TITLE PAGE
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATION 11
Abstract 12
Ⅰ. Introduction 13
Ⅱ. Experimental methods 18
Ⅱ.1. Materials 18
Ⅱ.1.1. Animals 18
Ⅱ.1.2. Constructionofexpressionvector 18
Ⅱ.1.3. Reagents 18
Ⅱ.1.4. Instruments 20
Ⅱ.2. Methods 21
Ⅱ.2.1. Preparation of plasmid DNA 21
Ⅱ.2.2. Constructive of the competitor 21
Ⅱ.2.3. Competitive and quantitative PCR-based assay 22
Ⅱ.2.4. Administration of plasmid DNA 25
Ⅱ.2.5. Preparation of biological samples 25
Ⅱ.2.6. Reverse transcription PCR analysis 26
Ⅱ.2.7. Statistics 29
Ⅲ. Results 30
Ⅲ.1. Competitive and quantitative PCR analysis for measurement of plasmid DNA 30
Ⅲ.2. Effect of sizes on intranasal absorption of plasmid DNA 32
Ⅲ.3. Brain targeting of plasmid DNA following intranasal or intravenous administration 34
Ⅲ.4. Brain regional distribution of plasmid DNA following intranasal or intravenous administration 38
Ⅲ.5. Brain regional mRNA expression of plasmid DNA 41
Ⅳ. Discussion 43
Ⅴ. Conclusion 46
References 47
Summary 54
Table 1. The abbreviation of mouse brain regional names 28
Figure 1. Pathway of intranasally administered therapeutic agents to the brain and spinal cord 16
Figure 2. Plasmid map of pCMVβ 19
Figure 3. Preparation of deleted mutant pCMVβ 23
Figure 4. Preparation of deleted mutant of the various pDNAs 24
Figure 5. Structures of mouse brain sections 27
Figure 6. A representative gel picture (A) and calibration curve (B) of quantitative PCR. (A) The amounts of IS were 1.0pg/ml (lane 1), 0.3pg/ml (lane 2), 0.1pg/ml (lane 3), 0.03pg/ml (lane 4), 0.01pg/ml (lane 5), 0.003pg/ml (lane 6). The constant amount of the target plasmid DNA was present in all the samples. (B) The ratio between the intensities of the bands at each lane was plotted against the amounts of IS added 31
Figure 7. Pharmacokinetics of intranasally administered plasmid DNA, After intranasal administration of plasmid DNA the, amount of plasmids in the serum was measured by quantiative PCR using corresponding IS. The expressd as the mean SE (n=6) 33
Figure 8. The levels of plasmids in brain and serum at 5min, 10min and 30min after intravenous (A) or intranasal (B) administration were measured by quantitative PCR. The results are expressed as the mean SE (n=5) 35
Figure 9. Brain targeting ratios. Determination by dividing the brain levels of plasmid DNA with serum levels of plasmid DNA 36
Figure 10. The levels of pasmids in the liver at 5min, 10min, 30min and 60min after intravenous or intranasal administration were measured by quantitative PCR. The results are expressed as mean SE (n=5) 37
Figure 11. Brain regional distribution of plasmid DNA following intranasal or intravenous administration. The distribution of administered plasmids to various brain regions at 5min post-dose were measured by quantitative PCR. The results are expressed as mean SE (n=5) 39
Figure 12. Brain regional distribution of plasmid DNA following intranasal or intravenous administration. The distriboution of administered plasmids to various brain regions at 10min post-dose were measured by quantitative PCR. The results are expressed as mean SE (n=5) 40
Figure 13. The mRNA expression levels of plasmid DNA in the brain. At 24hr after intranasal adminstration of plasmid DNA, the mice were sacrificed and the brain tissue were removed. As a control, the brain tissues of untreated mice were used 42