Kit for screening steroid-induced necrosis of femoral head

A femoral head necrosis and kit technology, applied in the fields of biomolecules and medicine, can solve the problems that cannot fully explain the pathological process of femoral head necrosis

Inactive Publication Date: 2019-04-19
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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  • Kit for screening steroid-induced necrosis of femoral head

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 is not to 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 f...

Embodiment 2

[0051] Example 2 Real-Time PCR verification of miR-100-5p 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)

miRNART Primer (RPrimer)

1μL

sonRNA202R Primer

1μL

dNTP (2.5mM)

1μL

DEPCwater

9-x μL

[0061] Mix gently, p...

Embodiment 3

[0079] Embodiment 3 kit preparation

[0080] Based on the primers obtained in Example 2, assemble the test kit for the prognostic diagnosis of steroid-induced necrosis of the femoral head according to the present invention, the kit includes (1) containing reverse transcriptase buffer, reverse transcription primers, reverse transcriptase, etc. Reverse transcription reagent; (2) The forward primer for specific amplification of miR-100 is shown in SEQ ID NO: 1, and the reverse primer is a universal primer such as SEQ ID NO: 2: and specific amplification of snRNA T6 (as an internal reference) The primer pair is shown as SEQ ID NO:5 and SEQ ID NO:6; (3) also includes SYBR Green polymerase chain reaction system, such as PCR buffer, SYBR Green fluorescent dye, dNTPs. The composition of described PCR buffer is 25mM KCl, 2.5mM MgCl 2 , 200mM (NH4) 2 SO 4 .

[0081] Through the optimization of primer concentration and annealing temperature, the reaction system was finally determined...

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Abstract

The invention discloses a kit for screening the steroid-induced necrosis of the femoral head. A molecular marker for screening the steroid-induced necrosis of the femoral head in the early period is proposed, and miR-100 expresses up-regulation in a sample of the steroid-induced necrosis of the femoral head; application of miR-100 in preparation of the kit for screening the steroid-induced necrosis of the femoral head and application of miR-100 in candidate medicine for screening or preventing or treating the steroid-induced necrosis of the femoral head are further provided. It is discovered that miR-100 is high in specificity and sensitivity, can be effectively applied to early detection of the steroid-induced necrosis of the femoral head and can be applied to clinical application of genetreatment, medicine treatment and the like.

Description

technical field [0001] The invention relates to the fields of biomolecules and medicine, in particular to a kit for screening steroid-induced necrosis of the femoral head. Background technique [0002] Osteonecrosis offemoral head (ONFH) is often called avascular necrosis of the femoral head (avascμLarnecrosis, AVN) because its main pathology is the blockage of blood supply to the femoral head and the destruction of the head bone ischemia. It is a common and refractory orthopedic disease that occurs in young and middle-aged people, and the disability rate is extremely high. 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,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/118C12Q2600/178
Inventor 边焱焱王英杰王龙超李曾程开源翁习生
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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