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Biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells

A technology of bone marrow mesenchyme and biological preparations, applied in the field of biomedicine, can solve problems such as unclear biological functions of lncRNA

Active Publication Date: 2019-04-05
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the biological roles of lncRNAs in the pathogenesis of AIS are still unclear

Method used

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  • Biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
  • Biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells
  • Biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0128] Example 1: Identification of LncAIS as a significantly differentially expressed lncRNA down-regulated in bone marrow mesenchymal stem cells (BM-MSC) of AIS patients

[0129] In order to identify the key lncRNA involved in adolescent idiopathic scoliosis (AIS), we performed microarray analysis on BM-MSCs from 5 healthy donors and 12 AIS patients. In the BM-MSC of normal BM-MSC and AIS patients, 1483 lncRNA showed differential expression, of which 718 were up-regulated and 765 were down-regulated. figure 1 shown in a. Among the most down-regulated lncRNAs in the BM-MSCs of AIS patients, we focused on the uncharacterized lncRNA that we called lncAIS (gene symbol: ENST00000453347). LncAIS is located on human chromosome 1, contains 4 exons and is a transcript of 476 nt in full length. lncAIS was identified as a conserved locus, such as figure 1 Shown in b.

[0130] Perform real-time qPCR according to routine operations to analyze LncAIS transcripts in BM-MSCs of normal healthy ...

Embodiment 2

[0136] Example 2: LncAIS knockdown inhibits osteogenic differentiation and bone formation

[0137] In order to explore the physiological role of lncAIS in the pathogenesis of AIS, we used lentivirus-mediated short hairpin RNA (shRNA) to knock down lncAIS in healthy normal BM-MSC (the shLncAIS gene sequence used for lncAIS knockdown is shown in Table 2 above Shown in SEQ ID NO: 4-6), and confirm knockdown efficiency by real-time quantitative PCR, as figure 2 shown in a. BM-MSC was infected with lentivirus expressing shlnAIS and cultured in MSC medium for 3 days, and then the mRNA level of lncAIS was detected by real-time qPCR. The primer pairs used for cDNA amplification in qRT-PCR are listed in Table 5 above. The relative gene expression fold change was normalized to endogenous β-actin and counted as the mean±S.D. **, P <0.01. The data are from three independent experiments using BM-MSCs derived from 3 healthy donors.

[0138] The cell proliferation of shlnAIS BM-MSC and shCtr...

Embodiment 3

[0146] Example 3: Overexpression of LncAIS promotes osteogenic differentiation and bone formation

[0147] We overexpress lncAIS in normal BM-MSC by lentivirus, first construct the target sequence into pSicoR plasmid, and simultaneously use pMD2.G and psPAX2 vectors to package the virus, and then infect the target BM-MSC. Overexpression of LncAIS (oeLncAIS) significantly increases the cell proliferation of normal BM-MSC ( image 3 a). Consistently, lncAIS overexpression enhanced the expression levels of self-renewal related genes (NANOG, POU5F1, and SOX2) and osteogenic differentiation genes (ALPL, BSP, RUNX2, LPL, and PPAR) ( image 3 b, 3c). In addition, compared with vector-transfected BM-MSCs during adult differentiation, lncAIS overexpression also increased osteogenic marker genes such as osteopontin (OPN), COL1A1 and IBSP ( image 3 d) Protein levels. Therefore, staining by ALP ( image 3 e) and Von Kossa stain ( image 3 f) Overexpression of lncAIS enhances the osteogenic...

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Abstract

The invention discloses a biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC), and a pharmaceutical composition for prevention and / or treatment ofadolescent idiopathic scoliosis (AIS). The inventor finds that lncAIS is key lncRNA involved in progress of AIS, wherein the lncAIS interacts with NF90 to promote HOXD8mRNA stability, thus transcription of RUNX2 in the BM-MSC is enhanced, and thus osteogenic differentiation of the normal BM-MSC is caused. By contrast, the NF90 cannot be collected due to down-regulating of the lncAIS in the BM-MSCof an AIS patient , thus the HOXD8mRNA stability is eliminated, and in this way, transcription of the RUNX2 for osteogenic differentiation is hindered. Thus, the BM-MSC of overexpression lncAIS, HOXD8or the RUNX2 can be used for preparing the biological agent for promoting osteogenic differentiation of the BM-MSC and the pharmaceutical composition for prevention and / or treatment of the AIS, and thus the new means is provided for prevention and / or treatment of the AIS.

Description

Technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a biological agent for promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSC) and a pharmaceutical composition for treating juvenile idiopathic scoliosis (AIS). Background technique [0002] Adolescent idiopathic scoliosis (Adolescent idiopathic scoliosis, AIS) is a complex three-dimensional deformity of the spine, which mainly occurs in girls aged 10 to 16 during puberty. The progression of scoliosis may appear deformities, back pain, and dysfunction. In severe cases, physical function problems such as cardiopulmonary function limitation may occur, which affects social activities to varying degrees. Epidemiological survey results show that the global incidence of AIS among adolescents is about 2-4%, and about 10% of those diagnosed with AIS need treatment. The current treatment of AIS includes brace therapy and surgical correction; among them...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61K45/06A61P19/08
CPCA61K35/28A61K45/06A61P19/08
Inventor 庄乾宇仉建国惠尚懿范祖森邱贵兴吴志宏叶步青赵春华李静李娜王升儒杨阳林莞峰张延斌
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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