臺灣博碩士論文加值系統

神經系統除了可以調控許多新陳代謝活性，也可對免疫反應進行調控。哺乳類動物的心臟活動可以受到自主神經系統的調控：交感神經系統的活化，可以促進心跳加速；反之，副交感神經系統的活化則會降低心跳速率。交感神經與副交感神經兩大系統相互拮抗作用影響之下，會使得心跳速率產生變化。這樣子的現象稱之為心率變異性。臨床上指出，心率變異性對於偵測病患生理狀態是個非常有用的工具。本論文將針對斑馬魚遭受到創傷弧菌感染之後，其心跳速率，心率變異性和促發炎基因的表現量變化進行觀察。斑馬魚的心電圖在感染前後的不同時間點都被記錄下來，並且利用軟體來進行心跳速率與心率變異性的分析。此外，我們也利用反轉錄-聚合��鏈鎖反應來觀察一些促進發炎反應產生的免疫基因之表現。同時也進行心電圖的記錄，並進行兩者之相關性分析。浸泡在高濃度菌液當中接受感染的斑馬魚，其心跳速率自感染後的三小時起會有增加的趨勢，並隨著感染時間的增加而比對照組有顯著性的上升。另外心率變異性在時域各方面參數(NN50, pNN50, SDNN和RMSSD)以及頻譜方面的分析，受感染的斑馬魚比起對照組也有顯著下降的趨勢。隨著感染時間增加，促發炎基因（TNFa, COX-2, and IL-1b）的mRNA表現量也有顯著上升的趨勢。免疫基因表現也與心跳速率呈現正相關，並與心率變異性的變化呈現負相關，以上的結果顯示了偵測魚類的心率變異性變化可做為一個偵測其生理狀態的便利工具。

Autonomic nervous system is able to regulate a variety of physiological activities, including the cardiovascular function and immune responses. The activation of sympathetic nervous system speeds up the heart rate (HR) and the activation of parasympathetic nervous system slows down the HR. The balances between sympathetic and parasympathetic tone change the intervals between the two neighboring heart beats and induced the heart rate variability (HRV). It has been indicated that the HRV is a useful tool for screening the physiological state of the subject under certain pathological condition. In addition, the stimulation of vagus nerve also reduces the activation of immune cell and the release of pro-inflammatory cytokines. This study investigated the change of HR, HRV and pro-inflammatory genes induced by the infection of Vibrio vulnificus (V. vulnificus) in zebrafish. The electrocardiograms (ECG) of zebrafish were recorded before and after infection for several time points to analyze the changes in HR and HRV. The expressions of pro-inflammatory genes were also monitored with semi-quantitative RT-PCR technique, and the change of HR and HRV were correlated to the gene expressions. The HR increased with the infection time and was significantly higher than the control group after 3 hr of infection. The time-domain measures (NN50, pNN50, SDNN and RMSSD) as well as the spectrum analysis showed that HRV were significantly lower in the infected zebrafish than the control group. The mRNA level of TNFa, COX-2, and IL-1b were also enhanced with the infection time. The expression of pro-inflammatory genes shows positive correlation with the change of HR and negative correlation with the change of HRV. These results suggest that HRV is a convenient method for monitoring the physiological states of zebrafish.

目錄
中文摘要 I
Abstract II
目錄 III
圖表目錄 V
Introduction 1
1. Homeostasis and the complex system 1
2. Neuroimmunomodulation 1
2.1. The immune system 1
2.2. Regulation of the immune system by the nervous system 4
3. Heart rate variability (HRV) analysis 8
4. The model for infectious disease 12
4.1. Zebrafish (Danio rerio) 12
4.2. Vibrio vulnificus 13
5. Motivation and Specific aims 14
Materials and Methods 15
1. Animals 15
2. Perfusion and establishment of the recording system 15
3. Bacterial preparation 17
4. Infection of zebrafish 17
5. ECG data analysis 18
6. Semi-quantitative RT-PCR 19
6.1. RNA extraction 19
6.2. Reverse transcription 19
6.3. Polymerase chain reaction (PCR) 20
7. Experimental protocols 20
7.1. The changes of HR and HRV during continuous V. vulnivicus infection 20
7.2. The Correlations between physiological parameters and immune responses. 21
8. Statistical analysis 21
Results 23
1. Electrocardiogram (ECG) of the zebrafish 23
2. The changes of heart rate during V. vulnificus infection. 24
3. The changes of Time-domain measures during V. vulnificus infection. 25
3.1. The effect of V. vulnificus infection on NN50 25
3.2. The effect of V. vulnificus infection on pNN50 26
3.3. The effect of V. vulnificus infection on SDNN 26
3.4. The effect of V. vulnificus infection on RMSSD 27
4. Spectrum analysis of the zebrafish ECG during V. vulnificus infection. 28
5. The relationship between cardiovascular function and immune responses to V. vulnificus infection 28
5.1. The expression of pro-inflammatory cytokine genes during V. vulnificus infection 28
5.2. Correlation between physiological parameters and immune responses 30
Discussion 33
Conclusion remarks 39
Figures 40
Table 55
References 56


圖表目錄
Figure 1. The recording system.
Figure 2. Representative ECG of adult zebrafish.
Figure 3. The changes of HR in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 4. The changes of NN50 in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 5. The changes of pNN50 in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 6. The changes of SDNN in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 7. The changes of RMSSD in zebrafish during the infection with different concentrations of V. vulnificus.
Figure 8. The spectrum analysis of the raw ECG in the control and the infected zebrafish (108 CFU/mL).
Figure 9. Induction of pro-inflammatory cytokine gene expressions in zebrafish by infection with V. vulnivicus (108 CFU/mL).
Figure 10. The average expressions of TNFa, COX-2 and IL-1b gene with semi-quantitative RT-PCR in zebrafish after different infection times.
Figure 11. Correlations between gene expressions and changes of heat rate in control and infection groups (108 CFU/mL).
Figure 12. Correlations between gene expressions and changes of NN50 in control and infection groups (108 CFU/mL).
Figure 13. Correlations between gene expressions and changes of pNN50 in control and infection groups (108 CFU/mL).
Figure 14. Correlations between gene expressions and changes of SDNN in control and infection groups (108 CFU/mL).
Figure 15. Correlations between gene expressions and changes of RMSSD in control and infection groups (108 CFU/mL)
Table 1. The PCR primer sequences used for semi-quantitative PT-PCR.

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