A shrna, carrier, kit and application thereof for knocking down tgf-β1

A TGF-, kit technology, applied in the field of TGF-β1 knockdown shRNA, carrier, can solve the problems of large organ differences, unfavorable diagnosis of neurogenic bladder fibrosis, development of therapeutic targeted drugs, unclear specific relationship, etc. , to achieve the effect of inhibiting fibrosis

Active Publication Date: 2021-10-29
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are large differences between different organs and tissues. At the same time, TGF-β1 involves complex signaling pathways, and its downstream effector molecules are numerous. At present, the specific relationship between TGF-β1 and neurogenic bladder fibrosis is still unclear, and its specific role The mechanism has not been revealed, which is not conducive to the diagnosis, treatment and development of targeted drugs for neurogenic bladder fibrosis

Method used

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  • A shrna, carrier, kit and application thereof for knocking down tgf-β1
  • A shrna, carrier, kit and application thereof for knocking down tgf-β1
  • A shrna, carrier, kit and application thereof for knocking down tgf-β1

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Experimental program
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Effect test

Embodiment 1

[0033]The siRNA target was designed, and the primer shRNA was synthesized. The siRNA sequence and the shRNA sequence are shown in Tables 1-2. The primers were annealed to form double-stranded fragments with cohesive ends, the expression vector was digested with restriction endonucleases BamHI and EcoRI, and the digested products were recovered. Interfering fragments were ligated into expression vectors, ligated products were ligated overnight at 16°C, transformed into competent cells DH5α, positive bacteria were picked, and confirmed by sequencing. A large number of lentiviral vector pHBLVTM and auxiliary plasmids pSPAX2 and pMD2G determined by sequencing were extracted, and the virus was packaged and the titer of the virus was determined.

[0034] Table 1 siRNA sequence

[0035] name sequence mock siRNA TTCTCCGAACGTGTCACGTAA (SEQ ID NO: 1) TGF-β1-siRNA1 ACAATTCCTGGCGTTACCTTGGTAA (SEQ ID NO: 2) TGF-β1-siRNA2 GCAAAGATAATGTACTCCACGTGGA (SEQ ID N...

Embodiment 2

[0040] Rats were killed by neck dissection, fresh bladder tissue specimens were cut under sterile conditions, primary rat bladder detrusor cells were isolated and purified, inoculated in 6-well plates, and added or not in the manner described in Table 3 Add the lentivirus and carry out the grouping test. After 48 hours of infection, observe under the fluorescent inverted microscope, the cells infected with the lentiviral vector carrying the GFP gene emit green fluorescence. After 72 hours of infection, observe the expression of green fluorescent protein in the GFP group, judge the transfection efficiency of the virus and take pictures (for specific transfection effects, see figure 1 ), the expression level of TGF-β1 protein in each group of cells was further detected by WB (for specific results, see figure 2 ), the mRNA transcription level of TGF-β1 in the cells of each group was detected by qRT-PCR (for specific results, see image 3 ).

[0041] according to figure 2 The...

Embodiment 3

[0045] Construct neurogenic bladder fibrosis model: After successful isoflurane anesthesia, the rats were fixed on the operating table in the prone position, and the sternum was found by hand as a bony landmark approximately at the level of the forelimbs, and the surgical site was determined. For the burrow towel, cut the back skin longitudinally about 2cm long, bluntly separate the subcutaneous and muscle tissue, and spread it with a spreader to fully expose the T9 spinal cord. Use scissors to cut the spinal cord transversely. The tail moved violently, and then the two stumps were lifted with tweezers, and the spinal canals at the two stumps were visible, which proved that the nerves at this part had been completely severed, and gelatin sponge was filled between the two stumps. After complete hemostasis, the layers are closed sequentially. The animals were put into the recovery room for observation, and then returned to the animal room after waking up. Method of the control ...

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Abstract

The present invention relates to a shRNA, carrier, kit and application thereof for knocking down TGF-β1. The shRNA of the present invention comprises two oligonucleotide chains, and the sequence of the oligonucleotide chains is as SEQ ID NO : Shown in 9~10. The shRNA, carrier or kit of the present invention can effectively reduce the expression of TGF-β1 from the mRNA level and protein level, inhibit the expression and Smads phosphorylation of TGF-β1, CTGF, α-SMA in detrusor muscle cells or bladder, thereby It effectively inhibits the fibrosis of detrusor muscle cells or bladder, and has a therapeutic effect on neurogenic bladder fibrosis.

Description

technical field [0001] The present invention relates to the field of gene knockout or knockdown, in particular to a shRNA, carrier, kit and application thereof for knockdown of TGF-β1. Background technique [0002] Neurogenic bladder (neurogenic bladder, NB) refers to bladder and (or) urethral dysfunction caused by damage to the central and (or) peripheral nervous system that regulates voiding function, resulting in bladder fibrosis, small volume, high pressure and upper bladder. Urinary tract hydrops, also known as neurogenic vesico-urethraldysfunction (neuropathic vesico-urethraldysfunction, NVD). Clinically, there are urinary retention, urinary incontinence and other abnormal urination. [0003] Decreased bladder compliance due to increased collagen fibers in the detrusor muscle of neurogenic bladder is the main cause of bladder dysfunction. Current studies have shown that transforming growth factor β1 (trasnforming growth factor-beta 1, TGF-β1) plays an important role ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867
CPCC12N15/113C12N15/86C12N2310/15C12N2740/15043
Inventor 贾春松崔昕欧彤文
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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