以細胞—細胞接觸模式及骨分化因子調控胚胎幹細胞增生與骨分化為類造骨細胞之研究

Osteoblast-like cells regulated by proliferation and osteogenesis of embryonic stem cells using cell — cell contact and osteogenic supplements model

林琇婷(Hsiu-Ting Lin)

指導教授 ： 張恒雄

中原大學/工學院/醫學工程研究所/碩士(2009年)

https://doi.org/10.6840/cycu200901214

摘要

造骨細胞之增生與分化在骨代謝與生長中扮演非常重要的角色，且造
骨細胞同時受到局部性激素與系統性激素之調控。在骨形成的過程中，分
化終期的造骨細胞會受到自體分泌製造之細胞外基質所包埋，進而形成骨
細胞。儘管幹細胞在特定刺激下，可分化成特定細胞之萬能分化能力已經
被廣為人知，目前對於幹細胞於體內多細胞之複雜環境下之骨分化路徑卻
仍未知。欲探討在骨分化過程中，胚胎幹細胞與造骨細胞彼此之間的交互
作用。在本研究中，首先使用胚胎幹細胞的小鼠胚胎幹細胞株C3H10T1/2，
建立一MC3T3-E1 與C3H10T1/2 細胞共同培養之系統，並比較在有無添加
骨誘導分化劑的培養環境中，探討細胞—細胞直接間接觸是否會誘使
C3H10T1/2 細胞逐漸走向骨生成；接著在體外建立一簡單的細胞共同培養
模式，將每日收集的小鼠造骨細胞培養上清液，依不同比例混合添加於小
鼠胚胎幹細胞中，進行間接式共同培養，以瞭解造骨細胞在此共同培養模
式下所扮演之角色。
在直接共同培養模式下，C3H10T1/2 與MC3T3-E1 以不同比例細胞密
度共同培養，結果發現細胞數目與ALP 活性增加，且增加鈣沈積。此結果
顯示直接共同培養不僅促進骨分化而且能產生細胞外基質的礦化。在間接
共同培養模式下，MC3T3-E1 以不同濃度條件培養基與C3H10T1/2 細胞共
同培養，結果發現細胞數目與ALP 活性皆增加，但是鈣沈積的量很少。所
以我們認為MC3T3-E1 CM 能使細胞與ALP 活性增加，但還不足以使細胞
外基質產生礦化。

關鍵字

共同培養 ； 造骨細胞 ； 胚胎幹細胞 ； 骨分化

並列摘要

The proliferation and differentiation of osteoblasts are important event
during bone turnover and are controlled both by local growth factors as well as
by systemic hormones. In their final phase of differentiation, osteoblasts become
embedded deep within the mineralized bone matrix during bone formation and
become osteocytes. Despite it is well known that the embryonic stem cells
(ESCs) are totipotent which can differentiate into a variety cell types, the
mechanism of stem cells osteogenesis in a multi-cellular environment is still
unknown. To further understand the interaction between ESCs and osteoblasts
during the osteogenesis, in this study, at first, we were interested in the
morphology of mouse embryonic stem cell line – C3H10T1/2 during the
osteogenic differentiation, cocultured with mouse MC3T3-E1 cell line in this
direct coculture model. Secondly, Different ratios of conditioned medium
collected from MC3T3-E1 were additional added into the medium for murine
C3H10T1/2 cell culture in this indirect coculture model. The osteoblast may
play a role on inducing or regulating the ESCs osteogenesis.
In the direct coculture system, direct coculture with different density
MC3T3-E1 cells and C3H10T1/2 resulted in increased cell number and alkaline
phosphatase activity expression, and measured by increased of deposited
calcium. This coculture system appears to enhance osteoblastic differentiateon
and produce extracellular mineralizing matrix.In the indirect coculture system,
there was a significant increase in cell number and alkaline phosphatase activity
expression in the presence of MC3T3-E1 CM in C3H10T1/2 cell. But the
amounts of deposited calcium were low. We propose MC3T2-E1 CM may
support initial proliferation and ALP activity but do not alter the ability of the
osteoblasts to produce extracellular mineralizing matrix.

並列關鍵字

Embryonic stem cells ； Coculture ； Osteoblast ； Osteogenic different

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