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Application of miRNA

A hsa-mir-4788, hsa-mir-196b-5p technology, applied in the field of molecular biology, can solve problems such as unclear roles

Active Publication Date: 2017-12-26
BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the role of miRNAs in the regulation of odontogenic differentiation of mesenchymal stem cells is still unclear.

Method used

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Embodiment 1

[0045] Example 1 Cell Culture and Osteogenic Differentiation

[0046] All research involving human stem cells adheres to the ISSCR "Guidelines for Conduct in Human Embryonic Stem Cell Research". Dental tissues were obtained with the consent of the patients and strictly followed the guidelines of the Beijing Stomatological Hospital Affiliated to Capital Medical University. Teeth were first sterilized with 75% alcohol and then rinsed with phosphate buffered solution. Periodontal ligament stem cells, dental pulp stem cells and apical papilla stem cells were isolated, cultured and identified as previously described. Briefly, apical papilla stem cells were isolated from the apical tooth papilla, periodontal ligament stem cells were isolated from the periodontal ligament in the middle third of the root, and dental pulp stem cells were isolated from the coronal pulp. Next, these stem cells were placed in a solution of 3 mg / ml collagenase I (Worthington Biochemical Corp., Lakewood, ...

Embodiment 2

[0048] Example 2 total RNA isolation and microarray hybridization

[0049] Three groups of total RNA samples from periodontal ligament stem cells, dental pulp stem cells, apical papilla stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells and adipose mesenchymal stem cells were obtained by using extraction reagents and RNA extraction. Extraction kits (Qiagen, Germany) were presented separately. Total RNA was quantified by a spectrophotometer ND-2100 (Thermo Fisher), and RNA integrity was assessed using an Agilent 2100 (Agilent Technologies). Each group of total RNA samples were further purified using RNA extraction kit and RNase-Free DNase Set (QIAGEN, GmBH, Germany). Microarray analysis used human MiRNAs 4.0 array (Affymetrix). Sample labeling, microarray hybridization, and washing were performed based on the manufacturer's standards. Briefly, total RNA was polyadenylated and subsequently labeled with biotin. Afterwards, the labeled RNA is...

Embodiment 3

[0050] Embodiment 3 biological information analysis

[0051] Bioinformatics analysis was completed (Compass biotechnology Ltd, Beijing, China). Affymetrix Microarray Command Console software (version 4.0, Affymetrix) was used to analyze microarray images to obtain raw data and then provide normalization for receptor modulation analysis. Then, R software was used to continue the data analysis below. Identification of differentially expressed miRNAs by fold changes. The target genes of differentially expressed miRNAs are the intersection points predicted by five databases (miRanda, miRDB, miRTarbase, TargetScan, and miRecords). KEGG analysis was used to determine the role of these target genes. Hierarchical clustering was used to reveal distinguishable expression patterns of miRNAs across samples.

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Abstract

The invention relates to the field of molecular biology, and particularly relates to an application of miRNA. 15 miRNAs non-odontogenic tissue derived mesenchymal stem cells and odontogenic tissue derived mesenchymal stem cells are differentially expressed, wherein compared with odontogenic tissue derived mesenchymal stem cells, 9 non-odontogenic tissue derived mesenchymal stem cells are highly expressed, while 6 odontogenic tissue derived mesenchymal stem cells are lowly expressed. Inhibition of candidate miRNAs(hsa-miR-615-3p, hsa-miR-196b-5p) can be used for improving the dentin differentiation potential of WJCMSCs. Furthermore, seven key target genes which are highly expressed in dental tissues derived from mesenchymal stem cells can be obtained, including TFAP2C, SEMA6D, PDGFRA, PAX9, NR4A3, SATB2 and ZNF367.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to the application of miRNA. Background technique [0002] Dental chipping and loss are common and recurring conditions that can affect chewing efficiency, speech, digestion, and even mental health. The current treatment is a non-biological therapy, and it is difficult to reconstruct the normal structure and function of natural teeth. With the development of biotechnology, the application of mesenchymal stem cells combined with tissue engineering technology to promote oral tissue regeneration has become a frontier and important scientific issue. [0003] Mesenchymal stem cells are postpartum stem cells that can be isolated from the mesenchymal tissue of the bone marrow, have the ability to self-renew, and differentiate into a variety of mesenchymal components that make up bone, dentin, adipose tissue, cartilage, tendon, and skeletal muscle Potential of cell lines. Nowadays, mesen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N5/077
CPCC12N5/0654C12N2500/40C12N2506/1346C12Q1/6888C12Q2600/158C12Q2600/178
Inventor 范志朋杨昊清曹杨杨林潇
Owner BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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