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研究生:吳博貴
研究生(外文):Po-kuei Wu
論文名稱:年輕代數間葉幹細胞藉由表現較高之PARP-1值來增加放射線抵抗性
論文名稱(外文):Early-passage MSCs increase irradiation-resistance through increased level of PARP-1
指導教授:陳威明陳威明引用關係洪士杰
指導教授(外文):Wei-Ming ChenShih-Chieh Hung
學位類別:博士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:英文
論文頁數:69
中文關鍵詞:間葉幹細胞脱氧核糖核酸損傷反應脱氧核糖核酸雙股斷裂放射線
外文關鍵詞:mesenchymal stem cellsDNA damage responsesDNA double strand breaksIrradiation
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骨髓間葉幹細胞爲多功能幹細胞,能分化為不同的細胞。因其『自我更新』與『分化』能力,在臨床上具有可應用於組織修補與再生的潛力;但目前幹細胞的臨床治療仍有所受限,從年齡較大病患取出之骨髓間葉幹細胞或年輕之骨髓間葉幹細胞經多代培養後,會有『老化』之現象。一旦老化後,基因與蛋白的表現絕對受到影響,其『自我更新』與『分化』能力也會下降。然而,骨髓間葉幹細胞運用於臨床之治療時,基因穩定性與一致性相當重要,需有嚴格的控制以確保治療的安全與有效。一旦基因發生突變,輕則影響治療的效力,重則造成癌化之可能。因此,了解骨髓間葉幹細胞老化所造成基因與蛋白表現不同的機制尤為重要。
本研究探討老化後之人類間葉幹細胞,其脫氧核醣核酸受傷後修復(DNA damage responses, DDR)之變化。在接受過放射線照射後,較老代數之人類間葉幹細胞的存活率與殖生能力均相對年輕代數人類間葉幹細胞明顯下降。在流式細胞儀實驗中,可以明顯的觀察到在接受過放射線照射72小時後,較老代數之人類間葉幹細胞之亞二倍體峰 (Sub-G1 phase)比例明顯上升。同時在TUNEL染色與慧星分析法(Comet assay Kit)中,亦可以同時發現年輕代數間葉幹細胞具有較高的抗放射線之能力。此外,年輕與較老代數之人類間葉幹細胞之多種與脫氧核醣核酸受傷後修復之蛋白,如phospho-ATM、-H2AX、RNF8 與 phospho-p53等,會在接受放射線照射後的1至12小時短時間內迅速上升,但是較老代數之間葉幹細胞之蛋白增加量明顯低於年輕代數之間葉幹細胞。
再藉由加入小分子特異性ATM激脢抑製劑KU55933,可以得知此不同蛋白表現之現象,為ATM激脢依賴性。依此,我們接者觀察ATM之上游蛋白,多聚腺甘二磷酸核糖聚合脢(poly (ADP-ribose) polymerase-1, PARP-1)在一般的環境之下,年輕代數之之表現量遠大於較老代數之人類間葉幹細胞。將年輕代數人類間葉幹細胞給予PARP-1基因剔除後,可以發現其脫氧核醣核酸受傷後修復能力下降。相反的,將較老代數人類間葉幹細胞給予PARP-1基因回補後,更可以發現其脫氧核醣核酸受傷後修復能力提升。最後,本研究發現較老代數之人類間葉幹細胞其多聚腺甘二磷酸核糖聚合脢會迅速的被蛋白脢所分解,進而導致其對抗放射線之能力較差。
藉此研究的發現,我們可以得知較老代數之人類間葉幹細胞,其多聚腺甘二磷酸核糖聚合脢表現量少,進而影響到自我修復之能力。未來在臨床幹細胞之應用上,可藉此恢復培養代數較久之幹細胞脫氧核醣核酸的修補機制,維持其增殖與分化的能力,以突破幹細胞之臨床應用之限制。
Human mesenchymal stem cells (MSCs) have received much attention because of their use in cell therapies, where cell expansion is required to generate a great number of cells. Long-term expansion in vitro, however, can lead to altered functions. To explore the changes in DNA damage responses (DDR) in expanded MSCs, DDR pathways following irradiation were characterized in early- and late-passage bone marrow MSCs. Following irradiation up to 72 h, the percentage of sub-G1 cells in early-passage MSCs remained unchanged, while late-passage MSCs increased in the sub-G1 phase. Reduced TUNEL staining was observed in early-passage MSCs compared to late-passage MSCs at 4 h post-irradiation. Comet assay also revealed that early-passage MSCs were more resistant to irradiation or other genotoxic agents induced DNA damages when compared to late-passage MSCs. ATM phosphorylation and increased levels of -H2AX and phospho-p53 were evident at 1 h, peaked at 2-12 h post-irradiation in early-passage MSCs, while reduction of these changes were observed in late-passage MSCs. Through inhibition by KU55933, we found the DDR pathway in early-passage MSCs was ATM-dependent. The levels of poly (ADP-ribose) polymerase-1 (PARP-1) and PAR synthesis were greater in early-passage MSCs than late-passage MSCs. Knockdown of PARP-1 in early-passage MSCs resulted in sensitization to irradiation-induced apoptosis, while overexpression of PARP-1 in late passage MSCs rendered irradiation resistance. Lower activity of DDR in late-passage MSCs was associated with rapid proteasomal degradation of PARP-1. In conclusion, early-passage MSCs are more irradiation-resistant and with increased DDR activity involving PARP-1, ATM and their downstream signals.
Contents
Contents……………………………………………………………………………………….. 1
English Abstract……………………………………………………………………………. 5
Chinese Abstract ….......……………………………………………………………… 6
List of Abbreviations……………………………………………………………………... 8
Introduction…………………………………………………………………………………... 9
I. Mesenchymal stem cells
II. The limitation of clinical applications of mesenchymal stem cells
III. DNA damage response process of mesenchymal stem cells
IV. Research motivation
Materials and Methods………………………………………………………………… 12
Results........................................................................................... 18
Discussion...................................................................................... 25
Conclusion..................................................................................... 29
Perspectives….....…………………………………………………………………………..31
Title: Reconstructor for Biological Recycle Autograft Bone Defect: Combined PPF
Scaffold and PARP-1-riched Mesenchymal Stem Cells
Background
Hypothesis
Study design
References………………………………………………………………………………….. 36
Figures and Tables…………………………………………………………………….... 42
Figure 1. Cell morphology of early- and late-passage MSCs after γ-irradiation.
Figure 2. Quantification of early- and late-passage MSCs un-treated and treated with γ-irradiation.
Figure 3. Quantification of the colony numbers for early- and late-passage MSCs un-treated and treated with γ-irradiation.
Figure 4. Quantification of the colony numbers for another two individual MSCs un-treated and treated with γ-irradiation.
Figure 5. Early-passage MSCs are more resistant to irradiation-induced apoptosis than late-passage MSCs.
Figure 6. TUNEL staining for analyzing apoptotic cells after γ-irradiation.
Figure 7. Early-passage MSCs are more resistant to γ-irradiation- and genotoxic agents-induced DNA damage than late-passage MSCs.
Figure 8. Early-passage MSCs isolated from another two individuals have greater DNA double strand break repair than late-passage MSCs.
Figure 9. Western blot of early- and late-passage MSCs were subjected to un-treated and treated with γ-irradiation.
Figure 10.Immune-fluorescence of early- and late-passage MSCs before or 2 h after irradiation.
Figure 11.Western blot analysis of early- and late-passage MSCs before or 2 h after irradiation.
Figure 12. Early-passage MSCs isolated from another two individuals increase in DNA damage responses.
Figure 13. Increased DNA double strand break repair in early-passage MSCs is ATM-dependent
Figure 14. Colony formation assay of early- and late-passage MSCs without or with 2-h KU55933 pretreatment.
Figure 15. DNA damage in olive tail moment of early- and late-passage MSCs without or with 2-h KU55933 pretreatment.
Figure 16. Increased PARP-1 expression in early-passage MSCs before or after irradiation revealed by western blot.
Figure 17. Western blot of MSCs lentivirally transduced with control or PARP-1 specific shRNAs without or with irradiation.
Figure 18. Cell number counting of MSCs lentivirally transduced with control or PARP-1 specific shRNAs after irradiation.
Figure 19. Colony formation assay of MSCs lentivirally transduced with control or PARP-1 specific shRNAs after irradiation.
Figure 20. DNA damage in olive tail moment of MSCs lentivirally transduced with control or PARP-1 specific shRNAs without or with irradiation.
Figure 21. PARP-1 is rapidly degraded in late-passage MSCs. Quantitative RT-PCR for analyzing the PARP-1 mRNA levels in early- and late- passage MSCs.
Figure 22. Western blots analysis of early- and late-passage MSCs were treated without or with MG132 in the presence of cycloheximide.
Figure 23. Western blot analysis of late-passage MSCs lentivirally transduced with control or PARP-1 without or with irradiation.
Figure 24. Colony formation assay of late-passage MSCs lentivirally transduced with control or PARP-1 without or with irradiation.
Figure 25. Measurement and quantification of DNA damage in olive tail moment of late-passage MSCs lentivirally transduced with control or PARP-1 without or with irradiation.
Table 1. Information on donor profile
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