源自於大白鼠嗅球組織的間葉幹細胞可以分化成具有心肌特性的細胞

間葉幹細胞廣泛分布在身體各部位，包括: 骨髓、脂肪組織、周邊血液、臍帶及羊膜組織。最近的研究顯示，間葉幹細胞也可能存在於嗅覺系統組織中。嗅球內含有類纖維母細胞（fibroblast-like cells），將其分離培養並定性，發現其具有類似間葉幹細胞特性，可在特定培養基誘導後，分化為硬骨細胞、軟骨細胞及脂肪細胞。雖然源自於骨髓、脂肪的間葉幹細胞在體內及體外的環境下可誘導成心肌細胞，然而來自嗅球的幹細胞是否能有分化為心肌細胞的潛能仍不清楚。在此研究中，我們評估嗅球的幹細胞是否可在體外環境分化成心肌細胞。本實驗從新生大白鼠的嗅球組織分離及培養類纖維母細胞，再分別進行以下四種誘導處理：（1）控制組：培養液培養。（2）培養液加入三種因子；神經調節素-1 （neuregulin-1）、鹼性基底纖維母細胞生長因子（b-FGF）和錦紫蘇萃取物（forskolin）。（3）與心肌細胞共培養。（4）與心肌細胞共培養，並同時在培養液中加入上述三種因子。心肌細胞分化潛能的方法評估包括光學顯微鏡、RT-PCR、西方墨點法、免疫螢光染色及細胞對藥物的反應。本實驗結果發現，從新生大白鼠嗅球分離出來的類纖維母細胞表現 CD29、CD44、CD90、CD105 和 CD166 等間葉幹細胞特有的表面標誌蛋白，但不會表現造血幹細胞的表面標誌蛋白 CD34 和 CD45，此結果顯示，培養的嗅球細胞中具有類似間葉幹細胞的分子特徵，進一步評估其分化成心肌細胞的能力，發現只有類纖維母細胞與心肌細胞共培養並且加入三種因子的這個組別在培養第七天的時候出現長柱狀型態並且會自發性收縮的細胞，以RT-PCR及西方墨點法分析顯示這些分化的細胞會表現心肌細胞特有標誌基因或蛋白，包括: Nkx2.5、GATA4、sarcomeric alpha-actinin、心肌肌鈣蛋白I （cardiac troponin I）、心肌肌凝重鏈蛋白（cardiac myosin heavy chain）、心房利鈉胜肽（atrial natriuretic peptide）及連接蛋白43（connexin 43），且呈現典型的肌節染色，相鄰細胞並具有間隙連結（gap junction）表現，此外，分化的細胞會受到腎上腺性和膽鹼性的促進劑及拮抗劑的影響而改變細胞收縮能力。綜合以上的結果顯示，源自於老鼠嗅球的類纖維母細胞具有間葉幹細胞的特性，在經過誘導分化處理之後能分化成似心肌細胞。

關鍵字

間葉幹細胞；嗅球組織；類纖維母細胞；心肌細胞；幹細胞誘導分化

並列摘要

Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC-like cells also exist in rat olfactory bulb and are capable to induce differentiate into mesenchymal lineages: osteocytes, chondrocytes and adipocytes. In vitro and in vivo studies have shown that MSCs derived from bone marrow and adipose tissue are capable of differentiate into cardiomyocytes; however, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb derived MSCs could differentiate into myocardial cells in vitro. Fibroblast-like cells isolated from the olfactory bulb of neonatal rats were grown under 4 conditions: no treatment; in the presence of growth factors (neuregulin-1, b-FGF, and forskolin); co-cultured with cardiomyocytes; co-cultured with cardiomyocytes plus neuregulin-1, b-FGF, and forskolin. Cell differentiation into myocardial cells was monitored by RT-PCR, light microscopy, immunofluorescence, Western blot analysis, and contractile response to pharmacological treatments. The isolated olfactory bulb-derived fibroblast-like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of MSC co-culture with rat cardiomyocytes plus neuregulin-1, b-FGF and forskolin. RT-PCR and Western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α-actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacologic treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb derived fibroblast-like cells with MSC characteristics can differentiate into myocardial-like cells.

並列關鍵字

mesenchymal stem cell ； olfactory bulb ； fibroblast-like cell ； cardiomyocyte ； cell differentiation

參考文獻

Plotnikov EY, Khryapenkova TG, Vasileva AK, Marey MV, Galkina SI, Isaev NK, Sheval EV, Polyakov VY, Sukhikh GT, Zorov DB :

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Yuan Y, Chen LF, Zhang SY, Wu W, Chen H & Yan XW : Differentiation of mesenchymal stem cells into cardio myogenic cells under the induction of myocardial cell lysate. Zhonghua Xin X ue Guan Bing Za Zhi 2005, 33(2):170-3

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Augello A, Kurth TB & De Bari C: Mesenchymal stem cells: a perspective from in vitro cultures to in vivo migration and niches. Eur Cell Mater 2010, 20:121-133.

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源自於大白鼠嗅球組織的間葉幹細胞可以分化成具有心肌特性的細胞

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