Name: Yazar Spotlight: Oral Potansiyel Malignitelerin Gizemlerini Çözmek
Uploaded: 2023-08-11T15:00:00
Description: Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

Bu çalışma, Çin Ulusal Doğa Bilimleri Vakfı (81671006, 81300894), CAMS Tıp Bilimleri İnovasyon Fonu (2019-I2M-5-038), Ulusal klinik anahtar disiplin inşaat projesi (PKUSSNKP-202102), Pekin Üniversitesi Sağlık Bilimleri Merkezi (BMU2022BSS001) Üstün Doktora Adayları için İnovasyon Fonu.

Bu çalışma, Pekin Üniversitesi Okul ve Hastanesi kurumsal inceleme kurulu tarafından onaylanmıştır. Hastalardan bilgilendirilmiş onam alındı. Bu çalışmada kullanılan doku örnekleri tanımlandı. Çalışma şeması Şekil 1'de gösterilmiştir.
1. Numune hazırlama
<ol><li>Formalinle sabitlenmiş parafine gömülü oral submukoz fibroz dokularını bir mikrotom üzerinde 3 μm ve 10 μm kalınlığında sürekli bölümler halind...

Mekansal-omik analiz için oral submukoz fibrozis doku örneklerinin alınması

<ol>
	<li>Cai, X., 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=Oral+submucous+fibrosis:+A+clinicopathological+study+of+674+cases+in+China.">Oral submucous fibrosis: A clinicopathological study of 674 cases in China.</a> Journal of Oral Pathology &amp; Medicine. 48 (4), 321-325 (2019).</li><li>Cai, X., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinicopathological+factors+associated+with+progression+of+oral+submucous+fibrosis:+A+population-based+retrospective+study.">Clinicopathological factors associated with progression of oral submucous fibrosis: A population-based retrospective study.</a> Oral Oncology. 130, 105949 (2022).</li><li>Ray, J. G., Chatterjee, R., Chaudhuri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+submucous+fibrosis:+A+global+challenge.+Rising+incidence,+risk+factors,+management,+and+research+priorities.">Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities.</a> Periodontology 2000. 80 (1), 200-212 (2019).</li><li>Shih, Y. H., Wang, T. H., Shieh, T. M., Tseng, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+submucous+fibrosis:+A+Review+on+etiopathogenesis,+diagnosis,+and+therapy.">Oral submucous fibrosis: A Review on etiopathogenesis, diagnosis, and therapy.</a> International Journal of Molecular Sciences. 20 (12), 2940 (2019).</li><li>Cai, X., 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=The+preliminary+exploration+of+immune+microenvironment+in+oral+leukoplakia+concomitant+with+oral+submucosal+fibrosis:+A+comparative+immunohistochemical+study.">The preliminary exploration of immune microenvironment in oral leukoplakia concomitant with oral submucosal fibrosis: A comparative immunohistochemical study.</a> Journal of Oral Pathology &amp; Medicine. , (2023).</li><li>Warnakulasuriya, S., 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=Oral+potentially+malignant+disorders:+A+consensus+report+from+an+international+seminar+on+nomenclature+and+classification,+convened+by+the+WHO+Collaborating+Centre+for+Oral+Cancer.">Oral potentially malignant disorders: A consensus report from an international seminar on nomenclature and classification, convened by the WHO Collaborating Centre for Oral Cancer.</a> Oral Diseases. 27 (8), 1862-1880 (2021).</li><li>Cai, X., 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=Development+and+validation+of+a+nomogram+prediction+model+for+malignant+transformation+of+oral+potentially+malignant+disorders.">Development and validation of a nomogram prediction model for malignant transformation of oral potentially malignant disorders.</a> Oral Oncology. 123, 105619 (2021).</li><li>Murthy, V., 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=Malignant+transformation+rate+of+oral+submucous+fibrosis:+A+systematic+review+and+meta-analysis.">Malignant transformation rate of oral submucous fibrosis: A systematic review and meta-analysis.</a> Journal of Clinical Medicine. 11 (7), 1793 (2022).</li><li>Kujan, O., Mello, F. W., Warnakulasuriya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+transformation+of+oral+submucous+fibrosis:+A+systematic+review+and+meta-analysis.">Malignant transformation of oral submucous fibrosis: A systematic review and meta-analysis.</a> Oral Diseases. 27 (8), 1936-1946 (2020).</li><li>Qin, X., Ning, Y., Zhou, L., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+submucous+fibrosis:+Etiological+mechanism,+malignant+transformation,+therapeutic+approaches+and+targets.">Oral submucous fibrosis: Etiological mechanism, malignant transformation, therapeutic approaches and targets.</a> International Journal of Molecular Sciences. 24 (5), 4992 (2023).</li><li>Sun, 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=Single-cell+and+spatial+dissection+of+precancerous+lesions+underlying+the+initiation+process+of+oral+squamous+cell+carcinoma.">Single-cell and spatial dissection of precancerous lesions underlying the initiation process of oral squamous cell carcinoma.</a> Cell Discovery. 9 (1), 28 (2023).</li><li>Zhu, J., 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=Delineating+the+dynamic+evolution+from+preneoplasia+to+invasive+lung+adenocarcinoma+by+integrating+single-cell+RNA+sequencing+and+spatial+transcriptomics.">Delineating the dynamic evolution from preneoplasia to invasive lung adenocarcinoma by integrating single-cell RNA sequencing and spatial transcriptomics.</a> Experimental &amp; Molecular Medicine. 54 (11), 2060-2076 (2022).</li><li>Ji, A. 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=Multimodal+analysis+of+composition+and+spatial+architecture+in+human+squamous+cell+carcinoma.">Multimodal analysis of composition and spatial architecture in human squamous cell carcinoma.</a> Cell. 182 (2), 497-514 (2020).</li><li>Van den Bossche, V., 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=Microenvironment-driven+intratumoral+heterogeneity+in+head+and+neck+cancers:+clinical+challenges+and+opportunities+for+precision+medicine.">Microenvironment-driven intratumoral heterogeneity in head and neck cancers: clinical challenges and opportunities for precision medicine.</a> Drug Resistance Updates. 60, 100806 (2022).</li><li>Li, X., 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=Improvement+in+the+risk+assessment+of+oral+leukoplakia+through+morphology-related+copy+number+analysis.">Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.</a> Science China. Life sciences. 64 (9), 1379-1391 (2021).</li><li>Watkins, T. B. K., 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=Pervasive+chromosomal+instability+and+karyotype+order+in+tumour+evolution.">Pervasive chromosomal instability and karyotype order in tumour evolution.</a> Nature. 587 (7832), 126-132 (2020).</li><li>Odell, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+and+loss+of+heterozygosity+as+risk+markers+for+malignant+transformation+in+oral+mucosa.">Aneuploidy and loss of heterozygosity as risk markers for malignant transformation in oral mucosa.</a> Oral Diseases. 27 (8), 1993-2007 (2021).</li><li>Wood, H. 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=The+genomic+road+to+invasion-examining+the+similarities+and+differences+in+the+genomes+of+associated+oral+pre-cancer+and+cancer+samples.">The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.</a> Genome Medicine. 9 (1), 53 (2017).</li><li>Venugopal, D. 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=Integrated+proteomics+based+on+2D+gel+electrophoresis+and+mass+spectrometry+with+validations:+Identification+of+a+biomarker+compendium+for+oral+submucous+fibrosis-An+indian+study.">Integrated proteomics based on 2D gel electrophoresis and mass spectrometry with validations: Identification of a biomarker compendium for oral submucous fibrosis-An indian study.</a> Journal of Personalized Medicine. 12 (2), 208 (2022).</li><li>Kundu, P., Pant, I., Jain, R., Rao, S. G., Kondaiah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+DNA+methylation+changes+in+oral+submucous+fibrosis.">Genome-wide DNA methylation changes in oral submucous fibrosis.</a> Oral Diseases. 28 (4), 1094-1103 (2022).</li><li>Cai, X., Zhang, H., Li, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-target+pharmacological+mechanisms+of+Salvia+miltiorrhiza+against+oral+submucous+fibrosis:+A+network+pharmacology+approach.">Multi-target pharmacological mechanisms of Salvia miltiorrhiza against oral submucous fibrosis: A network pharmacology approach.</a> Archives of Oral Biology. 126, 105131 (2021).</li><li>Xiao, X., Hu, Y., Li, C., Shi, L., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+abnormality+in+oral+submucous+fibrosis+concomitant+leukoplakia:+A+preliminary+evaluation+of+the+diagnostic+and+clinical+implications.">DNA content abnormality in oral submucous fibrosis concomitant leukoplakia: A preliminary evaluation of the diagnostic and clinical implications.</a> Diagnostic Cytopathology. 48 (11), 1111-1114 (2020).</li><li>Zhou, S., 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=Long+non-coding+RNA+expression+profile+associated+with+malignant+progression+of+oral+submucous+fibrosis.">Long non-coding RNA expression profile associated with malignant progression of oral submucous fibrosis.</a> Journal of Oncology. 2019, 6835176 (2019).</li><li>Lunde, M. 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Bu protokol, lazer mikrodiseksiyon yoluyla daha fazla mekansal-omik analiz için morfolojik ve mekansal bilgilerle OSF doku örneklerini yakalamak için bir boru hattı bildirdi. Temsili sonuçlardan, morfoloji ile ilgili çeşitli örnekler arasında farklı CNA kalıpları belirledik.
OPMD'nin bir türü olan OSF, oral skuamöz hücreli karsinomun yaygın bir prekanseröz durumudur6. Genomik instabilitenin OPMD17,18'in<sup class="xre...

Oral submükoz fibrozis (OSF), esas olarak bukkal mukozada gelişen ve ağız açıklığının kısıtlanması ile sonuçlanan kronik, sinsi bir hastalıktır1. OSF multifaktöriyel bir hastalık iken, areca fıstığı veya tembul fıstığı çiğneme OSF 2,3'ün ana nedenidir. Coğrafi olarak spesifik olan bu alışkanlık nedeniyle, OSF ağırlıklı olarak Güneydoğu ve Güney Asya'daki popülasyonlarda yoğunlaşmıştır3. OSF'nin yaygın histolojik özellikleri arasında oral mukozal epitelin altındaki bağ dokusunda anormal kollajen birikimi, vasküler stenoz ve oklüzyon1 yer alır. OSF epitel dokusu, oral lökoplaki ile birlikte olduğunda atrofi veya hiperplazi ve hatta displazi belirtileri ile ortaya çıkabilir 4,5.
OSF, Dünya Sağlık Örgütü tarafından %4-%6 malign dönüşüm oranı ile oral skuamöz hücreli karsinomlara ilerleme potansiyeli sergileyen yaygın bir oral potansiyel malign bozukluk (OPMD) olarak tanımlanmaktadır6,7,8,9. OSF'nin malign dönüşümünün altında yatan mekanizma kompleks10'dur. Hem displazi hem de atrofi dahil olmak üzere epitelin anormal büyümesi, karsinogenez potansiyelini arttırır ve stromadaki yaşlanan fibroblastlar, reaktif oksijen türleri (ROS) ve diğer moleküller yoluyla epitelyal-mezenkimal geçişi (EMT) indükleyerek OSF'nin malign ilerlemesinde rol oynayabilir10.
Mekansal-omik analizler için teknolojiler, kanser mekanizmaları hakkında içgörü sağlayan morfolojik ve mekansal bilgilerle multi-omik veriler üretti11,12,13. Burada, lazer mikrodiseksiyon ile formalinle sabitlenmiş parafine gömülü OSF dokusundan morfoloji ile ilişkili hücre popülasyonlarını yakalamak için bir protokol sunuyoruz. Bu örneklerin multi-omik analizleri, doku içi heterojenlik ile ilgili zorlukların üstesinden gelebilir ve OSF14'teki moleküler patolojinin ve malign transformasyon mekanizmalarının anlaşılmasını artırabilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion microscope slides</td>
			<td>CITOTEST</td>
			<td>REF.188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Div-haematoxylin</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin solution</td>
			<td>BASO</td>
			<td>BA4098</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>PEKING REAGENT</td>
			<td>No.32061</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin dye solution</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>LEICA</td>
			<td>HI1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser capture microdissection system</td>
			<td>LEICA</td>
			<td>LMD7</td>
			<td>Machine</td>
		</tr>
		<tr>
			<td>Laser microdissection microsystem</td>
			<td>LEICA</td>
			<td>8.2.3.7603</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Micromount mounting medium</td>
			<td>LEICA</td>
			<td>REF.3801731</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>CITOTEST</td>
			<td>REF.10212450C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>LEICA</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>AXYGEN</td>
			<td>16421959</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane slides</td>
			<td>LEICA</td>
			<td>No.11505158</td>
			<td></td>
		</tr>
		<tr>
			<td>Re-blue solution</td>
			<td>YiLi</td>
			<td>20230326</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>REF.10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>PEKING REAGENT</td>
			<td>No.33535</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of cells with morphological and spatial information from oral submucous fibrosis samples by laser capture microdissection

Oral submukoz fibrozis (OSF), ağız boşluğunda yaygın olarak görülen potansiyel olarak malign bir bozukluk türüdür. Epitel atrofisi ve lamina propria ve submukoza fibrozisi sıklıkla histopatolojik slaytlarda bulunur. Epitelyal displazi, epitelyal atrofi ve yaşlanan fibroblastların OSF'nin malign transformasyonu ile ilişkili olduğu öne sürülmüştür. Bununla birlikte, potansiyel olarak malign oral bozuklukların ve oral skuamöz hücreli karsinomun heterojenliği nedeniyle, OSF'de malign transformasyonun spesifik moleküler mekanizmalarını tanımlamak zordur. Burada, formalinle sabitlenmiş parafine gömülü doku preparatları üzerinde lazer yakalama mikrodiseksiyonu ile morfolojik veri ve uzamsal bilgi taşıyan az sayıda epitelyal veya mezenkimal hücre elde etmek için bir yöntem sunuyoruz. Mikroskop kullanarak, mikro ölçekli (~ 500 hücre) displastik veya atrofik epitel dokusunu ve fibrotik subepitel dokusunu hassas bir şekilde yakalayabiliriz. Ekstrakte edilen hücreler, morfolojik ve mekansal bilgilerle genomik ve transkriptomik veriler elde etmek için genom veya transkriptom dizilimi ile değerlendirilebilir. Bu yaklaşım, toplu OSF doku diziliminin heterojenliğini ve lezyonsuz alanlardaki hücrelerin neden olduğu girişimi ortadan kaldırarak, OSF dokusunun hassas uzamsal-omik analizine izin verir.

Oral submucous fibrosis (OSF) is a chronic, insidious disease that develops mainly in the buccal mucosa and results in restricted mouth opening1. While OSF is a multifactorial disease, areca nut or betel nut chewing is the main cause of OSF2,3. Because of this geographically specific habit, OSF is predominantly concentrated in populations in Southeast and South Asia3. The common histological features of OSF include abnormal collagen deposition in the connective tissue beneath the oral mucosal epithelium, vascular stenosis, and occlusion1. OSF epithelial tissue can present with manifestations of atrophy or hyperplasia and even dysplasia when concomitant with oral leukoplakia4,5.
OSF is defined by the World Health Organization as a common oral potentially malignant disorder (OPMD) that exhibits the potential to progress to oral squamous cell carcinoma with a malignant transformation rate of 4%-6%6,7,8,9. The mechanism underlying the malignant transformation of OSF is complex10. Abnormal growth of the epithelium, including both dysplasia and atrophy, increases the potential for carcinogenesis, and senescent fibroblasts in the stroma may be involved in the malignant progression of OSF by inducing epithelial-mesenchymal transition (EMT) through reactive oxygen species (ROS) and other molecules10.
Technologies for spatial-omic analyses generated multi-omic data with morphological and spatial information that have provided insights into cancer mechanisms11,12,13. Here, we present a protocol to capture morphology-related cell populations from formalin-fixed paraffin-embedded OSF tissue by laser microdissection. Multi-omic analyses of these samples can overcome challenges with intratissue heterogeneity and increase understanding of the molecular pathology and mechanisms of malignant transformation in OSF14.

Yazar Spotlight: Oral Potansiyel Malignitelerin Gizemlerini Çözmek 

Lazer Yakalama Mikrodiseksiyonu ile Oral Submukoz Fibrozis Örneklerinden Morfolojik ve Mekansal Bilgilerle Hücrelerin İzolasyonu

Our main research interests are the mechanisms of malignant transformation under the development of predictive models of oral, potentially malignant disorders such as oral leukoplakia and oral submucous fibrosis. Genomic transcriptomic, proteomic, and other multi-omic sequencing analysis under deep learning artificial intelligence models are used to study the progression from oral, potentially malignant disorders to oral cancer. Current sequencing and multi-omics analysis are mostly based on frozen samples, but oral, potentially malignant disorders are mostly formally fixed paraffin embedded tissue, often by biopsy, and they the need to develop experimental techniques based on paraffin samples.Paraffin tissue samples from oral, potentially malignant disorders, and the squamous cell carcinoma are available in large numbers and from a variety of sources, making them a very valuable resource for study. All protocol can help fully use these resources to better understand the mechanisms of disease development and malignant transformation.

OSF dokularının lazer mikrodiseksiyonu ile displastik epitel, displastik epitel altında stroma, atrofik epitel ve atrofik epitel dokusu altında stroma örnekleri elde edildi (Şekil 1). DNA'nın çıkarılması ve düşük derinlikli tüm genom dizilimi yoluyla, morfolojiyle ilgili kopya sayısı değişikliklerini (CNA) analiz edebildik15. CNA, OPMD15,16'da artmış malign transformasyon riski ile ilişki...

Watch this Scientific Journal Video about Unlocking the Mysteries of Oral Potential Malignancies at JoVE.com

Oral submukoz fibroz dokularının lazer yakalama mikrodiseksiyonu, morfolojik ve mekansal bilgilerle multi-omik verilerin analizi için ilgilenilen histolojik bölgelerden hücrelerin hassas bir şekilde çıkarılmasına izin verir.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

Başlıca araştırma alanlarımız, oral lökoplaki ve oral submukoz fibrozis gibi oral, potansiyel olarak malign bozuklukların prediktif modellerinin geliştirilmesi altında malign dönüşüm mekanizmalarıdır. Derin öğrenme yapay zeka modelleri altında genomik transkriptomik, proteomik ve diğer multi-omik dizileme analizleri, ağız, potansiyel olarak malign bozukluklardan ağız kanserine ilerlemeyi incelemek için kullanılır. Mevcut dizileme ve multi-omik analizler çoğunlukla donmuş örneklere dayanmaktadır, ancak oral, potansiyel olarak malign bozukluklar çoğunlukla biyopsi ile resmi olarak sabitlenmiş parafine gömülü dokudur ve parafin örneklerine dayalı deneysel teknikler geliştirme ihtiyacı vardır.Oral, potansiyel olarak malign bozukluklardan ve skuamöz hücreli karsinomdan alınan parafin doku örnekleri çok sayıda ve çeşitli kaynaklardan temin edilebilir, bu da onları çalışma için çok değerli bir kaynak haline getirir. Tüm protokoller, hastalık gelişimi ve malign dönüşüm mekanizmalarını daha iyi anlamak için bu kaynakları tam olarak kullanmaya yardımcı olabilir.

isolation-cells-with-morphological-spatial-information-from-oral

Research

JoVE Journal

Medicine

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Tıp

Oral Submucous Fibrosis Tissue Sample Preparation for Hematoxylin-Eosin Staining

To begin, using a microtome, cut the formalin fixed paraffin embedded oral submucous fibrosis tissues into continuous sections of three in 10 micrometers thickness, then unfold the sections in water. Place three micron sections on the adhesion microscope slide or slide A, and 10 microns on pen membrane slide or slide B.Place both slides on a hot plate at 60 degrees Celsius. After two hours, place slides A and B in xylene for 10 minutes, then hydrate the slides in graded ethanol concentrations for one minute in each concentration.Next, place the slides in ultrapure distilled water for 30 seconds. Soak slides in Harris hematoxylin dye solution for 90 seconds. Again wash the slides thrice in ultrapure distilled water for 30 seconds each.After that, submerge the slides in div-hematoxylin solution for two seconds. Return slides into ultrapure distilled water for 30 seconds, repeating three times. Next, immerse slides A and B in 1%re-blue solution for two seconds.After washing the slides with distilled water, place them in an eosin solution for two seconds. Again immerse slides in ultrapure distilled water for 30 seconds before dehydrating and increasing ethanol concentrations, then immerse slide A in xylene for three minutes. Finally, apply a drop of the mounting medium to slide A, and cover it with a glass cover.

Hematoksilen-Eozin Boyama için Oral Submukoz Fibrozis Doku Örneği Hazırlama

Laser Capture Microdissection of Oral Submucous Fibrosis Tissues for Morphology and Spatial Information Analysis

To begin, turn on the Laser Microdissection system and software. Place slide A containing stained three-micron sections of oral submucous fibrosis tissues on the slide stage. Observe the histological morphology to identify the area of interest for laser microdissection.Remove slide A and place slide B with the tissue side facing down on the slide stage. Next, click the Unload button to push out the collection device. Insert a PCR tube into the collection device and ensure the tube is fixed.Then click the Okay button. Select a cap by clicking the corresponding red circle, marking the collector device to tube caps, and wait until the selected circle turns green. Then click the Draw button and Draw plus Cut button and use the mouse to sketch the area of interest.Click on Start Cut to capture the area of interest, which will be collected by the cap. Then press the lower button to ensure the captured sample is collected. Click the specimen button to capture the next sample.Finally, click Unload to unload the PCR tube with the captured sample. In this study, using laser microdissection of oral submucous fibrosis, tissues of epithelium and stroma with different patterns are captured. Copy number alternations were present in epithelial samples but not in stroma samples.The copy number alternations pattern in the dysplastic epithelium was observed in chromosomes three and eight, while copy number alternations were detected at a lower frequency in chromosome eight in the atrophic epithelium.