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生物醫學暨環境生物系 助理教授 張學偉 genomed.dlearn.kmu.tw

生物醫學暨環境生物系 助理教授 張學偉 http://genomed.dlearn.kmu.edu.tw. Survival curve = radiation dose & cell survival fraction. Reproductive Integrity. Cell survival  cell death. For differentiated cells that do not proliferate e.g., nerve, muscle  lose of specific function (death).

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生物醫學暨環境生物系 助理教授 張學偉 genomed.dlearn.kmu.tw

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  1. 生物醫學暨環境生物系 助理教授 張學偉 http://genomed.dlearn.kmu.edu.tw

  2. Survival curve = radiation dose & cell survival fraction

  3. Reproductive Integrity Cell survival  cell death For differentiated cells that do not proliferate e.g., nerve, muscle  lose of specific function (death) For proliferating cells e.g., hematopoietic stem cells, culture cells  lose for sustained proliferation (death)  lose of reproductive integrity (reproductive death)

  4. Definition of survival for radiobiology Retain reproductive integrity Proliferate indefinitely to produce colony (a large clone = colony =clonogenic)

  5. The in vitro survival curve Cell culture Established cell lines Tissue  trypsin cell culture in vitro Dose-survival curve

  6. PE = plating efficiency

  7. Serial dilution

  8. The shape of the survival curve Width of shoulder = Dq or n =semi-threshold dose log Linear –quadratic function

  9. Multi-target model Initial slope  single-event killing = D1 final slope  multiple-event killing = D0

  10. D1 & D0 = the dose required to reduce the fraction of surviving cells to 37% of it previous value. D0 (straight at high dose) = the average dose required to deliver one inactivating event (one hit) per cell. = the dose required to reduce survival from0.1 to 0.037 or 0.01 to 0.0037. = D0 (37% survival) dose required to reduce survival to e-1 (0.37)

  11. Dq (quasithreshold dose) = Width of shoulder=semi-threshold dose (almost the threshold dose) n = extrapolation number Logen = Dq/D0 D0/0.37(e-1) = Dq/n D0/e-1 = Dq/n n= e-1xDq/D0 ln n = -Dq/D0 Threshold dose=the dose below which there is no effect.

  12. Linear –quadratic model Two components to cell killing by radiation One that is proportional to dose One that is proportional to square of dose (dual-radiation action; two separate breaks) S = e-aD-bD2

  13. S = e-aD-bD2 S = the fraction of surviving a dose D a,b = constant When aD = bD2 D = a/b Linear & quandratic contribution equally to cell killing at same dose

  14. The mechanism of cell killing Target : DNA (nucleus), nuclear membrane Chromosome, specifically the DNA, as the primary target for radiation-induced lethality.

  15. Apoptotic and mitotic death: AD (falling off) http://www-micro.msb.le.ac.uk/3035/kalmakoff/baculo/baculohostinteract.html

  16. http://www.niaaa.nih.gov/publications/arh25-3/image01.gif

  17. http://www.copewithcytokines.de/cope.cgi?000638

  18. http://www.ucihs.uci.edu/anatomy/histo/corenotes/celldeath2004.pdfhttp://www.ucihs.uci.edu/anatomy/histo/corenotes/celldeath2004.pdf

  19. The detection of this DNA ladder is still currently used to distinguish at the molecular level apoptosis from necrosis. • Apoptosis: Dsb in linker DNA between nucleosome 185bp [“ladder” in Gel] • nercosis [“smear” in Gel] Fig.3-9

  20. Radiation-induced apoptosis is highly cell type dependent. Hemopoietic cells & lymphoid cells  prone to rapid radiation-induced apoptosis. Most tumors  mitotic cell death & apoptosis. or mitotic cell death only.

  21. Apoptotic and mitotic death: MD • The most common form of cell death from radiation is mitotic death (MD).  Cell die in attempting to divide because of damaged chromosomes. http://www.nature.com/cgi-taf/DynaPage.taf?file=/onc/journal/v23/n16/abs/1207528a.html&dynoptions=doi1097246946

  22. Asymmetric exchange-type chromosome aberrations (i.e., dicentrics and rings ) • Loss reproductive integrity • Unable to proliferate • death Asymmetric exchange-type chromosome aberrations represent the principle mechanism for radiation-inducedmitotic death in mammalian cells.

  23. No apoptosis Cell surviving & cell without visible aberration  correlation

  24. Exchange type aberrations require two chromosome breaks. The probability of an interaction between the two breaks is related to D (low dose) or D2 (high dose).

  25. Chromosome aberrations in human lymphocytes Ch 2

  26. Survival curves for various mammalian cells in culture First in vitro survival curve All mammalian cells, normal & malignant, exhibit similar x-ray survival curve (initial shoulder but size vary) The Do of X-ray survival curves for most culture cells range from 1 to 2 Gy (100-200 rad or cGy). (page 41)

  27. radiosensitivity

  28. Survival-curve shape and mechanism of cell death Most cells fall between apopototic & mitotic death mitotic death Note! Shoulder

  29. Mitotic death results (principally) from exchange-type chromosomal aberration. • log-linear plot with broad shoulder • Characterized by subsequently dose-rate effect (page 74). • Apoptotic death result unknow mechanism. • straight line on log-linear plot. • Characterized by expotential function of dose. • little or no dose-rate effect.

  30. S = e-aD-bD2 Linear –quadratic model mitotic death apoptotic death S = e-(aM+aA)D-bMD2 aA = cell killing from apoptotic death (vary linear) aM = cell killing from mitotic death (vary linear) bM= cell killing from mitotic death (vary square)

  31. Oncogenes and radioresistance Transfection of activated oncogenes to culture cells  Increase radioresistance

  32. Genetic control of radiosensitivity ATM (AT-mutated) gene

  33. Intrinsic Radiosensitivity and predictive assay Courtenary assay Semisolid agar gel with growth factor Nonclonogenic assay Cell growth in multi-wells plate, e.g. MTT assay or chapter 15

  34. Surviving cell number is then determined indirectly by MTT dye reduction.(Fig23.4) The amount of MTT-formazan produced can be determined spectrophotometrically once and solublilized it in a suitable solvent. 補充

  35. The effective survival curve for a multifraction regimen multifraction regimen are most often used in clinical radiotherapy What is multifraction regimen? Dilute dose to fraction at time intervals sublethal damage & time for repair  Shoulder The effective survival curve

  36. For human, effective D0 (37% survival) = 300cGy = 3Gy D10 (the dose required to kill 90% = 10 % survival) = one decade of cell killing = 2.3 X D0 • Natural log 10 • = 2.3 • equal Slope Ps: equal Slope Logen = Dq/D0 (F3.3 & page 37)

  37. The Do of effecitive survival curve (slope) has a value close to 300cGy for cells of human origin. This is an avarage value and can differ significantly for different tumors.

  38. Calculations of tumor cell kill 109

  39. The radiosensitivity of mammalian cells compared with microorganisms

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