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A kind of inhibitor of circRNA-cer gene and application thereof

An inhibitor and gene technology, applied in the field of bioengineering, can solve the problems of cartilage damage, increase the difficulty of extracellular matrix synthesis, and reduce the level of

Active Publication Date: 2019-09-17
PEKING UNIV THIRD HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under the action of biomechanics, chondrocytes produce matrix metalloproteinase MMP13 (matrix metalloproteinase-13), which can especially cause the formation of type II collagen (Collagen Type II, COL2) and aggrecan (AGGRECAN) in the extracellular matrix in cartilage tissue. Degradation, thereby reducing the level of chondrocytes to synthesize extracellular matrix, further increasing the difficulty of extracellular matrix synthesis, leading to a vicious cycle, causing cartilage tissue destruction and cartilage damage

Method used

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  • A kind of inhibitor of circRNA-cer gene and application thereof
  • A kind of inhibitor of circRNA-cer gene and application thereof
  • A kind of inhibitor of circRNA-cer gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Step 1. Culture chondrocytes

[0062] Under sterile conditions, cut the cartilage tissue to less than 1cm with scissors 3 Size, and washed repeatedly with PBS buffer containing double antibody. Then, digest with trypsin in an amount 10 times the mass of cartilage tissue at 37° C. for half an hour, and discard the enzyme solution. Then utilize the PBS buffer solution of 0.2% (g / ml) type Ⅱ collagenase at the concentration of 10 times of the mass of cartilage tissue to continue to stir and digest at 37°C for 4 hours, and filter the treated cartilage after removing impurities. The tissue fragments are planted on the bottom surface with an area of ​​10cm 2 In a new petri dish, the culture medium is low-sugar DMEM complete medium, at 37°C, containing 5% volume concentration of CO 2 CO at saturated humidity 2 Constant temperature incubator culture.

[0063] Step 2. Stimulate chondrocytes with inflammatory factors

[0064] Inflammatory factors: interleukin-1 (IL-1) and tu...

Embodiment 2

[0096] Step 1. Culturing chondrocytes: the chondrocyte culture process in this example is the same as the chondrocyte culture process in Step 1 in Example 1.

[0097] Step 2, in vitro transfection of siRNA molecules

[0098] Negative control siRNA: The selected negative control siRNA is the siRNA molecule of product number siN05815122147-1-5 sold by Guangzhou Ruibo Biotechnology Co., Ltd.

[0099] Experimental siRNA: Using siCatch TM siRNA design intelligently designs the siRNA sequence targeting the circRNA-CER gene, that is, the siRNA sequence that can inhibit the expression of the circRNA-CER gene. The sequence is as follows:

[0100] Sense strand (5'-3') 5'CCCACGCUCCUACAAUGUU dTdT 3'

[0101] Antisense strand (3'-5') 3'dTdT GGGUGCGAGGAUGUUACAA 5'

[0102] 2.1. One day before transfection, inoculate 2×10 5 Put the chondrocytes cultured in Step 1 into a 6-well cell culture plate, and add antibiotic-free medium to each well so that the cell density at the time of transfe...

Embodiment 3

[0112] The present embodiment utilizes Western Blot (Western Blot) to detect the expression levels of each protein in the experimental chondrocytes and negative control chondrocytes, and the specific steps are as follows:

[0113] Step 1. Prepare experimental chondrocytes and negative control chondrocytes, wherein the cultivation and transfection of the experimental chondrocytes and negative control chondrocytes are the same as in Example 2.

[0114] Step 2, determination of total cell protein concentration and Western Blot experiment. Wherein, protein extracted from cultured chondrocytes is used as protein sample. The medium in the chondrocytes was washed twice with PBS buffer solution, and then 200 ul of protein lysate with a mass concentration of 2% SDS was added to each well of a six-well plate to obtain protein samples.

[0115] 2.1. Determination of total cell protein concentration

[0116] The preparation of bovine serum albumin BSA standard substance and the determin...

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Abstract

The invention discloses a circRNA-CER gene inhibitor and application thereof, belonging to the field of bioengineering. The inhibitor can inhibit the expression of the circRNA-CER gene so as to lower the circRNA-CER gene expression product level. The nucleotide sequence of the circRNA-CER gene is disclosed as SEQ ID NO:1. The inhibitor capable of inhibiting circRNA-CER gene expression can prevent the degradation of the extracellular matrix in the cartilaginous tissue due to the expression of the circRNA-CER gene, and has important meanings in preventing and treating osteoarthritis cartilage injury.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an inhibitor of circRNA-CER gene and application thereof. Background technique [0002] RNA interference (RNAi) refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsRNA), which leads to the silencing of the expression of target genes and the loss of corresponding functional phenotypes. Phenomenon. After the double-stranded RNA enters the cell, it is combined and cleaved by Dicer enzyme (an enzyme specific to double-stranded RNA in the RNAase III family), forming a product with a length of about 20-25bp, and there are two at the 3' end of each strand Nucleotide-based small interfering RNA molecules (small interference RNA, siRNA). One strand of the siRNA is incorporated into the RNA-induced silencing complex (RISC) and pairs with the sequence of the complementary RNA. RISC first mediates the unwinding of the double str...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P19/02C12N15/113
Inventor 敖英芳刘强张辛胡晓青周春燕
Owner PEKING UNIV THIRD HOSPITAL
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