مقايسه روش‌هاي MTT، تريپان‌بلو و سنجش كلونوژنيك در تعيين بقاي رده سلولي سرطان تيروييد آناپلاستيك انساني

Comparison of MTT, trypan blue, and clonogenic assay, to determine the viability in human anaplastic thyroid cancer cell line

زمينه و هدف: ارزيابي بقاي سلولي در مطالعات دارويي و سرطان براي تعيين حساسيت سلولي مهم و تعيين‌كننده نتيجه درمان است. بنابراين روش‌هاي مختلفي استفاده مي‌گردد كه نقطه پاياني متفاوتي را ارزيابي مي‌كنند. تعيين وجود همبستگي بين روش‌ها داراي اهميت مي‌باشد. در اين مطالعه، حساسيت روش‌هاي رنگ‌سنجي 3-(4و5-دي متيل تيازول-2-ايل)-2و5-دي فنيل تترازوليوم برمايد (MTT)، تريپان‌بلو و سنجش كلونوژنيك در تعيين بقاي رده سلولي سرطان تيروييد آناپلاستيك ارزيابي گرديد. روش بررسي: مطالعه تجربي كنوني از مهر تا اسفند 1395 در مركز تحقيقات سلولي و مولكولي دانشگاه علوم پزشكي گيلان انجام پذيرفت. رده سلولي سرطان تيروييد آناپلاستيك انساني كشت شده در محيط Dulbecco;s modified Eagle;s medium (DMEM) حاوي 10% Fetal bovine serum (FBS)، به‌مدت 24 ساعت با ملاتونين تيمار گرديد، سپس بقاي سلولي توسط تست‌هاي MTT، تريپان‌بلو و سنجش كلونوژنيك بررسي و از راندمان كشت و درصد كسر بقا جهت رسم منحني بقا در سنجش كلونوژنيك استفاده گرديد. يافته‌ها: غلظت ملاتونين در نقطه IC50 در روش‌هاي MTT، تريپان‌بلو و سنجش كلونوژنيك به‌ترتيب، شامل 0/117±4/794، 0/894±4/375 و 0/326±2/246 ميلي‌مولار بود كه مقايسه مقادير IC50 در آزمون MTT و تريپان‌بلو اختلاف معناداري را نشان نداده (0/6446P=)، در‌حالي‌كه بين MTT-سنجش كلونوژنيك (0/0032P=)، تريپان‌بلو-سنجش كلونوژنيك (0/0078P=) اختلاف معنادار بود. نتايج تحليل رگرسيون بقاي سلولي، نشان‌دهنده همبستگي خطي، مثبت و معنادار بين روش‌ها بوده كه بين روش‌هاي MTT و تريپان‌بلو همبستگي بيشتر بود (0/99r=، 0/001P<). نتيجه‌گيري: نتايج نشان دادند، هر س

Background: Evaluation of cell viability is momentous in pharmacologic and oncological research. Cell viability evaluation determines cell sensitivity and consequently treatment outcome. Various methods are available to determine cell survival. Each of these methods evaluates different endpoints. Accordingly, determining the correlation between these methods is important. In this study, in order to determine the viability of human anaplastic thyroid cancer cell line, the sensitivity of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, trypan blue test and clonogenic assay were compared. Methods: This experimental study was performed in the Cellular and Molecular Research Center at Guilan University of Medical Sciences, Rasht, Iran from October 2016 to March 2017. The human anaplastic thyroid cancer cell line was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The cultured cells were treated with melatonin, for 24 hours. Then, the viability of the cells was evaluated by MTT assay, trypan blue test and clonogenic assay. Furthermore, plating efficiency and surviving fraction were used in order to draw survival curve in the clonogenic assay. Results: The concentration of melatonin at IC50 point was 4.794±0.117 millimolar (mM) in MTT assay, 4.375±0.894 mM in trypan blue test and 2.246±0.326 mM in clonogenic assay. Comparing the IC50 values of these test revealed that C50 values obtained from MTT assay and trypan blue test had no significant difference (P=0.6446), while there was a significant difference between IC50 values obtained from MTT and clonogenic assays (P=0.0032). Moreover, the IC50 values obtained from trypan blue test and clonogenic assay were also significantly different (P=0.0078). The results of the regression analysis of cell viability were shown a linear, positive and significant correlation between these three methods and MTT assay and trypan blue test showed higher correlation (r=0.99, P<0.001). Conclusion: Based on our results, all these methods were effective to identify cytotoxicity in human anaplastic thyroid cancer cell line, while MTT assay and trypan blue test were more sensitive than clonogenic assay.