Application of LILRA2 gene and expression product thereof as diagnosis and treatment target of multiple myeloma

A multiple myeloma and genetic technology, applied in the determination/testing of microorganisms, antineoplastic drugs, drug combinations, etc.

Active Publication Date: 2016-10-12
QINGDAO MEDINTELL BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, hormones, proteasome inhibitors, and immunomodulators are mainly used in the treatment of myeloma. The treatment of these drugs can make MM remission, but it is inevitable to relapse in the end. Therefore, new diagnostic and therapeutic strategies need to be discovered

Method used

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  • Application of LILRA2 gene and expression product thereof as diagnosis and treatment target of multiple myeloma
  • Application of LILRA2 gene and expression product thereof as diagnosis and treatment target of multiple myeloma
  • Application of LILRA2 gene and expression product thereof as diagnosis and treatment target of multiple myeloma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Gene Chip Screening for Differentially Expressed Genes

[0070] 1. Materials:

[0071] Multiple myeloma tissue: A total of 10 bone marrow biopsy specimens were collected from confirmed MM patients. The diagnostic criteria for MM refer to the "Chinese Guidelines for the Diagnosis and Treatment of Multiple Myeloma 2011 Revised Edition". Among the patients, there were 5 males and 5 females, with a median age of 59 years.

[0072] Normal bone marrow tissue: 10 bone marrow biopsy specimens were collected from patients with malnutrition anemia at the same period as the control group, including 5 males and 5 females, with a median age of 53 years.

[0073] 2. Obtaining tissue RNA

[0074] Total tissue RNA was extracted using the Trizol one-step method.

[0075] 3. Determination of RNA purity and concentration

[0076] Take 1 μl of RNA solution, measure OD260 and OD280 with the instrument, the RNA concentration is OD260 value × dilution factor × 40 / 1000, calculate...

Embodiment 2

[0089] Example 2 Large sample verification screened out differentially expressed genes

[0090] Considering that there is no gene in the prior art that has been studied on the correlation between this gene and multiple myeloma as a candidate gene, and considering the results of gene sequencing, the LILRA2 gene (its expression is down-regulated in multiple myeloma tissues) is selected for verification .

[0091] 1. Sample collection

[0092] According to the method of Example 1, 50 cases of multiple myeloma tissues and 60 cases of normal bone marrow tissues were collected.

[0093] 2. Validation at the mRNA level

[0094] 2.1 Extract tissue RNA

[0095] Step is with embodiment 1.

[0096] 2.2 Reverse transcription

[0097] A total of 20 μL of reverse transcription system, including 2 μg / 2 μL of total cell RNA, 1 μL of 50 U / μL Rnasin, 4 μL of 5× reverse transcription reaction buffer, 2 μl of 10 mM d NTP, 2 μL of 50 μg / mL random primer (promega), 200 U / μL M- MLV reverse tra...

Embodiment 3

[0120] Example 3 LILRA2 Gene Overexpression

[0121] 1. Plasmid construction

[0122] Amplification primers were designed according to the coding sequence of LILRA2 gene, and the design of primers is well known to those skilled in the art. Amplify the coding sequence of the full-length LILRA2 gene from the cDNA library of adult fetal brain (clontech company, article number: 638831), insert the above cDNA sequence into the eukaryotic cell expression vector pcDNA3.1, and connect the obtained recombinant vector pcDNA3.1 -LILRA2 was used in subsequent experiments.

[0123] 2. Culture and transfection of multiple myeloma cells

[0124] 2.1 Cell culture

[0125] RPMI8226 cells were placed in RPMI 1640 medium containing 15% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C, 5% CO 2 , cultivated in a saturated humidity environment.

[0126] 2.2 Cell transfection

[0127] (1) The day before transfection, 0.5-2*10 5 Tumor cells were suspended in 50...

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Abstract

The invention discloses application of an LILRA2 gene as a molecular marker for diagnosing multiple myeloma. Through adoption of a gene chip, a QPCR experiment shows that an LILRA2 gene expression product has significant difference between expression in normal myeloid tissue and expression in multiple myeloma tissue, namely the detection of expression condition of the LILRA2 gene in the myeloid tissue can be used for determining whether a tested person suffers from multiple myeloma or has the risk of suffering from multiple myeloma. A kit for diagnosing multiple myeloma is developed according to the relevance between the LILRA2 gene and the multiple myeloma; the kit can be applied to early diagnosis of diseases and has an extensive application prospect in clinic. In addition, the invention also discloses application of the LILRA2 gene as a molecular target for treating multiple myeloma and provides the basis for development work of medicines for treating multiple myeloma.

Description

technical field [0001] The present invention relates to the field of tumor diagnosis, treatment and prognosis prediction, more specifically, the present invention relates to a method for tumor diagnosis and prognosis prediction by means of detecting LILRA2 abnormality; and a tumor therapeutic agent for activating LILRA2 gene or protein. Background technique [0002] Multiple myeloma (multiple myeloma, MM) is a hematological malignancy in which plasma cells in the bone marrow abnormally proliferate. It accounts for about 10% of hematological malignancies. The number has increased. Its main feature is the abnormal proliferation of clonal plasma cells that invade the bone marrow and produce clonal immunoglobulins. The clinical manifestations are the proliferation and infiltration of myeloma cells and the destruction of the bone marrow microenvironment, leading to anemia, bleeding, bone pain or fracture, hypercalcemia, renal insufficiency, and low immune function. β2-MG is a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/574A61K45/00A61P35/00
Inventor 杨承刚宋宏涛
Owner QINGDAO MEDINTELL BIOMEDICAL CO LTD
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