利用肌腱细胞进行肌腱组织工程的研究

发布时间：2018-05-15 03:35

  本文选题：肌腱细胞 + 生长因子　； 参考：《天津医科大学》2010年博士论文

【摘要】：人肌腱细胞用含有不同浓度的胎牛血清和生长因子组合(PDGFBB、IGF-1, bFGF和IGFβ-3)在α-MEM培养基中培养。实验部分析因设计系用于筛选具有下述功能的生长因子组合(1)在最少量胎牛血清条件下,促进细胞增殖且保持细胞处于未分化状态；(2)在无胎牛血清条件下维持细胞存活并促进细胞分化；(3)序贯使用上述两种培养方法在二维和三维条件下对肌腱细胞进行培养,观察肌腱细胞分化能力；(4)序贯使用上述培养方法对肌腱细胞进行培养,并将其移植入裸鼠股四头肌内,观察这种方法培养的肌腱细胞在体内的分化能力。用含胎牛血清(0%,1%,5%和10%)和不同浓度PDGFBB (0ng/ml,5ng/ml,10ng/ml和50ng/ml), IGF-1(0ng/ml,10ng/ml和50ng/ml), bFGF (0ng/ml,5ng/ml,10ng/ml和50ng/ml)和TGF β-3(Ong/ml,1ng/ml和50ng/ml)组合添加至a-MEM培养基,培养肌腱细胞。结果显示,(1)含有1%胎牛血清,50ng/ml PDGFBB和50ng/ml bFG的培养基培养肌腱细胞14天后的细胞数量和含10%胎牛血清培养基的培养结果相当。然而胶原合成量和肌腱细胞分化标志物mRNA的表达却较10%胎牛血清组显著下调。虽然低浓度IGF-1血清不能促进细胞增殖,但能有效促进细胞分化。肌腱细胞形态学同样证实,含有1%胎牛血清,50ng/ml PDGFBB和50ng/ml bFGF的培养基培养的细胞处于未分化状态；(2)在无血清培养基中(含50ng/mⅡGF-1和10ng/ml TGF β-3),肌腱细胞存活了14天且细胞处于分化状态。与含有10%的胎牛血清组的培养基组相比,实验组的胶原合成及肌腱细胞分化标志物mRNA表达明显上调。细胞形态学也证实,实验组细胞分化程度要明显高于对照组(10%胎牛血清组)。(3)先后应用加入生长因子组的低/无血清培养基二维培养各14天(共28天),肉眼即可见到致密的胶原结构形成。在10%胎牛血清组(对照组,无生长因子),即使延长培养至45天,也未见肉眼可见的胶原结构的形成。(4)以脱胶Bombix蚕丝为细胞支架,序贯应用低/无血清培养基进行三维培养各14天(共28天),可见与人正常肌腱微结构相似的肌腱样结构产生,且人工肌腱力学测试结果优于人正常肌腱；(5)先后应用不同的培养基方法培养出来的肌腱细胞在体内也具备分化能力,与10%的胎牛血清组培养的肌腱细胞相比,序贯应用生长因子的方法培养的肌腱细胞为定向分化,所以没有产生我们不需要的骨与软骨组织。 这是一项创新性研究。我们首次展示了(1)培养基内加入PDGFBB与bFGF可以把胎牛血清的使用量降至1%,而同样达到10%胎牛血清的增殖速率；(2)在无胎牛血清条件下,培养基内加入TGFβ与IGF-1可以使肌腱细胞能存活14天以上,且可以促进细胞的分化；(3)序贯用上述方法在二维和三维培养环境中培养的肌腱细胞具有显著的细胞分化能力；(4)序贯用上述方法培养的肌腱细胞在动物体内移植试验中具有定向分化成肌腱样组织的能力。
[Abstract]:Human tendon cells were cultured in 伪 -MEM medium with different concentrations of fetal bovine serum and growth factor combination PDGF BBF IGF-1, bFGF and IGF 尾 -3. The experiment section analyzed that because the design department was used to screen growth factor combination 1, which had the following functions, under the condition of minimal fetal bovine serum, Promoting cell proliferation and keeping the cells in an undifferentiated state) maintaining cell survival and promoting cell differentiation in the absence of fetal bovine serum.) the two methods mentioned above were used to culture tendon cells in two and three dimensional conditions. To observe the differentiation ability of tendon cells and to observe the differentiation ability of tendon cells in vivo, the tendon cells were cultured and transplanted into the quadriceps femoris muscle of nude mice. 5% and 10% with fetal bovine serum and 10% with different concentrations of PDGFBB. Add 10 ng / ml 10ng / ml and 50ng / ml / ml IGF-1ngml / ml 10ng / ml and 50ng / ml / ml, bFGF 0ngr / ml 5nggrml / ml and TGF 尾 -3Ong / ml 1ngrml and 50ngmlr / ml) to a-MEM medium to culture tendon cells. The results showed that the number of tendon cells cultured in the medium containing 1% fetal bovine serum 50 ng / ml PDGFBB and 50ng/ml bFG for 14 days was the same as that in the medium containing 10% fetal bovine serum. However, collagen synthesis and mRNA expression of tendon cell differentiation markers were significantly decreased compared with 10% fetal bovine serum group. Although low concentration of IGF-1 serum can not promote cell proliferation, but can effectively promote cell differentiation. The morphology of tendon cells also confirmed that the cells cultured in the culture medium containing 50 ng / ml PDGFBB and 50ng/ml bFGF of 1% fetal bovine serum were in undifferentiated condition (50ng/m 鈪，

本文编号：1890815