熱休克蛋白的分子機制 : 熱休克蛋白於磁性奈米顆粒引發之熱療的影響

The molecular mechanism of heat shock protein : its impact on magnetic nanoparticles-induced hyperthermia therapy

林弘翊(Hung-I Lin)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02643

摘要

磁性氫氧基磷灰石是一種生物相容性良好的材料，利用共沉法可順利合成磁性氫氧基磷灰石，且不破壞氫氧基磷灰石的結構，磁性氫氧基磷灰石具備超順磁的特性，於高周波加熱器作用下，可快速升溫至熱療所需溫度 ( 42 ~ 47℃ ) ，非常適合當作熱療之磁性材料。
細胞與磁性氫氧基磷灰石共養後，會胞吞磁性氫氧基磷灰石，利用高周波加熱器使磁性氫氧基磷灰石於細胞內發熱，達到熱療之效果。人類纖維母細胞 ( human fibroblast, HF ) 以磁性氫氧基磷灰石進行熱療之處理 ( 43℃ 持續 30 分鐘 ) ，熱休克蛋白-70 ( heat shock protein 70, HSP70 ) 的表現量在 24 小時內會持續穩定上升，保護及修復 HF 因熱處理所受到的傷害；以相同熱療條件處理肺癌細胞 ( A-549 ) 與骨癌細胞 ( MG-63 ) ， A-549 在熱處理後 6 小時開始表現熱休克蛋白-27 ( heat shock protein 27, HSP27 ) ，然而 HSP27 的表現量在熱處理後 12 與 24 小時逐漸減少，無法有效的彌平熱所造成 A-549 的傷害； MG-63 在熱處理後 0 小時表現 HSP70 與熱休克蛋白-90 ( heat shock protein 90, HSP90 )，立即保護 MG-63 因熱所造成的損傷，但在熱處理後 6 小時、 12 小時與 24 小時， HSP70 與 HSP90 並無明顯表現，無法持續的修復 MG-63 的損傷，導致 MG-63 在熱處理後 12 小時其生長停滯。
細胞死亡分為凋亡與壞死，利用膜外翻、 DNA 斷裂與 procaspase-3 活化的方式，可分辨細胞走向凋亡亦或壞死。 HF 在熱處理後因為 HSP70 表現量的穩定上升，在 24 小時其凋亡細胞佔 8.3% ± 0.85% ，壞死細胞佔 5.1% ± 0.57% ， A-549 因 HSP27 的表現量在 24 小時逐漸減少，在 24 小時其凋亡細胞佔 57.96% ± 5.09% ，壞死細胞佔 26.86% ± 1.38% ， MG-63 雖因熱能立即表現 HSP70 與 HSP90 ，但在 24 小時 HSP 並無明顯表現，在 24 小時凋亡細胞佔 60.35% ± 5.85% ，壞死細胞佔 4.2% ±0.85% 。在熱處理後 6 小時， HF 、 A-549 與 MG-63 皆觀測到 DNA 斷裂的情形。 HF 因 HSP70 的表現量穩定上升，故只在熱處理後 0 小時觀測到 procaspase-3 被活化， A-549 因 HSP27 在熱處理後 6 小時才逐漸被表現，其 procaspase-3 在熱處理後 0 小時明顯被活化， MG-63 因 HSP70 與 HSP90 迅速被熱所誘導表現，所以在熱處理後 0 小時並無 procaspase-3 被活化，然而 HSP70 與 HSP90 無法持續表現，故在熱處理後 6 與 12 小時可觀察到 procaspase-3 被活化。

關鍵字

磁性氫氧基磷灰石 ； 熱療 ； 熱休克蛋白 ； 膜外翻 ； DNA 斷裂 ； procaspase-3 活化

並列摘要

Magnetic hydroxyapatite ( mHAP ) is a material with good biocompatibility. By using the co-precipitation method, mHAP can be easily synthesized and kept the structure of hydroxyapatite ( HAP ). mHAP is super-paramagnetic and can be rapidly raised to the temperature of hyperthermia ( 42 ~ 47℃ ) by using the high frequency inductive heat generator. Therefore mHAP is very suitable to magnetic material for hyperthermia.
When cells cultured with mHAP, the cells would engulf some mHAP by endocytosis process. By using the high frequency inductive heat generator, mHAP generated heat in the cells to make the effects of hyperthermia. Treating the human fibroblast ( HF ) with the mHAP-induced hyperthermia ( 43℃ for 30 minutes ), the expression of heat shock protein 70 ( HSP70 ) stably increased in 24 hours in order to protect and repair the damages caused by hyperthermia. The same hyperthermia condition was treated to lung adenocarcinoma cells ( A-549 ) and osteosarcoma cells ( MG-63 ), the A-549 cells expressed heat shock protein 27 ( HSP27 ) at 6-hour after the heat treatment but the expression of HSP27 was gradually decreased at 12-hours and 24-hours. Therefore the damages of A-549 cells caused by hyperthermia could not be effectively repaired. MG-63 cells immediately expressed HSP70 and heat shock protein 90 ( HSP90 ) at 0-hour after hyperthermia in order to protect the cells from the heat damages. However, there was no obvious expression of HSP70 and HSP90 at 6-hours, 12-hours and 24-hours after hyperthermia. Therefore the repairs could not be sustained, which caused the growth arrest of MG-63 cells at 12-hours after hyperthermia.
Cell death can be classified as apoptosis and necrosis. By using the cell membrane externalization, DNA fragmentation and procaspase-3 activation, we could distinguish the cell death was through apoptosis or necrosis. After hyperthermia, the apoptotic cells of HF at 24-hours were 8.3% ± 0.85% and the necrotic cells were 5.1% ± 0.57% due to the stably increased HSP70. Because the expression of HSP27 gradually decreased at 24-hours, the apoptotic cells of A-549 were 57.96% ± 5.09% and the necrotic cells were 26.86% ± 1.38%. MG-63 cells expressed HSP70 and HSP90 immediately but no obvious expressions at 24-hours after hyperthermia. Therefore the apoptotic cells at 24-hours were 60.35% ± 5.85% and the necrotic cells were 4.2% ±0.85%. 6 hours after hyperthermia, we could observe that the DNA of HF cells, A-549 cells and MG-63 cells was degraded into fragments. The procaspase-3 of HF cells did not be activated at 6-hours and 24-hours after hyperthermia because HSP70 was stably increased and cells were protected from damages. Because the HSP27 of A-549 cells was slowly and gradually expressed at 6-hours after hyperthermia, the procaspase-3 was obviously activated at 0-hour and 6-hours. On the other hand, the HSP70 and HSP90 of MG-63 cells were rapidly induced by hyperthermia so the procaspase-3 was not activated at 0-hour after hyperthermia. But the HSP70 and HSP90 could not be expressed consecutively, and hence the procaspase-3 was activated at 6-hours and 12-hours after hyperthermia.

並列關鍵字

magnetic hydroxyapatite ( mHAP ) ； hyperthermia ； heat shock protein ( HSP ) ； cell membrane externalization ； DNA fragmentation ； procaspase-3 activation

參考文獻

[2] Wolfgang Arthur Schulz "Molecular biology of human cancers : an advanced student's textbook" Springer, 2005

[3] Armstrong, P., J. Congleton, et al. "Guidelines on the selection of patients with lung cancer for surgery." Thorax 56(2): 89-108, 2001

[4] Alexakis, N., C. Halloran, et al. "Current standards of surgery for pancreatic cancer." British Journal of Surgery 91(11): 1410-1427, 2004

[5] Kurtz, J. "Oral chemotherapy in colorectal cancer treatment: review of the literature." European Journal of Internal Medicine 14(1): 18-25, 2003

[6] Han, E., P. Lin, et al. "Current Status on Biologic Therapies in the Treatment of Epithelial Ovarian Cancer." Current treatment options in oncology 10(1): 54-66, 2009