SDF-1α復合Pluronic F-127對BMSCs體外誘導成骨的實驗觀察
發(fā)布時間：2018-08-17 09:23
【摘要】：目的以Pluronic F-127為支架材料，構建裝載基質(zhì)細胞衍生因子-1α（stromal cellderived factor-1α，SDF-1α）的Pluronic F-127新型復合材料，探討其對大鼠骨髓間充質(zhì)干細胞（bone marrow stromal cells，BMSCs）的體外趨化作用，并將PluronicF-127與BMSCs復合培養(yǎng)，觀察兩者間生物相容性，進而研究復合SDF-1α水凝膠對BMSCs誘導分化成成骨細胞的影響。 研究方法構建裝載不同濃度SDF-1α（200、50ng/ml）的Pluronic F-127復合水凝膠，采用Transwell小室法進行復合水凝膠對BMSCs的體外趨化實驗，通過對遷移BMSCs的計數(shù)與統(tǒng)計分析，篩選出趨化作用較好的復合水凝膠。將BMSCs與篩選出的復合水凝膠均勻混合培養(yǎng)，用MTT法檢測復合培養(yǎng)后細胞的活性狀況。將BMSCs與篩選出的復合水凝膠混合均勻，待凝膠形成后加成骨誘導液進行培養(yǎng)，同時分別設置單純BMSCs的成骨誘導液培養(yǎng)和完全培養(yǎng)基培養(yǎng)，對以上各行堿性磷酸酶（alkaline phosphatase，AKP）活性測定及茜素紅染色，觀察并記錄各檢測結果，分析各BMSCs的成骨誘導分化情況。 結果趨化結果顯示，含SDF-1α濃度為200ng/ml的復合水凝膠對BMSCs的募集效果顯著，與含SDF-1α濃度為50ng/ml的復合水凝膠相比，其趨化下室濾膜表面的細胞轉(zhuǎn)移數(shù)目明顯增多（P0.05）。MTT法證實Pluronic F-127對細胞增殖不產(chǎn)生影響，BMSCs-水凝膠復合培養(yǎng)與單純BMSCs培養(yǎng)相比，兩者間細胞活性差異并無統(tǒng)計學意義（P0.05）。AKP活性測定表明，，BMSCs-復合水凝膠、單純BMSCs在成骨誘導培養(yǎng)條件下，兩者中細胞的AKP表達量均明顯增高，并且都高于非成骨誘導組（P0.05），同時經(jīng)成骨誘導液培養(yǎng)的細胞，在鏡下均可見明顯的橘紅色鈣結節(jié)團塊，而非加成骨誘導液培養(yǎng)的細胞，鏡下則未見鈣結節(jié)形成。 結論成功構建裝載SDF-1α的Pluronic F-127新型復合材料，其在體外對BMSCs有明顯的趨化作用，在Pluronic F-127水凝膠中，BMSCs能夠存活較好并能進行正常的生長及增殖，并且在細胞-復合水凝膠復合培養(yǎng)的三維支架中，BMSCs能夠被成功誘導分化為成骨細胞。
[Abstract]:Objective to construct a novel Pluronic F-127 composite loaded with stromal cell derived factor 1 偽 (stromal cellderived factor-1 偽 (SDF-1 偽) and investigate its chemotactic effect on rat bone marrow mesenchymal stem cells (bone marrow stromal cells) in vitro, and to co-culture PluronicF-127 with BMSCs. The effect of SDF-1 偽 hydrogel on the differentiation of osteoblasts induced by BMSCs was studied. Methods Pluronic F-127 hydrogels loaded with different concentrations of SDF-1 偽 (200ng / ml) were constructed. The chemotactic experiments of BMSCs were carried out by Transwell chamber method. The chemotactic complex hydrogels with good chemotaxis were screened by counting and statistical analysis of BMSCs migration. The BMSCs and the selected composite hydrogels were cultured uniformly and the activity of the cells was detected by MTT method. The mixture of BMSCs and the selected composite hydrogels was mixed evenly. After the gel was formed, the osteoblast was cultured with osteogenic inducer, and the pure BMSCs osteogenic medium and the complete culture medium were set up respectively. The activity of alkaline phosphatase (alkaline) and alizarin red staining were measured, the results were observed and recorded, and the osteogenic differentiation of each BMSCs was analyzed. Results the chemotaxis results showed that the composite hydrogel containing SDF-1 偽 concentration of 200ng/ml had significant effect on the recruitment of BMSCs, compared with the composite hydrogel containing SDF-1 偽 concentration of 50ng/ml. The number of cell metastases on the surface of the membrane increased significantly (P0.05). The results showed that Pluronic F-127 had no effect on cell proliferation. Compared with BMSCs culture, Pluronic F-127 had no effect on cell proliferation. There was no significant difference in cell activity between the two groups (P0.05) .AKP activity test showed that the expression of AKP was significantly increased in osteogenic culture with BMSCs alone. Both of them were higher than those of non-osteogenic induction group (P0.05). At the same time, the cells cultured with osteoblast could be seen the obvious orange calcium nodule mass under the microscope, but the cells cultured with non-additive osteoinductive fluid had no calcium nodule formation under the microscope. Conclusion the novel Pluronic F-127 composite loaded with SDF-1 偽 has obvious chemotactic effect on BMSCs in vitro. It can survive well and can grow and proliferate normally in Pluronic F-127 hydrogel. BMSCs could be successfully induced to differentiate into osteoblasts in three dimensional scaffolds cultured with cell-composite hydrogel.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783
[Abstract]:Objective to construct a novel Pluronic F-127 composite loaded with stromal cell derived factor 1 偽 (stromal cellderived factor-1 偽 (SDF-1 偽) and investigate its chemotactic effect on rat bone marrow mesenchymal stem cells (bone marrow stromal cells) in vitro, and to co-culture PluronicF-127 with BMSCs. The effect of SDF-1 偽 hydrogel on the differentiation of osteoblasts induced by BMSCs was studied. Methods Pluronic F-127 hydrogels loaded with different concentrations of SDF-1 偽 (200ng / ml) were constructed. The chemotactic experiments of BMSCs were carried out by Transwell chamber method. The chemotactic complex hydrogels with good chemotaxis were screened by counting and statistical analysis of BMSCs migration. The BMSCs and the selected composite hydrogels were cultured uniformly and the activity of the cells was detected by MTT method. The mixture of BMSCs and the selected composite hydrogels was mixed evenly. After the gel was formed, the osteoblast was cultured with osteogenic inducer, and the pure BMSCs osteogenic medium and the complete culture medium were set up respectively. The activity of alkaline phosphatase (alkaline) and alizarin red staining were measured, the results were observed and recorded, and the osteogenic differentiation of each BMSCs was analyzed. Results the chemotaxis results showed that the composite hydrogel containing SDF-1 偽 concentration of 200ng/ml had significant effect on the recruitment of BMSCs, compared with the composite hydrogel containing SDF-1 偽 concentration of 50ng/ml. The number of cell metastases on the surface of the membrane increased significantly (P0.05). The results showed that Pluronic F-127 had no effect on cell proliferation. Compared with BMSCs culture, Pluronic F-127 had no effect on cell proliferation. There was no significant difference in cell activity between the two groups (P0.05) .AKP activity test showed that the expression of AKP was significantly increased in osteogenic culture with BMSCs alone. Both of them were higher than those of non-osteogenic induction group (P0.05). At the same time, the cells cultured with osteoblast could be seen the obvious orange calcium nodule mass under the microscope, but the cells cultured with non-additive osteoinductive fluid had no calcium nodule formation under the microscope. Conclusion the novel Pluronic F-127 composite loaded with SDF-1 偽 has obvious chemotactic effect on BMSCs in vitro. It can survive well and can grow and proliferate normally in Pluronic F-127 hydrogel. BMSCs could be successfully induced to differentiate into osteoblasts in three dimensional scaffolds cultured with cell-composite hydrogel.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R783
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