MiRNA with cell corpuscule as vector and preparation research approach thereof and application

A technology of microRNA and cell microparticles, applied in biochemical equipment and methods, DNA preparation, extracellular fluid diseases, etc., can solve problems such as low efficiency, unclear target genes, poor specificity of microRNA delivery, etc.

Inactive Publication Date: 2009-03-18
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the unclear target gene of microRNA, the poor specificity and low efficiency of delivery of microRNA are the main reasons hindering its application

Method used

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  • MiRNA with cell corpuscule as vector and preparation research approach thereof and application
  • MiRNA with cell corpuscule as vector and preparation research approach thereof and application
  • MiRNA with cell corpuscule as vector and preparation research approach thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Separation and observation of cell microparticles in serum / plasma

[0048] There are three main methods for the separation of cell microparticles in serum / plasma:

[0049] 1) Fractional centrifugation. Firstly, the serum / plasma (3000 rpm) was centrifuged for 30 minutes to remove various cells and debris, and then the supernatant was ultracentrifuged (80000 rpm) for 4 hours, and the precipitate was the total cell particles of the serum / plasma;

[0050] 2) Immunoadsorption method. Adsorb blood cell-specific antibodies on tissue culture dishes or directly use immunomagnetic beads, and incubate the serum / plasma after removing various cells and debris directly with culture dishes or immunomagnetic beads (30 minutes or 1 hour), different The cell microparticles of cells can be directly adsorbed and recycled;

[0051] 3) Filtration method. The serum / plasma after removing all kinds of cells and debris is directly placed in a concentrated centrifuge tube with a 100...

Embodiment 2

[0053] The RT-PCR experiment of microribonucleic acid carried by cell microparticles in embodiment 2 serum / plasma

[0054] Using RT-PCR technology to discover and prove that a large part of various microRNAs in human serum / plasma come from cell microparticles, and their expression is quite abundant. The specific steps are:

[0055] (1) Collect serum / plasma from normal people and certain patients;

[0056] (2) Separation of cell microparticles in serum / plasma. There are two schemes for this operation, one scheme is to ultracentrifuge the serum / plasma (120000g, 3 hours), and then take the fine particles to precipitate. Another method is to use immunoprecipitation, immobilizing antibodies or ligands against specific receptors on the surface of cell microparticles on specific particles, and then incubating with a solution containing microparticles or serum / plasma to adsorb various types of microparticles.

[0057] (3) Separate and prepare cDNA samples. There are two schemes fo...

Embodiment 3

[0059] Example 3 Real-time PCR experiment of microRNA carried by cell microparticles in serum / plasma

[0060] Quantitative PCR will be used to study microRNA contained in cells and cell microparticles. The experimental principle and experimental steps of quantitative PCR are the same as RT-PCR, the only difference is that the fluorescent dye EVA GREEN is added during PCR. The instrument used was ABI Prism 7500 fluorescent quantitative PCR instrument, and the reaction conditions were 95°C, 1 cycle in 5 minutes → 95°C, 15 seconds; 60°C, 40 cycles in 1 minute. Data processing method is ΔΔCT method, and CT is set as the number of cycles when the reaction reaches the threshold value, then the expression of each microRNA relative to the standard internal reference can be represented by equation 2-ΔCT, where ΔCT=CT 样品 -CT 内参 . U6 was used as an internal reference in the experiment. The results are shown in the table below:

[0061] miRNA / U6 SD miR-1 0.002838 ...

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Abstract

The invention discloses micro ribonucleic acids (microRNA, miRNA) carried by cell microparticles (Microparticle, MP), a method for preparing the same, and application thereof in the technical field of biotechnological pharmacy. The invention provides a combination of the micro ribonucleic acids for evaluating the physiological and / or pathological states of a participant, and the combination contains all the micro ribonucleic acids which exist stably in serum / plasma particles of the participant and are detectable. At the same time, the invention provides an experimental method for preparing the cell microparticles containing specific micro ribonucleic acids and using the cell microparticles to perform gene-level regulation and control as well as modification on other cells and tissues. The combination and the method can be used for detecting and treating various diseases, including the aspects of the diagnosis and the differential diagnosis of various tumors, various acute and chronic infectious diseases and other acute and chronic diseases, the prediction and the curative effect evaluation of the occurrences of disease complications and the recurrences of malignant diseases, as well as the active ingredient screening, the efficacy evaluation and the judicial authentication of drugs, the detection of prohibited drugs and the like; besides, the combination and the method have the advantages of wide detection pedigree, high sensitivity, low detection cost, convenient available material, easy storage of samples and the like.

Description

a technical field [0001] The invention belongs to the technical field of biotechnology and pharmacy, and in particular relates to a preparation method of microribonucleic acid carried by cell microparticles and its application in disease treatment. Two background technology [0002] Micro ribonucleic acid, the English name is microRNA, is a kind of non-coding single-stranded small ribonucleic acid molecule with a length of about 19 to 23 nucleotides. They are highly conserved in evolution and are closely related to many normal physiological activities of animals, such as individual development, tissue differentiation, cell apoptosis and energy metabolism, and are also closely related to the occurrence and development of many diseases. Recent studies have found that the expression levels of several microRNAs in chronic lymphocytic leukemia and Burkitt lymphoma are down-regulated to varying degrees; when analyzing and comparing the expression of microRNAs in human lung cancer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12Q1/68A61K48/00A61P31/00A61P11/00A61P37/00A61P7/00A61P9/00A61P3/00A61P5/00A61P1/00A61P25/00A61P13/00A61P15/00A61P19/00C12N15/113
Inventor 曾科张辰宇李丽民张峻峰陈熹刘丹青陈江宁
Owner NANJING UNIV
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