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標題: 探討量子點細胞標定技術與功能性RGD融合蛋白結合褐藻膠三維培養模式對脂肪間葉幹細胞特性與分化之影響
Effects of quantum dots and functional RGD-chimeric protein on the properties and differentiation of adipose-derived adult stem cells in an alginate three-dimensional culture system
作者: 張瑞芝
Chang, Jui-Chih
關鍵字: 量子點
Quantum dots
Adult adipose-derived stem cells
Gap junction intercellular communication
Alginate three-dimensional culture
RGD-chimeric protein
出版社: 生命科學系所
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摘要: 活體追蹤幹細胞技術能有助於釐清細胞於體內修復之機轉, 提升動物實驗之精準度與成效,但利用量子點 (CdSe/ZnS Quantum dots) 標定細胞已證實會抑制人類之骨髓幹細胞分化,其機轉未明。本研究將比較不同途徑運,Pep-1胜肽與PolyFect轉殖,運送10 nM量子點 (655 nm) 標定人類脂肪幹細胞 (human adipose-derivated adult stem cells) ,探討其對細胞標定成效、細胞特性與分化能力之影響。研究發現,相較於量子點轉殖,細胞標定效率並無顯著差異(98.2-94.4%),但Pep-1運送會促進細胞質內量子點聚集,顯著提升螢光強度,儘管Pep-1運送之量子點容易隨細胞分裂而釋出或分散,但經過二十八天培養,其螢光強度仍顯著高於轉殖組約三倍;此外,Pep-1可促使量子點攝入後一天逃離溶小體 (lysosome) 分解途徑(endocytosis-independent pathway),但相反地,PolyFect轉殖則會顯著增加溶小體表現,但經過二十一天培養,不論經由何種途徑運送,量子點均會於溶小體代謝;Pep-1運送能改善量子點經由轉殖攝入對細胞特性抑制之影響,包含維持表面標誌蛋白(CD29 和CD105) 、細胞增生與維生素D (1,25 dihydroxyvitamine D) 和轉化生長子(Transforming growth factor β3, TGF-β3) 所誘導之硬軟骨分化。推測其機轉可能與早期胞內量子點降解途徑有關, 因於溶小體內之量子點可能阻礙的胞內核內體/溶小體途徑 (endo-/lysome pathway) 所調控之訊息傳遞。 本研究同時發現,Pep-1運送之量子點會經由同時活化細胞骨架蛋白 (F-actin) 和增加extracellular signal-regulated kinase (ERK) 激酶磷酸化,促進43型接合素 (Connexin 43, Cx43) 磷酸化而導致蛋白降解,進而抑制Cx43所調控之細胞間縫隙連接(Cx43-mediated gap junction intracellular communication, GJIC) ;然而,以高密度三維培養Pep-1運送量子點標定之細胞七天,原先受抑制之縫隙連接 (gap junction) 則會由於 Cx43蛋白質合成顯著增加而促使GJIC功能恢復;此外,轉殖之量子點會抑制細胞分化但並不影響細胞Cx43蛋白與gap junction表現。儘管gap junction在幹細胞的調控角色仍未釐清,然而多數研究證實原始未分化之間葉幹細胞僅表現少量Cx43和gap junction,本研究結果推測暫時性抑制GJIC可能有助於減少量子點攝入所導致的細胞毒性。 褐藻膠(alginate hydrogel)具有可降解與良好生物相容特性,已廣泛應用於藥物釋放與細胞包覆。研究亦證實其包覆細胞培養系統有助軟骨細胞維持其型態與活性。本研究將利用大分子 (25 KDa)、具電中性 (等電6~6.25) 之已被證實具有生物活性含纖維素結合區域之RGD (cellulose-binding domain linked Arg-Gly-Asp, CBD-RGD) 之融合蛋白 ( RGD-Chimeric protein),結合此褐藻膠培養系統,測試其對大鼠脂肪幹細胞分化為軟骨之影響。研究證實CBD-RGD能專一性結合於細胞表面,經七天培養仍可分別保留20.18±0.73%和8.6±2.17% 蛋白於10mg/g 和 20 mg/g包裹濃度。比較包裹不同濃度之蛋白 (10 and 20 mg/g),濃度10 mg/g能經由結合膜表面專一受器(integrin α5 and β1-dependent pathway) 顯著提升生長因子 (TGF-3β) 誘導軟骨細胞之分化成效,包含促進早期軟骨分化基因表現 (Sox9, Aggrecan, fibronectin and collagen type II) 與胞外基質 (fibronetin、collagen type II and glycosaminoglycans) ,並同時抑制經由TGF-β3誘導之肥大軟骨細胞基因Collagen type X表現。相反地,濃度20 mg/g則會顯著抑制分化效果。研究發現,CBD-RGD 促進分化程度與Sox9 基因表現之相關性高於其它軟骨基因,且會暫時抑制第一天fibronetin 蛋白和持續性抑制RhoA 活性表現,故實驗結果推測,CBD-RGD促進細胞分化基轉可能與調控RhoA訊息傳遞有關。因此,本實驗成功建立有效便利之CBD-RGD結合褐藻膠三維培養系統,利用蛋白能保持於水膠中 (protein-depot)亦能緩慢釋放,促使軟骨分化過程中包裹之細胞能經由彈性調分化環境有效促進其分化之表現。
Label of human bone mesenchymal stem cells with CdSe/ZnS quantum dots (QDs) had been demonstrated to impair cell functions and activities. In the present study, QDs delivered by two different routes, Pep-1-labeled QDs (LQ) and PolyFect transfected QDs (TQ), were utilized to assess the effects of delivery mechanisms on various cellular responses of the QDs-internalized human adipose-derived adult stem cells (hADAS). Examination of labeled cells by flow cytometry and laser scanning confocal microscopy showed that LQ had higher fluorescence intensity due to the culster formation and their distribution in cytoplama while TQ were preferentially accumulated at peri-nuclear regions. The fluorescence intensity of the LQ group was still higher than that of the TQ group at 28 days after labeling, though cellular LQ were partitioned after initial cell division. Pep-1 but not PolyFect delivery facilitated QDs to escape from lysosome degradation. Pep-1 delivery of QDs rescued the cells from the negative effects caused by the internalized QDs on cell proliferation and on the expressions of CD29 and CD105 as well as osteogenic and chondrogenic associated lineage markers. The same effect was also observed in the expression of alkaline phosphatase activity, calcium deposition and secretion of chondrogenic matrices (GAG and collagen type II) in micromass culture. These indicated that Pep-1-delivered QDs may serve appropriately to track the hADAS employed in cell therapy/tissue engineering applications. A further study was conducted to reveal the associated mechanism. By comparing Pep-1- and PolyFect-mediated QD internalizations, the connexin 43 (Cx43)-mediated gap junction intercellular communication (GJIC) of hADAS was investigated in monolayer and in three-dimensional (3D) culture (alginate hollow spheres). The latter system offered cells more mobility, which was more similar as in vivo. The results showed that Pep-1-coated QDs, which escaped from the endo-/lysosome degradation, could activate the F-actin assembly and the ERK-dependent phosphorylation of Cx43. The consequence was a reduction in Cx43-mediated GJIC. When the cells were grown in high density 3D alginate hollow spheres instead of in monolayer, the decrease of GJIC caused by the QD internalization was restored. These results indicated that the adaptability in QDs-mediated regulation of GJIC with different delivery coatings depended on the culture systems. The study also suggested that the regulation of gap junction may play a key role in QD cytotoxicity. On the other hand, the role of integrin-binding peptides RGD on chondrogenesis of mesenchymal stem cells is controversial. We revealed the feasibility for flexible modification of RGD by embedding a large molecular weight and slightly charged (isoelectric point, 6-6.25) RGD-chimeric protein (CBD-RGD) with cellulose-binding domain (CBD) in three dimensional (3D) alginate beads to evaluate the chondrogenesis of ADAS. The binding of CBD-RGD with cells and its diffusion from alginate beads were studied on fluorescein isothiocyanate (FITC)-conjugated CBD-RGD. The increases in gene expression (Sox9, Aggrecan, fibronectin and collagen II), accumulation of chondrogenic matrice and decrease of collagen X gene expression during TGF-β3 induction were only observed for those beads containing 10 mg/g CBD-RGD initially, with 20.18±0.73% of that released in a week. The contrary was observed for beads with CBD-RGD 20 mg/g initially and having higher persistence (only 8.6±2.17% relased in a week). The 10 mg/g CBD-RGD-meiated enhancement was demonstrated via the activation of integrin α5 and β1-dependent pathway, and especially related to the upregulation of Sox9 gene and the temporary block of fibronectin expression as well as sustained inhibition of RhoA activity in the early differentiation stage. Thus, we speculated that the dynamic mobility of CBD-RGD may account for the enhanced chondrogenesis. It was concluded that the CBD-RGD-alginate culture system promoted the chondrogenesis of mesenchymal stem cells coordinated with TGF-β3 induction in an RGD dose-dependent manner.
其他識別: U0005-2910200902395300
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