کشت و تکثیر سلول های بنیادی واقع در لیمبوس قرنیه انسانی

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی پسادکتری پزشکی مولکولی، پژوهشکده بوعلی، دانشگاه علوم پزشکی مشهد، مشهد، ایران

2 کارشناسی ارشد بیوشیمی بالینی، دانشکده پزشکی، دانشگاه علوم پزشکی مشهد، مشهد، ایران

3 دانشیار گروه بیوشیمی، دانشکده پزشکی، دانشگاه علوم پزشکی مشهد، مشهد، ایران

چکیده

مقدمه
سلول های اپیتلیوم قرینه به طور طبیعی از بین می روند و سلول های بنیادی ناحیه لیمبوس با تکثیر و تمایز جایگزین آن ها می گردند. در صورت نقص در این سلول ها امکان جایگزینی وجود ندارد و در بسیاری از موارد بیمار بینایی خود را از دست می دهد. یکی از جدیدترین روش های درمان نقص سلول های بنیادی لیمبال، کشت آن ها در آزمایشگاه و انتقالشان روی چشم بیمار است. بدین منظور در این مطالعه به ارتقاء کیفیت کشت آزمایشگاهی سلول های لیمبال پرداختیم.
روش کار
ابتدا ناحیه ی لیمبوس نمونه های چشم های اهدایی جدا شدند. سپس قطعات جدا شده با روش explant بر روی پرده آمینوتیک کشت داده شدند. حضور و تکثیر سلول های بنیادی لیمبال با روش ایمونوفلورسنت وآنتی بادی علیه مارکرهای ΔNP63α و ABCG2 در روزهای دهم، چهاردهم و بیست و یکم بررسی گردید. همچنین با استفاده از روش فلوسایتومتری و آنتی بادی علیه مارکر ΔNP63α درصد سلول های لیمبال در بهترین روز کشت آن ها بر اساس نتایج ایمونوفلورسنت تعیین گردید.
نتایج
نتایج ایمونوفلورسنت هر سه روز تایید کننده ی کشت موفق سلول های بنیادی لیمبال بودند. با توجه به نتیجه بهتر کشت در روز چهاردهم آزمون فلوسایتومتری برای کشت های روز چهاردهم انجام شد. به طور متوسط 5.8 درصد از سلول های کشت داده شده، سلول های بنیادی لیمبال بودند.
نتیجه گیری
نتایج حاکی از کشت موفق سلول های لیمبال در شرایط آزمایشگاهی بود اما به منظور کاربرد بالینی آن ها بهینه سازی بیشتر کشت مورد نیاز می باشد.

کلیدواژه‌ها

عنوان مقاله [English]

Culture and expansion of stem cells located at the corneoscleral limbus

نویسندگان [English]

  • Elaheh Emadi 1
  • Danial Taherzadeh 2
  • Daryoush Hamidi Alamdari 3
1 Bo Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran
2 Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
3 Department of Clinical Biochemistry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
چکیده [English]

Introduction:
Corneal epithelial cells are destroyed naturally and the limbal stem cells replace them by multiplying and differentiating. In case of defects in these cells, it is impossible to replace them, and in many cases, the patients lose their vision. One of the newest methods of treating limbal stem cell deficiency (LSCD) is to cultivate them in the laboratory and transfer them to the patient's eye. For this purpose, in this study, we aimed to culture limbal stem cells in vitro and optimized the culture quality.
Methods:
First, the limbus region of the donated eye samples was separated. Then the separated parts were cultured on the amniotic membrane by the explant method. The presence and proliferation of limbal stem cells were investigated by immunofluorescent method and antibody against ΔNP63α and ABCG2 markers on the 10th, 14th, and 21st days. Also, by using the flow cytometry method and antibody against ΔNP63α marker, the percentage of limbal cells on the best day of their culture based on immunofluorescent results was determined.
Results:
Findings showed that limbal stem cells could be cultured properly in vitro. All immunofluorescent assays confirmed limbal stem cell attendance, however, the result of day 14th of culture was more considerable. Therefore, the flow cytometry assay was performed on the 14th day and proved that there were 5.8% limbal stem cells using explant culture.
Conclusion:
The results indicated the successful cultivation of limbal cells in laboratory conditions, but for their clinical application, further optimization of cultivation is required.

کلیدواژه‌ها [English]

  • Corneal limbus stem cells
  • Limbal stem cell deficiency
  • Stem cell culture

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مجله دانشکده پزشکی دانشگاه علوم پزشکی مشهد
دوره 66، شماره 1
فروردین-اردیبهشت1402
فروردین و اردیبهشت 1402
صفحه 1-10

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