刺头复叶耳蕨总黄酮对人脐带间充质干细胞成骨分化的作用及机制研究
发布时间：2018-09-12 06:44
【摘要】：目的:研究刺头复叶耳蕨总黄酮(total flavonoids from arachniodes exilis,TFAE)在人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)成骨分化中的作用,并进一步研究雌激素受体(estrogen receptor,ER)信号途径在其中的作用。方法:(1)采用组织块贴壁法分离、培养hUCMSCs,倒置相差显微镜观察细胞形态及生长。以下所有实验均采用第3代对数生长期hUCMSCs进行。(2)TFAE对hUCMSCs活性的影响:实验分为5组:0μg/mLTFAE组(对照组),1μg/mLTFAE组,5μg/mLTFAE组,10μg/mLTFAE组,20μg/m LTFAE组,分别作用24h、48h、72h后,CCK-8法检测细胞活性。(3)TFAE对hUCMSCs成骨分化的影响:根据细胞活性检测结果,实验分为4组:对照组(常规培养基),0μg/mLTFAE组(0μg/mLTFAE的成骨诱导培养基),1μg/mLTFAE组(1μg/mLTFAE的成骨诱导培养基),5μg/mLTFAE组(5μg/m LTFAE的成骨诱导培养基),诱导培养3d、7d后,AMP法测定ALP活力;诱导培养14d后,茜素红染色检测钙结节,RT-PCR检测成骨相关基因Ⅰ型胶原a1(collagen type I alpha1,Col1a1)、骨桥蛋白(osteopotin,OPN)、Runx2、Osterix(Osx)mRNA表达水平。(4)ER在hUCMSCs成骨分化中的作用:实验分为4组:对照组(0μg/m LTFAE的成骨诱导培养基),TFAE组(5μg/mLTFAE的成骨诱导培养基),TFAE+S组(5μg/mLTFAE的成骨诱导培养基+S1191),S组(S1191),诱导培养3d、7d后,AMP法测定ALP活力;诱导培养14d后,茜素红染色检测钙结节,RT-PCR检测成骨相关基因Col1a1、OPN、Runx2、OsxmRNA表达水平。结果:(1)脐带组织贴壁培养3-5d后,培养皿中有细胞长出,培养10-14d,组织块周围长满成纤维样细胞,经传代培养后,细胞形态均一,贴壁良好。(2)细胞活性检测结果:CCK-8检测结果显示,与对照组相比,1μg/mLTFAE组、5μg/mLTFAE组细胞活性明显增强(P0.05或P0.01),10μg/mLTFAE组、20μg/mLTFAE组细胞活性明显减弱(P0.05或P0.01)。(3)hUCMSCs成骨分化检测结果:1)ALP检测结果显示:与对照组相比,0μg/mLTFAE、1μg/m LTFAE组及5μg/m LTFAE组细胞ALP活力明显增强(P0.05或P0.01),与0μg/mLTFAE组,1μg/mLTFAE组及5μg/m LTFAE组细胞ALP活力也明显增强(P0.05或P0.01);2)茜素红染色结果显示:不同浓度TFAE组均有钙结节形成,而对照组未见钙结节,与0μg/mLTFAE组相比,1μg/mLTFAE组及5μg/mLTFAE组钙结节数量明显增多(P0.01);3)RT-PCR检测结果显示:与对照组相比,0μg/mLTFAE组、1μg/mLTFAE组及5μg/m LTFAE组成骨相关基因Col1a1、OPN、Runx2、OsxmRNA表达量明显增加(P0.01),与0μg/mLTFAE组相比,1μg/mLLTFAE组及5μg/mLTFAE组Col1a1、OPN、Runx2、OsxmRNA表达量也明显增加(P0.05或P0.01)。(4)ER抑制剂作用下,hUCMSCs成骨分化检测结果:1)ALP检测结果显示:与对照组相比,TFAE组细胞ALP活力明显增强(P0.01),与TFAE组相比,TFAE+S组细胞ALP活力明显减弱(P0.05或P0.01),对照组与S组无差异;2)茜素红染色结果显示:各组均有钙结节形成,与对照组相比,TFAE组钙结节数量增多(P0.01),而与TFAE组相比,TFAE+S组钙结节数量明显减少(P0.01),对照组与S组无差异;3)RT-PCR检测结果显示:与对照组相比,TFAE组成骨相关基因Col1a1、OPN、Runx2、OsxmRNA表达量明显增加(P0.01),与TFAE组相比,TFAE+S组Col1a1、OPN、Runx2、OsxmRNA表达量明显减少(P0.05或P0.01),对照组与S组无差异。结论:(1)一定浓度的TFAE增强hUCMSCs活性及促进其成骨分化。(2)TFAE通过ER信号途径促进hUCMSCs成骨分化。
[Abstract]:AIM: To investigate the role of total flavonoids from Arachniodes exilis (TFAE) in the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) and the role of estrogen receptor (ER) signaling pathway in the osteogenic differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs). HUCMSCs were isolated and cultured by tissue adherence method and observed by inverted phase contrast microscope. All the following experiments were carried out by the third generation of logarithmic growth hUCMSCs. (2) The effect of TFAE on the activity of hUCMSCs was divided into five groups: 0 ug/mLTFAE group (control group), 1 ug/mLTFAE group, 5 ug/mLTFAE group, 10 ug/mLTFAE group and 20 ug/mLTFAE group. (3) Effect of TFAE on osteogenic differentiation of hUCMSCs: According to the results of cell activity test, the experiment was divided into four groups: control group (conventional medium), 0 ug/mLTFAE group (0 ug/mLTFAE osteogenic induction medium), 1 ug/mLTFAE group (1 ug/mLTFAE osteogenic induction medium), 5 ug/mLTFAE group (5 ug/mLTFAE osteogenic induction medium). Alizarin red staining was used to detect calcium nodules, and RT-PCR was used to detect the expression of collagen type I alpha 1 (Col1a1), osteopontin (OPN), Runx2, Osterix (Osx) mRNA in hUCMSCs. Four groups: control group (0 ug/m LTFAE osteogenic induction medium), TFAE group (5 ug/m LTFAE osteogenic induction medium), TFAE+S group (5 ug/m LTFAE osteogenic induction medium + S1191), S group (S1191), induction culture 3 days, 7 days later, the ALP activity was measured by AMP method; 14 days after induction culture, alizarin red staining was used to detect calcium nodules, and RT-PCR was used to detect the osteogenic related gene Col1a1, OPN-PCR. Results: (1) Cells grew in the culture dish for 10-14 days, and fibroblast-like cells grew around the tissue blocks. After subculture, the cells were uniform in morphology and adherent well. (2) Cell viability test results: CCK-8 test results showed that compared with the control group, 1 ug / mLTFAE group, 5 UG / mLTFAE group. Cell viability was significantly increased in group A (P 0.05 or P 0.01), significantly decreased in group A (P 0.05 or P 0.01), and significantly decreased in group B (P 0.05 or P 0.01). (3) Osteogenic differentiation of hUCMSCs: 1) ALP assay showed that compared with control group, ALP activity was significantly increased in group B (P 0.05 or P 0.01), and group B (P 0.05 or P 0.01). Alizarin red staining showed that calcium nodules were formed in all TFAE groups, but no calcium nodules were found in the control group. Compared with 0 ug/mLTFAE group, the number of calcium nodules in 1 ug/mLTFAE group and 5 ug/mLTFAE group increased significantly (P 0.01). Compared with the control group, the expressions of Col1a1, OPN, Runx2 and Osx mRNA in 0 ug/mLTFAE group, 1 ug/mLTFAE group and 5 ug/mLTFAE group were significantly increased (P 0.01). Compared with 0 ug/mLTFAE group, the expressions of Col1a1, OPN, Runx2 and Osx mRNA in 1 ug/mLLTFAE group and 5 ug/mLTFAE group were also significantly increased (P 0.05 or P 0.01). (4) Under the effect of ER inhibitors, the expression of hUCMSCs in osteogenic differentiation was detected. Results: 1) Alizarin red staining showed that: compared with the control group, TFAE group cells ALP activity significantly increased (P 0.01), compared with TFAE group, TFAE + S group cells ALP activity significantly decreased (P 0.05 or P 0.01), the control group and S group no difference; 2) Alizarin red staining showed that all groups had calcium nodules formation, compared with the control group, TFAE group increased the number of calcium nodules (P 0.01). Compared with TFAE group, the number of calcium nodules in TFAE+S group decreased significantly (P 0.01), but there was no difference between the control group and S group; 3) RT-PCR results showed that compared with the control group, the expression of bone-related genes Col1a1, OPN, Runx2, OsxmRNA in TFAE+S group increased significantly (P 0.01), and the expression of Col1a1, OPN, Runx2, OsxmRNA in TFAE+S group decreased significantly (P 0.05 or P CONCLUSION: (1) TFAE at a certain concentration enhances the activity of hUCMSCs and promotes osteogenic differentiation. (2) TFAE promotes the osteogenic differentiation of hUCMSCs through ER signaling pathway.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68
本文编号：2238216
[Abstract]:AIM: To investigate the role of total flavonoids from Arachniodes exilis (TFAE) in the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) and the role of estrogen receptor (ER) signaling pathway in the osteogenic differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs). HUCMSCs were isolated and cultured by tissue adherence method and observed by inverted phase contrast microscope. All the following experiments were carried out by the third generation of logarithmic growth hUCMSCs. (2) The effect of TFAE on the activity of hUCMSCs was divided into five groups: 0 ug/mLTFAE group (control group), 1 ug/mLTFAE group, 5 ug/mLTFAE group, 10 ug/mLTFAE group and 20 ug/mLTFAE group. (3) Effect of TFAE on osteogenic differentiation of hUCMSCs: According to the results of cell activity test, the experiment was divided into four groups: control group (conventional medium), 0 ug/mLTFAE group (0 ug/mLTFAE osteogenic induction medium), 1 ug/mLTFAE group (1 ug/mLTFAE osteogenic induction medium), 5 ug/mLTFAE group (5 ug/mLTFAE osteogenic induction medium). Alizarin red staining was used to detect calcium nodules, and RT-PCR was used to detect the expression of collagen type I alpha 1 (Col1a1), osteopontin (OPN), Runx2, Osterix (Osx) mRNA in hUCMSCs. Four groups: control group (0 ug/m LTFAE osteogenic induction medium), TFAE group (5 ug/m LTFAE osteogenic induction medium), TFAE+S group (5 ug/m LTFAE osteogenic induction medium + S1191), S group (S1191), induction culture 3 days, 7 days later, the ALP activity was measured by AMP method; 14 days after induction culture, alizarin red staining was used to detect calcium nodules, and RT-PCR was used to detect the osteogenic related gene Col1a1, OPN-PCR. Results: (1) Cells grew in the culture dish for 10-14 days, and fibroblast-like cells grew around the tissue blocks. After subculture, the cells were uniform in morphology and adherent well. (2) Cell viability test results: CCK-8 test results showed that compared with the control group, 1 ug / mLTFAE group, 5 UG / mLTFAE group. Cell viability was significantly increased in group A (P 0.05 or P 0.01), significantly decreased in group A (P 0.05 or P 0.01), and significantly decreased in group B (P 0.05 or P 0.01). (3) Osteogenic differentiation of hUCMSCs: 1) ALP assay showed that compared with control group, ALP activity was significantly increased in group B (P 0.05 or P 0.01), and group B (P 0.05 or P 0.01). Alizarin red staining showed that calcium nodules were formed in all TFAE groups, but no calcium nodules were found in the control group. Compared with 0 ug/mLTFAE group, the number of calcium nodules in 1 ug/mLTFAE group and 5 ug/mLTFAE group increased significantly (P 0.01). Compared with the control group, the expressions of Col1a1, OPN, Runx2 and Osx mRNA in 0 ug/mLTFAE group, 1 ug/mLTFAE group and 5 ug/mLTFAE group were significantly increased (P 0.01). Compared with 0 ug/mLTFAE group, the expressions of Col1a1, OPN, Runx2 and Osx mRNA in 1 ug/mLLTFAE group and 5 ug/mLTFAE group were also significantly increased (P 0.05 or P 0.01). (4) Under the effect of ER inhibitors, the expression of hUCMSCs in osteogenic differentiation was detected. Results: 1) Alizarin red staining showed that: compared with the control group, TFAE group cells ALP activity significantly increased (P 0.01), compared with TFAE group, TFAE + S group cells ALP activity significantly decreased (P 0.05 or P 0.01), the control group and S group no difference; 2) Alizarin red staining showed that all groups had calcium nodules formation, compared with the control group, TFAE group increased the number of calcium nodules (P 0.01). Compared with TFAE group, the number of calcium nodules in TFAE+S group decreased significantly (P 0.01), but there was no difference between the control group and S group; 3) RT-PCR results showed that compared with the control group, the expression of bone-related genes Col1a1, OPN, Runx2, OsxmRNA in TFAE+S group increased significantly (P 0.01), and the expression of Col1a1, OPN, Runx2, OsxmRNA in TFAE+S group decreased significantly (P 0.05 or P CONCLUSION: (1) TFAE at a certain concentration enhances the activity of hUCMSCs and promotes osteogenic differentiation. (2) TFAE promotes the osteogenic differentiation of hUCMSCs through ER signaling pathway.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68
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