflammation is involved in these diseases, the development and outcome of the whole process. Macrophage is a kind of important cells in the immune system, not only has a strong phagocytic function, but also is the main antigen-presenting cells that play a key role in a variety of inflammatory and immune responses. Activated macrophages can secrete a variety of biological active substances, such as nitric oxide, interleukin -1, tumor necrosis factor alpha, ROS and prostaglandins, leukotrienes four arachidonic acid metabolites. Inducible ring Enzymes (cyclooxygenase, COX) - COX-2 can be highly expressed in macrophage. Many studies have suggested that COX-2 is not specific to participate in inflammation, also protect gastric mucosa, protect renal function, plays an important role in learning and memory, there may be other subtypes. A large number of inflammatory factors in different periods of inflammation the composition of the complex network, interaction, to control the process of inflammatory response and prognosis. The important role played in macrophage inflammatory immune responses, and the state can induce the expression of COXs, so we plan to COX conserved sequences of degenerate primers were designed to choose the acute inflammation induced by macrophages cDNA as template cloning of novel COX subtype or inflammation related genes, and preliminary study of its expression characteristics and functions.
Method
1. the conservative sequences of different species and subtypes of COX were analyzed, and the degenerate primers were designed.
2., a SD rat acute peritonitis model was established by intraperitoneal injection of yeast polysaccharide A. The peritoneal macrophages were collected, and the total RNA was extracted. RT-PCR was amplified. The amplified products were purified, then connected to T vector and transformed into DH5 alpha bacteria, and plasmid was extracted and sequenced.
3. a new sequence was selected, and 3 ', 5' rapid terminal reaction was used to obtain the full length gene sequence.
4. Realtime RT-PCR method was used to detect the expression of new gene CNT2 mRNA, CNT2b mRNA in different organ tissues of rats, as well as the temporal characteristics of expression in peritoneal macrophages of rats with acute peritonitis induced by zymosan.
5. the effect of anti inflammatory drugs aspirin (12mg/kg), celecoxib (0.13mg/kg) and prednisone (0.4mg/kg) on the expression of CNT2b mRNA and CNT2 mRNA in peritoneal macrophages of rats was observed by Realtime RT-PCR.
6., siRNA interference sequences were designed for CNT2b sequences. Liposomes were transfected into NR8383 rat macrophages in vitro. Realtime RT-PCR method was used to detect the expression of inflammatory factors IL-1 alpha, IL-1 IL-1, IL-6, TNF-, COX-2, IL-10, and TNF- and other molecules in the cells after interference.
Result
1. the COX-1, COX-2 and COX-3 sequences of different species exist in the sequence of two segment amino acids.
2. from rat peritoneal macrophages found and cloned a new gene, full-length 13330nt, a mRNA sequence corresponding to DNA is located on chromosome third in 3q35 region of rat. By BLAST analysis and sequence alignment, the gene of Na+ dependent nucleoside transporters (Na+/nucleoside, cotransporter, CNT) 2 have higher homology, retention the introns of 1,7~14,16,17 CNT2; and CNT2, in the last (eighteenth) exon, and additional sequence of a 3169nt long. Because of the CNT2 gene and contain the polyadenylation signals and polyA tail are transcripts complete, therefore identified as the splicing isomers of CNT2 that has been submitted to the Genbank database and the accession number: EU032627.2, renamed CNT2b.
3. CNT2 mRNA was expressed in all organs of the body, and the expression was higher in heart, liver, smooth muscle and spleen. The expression of CNT2b mRNA in normal organs was much less than that in CNT2 mRNA, and the expression of heart, smooth muscle and liver was slightly higher.
The expression of CNT2b mRNA in 4. peritonitis rat peritoneal macrophages has obvious phase characteristics. After zymosan injection, its expression was significantly increased and reached the peak point at 2h; subsequently, the expression level was gradually decreased, and 24h recovered to the level of 0.5h; after 36h inflammation, scale up again the expression of 48h increased to 47.52%.CNT2 and to 36h mRNA in rats with peritonitis in peritoneal macrophages also have similar characteristics, when the phase change amplitude is far less than the CNT2b mRNA.
5., three kinds of anti-inflammatory drugs, aspirin, prednisone and celecoxib can significantly reduce the expression of CNT2b mRNA. Under the clinical dosage, prednisone is the most effective, aspirin is the second and celecoxib is the weakest.
6. the expression of IL-1 beta mRNA, IL-6 mRNA, TNF- a mRNA and COX-2 mRNA increased significantly in NR8383 cells after inhibiting the expression of CNT2b gene, and the expression of IL-10 mRNA decreased significantly.
conclusion
1. a new gene, CNT2b., was found and cloned in rat peritoneal macrophages
2. CNT2b灞炰簬鐐庣棁璇卞鎬ч珮琛ㄨ揪鍩哄洜,鍦ㄦ甯哥粍緇囦腑寰堝皯琛ㄨ揪,鑰屽湪鎬ユ，

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