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研究生:陳彥樺
研究生(外文):CHEN, YAN-HUA
論文名稱:CNPs-AC、CNPs-Spd及CNPs-AC/Spd奈米碳材料之腎臟/肝臟毒性評估
論文名稱(外文):Renal / Hepato-toxicity evaluations of CNPs-AC、CNPs-Spd and CNPs-AC/Spd nanocarbon materials.
指導教授:林家驊
指導教授(外文):LIN, CHIA-HUA
口試委員:林坤儀彭彥彬張耿崚
口試委員(外文):LIN, KUN-YIPENG, YAN-BINCHANG, KENG-LING
口試日期:2020-07-22
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:生物科技系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:77
中文關鍵詞:奈米碳材料細胞毒性發炎因子慢性腎臟病α1-抗胰蛋白酶缺乏症
外文關鍵詞:carbon nanomaterialscytotoxicityinflammatory factorschronic kidney diseaseα1-antitrypsin deficiency
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本研究利用檸檬酸二銨(diammonium citrate;AC)及亞精三鹽酸鹽(spermidine trihydrochloride;Spd)製備三種已知具抗菌功效之奈米碳材料(Cabon nanoparticles;CNPs ),標示為CNPs-AC、CNPs-Spd和CNPs-AC/Spd,並針對肝臟、腎臟進行生物安全評估。依據FE-TEM的結果,材料中包含了較大的碳奈米顆粒及碳量子點。FTIR的分析顯示CNPs-AC/Spd包含了AC、Spd的特徵峰結構。動物實驗部分,以2g/kg的劑量對Balb/c小鼠進行口服試驗,其中CNPs-Spd組別在三小時內接續死亡,經病理判讀證實腺胃粘膜上皮細胞質壞死(necrosis)是死亡主要原因。不過針對腎、肝部位的組織病理切片的結果,則並未觀察到CNPs-AC、CNPs-Spd及CNPs-AC/Spd引起顯著的危害。體外細胞實驗部分,利用HEK-293細胞作為罹患慢性腎臟病(Chronic kidney disease;CKD)風險的評估。CCK-8細胞存活率、活性氧化物(Reactive oxygen species;ROS)以及HO-1的檢測結果顯示,暴露於CNPs-Spd(100μg/ml)及CNPs-AC/Spd(100μg/ml)24小時後,與控制組呈顯著差異(細胞存活率僅剩70-80%,ROS上調2-4倍、HO-1上調3-5倍)。後續測試發炎因子(IL-8),NLRP3發炎小體,結果並未顯著受到上調。因此CNP-Spd及CNP-AC/Spd儘管有造成細胞損傷的可能,我們仍推測材料短期刺激下罹患CKD的風險可能是較低的。肝臟部分,利用Hep-G2細胞作為罹患α1-抗胰蛋白酶缺乏症(α1- Antitrypsin Deficiency;AATD) 風險的評估。在CCK-8細胞存活率、ROS以及HO-1的檢測下,暴露於CNPs-Spd(100μg/ml)24小時後的細胞與控制組對照呈顯著差異(細胞存活率僅剩80%、HO-1顯著上調約4倍),後續進一步評估AATD的損傷路徑,CNPs-Spd(100μg/ml)在暴露 24小時後,引起IL-8、NF-κB顯著上調(高於正常表達約1.75倍),並明顯抑制了AAT的表達(僅剩正常水平30%),基本符合我們認為的損傷路徑,推測引起AATD的風險增加。整體而言,在動物實驗中並未觀察到材料對於肝、腎明顯的異狀,後續細胞實驗發現,CNPs-Spd引起顯著細胞損傷,CNPs-AC/Spd次之,CNPs-AC則相對無影響。對於在測試中沒有明顯差異的組別(AC,AC/Spd),在長期暴露下不排除仍有損傷風險,而短期內,造成細胞損傷的CNPs-Spd則更應關注長期的暴露其潛在的危害。
In this study, diammonium citrate (AC) and spermidine trihydrochloride (Spd) were used to prepare three known antibacterial nanocarbon materials, labeled CNPs-AC, CNPs-Spd, and CNPs-AC /Spd. According to the results of FE-TEM, all three materials contain larger carbon nanoparticles and carbon quantum dots. FTIR analysis shows that CNPs-AC contains nitrogen,CNPs-Spd contains nitrogen,chlorine characteristic peaks and CNPs-AC/Spd observed the peak of both. In the animal experiment, Balb/c mice were orally tested at a dose of 2g/kg. The CNPs-Spd group die within three hours. that caused cytoplasmic vacuolation, pyknosis and necrosis of the mucosal epithelium in the glandular stomach. However, for the results of histopathological sections of the kidney and liver. CNPs-AC, CNPs-Spd, and CNPs-AC/Spd caused no significant damage. It is speculated that the short-term exposure of the material may cause a limited risk of kidney and liver damage. In vitro cell experiments, using HEK-293 cells as an assessment of the risk of chronic kidney disease (CKD). The results of CCK-8 cell survival rate, reactive oxides and HO-1 showed that after exposure to CNPs-Spd (100μg/ml) and CNPs-AC/Spd (100μg/ml) for 24 hours, the control group There was a significant difference (cell survival rate was only 70-80%, ROS was increased 2-4 times, HO-1 was increased 3-5 times). Subsequent testing of the inflammatory factor (IL-8), NLRP3 inflammasome, did not significantly increase the results. Therefore, although CNP-Spd and CNP-AC/Spd may cause cell damage, we still speculate that the risk of chronic kidney disease under short-term exposure may be lower. In the liver part, Hep-G2 cells were used as an assessment of the risk of developing α1-antitrypsin deficiency (AATD). Under the detection of CCK-8 cell survival rate, reactive oxides (ROS) and HO-1 protein, the cells exposed to CNPs-Spd (100 μg/ml) for 24 hours showed a significant difference from the control group (the cell survival rate was only 80% left, HO-1 was significantly increased by about 4 times), follow-up further evaluation of the damage path of AAT deficiency, CNPs-Spd (100μg/ml) caused a significant increase in IL-8 and NF-κB after 24 hours of exposure (high It is approximately 1.75 times normal expression), and significantly inhibits the expression of AAT (only 30% of normal level is left), which basically conforms to the damage path we think, and speculates that the risk of α1-antitrypsin deficiency (AATD) is increased. Overall, in animal experiments, we did not observe obvious abnormalities of the material for liver and kidney. Subsequent cell experiments found that CNPs-Spd caused significant cell damage, followed by CNPs-AC/Spd, and CNPs-AC was relatively absent. influences. For groups with no obvious difference in the test (AC, AC/Spd), there is still no risk of damage under long-term exposure, and in the short-term, CNPs-Spd, which may cause cell damage, should pay more attention to the potential of long-term exposure.
摘要………i
Abstrate………ii
誌謝………iv
目錄………v
表目錄………viii
圖目錄………ix
縮寫對照表………xi
第一章 緒論………1
1-1研究緣起………1
1-2研究目的………1
第二章 文獻回顧………2
2-1奈米結構材料(Nanostructured materials)………2
2-1-1奈米複合材料(nano-composite materials)………2
2-1-2奈米聚合系統(Nanostructured assembling system)………2
2-2奈米碳材料(carbon nanoparticles, CNPs)簡介………3
2-2-1奈米碳材料-碳量子點(Carbon Quantum Dots;CQDs)………4
2-3奈米碳材料的生物應用性………5
2-3-1 抗菌………5
2-3-2生物成像………5
2-3-3 藥物載體………6
2-3-4 癌症治療………6
2-3-5 生物傳感器 ………6
2-4奈米碳材料生物毒性研究的評估………7
2-4-1生體可用率(bioavailability)………7
2-4-2藥物動力學及毒性期評估………7
2-5以生理為基礎之藥物動力學(Physiologically Based Pharmacokinetic;PBPK)………8
2-6腎臟生理模式(Kidney Physiological Model)………8
2-6-1慢性腎臟病(Chronic kidney disease;CKD)………9
2-6-2慢性腎臟疾病潛在風險………10
2-6-3 NLRP3炎性體與慢性腎臟病(CKD)之間的關聯………10
2-7肝臟生理模式(Liver Physiological Model)………11
2-7-1 alpha-1 antitrypsin (AAT)………11
2-7-2 AAT與抗胰蛋白酶缺乏症之間的關聯………12
2-7-3 ER overload response (EOR)………12
2-7-4 α1-抗胰蛋白酶缺乏症(AATD)遺傳及流行病學………13
2-8奈米碳材料細胞毒性安全評估………14
2-8-1自由基及氧化壓力的危害………14
2-8-2發炎因子(Inflammatory factor)………15
2-8-3緊密連接蛋白(tight junction protein)………16
2-8-4血鐵質氧化酶( heme oxygenase, HO)………16
2-8-5 NLRP3炎性體………16
2-8-6 甲型抗胰蛋白酵素(α1-Antitrypsin ; AAT)………17
第三章 材料方法………18
3-1 實驗藥品………18
3-2 實驗儀器………20
3-3實驗架構………21
3-4 抗菌奈米碳材料的製備及分析………22
3-4-1抗菌奈米碳材料的製備………22
3-4-2場發射式穿透顯微鏡(FE-TEM)………22
3-4-3動態光散射粒徑分析儀(DLS)………22
3-4-4介面電位分析儀(Zeta potential)………23
3-4-5傅立葉紅外光譜(FTIR)………23
3-5細胞株種類………23
3-6細胞培養/繼代………24
3-7細胞存活率試驗 ………24
3-8活性氧化壓力試驗………25
3-9酵素連結免疫吸附分析法(ELISA)………25
3-9-1發炎因子IL-6 測定………25
3-9-2發炎因子IL-8測定………25
3-10西方墨點法(Western blot)………26
3-11抗菌奈米碳材料口服急毒性試驗………27
3-12統計分析………27
第四章 結果與討論………28
4-1 CNPs-AC、CNPs-Spd、CNPs-AC/Spd之物化分析………28
4-1-1場發射式電子穿透顯微鏡(FE-TEM)之粒徑分析………28
4-1-2 X-射線繞射(XRD)之材料晶體結構分析………29
4-1-3奈米粒徑及介面電位測量(DLS & zeta potential)………30
4-1-4傅里葉轉換紅外光譜(FTIR)之定性分析………31
4-2動物口服急毒性試驗………32
4-2-1死亡率………32
4-2-2血液生化分析………33
4-2-3組織病理學評估………35
4-3體外細胞實驗(HEK-293)………38
4-3-1細胞存活率(CCK-8試驗)………38
4-3-2氧化壓力測試-活性氧化物(ROS試驗)………39
4-3-3氧化壓力測試-血紅素氧化酶(HO-1)………40
4-3-4發炎因子/趨化因子對細胞的生理意義(IL-6,IL-8)………41
4-3-5細胞皮膜屏障-緊密連接蛋白(ZO-2)………43
4-3-6 NLRP3發炎小體………44
4-4體外細胞實驗(Hep-G2)………45
4-4-1細胞存活率(CCK-8試驗)………45
4-4-2氧化壓力測試-活性氧化物(ROS試驗)………46
4-4-3氧化壓力測試-血紅素氧化酶(HO-1)………47
4-4-4發炎因子/趨化因子對細胞的生理意義(IL-6,IL-8)………48
4-4-5發炎反應檢測(NF-κB)………50
4-4-6細胞皮膜屏障-緊密連接蛋白(ZO-2)………51
4-4-7 α1-抗胰蛋白酶 (AAT)………52
第五章 結論………54
第六章 建議………55
參考文獻 ………56
Extended Abstract ……………………………………………………………………71



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