Name: experimental and imaging techniques for examining fibrin clot structures in normal and diseased states
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Description: Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

The authors would like to thank the Lam Lab at Georgia Tech for many helpful discussions in developing the experimental assays. Research reported in this publication was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL115486 and by New Innovator Grant 1DP2OD007433-01 from the Office of the Director, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Not: Aşağıdaki protokol Georgia Tech Kurumsal Değerlendirme Kurulu (KİK) tarafından belirlenen kurallara uyar. 1. Kan Toplama ve Eritrosit İzolasyon Prosedürü <ol><li> 10 ml heparinli boşaylı tüpler içinde bağışçılardan kan 40-120 ml toplayın. Toplama 4 saat içinde PBMC (periferal kan mononükleer hücre) izolasyon başlatın. Bu süre boyunca, oda sıcaklığında kan tutun. NOT: Bu alt PBMC verim neden olacak gibi RT ya da 4 ° C kan O / N depolama tavs...

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Hastalık durumlarında, pıhtılaşma mekanizmalarının yapısı anlamlı veriler elde etmek için, bu koşullar altında, protein ve hücre etkilerini belirlemek için pıhtılaşma faktörleri izole etmek için önemlidir. Bu protokol in vitro diyabetik ve OHA devletler fibrin pıhtı yapısını araştıran amacıyla geliştirilmiştir. Bu değiştirilmiş koşullar hiperkoagülasyon, Aterotrombozu ve KVH neden beri hastalık durumlarında fibrin oluşumu ve fibrinoliz yer alan...

Bir kan geminin endotel astar yaralanması hemostatik yanıt, ya da bir kan pıhtısı oluşumu ile tamir edilir. Kan hücre dışı matris içine nüfuz zaman, doku faktörleri koagülasyon kaskad başlatılmasını kolaylaştırmak, kan akışında trombositleri aktive. Bu iyileşme sürecinin önemli mekanik parça son derece esnek olan fibrin liflerden oluşan fibrin matrisi, ve büyük kuvvetler 1-4 muhafaza eder. Birçok araştırmacı yoğun geçmiş yıllarda 5-13 fibrin oluşumu yapısı ve fonksiyonu inceledik. Diabetes mellitus ve orak hücre olarak hastalıkları olan hastalar gibi miyokard enfarktüsü, iskemik kalp hastalığı gibi trombotik komplikasyonların gelişme riski, ve 14-19 okşar. 2 milyondan fazla insan yeni Amerika Birleşik Devletleri&#39;nde her yıl diyabet tanısı konur. Tip I nerede,: diyabet iki türü vardırvücut insülin dirençli hale insülin ve Tip II, yeterli miktarda üretemez. Diyabetik hastalarda, kardiyovasküler hastalık (CVD) hastalığı 20,21 ile ilişkili morbidite ve mortalitenin% 80 nedenidir. Orak hücre hastalığı (SCD) Amerika Birleşik Devletleri 22 100.000&#39;den fazla insanı etkileyen bir genetik kan hastalığıdır. OHA zor hücreler, kan damar 23 geçmesi için yapım, hilal şeklinde olmak üzere, kırmızı kan hücrelerinin neden olan bir nokta mutasyon hastalıktır. Bu hastalık durumlarının her iki gövde aterosklerotik durumları geliştirme ihtimalini artırır. Bunun nedenlerinden biri, hastalıklı devletlerin 14,24-26 değişmiş fibrin yapısı ve fonksiyonu bir sonucudur. Her iki diyabet ve orak hücre hastalığında, hiperkoagülasyon ve Aterotrombozu ve kardiyovasküler hastalık neden olur hipofibrinolizin aktivitesi (C varVD) sağlıklı hastalarda 17,27,28 ile karşılaştırıldığında. Bu hipofibrinolizin aterosklerozun ilerlemesini teşvik eder ve erken koroner arter hastalığı 29 olan hastalar için tekrarlayan iskemik olayları doğurur bilinmektedir. Mevcut yazıda, bu özel ortamda fibrin fiziksel özelliklerinin rolü araştırıldı. Olmayan hastalıklı hastalarda fibrin pıhtısı yapıları, ince lifler, daha büyük gözenekleri ve daha az yoğun, genellikle 14,24 oluşmaktadır. Sağlıklı hastalarda artan bir geçirgenliğe ve daha az yoğun bir fibrin pıhtısı fibrinoliz 16 kolaylaştırmak için tespit edilmiştir. Diyabetik ve orak hücre hastalığı gibi hyperthrombotic koşullarda sağlıklı hastalarda 30-33 2.5 mg / ml, normal seviyelerine artış fibrinojen neden fibrinojen üretiminde bir artış vardır. Sağlıklı, sigara dia göre diyabetik hastalarda oluşan fibrin pıhtılar daha fazla dal noktalarını, ve daha yoğun, daha sert ve daha az gözenekli olduğu tespit edilmiştirdiyabetik hastalarda 14,24,33-35. değiştirilmiş fibril bağları pıhtı oluşumunda rol oynayan proteinlerde meydana gelen glikozilasyon mekanizmasının bir sonucudur. Glukoz molekülleri, uygun bir şekilde çapraz bağlama glutamin ve lisin tortuları 33,36,37 insan faktör XIIIa (FXIIIa) inhibe fibrinojen molekülü, lizin artıklarının bağlanan zaman Enzimatik olmayan (geri dönüşümsüz) glikasyonu oluşur. Fibrin ağlarının yapısal analiz son zamanlarda yoğun çalışılmıştır. Özellikle, araştırmacılar elektron mikroskobu ve intravasküler (endotel) hücreleri ve ekstravasküler (fibroblastlar ve düz kas) hem hücreler fibrin yapıları 40 analiz fibrin yapısı 39, kullanılan viskoelastik ve spektral analiz nasıl etkilediğini araştırdık fibrin ağları 38, 3D rekonstrüksiyon ve kullanmıştır Fibrin yapısı ve mekanik özellikleri arasında gelişmiş korelasyon deneysel kullanarak ve hesaplamalı yaklaşımlar 41 </sup&gt;. Bu çalışmanın odak simüle diyabetik ve orak hücreli tromboz koşulları altında pıhtı yapıları formüle etmek ve hastalıklı devletlerin pıhtı yapısının incelenmesi ve işlevi için konfokal mikroskopi kullanımı idi. Fibrin pıhtılar insan fibrinojen, insan trombini ve FXIIIa oluşmuş. pıhtılar plazmin kullanılarak lize edilmiştir. Diyabetik koşulların simüle edilmesi için, fibrinojen artan konsantrasyonu, in vitro olarak fibrinojen glikasyonunu uyarılması için glikoz çözeltisi içinde kuluçkalanmıştır. Orak hücre hastalığı pıhtılaşma koşulları simüle etmek için, artmış fibrinojen konsantrasyonları grubumuza 42 tarafından daha önce yapıldığı gibi hastalardan toplanan orak hücre hematokrit ile karıştırıldı. Bu yöntemler, yapı ve hastalıklı koşullar altında, fibrin pıhtısı oluşumu ve fibrinolizde rol alan ve fonksiyonları, yanı sıra CVD neden mekanizmaları incelemek için kullanılmıştır. Bu hastalıklar hakkında güncel bilgilere dayanarak, glike fibrin pıhtı yapıları daha az a ile yoğun olduğunund küçük gözenekler. Orak hücre hastalarında (eritrositler) kırmızı kan hücreleri ile fibrin pıhtıları da yoğun ve eritrosit agregasyonunu ve aglomere fibrin kümeleri görüntülenir. Bu, daha önce 43 tespit edilmiştir köklü bir olgudur. Ayrıca fibrinoliz oranı ile sağlıklı, normal bir fibrin kıyasla azalmış plazmin olmadan glike fibrin pıhtıları anlamlı olarak düşük olacağı varsayılmıştır. Sonuçlar, glikolize fibrin pıhtılaşması için önemli ölçüde farklı lisis oranı sonuçları yalnızca sınırlı plazmin konsantrasyonu durumunda görüldüğünü göstermiştir. Hücreler ve proteinler pıhtılaşma faaliyetinin gerçek zamanlı video yakalama sağlayan kendi ana devlet kalır, çünkü konfokal mikroskopi kullanılarak bu deneysel tekniği diğer görüntüleme yöntemlerine göre önemli avantajlar sunmaktadır. Sentetik indükleyici pıhtılaşma Bu yöntem, hasta örnekleri elde etmek ve bireysel filtreleyerek daha da ucuz ve daha fazla zaman verimliproteinler ve enzimler. Örnekler arasında değişkenlik plazma diğer proteinler sonucu olmadığını Bundan başka, pıhtı sentezlenmesi için ayrılmış proteinler ve enzimler kullanarak, pıhtılar standardize edildi.

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experimental and imaging techniques for examining fibrin clot structures in normal and diseased states

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

Glike Fibrin pıhtı Yapıların Konfokal Mikroskopi Analizi Normal ve glikolize pıhtı konfokal mikroskobu görüntüleri. Şekil 3&#39;te sunulmuştur normal ve glikolize pıhtı mikroskopisi analizi Glike pıhtılar daha yoğun olan ve pıhtı polimerizasyon sırasında FXIIIa ilavesi olmadan hem normal pıhtıları daha küçük gözeneklere sahip olduğunu ortaya koymaktadır. Şekil 3A ve 3B, daha yüksek bir fibrin konsantrasyonu <st...

Watch this Scientific Journal Video about Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States at JoVE.com

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Normal ve Hastalıklı Devletleri&#39;nde Fibrin pıhtısı Yapıları incelenmesi için deneysel ve Görüntüleme Teknikleri

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

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