영양인자를 ex vivo 처리한 중간엽 줄기세포에서의 영양인자 지지효과
Trophic Supports in Mesenchymal Stem Cells; effects of ex vivo-treatment
초록/요약
Several studies have examined the enhanced efficacy of neurotrophic factor - transfected mesenchymal stem cells (MSCs) in ischemic rat models. However, gene therapy, i.e., the application of MSCs transfected with neurotrophic factors, is not feasible in clinical practice for safety issue. Thus, these hypothesized that besides genetic manipulation, ex vivo cultivation with specific trophic factors per se might also enhance the efficacy of human MSCs (hMSCs) in an animal model of ischemic stroke. The stroke rat model was performed from 2 hrs transient middle cerebral artery occlusion (tMCAo). The intravenous application of ex vivo - cultured hMSCs was performed at 24 hrs after tMCAo. The ischemic brains were extracted from the injured hemisphere at one day after tMCAo. The ischemic brain-extracts were treated after 2 days of hMSCs culture, and media of cultured hMSCs with ischemic brain-extracts was collected for 7 days. The trophic factors, such as BDNF, NGF, VEGF, HGF, and bFGF, levels were analyzed using ELISA. The each trophic factor was pretreated in ex vivo culture conditions of hMSCs. Trophic factor treated hMSCs were re-seeded and cultured with ischemic brain extracts. Concentrations of each trophic factor were measured and compared between the trophic factor - treated and the non - treated hMSCs. Tissue levels of trophic factor, in all case, were higher in hMSCs - received ischemic brain than in non - received ischemic brain. In addition, hMSCs cultured with ischemic rat brain extract increased production of BDNF, VEGF, and HGF. When the BDNF, VEGF, and HGF - treated hMSCs received ischemic brain -extracts, the released levels of trophic factor were significantly high than those that trophic factor non - treated hMSCs. These data suggested that hMSCs provide the trophic support to ischemic brain and it can be enhanced by ex vivo treatment of trophic factor. These results indicate that hMSCs transplantation may be clinically useful as a potential therapy for stroke.
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ABSTRACT ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅰ
TABLE OF CONTENTS ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅲ
LIST OF FIGURES ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥ⅴ
Ⅰ. INTRODUCTION ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥1
Ⅱ. MATERIALS AND METHODS ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3
A. Isolation of human MSC and Cell Culture ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3
B. Animal model of transient
middle cerebral artery occlusion ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥3
C. Tissue preparation ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥4
1. Preparation of ischemic brain – extracts for treatment into hMSCs ‥‥‥‥‥‥4
2. Preparation of quantitative for trophic factors into ischemic brain tissues ‥‥‥5
D. ELISA for trophic factor levels ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
1. In vitro ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥5
2. In vivo ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥6
E. Characteristics of human MSCs ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥7
1. Cell viability test ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥7
2. Fluorescence-Activated Cell Sorting (FACS) Analysis
of hMSCs ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥8
F. Statistical analysis ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥8
Ⅲ. RESULTS ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
A. Brain levels of trophic factors increased after application of
hMSCs in ischemia rat. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
B. Trophic factors produced from hMSCs
in the response to the ischemic insult. ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥9
C. Characteristics of trophic factor – pretreated hMSCs. ‥‥‥‥‥‥‥‥‥‥10
D. Enhancing production of trophic factors by
ex vivo treatment with trophic factors ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥11
Ⅳ. DISCUSSION ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥12
Ⅴ. REFERENCES ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥23
국문요약 ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥31
Fig. 1. TTC stained coronal brain section ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥17
Fig. 2. Characterization of isolated hMSCs ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥18
Fig. 3. In vivo levels of trophic factor ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥19
Fig. 4. Trophic factors secretion by hMSCs ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥20
Fig. 5. Growth characteristics of hMSCs – trophic factor ‥‥‥‥‥‥‥‥‥‥‥21
Fig. 6. Morphology of cultured hMSCs with trophic factor ‥‥‥‥‥‥‥‥‥‥22
Fig. 7. The cellular phenotypes of undifferentiated
hMSCs – trophic factors by flow cytometry ‥‥‥‥‥‥‥‥‥‥‥‥‥23
Fig. 8. Enhancing trophic support by ex vivo – treated hMSCs
in ischemic environment ‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥‥24
