2026-09-22T03:15:31Zhttps://repository.ajou.ac.kr/oai/requestoai:repository.ajou.ac.kr:201003/12622024-10-11T02:17:38Zcom_201003_14369com_201003_14368col_201003_14371
윤, 준기
안, 영환
박, 복남
대학원 의학과
105798
박, 복남
2011-01-26
2011-01-26
2010
2010
http://repository.ajou.ac.kr/handle/201003/1262
http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000010981
000000010981
This study was to evaluate the in vivo distribution of intravenously transplanted bone marrow-derived mesenchymal stem cells (BMSCs) in an acute brain trauma model by 111In-tropolone labeling and to perform the effect of 111In-labeling on the viability and functions of BMSCs. Rat BMSCs were labeled with 37 MBq 111In-tropolone. Their labeling efficiency and in vitro retention rate were measured. To evaluate dose-dependent effect of 111In-labeling, BMSCs were labeled with various doses (0.4-11.1 Bq/cell) of 111In-tropolone, and growth curve analysis, fluorescent activated cell sorter (FACS) analysis after staining with 5-bromo-2-deoxy-uridine (BrdU), and microscopic evaluation after 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) staining were performed until the 14th day. FACS analysis after staining with Annexin V- fluorescein isothiocyanate (FITC) and propidium iodide (PI) was performed at early (3 and 12 hr) and late (7 days) stages with higher doses of 111In (11.1 and 33.3 Bq/cell) to evaluate apoptotic or necrotic change of labeled BMSCs. The biodistribution of 111In-BMSCs in trauma models was compared with those in sham-operated rats and normal rats by gamma camera images. The migration of 111In-BMSCs to the traumatic brain was evaluated using confocal microscope. The labeling efficiency of 111In-BMSCs was 66 ± 5%, and their retention rate was 85.3% at 1 h after labeling. There was no difference in the number of viable cells between 111In-BMSCs and controls at 48 h after labeling. However, the proliferation of 111In-BMSCs was inhibited after the third day of labeling, and it did not reach confluency. For lower doses of 111In (0.4 and 1.1 Bq/cell), the growth of labeled stem cells was not significantly different from that of control, whereas, labeling with higher doses of 111In (4.4 and 11.1 Bq/cell) led to a significant proliferative inhibition from the 3rd day to the 14th day. FACS analysis also revealed less BrdU positive cells in BMSCs labeled with 1.1, 4.4 and 11.1 Bq/cell compared with controls on the 3rd day after labeling. Of these, the patterns of cell cycle in BMSCs labeled with 0.4 and 1.1 Bq/cell of 111In were restored similar to controls on the 14th day. On the contrary, BMSCs labeled with 4.4 and 11.1 Bq/cell of 111In could not recover from cell cycle arrest. Senescence-associated β-gal (SA- β-gal) staining was not prominent in all concentrations until the 14th day after labeling. FACS analysis with Annexin V-FITC and PI also revealed no significant apoptosis or necrosis in both early and late stages. On gamma camera images, most of the 111In-BMSCs uptake was observed in the liver and spleen at the second day of injection. The brain uptake of 111In-BMSCs was more prominent in trauma models (1.4%) than in sham-operated (0.5%) or normal rats (0.3%). Radiolabeled BMSCs were observed at the marginal region of traumatic brain on the confocal microscope. We observed the dose-dependent growth inhibition of BMSCs by 111In-labeling, which was developed by dose-dependent, transient cell cycle arrest, not by cellular senescence or apoptosis/necrosis. Although growth inhibition by 111In-labeling need to be evaluated further prior to use in humans, 111In-BMSCs are useful for the tracking of intravenously transplanted mesenchymal stem cells in brain disease models.
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"ABSTRACT ----------------------------------------- i
TABLE OF CONTENTS ------------------------------ iv
LIST OF FIGURES ----------------------------------- vi
I. INTRODUCTION ---------------------------------- 1
II. MATERALS AND METHODS ----------------------- 3
A. Isolation and culture of rat BMSCs ----------------- 3
B. Generation of an animal model of acute brain trauma 4
C. Synthesis and radioabeling of mesenchymal stem cells
with 111In-tropolone ------------------------------- 4
D. In vitro stability and cell viability of mesenchymal stem
cells with 111In-tropolone-------------------------- 5
E. Dose-dependent effect of 111In on the growth
of BMSCs ---------------------------------------- 6
F. In vivo tacking of 111In-BMSCs by gamma camera in
animal models ----------------------------------- 6
G. PKH 26 labeling of mesenchymal stem cells -------- 7
H. Tissue preparation with DAPI staining for confocal
microscopy -------------------------------------- 7
I. In vitro BrdU labeling for 111In-BMSCs -------------- 8
J. Annexin V-FITC/PI double staining for 111In-BMSCs 9
K. Cytochemical staining with SA-β-galactosidase
for 111In-BMSCs ---------------------------------- 9
L. Statistical analysis -------------------------------- 10
III. RESULTS --------------------------------------- 11
A. Radiolabeling efficiency and viability of 111In-BMSCs 11
B. Dose-dependent growth of 111In-BMSCs ----------- 12
C. In vivo tracking of 111In-BMSCs by gamma camera
in trauma models and controls --------------------- 13
D. Histological analysis of transplanted BMSCs in animal
model of trauma ---------------------------------- 14
E. Cell cycle analysis by flow cytometry -------------- 16
F. Annexin V-FITC/PI double staining flow cytometry -- 18
G. Senescence-associated-β-galactosidase
histochemistry ------------------------------------ 20
IV. DISCUSSION ------------------------------------ 22
V. CONCLUSION ----------------------------------- 29
REFERENCES -------------------------------------- 30
국문요약 -------------------------------------------- 38
"
Doctor
en
Homing of 111In-Labeled Bone Marrow Mesenchymal Stem Cells in Acute Brain Trauma Model
급성 brain trauma 모델에서 111In으로 표지된 중간엽 줄기세포의 귀소성
Thesis
Theses
In-111 tropolone
Bone marrow mesenchymal stem cells
Cell tracking
Growth arrest
2010
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