術前のワークアウト<ol><li>腹部CTスキャンでは、簡単かつ大動脈から直接発信右肝動脈の存在を明らかに肝臓.3 D再構成の左側の胆管が（暗い色の肝実質内に記述されて）広く拡大を見ることができます。それ以上の血管異常なし。 </li><li>肝臓は機能的にビデオに示すように8つのセグメントに分割し、拡張、セグメント2と3（左セクター）に限られていた、と腹腔鏡下左sectoriectomyが計画されていることを考慮することができます。 </li></ol>術中の手順<ol><li>患者は仰臥位と患者の脚の間に外科医のスタンドに配置されます。ビデオ5に示すようトロカールを使用して配置されています</li><li>血管クランプは（プリングルのmanouvre）出血を減らすために門脈三管に配置され</li><li>肝実質の分割は、セグメントIIIとIVの間に肝鎌状間膜に沿ってメスの組み合わせ、またはバイポーラ電気メスとハーモニックを使用して実行されます</li><li>茎セグメントIIIは完全にそれが血管のカートリッジで45ミリメートルエンドGIAを使用して分割することができる解剖されると。 </li><li>セグメントIIの主茎に達するまで、単離され、最終的に血管のカートリッジで45ミリメートルendoGIAを使用して分割されるまで彼は、プロシージャは実質のさらなる分裂とに運ぶ</li><li>左の三角靱帯は、現在電気焼灼器を使用して、左肝静脈は血管のステープラーを使用して分離し、分割された分割されています</li><li>肝臓表面が出血のためにチェックされ、アルゴンビームは、肝臓の表面に適用され、血管クランプが削除されます。この時点で、フィブリン糊は、術後の胆汁の漏れのリスクを軽減するために適用されます。試料はendobagに置かれ、除去され</li></ol>

<ol>
	<li>Nguyen, K. T., Gamblin, T. C., Geller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+for+cancer.">Laparoscopic liver resection for cancer.</a> Future Oncol. 4 (5), 661-670 (2008).</li><li>Gumbs, A. A., Gayet, B., Gagner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection:+when+to+use+the+laparoscopic+stapler+device.">Laparoscopic liver resection: when to use the laparoscopic stapler device.</a> HPB (Oxford). 10 (4), 296-303 (2008).</li><li>Pulitan&#242;, C., Catena, M., Arru, M., Guzzetti, E., Comotti, L., Ferla, G., Aldrighetti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+liver+resection+without+portal+clamping:+a+prospective+evaluation.">Laparoscopic liver resection without portal clamping: a prospective evaluation.</a> Surg Endosc. 22 (10), 2196-2200 (2008).</li><li>Yonem, O., Bayraktar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics+of+Caroli's+disease.">Clinical characteristics of Caroli's disease.</a> World J Gastroenterol. 13 (13), 1934-1937 (2007).</li></ol>

腹腔鏡手術を安全に肝切除のために両方の良性および悪性疾患に対しても行うことができます。低侵襲的アプローチを用いて外科手術による外傷を減らすことが術後合併症と術後の入院の発生率を最小限に抑えることができます。

laparoscopic left liver sectoriectomy of caroli's disease limited to segment ii and iii

カロリー病は、イントラ肝内胆管の異常な拡張として定義されています：その発生率は極めて低いです（1,000,000人口の1）とほとんどの場合には肝臓全体が興味を持っており、肝移植が選択される治療である。左または右葉に限定膨張の場合には、肝切除を行うことができます。多くの年のために肝切除のための標準的なアプローチは重要なポストoperatieの罹患率によって特徴づけられる腹部の大切開により正式な開腹手術をしている。さらに最近では、低侵襲、腹腔鏡下のアプローチは、両方の良性および悪性疾患に対する肝切除の可能な手術方法として提案されている。低侵襲的アプローチの主なメリットは少ない術後合併症迅速な復旧を可能にする外科的外傷の大幅な減少によって表されます。 このビデオでは、Caroliは病の場合を示して発熱に関連付けられている腹痛の突然の発症のために消化器科で入院した58歳の雄に発生した（&gt; 38C °）、吐き気、震え。腹部超音波検査では胆嚢または総胆管結石のない証拠を使用して、イントラ肝左据え付け胆管の著しい拡張を示した。このような所見は、腹部の高解像度のコンピュータトモグラフィを確認した。腹腔鏡下左sectoriectomyが計画されました。五トロカールと30 °光が使用されて、腹腔の探査は、他の疾患のない癒着や証拠を示さなかった。 肝臓への血液流入を制御するために、血管クランプが茎肝（プリングルのmanouvre）に配置され、実質部門は5mmバイポーラ鉗子と5mm超音波解剖を併用することで行われます。深刻な拡張した左肝管を単離し、45ミリメートル内視鏡血管ステープラーを使用して分割されました。肝臓の解剖は、45ミリメートルの血管ステープラーのさらなるカートリッジで分割されたメインの左のポータルの枝の分離まで継続した。 彼の時点では左肝は左肝静脈によって接続されたままです：三角靱帯の分割は、モノポーラのフックと肝静脈分離し、血管のステープラーを使用して分割を用いて行った。 Haemostatisは、アルゴンビーム凝固のアプリケーションによって洗練され、出血であっても血管クランプの取り外し（合計プリングルの時間27分）後に明らかにされなかった。 術後経過は良好であった、肝機能検査の最小限の上昇は、術後1日目に記録が手術後3日目で退院で正常に返されました。

Watch this Scientific Journal Video about Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III at JoVE.com

Laparoscopic Left Liver Sectoriectomy of Caroli&#039;s Disease Limited to Segment II and III

このビデオでは、肝臓のセグメントIIとIIIに限定カロリー病を患っている53歳の男性で付け方腹腔鏡下左肝sectoriectomyのケースを提示。

セグメントIIおよびIIIに制限カロリー病の腹腔鏡下左肝Sectoriectomy

Caroli's disease is defined as a abnormal dilatation of the intra-hepatica bile ducts: Its  incidence is extremely low  (1 in 1,000,000 population) and in ...

laparoscopic-left-liver-sectoriectomy-caroli-s-disease-limited-to

Research

JoVE Journal

Biology

Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

14.1K ビュー.  University of Insubria. このビデオでは、肝臓のセグメントIIとIIIに限定カロリー病を患っている53歳の男性で付け方腹腔鏡下左肝sectoriectomyのケースを提示。

ビデオ: Laparoscopic Left Liver Sectoriectomy of Caroli's Disease Limited to Segment II and III

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

腫瘍細胞のVasculogenic活性を評価するためにマトリゲルベースの​​チューブ形成アッセイ

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

生物学

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

肝灌流による肝細胞の精製した生体肝エンドサイトーシスに

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

の用法<em&gt; ex vivoで</em&gt;全体の臓器イメージングと定量的組織組織学は、蛍光標識分子のバイオ分布を決定します

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell motility, or cell signaling. The green algae Chlamydomonas reinhardtii has proven to be an insightful model in the study of centriole architecture, function, and protein composition. Despite great advances toward understanding centriolar architecture, one of the current challenges is to determine the precise localization of centriolar components within structural regions of the centriole in order to better understand their role in centriole biogenesis. A major limitation lies in the resolution of fluorescence microscopy, which complicates the interpretation of protein localization in this organelle with dimensions close to the diffraction limit. To tackle this question, we are providing a method to purify and image a large number of C. reinhardtii centrioles with different orientations using super-resolution microscopy. This technique allows further processing of data through fluorescent single-particle averaging (Fluo-SPA) owing to the large number of centrioles acquired. Fluo-SPA generates averages of stained C. reinhardtii centrioles in different orientations, thus facilitating the localization of distinct proteins in centriolar sub-regions. Importantly, this method can be applied to image centrioles from other species or other large macromolecular assemblies.

Isolation and Fluorescence Imaging for Single-particle Reconstruction of Chlamydomonas Centrioles

We have developed a strategy to purify and image a large number of centrioles in different orientations amenable for super-resolution microscopy and single-particle averaging.

分離とクラミドモナス由来の単一粒子再構成のための蛍光イメージング

Centrioles are large macromolecular assemblies important for the proper execution of fundamental cell biological processes such as cell division, cell ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

in vitro疾患モデリングのためのヒト初代弁細胞の単離

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective treatments. Considerable limiting factors hindering the timely and accurate diagnosis (including grading) and monitoring of NAFLD, as well as the development of potential therapies, are the current inadequacies in the characterization of the hepatic microenvironment structure and the scoring of the disease stage in a spatiotemporal and non-invasive manner. Using a diet-induced NAFLD mouse model, we investigated the use of in vivo micro-computed tomography (CT) imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation. This imaging methodology allows for longitudinal analysis of liver steatosis and functional tissue uptake, as well as the evaluation of the relative blood volume, portal vein diameter, and density of the vascular network. Understanding the adaptations of the hepatic vascular network during NAFLD progression and correlating this with other ways of characterizing the disease progression (steatosis, inflammation, fibrosis) using the proposed method can pave the way toward the establishment of new, more efficient, and reproducible approaches for NAFLD research in mice. This protocol is also expected to upgrade the value of preclinical animal models for investigating&#160;the development of novel therapies against disease progression.

Novel In Vivo Micro-Computed Tomography Imaging Techniques for Assessing the Progression of Non-Alcoholic Fatty Liver Disease

Using a diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we describe the use of novel in vivo micro-computed tomography imaging techniques as a non-invasive method to assess the progression stages of NAFLD, focusing predominantly on the hepatic vascular network due to its significant involvement in NAFLD-related hepatic dysregulation.

非アルコール性脂肪性肝疾患の進行を評価するための新しい in vivo マイクロコンピュータ断層撮影イメージング技術

Non-alcoholic fatty liver disease (NAFLD) is a growing global health issue, and the impact of NAFLD is compounded by the current lack of effective ...

ヒト血清中のB型肝炎ウイルスDNAの検出と定量

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

著者スポットライト:B型肝炎ウイルス検出の進歩と課題 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.