{"created":"2023-06-19T07:18:05.923518+00:00","id":3223,"links":{},"metadata":{"_buckets":{"deposit":"49433d96-d1ea-4704-b8aa-6ff5a84565b6"},"_deposit":{"created_by":4,"id":"3223","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3223"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003223","sets":["370:15:392"]},"author_link":["16282","16281"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"1994-07-06"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Cytokine is a general term for certain high-molecule peptides produced and secreted by cells, mainly by immune system cells, and it has an important function in living organisms as a messenger between various cells. To be more specific, when a foreign object enters a living body, cytokine acts as a nonspecific protective factor in preventing infection, in immune response, and in inflammatory reactions. It also plays an important role as an initiator of humoral immunity and cellular immunity.\n Interleukin (IL)-1 is one cytokine and has been reported in such animal species as human, mice, rabbits, cats, pigs and cows. In particular, human IL-1 has been studied in detail. According to such study, IL-1 is made up of proteins whose molecular mass are approximately 17 kDa and that are mainly secreted by monocytes and macrophages, and there exist two kinds of IL-1 with different isoelectric points: IL-1α (isoelectric point: approx. pI≒5) and IL-1β (isoelectric point: approx. pI≒7). It is said that both IL-1α and IL-1βcombine with the same IL-1 receptor and have the same biological functions. For human and mice, a kit to measure IL-1α and β with ELISA (Enzyme linked immunosorbent assay) using monoclonal antibodies has already been developed and used. Also, with regard to IL-1 gene expression and their regulation mechanism, detailed studies on regulatory gene and intranucleus trsanscription factor are being carried out. Like other cytokines, IL-1 has a wide range of bioactivities. Its most important function is inducing the production of IL-2 from helper T cells and promoting division and propagation of T cells through this IL-2. It also influences the division and propagation of B cells. IL-1 acts not only on immune system cells but also is involved in inflammatory reactions. For example, in the brain, it works as an endogeneous pyrogen; in hepatic cells, it promotes production of acute phase proteins which reveal inflammatory reaction; and in synovial cells, it induces production of collagenase and prostaglandins.\n As for bovine IL-1, Maliszewski et al. succeeded in cDNA cloning and determined its sequence in 1988. Protein expression by E. coli was also reported. Their report, however, is limited to molecular biological study of IL- 1 as a substance and dose not touch upon the dynamic state of cytokine regarding protective reactions in living bodies, i.e., from gene expression in the producing cell to secretion and its kinetics. Also, bioactivity of IL-1 in both human and mice is measured using an index a propagation of reinforcing action in the presence of mitogen in mice thymocytes. But it is impossible to distinguish IL-1α from β with this method, which means that comprehensive activities of IL-1α and β is measured. Thus it is impossible to determine, with a biological measuring method, whether IL-1 activity is caused by α or β.\n This study was conducted to clarify the action of bovine IL-1 as an immunological initiator (the significance of IL-1 production during illness in particular) and the relation between individual differences of IL-1 production and the induction of immune response. For this purpose, I established a method to detect small amounts of bovine IL-1 mRNA by using RT-PCR (Reverse transcription-polymerase chain reaction) and ELISA, the latter of which uses monoclonal antibody as a method to measure IL-1β which is said to be produced in large amount. I used these methods to examine IL-1 gene expression and productivity.\n\n The results of this study are summarized as follows.\n\n1. Separation of bovine peripheral mononuclear cells, monocytes and alveolar macrophages\n\n Bovine peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Conray (s.g.=1.081) gradient centrifugation of heparinized peripheral blood. As a result, about 1×10^7 mononuclear cells were obtained from 10 ml of the heparinized blood, and a two-hour adhesive operation yielded about 1-1.5×10^6 monocytes. To prepare the bovine alveolar macrophages (AMs) the lungs of healthy cow were lavaged using sterile phosphate-buffered saline (PBS) and AMs were separated by an adhesive method. As a result, about 1-2×10^8 alveolar macrophages per adult cow were separated. Both PBMCs and AMs were cultured in RPMI-1640 medium supplemented with 2mM of L-glutamine, 25mM of HEPES, 5×10^-5 M of 2ME, 100IU/ml of penicillin, 100μg/ml of streptomicin, and 10% heat inactivated fetal calf serum (FCS). These cells were proven to be macrophages, because of their phagocytosis of fixed sheep erythrocytes, positive stain of non-specific esterase and acid-phosphatase.\n\n2. Detection of bovine IL-1α and IL-1β mRNA by RT-PCR method\n\n Poly(A)^+ RNA from PBMCs and AMs stimulated with 20μg/ml lipopolysaccharide (LPS) were isolated using a mRNA purification kit. Using poly(A)^+ RNA as a template, RT-PCR method to detect IL-1α and IL-1β mRNA was established. RT reaction was employed at 42℃ for 15 min and then at 99℃ for 5 min to inactivated reverse transcriptase. The following PCR reaction was employed as following condition: 1) denaturation: 95℃, 1 min. (except for the 1st denaturation, which was 3 min), 2) primer annealing: 55℃, 1 min, and 3) extension: 72℃, 1 min. PCR reaction was coducted generally in cycles of 35. For a primer at RT reaction, generally 3 kinds can be used: 1) random hexamers, 2) oligo d(T)16, and 3) specific downstream primer. In this experiment also, each of the primers produced good results. That is, the fragments of predicted size (IL-1α: 424bp, and IL-1β: 394bp) were detected as a result of 4% agarose gel electrophoresis. These RT-PCR products were identified by restriction enzyme analysis. The enzymes used and diagnostic fragments were 317/107bp; (IL-1α, HindIII), 154/146/124bp; (IL-1α, HinfI), 301/93bp; (IL-1β, HindIII), 326/68bp; (IL-1β, PstI). These results were in agreement with the sequence data of bovine IL-1α and IL-1β by Maliszewski et al. To evaluate the sensitivity of RT-PCR, serial ten-fold dilutions (0.1 ng to 1 fg) of poly(A)^+ RNA isolated from PBMCs stimulated with LPS for 24 hours were amplified by using the IL-1α and IL-1β specific primers. After 35 cycles of PCR, IL-1α specific fragment was detected at 0.01ng of template poly(A)^+ RNA, whereas, IL-1β specific fragment was detected at 0.1 pg. Furthermore, to evaluate the efficiency of poly(A)^+ RNA isolation, poly(A)^+ RNA was isolated from serial ten-fold dilutions (1×10^7 to 1×10^2) of PBMCs and subjected to RT-PCR using the IL-1α specific primer. After 35 cycles of PCR, IL-1α specific fragment could be detected in as few as ten PBMCs. Furthermore, for detection sensitivity by the number of PCR cycles, where 5 ng of poly(A)^+ RNA was used as template, the IL-1α specific fragment was detected using more than 25 cycles of PCR. Also, the RT-PCR method was about 10^6 times more sensitive than the norther blotting method. Evaluation of application of RT-PCR products proved them quite useful as a probe in the northern blotting, dot blotting and in situ hybridization methods.\n Evaluation of the dynamic expression state of IL-1 mRNA in vitro using the RT-PCR method confermed that, in peripheral blood mononuclear cells, the expression of IL-1α and IL-1β mRNA is seen structurally (continuously) even in the so-called resting situation, although it is quite low in level. And also, expression of IL-1α and IL-1β mRNA is increased in a shorter time period (within 3 hours) due to the LPS stimulation, and the expression of IL-1α mRNA is transient in nature. As a result of consideration of the dynamic expression state of IL-1 mRNA in cattle administered LPS, it was indicated that, also in vivo, mRNA was found in quite a short time period after LPS was administered, and that it is deeply involved in inflammation as so-called early-phase cytokine. Also, as in the case in vitro, the transient expression of IL-1α mRNA was found. Forther examination about individual differences in the expression of IL-1β mRNA demonstrated individual differences in its expression level.\n\n3. Measerement of bovine IL-1 activity and monoclonal antibody against IL-1β\n\n A series of monoclonal antibodies was producted by an in vitro stimulation method with IL-1β refined from a supernatant of the macrophage culture by purified by isoelectric focusing as antigen. To measure IL-1β, ELISA using this monoclonal antibody was established and be compared the measurement with result of bioassay using mouse thymocytes. As a result, IL-1 in the supernatant of the culture with almost the same level of sensitivity as the bioassay. IL-1 produced by peripheral mononuclear cells was measured both by bioassay using mouse thymocytes and by ELISA, and activity was detected in 3 hours after the stimulation, and high activity in 24 and 48 hours.\n\n As explained above, expression of IL-1α and IL-1β mRNA was found in quite a short time (30 minutes-3 hours) after LPS stimulation. This indicates that IL-1α and IL-1β are deeply involved in early phase, non-specific inflammation of protective reaction of living bodies as so-called \"early phase cytokines\". That is, protective reaction of living bogies following infection can be divided into an early phase, non-specific inflammation reaction and a late phase, specific immune response. Non-specific inflammation reaction such as fever in the early reaction, production of acute phase protein, and infiltraion of phagocytes involve so-called early cytokines, of which IL-1 is typical. Antigens processed by macrophages and B cells early in reaction are presented to corresponding T cells along with class II molecules of MHC (Major histocompatibility complex), and the stimulated T cells produce late-phase cytokines such as IL-2, IL-4, IL-5 and IFNγ. I also detected the expression of IL-1β mRNA in peripheral mononuclear cells in so-called resting state of healty cows, and confermed individual differences. This result, though it is not clear whether it is simply due to differences in situatioin including the breeding environment of individual cows or to immunogenetic individual differences, is interesting. In other words, if the difference in expression of IL-1β at a time of infection appears as an individual difference in the immune response which takes part afterward and it can be analyzed as one of the things regulating the sensitivity and resistance to disease, it is significant as a direction of future cattle clinical immunological study.\n\n The report on the detection of the expression of bovine IL-1α and IL-1β by the RT-PCR method follows that of on IL-2 and IL-6, will be applied to many cytokins, and will help clarify details of the cytokine network. The IL-1β measurement method by ELISA using monoclonal antibodies is also useful as an easy measurement method of bovine IL-1β.\n\n Recently, cDNAs such as bovine IL-1, IL-2, IFN, TNFα and CSF have been isolated, which has made possible studies on clarification of immune system using recombinant cytokine. Clinical application of cytokine to cattle is designed to prevent or cure infections. In particular, cytokine can be used for calves which have a high risk of becoming unusable due to infection in order to improve the protective ability of their immature bodies. It can also be used to restore immune systems which have deteriorated during the perinatal period or times of stress. Furthermore, the adjuvant effect of cytokine to intensify the immune effect of recombinant vaccines has also been indicated. However, in order to utilize cytokines as medicine for preventing or curing infections, it is necessary to clarify each one's effect and network in detail. With the production of many recombinant cytokines, and the establishment of a detection method of the expression of cytokine genes and a cytokine activity measurement method, there will be remarkable progress in the clarification of the cattle immune system.","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"サイトカインとは細胞，主として免疫系細胞が産生，分泌する高分子ペプチドの総称であり，各種細胞間情報伝達物質として，生体において重要な機能を担っている。すなわち，生体への異物の侵入に際し，感染防御，免疫応答あるいは炎症反応等の場において非特異的防御因子として，または，液性免疫，細胞性免疫に働きかけるイニシエーターとして重要な役割を果たしている。その内のひとつ，インターロイキン1(IL-1)はヒト，マウス，ウサギ，ネコ，ブタ，ウシ等の動物種で報告されている。特にヒトIL-1に関しては詳細な研究がなされている。それによると，IL-1は主として単球・マクロファージ系の細胞から分泌される，約17kDaの蛋白質であり，等電点の異なるIL-1α(等電点約5)とIL-1β(等電点約7)の2種類が存在する。IL-1αとIL-1βは共に同一のIL-1レセプターに結合し，同一の作用を発揮するとされている。ヒトやマウスにおいては既に単クローン抗体を用いたELISAによるIL-1α及びβの測定キットが開発され，利用されている。さらに，IL-1遺伝子発現及び発現調節機構についても調節遺伝子や核内転写因子等の詳細な研究が進んでいる。IL-1は他のサイトカインと同様，多様な生物活性を持っている。最も重要な機能はヘルパーT細胞からのIL-2の産生を誘導し，このIL-2を介してT細胞の分化・増殖を促進する点にある。また，B細胞の分化・増殖にも影響している。IL-1は免疫系細胞に対して作用を示すばかりでなく，脳に対しては内因性発熱因子として作用したり，肝細胞に対しては炎症作用を発揮する急性期蛋白質の産生を促進し，更に，滑膜細胞に対してはコラゲナーゼやプロスタグランジン産生を誘導するなど，炎症反応にも関与している。\n　ウシIL-1に関しては1988年，MaliszewskiらによりcDNAがクローニングされ，その塩基配列が決定された。また，大腸菌による蛋白の発現も報告されている。しかし，彼らの報告はIL-1の物質としての分子生物学的研究にとどまり，生体防御反応に関わるサイトカインとしての動態，すなわち，産生細胞内での遺伝子発現から分泌およびそのカイネティクス等についてはふれられていない。また，IL-1の生物活性はヒト，マウスにおいてもマウス胸腺細胞のマイトジェン存在下での増殖増強作用を指標として測定されているが，この方法ではIL-1α及びβの区別が不可能であり，IL-1αおよびβの総合的な活性を測定していることになる。したがって生物学的測定法ではIL-1活性がαによるものであるのかβによるものであるのかの区別が不可能である。\n　本研究はこのような背景に基づいて，IL-1の免疫学的イニシエーターとしての作用，特に，疾病におけるIL-1産生の意義，さらにIL-1産生の個体差と疾病に対する免疫応答誘導との関連性等を解明するための新技法の確立を目的として行われたものである。そのためにRT-PCR法を用いたウシIL-1遺伝子発現の微量検出法を確立し，また，その遺伝子産物，特に産生量の多いといわれているIL-1βの測定法として単クローン抗体を用いたELISAを確立し，これらの手法を用いてIL-1遺伝子発現および産生性について検討を加えた。\n\n　以下に得られた主要成果を要約する。\n\n1. 牛末梢血単核球，単球及び肺胞マクロファージの分離\n　健康牛の頸静脈からヘパリン加血液を採取し，フィコール・コンレイ比重遠心法(比重=1.081)により単核球を分離した。その結果，ヘパリン加血液10mlから約1×10^7個の単核球が得られ，さらに2時間の付着操作により約1-1.5×10^8個の単球が得られた。肺胞マクロファージは健康牛の気管支肺胞洗浄液より細胞を回収し，付着法により分離した。その結果，成牛1頭当り約1-2×10^8個の肺胞マクロファージが分離された。これらの付着細胞はグルタールアルデヒド固定羊赤血球の貪食能陽性，非特異的エステラーゼ染色陽性，酸フォスファターゼ染色陽性によりマクロファージであることが確認された。\n2. RT-PCR法による牛IL-1α及びIL-1βmRNAの検出\n　牛末梢血単核球および肺胞マクロファージを20μg/mlのLPSにより一定時間刺激し，両細胞から分離，精製したpoly(A)^+ RNAをテンプレートとしてRT-PCR法によるIL-1α及びIL-1βmRNAの検出法を確立した。RT反応条件は42℃，15分で行い，続いて99℃，5分で逆転写酵素を失活させた。そしてPCR反応条件は，1)熱変性:95℃，1分(1回目の熱変性のみは3分)，2)プライマーアニーリング:55℃，1分，3)伸長反応:72℃，1分で行い，原則として35サイクルで実施した。RT反応時のプライマーとしては一般に1)ランダムヘキサマー，2)オリゴd(T)_16，3)特異的下流プライマーの3種類が使用可能であるが本実験においてもこの3種類のプライマーの何れを用いてもよい結果が得られた。すなわち，4%アガロースゲル電気泳動の結果，予想したサイズのバンドを検出した(IL-1α:424bp，およびIL-1β:394bp)。これらのRT-PCR生成物は制限酵素切断パターンにより同定した。\n　RT-PCR法の検出感度を検討したところ，LPSで24時間刺激した末梢血単核球において，IL-1αでは最少poly(A)^+ RNA，0.01ng，また，IL-1βでは最少0.1pgの濃度で検出可能であった。また，サンプル細胞数による検出感度としては，最少1×10の細胞から抽出したpoly(A)^+ RNAで検出可能であった。さらに，PCRサイクル数による検出感度としては，50ngのpoly(A)^+ RNAをテンプレートとした場合，サイクル数25以上で検出可能であった。また，ノーザンブロット法に比べ約10^6倍，RT-PCR法の方が高感度であった。\n　RT-PCR産物の応用について検討した結果，ノーザンブロット法，ドットブロット法およびin situ hybridization法におけるプローブとして極めて有用であった。\n　RT-PCR法を用いてin vitroにおけるIL-1遺伝子発現動態を検討した結果，末梢血単核球ではLPS等の刺激のない，いわゆるrestingの状態であってもきわめて低レベルではあるが，構成的(持続的)にIL-1αおよびIL-1βmRNAの存在が認められることが明らかとなった。さらに，LPS刺激により短時間(3時間以内)でIL-1αおよびβmRNA濃度の上昇が認められ，また，IL-1αmRNAの発現が一過性を示すことが明らかとなった。また，LPS投与牛におけるIL-1mRNA発現動態について検討した結果，in vivoにおいてもLPS投与後，極めて短時間でmRNAの出現を認め，いわゆるearly phaseのサイトカインとして炎症に深く関わることが示された。また，in vitroにおいても認められたIL-1αmRNA出現の一過性も認められた。さらに，IL-1βmRNA生成の個体差を検討した結果，その量は個体により差を認めた。\n3. 牛IL-1活性測定とIL-1βに対する単クローン抗体\n　肺胞マクロファージの培養上清から液体等電点電気泳動法により精製したIL-1βを免疫原としてin vitro stimulation法により単クローン抗体を作成した。この単クローン抗体を用いたELISAによりIL-1βの測定を試み，マウス胸腺細胞を用いたバイオアッセイの結果と比較した。その結果，バイオアッセイと同程度の感度で培養上清中の1L-1の測定が可能であった。マウス胸腺細胞を用いたバイオアッセイおよびELISAを用いて末梢血単核球の産生するIL-1を測定した結果，刺激後3時間以降で活性が認められ，24，48時間後には高活性が認められた。\n\n　本研究は牛IL-1の免疫学的イニシエーターとしての作用，特に，疾病におけるIL-1産生の意義，さらにIL-1産生の個体差と疾病に対する免疫応答誘導との関連性等を解明することを目的として行われたものである。そのために，RT-PCR法を用いた牛IL-1αおよびIL-1β遺伝子発現の高感度検出法及び，単クローン抗体を用いたELISAによるIL-1β測定法を確立した。RT-PCR法による牛IL-1αおよびIL-1βmRNA検出に関する報告はIL-2およびIL-6につづくものであり，今後，さらに数多くのサイトカインに応用され，サイトカインネットワークの詳細の解明に役立つであろう。また，単クローン抗体を用いたELISAによるIL-1β測定法も牛IL-1βの簡易測定法として有用である。\n　近年，牛のIL-1，IL-2，IFN，TNFα，CSFなどの遺伝子やcDNAが単離され組換体サイトカインを用いた免疫機構の解明に関する研究が可能となってきた。牛におけるサイトカインの臨床応用は，感染症の予防，治療効果を目的としたものである。特に感染症による廃用率が高い子牛に対し，未熟な生体防御機能の増強を目的としたサイトカインの利用が考えられる。また，周産期やストレス負荷時などに低下した免疫機能を回復させるための利用も考えられる。さらに，組換体ワクチンの免疫効果の増強を目的としたサイトイカインのアジュバント効果が提示されている。しかし，感染症の予防，治療薬としての有効利用をはかるためには個々のサイトカインの詳細な作用に加えて，サイトカインネットワークの詳細の解明が必要であろう。多くの組換体サイトカインの作出，サイトカイン遺伝子発現の検出法あるいはサイトカイン活性測定法の確立により家畜の免疫機構解明はめざましく発展するであろう。","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第332号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"伊藤, 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