臺灣博碩士論文加值系統

僅藉由四個轉錄因子體細胞產生的誘導性多能幹細胞（iPS），已將細胞的再程式化(reprogramming)帶領進入另一領域。由於傳統iPS需要載體，但載體基因對於母體的嵌入等影響無法完全預測，已有源源不絕的研究，報導包括利用蛋白質而不直接參與DNA等方法來改善此問題。這種方法需要大量蛋白質萃取物，但目前的研究多利用老鼠胚胎幹細胞或畸胎瘤細胞株等作為來源，較難在日後應用於臨床。臍帶血已廣為認可為一穩定的具潛能性的間質幹細胞來源;人類牙齦纖維母細胞有極高的增生能力，且對於牙醫師為一方便收集的體細胞。儘管人類牙齦纖維母細胞在最近已被證實為傳統iPS方法進行再程式化的可行細胞來源，臍帶血間質幹細胞和人類牙齦纖維母細胞目前都未曾被使用於此種蛋白萃取方式進行的再程式化。鏈球菌溶血素O (SLO)為一種會造成細胞膜穿孔的細菌內毒素，已被廣泛使用於此種蛋白萃取方式進行的再程式化。然而，鏈球菌溶血素O在非致命劑量下對牙齦纖維母細胞的細胞毒性與訊息傳遞等影響目前仍未知。因此，本實驗研究目的為探討臍帶血間質幹細胞蛋白質萃取的方法使人類牙齦細胞進行再程式化及其表現。研究假設人類牙齦纖維母細胞可被幹細胞萃取再程式化。
根據上述問題進行以下研究方法：利用MTT和螢光染色評估SLO對牙齦纖維母細胞生存率和細胞膜穿孔效率的影響，利用西方墨點法分別SLO對牙齦纖維母細胞的訊息傳遞影響。加入臍帶血間質幹細胞萃取物的細胞，則利用反轉錄-聚合酶酵素鏈鎖反應和DNA微陣列，測量再程式化造成的基因的改變。此外，也透過多種細胞分化的方式確認再程式化的表現。
研究結果:低濃度SLO 對於牙齦和皮膚纖維母細胞的生存程度影響不大，但仍會達成一定程度的細胞膜開孔，且會誘導ERK1/2與P38的磷酸化表現。加入臍帶血間質幹細胞萃取物的牙齦纖維母細胞，會表現OCT4和NANOG等幹細胞基因; 且在後續成脂、成骨、神經等誘導分化上有較理想的表現。
結論:臍帶血間質幹細胞萃取物可使牙齦纖維母細胞在適當SLO濃度作用下進行再程式化，造成基因與分化能力的改變。

Induced pluripotent stem cell (iPS), generated from somatic cells by the four transcription factors, has been startling development of reprogramming technology. Since the integration of vectors is not fully predictable, a flood of studies have been publishing, including protein based-DNA free method. This method required abundant protein extracts, which were often collected by embryonic stem cells or teratocarcinoma, were harder to be applied clinically. Human cord blood has been established as a stable and potential source of mesenchymal stromal cells (cbMSC). Human gingival fibroblasts (GF) characterize as high proliferation rate and friendly collected by dentists. Although GF has recently been proved as a potential source by the traditional four-factor reprogramming via retrovirus, both cbMSC and GF have never been used in any DNA free reprogramming methods. Streptolysin O (SLO), a pore forming endotoxin, is widely used in protein delivery for DNA free reprograming. However, the response of GF treated with low sublytic dose has never been studied as well. Thus, we hypothesize that GF could be reprogrammed by protein extracs, and the research goal of the study is to reprogram GF by cbMSC’s protein extracts.
Materials and methods: The questions remained were evaluated as follows: SLO caused cell viability change was evaluated by MTT assay, the membrane permeabiliting efficiency was evaluated by fluorescence stain, and phosphorylation of signal pathways were evaluated by Western Blot. After adding stem cell extracts, the reprogramming effect was examined by RT-PCR and DNA microarray for mRNA level. The differentiation ability was also checked as well.
Results: Though SLO showed little effect at the concentration below 1000ng/mL, it caused membrane permeabilization in both GF and SF. Phosphorylation of ERK1/2 and P38 were also induced when SLO was treated. Adding cbMSC extracts in SLO treated GF increased the mRNA expression of OCT4 and NANOG, which were regarded as stem cell markers. Furthermore, these treated cells differentiated better in adipogenic, osteogenic, and neurogenic induction.
In conclusion, cbMSC extracts could reprogram SLO treated GF causing changes in genetic level and differentiation ability.

中文摘要 ii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES x
LIST OF TABLES xii
Abbreviations xiii
Chapter 1 Introduction 1
1.1 Periodontitis and its tissue-engineering strategies 1
1.2 Reprogramming and the assessments 1
1.3 Reprogramming via cell extracts 3
1.4 Gingiva and Gingival fibroblast (GF) 5
1.5 Streptolysin O (SLO) 5
1.6 SLO and MAPK signalings 6
1.6.1 p38: 6
1.6.2 ERK: 6
1.6.3 JNK: 7
Chapter 2 Design of the experiments 8
2.1 Hypothesis 8
2.2 Goal 8
2.3 Specific Aims 8
Chapter 3 Materials and Methods 10
3.1 Isolation and culture of GF 10
3.2 Culture of cbMSC 10
3.3 Culture of SF 11
3.4 Flow cytometry analysis 11
3.5 Evaluation of cell viability (MTT assay) 11
3.6 Preparation of stem cell extracts 12
3.7 Cell permeabilization and cell extract treatment 12
3.8 Permeability efficiency of SLO 13
3.9 RNA extraction and quantification 13
3.10 Reverse Transcription (RT) for cDNA preparation 14
3.11 Polymerase Chain Reaction (PCR) 14
3.12 Agarose gel electrophoresis 15
3.13 cDNA microarray 15
3.14 Total protein quantification 16
3.15 Western Blot 16
3.16 Adipogenic differentiation 17
3.17 Osteogenic differentiation 17
3.18 Neural differentiation 18
3.19 Immunofluorence Stain 18
3.20 Oil Red O Stain 18
3.21 ARS Stain 19
3.22 Data Normalization and Statistical Analysis 19
Chapter 4 Results 21
4.1 Cell morphology of cbMSC, primary-cultured human GF, SF, and stem cell extract treated GF 21
4.2 Purification and characterization of primary cultured GF 21
4.3 Different concentrations of SLO influenced cell viability 21
4.4 Different concentrations of SLO influenced cell membrane’s permeability 22
4.5 Different treatment times of SLO influenced MAPK phosphorylation 23
4.5.1 SLO induced ERK1/2 phosphorylation 23
4.5.2 SLO induced P38 phosphorylation 23
4.5.3 SLO didn’t induce JNK phosphorylation 24
4.6 mRNA level change in stem extracts treated GF 24
4.7 cDNA microarray 24
4.8 Adipogenic differentiation 25
4.9 Osteogenic differentiation 26
4.10 Neurogenic differentiation 26
Chapter 5 Discussion 66
5.1 GF as a potential cell to induce reprogramming 66
5.2 SLO effect 67
5.2.1 Effect of cell viability 67
5.2.2 The effect of SLO induced MAPK signaling 67
5.3 Gingival fibroblast is an acceptable candidate cell source for stem cell extracts delivery comparing to skin fibroblast 68
5.3.1 Comparison of cell viability in SLO treatment 68
5.3.2 Comparison of cell permeability in SLO treatment 68
5.3.3 Comparison of signaling in SLO treatment 69
5.4 Stem cell related mRNA level changed in stem cell extracts induced reprograming 70
5.5 Stem cell induced reprograming promoted cell differentiation 71
5.6 What really works as the key factors for reprogramming induced by stem cell extracts? 72
5.6.1 Proteins 72
5.6.2 microRNA/siRNA 73
Chapter 6 Conclusion 74
Chapter 7 References 76

LIST OF FIGURES

Fig. 2.1 Experimental Design 9
Fig. 4.1 Morphology of the cells 27
Fig. 4.2 Kl;kl 28
Fig. 4.3 MTT assay of GF and SF treated with SLO. 29
Fig. 4.4 Permeating efficiency of GF and SF treated with SLO. 32
Fig. 4.5 SLO induced ERK1/2 phosphorylation in GF and SF. 33
Fig. 4.6 U0126 inhibited SLO induced ERK1/2 phosphorylation in GF and SF. 36
Fig. 4.7 SLO induced P38 phosphorylation in GF and SF. 37
Fig. 4.8 SLO didn’t induce JNK phosphorylation in GF or SF. 38
Fig. 4.9 cbMSC extract induced mRNA expression level changed in GF. 40
Fig. 4.10 Quantile normalization of microarray samples. 41
Fig. 4.11 Correlation relationship of microarray samples. 41
Fig. 4.12 Principle component analysis (PCA) of microarray samples. 42
Fig. 4.13 Heatmap of ≥12X fold change comparing to GF in E1&E2&cbMSC but NOT E0 group 59
Fig. 4.14 Adipogenic differentiation. 60
Fig. 4.15 Osteogenic differentiation. 61
Fig. 4.16 Neurogenic differentiation for 7 days. 62
Fig. 4.17 Neurogenic differentiation for 10days. 63
Fig. 4.18 Neurogenic differentiation for 10days. 64
Fig. 4.19 Neurogenic differentiation for 10days. 65
Fig. 5.1 Protein proportion changed after stem cell treated reprogramming in mouse SF. 72

LIST OF TABLES

Table 1.1 Methods for reprogramming somatic cells to iPS cells 2
Table 1.2 Stem cell extracts promoting reprogramming by direct delivery 4
Table 1.3 Stem cell extracts promoting SCNT reprogramming 4
Table 3.1 Primer sequence 20
Table 3.2 Fluorescence antibodies or markers 20
Table 4.1 70 genes presented significant (12X) change due to stem cell extract treatment. 43
Table 4.2 GO 50
Table 4.3 Genes most influenced in the cbMSC extract treatment 58

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