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研究生:尤偉勳
研究生(外文):You, Weihsun
論文名稱:豬血液血小板濃厚血漿之最佳化製程之確立及無血清幹細胞培養之應用
論文名稱(外文):Optimization For Processing Porcine Platelet-Rich Plasma And Application On Serum-Free Stem Cell Cultivation
指導教授:林詠凱
指導教授(外文):Lin, Yangkai
口試委員:馬旭王盈錦
口試委員(外文):Ma, HsuWang, Yingjin
口試日期:2012-06-26
學位類別:碩士
校院名稱:中國文化大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:97
中文關鍵詞:豬血液血小板濃厚血漿胎牛血清
外文關鍵詞:Porcine platelet-rich plasma
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幹細胞於臨床及組織應用最大的瓶頸是無法在體外快速培養至治療所需之數量,目前許多研究使用胎牛血清 (fetal bovine serum, FBS) 做為培養基補充劑,但胎牛血清的生產與使用始終伴隨著倫理及科學之爭議,在倫理上剝奪胎牛的生命與不人道的採集常被詬病;在科學上,血清的組成複雜且易變動,常對研究品質造成影響。而在台灣,豬血液的利用效率不佳,僅於食品加工及飼料添加使用,故本研究擬自豬血液開發一合適於幹細胞培養用之胎牛血清替代物,自豬血液以不同離心方式濃縮製成豬血液血小板濃厚血漿 (porcine platelet-rich plasma, P-PRP),加入不同濃度的膠原蛋白 (collagen)、凝血酶 (thrombin)、氯化鈣 (CaCl2) 配方組合活化血小板並以酵素結合免疫吸附分析 (enzyme-linked immunosorbent assay, ELISA) 測定類胰島素生長因子 (insulin-like growth factor , IGF) 、角質化細胞生長因子 (keratinocyte growth factor, KGF)、血小板衍生生長因子AB (platelet-derived growth AB, PDGF-AB)、轉形生長因子-β (transforming growth factor, TGF-β)之釋出量。經定量分析後以Design-Expert軟體設計最佳之活化劑配方,並檢測重金屬及纖維連結素含量。配製成培養基與胎牛血清及市售無血清培養基,以比較對人類脂肪間葉幹細胞貼附率、增生率及存活率之影響。並以CD73、CD105、CD90、CD34、CD45及HLA-DR鑑定間葉幹細胞經P-PRP培養,是否會因而改變細胞特性。結果顯示P-PRP經活化後所有生長因子分泌量皆顯著提高 (p<0.05),TGF-β、PDGF、KGF的分泌量高於FBS,其中經膠原蛋白、氯化鈣活化會增加TGF-β及PDGF的釋放,氯化鈣則增加KGF的分泌,特別是TGF-β達顯著的差異 (p<0.05)。以生長因子分泌量經Design-Expert軟體藉反應區面法判定Co-T+Ca-、Co+T-Ca-及Co+T+Ca-為最佳化活化組合。含量分析結果顯示P-PRP不含有害之重金屬,而較高之硒離子濃度可增加細胞抗氧化之能力。P-PRP纖維連結素含量略低於市售胎牛血清,更影響並表現較低細胞貼附率。細胞培養結果顯示三種P-PRP可促進細胞增生率及存活率,並佔有數量上之優勢。此外,間葉幹細胞經P-PRP培養後仍保持原有細胞之特性。未來希望開發出單價合理、成份穩定、符合動物福祉且能促進幹細胞體外增生且維持原始特性之胎牛血清替代品並應用於再生醫療領域。
關鍵詞:胎牛血清,豬血液血小板濃厚血漿,間葉幹細胞,胎牛血清替代物

Efficient In vitro cell expansion is an obstacle for stem cell research in cell therapy and tissue engineering. Conventionally, stem cell culture medium contains 5-20% fetal bovine serum (FBS). However, due to ethical issues and scientific problems, the FBS concentration of stem cell culture medium must be minimized. On ethical aspect, the preparing procedures of FBS are inhumane. Furthermore, the batches of serum display quantitative and qualitative variations among their compositions and may induce the scientific problems. In Taiwan, the utilizations of porcine blood is mainly used in food manufacturing and animal feed staff. Therefore, the aim of this study is to develop porcine platelet-rich plasma (P-PRP) as the fetal bovine serum substitute in the mesenchymal stem cell cultivation. The effect of platelet-rich plasma will be activated by different formulations (collagen/thrombin/calcium chloride) on release amount of insulin-like growth factor (IGF), keratinocyte growth factor (KGF), platelet-derived growth AB (PDGF-AB), and transforming growth factor (TGF-β) were determined by ELISA (enzyme-linked immunosorbent assay) methodology. The ELISA results were analyzed by Design-Expert software to develop optimal agonist formulations. Moreover, the amount of heavy metal and fibronectin of P-PRP were quantified. P-PRP, fetal bovine serum and serum-free medium were utilized to stem cell cultivation media, and then treated human adipose-derived mesenchymal stem cells (MSC) to observe the efficiency for cell attachment, proliferation and viability. Futhermore, we characterized these MSCs by surface marker expressions, including CD73, CD105, CD90, CD34, CD45 and HLA-DR. Results demonstrated all release amount of growth factors of P-PRP were significantly higher than non-activated P-PRP (p<0.05). The releasing amount of PDGF-AB and KGF of P-PRP did not reach significant difference from FBS. Furthermore, the levels of TGF-β and PDGF-AB in P-PRP could be increased based on collagen and calcium chloride activation, and calcium chloride could enhance the releasing of KGF in P-PRP. Notably, TGF-β in activated P-PRP was significantly higher than in FBS (p<0.05). According to the results of growth factor, we used response surface methodology to establish Co-T+Ca-, Co+T-Ca- and Co+T+Ca- as the optimal agonist formulations for P-PRP activation by Design-Expert program. We determined Co+T+Ca-, Co+T-Ca- and Co-T+Ca- as the optimal agonist formulations. These three P-PRP formulations contained no harmful heavy metals but with higher selenium concentration, which would improve the cell ability of anti-oxidation. The fibronectin in P-PRPs were comparatively less than in FBS, which may cause the lower cell attachment on MSCs morphology. P-PRP cell cultivation revealed a higher cell proliferation rate and viability. Furthermore, the results of MSC surface marker expressions presented MSCs do not alter characteristic under P-PRPs cultivation. In summary, we succeed to develop the FBS substitute which would improve in vitro expansion and maintain the differentiation capacity of mesenchymal stem cell with reasonable price, stable composition, and high ethical quality. Further, the P-PRP could be used in human regenerative medicine.

Key words: Fetal bovine serum, Porcine platelet-rich plasma, Mesenchymal stem cell, FBS substitute

壹、 摘要……………………………………………………………………………………..............5
一、中文摘要……………………………………………………………………………………...5
二、英文摘要……………………………………………………………………………………...6

貳、 文獻檢討……………………………………………………………………………………......8
一、幹細胞研究及再生醫學……………………………………………………………………...8
(一) 再生醫學定義……………………………………………………………………………...8
(二) 幹細胞生理………………………………………………………………………………...8
(三) 幹細胞之分化…………………………………………………………………………….10
1. 間葉幹細胞 (Mesenchymal stem cell, MSC) …………………………………………10
2. 造血幹細胞 (Hematopoietic stem cell, HSC) ………………………………………...11
(四) 幹細胞療法之應用…………………………………………………………………….....11
二、胎牛血清 (Fetal bovine serum, FBS) ……………………………………………………...12
(一) 胎牛血清的使用……………………..………………………………………….………..12
(二) 胎牛血清之收集……………………….…………………..…………………...………...13
(三) 胎牛血清之生化組成...………………...………………………………………………...15
(四) 使用胎牛血清之倫理與科學問題………….…………………………………………....19
三、無血清幹細胞培養…..……………………………………………………………………...20
四、濃厚血小板血漿(platelet-rich plasma, PRP) ...…………………………………………...23
(一) 定義…………………………………………………………………………………….....23
(二) 國內豬血液使用現況……………….……………………………………………………29
五、反應曲面法 (response surface methodology, RSM) ………………………...……………30
六、二因子設計法 (Two level factorial design) …………………...…….…….……………….32
七、血小板濃厚血漿與幹細胞體外培養…………………………………………………….....32
(一) 以豬血液血小板濃厚血漿中取代幹細胞培養用胎牛血清之優勢…………..………...33

參、目的與動機……………………………………………………………………………………..36
肆、材料與方法……………………………………………………………………………………..37
一、材料…………………………………………………………………………..……………...37
二、實驗方法……………………………………………………………………..……………...39
(一) 不同製備方式對豬血液血小濃縮指數之影響……………………………………..…...41
1. 血液抗凝劑 (Citrate-phosphate-dextrose, CPD)之配置………………………………41
2. 豬血液的採集……………………………………………….…………………………...41
3. 不同離心方式對P-PRP中血小板濃度之影響………………...………………………41
4. 血小板數量分析…………………………………………………………………………41
(二) 不同激活方式對P-PRP中生長因子釋放量之影響…………………………………...42
1. 激活方式…………………………………………………………………………………42
2. 不同激活方式對P-PRP中生長因子釋放量之影響…………………………………..43
3. 反應曲面分析……………………………………………………………………………44
(三) P-PRP生化組成檢測……………………………………………………………………44
1. 纖維連結素 (fibronectin) 分析…………………………………………………………44
2. 重金屬含量之分析……………………………………………………………………...45
3. 內毒素含量之分析……………………………………………………………………...45
4. 胰蛋白酶 (Trypsin) 抑制反應試驗……………………………………………...……...45
(四) 比較P-PRP、FBS及無血清培養基對細胞生長之影響…….………………………...46
1. 培養基製備……………………………………………………………………………...46
2. 人類脂肪間葉幹細胞分離與培養條件………………………………………………...46
3.細胞貼附率 (cell attachment)….……………………….………………………………..47
4. 細胞增生率 (Proliferation) …………………………….…………………………..…...47
5. 細胞存活率 (Viability) ……………………….…………………………………………47
(五) 不同代數細胞之鑑定…………………………………………………………………….48
1. 經不同代數培養後MSC細胞表面抗原之鑑定………………………….……………..48
(六) 統計分析………………………………………………………………………………….49

伍、結果與討論……………………………………………………………………………………..50
一、豬血液濃縮條件…………………………………………………………………………….50
二、生長因子含量檢測……………………………………………………………………….....54
三、最佳活化配方設計………………………………………………………………..………...56
四、纖維連結素 (fibronectin) 分析…………………………………………………...………...60
五、重金屬分析…………………………………………………………………..……………...62
六、內毒素含量檢測試驗……………………………………………………………..………...63
七、酵素抑制能力………………………………………………..……………………………...65
八、幹細胞貼附率試驗………………………………..………………………………………...67
九、幹細胞增生率試驗…………………………………………..……………………………...69
十、幹細胞存活率試驗………………………………………..………………………………...71
十一、不同血液製劑對不同代數細胞表面抗原表現之影響…………………..……………...73

陸、 結論………………………………………………………………………………………….....77

柒、 參考文獻………………………………………………………………..……………………...79

捌、附錄…………………………...……………….......................…….…………………………...86

玖、致謝……………………………………..………………………………………………………95

拾、作者介紹……………………………………………..…………………………………………96

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