【摘要】： 目的研究周期性壓力培養(yǎng)對兔骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)增殖活性及基質(zhì)金屬蛋白酶-2(matrix metalloproteinase 2, MMP-2)的影響,探索影響B(tài)MSCs增殖的最佳力學(xué)參數(shù),達(dá)到大量擴(kuò)增的目的。 方法密度梯度離心法分離培養(yǎng)新西蘭大白兔BMSCs,取生長良好的第3代細(xì)胞,按4×104個(gè)/孔接種于6孔板,設(shè)立5組:5.32 kPa, 10.64 kPa, 21.28 kPa, 29.26 kPa (1 kPa=7.5 mmHg)壓力組、對照組(正常大氣壓培養(yǎng))。分別給予對應(yīng)的壓力培養(yǎng),每日加壓6 h×3 d;正常培養(yǎng)組細(xì)胞只接受正常大氣壓刺激,不予額外加壓。通過四甲基偶氮唑(MTT)比色試驗(yàn),流式細(xì)胞儀(flow cytometer, FCM)鑒定細(xì)胞表面抗原陽性表達(dá)及檢測細(xì)胞增殖周期,同時(shí)收集培養(yǎng)液作MMP-2質(zhì)量濃度檢測。觀察不同周期性壓力對BMSCs增殖及MMP-2的影響。 結(jié)果6 h/d壓力干預(yù)BMSCs,3 d后細(xì)胞形態(tài)無異常變化,呈集落樣生長。隨著壓力的增加,細(xì)胞吸光度值逐漸升高,以21.28 kPa壓力組最高,但升高至29.26 kPa壓力時(shí)吸光度值明顯降低(F=3.731, P=0.008)。與正常培養(yǎng)組比較,5.32, 10.64, 21.28 kPa壓力組細(xì)胞周期發(fā)生改變,增殖指數(shù)(proliferation index, PI)均明顯升高(P 0.05或0.01),但29.26 kPa壓力組PI則明顯下降。細(xì)胞培養(yǎng)上清液MMP-2質(zhì)量濃度以21.28 kPa壓力組最低,29.26 kPa壓力組最高,組間比較差異有顯著性意義(t=213.214,P 0.001)。 結(jié)論5.32～21.28 kPa的周期性壓力可以提高BMSCs的增殖能力,特別是21.28 kPa壓力刺激促增殖作用尤為明顯,并降低培養(yǎng)液中的MMP-2的濃度。但壓力過高則抑制細(xì)胞增殖,培養(yǎng)液中的MMP-2的濃度升高,導(dǎo)致細(xì)胞死亡增多。
[Abstract]:Objective to study the effect of cyclic pressure culture on (bone marrow mesenchymal stem cells, BMSCs) proliferation activity and matrix metalloproteinase-2 (matrix metalloproteinase 2 (MMP-2) of rabbit bone marrow mesenchymal stem cells (BMSCs), and to explore the best mechanical parameters affecting the proliferation of BMSCs. To achieve the purpose of a large number of amplification. Methods New Zealand white rabbit BMSCs, cells were isolated and cultured by density gradient centrifugation. The third passage cells were inoculated into 6 well plates according to 4 脳 104 cells per well. The cells were divided into 5 groups: 5.32 kPa, 10.64 kPa, 21.28 kPa,. 29.26 kPa (1 kPa=7.5 mmHg) pressure group and control group (normal atmospheric pressure culture). The cells in the normal culture group received only normal atmospheric pressure stimulation, but no additional pressure. The positive expression of cell surface antigen and cell proliferation cycle were identified by flow cytometry (flow cytometer, FCM) and tetramethyl azo (MTT) colorimetric assay. Meanwhile, the culture medium was collected to detect the mass concentration of MMP-2. The effects of different periodic pressures on BMSCs proliferation and MMP-2 were observed. Results there was no abnormal change in cell morphology after 6 h / d pressure intervention on BMSCs,3 d, and the cells grew like colony. With the increase of the pressure, the absorbance value of the cells increased gradually, especially in the 21.28 kPa pressure group, but the absorbance value decreased significantly when the pressure reached 29.26 kPa (FN 3.731, P0. 008). Compared with the normal culture group, the cell cycle of 5.32, 10.64 and 21.28 kPa stress groups changed, and the proliferation index (proliferation index, PI) increased significantly (P 0.05 or 0.01), but the PI decreased significantly in 29.26 kPa pressure group. The concentration of MMP-2 in the supernatant of cell culture was the lowest in 21.28 kPa pressure group and the highest in 29.26 kPa pressure group. There was a significant difference between the two groups (t _ (213.214) P _ (0.001). Conclusion the periodic pressure of 5.32 ~ 21.28 kPa can increase the proliferative ability of BMSCs, especially when stimulated by 21.28 kPa, and decrease the concentration of MMP-2 in the culture medium. However, high pressure inhibited cell proliferation and increased MMP-2 concentration in the culture medium, resulting in the increase of cell death.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R329.28

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