hMSCs成骨分化相关IncRNAs的筛选和鉴定
发布时间：2019-02-12 11:49
【摘要】：目的：筛选和鉴定人骨髓间充质干细胞(hMSCs)成骨分化相关的lncRNAs。方法：利用Refseq, UCSC knowngenes, Ensembl,H-invDB7.0, RNAdb2.0, NRED生物软件分析成骨基因相关的lncRNAs，综合获得可能的成骨相关lncRNAs；密度梯度法分离hMSCs，地塞米松、维生素C、β-甘油磷酸钠诱导hMSCs成骨分化，，通过ALP测定及钙结节茜素红染色进行成骨诱导鉴定；qRT-PCR验证hMSCs成骨分化前、后差异表达的lncRNAs，以及成骨分化前、后差异表达的基因BMP1、MSX1、Runx2、Smurf-1、ALP。 结果：通过上述生物软件综合分析发现3个成骨基因MSX1、BMP1和Smurf1周围存在相关lncRNAs。4个lncRNAs (AK129811、AK024937、AK096529、uc003ups)与成骨基因Smurf-1相关；2个lncRNAs(AF289591和uc003xbe)与成骨基因BMP1相关；1个lncRNAs(AK056311)与成骨基因MSX1相关。ALP检测示：与对照组细胞相比，成骨诱导组细胞1周ALP值最高，达到16.82±1.64nmol/min·OD450，随着诱导时间的延长，ALP值逐渐降低，3周降至8.37±1.01nmol/min·OD450。钙结节染色显示hMSCs成骨诱导分化2周后胞外开始出现少量钙结节，随着诱导时间延长钙结节数量逐渐增加，成骨诱导至第3周钙结节最多，定量测定钙浓度达716.10±49.46mg/ml。RT-qPCR结果显示绝大部分lncRNAs在hMSCs成骨分化过程中表达均下调，其中2个与Smurf-1相关lncRNAs (AK096529和uc003ups)与成骨诱导前相比，存在显著性下调。1个与MSX1相关的lncRNAs(AK056311)与成骨诱导前相比，也存在显著性下调。同时，成骨分化基因Runx2、ALP表达显著性上调，MSX1、Smurf-1表达显著性下调。 结论：结合既往研究结果分析：AK096529和uc003ups通过正向调控Smurf1，促使Smurf1下调，减少Runx2的降解，继而促进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 卤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 卤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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2420414
[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 卤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 卤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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前1条
1 张维成;;不同浓度地塞米松对骨髓基质细胞成脂、成骨分化的影响[J];中国临床康复;2006年29期
本文编号：2420414
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