【摘要】：目的:研究刺頭復葉耳蕨總黃酮(total flavonoids from arachniodes exilis,TFAE)在人臍帶間充質干細胞(human umbilical cord mesenchymal stem cells,hUCMSCs)成骨分化中的作用,并進一步研究雌激素受體(estrogen receptor,ER)信號途徑在其中的作用。方法:(1)采用組織塊貼壁法分離、培養(yǎng)hUCMSCs,倒置相差顯微鏡觀察細胞形態(tài)及生長。以下所有實驗均采用第3代對數(shù)生長期hUCMSCs進行。(2)TFAE對hUCMSCs活性的影響:實驗分為5組:0μg/mLTFAE組(對照組),1μg/mLTFAE組,5μg/mLTFAE組,10μg/mLTFAE組,20μg/m LTFAE組,分別作用24h、48h、72h后,CCK-8法檢測細胞活性。(3)TFAE對hUCMSCs成骨分化的影響:根據(jù)細胞活性檢測結果,實驗分為4組:對照組(常規(guī)培養(yǎng)基),0μg/mLTFAE組(0μg/mLTFAE的成骨誘導培養(yǎng)基),1μg/mLTFAE組(1μg/mLTFAE的成骨誘導培養(yǎng)基),5μg/mLTFAE組(5μg/m LTFAE的成骨誘導培養(yǎng)基),誘導培養(yǎng)3d、7d后,AMP法測定ALP活力;誘導培養(yǎng)14d后,茜素紅染色檢測鈣結節(jié),RT-PCR檢測成骨相關基因Ⅰ型膠原a1(collagen type I alpha1,Col1a1)、骨橋蛋白(osteopotin,OPN)、Runx2、Osterix(Osx)mRNA表達水平。(4)ER在hUCMSCs成骨分化中的作用:實驗分為4組:對照組(0μg/m LTFAE的成骨誘導培養(yǎng)基),TFAE組(5μg/mLTFAE的成骨誘導培養(yǎng)基),TFAE+S組(5μg/mLTFAE的成骨誘導培養(yǎng)基+S1191),S組(S1191),誘導培養(yǎng)3d、7d后,AMP法測定ALP活力;誘導培養(yǎng)14d后,茜素紅染色檢測鈣結節(jié),RT-PCR檢測成骨相關基因Col1a1、OPN、Runx2、OsxmRNA表達水平。結果:(1)臍帶組織貼壁培養(yǎng)3-5d后,培養(yǎng)皿中有細胞長出,培養(yǎng)10-14d,組織塊周圍長滿成纖維樣細胞,經傳代培養(yǎng)后,細胞形態(tài)均一,貼壁良好。(2)細胞活性檢測結果:CCK-8檢測結果顯示,與對照組相比,1μg/mLTFAE組、5μg/mLTFAE組細胞活性明顯增強(P0.05或P0.01),10μg/mLTFAE組、20μg/mLTFAE組細胞活性明顯減弱(P0.05或P0.01)。(3)hUCMSCs成骨分化檢測結果:1)ALP檢測結果顯示:與對照組相比,0μg/mLTFAE、1μg/m LTFAE組及5μg/m LTFAE組細胞ALP活力明顯增強(P0.05或P0.01),與0μg/mLTFAE組,1μg/mLTFAE組及5μg/m LTFAE組細胞ALP活力也明顯增強(P0.05或P0.01);2)茜素紅染色結果顯示:不同濃度TFAE組均有鈣結節(jié)形成,而對照組未見鈣結節(jié),與0μg/mLTFAE組相比,1μg/mLTFAE組及5μg/mLTFAE組鈣結節(jié)數(shù)量明顯增多(P0.01);3)RT-PCR檢測結果顯示:與對照組相比,0μg/mLTFAE組、1μg/mLTFAE組及5μg/m LTFAE組成骨相關基因Col1a1、OPN、Runx2、OsxmRNA表達量明顯增加(P0.01),與0μg/mLTFAE組相比,1μg/mLLTFAE組及5μg/mLTFAE組Col1a1、OPN、Runx2、OsxmRNA表達量也明顯增加(P0.05或P0.01)。(4)ER抑制劑作用下,hUCMSCs成骨分化檢測結果:1)ALP檢測結果顯示:與對照組相比,TFAE組細胞ALP活力明顯增強(P0.01),與TFAE組相比,TFAE+S組細胞ALP活力明顯減弱(P0.05或P0.01),對照組與S組無差異;2)茜素紅染色結果顯示:各組均有鈣結節(jié)形成,與對照組相比,TFAE組鈣結節(jié)數(shù)量增多(P0.01),而與TFAE組相比,TFAE+S組鈣結節(jié)數(shù)量明顯減少(P0.01),對照組與S組無差異;3)RT-PCR檢測結果顯示:與對照組相比,TFAE組成骨相關基因Col1a1、OPN、Runx2、OsxmRNA表達量明顯增加(P0.01),與TFAE組相比,TFAE+S組Col1a1、OPN、Runx2、OsxmRNA表達量明顯減少(P0.05或P0.01),對照組與S組無差異。結論:(1)一定濃度的TFAE增強hUCMSCs活性及促進其成骨分化。(2)TFAE通過ER信號途徑促進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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R68

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