- 學位論文
紅螢光蛋白質基因轉殖豬之骨髓間葉幹細胞及脂肪幹細胞特性分析
Characterization of bone marrow-derived stem cells and adipose tissue-derived stem cells isolated from transgenic pigs expressing red fluorescent protein
摘要
成體幹細胞為生物體內能夠自我更新並分化成其他成熟體細胞且進行組織及器官之修復。近年來發現間葉幹細胞（mesenchymal stem cells）可分離自哺乳動物體內許多組織中，例如骨髓、脂肪、關節滑液、臍帶、肌肉及胎盤等，雖然來自不同組織中之間葉幹細胞有許多相似特性，但也因為來源不同，在各種分化及生長特性亦有所差異。由於具多分化潛能且道德爭議較胚胎來源幹細胞小等優點，間葉幹細胞在未來細胞治療之再生醫學領域有著極大的潛力。但在應用於臨床試驗前尚有許多未知的挑戰尚待解決，因此需要有良好的研究工具來釐清問題，以增加人們對幹細胞基本知識之了解，另外可針對特定之人類疾病或組織損傷，透過動物模式之幹細胞治療研究結果，探討移植入的細胞對該疾病是否有改善的功效。 本研究分離同一紅螢光蛋白質（red fluorescent protein, RFP）基因轉殖豬之骨髓間葉幹細胞及脂肪幹細胞，此基因轉殖豬全身皆表現 RFP，RFP 基因為一廣泛運用之報導基因，可應用於細胞治療後之追蹤試驗。本研究之目的係分離同一紅螢光豬之骨髓間葉幹細胞（RFP porcine bone marrow mesenchymal stem cells, rpBMMSCs）及脂肪幹細胞（RFP porcine adipose tissue-derived stem cells, rpADSCs），並比較二種來源之幹細胞之增殖特性、免疫表現型、遷移能力、免疫調節作用及分化潛能，且進行其分化前及後之特性分析，做為未來幹細胞活體移植試驗之參考。 針對 rpADSCs 及 rpBMMSCs 增殖試驗之比較結果顯示，rpADSCs在連續繼代至 passage 6（p6）時，細胞倍增時間明顯高於 p3（p < 0.01） ，但 rpBMMSCs 則無此現象出現。在產生成纖維母細胞聚落之能力測定上， rpADSCs 明顯的具有較 rpBMMSCs 為高之細胞聚落形成能力（p < 0.05） 。此外，藉由流式細胞儀分析結果，證明其兩種細胞皆表現間葉幹細胞之表面抗原包括 CD29、CD44、CD90；惟不表現造血幹細胞之表面抗原諸如 CD4a、CD31、CD45 。以混合淋巴球反應（mix lymphocyte reaction, MLR）及裂殖原增生反應（mitogen proliferation assay, MPA）探討 rpBMMSCs 及 rpADSCs 之免疫調節特性，結果顯示兩種細胞皆能有效地降低淋巴球之增生（p < 0.01） 。此外，在細胞傷口癒合能力分析中，rpADSCs 在遷移能力上較 rpBMMSCs 迅速。分化潛能方面 rpBMMSCs 及 rpADSCs 皆能分化為中胚層之硬骨及脂肪細胞，其中 rpBMMSCs 在分化為硬骨效率明顯較高，而脂肪分化的效率則以 rpADSCs 較高。從 RT-PCR 結果亦可顯現其相關脂肪分化基因 FABP4 （fatty acid binding protein 4）及硬骨分化基因 osteonectin 及 osteocalcin 表達。 除了針對 rpBMMSCs 及 rpADSCs 進行基本間葉幹細胞特性之分析外，本試驗進一步規劃進行rpADSCs 跨胚層之分化試驗，將 rpADSCs 以神經分化培養液處理後證實可表現相關神經前軀細胞之基因，且透過細胞免疫染色可觀察到專一表現於神經前軀細胞之 βIII-tubulin，證明其具有分化成特定外胚層來源細胞之能力，且持續表現紅螢光蛋白質。至於分化為內胚層來源細胞能力，則可將 rpADSCs 預培養為立體結構之細胞團塊下，以高葡萄糖濃度之分化培養液培養十天後，可觀察其表現特定胰島細胞之基因，往後應更近一步確認其是否具有分泌胰島素且具有調節血糖之功能，若未來改善分化條件以引導細胞能更往成熟之胰島細胞分化，將來 ADSCs 可作為分泌胰島素功能之細胞取代胰島移植之細胞來源，以解決胰島素依賴型糖尿病之問題 。 綜合上述結果顯示，本試驗分離之 rpBMMSCs 及 rpADSCs 殊能符合多項間葉幹細胞所具有之特性且 RFP 基因於分化前後之表現穩定，雖然兩種不同來源之間葉幹細胞在各種特性上有所差異，惟此差異也可供未來不同細胞治療之幹細胞來源參考，此兩種細胞可望在未來應用於幹細胞治療之動物模式研究。
並列摘要
Adult stem cells are undifferentiated cells found in living organisms that can selfrenew and can differentiate to yield major specialized cell types of the tissue or organ. Mesenchymal stem cells (MSCs) are adult stem cells found in various tissues, such as bone marrow, adipose tissue, umbilical cord, synovium, skeletal muscle and placenta. Although MSCs isolated from different sites have similar phenotype, they may exhibit heterogeneity in differentiation potentials, which related to the tissue sources. Several characteristics, such as multipotency and less ethical concerned make MSCs hold the promise to restore human damaged tissue or organs, but it also faces tremendous challenges on the way to reach clinical application. To elucidate all these problems, improvement of research tools should be applied to gain knowledge from basic cell culture system and model organism. To understand which tissue derivative stem cells will be proper graft materials for repairing target tissue in the future. MSCs were isolated from bone marrow and adipose tissue of transgenic pigs expressing red fluorescent proteins (RFP) gene, an ideal tracing marker used to monitor cellular processes in living systems. Herein, we aim to isolate two kinds of adult stem cells, RFP porcine bone marrow mesenchymal stem cells (rpBMMSCs) and RFP porcine adipose tissue-derived stem cells (rpADSCs) from the same RFP transgenic pigs, and verify their characterizations of proliferation, differentiation, migration, immunosupression properties and expression of surface markers. In proliferation capacity, rpADSCs showed higher colony forming unit fibroblasts (CFU-Fs) than rpBMMSCs (p < 0.05), but rpBMMSCs exhibited the same population doubling time in serial passage to p6. In contrast, proliferation ability of rpADSCs decreased in p6 (p < 0.01). The result in surface markers assay indicated that both rpBMMSCs and rpADSCs are positive in CD29, CD44 and CD90 mesenchymal stem cell surface markers and negative in CD31, CD45 and CD4a hematopoietic cell markers. In mitogen proliferation assay and mix lymphocyte reaction, peripheral blood mononuclear cell proliferation was eased by these two cell types (p < 0.01), which validated the immunomodulatory feature of mesenchymal stem cells. Wound healing assay revealed that rpADSCs migrated faster than rpBMMSCs. Differentiation potency were investigated by osteogenic and adipogenic differentiation. Extracellular calcium produced by rpBMMSCs was higher in osteogenic induction, whereas oil accumulation of induced rpADSCs was higher in adipogenic culture. Adiogenic related gene, adipocyte fatty acid binding protein (FABP4) and osteogenic related gene, osteonectin and osteocalsin gene expressions were also confirmed by RT-PCR. All of differentiated cells expressed RFP gene stably. These results might represent that characteristics of stem cells are tend to fit their original niche. Except characterizing the properties of mesenchymal stem cells for these two cell types, we also proceeded trans-differentiation experiments for rpADSCs. After neural induction medium treatment for three days, rpADSCs expressed several genes related to neural progenitor cell and neural specific βIII-tubulin could be observed in some cells. On the other hand, pancreatic islet differentiation of rpADSCs was induced by high glucose induction medium and three-dimensional culture. Several pancreatic genes, such as Ngn3, NeuroD and Nkx6.1, were detected by RT-PCR in differentiated cells. The next step is to check the function of these cells comparing with mature islet to demonstrate wether ADSCs is a good candidate cell type for islet transplantation. Taken together, both rpBMMSCs and rpADSCs possess regular mesenchymal stem cell properties that can be applied to cell tracing in model animal and some different characteristics of them may influence their future application in regenerative medicine.
參考文獻
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