【摘要】：目的：篩選和鑒定人骨髓間充質(zhì)干細(xì)胞(hMSCs)成骨分化相關(guān)的lncRNAs。方法：利用Refseq, UCSC knowngenes, Ensembl,H-invDB7.0, RNAdb2.0, NRED生物軟件分析成骨基因相關(guān)的lncRNAs，綜合獲得可能的成骨相關(guān)lncRNAs；密度梯度法分離hMSCs，地塞米松、維生素C、β-甘油磷酸鈉誘導(dǎo)hMSCs成骨分化，，通過ALP測(cè)定及鈣結(jié)節(jié)茜素紅染色進(jìn)行成骨誘導(dǎo)鑒定；qRT-PCR驗(yàn)證hMSCs成骨分化前、后差異表達(dá)的lncRNAs，以及成骨分化前、后差異表達(dá)的基因BMP1、MSX1、Runx2、Smurf-1、ALP。 結(jié)果：通過上述生物軟件綜合分析發(fā)現(xiàn)3個(gè)成骨基因MSX1、BMP1和Smurf1周圍存在相關(guān)lncRNAs。4個(gè)lncRNAs (AK129811、AK024937、AK096529、uc003ups)與成骨基因Smurf-1相關(guān)；2個(gè)lncRNAs(AF289591和uc003xbe)與成骨基因BMP1相關(guān)；1個(gè)lncRNAs(AK056311)與成骨基因MSX1相關(guān)。ALP檢測(cè)示：與對(duì)照組細(xì)胞相比，成骨誘導(dǎo)組細(xì)胞1周ALP值最高，達(dá)到16.82±1.64nmol/min·OD450，隨著誘導(dǎo)時(shí)間的延長(zhǎng)，ALP值逐漸降低，3周降至8.37±1.01nmol/min·OD450。鈣結(jié)節(jié)染色顯示hMSCs成骨誘導(dǎo)分化2周后胞外開始出現(xiàn)少量鈣結(jié)節(jié)，隨著誘導(dǎo)時(shí)間延長(zhǎng)鈣結(jié)節(jié)數(shù)量逐漸增加，成骨誘導(dǎo)至第3周鈣結(jié)節(jié)最多，定量測(cè)定鈣濃度達(dá)716.10±49.46mg/ml。RT-qPCR結(jié)果顯示絕大部分lncRNAs在hMSCs成骨分化過程中表達(dá)均下調(diào)，其中2個(gè)與Smurf-1相關(guān)lncRNAs (AK096529和uc003ups)與成骨誘導(dǎo)前相比，存在顯著性下調(diào)。1個(gè)與MSX1相關(guān)的lncRNAs(AK056311)與成骨誘導(dǎo)前相比，也存在顯著性下調(diào)。同時(shí)，成骨分化基因Runx2、ALP表達(dá)顯著性上調(diào)，MSX1、Smurf-1表達(dá)顯著性下調(diào)。 結(jié)論：結(jié)合既往研究結(jié)果分析：AK096529和uc003ups通過正向調(diào)控Smurf1，促使Smurf1下調(diào)，減少Runx2的降解，繼而促進(jìn)hMSCs的成骨分化。
[Abstract]:Objective: to screen and identify lncRNAs. associated with osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs) Methods: possible osteoblast related lncRNAs; was synthesized by Refseq, UCSC knowngenes, Ensembl,H-invDB7.0, RNAdb2.0, NRED analysis of lncRNAs, associated with osteogenic gene. HMSCs, dexamethasone, vitamin C, 尾 -glycerophosphate were isolated by density gradient method. The osteogenic differentiation of hMSCs was induced by ALP and calcium nodule alizarin red staining. QRT-PCR was used to verify the differential expression of lncRNAs, before and after osteogenic differentiation of hMSCs and the gene BMP1,MSX1,Runx2,Smurf-1,ALP. before and after osteogenic differentiation. Results: through the comprehensive analysis of the above biological software, it was found that there was a correlation between lncRNAs.4 lncRNAs (AK129811,AK024937,AK096529,uc003ups) and osteogenic gene Smurf-1 around the three osteogenic genes MSX1,BMP1 and Smurf1, two lncRNAs (AF289591 and uc003xbe) associated with the osteogenic gene BMP1, and two lncRNAs (AF289591 and uc003xbe) were associated with the osteogenic gene BMP1. One lncRNAs (AK056311) was associated with osteogenic gene MSX1. ALP analysis showed that the ALP value of osteoblast induction group was the highest in 1 week compared with the control group, and the ALP value decreased gradually with the prolongation of induction time. 3 weeks down to 8.37 鹵1.01nmol/min OD450. Calcium nodule staining showed that a small number of extracellular calcium nodules began to appear 2 weeks after osteogenic differentiation of hMSCs, and the number of calcium nodules increased gradually with the prolongation of induction time, and the number of calcium nodules increased to the third week after osteogenesis induction. The results of quantitative determination of calcium concentration up to 716.10 鹵49.46mg/ml.RT-qPCR showed that most of lncRNAs expression was down-regulated during hMSCs osteogenic differentiation, two of which were compared with Smurf-1 related lncRNAs (AK096529 and uc003ups) and pre-osteogenic induction. A lncRNAs (AK056311) associated with MSX1 was significantly down-regulated compared with that before osteogenesis. At the same time, the expression of osteogenic differentiation gene Runx2,ALP was significantly up-regulated and the expression of MSX1,Smurf-1 was significantly down-regulated. Conclusion: according to the analysis of previous studies, AK096529 and uc003ups can promote the down-regulation of Smurf1 through the positive regulation of Smurf1, reduce the degradation of Runx2, and then promote the osteogenic differentiation of hMSCs.
【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】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張維成;;不同濃度地塞米松對(duì)骨髓基質(zhì)細(xì)胞成脂、成骨分化的影響[J];中國臨床康復(fù);2006年29期

本文編號(hào)：2420414