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研究生:林才民
研究生(外文):Tsai-Ming Lin
論文名稱:人類脂源性和源自脂肪瘤的間葉幹細胞之培養及特性探討
論文名稱(外文):Isolation and Characterization of Human Adipose and Lipoma Tissue-derived Mesenchymal Stem Cells
指導教授:林幸道林幸道引用關係
指導教授(外文):Sin-Daw Lin
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:醫學研究所博士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:109
中文關鍵詞:間葉幹細胞脂肪組織脂肪瘤多潛能性
外文關鍵詞:Mesenchymal stem cellAdipose tissueLipomaMultipotential
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人類間葉幹細胞(Mesenchymal Stem Cells, MSCs)已經從骨髓(Bone marrow)及其他許多成體組織被培養出來,並且有潛力成為組織工程學(Tissue engineering)之重要角色。就脂肪組織(Adipose tissue)而言,它具有一些優點如來源豐富、容易大量獲得、相對上較少宗教道德考量及法律限制,使它成為獲取幹細胞的良好起始材料。然而,現有擴張脂源性間葉幹細胞(Adipose-derived Mesenchymal Stem Cells, ADMSCs)數目的方法卻不甚理想!
因此,我們提出一個新的幹細胞培養技術,藉以大量加快脂源性間葉幹細胞的生長速度並延長其生命期限(lifespan);這方法使用低鈣離子濃度的細胞生長培養基並添加了N-acetyl-L-cysteine及L-ascorbic acid 2-phosphate,在這些間葉幹細胞的培養過程中,早期的細胞便呈現多潛能性(multipotent)的幹細胞特性;包括了在軟凝膠(soft agar)上表現高頻率的非停泊依賴性生長(anchorage independent growth, AIG)、外表呈現匐行狀(serpiginous)的細胞缺乏細胞間裂口接合性交流(gap junctional intercellular communication, GJIC)及在短期間內有極高的累計性細胞族群總數倍增程度(cumulative population doubling level, cpdl)之現象等;此外這些間葉幹細胞可輕易地被誘導分化成脂肪細胞(adipocytes)、骨細胞(osteoblasts)及軟骨細胞(chondrocytes)等。因此,藉由對細胞生長培養基的改變-即使用低濃度鈣離子和添加抗氧化物,我們大大地提高這些多潛能性(multipotential)脂源性間葉幹細胞(ADMSCs)的生長速度及延長了他們的生命期限。
根據這個新的幹細胞培養技術,我們也成功地從人類良性脂肪瘤(lipoma)培養出間葉幹細胞(Mesenchymal Stem Cells, MSCs),並且發現同樣具有各種間葉幹細胞的特性:如在軟凝膠上表現高頻率的非停泊依賴性生長(AIG)、外表呈現匐行狀之細胞缺乏細胞間裂口接合性交流(GJIC)、可輕易地被誘導分化成脂肪細胞(adipocytes)、骨細胞(osteoblasts)及軟骨細胞(chondrocytes)及具備在短期間內有極高的累計性細胞族群總數倍增程度(cpdl)。
我們利用5-Azacytidine誘導脂源性間葉幹細胞(ADMSCs)二十四小時後,可見有如心肌狀的細胞出現,並且在免疫螢光染色下呈現α- actinin及Troponin I正反應現象。並且這兩種間葉幹細胞-脂源性間葉幹細胞(ADMSCs)及源自脂肪瘤之間葉幹細胞(Lipoma-Derived Mesenchymal Stem Cells, LDMSCs)在Statin類的藥物Fluvastatin之誘導下轉分化(trans-differentiation)成表現出GFAP(glial fibrillary acidic protein)及MAP (microtubule-associated protein)螢光正反應的神經膠質細胞(neuroglial cells)。除此之外,這兩種間葉幹細胞都明確地表現出Oct-4 (Octamer-4)及h-TERT (human telomerase reverse transcriptase)的基因型。
我們建立一種新的幹細胞培養方法,成功地從人類脂肪組織及脂肪瘤培養出間葉幹細胞,除了可以在短期間內提高這些多潛能性(multipotential)間葉幹細胞(MSCs)的生長速度及延長了他們的生命期限外,更可以將所培養出的間葉幹細胞(MSCs)分化成心肌細胞(cardiomyocyte)及神經膠質細胞(neuroglial cell),為未來的幹細胞研究(stem cell research)、組織工程學(tissue engineering)、移植醫學(transplantation medicine)、腫瘤醫學(oncology Medicine)及再生醫學(reparative medicine)架構一個有效可用的研究平臺。
Human mesencyhmal stem cells have been isolated from bone marrow and other adult tissues and are potentially useful for tissue engineering. Adipose tissue has several clear advantages as a starting material for harvesting stem cells, as it is abundant and relatively easy to procure, less restriction in law, and molarity or ethic concerns. However, existing methods to expand adipose-derived mesenchymal stem cells are less than optimal.
Here we describe a new cell-culture method that accelerates greatly the growth rate and prolongs the lifespan of adipose mesenchymal stem cells. This was accomplished by using a growth medium with low calcium and supplementing with N-acetyl-L-cysteine and L-ascorbic acid 2-phosphate. Cells produced early in these cultures displayed characteristics similar to those previously reported for pluripotential stem cells, including high frequency of anchorage-independent growth in soft agar, a lack of gap junctional intercellular communication in a cell type with serpiginous morphology, and high cumulative population doubling level in a shorter time. These cells could readily be induced to differentiate into adipocytes, osteoblasts, and chondrocytes. Thus, modification of the growth medium by reduction of calcium and addition of antioxidants greatly enhanced the growth rate and extended the lifespan of adipose-derived multi-potential human mesenchymal stem cells.
Applying this novel technique, we successfully isolated mesenchymal stem cells from human lipoma possessing variable-characteristic stem cell features including high frequency of anchorage-independent growth in soft agar, lack of gap junctional intercellular communication in serpiginous cells, ready differentiation into adipocytes, osteoblasts, chondrocytes, and high cumulative population doubling level in a shorter time.
Twenty four hours after induction by 5-Azacytidine, adipose-derived mesenchymal stem cells (ADMSCs) appeared to be cardiomyocyte-like in morphology with positive α-actinin and Troponin I immunostain. When induced by statin drug (Fluvastatin), these two kinds of mesenchymal stem cells – adipose-derived mesenchymal stem cells (ADMSCs) and lipoma-derived mesenchymal stem cells (LDMSCs) -- were trans-differentiated into neuroglial cells stained positively with GFAP (glial fibrillary acidic protein) and MAP (microtubule-associated protein). In addition, these mesenchymal stem cells expressed genotype of Oct-4 (Octamer-4) and h-TERT (human telomerase reverse transcriptase).
We developed a novel method for cultivating mesenchymal stem cells from human adipose tissue and lipoma. Not only could high growth rate and extended life expectancy of those mesenchymal stem cells be observed, but these mesenchymal stem cells could be induced into cardiomyocytes and neuroglial cells. In this way, the study provides an effective and useful platform for future study in stem cell research, tissue engineering, transplantation medicine, oncology medicine, and reparative medicine.
目錄
中文摘要 1
英文摘要 4
第一章:研究背景 6
第二章: 材料與方法 13
A. 培養及特性化人類脂源性幹細胞 13
1. 細胞培養基質的選擇 (CELL CULTURE MEDIA) 13
2. 從人類脂肪組織發展出初步細胞培養的方法 14
(DEVELOPMENT OF PRIMARY CELL CULTURES FROM HUMAN ADIPOSE TISSUE) 14
3. 細胞間裂口接合性交流 15
(GAP JUNCTIONAL INTERCELLULAR COMMUNICATION, GJIC) 15
4. 累計性細胞族群總數倍增程度 18
(CUMULATIVE POPULATION DOUBLING LEVEL, CPDL) 18
5. 非停泊依賴性生長 19
(ANCHORAGE INDEPENDENT GROWTH, AIG ) 19
6. 多族系分化–脂肪生成、軟骨生成、骨生成、肌肉生成 20
(MULTILINEAGE DIFFERENTIATION–ADIPOGENESIS、CHONDROGENESIS、OSTEOGENESIS、AND MYOGENESIS) 20
7. 鈣化的細胞外基質之鈣離子定量測定 27
(QUANTITATIVE ASSAY OF CALCIUM IN CALCIFIED EXTRACELLULAR MATRIX) 27
B. 從人類脂肪瘤培養出間葉幹細胞 28
1. 細胞培養基質的選擇 28
(CELL CULTURE MEDIA) 28
2. 細胞間裂口接合性交流 29
(GAP JUNCTIONAL INTERCELLULAR COMMUNICATION, GJIC) 29
3. 累計性細胞族群總數倍增程度 29
(CUMULATIVE POPULATION DOUBLING LEVEL, CPDL) 29
4. 非停泊依賴性生長 29
(ANCHORAGE INDEPENDENT GROWTH, AIG ) 29
5. 多族系分化–脂肪生成、軟骨生成、骨生成 30
(MULTILINEAGE DIFFERENTIATION–ADIPOGENESIS、CHONDROGENESIS、AND OSTEOGENESIS) 30
6. OCT-4及H-TERT基因型的表現測定 31
(EXPRESSION OF OCT-4 AND H-TERT IN ADMSCS AND LDMSCS) 31
C. 從人類脂源性間葉幹細胞到心肌細胞 34
1. 細胞誘導分化成心肌細胞 34
(INDUCTION OF ADMSCS INTO CARDIOMYOCYTE) 34
2. 推定的心肌細胞之ΑLPHA- ACTININ AND TROPONIN I免疫螢光染色 34
(ΑLPHA- ACTININ AND TROPONIN I IMMUNOSTAIN OF PUTATIVE CARDIOMYOCYTES) 34
D. 從人類間葉幹細胞到神經膠質細胞 36
1. 轉分化誘導脂源性及源自脂肪瘤之間葉幹細胞成神經膠質細胞 36
(TRANS-DIFFERENTIATION OF ADMSCS AND LDMSCS INTO NEUROGLIAL CELLS) 36
2. 推定性的神經膠質細胞之GFAP及MAP免疫螢光染色 36
(GFAP AND MAP IMMUNOSTAIN OF PUTATIVE NEUROGLIAL CELLS) 36
第三章: 結果 38
A. 培養及特性化人類脂源性幹細胞 38
1. 建立從人類脂肪組織的原始細胞培養 38
(DEVELOPMENT OF PRIMARY CELL CULTURES FROM HUMAN ADIPOSE TISSUE) 38
2. 推定的脂源性幹細胞/源袓細胞之對稱性與不對稱性 分裂 39
(SYMMETRIC AND ASYMMETRIC DIVISION OF PUTATIVE ADIPOSE-DERIVED STEM/PROGENITOR CELLS) 39
3. 推定的脂源性幹細胞/源組細胞之增生潛能 40
(PROLIFERATION POTENTIAL OF PUTATIVE ADIPOSE-DERIVED STEM/PROGENITOR CELLS) 40
4. 非停泊依賴性生長的能力 41
(ABILITY OF ANCHORAGE INDEPENDENT GROWTH) 41
5. 細胞間裂口接合性交流 42
(GAP JUNCTIONAL INTERCELLULAR COMMUNICATION, GJIC) 42
6. 誘導多族系間葉細胞分化 43
(INDUCTION OF MULTILINEAGE MESENCHYMAL CELL DIFFERENTIATION) 43
B. 從人類脂肪瘤培養出間葉幹細胞 50
1. 建立從人類脂肪瘤組織的原始細胞培養 50
(DEVELOPMENT OF PRIMARY CELL CULTURES FROM HUMAN LIPOMA TISSUE) 50
2. 推定的源自脂肪瘤之間葉幹細胞脂之對稱性與不對稱性分裂 51
(SYMMETRIC AND ASYMMETRIC DIVISION OF PUTATIVE LIPOMA-DERIVED MESENCHYMAL STEM CELLS) 51
3. 推定的脂源性幹細胞/源組細胞之增生潛能 52
(PROLIFERATION POTENTIAL OF PUTATIVE ADIPOSE-DERIVED STEM/PROGENITOR CELLS) 52
4. 非停泊依賴性生長的能力 54
(ABILITY OF ANCHORAGE INDEPENDENT GROWTH) 54
5. 細胞間裂口接合性交流 54
(GAP JUNCTIONAL INTERCELLULAR COMMUNICATION, GJIC) 54
6. 誘導多族系之間葉細胞分化 55
(INDUCTION OF MULTILINEAGE MESENCHYMAL CELL DIFFERENTIATION) 55
7. OCT-4及H-TERT基因型的表現測定 60
(EXPRESSION OF OCT-4 AND H-TERT IN ADMSCS AND LDMSCS) 60
C. 從人類脂源性幹細胞到心肌細胞 63
D. 從人類間葉幹細胞到神經膠質細胞 65
第四章: 討論 68
A. K-NAC培養液的優點及可運用之延伸 68
B. 人類脂源性間葉幹細胞之特色及獨特之基因表現 70
C. 人類脂源性間葉幹細胞在醫學領域的運用 71
D. 發現源自人類脂肪瘤之間葉幹細胞所提供的訊息與運用 72
E. 心肌細胞的分化與神經膠質細胞的轉分化之臨床潛力 74
第五章: 結論 76
參考文獻 78
附錄: 發表之論文 98
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