【摘要】：背景:目前已有較多實(shí)驗(yàn)研究證實(shí)低頻脈沖電磁場在二維支架下促進(jìn)多種干細(xì)胞向成骨細(xì)胞分化,但鮮有研究磁場在三維支架下對干細(xì)胞的增殖及成骨分化的影響。目的:探討在聚已內(nèi)酯3D培養(yǎng)支架環(huán)境下,外加低頻脈沖電磁場對人脂肪源干細(xì)胞增殖及成骨分化的影響。方法:將第3代人脂肪源干細(xì)胞接種于聚已內(nèi)酯3D培養(yǎng)支架上,分2組培養(yǎng),實(shí)驗(yàn)組給予50 Hz、1 m T的低頻脈沖電磁場干預(yù),2 h/d,連續(xù)干預(yù)14 d;對照組不給于低頻脈沖電磁場干預(yù)。培養(yǎng)7 d后,Live/Dead染色觀察細(xì)胞存活情況;培養(yǎng)1,3,5,7 d后,MTT法檢測細(xì)胞增殖;培養(yǎng)7,14 d,堿性磷酸酶染色及qR T-PCR檢測細(xì)胞成骨分化水平。結(jié)果與結(jié)論:(1)Live/Dead染色:人脂肪源干細(xì)胞已長入到支架材料的空隙結(jié)構(gòu)內(nèi)部,在材料中可保持較高的活性;(2)細(xì)胞增殖:隨著培養(yǎng)時間的延長,兩組細(xì)胞A值均逐漸升高,實(shí)驗(yàn)組培養(yǎng)1,3 d的細(xì)胞A值高于對照組(P0.05);(3)堿性磷酸酶染色:實(shí)驗(yàn)組培養(yǎng)7,14 d的堿性磷酸酶陽性染色強(qiáng)于對照組;(4)qR T-PCR檢測:培養(yǎng)7 d時,實(shí)驗(yàn)組堿性磷酸酶、Ⅰ型膠原基因表達(dá)量高于對照組(P0.01);培養(yǎng)14 d時,實(shí)驗(yàn)組骨橋蛋白、Runt相關(guān)轉(zhuǎn)錄因子2、堿性磷酸酶及Ⅰ型膠原的表達(dá)量均明顯高于對照組(P0.05);(5)結(jié)果表明:在聚已內(nèi)酯3D培養(yǎng)支架環(huán)境下,外加低頻脈沖電磁場可促進(jìn)人脂肪源干細(xì)胞的增殖及成骨分化。
[Abstract]:Background: at present, many experimental studies have confirmed that low-frequency pulsed electromagnetic fields can promote the differentiation of stem cells into osteoblasts under two-dimensional scaffolds, but little has been done on the effects of magnetic field on the proliferation and osteogenic differentiation of stem cells under three-dimensional scaffolds. Aim: to investigate the effects of low frequency pulsed electromagnetic field on proliferation and osteogenic differentiation of human adipose-derived stem cells in polycaprolactone 3D culture scaffold. Methods: the third generation of human adipose derived stem cells were inoculated on polycaprolactone 3D culture scaffold and cultured in two groups. The experimental group was treated with 50 Hz,1 Mt low frequency pulsed electromagnetic field for 2 h / d for 14 days. The control group was not treated with low frequency pulsed electromagnetic field. After 7 days of culture, the survival of cells was observed by Live/Dead staining, the proliferation of cells was detected by MTT assay, and the osteogenic differentiation level of cells was detected by alkaline phosphatase staining and qR T-PCR at 714 days after culture. Results and conclusions: (1) Live/Dead staining: human adipose-derived stem cells (HSCs) had grown into the interstitial structure of the scaffold and could maintain high activity in the scaffold. (2) Cell proliferation: with the extension of culture time, the A value of the two groups increased gradually, and the A value of the experimental group was higher than that of the control group (P0.05). (3) Alkaline phosphatase staining: the alkaline phosphatase positive staining in the experimental group was stronger than that in the control group for 714 days. (4) qR T-PCR detection: after 7 days of culture, the expression of alkaline phosphatase and collagen 鈪，

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