{"created":"2023-06-19T07:18:01.950812+00:00","id":3162,"links":{},"metadata":{"_buckets":{"deposit":"51f286c5-5d12-49ca-a2b3-a9ae55fdecae"},"_deposit":{"created_by":4,"id":"3162","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3162"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003162","sets":["370:15:391"]},"author_link":["16169"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2004-03-13"}]},"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":"[Chapter I]\n Glomerulonephritis is very important disease and many cytokines such as platelet derived growth factor (PDGF), transforming growth factor (TGF-β) or vascular endothelial growth factor (VEGF) are involved in the mechanisms of progression and recovery of this disease. Analyzing the expression of these factors in the glomeruli is very important to understand the pathogenesis and to provide new approaches to the therapy of glomerulonephritis. To isolate renal glomeruli for the analysis of gene expression, some methods including sieving method have been used, and recently, laser microdissection (LMD) or laser capture microdissection (LCM) system which enable us to dissect target tissue such as glomeruli from the tissue sections have developed. In this chapter, these three different methods (sieving method, LMD and LCM system) were compared to find the best method to isolate glomeruli for the analysis of gene expression in the glomeruli efficiently and exactly. At first, sieving method was performed using three different sizes of stainless steel meshes and glomeruli were isolated from the renal cortex of normal male CRJ/CD rats. Second, frozen sections of the renal cortex derived from normal male Fischer 344 rats were made and after fixation and washing, they were air-dried. And then, the glomeruli were dissected from their sections and collected to the transfer films on the tube cap using LCM system. Third, frozen sections of the renal cortex derived from normal male Wistar rats and after fixation, staining and washing, they were air-dried. And then glomeruli were dissected from their sections and dropped into the tube cap filled with RNA extraction regent using LMD system. After confirming the quality of the samples, total RNA was extracted from the collected glomeruli and analyzed their concentration, and RT-PCR was performed to detect GAPDH gene expression in the glomeruli. The glomerular samples obtained by sieving method contained other renal component and quantity of total RNA obtained was not stable. With sieving method, GAPDH could be amplified and confirmed its specific size band by electrophoresis. It was possible to dissect glomeruli exactly and to collect them easily from the frozen sections using LMD and LCM systems. However, it was difficult to take the collected glomeruli away from the films and to amplified GAPDH gene by PCR although 1000 glomeruli were collected using LCM system, while dissected 50 glomeruli using LMD system were collected efficiently and GAPDH gene expression was easily detected. In conclusion, LMD system is the best method to analyze gene expression in the renal glomeruli. The combination of this system and other technique such as real-time PCR would contribute to the resolution of the mechanisms of the disease.\n\n[Chapter II]\n The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. In the renal glomeruli, VEGF is produced by the podocytes in normal and diseased kidneys, activated mesangial cells, infiltrating leukocytes in vivo and endothelial cells in vitro. The receptor flk-1 is expressed on normal, damaged and regenerating glomerular endothelial cells as well as mesangial cells. The author suspected that VEGF might contribute the reconstruction of damaged glomeruli to stimulate glomerular cells such as endothelial cells as well as mesangial cells. Therefore, it is important to analyze the gene expression of VEGF and its receptors in the glomeruli for understanding the mechanisms of glomerular diseases and for establishing the new therapeutic strategies. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system in order to show the usefulness of this system for further applications to renal pathological research. The frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. RT-PCR revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. And it was possible to perform quantitative evaluation of flk-1 mRNA using twice as much cDNA derived from 200 glomeruli as it was used for VEGF and GAPDH with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms of kidney diseases.\n\n[Chapter III]\n One of the angiogenic growth factors, vascular endothelial growth factor (VEGF) and its specific receptor, flk-1 are strongly involved in endothelial cell proliferation. In the renal glomeruli, VEGF is produced by the podocytes in normal and diseased kidneys, activated mesangial cells and infiltrating leukocytes, and flk-1 is expressed on normal, damaged and regenerating glomerular cells including mesangial cells in human renal diseases and in rat anti-Thy-1 nephritis. Recent studies indicated that the glomerular endothelial cells were involved in the capillary repair and restoration of glomerular architecture, and VEGF played an important role in endothelial cell proliferation and capillary repair in a resolution phase of anti-Thy-1 nephritis. The author also suspected that VEGF might contribute to the reconstruction or repair of injured glomeruli. However, suitable method has not been performed as regards the quantitative analysis of VEGF and flk-1 mRNA expression in the glomeruli of anti-Thy-1 nephritis in the previous studies. In this chapter, the detailed quantitative evaluation was performed using laser microdissection system (LMD) technique and real-time PCR to clarify the role of VEGF and its receptor in the glomeruli of anti-Thy-1 nephritis. Furthermore, the author examined their localization using immunohistchemistry and in situ hybridization analysis.\n Anti-Thy-1 nephritis was induced in 8-week-old male Wistar rats and their kidneys were collected on days 3, 7, 14, 28 and 56 after the administration of anti-Thy-1 antibody. Histopathological and immunohistochemical examinations for ED-1, α-SMA, PCNA, VEGF and flk-1 were performed. For quantitative analysis of VEGF and flk-1 mRNA in the glomeruli, 200 glomeruli were obtained using LMD system and total RNA was extracted followed by reverse transcript (RT) reaction to make first strand cDNA , and real-time PCR was performed using primer pairs and TaqMan probes for VEGF164, flk-1 and GAPDH. Moreover, in situ hybridization was performed to examine the localization of VEGF164 mRNA using digoxigenin-labeled riboprobes.\n In anti-Thy-1 nephritis, mesangial lesions including mesangiolysis were observed on day 3, and the glomeruli on day 7 showed microaneurysm and intensive mesangial cell proliferation, and almost glomeruli had been reconstructed by day 56. VEGF protein and mRNA were located only in the podocytes, and there were no positive cells in proliferating foci in which α-SMA was positive. Flk-1 was located in the glomerular endothelial cells in control and nephritic kidneys, and there were no positive cells in proliferating foci. The level of VEGF and flk-1 mRNA expression were lower than that of control group on day 3, significantly reduced to the minimum on day 7, and then gradually increased. The level of VEGF mRNA returned to the control level by day 28. The level of flk-1 mRNA increased to the control level on day 14 and became significantly high on day 28 and then returned to the control level on day 56. The number of ED-1 positive cells infiltrating into the glomeruli was maximum on day 3 and then gradually reduced. However, none of these cells showed positive reaction of VEGF by immunohistochemistry and in situ hybridization.\n These results suggest that there may be little possibility of autocrine mechanism of VEGF/flk-1 in rat mesangial cells in vivo. And the mesangial cells might not be involved in endothelial cell proliferation by paracrine mechanisms of VEGF/flk-1. VEGF mRNA level in the glomeruli of nephritic kidneys was gradually increased, which was accompanied with the glomerular reconstruction suggesting that VEGF might play a role on the recovery of glomerular structure in anti-Thy-1 nephritis in the paracrine mechanism.\n\n[Chapter IV]\n Fibroblast growth factor-2 (FGF-2) belongs to a large family of fibroblast growth factors and there are four different forms (18-, 22.5-, 23.1- and 24.2- kDa). FGF-2 is expressed in many kinds of cells including fibroblasts, endothelial cells, vascular smooth muscle cells and macrophages. In the kidney, FGF-2 exhibits mitogenic actions on glomerular cells in vitro and may be involved in many phenomenons including mesangial cell proliferation, proteinuria, glomerulosclerosis and cytotoxic damage in vivo. FGF-2 is also known as the angiogenic factor and it might stimulate the proliferation of glomerular endothelial cells in vitro. Anti-Thy-1 nephritis is the reversible model of mesangioproliferative glomerulonephritis and in a \"resolution\" phase of this model, glomerular endothelial cells might be active participants in the capillary repair and restoration of glomerular architecture. Recent studies have suggested that VEGF would play an important role in endothelial cell proliferation and capillary repair in anti-Thy-1 nephritis. However, the author suggested that endogenous VEGF might have little importance on the recovery of the glomerular structure in chapter-3. Therefore, in this chapter, the author selected another angiogenic factor, FGF-2 and FGF receptor (FGFR) and examined their localization and FGF-2 mRNA expression by real-time PCR to clarify their role in anti-Thy-1 nephritis because there are a few studies on the role of FGF-2 in the recovery of injured glomeruli.\n Anti-Thy-1 nephritis was induced in 8-week-old male Wistar rats and their kidneys were collected on days 3, 7, 14, 28 and 56 after the administration of anti-Thy-1 antibody. Histopathological and immunohistochemical examinations for FGF-2, FGFR and RECA-1 were performed. For quantitative analysis of FGF-2 mRNA in the glomeruli, 200 glomeruli were obtained using LMD system and total RNA was extracted followed by reverse transcript (RT) reaction to make first strand cDNA, and real-time PCR was performed using primer pair and TaqMan probe for FGF-2.\n In anti-Thy-1 nephritis, mesangial lesions including mesangiolysis were observed on day 3 and the glomeruli on day 7 showed microaneurysm and intensive mesangial cell proliferation, and almost glomeruli had been reconstructed by day 56. RECA-1 positive cells decreased from day 3 and there were no RECA-1 positive cells in the proliferating foci on days 7 and 14. The number of RECA-1 positive cells distinctly increased and many RECA-1 positive cells and capillary lumens were observed by day 56. FGF-2 was observed in glomerular endothelial cells outlined the opened capillaries and in some podocytes. FGFR was located only in endothelial cells. There were no FGF-2 and FGFR positive cells in the proliferating foci in the glomeruli on days 7 and 14. FGF-2 mRNA level was minimum on day 3 and then gradually increased and became to be control levels as early as on day 14. These results suggest that FGF-2 expressed in endothelial cells and the podocytes might act on FGFR in endothelial cells. They might be greatly involved in the recovery of glomerular structure in autocrine/paracrine manner. Moreover, there might be little possibility which proliferating mesangial cells produce FGF-2 and play an important role on the proliferation of endothelial cells as well as mesangial cells.\n","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"［背景］\n　糸球体腎炎は、ヒトや動物の腎臓病の中で非常に重要な疾患の一つであり、その発症や進行のメカニズムは複雑である。近年、様々なサイトカインが本疾患の進行に関わっていることが示唆されており、これらサイトカインの役割を明らかにすることは、その進行のメカニズムの解明や、治療法の確立に重要であると考えられている。腎炎研究においては、従来、これらサイトカインが、その進行・進展にどのような役割を果たしているかに焦点が当てられてきたが、近年、本疾患の寛解メカニズムやそれに関わるサイトカインについても注目されつつある。そこで、著者は、障害された糸球体の再構築にとって重要と考えられる内皮細胞およびメサンギウム細胞（Mes細胞）に増殖刺激を持つとされるサイトカインの中から、血管新生因子である血管内皮増殖因子（VEGF）と線維芽細胞増殖因子-2（FGF-2）に注目した。\n　VEGFは、血管内皮に存在する2つの特異的レセプター、flk-1、flt-1に結合し、血管内皮細胞増殖、血管透過性亢進作用を示すサイトカインである。ヒトVEGFは、アミノ酸数が異なる少なくとも5つのアイソフォーム（VEGF_121, VEGF_145, VEGF_165, VEGF_189, VEGF_206）、の存在が示されているが、ラットでは少なくとも3つの、ヒトよりアミノ酸が1つ少ないアイソフォーム（VEGF_120,VEGF_164,VEGF_188）が存在する。一方、FGF-2は、様々な細胞での発現が認められており、高新和性レセプターFGFR-1～4に結合し、増殖因子、血管新生因子、走化性因子などとして作用する。\n　ラット抗Thy-1腎炎は、Mes細胞表面に存在するThy-1抗原に対する抗体を単回投与すると、メサンギウムの融解病変が生じ、次いでMes細胞を中心とした高度な細胞増殖反応が認められた後、糸球体がほぼ元の形態に修復する寛解型腎炎モデルである。VEGFは糸球体上皮細胞、活性化、Mes細胞や浸潤白血球に、flk-1は糸球体内皮細胞、活性化Mes細胞に局在し、本モデルの糸球体再構築に関わっていることが示唆されている。特に、本モデルでは、増殖病変の主体を成す活性化Mes細胞で産生されたVEGFが、糸球体構造の修復に深く関わるとの報告がある。しかし、これまでの報告では、糸球体局所におけるVEGF、flk-1の解析は厳密に行われておらず、その役割は必ずしも明らかではない。また、FGF-2は、糸球体修復の早期に内皮細胞増殖に関わることが報告されているものの、詳しい検討は行われていない。\n　そこで本研究では、厳密な糸球体分離法と糸球体局所での遺伝子発現の定量的解析条件を確立し、抗Thy-1腎炎の糸球体修復におけるVEGF/flk-1およびFGF-2/FGFRの動態を明らかにし、その役割について考察した。\n\n［第1章］\nThe methodological investigation for isolation of the renal glomeruli to evaluate local gene expression.（糸球体局所における遺伝子発現解析のための糸球体単離についての方法論的研究）\n\n　腎臓からの糸球体単離法として、以前から主に糸球体細胞培養のために利用されてきたSieving method（S法）や、近年、レーザーによる組織切片からの組織採取法として開発された異なる2種の方法、Laser Capture Microdissection（LCM）法、Laser Microdissection（LMD）法があるが、これらはmRNAの定量的解析や糸球体障害の研究にはほとんど応用されていない。そこで、本章ではこれらの方法により正常ラット腎糸球体を単離し、遺伝子発現解析までの操作、結果について比較検討した。\n　S法では、大きさが異なる3種のステンレスメッシュを用いて糸球体を回収した。また、新鮮凍結切片を作製し、LCMまたはLMDシステムにより、糸球体を30～1000個採取した。各々回収した糸球体を材料として、reverse transcribed-polymerase chain reaction（RT-PCR）法によりハウスキーピング遺伝子であるglyceraldehyde-3-phosphate-dehydrogenase（GAPDH）mRNAを検出した。\n　その結果、S法では多くの糸球体を回収できたが、他組織の混入を回避できなかった。また、GAPDH mRNAの発現は確認されたが、全RNAの回収量は一定せず、糸球体の単離過程でRNAの変性が起こり易いと思われた。LMD法では、正確に糸球体を回収でき、50個の糸球体から高い再現性をもってGAPDH mRNA発現を検出できた。一方、LCM法では正確に糸球体を回収できたが、糸球体からのRNA抽出効率やRT-PCR法の結果もLMD法に劣っており、糸球体サンプル採取法としてLMD法が最も優れていた。\n\n［第2章］\nDetection of gene expression of VEGF and flk-1 in the renal glomeruli of the normal rat kidny using the laser microdissection system.（レーザーマイクロダイセクション法を用いた正常ラット腎糸球体におけるVEGFとflk-1遺伝子発現の検出）\n\n　VEGFとその特異的レセプターflk-1の障害糸球体再構築時における動態を知るため、本章では糸球体におけるVEGF/flk-1遺伝子発現の定性的、定量的解析の条件を検討した。\n　正常ラット腎皮質から、LMD法により糸球体10～200個を採取し、全RNA抽出後、RT-PCR法およびリアルタイムPCR法によりVEGF、flk-1、GAPDH mRNA発現の定性的、定量的解析の条件を検討した。\n　その結果、糸球体10個以上でVEGF、GAPDHについて定量的解析は可能で、100個以上で安定したデータが得られることが明らかになった。また、flk-1の定量的解析には、VEGF、GAPDH解析時の2倍量の糸球体200個由来全RNAが必要であった。\n\n［第3章］\nThe role of VEGF and flk-1 on the recovery of the glomerular structure in anti-Thy-1 nephritis.（抗Thy-1腎炎の腎糸球体再構築におけるVEGFとflk-1の役割）\n\n　第1、2章により確立した方法により、本章では、ラット抗Thy-1腎炎におけるVEGF、flk-1の糸球体内での動態を検索した。\n　8週齢雄のWistarラットにウサギ抗Thy-1抗体を0.5ml/100g/B.W.尾静脈より単回投与し、抗Thy-1腎炎を惹起した。対照群には、正常ウサギ血清を同量投与した。投与後3、7、14、28、56日に腎臓を採取し、病理組織学的検索、免疫染色によるVEGF、flk-1の局在などの検索、LMD法による糸球体採取（200個/1個体）とリアルタイムPCR法によるVEGF、flk-1 mRNA発現の定量、in situ hydridization（ISH）法によるVEGF mRNAの局在検索を行った。\n　抗体投与後3日には糸球体メサンギウムに融解性変化、7日には顕著なMes細胞主体の細胞増殖性変化が確認された。14日の糸球体には増殖病変が認められるものや構造が修復されつつあるものが混在し、56日には糸球体構造はほぼ修復されていた。免疫染色により、実験期間を通して対照群、実験群ともにVEGFは糸球体上皮細胞のみに、flk-1は血管腔が開いている部分の糸球体内皮細胞のみに認められた。また、投与後7日のMes細胞主体の増殖病変内にはVEGF、flk-1ともに認められなかった。VEGF mRNA発現量は、抗体投与後3日で対照群より有意に減少し、7日には最低レベルを示した。その後有意な増加を示し、28日に対照群と同レベルとなった。flk-1のmRNA量は、投与後7日に有意に減少したが、14日には対照群と同じレベルに戻り、28日には対照群より有意に高くなり、56日には再び対照群と同レベルに戻った。ISH法による観察では、VEGF mRNAは、観察期間を通して対照群、実験群ともに糸球体上皮細胞のみに認められたが、シグナル強度の変化は認められなかった。また、Mes細胞主体の増殖病変内には発現していなかった。\n　以上より、本モデルの糸球体再構築において、Mes細胞にはVEGF、flk-1の発現を認めず、細胞増殖期にこれらの遺伝子発現レベルが最低であったことから、Mes細胞によるVEGFのオートクライン的作用と内皮細胞に対するパラクライン的作用はほとんどなく、VEGF発現を認めた上皮細胞とflk-1発現を認めた内皮細胞間でのパラクライン的作用が作動し、内皮細胞増殖を促進し、糸球体修復に関与することが示唆された。\n\n［第4章］\nThe role of fibroblast growth factor-2 and its receptor on the recovery of the glomerular structure in anti-Thy-1 nephritis.（抗Thy-1腎炎の糸球体再構築におけるFGF-2とその特異的レセプターの役割）\n\n　VEGFと同様に、血管新生因子のFGF-2とそのレセプターについて、抗Thy-1腎炎における動態を検討した。\n　ラット抗Thy-1腎炎の腎サンプルを用いて、血管内皮マーカーRECA-1、FGF-2、FGFRの免疫染色を行い、糸球体における内皮細胞の変化とFGF-2/FGFRの局在を観察した。また、LMD法で採取した糸球体を用いて、リアルタイムPCR法によりFGF-2 mRNA発現を定量した。\n　RECA-1陽性細胞は、抗体投与後3日より糸球体構造の崩壊とともに減少し、投与後7日には増殖性病変辺縁に残存する糸球体毛細血管にわずかに認められたが、投与後14日以降にみられる糸球体の修復とともに増加し、多くの小さな血管腔が徐々に認められるようになった。形態学的に糸球体構造がほぼ修復した投与後56日には、RECA-1陽性細胞に囲まれた多くの血管腔が認められ、対照群とほぼ同様の所見を示した。FGF-2は対照群、実験群の内皮細胞、一部の糸球体上皮細胞に、FGFRは両群の糸球体内皮細胞のみに認められた。Mes細胞主体の増殖病変部にはFGF-2、FGFRともに認められなかった。FGF-2 mRNAは投与後3日に最低レベルを示し、その後有意に増加し投与後14日以降対照群と同レベルとなった。\n　以上より、ラット抗Thy-1腎炎においてFGF-2、FGFRは、ともにMes細胞に発現していなかったことから、VEGF同様、Mes細胞におけるオートクライン的作用と内皮細胞に対するパラクライン的作用はほとんどなく、FGF-2がメサンギウム増殖に関与する可能性が低いことが明らかになった。また、FGF-2は糸球体内皮細胞と上皮細胞に、FGFRは糸球体内皮細胞に発現し、FGF mRNAレベルが糸球体修復の早期から増加していたことから、本モデルの寛解過程においてFGF-2は内皮細胞に対しオートクライン/パラクライン的に作用してその増殖を促し、糸球体修復に関わることが示唆された。\n\n［総括］\n　腎皮質からの有効な糸球体分離法と糸球体局所における遺伝子発現量の厳密な解析法を確立した。この方法を用いて、従来の研究から血管再生、血管内皮増殖に強い作用を持つとされるVEGF、FGF-2の抗Thy-1腎炎における障害糸球体の再構築に対する役割を解析し、糸球体局所におけるこれらの遺伝子発現量が、本モデルの病変の推移とともに変化することを初めて示した。また、過去の報告では、この2つの増殖因子がMes細胞により産生され、内皮細胞やMes細胞に増殖性変化をもたらすとされていたが、そうした可能性は低いことを示した。さらに、VEGFは、糸球体上皮細胞からパラクライン的に、FGF-2はオートクライン/パラクライン的に糸球体内皮細胞に作用してその増殖を促し、糸球体再構築に役割を担うことが示唆された。","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"甲第 99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":"井上, 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nephritis","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["391"],"pubdate":{"attribute_name":"公開日","attribute_value":"2013-01-22"},"publish_date":"2013-01-22","publish_status":"0","recid":"3162","relation_version_is_last":true,"title":["抗Thy-1腎炎の進行・寛解における糸球体での血管新生因子の役割に関する研究"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:15:17.076968+00:00"}