Bu çalışma &quot;Interfaces inflamasyon&quot; Mükemmeliyet DFG-Küme (RA-Eğer, RA-D) tarafından desteklenmiştir. Biz Kristin Wischnat mükemmel teknik destek için minnettarız.

1. Chlamydia trachomatis serovar D Stok hazırlanması <ol><li> 4 ml C. ile HeLa-229 hücre içeren konfluent 175 cm 2 hücre kültürü şişesi Infect tampon SPG içinde trachomatis D. </li><li> Sürekli sarsıntı sırasında 37 ° C&#39;de bir saat inkübe edin. </li><li> % 10 FCS ve 240 ul Cycloheximide ile 20 ml DMEM ekleyin. </li><li> 37 de 48 saat süre ile nemlendirilmiş bir inkübatörde inkübe ° C&#39;de ve% 5 CO2. </li><li> C. ayırmak ve yok etmek için 5 ml steril cam boncuk, güçlü sarsıntı ekle HeLa-229 hücreleri barındıran trachomatis. </li><li> Süpernatant toplayın ve steril cam boncuk başka bir 5 ml ekleyin. </li><li> 1 dakika Kaya üç kez tüm HeLa-229 hücrelerini yok ve serbest C&#39;ye trachomatis. </li><li> 5 1000 RPMI dk ve 4 santrifüjleyin ° C hücresel enkaz kurtulmak için. </li><li> 8-10 175 cm 2 hücre kültürü enfeksiyonu için supernatant toplamak konfluent g şişelerHeLa 229 hücrelerle rown. </li><li> 37 az bir saat için 4 ml SPG tampon ve önceki enfeksiyondan 4 ml supernatant ° sürekli sarsıntı ile C hastası olan her şişesi inkübe edin. </li><li> Bir saat% 10 FCS ile 37 ° C ve% 5 CO2 az 48 saatlik inkübasyondan ardından 230 ul sikloheksimid ile 15 ml DMEM ekledikten sonra. </li><li> , 5 ml steril cam boncuk ekle ayırmak için sallayın ve C. yok HeLa-229 hücreleri barındıran trachomatis. </li><li> Süpernatant toplayın ve steril cam boncuk başka bir 5 ml ekleyin. </li><li> 1 dakika Kaya üç kez tüm HeLa-229 hücrelerini yok ve serbest C&#39;ye trachomatis. </li><li> 4 1000 RPMI az 5 dakika boyunca santrifüj ° C hücresel enkaz kurtulmak için. </li><li> Süpernatant toplayın. </li><li> 11000 RPMI ile 99 dakika boyunca 50 ml Falcon tüpleri, santrifüj süpernatant 40 ml doldurun. </li><li> Santrifüj sonra süpernatan atmak ve 1 ml tampon SPG ile pelet yıkayın. </li><li> Homojenizepelet -70 az 1, 5 ml Eppendorf tüp, mağaza ° C&#39;de kullanılıncaya kadar 1 ml SPG ve tablet 20 ul içinde. Hazırlanan stok biyolojik aktivite kurulu bir HeLa-229 hücre enfeksiyon modelinde doğrulandı. </li></ol> 2. İnsan Fallop Tüp hazırlanması <ol><li> Hemen RPMI hazırlık kadar 4 antibiyotik olmadan ° C% 5 FCS içeren cerrahi sonrası histerektomi geçiren kadınlarda Store&#39;dan insan fallop tüpleri (HFT). Protokolü Lübeck Üniversitesi (09-153) etik komitesi tarafından onaylandı. (53 yaşına kadar 34) çalışmaya dahil Kadınlar önceden C. öyküsü yoktu trachomatis enfeksiyonu. Fallop tüplerinin Doku sezaryen sırasında veya luteal faz semptomatik miyom nedeniyle histerektomi sırasında annenin isteği üzerine peripartum sterilizasyon toplanmıştır. Tüm HFT C. için negatif test edildi PCR ile trachomatis. </li><li> RPMI içeren Petri içinde HFT teşrihAntibiyotikler olmadan% 5 FCS. Bütün işleme sırasında doku kurumasını önlemek için ortam içine daldırılmış edilmelidir. </li><li> Kesiniz ve ameliyat sırasında tahrip bağ dokusu ve doku atın. </li><li> Diseksiyon sonra küçük bir bistüri ile dikkatlice HFT açın. </li><li> Daha ayrıntılı bilgi için deneyler x 0,5 cm yukarı 1 cm x 1 cm ile 0.5 cm büyüklüğünde doku parçalarını hazırlamak. </li><li> Enfeksiyon mağaza HFT numune için 1 ml RPMI antibiyotikler olmadan% 5 FCS içeren. HFT örneklerinin C. 5.5x10 5 IFU (dahil oluşturan birim) ile enfekte olduğunu trachomatis. </li><li> 37 içinde HFT numunesi inkübe ° C,% 5 CO2 ve% 21 O2 gibi 37 ° C,% 5 CO2 ve 2 saat süre ile% 24 O2. </li><li> İnkübasyon süresi 4 en az üç gün Monti en sabitleştirici olarak örnek ve mağaza toplamak sonra SEM / TEM hazırlık kadar ° C. </li></ol> 3. TEM için HFT Numune Hazırlanması <ol><li> IçinTEM Monti en sabitleştirici gelen örnek kaldırmak ve 4 x 5 mm adet 2 x 2 mm kesti. Geriye kalan doku SEM için kullanılabilir. </li><li> 1 saat süreyle sodyum cacodylate tamponu, pH 7.35 ile parçalara yıkanır. </li><li> % 1 (w / v) su içinde hedef osmiyum tetroksit de inkübe. gecede. </li><li> Sodyum cacodylate tamponu, pH 7.35 olarak 6x5 dakika yıkanması ile aşırı osmiyum tetroksit çıkarın. </li><li> Su içinde etanol artan yoğunluklarda (v / v) (2 saat süreyle% 30, 2 saat süreyle% 40, 2 saat süreyle% 50, 2 saat süreyle 60%, gece boyunca 70%, 2 için 80% in kuluçkaya suyu uzaklaştırmak s, 1 saat boyunca 90%, 1 saat boyunca 95%, 1 saat boyunca 100%. </li><li> Propilen oksit in 2 x 15 dakika boyunca etanol dışarı yıkanır ve daha sonra% 50 oranında bir karışımı (v / v) içinde propilen taze hazırlanmış araldit (tüm bileşenler de dahil olmak üzere) aktarmak ve bir gece boyunca inkübe edilir. </li><li> En az 1 saat için taze hazırlanmış araldit (tüm bileşenler de dahil olmak üzere) transfer edilir. </li><li> Düz gömme kalıpları transfer ve araldit ile doldurun. </li><li> Için araldit sertleştiriniz60 ° C&#39;de en az 48 saat </li><li> Gömülü örnek Kesimden sonra cam bıçak veya histo elmas bıçak kullanılarak bir ultramicrotome üzerine yarı ince kesitler (700 nm) kesilmiş ve Richardson ile leke leke. </li><li> Bir elmas bıçak ve bakır ızgaralar (örneğin 150 kare gözlü) transfer kullanan ultra-ince kesitler (yaklaşık 70 nm) kesin. </li><li> Aqua dest% 0.5 (w / v) uranil asetat ile ultra-ince kesitler Leke. % 3 (w / v) su hedef kurşun sitrat izledi. otomatik bir bölümü yapıcısı içinde. Alternatif olarak, bölüm ızgaraları su hedef olarak uranil asetat ile doymuş bir çözelti santrifüje üzerinde 15 dakika bırakarak elle boyanmış edilebilir. su hedef olarak% 0.3 kurşun sitrat (w / v) izledi. </li></ol> 4. SEM için HFT numune hazırlanması <ol><li> SEM epitel 1.5 x 1 cm mantar levha üzerinde yukarı bakacak şekilde Monti en fiksatörü, orient örnek kaldırmak ve böcek iğneleri kullanarak konumunu düzeltmek için. </li><li> Uygun metal sepet mantar üzerinde örnek aktarın vesodyum cacodylate tamponu, pH 7.35 olarak 30 dakika süre ile yıkanır. </li><li> Aseton / su karışımları artan konsantrasyonları (v / v) 6 saat süreyle% 30, 6 saat süre ile% 40, 8 saat boyunca 60%, gece boyunca 70%, 2 saat süreyle% 80,% 90 oranında içinde Örnek kuluçkaya suyu uzaklaştırmak Gece boyunca 2 saat ve% 100 (numune sürekli su altında tutmak son derece dikkatli olmalı). </li><li> Kritik nokta kurutma (sıcaklık 40 ° C, basıncı 80 bar) tarafından taze% 100 aseton ve kuru numune transfer. </li><li> Iletken karbon çimento kullanarak bir iletken karbon sekmesi ile donatılmış SEM numune montaj bakacak şekilde epitel ile iğne, mantar ve tutkal örnek çıkarın. Montaj ve iletken karbon sekmesini arasındaki iletkenliği artırmak için sıvı iletken gümüş kullanın. </li><li> Bir lak sputter kullanılan numunenin iletken bir platin, altın veya paladyum kaplama hazırlayın. </li></ol> 5.. Richardson&#39;ın Leke Yarı İnce Kesitler boyanması <ol><li> Slaytta yarı ince kesitler kurumasını bekleyin. </li><li> 1-2 dakika süreyle 60 ° C sıcaklıkta çözelti boyama ile bölümlere leke. </li><li> Aqua dest yıkayın. </li><li> Kuru bölümleri ve kapak. </li></ol> 6. Temsilcisi Sonuçlar Insan fallop tüpü enfeksiyonu modeli içinde biz enfekte olmayan kontroller (Şekil 1) ve C arasında epitelyal morfoloji farklılıklar görselleştirmek başardık trachomatis - normoksik koşul altında enfekte tüpleri (Şekil 2). Karakteristik morfolojik farklılıklar enfekte olmayan kontroller tespit edilemedi fallop tüpü epitel hücrelerinin patojen bağlı şişme ve lizis ima etti. Yarı ince kesit ve transmisyon elektron mikroskobu kullanarak bizim hücre içi C ispat başardık insan fallop tüpü doku enfekte ex vivo trachomatis dahil oluşumu (Şekil 3, 5) ancak enfekte olmayan kontrollerde (Şekil 4). Oksijen konsantrasyonu gibikadın genital yollarında ASIZ biz de hipoksik koşullarda bizim modeli incelenmiştir inflamatuar sürecinde fizyolojik koşullarda 11 ve daha fazla azalma altında zaten düşük. Ön sonuçlar modeli oksijen biyokatalist altında klamidyal büyüme ve döl analiz etmek yararlı olduğunu göstermektedir. Chlamydia-indüklenen epitel hücre hasarına enfeksiyonu önce fallop tüpleri (Şekil 6) dikkatsizce kullanımı ve kullanım aracılığı ile ortaya eserler ayrılması gerekir. <img src="/files/ftp_upload/4036/4036fig1.jpg" alt="Şekil 1" /> Şekil 1. Kontrolü ortamda bir gün inkübasyon (yeşil ok silli hücre, mavi ok olmayan silli hücre hepsini göster) sonra enfekte olmayan insan fallop elektron mikroskobu (SEM). <img src="/files/ftp_upload/4036/4036fig2.jpg" alt="Şekil 2" /> Şekil 2. Tarama elektron mikro<html> insan fallop tüpü bir gün sonrası enfeksiyon (beyaz ok işareti rüptüre dahil) enfekte bir ex vivo C. trachomatis kopyası (SEM). <img src="/files/ftp_upload/4036/4036fig3.jpg" alt="Şekil 3" /> Şekil 3. Yarı ince kesitler C ile insan tuba enfeksiyondan sonra tipik intraselüler inklüzyon (beyaz oklar) 1d göstermek trachomatis ex vivo. <img src="/files/ftp_upload/4036/4036fig4.jpg" alt="Şekil 4" /> Şekil 4. Kontrolü ortamda bir gün inkübasyondan sonra non-enfekte insan tuba transmisyon elektron mikroskobu (TEM). <img src="/files/ftp_upload/4036/4036fig5.jpg" alt="Şekil 5," /> Bir ex vivo C. Şekil 5. Transmisyon elektron mikroskobu (TEM) trachomatis insan fallop tüpü bir gün sonrası enfeksiyon (beyaz oklar klamidyal kapanım işaretleyin) bulaşmış. ve_content &quot;&gt; <img src="/files/ftp_upload/4036/4036fig6.jpg" alt="Şekil 6" /> Şekil 6. Numunenin dikkatsizce hazırlama ve işleme (beyaz ok işareti epitel doku tahrip) yoluyla zarar fallop tüpü epitelin elektron mikroskobu (SEM).

<ol>
	<li>Dean, D., Suchland, R. J., Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+long-term+cervical+persistence+of+Chlamydia+trachomatis+by+omp1+genotyping.">Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping.</a> J. Infect. Dis. 182, 909-916 (2000).</li><li>Campbell, L. A., Patton, D. L., Moore, D. E., Cappuccio, A. L., Mueller, B. A., Wang, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Chlamydia+trachomatis+deoxyribonucleic+acid+in+women+with+tubal+infertility.">Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility.</a> Fertil. Steril. 59, 45-50 (1993).</li><li>Peipert, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Genital+chlamydial+infections.">Clinical practice. Genital chlamydial infections.</a> N. Engl. J. Med. 349, 2424-2430 (2003).</li><li>Mardh, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubal+factor+infertility,+with+special+regard+to+chlamydial+salpingitis.">Tubal factor infertility, with special regard to chlamydial salpingitis.</a> Curr. Opin. Infect. Dis. 17, 49-52 (2004).</li><li>Ertel, A., Verghese, A., Byers, S. W., Ochs, M., Tozeren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-specific+differences+between+tumor+cell+lines+and+normal+and+tumor+tissue+cells.">Pathway-specific differences between tumor cell lines and normal and tumor tissue cells.</a> Mol. Cancer. 5, 55 (2006).</li><li>Roshick, C., Wood, H., Caldwell, H. D., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gamma+interferon-mediated+antichlamydial+defense+mechanisms+in+human+and+mouse+cells.">Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells.</a> Infect. Immun. 74, 225-238 (2006).</li><li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">Of mice and not men: differences between mouse and human immunology.</a> J. Immunol. 172, 2731-2738 (2004).</li><li>Hvid, M., Baczynska, A., Deleuran, B., Fedder, J., Knudsen, H. J., Christiansen, G., Birkelund, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+is+the+initiator+of+Fallopian+tube+destruction+during+Chlamydia+trachomatis+infection.">Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection.</a> Cell Microbiol. 9, 2795-2803 (2007).</li><li>Roth, A., Konig, P., van, Z. G., Klinger, M., Hellwig-Burgel, T., Daubener, W., Bohlmann, M. K., Rupp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+abrogates+antichlamydial+properties+of+IFN-gamma+in+human+fallopian+tube+cells+in+vitro+and+ex+vivo.">Hypoxia abrogates antichlamydial properties of IFN-gamma in human fallopian tube cells in vitro and ex vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19502-19507 (2010).</li><li>Wyrick, P. B., Knight, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-exposure+of+infected+human+endometrial+epithelial+cells+to+penicillin+in+vitro+renders+Chlamydia+trachomatis+refractory+to+azithromycin.">Pre-exposure of infected human endometrial epithelial cells to penicillin in vitro renders Chlamydia trachomatis refractory to azithromycin.</a> J. Antimicrob. Chemother. 54, 79-85 (2004).</li><li>Van Voorhis, W. C., Barrett, L. K., Sweeney, Y. T., Kuo, C. C., Patton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Chlamydia+trachomatis+infection+of+Macaca+nemestrina+fallopian+tubes+produces+a+Th1-like+cytokine+response+associated+with+fibrosis+and+scarring.">Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring.</a> Infect. Immun. 65, 2175-2182 (1997).</li></ol>

Çıkar çatışması ilan etti.

Enfekte dokuya patojen ile endüklenen zarar görselleştirme zahmetli ve farelerde deney prosedürleri genellikle sınırlı değildir. Biz C. analiz etmek, insan fallop tüpleri bir ex vivo enfeksiyon modelinde kurulan Üst kadın genital sistem enfeksiyonları trachomatis. Bu metodun kullanılması suretiyle, C. görsel mümkün olduğu trachomatis - bir gün sonrası enfeksiyon içinde insan fallop tüpü epitel hasarını. Hücresel şişme ve lizis C. gözlendi tipik morfolojik değişiklikler vardı trachomatis - enfekte fallop tüpleri ancak enfekte olmayan kontrol. TEM analizleri klamidyalar yeniden farklılaşmış temel organları değil, aynı zamanda kalıcılık gösterebilir genişlemiş retiküler organları ile küçük inklüzyonlar büyük inklüzyonları gösteren, enfekte dokuların içinde tipik bir hücre içi gelişim döngüsünü gerçekleştirirken ortaya koymuştur. Ancak tek başına morfolojik resmi repl arasında ayrım yeterli değilazalmış metabolizma ile karakterize edilir ve icating ve inatçı enfeksiyonlar, antibiyotik 10 dayanıklılığı artar. Enfekte fallop tüpleri epitel tahribatı C. bozulması için iki nedeni trachomatis - hücre içi gelişim döngüsü ve enfeksiyöz elementer cisimlerin serbest bırakılması veya pro-inflamatuvar 8 sürümü tamamlanmasından sonra enfekte olmuş epitel hücreleri. Patojen bağlı doku hasarı ve C&#39;ye karşı konak bağlı immün reaksiyonlar trachomatis epitel hücre fonksiyonu, fibroblast şekillenmesi ve in vivo 8,11 nihayet tubal kaybına yol açan başlıca faktörler olması gerekiyordu. Bu model önemli kısıtlamaları biri başlangıç ​​C. sekonder etkileri analizi engellemektedir inflamatuar hücrelerin yokluğudur Fallop tüpü epitel trachomatis enfeksiyonu. Bununla birlikte, modeli ap olduğufarklı çevresel koşullarda (örneğin oksijen içeriği) ve akut C. ilk ana-immün yanıtı araştırmak için propriate trachomatis enfeksiyonları 8,9. Daha planları C&#39;ye karşı antibiyotik test için bir model oluşturmaktır Tüm insan fallop tüpleri ve 2-foton lazer tarama mikroskop ile tuba epiteli gözlenen farklı gelişim aşamalarında metabolik durumları karakterize etmek trachomatis.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> DMEM </td><td> PAA </td><td> E15-843 </td><td></td></tr><tr><td> RPMI </td><td> PAA </td><td> R15-802 </td><td></td></tr><tr><td> FCS </td><td> PAA </td><td> A15-101 </td><td></td></tr><tr><td> Cycloheximide </td><td> Sigma - Aldrich </td><td></td><td></td></tr><tr><td> Tampon SPG </td><td> çeşitli </td><td></td><td> aşağıdaki tarife bakınız </td></tr><tr><td> Monti en fiksatif </td><td> çeşitli </td><td></td><td> aşağıdaki tarife bakınız </td></tr><tr><td> sodyum cacodylate tampon </td><td> çeşitli </td><td></td><td> aşağıdaki tarife bakınız </td></tr><tr><td> Richardson en leke </td><td> çeşitli </td><td></td><td> aşağıdaki tarife bakınız </td></tr></tbody></table> Tarifler: 1. Tampon SPG <ol><li> 75 g sakaroz ekleyin. </li><li> 2.47 g disodyum fosfat ekleyin. </li><li> 0.36 g monosodyum fosfat ekleyin. </li><li> 0.72 g glutamik asit ekleme. </li><li> Aqua dest ile 1 l doldurun. </li><li> 7.3 pH ayarlayın. </li></ol> 2. 0.1 M sodyum cacodylate tamponu, pH 7.35. <ol><li> 42.80 g dimethylarsenic asit sodyum tuzu ekleyin. </li><li> 68.46 g sakaroz ekleyin. </li><li> Aqua dest ile 1 l doldurun. 0.2 M sodyum cacodylate tamponu almak için. </li><li> Aqua dest ile çözelti kullanmadan önce. istenen konsantrasyonu ve 7,35 gerekirse üzere pH&#39;ı ayarlamak için. </li></ol> 3. Monti en fiksatif <ol><li> 156 ml 0,12 M sodyum cacodylate tamponunu ekleyin. 3,2 su hedef olarak 25 mL% 25 Glutaraldehit ekleyin. </li><li> 19 ml ekleAqua dest% 10 paraformaldehit. </li><li> Aqua dest 3 ml% 3 CaCI2 ekleyin. </li><li> 7,35 pH ayarlayın ve aqua dest ile doldurun. 312 ml nihai bir çözüm için. </li></ol> 4. Richardson&#39;ın leke <ol><li> Aqua dest% 1 sulu boraks çözeltisi ve% 1 Azur II% 1 metilen mavisi eşit hacimde karıştırılır. </li></ol>

a human fallopian tube model for investigation of c. trachomatis infections

Chlamydia trachomatis ile genital sistem enfeksiyonları (C. trachomatis) tüm dünyada kadınlarda en sık görülen cinsel yolla bulaşan hastalık vardır. Patojenlerin Verimsiz klirensi veya kalıcılık üst genital sistem enfeksiyonları artan ve enfekte dokuların 1,2 kronik inflamatuar zarar gerekiyordu yol açabilir. Sonuç olarak, pelvik inflamatuar hastalık (PID), tubal oklüzyon ve infertilite gibi ciddi klinik sekeller 3,4 oluşabilir. C ile araştırmaların çoğu trachomatis epitel hücre çizgilerinden (örneğin HEp-2 hücreleri ve HeLa-229) veya farelerde yapılmıştır. Bununla birlikte, hücre kültürü bazlı modelleri ile olduğu gibi, ne de ana doku fizyolojisini ya da C&#39;nin patofizyolojisini yansıtır yok in vivo 5 trachomatis genital sistem enfeksiyonları. Başka sınırlamalar gerçeği ile verilir, merkez sinyal şelaleri (ör. IFN-γ mediatintrasellüler klamidyal büyümesini kontrol ed JAK / STAT sinyal yolağı) temelde farelerde ve insanlarda 6,7 arasında farklılık. Biz ve diğerleri, bu nedenle C arasında doğrudan etkileşimleri araştırmak için bütün bir organın fallop tüpü model oluşturdu trachomatis ve insan fallop tüpü hücrelerinin ex vivo 8,9. Bu amaçla, histerektomi geçiren kadınlarda insan fallop tüpleri C ile toplanır ve enfekte edildi 24 saat sonrası enfeksiyon içinde trachomatis serovar D., örnek nerede Chlamydia trachomatis algılamak için taramalı elektron mikroskobu (SEM) ve transmisyon elektron mikroskobu (TEM) kullanılarak epitel hasarı gibi aracılık analiz C. Fallop dokusunda trachomatis dahil oluşumu.

Watch this Scientific Journal Video about A Human Fallopian Tube Model for Investigation of C. trachomatis Infections at JoVE.com

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Biz bir tarif<em&gt; Ex vivo enfeksiyon</emInsan fallop tüpü hücreleri ile bakteriyel patojenler doğrudan etkileşim görselleştirme&gt; modeli. Tüm organ doku modeli araştırmak için kuruldu<em&gt; C. trachomatis</em&quot;Hayat&quot; gibi koşullar altında kadın fallop tüpüne&gt; bağlı patoloji.

Incelenmesi için İnsan Fallop Tüp Modeli<em&gt; C. trachomatis</em&gt; Enfeksiyonlar

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

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Medicine

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

12.9K Görüntüleme.  University of Lübeck. Biz bir tarif Ex vivo enfeksiyon modeli. Tüm organ doku modeli araştırmak için kuruldu C. trachomatis bağlı patoloji.

Video: A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Hücre Kültürü İnsan Azaltma Meme Cerrahisi ve Mastektomi Doku İşleme

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Tıp

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Yara Şifa murin Modeli

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

İnsan Prostat Kanser metastaz Ortotopik fare modeli

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Farelerde Hızlandırılmış Ateroskleroz Indüksiyonu: "Tel-Yaralanma" Modeli

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Fosfor-31 Manyetik Rezonans Spektroskopisi: Ölçme için Aracı<em&gt; İn Vivo</emİnsan İskelet Kası içinde&gt; Mitokondriyal Oksidatif Fosforilasyon Kapasitesi

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Yumurtalık Kanseri Erken Teşhisi İçin Umut Verici Bir Araç Olarak Fallop Tüpünün Tubal Sitolojisi

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Vitro ve In Vivo insan hücreleri, C. Elegansve fareler Mitophagy tespiti

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Split Korneal Düğmeleri Kullanarak Bir Domuz Korneal Endotel Organ Kültür Modeli

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Incelenmesi için İnsan Fallop Tüp Modeli

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