臺灣博碩士論文加值系統

慢性腎臟病 (CKD) 是一種緩慢喪失腎功能的疾病，目前是台灣主要的健康問題之一，其病程會引發許多併發症，其中一個為貧血。儘管在任何的CKD分期中都可以被診斷出貧血，然而貧血與CKD的嚴重程度有極大的相關性。腎所製造的紅血球生成素(EPO)產量減少是產生慢性腎臟病相關貧血的主因，因為EPO對於紅血球生長和分化是必須的。臨床上使用基因重組合成的人類EPO治療慢性腎臟病相關貧血，不幸的是有部分病人對於此藥物沒有反應或者產生EPO抗性，研究其他的機制對於發展新的診斷方式和療法是關鍵的。
鄰苯二甲酸二(2-乙基己基)酯 (DEHP) 是一種廣泛使用於醫療器材的塑化劑，先前的報告指出洗腎的CKD的病人血漿中被偵測出高濃度的DEHP，因此我們假設DEHP會影響紅血球分化。人類K562和HEL細胞具有可以分化成紅血球系的能力，被用於測試我們的假說。此外DEHP在兩個細胞模型中明顯的抑制紅血球分化，由於表觀遺傳學對於細胞表現非常重要，其中包括DNA甲基化，其會被環境所影響。因此我們進一步分析DNA甲基轉移酶 (DNMT) 的表現，我們假定DEHP對於不同的DNMT表現影響不同。而我們將重點放在DNMT3A和DNMT3B，降低DNMT3A和DNMT3B可以減少DEHP的影響，但是過度表達DNMT3A時，DEHP的影響會存在。另一方面，我們也使用DNA微陣列 (microarray) 去分析在DEHP環境培養的K562細胞，進而去確定其他的分子機制。綜合以上結果，本篇研究證實DNMT3A和DNMT3B在DEHP作用中扮演重要的角色。

Chronic kidney disease (CKD) is the slow loss of kidney function over time, and is a major health problem in Taiwan. Its progression is associated with many serious complications, such as anemia. Although anemia may be diagnosed at any CKD stage, there is a strong correlation between anemia and the severity of CKD. Decreased erythropoietin (EPO) synthesis in kidney is the most important for CKD-associated anemia, because it is essential for growth and differentiation of red blood cells. Therefore, recombinant human EPO is used to treat CKD patients with anemia. Unfortunately, some patients are not response, or develop the resistance to EPO (Thomas, et al., 2008). Identifying other mechanisms is importance for developing new diagnosis or therapy. Di-(2-ethylhexyl) phthalate (DEHP) is widely used as plasticizers in manufacturing numerous medical devices. Previous report demonstrated that high plasma levels of DEHP were detected in CKD patients undergoing hemodialysis (Faouzi, et al., 1999). Thus, we hypothesized DEHP can affect erythropoiesis. Human K562 or HEL cells with capacity differentiating into erythroid cells were used to test our hypothesis. 1 g/ml of DEHP, detected in CKD patients undergoing hemodialysis, decreased proliferation at 96 hrs after DEHP treatment, but did not induce cell death.
In addition, DEHP significantly suppressed erythrocytic differentiation in both cell models. Due to the importance of epigenetics, such as DNA methylation, in responding to environmental stimuli, the expression of DNA methyltransferases (DNMTs) was analyzed. As we assumed, DEHP differentially modulated expressions of DNMTs. We focused on DNMT3B and DNMT3A. Knockdown of DNMT3A and DNMT3B abolished DEHP effects. While overexpression of DNMT3A showed DEHP effect significantly. On the other hand, we also used microarray to characterize gene expression signatures in DEHP-treated K562 cells to identify other molecular mechanisms. Taken together, we demonstrated the DNMT3B and DNMT3A play important role in DEHP effect.

中文摘要.......i
Abstract.......ii
Contents.......iv
Chapter 1. Introduction.......1
1.1 Di-(2-ethylhexyl) phthalate (DEHP) is the commonly used phthalates.......1
1.2 The influence of DEHP in hematopoietic system is not fully elucidated.......2
1.3 DNA methylation is an important epigenetic regulation.......5
1.4 The role of DNMTs in hematopoietic system is not fully understood.......6
1.5 Erythropoiesis.......7
1.6 Ara-C and Nab are used as erythropoiesis differentiation stimulators.......9
1.7 Human leukemic cell lines are used as models for hematopoietic differentiation study.......9
Chapter 2. Research aims.......11
Chapter 3. Materials and Methods.......12
3.1 Materials.......12
3.1.1 Drugs and reagents.......12
3.1.2 Cell lines and bacterial strains.......15
3.1.3 Plasmids.......15
3.1.4 Antibodies.......15
3.1.5 Instruments.......15
3.2 Cells and Cell culture.......16
3.3 Western analysis.......17
3.4 Benzidine staining.......17
3.5 Selection of K562 stable cell clones.......18
3.6 RNA extraction.......18
3.7 Microarray analysis.......18
3.8 Gene set enrichment analysis (GSEA).......19
3.9 Ingenuity pathway analysis (IPA).......19
3.10 Statistical analysis.......20
Chapter 4. Results....... 21
4.1 The cell proliferation was slightly decreased at 96 hours after DEHP treatment.......21
4.2 DEHP suppressed erythrocytic differentiation.......21
4.3 DEHP influenced some DNMT expression.......21
4.4 Knockdown of DNMT3B abolished DEHP effect in erythroid differentiation by transiently mix clone.......22
4.5 Overexpression of DNMT3B abolished DEHP effect in erythroid differentiation.......23
4.6 The role of DNMT3A in the effect of DEHP during erythrocytic differentiation.......23
4.7 Microarray and bioinformatic analysis revealed DEHP modulated erythrocytic differentiation.......24
Chapter 5. Conclusion and Discussion.......24
Chapter 6. References.......25
Chapter 7.Tables.......34
Table 1. Primary antibodies list.......34
Table 2. Secondary antibodies list.......35
Table 3. KEGG: Genetic Information Processing.......35
Table 4. KEGG: Human Diseases.......35
Table 5. KEGG: Organismal Systems.......36
Table 6. KEGG: Environmental Information Processing.......37
Table 7. KEGG: Metabolism.......37
Table 8. KEGG: Cellular Processes.......38
Chapter 8. Figures.......39
Figure 1. Treatment with 1 μg/ml DEHP slightly influenced the proliferation of K562 cells, but not affect survival........39
Figure 2. DEHP suppressed erythrocytic differentiation of K562 cells........40
Figure 3. DEHP suppressed erythrocytic differentiation of HEL cells........41
Figure 4. The effect of DEHP in DNMT expression........42
Figure 5. Transfection efficiency in K562 cells .......44
Figure 6. Selection of DNMT3B knockdown clones.......45
Figure 7. Knockdown of DNMT3B abolished DEHP effect in erythroid differentiation. .......46
Figure 8. Transient expression of two DNMT3B shRNAs in K562 cells suppressed erythrocyte differentiation. .......47
Figure 9. Overexpression of DNMT3B also abolished DEHP-mediated suppression in erythrocyte differentiation........48
Figure 10. DEHP did not further decrease erythrocytic differentiation in DNMT3A deficient cells........49
Figure 11. Overexpression DNMT3A cells showed less function in DEHP effect........50
Figure 12. Correlation between DEHP mechanism and erythroid differentiation genes in K562.......51

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