Title Page
ACKNOWLEDGEMENT
CONTENTS
ABSTRACT 8
Ⅰ. Introduction 9
Ⅱ. Experimental methods 12
Ⅱ.1. Materials 12
Ⅱ.1.1. Animals 12
Ⅱ.1.2. Construction of expression vector 12
Ⅱ.1.3. Reagents 12
Ⅱ.1.4. Instruments 14
Ⅱ.2. Methods 15
Ⅱ.2.1. Preparation o fplasmid DNA 15
Ⅱ.2.2. Constructive of the competitor 15
Ⅱ.2.3. Competitive and quantitative PCR-based assay 15
Ⅱ.2.4. MSC culture 18
Ⅱ.2.5. Differentiation assay 18
Ⅱ.2.6. Flow cytometry 19
Ⅱ.2.7. Transfection of plasmid DNA using PEI in MSC and administration of transfected MSCs 19
Ⅱ.2.8. Preparation of biological samples 19
Ⅱ.2.9. Reverse transcription PCR analysis 20
Ⅲ. Results 22
Ⅲ.1. Competitive and quantitative PCR analysis for measurement of plasmid DNA 22
Ⅲ.2. MSC culture 24
Ⅲ.3. Differentiation of MSC 26
Ⅲ.4. Flow cytometry 28
Ⅲ.5. Transfection efficiency 30
Ⅲ.6. Distribution & life span of plasmid DNA in transfected mesenchymal progenitor cell 31
Ⅲ.6.1. Kinetics 31
Ⅲ.6.2. Biodistribution of plasmid DNA following transfected MSC injection 31
Ⅲ.6.3. Expression of plasmid DNA following transfected MSC injection 35
Ⅳ. Discussion 38
Ⅴ. Conclusion 41
References 42
Summary 48
Figure 1. Plasmid map of pCEP4 13
Figure 2. Preparation of deleted mutant pCEP-GFP 17
Figure 3. 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 23
Figure 4. MSC morphological charateristics. MSC cultured 7days in culture medium. Phase contrast magnification was100× 25
Figure 5. In vitro differentiation of murineMSC from ICR strain. Phase contrast photomicrographs of culture of mMSC were taken after incubating cells in containing adipogenic or osteogenic stimuli. a) mMSC cultured in osteogenic medium for 2weeks and presence of osteoblasts were evidenced by assessing calcium accumulation, after staining Alizarin Red S, b) mMSC cultured in adipogenic medium for 2weeks and presence of adipocytes were evidenced by accumulation of fat droplets, after Oil Red O staining. Phas 27
Figure 6. Expression of surface markers by mMSC from ICR strain. Primary MSC cells were incubated with antibodies for CD9, CD45, CD11b or Sca-1 and assayed by FACS 29
Figure 7. Serum concentration of plasmid DNA. Naked plasmid DNA and transfected MSC was injected intravenous route to 6-to-7-week-old ICR mice. After infection, 30㎕ of blood were sampled from tailvein ateach time pointand serum levels were measured by quantitative PCR. The results are expressed as the mean±SE (n=4) 33
Figure 8. Tissue distribution of plasmid DNA after transfected MSC intravenous injection. 34
Figure 9. mRNA expression levels of plasmid DNA in the liver, lymph node, lung, heart, kidney, spleen (a), bone marrow cell (b). At 1, 2, 4, and 8days after intravenous injection transfected MSC, the ICR mice were sacrificed and the tissue were removed. Total RNA extracted from tissues and bone marrow cells. PCR amplification of the GFP (green florescence protein) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was performed specific primers. The relative expression levels ofGFP in tissues and bone m 36
Figure 10. Representative gel pictures of GFP mRNA expression. At 1 (a), 2 (b) and 4days (c)) after intravenous injection of transfected MSC, the mice sacrificed. Total RNA extracted from relavent tissues .After preparation of cDNA, GFP and GAPDH genes were PCR-amplificed and separated on a 1.5% agarosegel. Representative gel pictures of GFP mRNA expression are presented for the liver (ⅰ), inguinal lymph node (ⅱ), lung (ⅲ), heart (ⅳ), kidney (ⅴ), spleen (ⅵ), bone marrow cells (d)) 37