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بررسی استرس اکسیداتیو در رده سلولی K562 تیمار شده با ترکیب فعالی از خانواده پیرانوپیریدین

تعداد    93   صفحه در فایل   word

چکیده:

لوسمی، سرطان مغز استخوان یا سرطان خون می باشد. تقریبا %90 موارد بر اساس نوع سلول درگیر و سرعت رشد بیماری به چهار گروه اصلی (ALL، CLL، AML،CML) تقسیم بندی میشود. CML یک اختلال کلونال سلولهای بنیادی خونساز است که جزو اختلالات در تکثیر میلوئیدی کلونی تقسیم بندی می شود. در دهه­های گذشته اکثر روش­های درمان سرطان خون شامل شیمی درمانی و پرتو درمانی بوده است. داروهای شیمی درمانی اغلب از طریق آسیب DNA و تنش اکسیداتیو باعث القاء آپوپتوز می شوند، تنش اکسیداتیو حالتی است که درآن عدم تعادل درمیزان تولید رادیکالهای آزاد و سیستم دفاع آنتی اکسیدانی پیش می آید. دراین میان استفاده از ترکیباتی که با سیستم استرس اکسیداتیو در داخل سلول سرطانی عمل کنند مثل  مشتقات خانواده‌‌ی پیرانوپیردینی، مفید خواهد. دراین تحقیق فعالیت استرس اکسیداتیو مشتقی فعال از خانواده پیرانوپیریدین در رده سلولی  K562بعنوان مدلی برای بررسی های آزمایشگاهی  CML بررسی گردید. بدین منظور  سلولهای  K562در غلظت IC50 ( µM 30) تیمار شدند. بدنبال آن، فعالیت آنزیم های آنتی اکسیدانی کاتالاز (CAT)، سوپراکسیددیسموتاز (SOD)، میزان تیول تام، مقادیر پروکسیداسیون لیپید (MDA) و اکسیداسیون پروتئین ها (PCO) بعنوان نشانه های استرس درون سلولی به روش اسپکتروفوتومتری سنجیده شد. تشخیص  سطح ROS داخل سلولی با استفاده از دستگاه فلوسایتومتری مورد مطالعه قرار گرفت. داده های بدست آمده با استفاده از آزمون آماری SPSS  و نرم افزار excel 2010 مورد ارزیابی قرار گرفت. نتایج این مطالعه نشان داد که فعالیت  SODوسطوح پروکسیداسیون لیپیدی و اکسیداسیون پروتئین بطور معنی داری افزایش یافت، درحالیکه فعالیت CAT و محتوای تیول تام نسبت به گروه کنترل پایین تر بود. بعلاوه نتایج حاکی از آن است که ترکیب فعال در غلظت IC50 تولید ROS داخل سلولی را بالا می برد.

بنابراین با توجه به عملکرد SOD که تبدیل مولکولهای سوپراکسید به H2O2 میباشد و کاهش فعالیت CAT و نیز افزایش ROS داخل سلولی می توان نتیجه گرفت که تجمع رادیکالهای آزاد درون سلول وضعیت استرس اکسیداتیو  را ایجاد می کنند. بر پایه ی اطلاعات حاضر و نیز نتایج مطالعات قبلی که ویژگی القاء آپوپتوزی این ترکیبات بررسی و تائید شده مشتقات جدید پیرانوپیریدینی می توانند گزینه ی مناسبی برای تحقیقات بیشتر در زمینه ی شیمی درمانی باشند.

فصل اول: بررسی منابع

سرطان …………………………………………………………………………………………………………………………………………………. 1

اصول طبقه بندی سرطان …………………………………………………………………………………………………………………… 3

سرطان خون یا لوکمی ……………………………………………………………………………………………………………………….. 3

1-3-1 تقسیم بندی انواع  لوکمی ……………………………………………………………………………………………………… 3

1-3-2 بیماری CML …………………………………………………………………………………………………………………………3

1-3-2-1مراحل لوسمی میلوئیدی مزمن ………………………………………………………………………………………….. 4

1-4 انکوژن BCR-ABL ………………………………………………………………………………………………………………… 4

1-4-1 انکوپروتئین BCR-ABL و عملکردهای آن ………………………………………………………………………. 6

1-5 درمان لوکمی ………………………………………………………………………………………………………………………………. 7

1-5-1 پیوند سلول های بنیادی خونساز …………………………………………………………………………………………… 8

1-5-2 درمان با اینترفرون آلفا …………………………………………………………………………………………………………… 8

1-5-3 تمایز درمانی …………………………………………………………………………………………………………………………… 9

1-5-4 هدف درمانی …………………………………………………………………………………………………………………………… 9

1-5-5 درمان با SiRNA ………………………………………………………………………………………………………………. 11

1-5-6 شیمی درمانی ………………………………………………………………………………………………………………………. 11

1-6 آپوپتوز ………………………………………………………………………………………………………………………………………. 12

استرس اکسیداتیو………………………………………………………………………………………………………………………………. 12

1-8 معرفی رادیکالهای آزاد ……………………………………………………………………………………………………………… 13

1-8-1 منابع سلولی تولید ROS …………………………………………………………………………………………………… 14

1-8-2 هوموستازی پیام رسانی ROS …………………………………………………………………………………………… 17

1-8-2-1 تولید وکنترل ROS ………………………………………………………………………………………………………. 17

1-8-2-2 پیام رسانی ROS …………………………………………………………………………………………………………….20

1-8-3 استرس اکسیداتیو و سرطان ……………………………………………………………………………………………….. 21

1-8-4 عملکرد ROS در خونسازی نرمال …………………………………………………………………………………….. 22

1-8-5 تولید ROS در بیماریهای میلوئید …………………………………………………………………………………….. 23

1-8-6STAT5 ، تولید ROS را همراه با افزایش شکست دو رشته ای در DNA القا می کند… 25

1-8-7 پیام رسانی پروتئین های STAT5 در سلول سالم و سلول سرطانی ………………………………. 25

ارتباط  پروتئین های STAT با متابولیسم اکسیداتیو در سلول سرطانی ……………………………………… 26

1-9 اثرات ROS افزایش یافته در بیماری های میلوئید…………………………………………………………………. 28

1-10 هدف قرار دادن ROS در بیماری های میلوئید …………………………………………………………………… 29

1-11 رده سلولی k562 …………………………………………………………………………………………………………………. 32

1-12 تركيبات هتروسيكليك ………………………………………………………………………………………………………….. 33

1-12-1 پیرانوپیریدین ها ……………………………………………………………………………………………………………….. 34

فصل دوم: مواد و روش ها

2-1 مواد …………………………………………………………………………………………………………………………………………… 39

2-2 دستگاه های مورد استفاده ……………………………………………………………………………………………………….. 40

2-3 روش ها …………………………………………………………………………………………………………………………………….. 41

2-3-1 کشت سلول …………………………………………………………………………………………………………………………. 41

2-3-1-1 وسایل مورد نیاز کشت سلول …………………………………………………………………………………………. 41

2-3-1-2 مواد مورد نیاز کشت سلول …………………………………………………………………………………………….. 41

2-3-1-3 روش کشت سلول …………………………………………………………………………………………………………… 42

2-3-1-3-1 یخ زدایی سلول ها ……………………………………………………………………………………………………… 42

2-3-1-3-2 مراحل تعویض محیط کشت سلول ……………………………………………………………………………. 43

2-3-1-3-3 ذخیره کردن سلول ها در بانک سلول ……………………………………………………………………….. 43

2-3-2 آزمون دفع رنگ تریپان بلو ………………………………………………………………………………………………….. 44

2-3-2-1 وسايل و مواد مورد نياز …………………………………………………………………………………………………… 44

2-3-2-2 تهیه ی رنگ تریپان بلو …………………………………………………………………………………………………… 44

2-3-2-3 هموسایتومتر …………………………………………………………………………………………………………………… 45

2-3-2-4 شمارش سلول‌ها توسط لام هموسایتومتر ………………………………………………………………………. 45

2-3-3 تیمار سلول ها با ترکیبات پیرانوپیریدین ……………………………………………………………………………. 46

2-3-3-1 روش تهیه ی غلظت های مختلف دارو ………………………………………………………………………….. 46

2-3-4 سنجش سطح  ROS داخل سلولی …………………………………………………………………………………… 46

2-3-4-1 مواد مورد نیاز ………………………………………………………………………………………………………………….. 47

2-3-4-1-1 روش انجام آزمایش ……………………………………………………………………………………………………. 47

2-3-5 سنجش تیول تام …………………………………………………………………………………………………………………. 48

2-3-5-1 معرف ها ………………………………………………………………………………………………………………………….. 48

2-3-5-2 روش انجام آزمایش ………………………………………………………………………………………………………… 49

2-3-6 سنجش های آنزیمی ……………………………………………………………………………………………………………. 49

2-3-6-1 استخراج پروتئین …………………………………………………………………………………………………………….. 49

2-3-6-1-1 مواد مورد نیاز ……………………………………………………………………………………………………………. 49

2-3-6-1-2 مراحل استخراج پروتئین …………………………………………………………………………………………… 49

2-3-6-2 تهیه ی منحنی استاندارد (تعیین غلظت پروتئین به روش برادفورد) …………………………… 50

2-3-6-3 سنجش فعالیت آنزیم کاتالاز (CAT) ………………………………………………………………………….. 50

2-3-6-3-1 مواد و محلول های مورد نیاز …………………………………………………………………………………….. 51

2-3-6-3-2 مراحل سنجش آنزیم ………………………………………………………………………………………………….. 52

2-3-6-4 سنجش فعالیت آنزیم سوپراکسید دیسموتاز (SOD) …………………………………………………… 52

2-3-6-4-1 محلول ها و بافرهای مورد نیاز …………………………………………………………………………………… 52

2-3-6-5 مراحل انجام واکنش ……………………………………………………………………………………………………….. 53

2-3-7 اندازه گیری غلظت پروتئین کل به روش برادفورد ……………………………………………………………… 53

2-3-7-1 روش اندازه گیری ………………………………………………………………………………………………………….. ..53

2-3-8 تعیین پروکسیداسیون لیپید ……………………………………………………………………………………………….. 54

2-3-8-1 محلول های مورد نیاز ……………………………………………………………………………………………………… 54

2-3-8-1-1 مراحل آزمایش …………………………………………………………………………………………………………… 54

2-3-9 تعیین اکسیداسیون پروتئین ……………………………………………………………………………………………….. 55

2-3-9-1 مواد مورد نیاز ………………………………………………………………………………………………………………….. 56

2-3-9-1-1 مراحل انجام آزمایش ………………………………………………………………………………………………….. 56

فصل سوم: نتایج و بحث

3-1 اثرات مورفولوژیکی ترکیب 4-C.P.P بر روی رده ی سلولی K562 …………………………………… 59

3-2 اثر ترکیب 4-CPP بر تولید ROS داخل سلولی ………………………………………………………………….. 60

3-3 تعیین میزان تیول تام ………………………………………………………………………………………………………………. 61

4-3 سنجش فعالیت های آنزیمی ……………………………………………………………………………………………………. 62

5-3 تعیین پروکسیداسیون لیپید ……………………………………………………………………………………………………. 63

3-6 تعیین اکسیداسیون پروتئین ……………………………………………………………………………………………………. 64

بحث ………………………………………………………………………………………………………………………………………………….. 65

نتیجه گیری ……………………………………………………………………………………………………………………………………… 71

پیشنهادات ……………………………………………………………………………………………………………………………………….. 72

منابع و مأخذ ……………………………………………………………………………………………………………………………………. 73

فهرست اشکال و جداول

 

شکل 1-1. ساختار کروموزوم فیلادلفیا ………………………………………………………………………………………………. 5

شکل 1-2. مسیرهای پیام رسانی BCR-ABL ……………………………………………………………………………… 7

شکل 1-3. مکانیسم عمل ایماتینیب ………………………………………………………………………………………………. 10

شکل 1-4. ساختار الکترونی مولکول های ROS رایج …………………………………………………………………… 13

شکل 1-5. تولید ROS از کمپلکس یک ETC درغشای داخلی میتوکندری …………………………….. 15

شکل 1-6. ساختار پروتئین های NOX در غشای سلولی ……………………………………………………………. 16

شکل 1-7. نقش ROS در خودنوسازی HSCS ……………………………………………………………………………..23

شکل 1-8. ساختار دومین های  STATs………………………………………………………………………………………. 24

شکل 1-9.پیام رسانی STAT5 در سلول سالم و سرطانی ………………………………………………………….. 26

شکل 1-10. متابولیسم اکسیداتیو و STAT3/5 در سلول سرطانی …………………………………………… 27

شکل 1-11. درمان آنتی اکسیدانی و پروکسیدانی بعنوان روشهای درمانی برای بدخیمی های خونی. …….32

شکل 1-12. ساختارهای متفاوت حلقه پیران …………………………………………………………………………………. 34

شکل 1-13. حلقه پیردین ……………………………………………………………………………………………………………….. 35

شکل 1-14. حلقه پیردون و فرایند Lctam ,lactim …………………………………………………………………. 36

شکل 1-15. پیرانو[3,2-c] پیردین ………………………………………………………………………………………………… 36

شکل 1-16. ساختار پایه ای پیرانوپیریدین ها ……………………………………………………………………………….. 37

شکل 2-1. واکنش گروه های تیول با DTNB …………………………………………………………………………….. 48

شکل 2-2. واکنش مولکول مالون دی آلدئید با تیو باربیتوریک اسید …………………………………………… 55

شکل 3-1. تغییرات مورفولوژی سلول های K562 تیمار شده با ترکیب 4-C.P.P ……………………. 59

شکل3-2. آنالیز تاثیر ترکیب 4-C.P.P بر تولید ROS داخل سلولی با رنگ آمیزی 2و4 دی کلروفلورزین

دی استات (DCFH) …………………………………………………………………………………………………………………………. 60

شکل 3-3. وضعیت تیول تام در سلول های K562 تیمار شده با مشتق فعال در غلظت IC50 … 61

شکل 3-4. اثر 4-C.P.P بر فعالیت آنزیم های سوپراکسید دیسموتاز (الف) و کاتالاز(ب) در سلول های K562 …………………………………………………………………………………………………………………………………………………. 62

شکل 3-5. وضعیت مالون دی آلدئید (MDA) در سلول های K562 تیمار شده با ترکیب 4-C.P.P ……………… 63

شکل 3-6. تشکیل گروه های پروتئین-کربونیل (PCO) در سلول های تیمار شده با 4-C.P.P در سلول های K562 ………………………………………………………………………………………………………………………………………. 64

شکل 3-7. تأثیر پیرانوپیریدین مورد بررسی بر توانایی زیستی سلول های K562 ………………………. 67

شکل 3-8. اثرات القاء آپوپتوز ترکیب 4.CPP در سلول های K562  بعد از 72ساعت ……………… 68

جدول 1-1: نام شیمیایی مشتق فعال پیرانوپیریدین ها ………………………………………………………………… 37

2445 دیدگاه برای بررسی استرس اکسیداتیو در رده سلولی K562 تیمار شده با ترکیب فعالی از خانواده پیرانوپیریدین

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    En Trendencias | 13 faldas que son tendencia en 2023, palabra del street style Vestidos casuales Verano Moda Mujer Sexy Sin tirantes Marrón Rosa Plumas Bodycon Vendaje Vestido 2023 Elegante Midi Noche Club Fiesta Al hacer clic en “Hazte miembro”, acepto los Términos y Condiciones de la afiliación de H&M. VESTIDOS ENTERIZOS рџ’–✨ vestidoscasuales Ingresa tu ubicación “En vez de comprar, puedes intentar en lo posible acudir a algún sistema de arriendo de vestidos, algún closet sale por Instagram, asesorarse en una tienda vintage o armar un look de dos piezas”, analiza Pavez, siendo la creatividad el punto clave para no repetir Click & Collect Paga en línea y recoge tus productos en tienda. Servicio gratuito.«Muy buena atención por parte de la corresponsal del mostrador para recoger pedidos de la web en tienda.” 10 12 2022
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    Desde hace unos días se están realizando trabajos de limpieza en el emblemático establecimiento, que ha sufrido sucesivos cierres en los últimos años y salió a subasta por deudas. La ciudad ha honrado con la ubicación de monumentos en las calles a muchos de sus hijos más ilustres. La contraseña debe tener al menos 8 caracteres: Después de aprender a programar, no hay nada imposible en este mundo. Sin embargo, al igual que una línea de código, un pequeño error puede destruir toda una fórmula. Veamos con ejemplos de currículum para informática, como prestarle atención a cada detalle. Izumi Onuki, la gurú japonés del secado -sí, del secado, hay expertos en todos los campos-, propone la técnica de la toalla con la que asegura, se evita la formación de moho en las paredes, especialmente en casas con un aislamiento deficiente.

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