利用三維環境共培養探討人類間葉幹細胞與軟骨細胞間透過旁分泌效果於軟骨化之研究

Through paracrine stimulation effect on chondrogenesis between human mesenchymal stem cells and chondrocytes in 3D co-cultures

陳翔麟(Chen, Xiang Lin)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/碩士(2015年)

摘要

軟骨組織的自我再生修復能力非常有限，且軟骨細胞的取得來源也有其限制性，使得細胞來源難以足夠用於自體的細胞治療。而間葉幹細胞作為一具有多向分化能力的前驅細胞便在此提供了良好的取代細胞來源，有能力分化成軟骨細胞作為軟骨組織工程上的應用，同時又不像胚胎幹細胞在應用上存在著倫理與道德上的議題。
為了促進軟骨細胞的再分化，以及在刺激間葉幹細胞趨向軟骨化的同時避免細胞肥大化，在許多研究中皆採用共培養的系統證實了這些效果。而在本研究中，我們利用人類/兔子的軟骨細胞與人類骨髓來源間葉幹細胞(hBMSCs)於平面與3D環境建立培養系統，且不額外添加生長因子：平面培養為利用含有軟骨細胞所分泌之可溶性因子之培養基來培養hBMSCs(在本實驗中簡稱CM-culture)，以及軟骨細胞與hBMSCs直接接觸共培養(在本實驗中簡稱CO-culture)；3D環境培養則是將兩種細胞於分別的兩顆明膠支架中進行細胞非接觸的共培養(在本實驗中簡稱3D-culture)。目的在於觀察軟骨細胞與幹細胞間的旁分泌刺激效果之影響。因此，我們主要探討間葉幹細胞能否被促進趨向軟骨化，或者是軟骨細胞能夠經由幹細胞提供所謂的營養效果來刺激其再分化達到正常的軟骨細胞表型，即軟骨化基因表現量的上調。
本研究之結果顯示在經過CM-culture和3D-culture後，hBMSCs隨著培養的天數逐漸喪失軟骨化的表型，即軟骨化基因表現量的減少；而在3D-culture中，軟骨細胞則是趨向穩定的軟骨化基因表型。但在此兩種僅有依靠細胞分泌可溶性因子來相互作用的實驗中發現，hBMSCs會具有分泌醣胺多醣(GAGs)的能力。另一方面，CO-culture則顯示著hBMSCs軟骨化基因表型的逐漸增強，即軟骨化基因表現量的增加。

關鍵字

間葉幹細胞 ； 軟骨細胞 ； 共培養

並列摘要

While cartilage has very limited self-regeneration capacity, and the source of
chondrocytes is limited harvesting, mesenchymal stem cells (MSCs) provide an
attractive substitute of cartilage tissue engineering as the precursor or progenitor cells
that possess the ability to differentiate into functional chondrocytes without ethical and
moral concerns.
In order to promote re-differentiation without dedifferentiation of chondrocytes
as well as to prevent the cell hypertrophy while MSCs tend to undergo chondrogenic
differentiation, co-culture strategies were used in this study, and we establish a
co-culture system (monolayer and 3D non-contact) of human (rabbit) chondrocytes
and human MSCs without any other growth factors to observe the paracrine
stimulation effect between these two cells. Therefore, we explore whether MSCs
could be promoted chondrogenesis or chondrocytes could be stimulated toward
normal functional phenotype (re-differentiation) by so-called trophic effect of MSCs.
After the CM-culture (chondrocyte-cultured medium for MSCculturing) and the
3D non-contact co-culture, MSCs gradually loss the chondrogenic phenotype along
with culture days while the more stable chondrogenic phenotype of chondrocytes. But
the exchange of soluble factors between these two cells makes MSCs produce
cartilage-like ECM (GAGs). On the other hand, the direct co-culture shows the
gradual enhancement of chondrogenic phenotype oh MSCs.

參考文獻

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