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Inhibitor for lncRNA-MSR and application of inhibitor

An inhibitor and molecular technology, applied in the field of lncRNA-MSR inhibitors, can solve problems such as cartilage damage, cartilage tissue destruction, and level decline

Inactive Publication Date: 2016-11-23
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 metalloproteinases MMP-13 (matrixmetalloproteinase-13) and ADAMTS5 (ADAM metallopeptidase with thrombospondintype 1 motif, 5), these enzymes especially cause collagen type II in the extracellular matrix of cartilage tissue (CollagenType Ⅱ , COL2) and aggrecan (AGGRECAN) 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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  • Inhibitor for lncRNA-MSR and application of inhibitor

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, in vitro transfection of siRNA molecules

[0064] Negative control siRNA: The selected negative control siR...

Embodiment 2

[0107] 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:

[0108] 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 those in Example 1.

[0109] Step 2, determination of total cell protein concentration and Western Blot experiment, wherein the protein extracted from cultured chondrocytes is used as a 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.

[0110] 2.1. Determination of total cell protein concentration

[0111] The preparation of bovine serum albumin BSA standard substance and th...

Embodiment 3

[0134] 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 those in Example 1.

[0135] Step 2. Immunofluorescence staining of chondrocyte cytoskeleton

[0136] 2.1. At room temperature (about 25° C.), fix the cultured chondrocyte slide (cover glass) in a 4% paraformaldehyde solution for 10-15 minutes.

[0137] 2.2. Then rinse the chondrocytes with PBS buffer for 3 times, 5 minutes each time.

[0138] 2.3. Incubate the chondrocytes with 2% BSA solution at 37° C. for 30 minutes to block non-specific binding sites.

[0139] 2.4. Add the specific primary antibody diluted with 2% BSA solution (1:200 dilution), and then incubate overnight at 4°C.

[0140] 2.5. Add fluorescein-labeled secondary antibody (diluted at 1:200) diluted with 2% BSA solution, and incubate for 30 min in a wet box in the dark.

[0141] 2.6. Rinse the chondrocy...

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Abstract

The invention discloses an inhibitor for lncRNA-MSR and application of the inhibitor, and belongs to the field of bioengineering. The inhibitor can inhibit expression of the lncRNA-MSR to decrease the level of an expression product of the lncRNA-MSR, and a nucleotide sequence of the lncRNA-MSR is shown as SEQ ID NO:1. By providing the inhibitor capable of inhibiting expression of the lncRNA-MSR, degradation, caused by expression of the lncRNA-MSR, of an extracellular matrix in cartilage tissue can be blocked, and an important significance on prevention and treatment of osteoarthritis cartilage injuries is achieved.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to an inhibitor of lncRNA-MSR and its application. 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 strand of siR...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P19/02A61P19/08
Inventor 敖英芳刘强张辛胡晓青周春燕
Owner PEKING UNIV THIRD HOSPITAL
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