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研究生:夏安良
研究生(外文):Sabuna-Alan Charis
論文名稱:第二代和第三代樹狀高分子PAMAM對小鼠胚胎發育的影響
論文名稱(外文):Effects of PAMAM Dendrimer Generation 2 and 3 on ear1y mouse embryonic development
指導教授:詹文雄詹文雄引用關係
指導教授(外文):Wen-Hsiung Chan
學位類別:碩士
校院名稱:中原大學
系所名稱:生物科技研究所
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:2014
畢業學年度:102
語文別:英文
論文頁數:72
中文關鍵詞:樹枝狀高分子PAMAM、小鼠囊胚期胚胎、胚胎毒性、細胞存活率、凋亡
外文關鍵詞:embryotoxicitycell viabilitymouse blastocystsPoly(amidoamine) dendrimersapoptosis.
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摘要

  早期的研究指出,樹狀高分子PAMAM可以運送藥物或基因進入哺乳動物組織系統,然而在近幾年的研究下,發現樹狀高分子PAMAM會導致動物組織細胞受損,但樹枝狀高分子PAMAM對胚胎發育的影響則尚無相關研究資料。因此本研究主要為探討第二代和第三代樹枝狀高分子PAMAM對小鼠囊胚期胚胎的影響,分析樹枝狀高分子PAMAM之胚胎的毒性,探討其對胚胎型態、凋亡、增生、體外發育、氧化壓力變化及粒線體膜電位改變之影響。結果顯示出,樹枝狀高分子PAMAM所產生之毒性與表面所帶有之胺基數具正相關性(第二代 < 第三代);第二代樹枝狀高分子PAMAM與小鼠囊胚共培養下顯示出較低毒性,研究也發現第二代樹枝狀高分子PAMAM並不會改變小鼠囊胚細胞內氧化壓力與造成粒線體膜電位的改變。因此本研究明確指出第二代樹枝狀高分子PAMAM對於小鼠囊胚期胚胎具有相當低的毒性。


ABSTRACT

PAMAM dendrimer has been recently shown can deliver drug or gene into the targeted mammalian tissues. However, certain studies revealed that PAMAM dendrimers impaired mammalian cells. Moreover, in terms of teratogenicity study little information focused on the cytotoxicological study of PAMAM dendrimers on mammalian embryos. Thus, this study is conducted to examine the influence of generation 2 (G2) and generation 3 (G3) of PAMAM dendrimer on mouse blastocyst. To investigate the influence of PAMAM-induced cytotoxicity, cell morphology, apoptosis, proliferation, in vitro development, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) change were examined. The results indicate that a generation dependent cytotoxicity of the PAMAM dendrimers was found to be correlated well with the number of amine group at surface area (G2<G3). Nevertheless, PAMAM dendrimer G2 showed only minor toxicity with low ability to induce apoptosis, low suppress on cell proliferation and small effect on in vitro development. Interestingly, the intracellular ROS level and mitochondrial membrane potential on mouse blastocyst was not altered by PAMAM dendrimer G2. This study provided an important insight that PAMAM dendrimer G2 has low cytotoxicity at the cellular level.


Contents

摘要 I
ABSTRACT II
Acknowledgments III
Contents V
Figure List VIII
Table List IX
Chapter I. Introduction 1
1.1 Background 1
1.2 Dendrimer 2
1.3 PAMAM dendrimers 4
1.4 PAMAM dendrimers in drug delivery and cytotoxicity 6
1.5 Mouse embryonic development 8
1.6 Apoptosis 10
Chapter II. Materials and Methods 18
2.1 Materials 18
2.2 Methods 20
a. Preparation of PAMAM dendrimer G2 and G3 solution 20
b. Collection of mouse blastocyst 20
c. Apoptosis detection 21
d. Cell proliferation determination 21
e. Morphological analysis of early embryonic development 22
f. In vitro development study 22
g. Reactive Oxygen species (ROS) detection 23
h. Mitochondrial membrane potential (MMP) detection 23
i. Statistical analysis 24
Chapter III. Results 25
3.1 Effects of PAMAM dendrimer G2 and G3 on the morphology of
mouse blastocyst 25
3.2 In vitro effects of PAMAM dendrimer G2 and G3 exposure on
blastocyst grade 26
3.3 Effects of PAMAM dendrimer G2 and G3 on development potency
of 2-cell stage embryo 29
3.4 Effects of PAMAM dendrimer G2 and G3 on the cell viability of
mouse blastocyst 32
3.5 Effects of PAMAM dendrimer G2 and G3 on cell proliferation of
mouse blastocyst 34
3.6 Effects of PAMAM dendrimer G2 and G3 on in vitro development
of mouse blastocyst 36
3.7 ROS generation in mouse blastocyst treated with PAMAM
dendrimer G2 40
3.8 MMP change in mouse blastocyst treated with PAMAM dendrimer
G2 41

Chapter IV. Discussion 44
4.1 Effects of PAMAM dendrimers on blastocyst morphology 45
4.2 Effects of PAMAM dendrimers on 2-cell stage of mouse embryo 46
4.3 Effects of PAMAM dendrimers on blastocyst development 47
4.4 Effects of PAMAM dendrimers on the cell viability of mouse
blastocyst 48
4.5 Effects of PAMAM dendrimers on cell proliferation of mouse
blastocyst 49
4.6 Effects of PAMAM dendrimers on in vitro development of mouse
blastocyst 50
4.7 Effects of PAMAM dendrimer G2 on ROS generation in mouse
blastocyst 52
4.8 Effects of PAMAM dendrimer G2 on mitochondrial membrane
potential change in mouse blastocyst 53
Chapter V. Conclusion and Further Works 55
References 56


Figure List

Figure 1. Structure of PAMAM dendrimer G2 5
Figure 2. Early Mouse Development 9
Figure 3. The blastocyst 10
Figure 4. Apoptosis versus necrosis 12
Figure 5. Intrinsic and extrinsic pathways of apoptosis 14
Figure 6. Regulation of cell death and signal transduction pathways in
response to reactive oxygen species 16
Figure 7. Effects of PAMAM dendrimers on blastocyst morphology 26
Figure 8. The effect of PAMAM dendrimer G3 on the development of
mouse blastocyst 29
Figure 9. The effects of PAMAM dendrimer G3 on the develop potency of
2-cell stage embryo 32
Figure 10. The effects of PAMAM dendrimers on the cell viability of mouse
blastocyst 34
Figure 11. The effects of PAMAM dendrimers on cell proliferation of mouse
blastocyst 37
Figure 12. The three levels of blastocyst outgrowth grade 38
Figure 13. The effects of PAMAM dendrimer G2 on ROS generation in
mouse blastocyst 41
Figure 14. The effects of PAMAM dendrimer G2 on mitochondrial membrane
potential of mouse blastocyst 43

Table List

Table 1. The calculated properties of amine surface functional PAMAM
dendrimers by generation 6
Table 2. In vitro effects of PAMAM dendrimer G2 exposure on blastocyst
grade 27
Table 3. In vitro effects of PAMAM dendrimer G3 exposure on blastocyst
grade 28
Table 4. Early embryonic development of mouse embryo treated with
PAMAM dendrimer G2 30
Table 5. Early embryonic development of mouse embryo treated with
PAMAM dendrimer G3 31
Table 6. The effects of PAMAM dendrimer G2 on in vitro development of
mouse blastocyst 38
Table 7. The effects of PAMAM dendrimer G3 on in vitro development of
mouse blastocyst 39


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