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Application of miR-99a in steroid-induced femoral head necrosis

A mir-99a, 1.mir-99a technology, applied in the fields of biomolecular science and medicine, can solve the problems that cannot fully explain the pathological process of femoral head necrosis

Inactive Publication Date: 2019-04-16
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The research on SANFH has always been a hot spot in orthopedic research. Because of its complex etiology, there are many hypotheses about the pathogenesis of SANFH, such as coagulation disorder theory, lipid metabolism theory, femoral head high pressure theory, vascular endothelial injury theory, bone marrow The theory of mesenchymal stem cell dysfunction, etc., although these studies have some clinical and experimental research basis, they cannot fully explain the entire pathological process of femoral head necrosis

Method used

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  • Application of miR-99a in steroid-induced femoral head necrosis
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 High-throughput sequencing screening of differentially expressed genes

[0032] 1. Sample

[0033] Collect blood samples from 5 patients with steroid-induced necrosis of the femoral head, numbered F1-F5; collect blood samples from 3 relatively healthy subjects. Number N1 suffered from traumatic necrosis of the femoral head, and N2 and N3 suffered from acetabular dysplasia. The samples were collected from patients who visited the Department of Orthopedics of Peking Union Medical College Hospital from October 2015 to May 2018. All clinical samples in this study were informed to the patients and approved by the Hospital Ethics Committee.

[0034] 2. Extract total RNA from the sample

[0035] 2.1 RNA extraction sample processing

[0036] Mix EDTA anticoagulated whole blood with Trizol at a ratio of 1:1. If RNA extraction will not be performed within a short period of time, the sample can be quickly frozen in liquid nitrogen for 30s-1min, and stored at -80°C fo...

Embodiment 2

[0051] Example 2 Real-Time PCR verification of miR-99a-5p expression in patients with steroid-induced necrosis of the femoral head

[0052] 1. Materials

[0053] Fifteen blood samples from patients with steroid-induced necrosis of the femoral head and 5 blood samples from normal subjects were selected, and grouped and numbered. All samples were confirmed by pathological examination.

[0054] 2. See Example 1 for RNA extraction steps

[0055] 3. Reverse transcription

[0056] Using total RNA as a template, miRNA cDNA was synthesized using Transcript Reverse Transcriptase and Ribonuclease Inhibitor reverse transcription kits.

[0057] Synthesize the first cDNA strand:

[0058] Using a microvolume EP tube, add the following system A (12 μL) in sequence:

[0059] Table 1 System A

[0060] RNA (2μg)

XμL(x≤9)

miRNA RT Primer (RPrimer)

1μL

sonRNA 202R Primer

1μL

dNTP (2.5mM)

1μL

DEPCwater

9-x μL

[0061] Mix gently,...

Embodiment 3

[0079] Example 3 Isolation, culture, induction and identification of human bone marrow-derived mesenchymal stem cells

[0080] Isolation and culture: Human bone marrow mesenchymal stem cells (BMSC) are derived from the N1 patient in Example 1. During total hip replacement, a sterile bone marrow puncture needle was used to extract bone marrow tissue when the femoral prosthesis trial was opened. 10mL, the method of separation from bone marrow tissue refers to the method of Pittenger et al (Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al.Multilineage potential of adult human mesenchymalstem cell[J]. (5411):143-147) isolated BMSC cells and cultured to the third generation.

[0081] Induction of differentiation: replace the osteogenic induction medium (H-DMEM medium containing 10% fetal bovine serum, 10nm dexamethasone, 0.2mM ascorbic acid and 10mM β-glycerophosphate sodium) to continue culturing, and change the medium once every two days . Osteogenesis w...

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Abstract

The invention relates to application of miR-99a in steroid-induced femoral head necrosis, discloses application of miR-99a serving as a marker to prepare a product detecting steroid-induced femoral head necrosis, and discloses application of an miR-99a inhibitor in preparation of a drug for curing steroid-induced femoral head necrosis. The drug contains the miR-99a inhibitor which serves as an active ingredient. The miR-99a is high in specificity and sensitivity, can be effectively applied to early detection of steroid-induced femoral head necrosis and has clinical application in genetic therapy, drug therapy and the like.

Description

technical field [0001] The invention relates to the fields of biomolecules and medicine, in particular to the application of miR-99a in steroid-induced necrosis of the femoral head. Background technique [0002] Osteonecrosis of femoral head (ONFH) is often called avascular necrosis of the femoral head (avascμLarnecrosis, AVN) because the main pathology is the blockage of the blood supply of the femoral head and the damage to the head bone ischemia. It is a common and refractory orthopedic disease that occurs in young and middle-aged people, with a high disability rate. In my country, the number of ONFH patients who need treatment is 5-7.5 million each year, and the number of new patients is 150,000-200,000 each year. Most of the patients are young adults aged 37-45. The number of males is more than that of females. The ratio of male to female in my country It is 3.8:1. Steroid-induced necrosis of the femoral head (steroid-induced avascμLar necrosis of the femoral head, SAN...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883A61K45/00A61K31/7088A61P19/08
CPCA61K31/7088A61K45/00A61P19/08C12Q1/6883C12Q2600/158C12Q2600/178
Inventor 翁习生项永波边焱焱李曾程开源
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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