Application of long-chain non-coding RNA MRAK053938 in the preparation of drugs for treating pulmonary fibrosis
A long-chain non-coding, pulmonary fibrosis technology, applied in the direction of DNA / RNA fragments, gene therapy, drug combination, etc., can solve the problems of ineffective inhibition of pulmonary fibrosis and aggravation of pulmonary fibrosis
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Embodiment 1
[0021] Example 1. Expression of lncRNA-MRAK053938 in normal lung tissue and fibrotic tissue
[0022] 1. Materials SD rats, male, weighing 200 ± 20 grams, clean grade, provided by the Experimental Animal Center of Shandong Luye Pharmaceutical Co., Ltd., certificate number: SYXK (Lu) 20030020, in a clean grade animal observation room, adaptive feeding 3 days; normal lung epithelial cell line (RLE-6TN), fibroblast cell line (RL1) were purchased from ATCC cell bank; RPMI1640 medium, high-glucose DMEM medium (DMEM-LG), fetal bovine serum (fetal bovine serum, FBS) were purchased from Hyclone Company of the United States; TGF-β1 was purchased from Invitrogen Company; riboFECT TM CP transfection reagent (Ruibo Biotechnology Co., Ltd., Guangzhou, China); SYBR Green PCR Master Mix (TAKARA, Dalian, China).
[0023] 2. Method
[0024] 1. Prepare rat pulmonary fibrosis model: 30 SD rats are divided into normal group and model group at random, and rats are anesthetized with 4% chloral hy...
Embodiment 2
[0042] Example 2. Effects of siRNA interference fragments specific to MRAK053938 on transdifferentiation of myofibroblasts
[0043] 1. Cell culture and grouping: select RLE-6TN and RL1 cells in logarithmic growth phase, make single cell suspension with 0.25% trypsin, and inoculate in 6-well plate. A blank control group, a TGF-β1 stimulation group, a transfection group, and a transfection control group were set up; when the cells grew to 70-80% confluent, the transfection group and the transfection control group were treated with riboFECT respectively. TM CP transfection reagent transfected cells with siRNA interference fragment and control siRNA at a final concentration of 50nM. The blank control group was cultured with serum-free medium, and the other groups were stimulated with 5nM TGF-β1. Cells were collected after 72 hours of culture.
[0044] 2. Western blot detection of expression changes of α-SMA
[0045] Strictly follow the instructions of the Western and IP cell lys...
Embodiment 3
[0047] Compared with the blank control group, the expression of α-SMA in the TGF-β1 stimulation group was significantly up-regulated, indicating that TGF-β1 can induce the transformation of cells into myofibroblasts; compared with the TGF-β1 stimulation group, RLE-6TN / siRNA-MRAK053938 The expression of α-SMA in the group and RL1 / siRNA-MRAK053938 group was significantly down-regulated, with a difference of 3.43 and 1.79 times, respectively, suggesting that reducing the expression of lncRNA-MRAK053938 can inhibit the transdifferentiation of RLE-6TN cells and the activation of RL1 cells (see figure 2 ). Example 3. Effects of siRNA interference fragments specific to MRAK053938 on the migration of myofibroblasts
[0048] 1. Cell scratch test: select cells RLE-6TN and RL1 in the logarithmic growth phase, make a single-cell suspension with 0.25% trypsin, and inoculate them on a special culture dish of the living cell workstation. Set up TGF-β1 stimulation group, transfection contro...
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