本文選題：肌腱細(xì)胞 + 生長因子　； 參考：《天津醫(yī)科大學(xué)》2010年博士論文

【摘要】：人肌腱細(xì)胞用含有不同濃度的胎牛血清和生長因子組合(PDGFBB、IGF-1, bFGF和IGFβ-3)在α-MEM培養(yǎng)基中培養(yǎng)。實(shí)驗(yàn)部分析因設(shè)計(jì)系用于篩選具有下述功能的生長因子組合(1)在最少量胎牛血清條件下,促進(jìn)細(xì)胞增殖且保持細(xì)胞處于未分化狀態(tài)；(2)在無胎牛血清條件下維持細(xì)胞存活并促進(jìn)細(xì)胞分化；(3)序貫使用上述兩種培養(yǎng)方法在二維和三維條件下對(duì)肌腱細(xì)胞進(jìn)行培養(yǎng),觀察肌腱細(xì)胞分化能力；(4)序貫使用上述培養(yǎng)方法對(duì)肌腱細(xì)胞進(jìn)行培養(yǎng),并將其移植入裸鼠股四頭肌內(nèi),觀察這種方法培養(yǎng)的肌腱細(xì)胞在體內(nèi)的分化能力。用含胎牛血清(0%,1%,5%和10%)和不同濃度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)組合添加至a-MEM培養(yǎng)基,培養(yǎng)肌腱細(xì)胞。結(jié)果顯示,(1)含有1%胎牛血清,50ng/ml PDGFBB和50ng/ml bFG的培養(yǎng)基培養(yǎng)肌腱細(xì)胞14天后的細(xì)胞數(shù)量和含10%胎牛血清培養(yǎng)基的培養(yǎng)結(jié)果相當(dāng)。然而膠原合成量和肌腱細(xì)胞分化標(biāo)志物mRNA的表達(dá)卻較10%胎牛血清組顯著下調(diào)。雖然低濃度IGF-1血清不能促進(jìn)細(xì)胞增殖,但能有效促進(jìn)細(xì)胞分化。肌腱細(xì)胞形態(tài)學(xué)同樣證實(shí),含有1%胎牛血清,50ng/ml PDGFBB和50ng/ml bFGF的培養(yǎng)基培養(yǎng)的細(xì)胞處于未分化狀態(tài)；(2)在無血清培養(yǎng)基中(含50ng/mⅡGF-1和10ng/ml TGF β-3),肌腱細(xì)胞存活了14天且細(xì)胞處于分化狀態(tài)。與含有10%的胎牛血清組的培養(yǎng)基組相比,實(shí)驗(yàn)組的膠原合成及肌腱細(xì)胞分化標(biāo)志物mRNA表達(dá)明顯上調(diào)。細(xì)胞形態(tài)學(xué)也證實(shí),實(shí)驗(yàn)組細(xì)胞分化程度要明顯高于對(duì)照組(10%胎牛血清組)。(3)先后應(yīng)用加入生長因子組的低/無血清培養(yǎng)基二維培養(yǎng)各14天(共28天),肉眼即可見到致密的膠原結(jié)構(gòu)形成。在10%胎牛血清組(對(duì)照組,無生長因子),即使延長培養(yǎng)至45天,也未見肉眼可見的膠原結(jié)構(gòu)的形成。(4)以脫膠Bombix蠶絲為細(xì)胞支架,序貫應(yīng)用低/無血清培養(yǎng)基進(jìn)行三維培養(yǎng)各14天(共28天),可見與人正常肌腱微結(jié)構(gòu)相似的肌腱樣結(jié)構(gòu)產(chǎn)生,且人工肌腱力學(xué)測(cè)試結(jié)果優(yōu)于人正常肌腱；(5)先后應(yīng)用不同的培養(yǎng)基方法培養(yǎng)出來的肌腱細(xì)胞在體內(nèi)也具備分化能力,與10%的胎牛血清組培養(yǎng)的肌腱細(xì)胞相比,序貫應(yīng)用生長因子的方法培養(yǎng)的肌腱細(xì)胞為定向分化,所以沒有產(chǎn)生我們不需要的骨與軟骨組織。 這是一項(xiàng)創(chuàng)新性研究。我們首次展示了(1)培養(yǎng)基內(nèi)加入PDGFBB與bFGF可以把胎牛血清的使用量降至1%,而同樣達(dá)到10%胎牛血清的增殖速率；(2)在無胎牛血清條件下,培養(yǎng)基內(nèi)加入TGFβ與IGF-1可以使肌腱細(xì)胞能存活14天以上,且可以促進(jìn)細(xì)胞的分化；(3)序貫用上述方法在二維和三維培養(yǎng)環(huán)境中培養(yǎng)的肌腱細(xì)胞具有顯著的細(xì)胞分化能力；(4)序貫用上述方法培養(yǎng)的肌腱細(xì)胞在動(dòng)物體內(nèi)移植試驗(yàn)中具有定向分化成肌腱樣組織的能力。
[Abstract]:Human tendon cells were cultured in 偽 -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，

本文編號(hào)：1890815