{"created":"2023-06-19T07:18:08.337459+00:00","id":3260,"links":{},"metadata":{"_buckets":{"deposit":"76f95488-7757-4634-bab7-ac821b2a8349"},"_deposit":{"created_by":4,"id":"3260","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3260"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003260","sets":["17:254:334","370:15:392"]},"author_link":["16355"],"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":"Animals with gram-negative bacterial infection often fall into some fatal conditions, including shock. It is well known that endotoxin (LPS) is mainly responsible for these conditions; however, its pathophysiological mechanism is unclear.\n Platelet activation is one of the important vital reactions against LPS. Endotoxemic animals frequently develop disseminated intravascular coagulation (DIC) or shock lung due to excessive platelet activation. Therefore, appropriate control of platelet activation is very important in the treatment of severe gram-negative bacterial infection. The mechanism of LPS-induced platelet activation must be determined to establish a method for platelet control.\n It is presumed that a number of chemical mediators are involved in LPS-induced vital reactions, including platelet activation. Recently, platelet-activating factor (PAF), a phosphocholine, has been recognized as an important mediator of LPS-induced vital reactions. PAF is a potent stimulator for many kinds of cells, including platelets.\n Small animal clinicians often encounter thrombocytopenic patient dogs with gram-negative bacterial infection. LPS stimulates canine platelets in vitro; however, the reaction is weak. Possibly, another platelet stimuli must be produced in endotoxemic dogs. Therefore, the author hypothesized that PAF plays an important role in LPS-induced excessive platelet activation in dogs with gram-negative bacterial infection. Since intravascular platelet aggregation induced by some stimuli results in remarkably consumptive thrombocytopenia, the platelet count in peripheral blood may be a useful indicator of intravascular platelet activation.\n Therefore, to verify the hypothesis, the author performed the following experiments and studied the role of PAF in LPS-induced thrombocytopenia in dogs.\n\nI. Sample preparation for platelet aggregometry.\n\n Platelet aggregometry is the most basic screening test of platelet reaction. Intact platelets must be isolated from blood for successful platelet aggregometry. Therefore, sample preparation methods for platelet aggregometry were studied.\n\n1) The optimum centrifugal condition to obtain platelet rich plasma (PRP).\n The most common sample used in aggregometry is PRP obtained by centrifugation of whole blood. Since size and density of platelets are different among animal species, appropriate centrifugal conditions are needed for each animal species. Therefore, canine whole blood anticoagulated with 3.8 % sodium citrate solution was centrifuged at different spinning speeds and times.\n The best recovery of platelets was obtained at 3,500 rpm centrifugation for 1 minute. Platelets in PRP, separated by this centrifugal condition, aggregated well to adenosine diphosphate (ADP) and collagen stimulation.\n\n2) Platelet isolation from plasma.\n It is known that platelets interact with several plasma components. Thus, to study the effects of some substances, e.g. PAF, on platelet reaction, platelets need to be isolated from plasma to avoid the effects of plasma components. Therefore, appropriate procedure for platelet isolation from PRP was studied.\n Platelets washed by centrifugation with albumin gradient cushion could not be used in aggregometry because of spontaneous aggregation. In contrast, intact platelets were well isolated from plasma by gel filtration using Sepharose 4B and Tangen HEPES buffer solution. The platelets were aggregated by both ADP and collagen stimulation in a modified MgCl_2 buffer solution; the concentration of MgCl_2 was shifted to 1 mM.\n\nII. The role of PAF in LPS-induced thrombocytopenia.\n\n1) Blood PAF concentrations in LPS-injected dogs.\n 1 mg/kg body weight (BW) of LPS was injected intravenously (LPS large dose injection) into 3 mongrel dogs under general anesthesia. 40 μg/kgBW of LPS was injected intravenously (LPS small dose injection) into another 9 beagles. Blood PAF concentrations before and after LPS injection were analyzed by radio-immunoassay.\n PAF concentrations increased significantly (P<0.05) in both the LPS large- and small-dose injected dogs.\n\n2) Hematological changes in LPS- or PAF-injected dogs.\n 40 μg/kgBW of LPS was injected intravenously (IV) into 16 beagle or mongrel dogs. 0.5 or 1 μg/kgBW of PAF was injected IV into 6 beagles. PAF (5 μg/kgBW/hour for 90 minutes) was infused into 3 beagles. As a control, physiological saline, 1 ml/kgBW/hour, was infused for 90 minutes into 3 beagles. Platelet and leukocyte counts were done before and after injection. Transient thrombocytopenia, leukopenia, and delayed rebound leukocytosis were seen in the 3 treatment groups with either LPS or PAF injection. These changes in PAF-injected dogs were milder and recovered faster than those in LPS-injected dogs.\n\n3) The suppressive effects of a PAF antagonist against LPS-induced thrombocytopenia.\n At first, a specific effect of TCV-309 (a PAF antagonist provided by Takeda Chemical Industry Co.Ltd.) in vitro and in vivo on PAF stimulated platelet activation was confirmed by aggregometry and ATP-release test.\n Secondly, the appropriate injection dose of TCV-309 was studied using platelet aggregometry by PAF stimulation. The appropriate IV injection dose of TCV-309 was 300 μg/kgBW.\n Finally, 40 μg/kgBW of LPS was injected IV into 11 beagle or mongrel dogs after pretreatment with TCV-309 (300 μg/kgBW). Blood platelets were counted before and after LPS injection.\n LPS-induced thropretmbocytopenia was significantly (P<0.05) suppressed by TCV-309 pretreatment.\n\n4) Desensitization of platelets to PAF after LPS injection.\n LPS induces severe thrombocytopenia in dogs; however, many platelets recirculate 1 hour after the injection. Therefore, desensitization of platelets to PAF was studied to confirm any evidence of PAF stimulation in platelets. Aggregometry of recirculated platelets using PRP or gel filtrated platelet suspension was performed in 6 beagles.\n The disappearance of PAF stimulated platelet aggregation, which means desensitization to PAF, was found in only one case using PRP.\n\n The results above; 1-3), strongly suggested that PAF was associated with LPS-induced thrombocytopenia. In conclusion, the hypothesis above was justified.\n However, PAF injection could not induce severe thrombocytopenia as found in the LPS-injected dogs, and TCV-309 did not strongly suppress thrombocytopenia. Therefore, the author thought that some other factors may stimulate platelets synergicaly with PAF.\n"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"グラム陰性菌に感染した動物は，しばしばショックなど致死的な状態に陥る。これは細菌の産生するエンドトキシン(LPS)の作用によるといわれている。しかしながら，LPSがどのようにして生体に致死的な反応を起こさせるか，そのメカニズムは十分に解明されていない。\n　LPSに対する生体反応として，血小板の活性化も重要なものの一つに挙げられる。すなわち動物がLPS血症に陥ると，血小板は過剰に活性化して，血管内凝固症候群(DIC)やショック肺などを引き起こす。したがって，LPS血症に陥る危険性のある重篤なグラム陰性菌感染症においては，血小板の制御が治療上重要なポイントとなる。この制御法を確立するためにはまず，LPSによって引き起こされる血小板活性化メカニズムを解明することが不可欠である。\n　血小板の活性に限らず，LPSによる生体反応には数々の中間的化学伝達物質の存在が考えられ，近年その一つとして，フォスフォコリンの一種，血小板活性化因子(PAF)が注目されてきた。この物質は多種類の細胞に対する刺激性を持ち，血小板もその主要な標的細胞の一つである。\n　小動物臨床分野においてはしばしば，犬のグラム陰性菌感染によるDICなど，血小板の過剰活性化症例に遭遇する。LPS自体も犬血小板に対して刺激性を持っているが，in vitro実験でみる限りその刺激はそれ程強いものでないことから，LPSによって体内に産生される何らか別の血小板刺激因子の存在が考えられる。そこで，『犬のグラム陰性菌感染の際に見られる血小板活性化には，PAFが関与している』という仮説を立てた。\n　血小板が活性化して血管内で凝集すると，その結果として末梢血中の血小板数は消費性に著しく減少する。そのため血小板数の減少は，血小板活性化の一指標となる。そこで上記仮説を裏付けるために，実験的LPS血症犬を用い，血小板減少の発現にPAFが関与することを証明しようとした。\n　研究成績の概要を以下に示す。\n\n1.血小板凝集試験に用いる試料の前処理法\n　血小板反応の研究では，血小板凝集試験が最も基本的な血小板活性化のスクリーニング検査である。この試験を行うためには，血小板をできるだけ活性化させずに血液から分離する必要がある。そこで最初に，試料前処理に関する基礎的検討を行った。\n1)多血小板血漿(血小板に富む血漿;PRP)を得るための遠心条件\n　凝集試験に用いる最も一般的なサンプルは，全血から遠心によって分離するPRPである。動物種によって血小板のサイズや比重に大きな違いがあるので，その遠心条件は動物種別に設定しなければならない。そこで，3.8%クエン酸Na液で凝固阻止した全血を，いろいろな速度と時間の組み合わせで遠心した。\n　血小板回収率が最も良かったのは，3,500rpm(2000g)・1分間で，平均71.7%が回収された。このPRP中の血小板は，ADPおよびコラーゲンによる刺激に対して十分な凝集反応を示した。\n2)血漿からの血小板分離\n　いくつかの血漿成分が，血小板反応に影響を与えるといわれている。したがってPAFが血小板にどのような影響を与えるか検討する場合に，誤差要因となる血漿成分による影響を取り除くことが必要である。そこで，血小板をPRPから分離し，緩衝液に再浮遊させる方法を検討した。\n　アルブミン密度勾配をクッションにした遠心洗浄で分離した血小板には自然凝集が起きてしまい，凝集試験に使用できなかった。これに対して，セファロース4BとTangen HEPES緩衝液を用いたゲルろ過法では，活性化していない血小板が効率的に分離され，さらにこの緩衝液の組成を一部改良(MgCl_2を1mMに変更)することによって，凝集試験にも使用できた。\n2.LPS投与犬の血小板減少におけるPAFの関与\n1)LPS投与犬の血中PAF濃度\n　全身麻酔した3頭の雑種犬にLPSを1mg/kg(大量投与群)，およびビーグル犬9頭に40μg/kg(少量投与群)を静注して，その前後の血中PAF濃度をラジオイムノアッセイ法で測定した。\n　その結果，両群ともにLPS静注後の血中PAF濃度は有意に増加した。\n2)LPSおよびPAF投与犬の血液学的変化\n　16頭の犬にLPSを40μg/㎏，6頭にPAFを0.5ないし1μg/kg静脈注射（単回投与)した。また，3頭にPAFを5μg/kg/時，他の3頭（対照群)に生理食塩水を1ml/kg/時の速度で，それぞれ90分間連続静注した。\n　LPS投与群，PAF単回，およびPAF連続投与の3群に共通して，血小板数と白血球数の減少，およびその後の反動的な白血球数増加が観察された。白血球増減の主体は成熟好中球であった。これらの変化は，PAF投与犬の方がLPS投与犬より軽度で，回復も速かった。\n3)PAF阻害剤によるLPS誘導血小板減少の抑制\n　PAF阻害剤として武田製薬(株)が開発したTCV-309を用いた。\n　まず，PAFに対する血小板の反応が，TCV-309のin vitroおよびin vivoいずれの処理によっても特異的に阻害されることを，凝集試験およびATP放出試験を用いて確認した。次いで，TCV-309の投与適量を検討した。\n　最後に，適量のTCV-309を前投与した犬5頭および生理食塩水を前投与した犬6頭（対照)に，LPS40μg/kgを投与し，その前後の末梢血血小板数を測定した。その結果，TCV-309前投与群の血小板減少は，対照群に比べ有意に抑制された。\n4)LPS投与後再循環した血小板のPAF脱感作\n　犬にLPSを投与すると，ほとんどの血小板は末梢血から一旦消失し，1時間後には大半が再循環する。そこで次の手順によって，再循環血小板がPAFに感作された形跡(PAF脱感作)を確認しようと試みた。延べ6頭の犬にLPSを投与し，1時間あるいは40分後に採血して，PRPないしゲルろ過血小板を用いたPAF凝集試験を行った。しかしながら，いずれのサンプルを用いても，PAF脱感作を示す血小板のPAF凝集能低下は確認できなかった。\n\n　以上のうち1)～3)の結果から，犬のLPS血症における血小板減少すなわち血小板の活性化にPAFが関与していることはほぼ間違いなく，したがって，当初の仮説が正しいと考えた。\n　しかしながら，PAF投与犬の血小板減少はLPS投与犬のそれと比べて軽度であり，また，TCV-309を前投与して，LPSによる血小板減少が完全に阻止されないことから，血小板の過剰活性化は，PAFに対する反応のみではなく，他のなんらかの刺激物質との相乗作用によることが推察された。"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第346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":"土屋, 亮"},{"creatorName":"Tsuchiya, Ryo","creatorNameLang":"en"}],"nameIdentifiers":[{},{},{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-06-29"}],"displaytype":"detail","filename":"diss_dv_otsu0346.pdf","filesize":[{"value":"30.0 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0346","url":"https://az.repo.nii.ac.jp/record/3260/files/diss_dv_otsu0346.pdf"},"version_id":"2a68a1c1-d961-4c11-baa1-aa005b600b37"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-18"}],"displaytype":"detail","filename":"diss_dv_otsu0346_jab&rev.pdf","filesize":[{"value":"224.6 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0346_jab&rev","url":"https://az.repo.nii.ac.jp/record/3260/files/diss_dv_otsu0346_jab&rev.pdf"},"version_id":"fa3b0cb8-046f-4d86-9bae-51026b1909b6"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2013-02-20"}],"displaytype":"detail","filename":"diss_dv_otsu0346_jab.pdf","filesize":[{"value":"148.7 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0346_jab.pdf","url":"https://az.repo.nii.ac.jp/record/3260/files/diss_dv_otsu0346_jab.pdf"},"version_id":"28d5259c-4fc2-4932-bfeb-5d0947992b7c"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis"}]},"item_title":"エンドトキシン血症犬の血小板減少における血小板活性化因子(PAF)の関与","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"エンドトキシン血症犬の血小板減少における血小板活性化因子(PAF)の関与"},{"subitem_title":"The role of platelet-activating factor(PAF) in endotoxin(LPS)-induced thrombocytopenia i dogs","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["334","392"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-02-13"},"publish_date":"2013-02-13","publish_status":"0","recid":"3260","relation_version_is_last":true,"title":["エンドトキシン血症犬の血小板減少における血小板活性化因子(PAF)の関与"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:17:32.277675+00:00"}