【摘要】：目的:研究刺頭復(fù)葉耳蕨總黃酮(total flavonoids from arachniodes exilis,TFAE)在人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,hUCMSCs)成骨分化中的作用,并進(jìn)一步研究雌激素受體(estrogen receptor,ER)信號(hào)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(xì)胞形態(tài)及生長(zhǎng)。以下所有實(shí)驗(yàn)均采用第3代對(duì)數(shù)生長(zhǎng)期hUCMSCs進(jìn)行。(2)TFAE對(duì)hUCMSCs活性的影響:實(shí)驗(yàn)分為5組:0μg/mLTFAE組(對(duì)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(cè)細(xì)胞活性。(3)TFAE對(duì)hUCMSCs成骨分化的影響:根據(jù)細(xì)胞活性檢測(cè)結(jié)果,實(shí)驗(yàn)分為4組:對(duì)照組(常規(guī)培養(yǎng)基),0μg/mLTFAE組(0μg/mLTFAE的成骨誘導(dǎo)培養(yǎng)基),1μg/mLTFAE組(1μg/mLTFAE的成骨誘導(dǎo)培養(yǎng)基),5μg/mLTFAE組(5μg/m LTFAE的成骨誘導(dǎo)培養(yǎng)基),誘導(dǎo)培養(yǎng)3d、7d后,AMP法測(cè)定ALP活力;誘導(dǎo)培養(yǎng)14d后,茜素紅染色檢測(cè)鈣結(jié)節(jié),RT-PCR檢測(cè)成骨相關(guān)基因Ⅰ型膠原a1(collagen type I alpha1,Col1a1)、骨橋蛋白(osteopotin,OPN)、Runx2、Osterix(Osx)mRNA表達(dá)水平。(4)ER在hUCMSCs成骨分化中的作用:實(shí)驗(yàn)分為4組:對(duì)照組(0μg/m LTFAE的成骨誘導(dǎo)培養(yǎng)基),TFAE組(5μg/mLTFAE的成骨誘導(dǎo)培養(yǎng)基),TFAE+S組(5μg/mLTFAE的成骨誘導(dǎo)培養(yǎng)基+S1191),S組(S1191),誘導(dǎo)培養(yǎng)3d、7d后,AMP法測(cè)定ALP活力;誘導(dǎo)培養(yǎng)14d后,茜素紅染色檢測(cè)鈣結(jié)節(jié),RT-PCR檢測(cè)成骨相關(guān)基因Col1a1、OPN、Runx2、OsxmRNA表達(dá)水平。結(jié)果:(1)臍帶組織貼壁培養(yǎng)3-5d后,培養(yǎng)皿中有細(xì)胞長(zhǎng)出,培養(yǎng)10-14d,組織塊周圍長(zhǎng)滿成纖維樣細(xì)胞,經(jīng)傳代培養(yǎng)后,細(xì)胞形態(tài)均一,貼壁良好。(2)細(xì)胞活性檢測(cè)結(jié)果:CCK-8檢測(cè)結(jié)果顯示,與對(duì)照組相比,1μg/mLTFAE組、5μg/mLTFAE組細(xì)胞活性明顯增強(qiáng)(P0.05或P0.01),10μg/mLTFAE組、20μg/mLTFAE組細(xì)胞活性明顯減弱(P0.05或P0.01)。(3)hUCMSCs成骨分化檢測(cè)結(jié)果:1)ALP檢測(cè)結(jié)果顯示:與對(duì)照組相比,0μg/mLTFAE、1μg/m LTFAE組及5μg/m LTFAE組細(xì)胞ALP活力明顯增強(qiáng)(P0.05或P0.01),與0μg/mLTFAE組,1μg/mLTFAE組及5μg/m LTFAE組細(xì)胞ALP活力也明顯增強(qiáng)(P0.05或P0.01);2)茜素紅染色結(jié)果顯示:不同濃度TFAE組均有鈣結(jié)節(jié)形成,而對(duì)照組未見(jiàn)鈣結(jié)節(jié),與0μg/mLTFAE組相比,1μg/mLTFAE組及5μg/mLTFAE組鈣結(jié)節(jié)數(shù)量明顯增多(P0.01);3)RT-PCR檢測(cè)結(jié)果顯示:與對(duì)照組相比,0μg/mLTFAE組、1μg/mLTFAE組及5μg/m LTFAE組成骨相關(guān)基因Col1a1、OPN、Runx2、OsxmRNA表達(dá)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(dá)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(cè)結(jié)果:1)ALP檢測(cè)結(jié)果顯示:與對(duì)照組相比,TFAE組細(xì)胞ALP活力明顯增強(qiáng)(P0.01),與TFAE組相比,TFAE+S組細(xì)胞ALP活力明顯減弱(P0.05或P0.01),對(duì)照組與S組無(wú)差異;2)茜素紅染色結(jié)果顯示:各組均有鈣結(jié)節(jié)形成,與對(duì)照組相比,TFAE組鈣結(jié)節(jié)數(shù)量增多(P0.01),而與TFAE組相比,TFAE+S組鈣結(jié)節(jié)數(shù)量明顯減少(P0.01),對(duì)照組與S組無(wú)差異;3)RT-PCR檢測(cè)結(jié)果顯示:與對(duì)照組相比,TFAE組成骨相關(guān)基因Col1a1、OPN、Runx2、OsxmRNA表達(dá)量明顯增加(P0.01),與TFAE組相比,TFAE+S組Col1a1、OPN、Runx2、OsxmRNA表達(dá)量明顯減少(P0.05或P0.01),對(duì)照組與S組無(wú)差異。結(jié)論:(1)一定濃度的TFAE增強(qiáng)hUCMSCs活性及促進(jìn)其成骨分化。(2)TFAE通過(guò)ER信號(hào)途徑促進(jìn)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R68

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