Name: 著者スポットライト:脳血栓の包括的な細胞成分分析のための新しい方法
Uploaded: 2023-07-21T13:30:00
Description: Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

この研究は、厦門市科学技術局(3502Z20227197)と福建省科学技術局(No.2019J01070、No.2021Y0027)から資金提供を受けました。

この研究は、華橋大学の医療倫理委員会の機関ガイドラインに準拠して行われました。脳血栓は外科的に除去され、福建医科大学附属泉州第一病院で、患者のインフォームドコンセントを得て採取された。
1.血栓の前処理
<ol><li>血栓を清潔な皿の上に置き、ピンセットで生理食塩水5mLを加え、皿を静かに振って、ピペットで生理食塩水を取り除きます。 ?...

脳血栓細胞成分を研究するための線維素溶解酵素ベースの方法

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+updated+definition+of+stroke+for+the+21st+century:+a+statement+for+healthcare+professionals+from+the+American+Heart+Association/American+Stroke+Association.">An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association.</a> Stroke. 44 (7), 2064-2089 (2013).</li><li>Thalin, C., Hisada, Y., Lundstrom, S., Mackman, N., Wallen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+villains+and+targets+in+arterial,+venous,+and+cancer-associated+thrombosis.">Neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis.</a> Arteriosclerosis Thrombosis and Vascular Biology. 39 (9), 1724-1738 (2019).</li><li>Mocsai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+novel+functions+of+neutrophils+in+immunity,+inflammation,+and+beyond.">Diverse novel functions of neutrophils in immunity, inflammation, and beyond.</a> Journal of Experimental Medicine. 210 (7), 1283-1299 (2013).</li><li>Dhanesha, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKM2+promotes+neutrophil+activation+and+cerebral+thromboinflammation:+therapeutic+implications+for+ischemic+stroke.">PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.</a> Blood. 139 (8), 1234-1245 (2022).</li><li>Ducroux, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombus+neutrophil+extracellular+traps+content+impair+tpa-induced+thrombolysis+in+acute+ischemic+stroke.">Thrombus neutrophil extracellular traps content impair tpa-induced thrombolysis in acute ischemic stroke.</a> Stroke. 49 (3), 754-757 (2018).</li><li>Solomon, C., Ranucci, M., Hochleitner, G., Schochl, H., Schlimp, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+methodology+for+calculating+platelet+contribution+to+clot+strength+(platelet+component)+in+thromboelastometry+and+thrombelastography.">Assessing the methodology for calculating platelet contribution to clot strength (platelet component) in thromboelastometry and thrombelastography.</a> Anesthesia and Analgesia. 121 (4), 868-878 (2015).</li><li>Daraei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+fiber+diameter+and+porosity+measurements+of+plasma+clots+in+scanning+electron+microscopy+images.">Automated fiber diameter and porosity measurements of plasma clots in scanning electron microscopy images.</a> Biomolecules. 11 (10), 1536 (2021).</li><li>Abbasi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+thrombus+composition+in+thrombectomy+stroke+patients+with+longer+time+to+recanalization.">Diverse thrombus composition in thrombectomy stroke patients with longer time to recanalization.</a> Thrombosis Research. 209, 99-104 (2022).</li><li>C W Francis, a., Marder, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concepts+of+clot+lysis.">Concepts of clot lysis.</a> Annual Review of Medicine. 37 (1), 187-204 (1986).</li><li>Xu, R., Ma, G., Chen, L., Cui, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Preparation and application of natural fibrinolytic enzyme from peanut worm. , (2019).</li><li>Fotso Fotso, A., Drancourt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+Diagnosis+of+tick-borne+african+relapsing+fevers:+latest+developments.">Laboratory Diagnosis of tick-borne african relapsing fevers: latest developments.</a> Front Public Health. 3, 254 (2015).</li><li>Liou, G. Y., Byrd, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioliquid+markers+for+pancreatic+cancer:+What+we+have+vs.+what+we+need.">Diagnostic bioliquid markers for pancreatic cancer: What we have vs. what we need.</a> Cancers (Basel). 15 (9), 2446 (2023).</li><li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus.">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> Journal of Visualized Experiments. 196, e65631 (2023).</li><li>Ge, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+antithrombotic+protease+from+marine+worm+Sipunculus+Nudus.">A Novel antithrombotic protease from marine worm Sipunculus Nudus.</a> International Journal Of Molecular Sciences. 19 (10), 3023 (2018).</li><li>Talukder, M. A., Menyuk, C. R., Kostov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinguishing+between+whole+cells+and+cell+debris+using+surface+plasmon+coupled+emission.">Distinguishing between whole cells and cell debris using surface plasmon coupled emission.</a> Biomedical Optics Express. 9 (4), 1977-1991 (2018).</li><li>Shapiro, D. J., Hicks, L. A., Pavia, A. T., Hersh, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+prescribing+for+adults+in+ambulatory++care+in+the+USA,+2007-09.">Antibiotic prescribing for adults in ambulatory care in the USA, 2007-09.</a> Journal of Antimicrobial Chemotherapy. 69 (1), 234-240 (2014).</li></ol>

sFEは、フィブリンを直接かつ効果的に分解することができる線溶剤である12,16。ここでは、sFEを用いて、脳血栓の架橋フィブリンを分解し、血栓内の封入細胞を遊離させ、血栓の細胞成分を定性的および定量的に分析しました。顕微鏡データと定期的な血液検査により、同封された細胞が血栓から放出されたことが示されました。さらに、放出され...

脳血管疾患は、人間の健康を脅かす可能性のある3大疾患の1つであり、そのうち虚血性脳血管疾患は80%以上を占めています。脳血栓症と脳静脈血栓症は、主に脳血栓によって引き起こされる、今日最も懸念されている虚血性脳血管疾患です1,2。治療が適切に行われないと、障害率や死亡率が高くなり、退院後の再発率も高くなります3。
近年、脳血栓の細胞成分が脳血栓症の診断、治療、予後と密接な相関関係にあることを示す研究が増えています4,5,6。したがって、血栓組成、特に細胞成分に関するデータの可用性は、臨床診断と治療にとって重要です。残念ながら、現在利用可能な方法では、血栓成分を定量的にも定性的にも包括的に分析することはできません。例えば、Martius Scarlett Blueベースのin-situ染色では、血栓の特定のスライスの赤血球/白血球しか調べることができません7。免疫組織化学(IHC)に基づくin-situ染色では、抗体を用いて血栓の特定のスライスの限られた血液成分しか研究できません8。顕微鏡画像ベースの方法は、血栓9の特定の構造にのみ関係しています。さらに、これらの方法はすべて手間と時間がかかります10.現在まで、脳血栓細胞成分を定量的および定性的に研究するための手順は報告されていません。架橋されたフィブリンが血栓11内の血球をしっかりと包み込むことは広く認められている。したがって、架橋されたフィブリンの特異的な分解と無傷の細胞の放出は、細胞成分の正確な分析にとって重要です。
以前の研究では、シプンクルス・ヌーダス(sFE)から線溶酵素を単離し、フィブリンを特異的かつ迅速に分解することができます12。本明細書では、sFEの特異な活性に基づいて脳血栓の細胞成分を分析する方法が提案された。このプロトコルは血栓のフィブリンを最初に低下させるのにsFEを利用し、次にライトの汚損および定期的な血液検査13,14によって細胞部品を分析した。この方法によれば、脳血栓の細胞成分を定量的および定性的に研究することができる。このシンプルで効果的なプロトコルは、他の血栓の細胞成分分析にも適用できます。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Agglutination Reaction Plate</td>
			<td>ROTEST</td>
			<td>RTB-4003</td>
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			<td>Auto Hematology Analyzer</td>
			<td>SYSMEX</td>
			<td>XNB2</td>
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			<td>Automatic Vertical Pressure Steam Sterilizer&#160;</td>
			<td>SANYO</td>
			<td>MLS-3750</td>
			<td></td>
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			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
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			<td>Clean bench</td>
			<td>AIRTECH</td>
			<td>BLB-1600</td>
			<td></td>
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			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
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			<td>Culture Dish (100 mm)</td>
			<td>NEST</td>
			<td>704001</td>
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			<td>DHG Series Heating and Drying Oven&#160;</td>
			<td>SENXIN</td>
			<td>DGG-9140AD</td>
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			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
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			<td>Filter Membrane (0.22 &#181;m)</td>
			<td>Millex GP</td>
			<td>SLGP033NK</td>
			<td></td>
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			<td>Micro Refrigerated Centrifuge&#160;</td>
			<td>Cence</td>
			<td>H1650-W</td>
			<td></td>
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			<td>Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>01-30253-50</td>
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			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
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			<td>Normal Saline</td>
			<td>CISEN</td>
			<td>H37022337</td>
			<td></td>
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			<td>Optical Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE E100</td>
			<td></td>
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			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
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			<td>Phosphate-Buffered Saline</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td></td>
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			<td>Pipette Tip (1 mL )</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>MARTOR</td>
			<td>23111</td>
			<td></td>
		</tr>
		<tr>
			<td>Small-sized Vortex Oscillator</td>
			<td>Kylin-Bell</td>
			<td>VORTEX KB3</td>
			<td></td>
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			<td>Tweezer</td>
			<td>Hystic</td>
			<td>HKQS-180</td>
			<td></td>
		</tr>
		<tr>
			<td>Wright Staining Solution</td>
			<td>Beyotime</td>
			<td>C0135-500ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

a comprehensive approach to analyze the cell components of cerebral blood clots

脳動脈や静脈に血栓ができる脳血栓症は、脳梗塞の最も一般的なタイプです。脳血栓の細胞成分の研究は、診断、治療、および予後にとって重要です。しかし、血栓の細胞成分を研究するための現在のアプローチは、主に in situ 染色に基づいており、細胞が血栓にしっかりと包まれているため、細胞成分の包括的な研究には適していません。これまでの研究では、 Sipunculus nudusから線溶酵素(sFE)を単離することに成功しており、架橋されたフィブリンを直接分解して細胞成分を放出することができます。本研究により、sFEに基づく脳血栓の細胞成分を網羅的に調べる方法を確立した。このプロトコルには、血栓溶解、細胞解放、細胞染色、および定期的な血液検査が含まれます。この方法によれば、細胞成分を定量的および定性的に研究することができる。この手法を用いた実験の代表的な結果を示す。

Cerebrovascular disease is one of three major diseases that can threaten human health, among which ischemic cerebrovascular disease accounts for more than 80%. Cerebral thrombosis and cerebral vein thrombosis are the most concerned ischemic cerebrovascular diseases today, mainly caused by cerebral blood clots1,2. If the treatment is not done properly, it will have high disability and mortality rates and a high recurrence rate after discharge3.
Recently, a growing number of studies have shown that the cell components of cerebral blood clots are tightly correlated with the diagnosis, treatment, and prognosis of cerebral thrombosis4,5,6. Therefore, the availability of data on thrombus composition, especially the cell components, is important for clinical diagnosis and treatment. Unfortunately, the currently available methods cannot comprehensively analyze the blood clot component quantitatively and qualitatively. For example, Martius Scarlett Blue based in-situ staining can only study the red/white blood cells of certain slices of the clot7. Immunohistochemistry (IHC) based in-situ staining can only study limited blood components of certain slices of the clot using their antibodies8. The microscopic image-based methods are only concerned with the specific structure of the clot9. Moreover, all those methods are laborious and time-consuming10. To date, the procedures for quantitatively and qualitatively studying cerebral thrombi cell components have not been reported. It is widely acknowledged that the cross-linked fibrin tightly wraps the blood cells in the clots11. Consequently, the specific degradation of the cross-linked fibrin and release of the intact cells is critical for the accurate analysis of cell components.
Previous works isolated a fibrinolytic enzyme from Sipunculus nudus (sFE), which can degrade the fibrin specifically and quickly12. Herein, a method for analyzing the cell components of the cerebral thrombi based on the unique activity of sFE was proposed. This protocol utilized sFE to degrade the fibrin of clots first and then analyzed the cell components by Wright&#39;s Staining and routine blood examination13,14. According to this method, the cell components of cerebral thrombi can be quantitatively and qualitatively studied. This simple and effective protocol might be applied for the cell component analysis of other blood clots.

著者スポットライト:脳血栓の包括的な細胞成分分析のための新しい方法 

脳血栓の細胞成分を包括的に解析する

Our research mainly focuses on cerebral thrombosis. Many studies have shown that cerebral blood clot cell component correlate with the diagnosis, treatment, and prognosis of cerebral thrombosis. In this study, we established a novel method for the analysis of cell component of cerebral blood clots.This challenge in analyzing cell components of cerebral blood clots is the release of entire shaft from blood clots. This is because the clots leaked fibering tiry rash, the blood cells in the clots. Targeted approaches to starting the cell components of the clots are best done in C2 standing, which is unsuitable for the comprehensive starting of the cell components.On the contrary, our method enables both quantitative and qualitative enlightenings of the cerebral slope cell components. Our method utilizes fibrinolytic enzyme to delay the fibering in blood clots, releasing the intact cells. The data obtained from the analysis of the cell components will facilitate the study of the mechanisms of cerebral thrombosis at a molecular level.Our team will continue to focus on the study of fibrinolysis and coagulation system.

分解プロセスの初期段階では、血栓が赤い緻密な構造をしており、作業溶液は無色であることがわかりました。30分間インキュベートした後、作業溶液は淡赤色に変化し、交差した血球が作業溶液中に放出されたことを示しました。インキュベーション時間を5時間に延長するとほとんどの血栓が溶解し、作業溶液は淡赤色になった。それどころか、10時間のインキュベーション後も生理食塩?...

Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

この研究では、血栓の溶解、細胞染色、および定期的な血液検査による脳血栓の細胞成分分析のための迅速かつ効果的な方法について説明します。

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

主に脳血栓症を研究対象としています。多くの研究により、脳血栓細胞成分が脳血栓症の診断、治療、および予後と相関していることが示されています。本研究では、脳血栓の細胞成分を解析するための新しい手法を確立した。脳血栓の細胞成分を解析する上での課題は、血栓から軸全体が放出されることです。これは、血栓が繊維状の発疹、血栓内の血球を漏らしたためです。血栓の細胞成分を開始するためのターゲットを絞ったアプローチは、細胞成分の包括的な開始には適さないC2静置で行うのが最適です。それどころか、私たちの方法は、大脳斜面細胞成分の量的および質的な啓蒙を可能にします。私たちの方法は、線溶酵素を利用して血栓の繊維化を遅らせ、無傷の細胞を放出します。細胞成分の分析から得られたデータは、分子レベルでの脳血栓症のメカニズムの研究を容易にします。私たちのチームは、線溶と凝固系の研究に引き続き注力していきます。

a-comprehensive-approach-to-analyze-cell-components-cerebral-blood

Research

JoVE Journal

Biochemistry

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

生化学

Cerebral Thrombus Clot Dissolution and Cell Staining for Cell Component Analyses

To begin, place the collected cerebral blood clot samples on a clean dish using tweezers. Then, using a pipette, add five milliliters of physiological saline to it and gently shake the dish. Remove the saline using a pipette.Using a pair of scissors, chop the clots into small pieces. Once again, add five milliliters of physiological saline to the clots and shake the dish gently before aspirating the saline with a pipette. Then, using tweezers, transfer the pieces of clots to a new, clean U-plate.Prepare the SFE working solution by adjusting the concentration of SFE to 2, 000 units per milliliter using physiological saline. Add 300 microliters of the SFE working solution to the pretreated clots. To initiate the first round of degradation, incubate the mixture of SFE and clots at 37 degrees Celsius for half an hour.After that, gently shake to mix the SFE treated sample. And using a clean pipette, transfer the liquid portion to a sterile tube. Set aside the remaining clot for another round of degradation.Centrifuge the collected liquid at 200 G for five minutes at four degrees Celsius. Next, using a pipette, transfer the supernatant or the recovered SFE solution to another clean tube. Resuspend the resultant cell pellet in 50 microliters of physiological saline by gentle mixing.After this, perform subsequent rounds of degradation on the remaining clots using the recovered SFE solution as demonstrated previously. Gather the cell mixture from each round of degradation to prepare the blood cell sample. Add five microliters of the obtained cell sample to the poly-L-lysine-coated glass slide.And using a cover slip, smear the cells. Allow the liquid to evaporate at room temperature. To perform cell staining, gently add 100 microliters of rights dye to the cell smear.Allow the cells to stain for 30 minutes at room temperature before gently rinsing the dye off with clean water. Finally, examine the stained cells using a light microscope. Compact red blood clots with a colorless working solution were observed during the early stage of degradation.Clot dissolution was observed after a 30-minute incubation and a prolonged incubation of up to five hours dissolved most clots, while there was no significant change in the negative control group, even after a 10-hour incubation. After a right staining, mature red blood cells, platelets, and various granulocytes were clearly identifiable under a light microscope. The cell components could be quantitatively analyzed with the aid of autohematology successfully.