Bu çalışma Sağlık hibe R01DK080971 ve Juvenil Diyabet Araştırma Vakfı hibe 37-2009-44 Ulusal Enstitüleri tarafından desteklenmiştir. Biz UCSD Pediatrik Diyabet Araştırma Merkezi (PDRC) alınan destek de teşekkür ederiz.

1. HEK293 Katman Transfeksiyon <ol><li> 50 ml FBS, 5 ml 100X Penisilin / streptomisin solüsyonu, 5 ml 100x L-glutamin solüsyonu ve 500 ul amfoterisin: DMEM 500 ml&#39;lik şişelerde (4.5 ug / ml glükoz, fenol kırmızısı ile ve glutamin olmadan) ekleyerek HEK293 hücre ortamı hazırlayın B. </li><li> De başına HEK293 medya 0.5 ml kullanarak 24 plaka HEK293 hücreleri plaka. Hücrelerin plakanın alt üzerine yayılıyor emin olun. </li><li> HEK293 hücreleri plazmid Lipofectamine protokolüne uygun olarak ilgi duyulan proteini (bir memeli ifade vektörünün içine) ve Lipofectamine 2000 2 ul kodlama 0.8 mikrogram ile% 100 birbirine karışmış, transfekte ulaşır. Biz memeli hücrelerinde ifade için tasarlanmış pcDNA 3.3 omurga kullanmak için seçtiniz. </li><li> İsteğe bağlı olarak, 6 saat inkübasyon sonrasında değişimi transfeksiyon ortamı normal HEK293 ortam ile Lipofektamin protokolde tarif edildiği. </li><li> Transfekte prote ifade değerlendirmekolarak, transfekte edilmiş doku kültürü gözenekli bir alt kümesini ifade edilen protein immunofloresans tespiti için ya da standart teknikler kullanılarak protein ekspresyonu, Western blot analizi için kullanılabilir. </li></ol> 2. HEK293 hücreleri isteğe bağlı fiksasyonu Transfekte protein ifade Transfekte HEK293 hücreleri hafifçe HEK293 hücreleri (örneğin aşağıda adım 3.9) yaşayan veya (ayrıca Tartışma) tek seferde birçok deneyler için plakaların önceden etkin hazırlık sağlamak için zararlı olabilir medyada kokültürü kolaylaştırmak için sabitlenebilir. <ol><li> HEK293 hücrelerinde protein ifade zaman ders belirlemek için pilot çalışmalar yapmak. </li><li> HEK293 hücre tabakası transfecting sonra, ifade en üst düzeyde ilişkili sonrası transfeksiyon zaman noktasında hücrelerden medya aspire. Bu ilk 24 saat içinde meydana gelirse, transfeksiyondan 24 saat sonra aspire kadar yoktur. </li><li> HE YıkamaYavaşça D-PBS ile K293 hücreleri daha sonra 500 ul% 4 paraformaldehit (PFA) ekleyin ve 30 dakika boyunca oda sıcaklığında inkübe edin. (% 4 PFA çözüm yapmak için, 1X PBS içinde% 16 solüsyonu ile başlar ve 1X PBS 1:4 sulandırmak.) </li><li> Steril PBS kullanarak, yavaşça 4 ° C de PBS O / N hücreleri 3 kez yıkama ve inkübe </li><li> Hücreler steril PBS ile 3 kez yıkayın, daha sonra PBS ekleyin ve 4 bırakın ° C kadar ihtiyaç duyulan (maksimum saklama süresi ifade protein bağlı olarak değişebilir. Biz gözlenen etkileri önemli değişiklikler olmadan 2 hafta için neuroligin izoformlar ifade etmek için transfekte HEK293 hücreleri saklamak insülin salınımı üzerinde). </li></ol> 3. HEK293 Hücreler ile INS-1 Beta Hücreleri Co-kültür <ol><li> (Glikoz ve fenol kırmızı ve glutamin olmadan) 500 ml RPMI 1640 ekleyerek INS-1 ortamı hazırlayın: 50 ml FBS, 5 ml 100X Penisilin / streptomisin, 5 ml 100X L-glutamin çözeltisi, 5 ml sodyum piruvat, 500 ul amfoterisin B, 500 ul beta-merkaptoetanol (bu medi neredeyse aynıdırum tipik olarak beta-mersaptoetanol ilavesi) hariç olmak üzere birinci adacık kültürü için kullanılmıştır. </li><li> (Veya başka bir enzimatik olmayan hücre sökücü) Hücre-striptizci kullanarak, hasat INS-1 T75 kültürü şişesi altındaki kapalı confluency yaklaşık% 70-80 olarak hücreleri. Her şişe 48 kuyu için yaklaşık yeterli hücre sağlayacaktır. </li><li> INS-1 ve HEK medya bir 50/50 karışımı içinde tekrar süspansiyon 1,200 rpm&#39;de 3 dakika boyunca santrifüj hücre aşağı iplik sonra. INS-1 hücreleri, bir% 70-80 oranında konfluent şişeye, karma ortamın yaklaşık 25 ml süspansiyon haline getirin. </li><li> Yerine canlı HEK293 hücreleri daha paraformaldehid giderildi HEK293 hücreleri kullanıyorsanız,% 100 yerine karışık teknik daha INS-1 medya INS-1 hücreleri tekrar süspansiyon. </li><li> 10 ml&#39;lik bir pipet kullanarak, homojen bir hücre süspansiyonu için pelet hücreleri çıkarmak. </li><li> HEK293 hücreleri üzerinde ortamı çıkarmak için aspire. </li><li> Bir P1000 pipet kullanarak, yavaşça o kenarları boyunca HEK hücre tabakası üzerine INS-1 hücre süspansiyonu 500 ul ekleyinf iyi. Her 6 kuyu sonra, homojen bir hücre süspansiyonu sağlamak için tekrar INS-1 hücreleri tekrar süspansiyon 10 ml&#39;lik bir pipet kullanın. Kokültürü kurulum genel şeması Şekil 1 &#39;de tasvir edilmiştir. </li><li> Deneysel protokol bağlı olarak 24-48 saat boyunca inkübe hücreleri. </li><li> Fazla 48 saat kokültürü süresi gerekiyorsa, HEK293 hücreleri düzeltmek için bölüm yukarıdaki 2 isteğe bağlı adımları kullanın. </li><li> Birincil ayrışmış kemirgen ya da insan adacıklar kullanarak [örneğin vallahi 11-15 öncesinde protokolleri görmek] ise, 5 ml kalem / strep, 5 böyle RPMI gibi uygun bir ortam ml L-Glut, 0.5 ml amfoterisin-B yerine kullanılmasını sağlamak INS-1 medya. Bir çünkü potansiyel olarak gerekli adacıklar çok sayıda uygulamada, bir 24 plaka ayrışmış adacık hücreleri kullanabilirsiniz rağmen, göz bir 48 plaka (iyi başına daha az adacık hücre gerektiren) için ölçekleme aşağı verilmesi önerilir. Bir 48 plaka üzerinde, çözülme değerinde yaklaşık 100 adacık ekleyinbaşlatmak için plaka başına ted hücreleri. Ayrışma etkinliği ve kullanılan yönteme bağlı olarak, daha az sayıda adacık gerekebilir. </li></ol> 4. Glikoz insülin salgılanması Uyarılmış <ol><li> 1 saat arasında, en az KRB 2.5 mM glükoz (Krebs-Ringer bikarbonat tamponu), 250 ul hücre Preincubate. , Preincubate kokültürü orta kaldırmak ve hemen KRB ile değiştirmek için. Bu değişim ortamdaki oksijene INS-1 hücreleri etkisini en aza indirmek için, bir seferde bir tane de yapılmalıdır. Diğer düşük (2.5 mM) glukoz KRB pipetleme sırasında tek elle aspire. </li><li> 2.5 mM veya bir zamanda iyi KRB tamponu biri 20 mM glükoz ya da 250 ul inkübasyon öncesi değişimi KRB. </li><li> Belirli bir deney için uygun olduğu takdirde, örneğin 100 uM IBMX gibi ek salgı daha sağlam bir uyarılmış insülin salgılama tepkisi üretmek için ilave edilebilir. Özellikle ayrışmış birincil adacık beta hücreleri artmış glukoz konsantrasyonları Alon için kötü duyarlıE, yani IBMX veya diğer salgı burada özellikle ilave 16,17 dikkate alınmalıdır. </li><li> Inkübasyondan 1 saat sonra, ortam tüpler mikrofuge&#39;de ve 5 dakika boyunca 1500 x g&#39;de dönmeye aktarın. </li><li> Süpernatant 200 ul çıkarın ve insülin RIA veya ELISA ile analiz için kullanabilirsiniz. </li><li> Hücre lisatı hazırlamak için, (% 1.0 IGEPALCA-630 veya Nonidet P-40,% 0.5 sodyum deoksikolat,% 0.1 SDS, 50 mM Tris, pH 8.0, 150 mM NaCI), proteaz inhibitörleri için RIPA hücre parçalama tamponu 100 ul hemen 4 de medya ve inkübe kaldırarak ° C 20 dakika sonra plaka. </li><li> 10,000 x g &#39;de 15 dakika boyunca lizat Spin ve insülin RIA ve ELISA ile analiz için süpernatan 50 ul çıkarın. </li></ol>

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Biz ifşa hiçbir şey yok.

Burada açıklanan kokültürü yöntemi özel beta-hücre yüzey, transmembran proteinlerin fizyolojik önemini belirlemek için etkili bir yol sağlar, ve özel olarak ekstrasellüler etki. Hücre yüzeyi üzerinde bir ilgi protein görüntüleme HEK293 hücreleri ile temas içinde kültürlenmesi beta hücreleri ya da insülinoma hücrelerine (örneğin, burada kullanılan INS-1 hücreleri gibi) ile, deney doğrudan zarar vermeden hücre dışı protein-protein etkileşimleri etkilerini belirlemek için dizayn edileb...

Burada belirli bir transmembran proteinin hücre dışı etki insülin salgılanması nasıl etkilediğini araştırmalar kolaylaştırmak için bir yöntem açıklanmaktadır. Pankreatik beta hücre yüzeyi üzerinde proteinler (veya muhtemelen diğer moleküller) ile söz konusu protein etkileşimleri gibi bir yöntem problar etkiler. Gibi bir yöntem beta hücreleri tarafından veya diğer komşu hücreler (örneğin, endotel hücreleri, nöronlar, pankreatik alfa hücreleri) ifade eden hücre yüzeyi proteinlerinin transsellüler etkileşimler (örneğin, komşu yüzeyinde etkileşim ortakları ile etkileşim yoluyla yoluyla beta hücresi fonksiyonunu etkileyebilir nasıl araştırmalar sağlar: beta hücreleri). , Hücresel plazma membranında hücre dışı ortam arasında bir köprü olarak hizmet veren yapısal ve fonksiyonel proteinlerin kompleks bir dizi içerir. Transsellüler bağlantı oluşumu ile ya da plastik sinyal olaylarını başlatılarak, hücre yüzeyi proteinleri arasındaki etkileşimi koordine yardımcı olabilirKomşu hücrelerin fonksiyonu. Pankreas beta hücreleri pankreas adacık içinde bir araya kümelenmiş ve glikoz dengesini 1 korumak için koordineli bir şekilde hareket edilir. Hücre dışı epha-ephrinA ve glikoz ile uyarılan insülin salgılanmasının düzenlenmesinde neuroligin-2 etkileşimlerinin önemi, örneğin, ortaya çıkardı, bu daha da açık hale gelmektedir, bitişik yüzeyleri üzerinde proteinler arasında meydana gelen hücre dışı etkileşimler artan bilgi beta hücreleri insülin salgılanması, beta hücre fonksiyonel olgunlaşması ve 1-3 glikoz dengesinin bakım tam bir anlayış kazanmak için büyük önem taşıyacaktır. Burada açıklanan yöntemin amacı, belirli bir transmembran veya başka şekilde-hücre yüzeyi ile ilişkili proteinler içeren transsellüler etkileşimleri beta hücre fonksiyonu üzerindeki etkileri araştırmalar sağlamaktır. Farklı ifade yapıları ile transfekte HEK293 hücreleri, B üzerindeki etkileri ile birlikte-kültür beta hücreleri tarafındanFarklı hücre yüzey proteinleri ya da mutasyona uğramış varyantları eta hücre fonksiyonu bunların etkin bir şekilde araştırılabilir. Bu beta hücreleri transfekte etmek için kendilerini kalmadan gerçekleştirilir. Kültür beta hücreleri veya in vivo olarak demonte, knockout ya da aşırı çalışmalarla özellikle transsellüler etkileşimlerinin rol izahı potansiyel analizi etkilemesi için değişik şekillerde beta hücre sağlık ve / veya fonksiyonunu etkileyen, beta hücresi mRNA ve protein ekspresyonu, doğrudan pertürbasyon gerektiren özel hücre dışı etkileşim etkileri. Bu, aynı zamanda, hedeflenen proteinlerin hücre içi etki düzeyleri değiştirmek ve daha fazla, transsellüler etkileşimlerin etkileri ayırt edilmesi ya da aynı hücrede protein arasındaki etkileşimlere bağlı etkileri izin vermez yaklaşır. Burada, beta-hücrelerinin insülin salgılayan kapasitesi ve yanıt belirli transsellüler etkileşimlerin etkilerini belirlemek için bir yöntem olup DescriIBED. Bu yöntem, INS-1 hücreleri, 4 gibi insülin salgılayan beta-hücre hatları, ve ayrışmış primer veya kemirgen, insan beta hücreleri için de geçerlidir. Bu HEK293 (veya COS) hücre katmanları ifade belirli nöronal proteinlere kültürlü nöronların maruz kalma bulundu neurobiologists, tarafından geliştirilen kokültürü modelleri dayandığını sürücü sinaps oluşumu 5,6 proteinleri tespit olabilir. Nöronal sinaps salgı makine arasında ve beta hücrelerinin paralellikler göz önüne alındığında, beta-hücre fonksiyonu ve fonksiyonel olgunlaşma benzer transsellüler etkileşimleri 7-9 ile tahrik olabilir gerekçeli. Bu etkileşimler prob amacıyla, beta hücreleri ilgi 10 bir protein ifade eden HEK293 hücreleri bir tabaka üzerinde kültüre edildiği tarifnamede açıklanan sistem geliştirilmiştir. Bu sistem, hücre-dışı protein-protein etkileşimleri manipüle edilir ise, beta hücre sitoplazması bakir kalmasını sağlar.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

Hücre yüzey proteinleri arasındaki etkileşim, komşu hücrelerin fonksiyonu koordine yardımcı olur. Pankreas beta hücreleri pankreas adacık içinde bir araya kümelenmiş ve glikoz dengesini korumak için koordineli bir şekilde hareket edilir. Bu komşu beta hücrelerinin yüzeylerinde transmembran proteinler arasındaki etkileşimleri beta-hücre fonksiyonu önemli belirleyicileri olduğu giderek daha açık hale gelmektedir. Kültür beta hücreleri veya in vivo olarak demonte, knockout ya da aşırı çalışmalarla özellikle transsellüler etkileşimlerinin rol izahı potansiyel etkileri analiz karıştırabileceklerinden edemeyeceği şekilde beta hücresi, sağlık ve / veya fonksiyonunu etkileyen, mRNA ve protein ekspresyonu, doğrudan pertürbasyon gerektiren spesifik etkileşimler. Bu yaklaşım, aynı zamanda hedef proteinlerin hücre içi etki düzeyleri değiştirebilir ve dis olmak için aynı hücre zarı içindeki proteinler arasındaki etkileşimlere bağlı etkileri önleyebilirtranssellüler etkileşimlerin etkileri bir biçimde ayrılır. Burada beta hücrelerinin insülin salgılayan kapasitesi ve yanıt belirli transsellüler etkileşimlerin etkilerini belirlemek için bir yöntem sunulmuştur. Bu yöntem, INS-1 hücreleri gibi beta-hücre hatları, ve ayrışmış primer beta hücreleri için de geçerlidir. Bu HEK293 (veya COS) sinaps oluşumu sürüş için önemli hücre tabakaları tespit proteinler üzerinde ifade belirli nöronal proteinlere kültürlü nöronların maruz kalma bulundu neurobiologists, tarafından geliştirilen kokültürü modelleri dayanmaktadır. Nöronal sinapsların ve beta hücrelerinin salgı makine arasındaki paralellikler göz önüne alındığında, beta-hücre olgunlaşması benzer transsellüler etkileşimleri ile tahrik olabilir gerekçeli. Biz beta hücreleri ilgi bir protein ifade eden HEK293 hücreleri bir tabaka üzerinde kültive edildiğinde, bir sistem geliştirilmiştir. Bu modelde, beta hücre sitoplazması dokunulmamış bir hücre dışı protein-protein ETKİLEŞİM ikenns manipüle. Öncelikle glikoz ile uyarılan insülin sekresyonunda çalışmalar burada odak rağmen, diğer işlemler analiz edilebilir, örneğin, gen ekspresyonunda değişmeler gibi immünolojik veya QPCR tarafından belirlenir.

Burada açıklanan yöntem kullanılarak, insülin salgılanması üzerindeki protein neuroligin farklı türevleri etkisini test ettik. Bu beta hücresi işlevi, 10 neuroligin-2 etkisini araştıran eden yayımlanmış çalışma tamamlar. Şekil 2, örneğin, burada adlandırılan bir neuroligin izoformu ifade etmek için transfekte HEK293 hücreleri ile birlikte coculturing INS-1 beta hücrelerinden elde edilen sonuçları göstermektedir NL- X Bu deney NL-X insülin salgısını artırdı...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Transsellüler protein etkileşimleri pankreatik beta hücre fonksiyonu önemli belirleyicidir. Burada ayrıntılı bir yöntem uyarlanmış özel transmembran proteinler insülin salgılanmasını nasıl etkilediğini araştıran sinaptogenez-için bir kokültürü model. Transfekte edilmiş HEK293 hücreleri ilgili proteinlerin ifade eder; beta hücreleri transfekte edilmiş veya başka şekilde doğrudan tedirgin gerekmez.

Pankreas Beta Hücreleri ile insülin salgılanması Etkileyen Ekstrasellüler Protein etkileşimleri kokültürü Analizi

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

coculture-analysis-extracellular-protein-interactions-affecting

Research

JoVE Journal

Medicine

12.8K Görüntüleme.  University of California, San Diego. Transsellüler protein etkileşimleri pankreatik beta hücre fonksiyonu önemli belirleyicidir. Burada ayrıntılı bir yöntem uyarlanmış özel transmembran proteinler insülin salgılanmasını nasıl etkilediğini araştıran sinaptogenez-için bir kokültürü model. Transfekte edilmiş HEK293 hücreleri ilgili proteinlerin ifade eder; beta hücreleri transfekte edilmiş veya başka şekilde doğrudan tedirgin gerekmez.

Video: Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Bir Topluluk Hastanesi IV insülin uygulaması Geliştirilmesi

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Tıp

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

İnsan Diş Pulp Kök Hücre İzolasyonu, Karakterizasyonu ve Karşılaştırmalı Farklılaşma İki Farklı Yöntemler Kullanılarak Daimi Dişler türetilen

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Bir Organotipik Beyin Dilim ve kanser hücrelerinin 3D Sferoid ile Coculture Sistemi

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

İnsan Dupuytren en<em&gt; Ex vivo</em&gt; Miyofibroblastların Çalışması ve Ekstrasellüler Matriks Etkileşimleri Kültür

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pankreas Kanseri Yeni Tedavi Stratejileri Geliştirme Hücre Özellikleri Kök incelenmesi

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Kronik Amiloid beta Oligomer İnfüzyon bir hayvan Modelinde Nörodejenerasyon Osmotik Pompalar ile Antikor Tedavisi infüzyon ile giderilir

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

kantitatif 3D<em&gt; Siliko</emAlzheimer Hastalığının Kemirgen Modelleri Serebral Amiloid-beta Fagositoz&gt; Modelleme (q3DISM)

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Homogeneous Time-resolved F&amp;#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Homojen zaman çözüldü Förster rezonans enerji transferi tabanlı tahlil insülin salgılanması algılanması için

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Birincil fare tetkikine yalıtım doğmakta olan Protein sentez analiz radyoaktif L-azidohomoalanine etiketleme yöntemi tarafından için

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Candida albicans kullanarak gerçek zamanlı ekstraselüler akı analiz biyo-enerji incelenmesi

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...