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| 研究生： |
鍾孟靜 Meng-Ching Chung |
|---|---|
| 論文名稱： |
使用野生型秀麗線蟲-動物模式探討PM2.5之不良效應 Adverse toxicity of PM2.5 in a wild-type Caenorhabditis elegans |
| 指導教授： |
黃國林
Huang, Kuo-lin |
| 學位類別： |
博士 Doctor |
| 系所名稱： |
工學院 - 環境工程與科學系所 Department of Environmental Science and Engineering |
| 畢業學年度： | 108 |
| 語文別： | 英文 |
| 論文頁數： | 65 |
| 中文關鍵詞： | 秀麗線蟲 、細懸浮微粒(PM2.5) 、交通排放之空氣污染物 、壽命分析 、運動行為評估 、生殖毒性試驗與老化現象 |
| 外文關鍵詞： | C. elegans, traffic related pollutant (TRP), lifespan, ageing |
| DOI URL： | http://doi.org/10.6346/NPUST202000161 |
| 相關次數： | 點閱：13 下載：0 |
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近幾年來細懸浮微粒與PM2.5污染問題日益嚴重，對於人體的毒性危害也越來越受到重視，目前研究已證實暴露於PM2.5與心血管疾病、中風、心臟病與高血壓有關。而現今科學家長期關心在探討PM2.5有害空氣污染物（hazardous air pollutants, HAPs）對於人體健康所帶來之危害。本研究為使用動物模型-野生型秀麗線蟲（Caenorhabditis elegans , C. elegans）探討暴露PM2.5後所造成之不良效應，採集地點選自屏東縣-國立屏東科技大學（NPUST）、麟洛國中（LJSH）與台中市（traffic-related-air-pollutant, TRP）的交通要道與鄉村地區，同時進行多種毒性試驗，如：致死率（存活率試驗與壽命分析）、運動行為評估（頭部擺動與身體彎曲）與生殖毒性試驗（繁殖後代數量），來觀察不同地區之PM2.5對於秀麗線蟲之影響。根據2個地區採集之PM2.5濃度顯示，屏東縣2所學校皆符合標準，反之台中市採集之PM2.5濃度超過世界衛生組織與行政院環境保護署的法規標準值。在秀麗線蟲暴露實驗中的第一項研究結果顯示，暴露NPUST與LJSH的PM2.5皆對於存活率沒有顯著影響，但相對在壽命分析、運動行為評估與生殖毒性試驗方面，NPSUT所造成之毒性高於LJSH，因此PM2.5暴露濃度高低與線蟲之不良效應有顯著關係，另外長時間暴露所造成之毒性影響比急性暴露更加嚴重，透過這次實驗表明，雖然採集PM2.5濃度較低，但長期下來然仍可能會對周遭環境生物造成毒影響。
實驗結果亦顯示，急性暴露後並未對秀麗線蟲造成顯著致死影響在生殖毒性試驗方面，雖然Day A與Day C並未減少線蟲產生後代數量，但透過統計發現線蟲對於PM2.5劑量依賴性有顯著降低趨勢。根據運動行為評估（神經系統）與壽命分析，實驗結果發現 PM2.5濃度越高，對於線蟲神經毒性（頭部擺動與身體彎曲）與壽命影響也越顯著。與生殖毒性試驗結果相比，特定實驗對線蟲歲造成的毒性影響，如壽命分析與運動行為評估（神經行為）會更加嚴重。
關鍵詞：秀麗線蟲、細懸浮微粒(PM2.5)、交通排放之空氣污染物、壽命分析、運動行為評估、生殖毒性試驗與老化現象。
Fine particulate or PM2.5 has raised the public concern due to its toxicity to human. PM2.5 pollution is linked into cardiovascular disease, stroke, heart attack, and hypertension in science. Many environmental scientists have focused on PM2.5-bounded hazardous air pollutants (HAPs) and their toxic effects correlated to human health. In the present study, an in-vivo wild-type Caenorhabditis elegans (C. elegans) was used to examine the induced toxicity after C. elegans was exposed to PM2.5. Many PM2.5 toxicity assessments using in vitro nematodes’ models have focused on PM2.5-bounded hazardous pollutants. The study design of the in-vivo models were addressed to PM2.5 toxicity in the rural and traffic areas in Pingtung and Taichung, respectively. Firstly, the toxic effects of outdoor PM2.5, collected from National Pingtung University of Science and Technology (NPUST) and Linluo Junior High School (LJHS), Pingtung, Taiwan, on nematode C. elegans were investigated. PM2.5 from NPUST and LJHS, which did not meet the standard. Secondly, the toxicity of traffic-related-pollutant (TRP) PM2.5 was evaluated in the animal model C. elegans using different toxicological endpoints such as lethality, survivability (lifespan or ageing), behavioral (head thrashing and body bending), and reproduction (brood size). Most TRP PM2.5 concentrations collected in Taichung were exceeded the standards of Taiwanese Environmental Protrction Adminstration and World Health Organzation guidline.
No significant PM2.5 lethality on C. elegans was observed for the NPUST and LJHS samples in acute toxicity tests. The PM2.5 from NPUST exhibited higher toxicity to lifespan, locomotion, and reproduction in the C. elegans animal models than that from LJHS; therefore, adverse effects could be correlated with PM2.5 concentrations. Prolonged exposure to PM2.5 led to more severe toxicity in nematodes as compared to acute exposure.Therefore the long-term adverse effects of ambient PM2.5 on environmental organisms should be carefully considered even when PM2.5 is at low levels.
No immediate lethality was observed after acute exposure of the nematodes. On the other hand, sublethal endpoints of reproduction exhibited statistically significant dose-dependent reduction, although Day A and Day C did not decrease the egg-laying capability of the worms. For the neurological toxicity, it is inferred that the higher the PM2.5 concentrations, the more the adverse effects of neurobehavior (head trashing and body bending) it poses on the C. elegans. The lifespans of nematodes exposed to heavily TRP PM2.5 were significantly shortened compared with those of untreated ones based on survival rate. The nematodes exposed PM2.5 models not only posed potentially adverse health effects on human but also represented ecotoxic impacts on the ecosystem. Accordingly heavy concentrations of TRP PM2.5 significantly and severely disrupted toxicological endpoints of neurology and reproduction to C. elegans. TRP PM2.5 significantly shortened the lifespan of the nematodes compared with the control. TRP PM2.5 might more severely influenced the specific toxic endpoints, such as lifespan and neurobehavira, in this in-vivo models compared with the reproductive endpoints. C. elegans animals model is a sensitive one for the evaluation of PM2.5 ecotoxicity.
Keywords: C. elegans; PM2.5; traffic related pollutant (TRP); lifespan; locomotion; reproduction; ageing.
Content
中文摘要 I
Abstract III
Acknowledgments VI
Content VII
Figure List IX
Table List X
Chapter 1 Introduction 1
Chapter 2 Literature Review 3
2.1 PM2.5 and its sources 3
2.2 PM2.5 and its health adverse effects 4
2.3 C. elegans models 6
2.4. The toxicity of PM2.5 in C. elegans models 10
Chapter 3 Materials and Methods 11
3.1 Chemical Reagents 11
3.2 Sample collection 12
3.2.1 Traffic pollution sources in schools in southern Taiwan : 13
3.2.2 Sources of traffic and road pollution in the Taiwan Tunghai : 14
3.3 Extraction, purification, and sample preparation 15
3.4 C. elegans age synchronization for exposure experiments 16
3.5 Acute Exposure 18
3.6 Lethality Assay 18
3.7 Lifespan Assay (Ageing Assay) 18
3.8 Reproductive or Brood Size Assay 19
3.9 Growth Measurement Assay 19
3.10 Locomotion Assay (Head Thrash and Body Bend Assay) 20
3.11 Statistical analysis 20
Chapter 4 Results and discussion 21
4.1 Investigation of PM2.5 in a wild-type C. elegans model in the rural areas 21
4.1.1 Lethality effects of PM2.5 on C. elegans 21
4.2 Lifespan of C. elegans after PM2.5 exposure 23
4.3 Growth measurement assay 26
4.4 Effect of outdoor air PM2.5 levels on the reproductive system of C. elegans 28
4.5 Effects of ambient PM2.5 on locomotion behavior of C. elegans 31
4.2 Investigation of TRP PM2.5 of C. elegans in the heavily traffic areas in the spring season 34
4.1 Air pollution in the highly heavy traffic area 34
4.2 Lethal Effects of PM2.5 to C. elegans 37
4.3 Effects of TRP PM2.5 on the Brood size of C. elegans 39
4.4 Effects of TRP PM2.5 on the Locomotion behavior of C. elegans 42
4.5 Effects of TRP PM2.5 on the Life Span of C. elegans 45
Chapter 5 Conclusions and Suggestion 50
5.1 Conclusions 50
5.1.1 Low levels of PM2.5 in the rural areas 50
5.1.2 The high levels of PM2.5 in the traffic areas 51
5.2 Limitations and suggestions 51
5.2.1 Limitations 51
5.2.2 Suggestions 52
Reference 53
附錄 62
作者簡介 62
Figure List
Fig. 2.1 The temperature effects on molting (A) and the life cycle of C. elegans（B）. (Gonzalez-Moragas et al. 2015) 9
Fig. 3.1 The PM2.5 air sampling device of SIBATA HV-1000R 12
Fig. 3.2 (a) The sampling locations in Pingtung. NPUST Latitude and longitude：（22°38'49.8"N 120°36'32.6"E）and LJSH Latitude and longitude：（22°38'29.1"N 120°32'11.2"E） 13
Fig. 3.2 (b) The sampling locations in Taichung. THU Latitude and longitude：（24.18182, 120.59595） 14
Fig. 3.3 The PM2.5 air Extraction device of Soxhlet extractor 15
Fig. 3.4 The incubation procedure of C. elegans 16
Fig. 3.5 The flow chart of examining procedure in C. elegans models 17
Fig. 4.1 Dose-dependent lethality of PM2.5 from (Left) NPUST and LJHS after 24-hour exposure. 23
Fig. 4.2 Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans lifespan after 24-hour exposure. 26
Fig. 4.3 Effects of PM2.5 from (Left) NPUST and LJHS on C. elegans growth, observed as changes in body length, after 24-hour exposure. 28
Fig. 4.4 Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans brood size, observed (A and C) two days and (B and D) four days after 24-hour acute exposure. 31
Fig. 4.5 Effects of PM2.5 from NPUST and (Bottom) LJHS on C. elegans locomotion and movement after 24-hour acute exposure. A and C for head thrashing and B and D for body bending. 34
Fig. 4.8 Survival rates of C. elegans after 24 h exposure to heavily TRP PM2.5 obtained by selected three days (Days A (a), B (b), and C (c)). 39
Fig. 4.9 Reproductive effects of brood sizes in the C. elegans models after 24-hr exposure to heavily TRP PM2.5 obtained by selected three days (Days A (a), B (b), and C (c)). 42
Fig. 4.10 Dose-dependent reductions of locomotion (head thrashing (1) and body bending (2)) in the C. elegans models after 24-hr exposure to heavily TRP PM2.5 obtained in selected three days (Days A (a1 and a2), B (b1 and b2), and C (c1 and c2)). 45
Fig. 4.16. Survival rates (%) (a), mean lifespans (b), 50th percentile death days (c), 75th percentile death days (d), and 95th percentile death days (e) of C. elegans after 24-hr exposure to heavily TRP PM2.5 obtained by selected three days (a mixture of Days A, B,and C). 50
Table List
Table. 2.1 Summary of the advantages and disadvantages of C. elegans as a tool for in vivo evaluation of nanotoxicity. (Gonzalez-Moragas et al. 2015) 9
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校外公開2025/07/05