shRNA and vector for knocking down TGF-beta1 (Transforming Growth Factor-beta 1), kit and application of shRNA or vector or kit

A TGF-, kit technology, applied in the field of TGF-β1 knockdown shRNA, carrier, can solve the problem of large organ differences, undisclosed specific mechanism of action, unfavorable diagnosis of neurogenic bladder fibrosis, and development of targeted drugs for treatment, etc. problems, to achieve the effect of inhibiting fibrosis

Active Publication Date: 2018-12-21
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

Method used

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  • shRNA and vector for knocking down TGF-beta1 (Transforming Growth Factor-beta 1), kit and application of shRNA or vector or kit
  • shRNA and vector for knocking down TGF-beta1 (Transforming Growth Factor-beta 1), kit and application of shRNA or vector or kit
  • shRNA and vector for knocking down TGF-beta1 (Transforming Growth Factor-beta 1), kit and application of shRNA or vector or kit

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Example Embodiment

[0032] Example 1

[0033] Design siRNA target, synthesize primer shRNA, the siRNA sequence and shRNA sequence are shown in Tables 1 to 2. The primers are annealed to form a double-stranded fragment with sticky ends. The expression vector is digested with restriction enzymes BamHI and EcoRI, and the digested product is recovered. The interference fragment was ligated into the expression vector, and the ligation product was ligated overnight at 16°C and transformed into competent cells DH5α. The positive bacteria were picked and confirmed by sequencing. A large number of lentiviral vectors pHBLVTM and helper plasmids pSPAX2 and pMD2G confirmed by sequencing were extracted, and the virus was packaged and the virus titration was determined.

[0034] Table 1 siRNA sequence

[0035] Name

[0036] Table 2 shRNA sequence

[0037]

[0038]

Example Embodiment

[0039] Example 2

[0040] The rats were sacrificed by cervical dissection, fresh bladder tissue specimens were cut under aseptic conditions, and primary rat bladder detrusor cells were isolated and purified. They were inoculated into a 6-well plate and added or not in the manner described in Table 3. The lentivirus was added and the grouping experiment was performed. After 48 hours of infection, the cells infected with the GFP gene lentiviral vector emitted green fluorescence. After 72 hours of infection, observe the expression of green fluorescent protein in the GFP group, determine the efficiency of virus transfection and take pictures (for specific transfection effects, see figure 1 ), to further detect the expression level of TGF-β1 protein in each group of cells by WB (see figure 2 ), using qRT-PCR to detect the mRNA transcription level of TGF-β1 in each group of cells (see image 3 ).

[0041] according to figure 2 The results show that compared with the Control group and t...

Example Embodiment

[0044] Example 3

[0045] Construct a neurogenic bladder fibrosis model: After the rat is successfully anesthetized with isoflurane, fix the operating table in the prone position, touch and find the sternum as a bony marker approximately at the level of the forelimb, determine the surgical site, and routinely prepare skin, sterilize, and cover Bacterial hole towel, longitudinally cut the back skin about 2cm, bluntly separate the subcutaneous and muscle tissue, and use a stretcher to fully expose the T9 spinal cord. Use scissors to cut the spinal cord horizontally. Rats will be seen at the moment of separation. After the tail moved vigorously, the two stumps were lifted with tweezers, and the spinal canals at the two stumps were visible, proving that the nerves in this part had been completely severed, and gelatin sponge was filled between the two stumps. After complete hemostasis, close each layer in turn. The animals were placed in the recovery room for observation, and then re...

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Abstract

The invention relates to shRNA and a vector for knocking down a TGF-beta1 (Transforming Growth Factor-beta 1), a kit and application of the shRNA or the vector or the kit. The shRNA disclosed by the invention comprises two oligonucleotide chains; a sequence of the oligonucleotide chain is as shown in SEQ ID NO:9 to 10. The shRNA, the vector or the kit disclosed by the invention can effectively reduce expression of the TGF-beta1 and inhibit expression of the TGF-beta1, CTGF (Connective Tissue Growth Factor) and alpha-SMA and Smads phosphorylation in detrusor smooth muscle cells or urinary bladder from the mRNA level and the protein level so as to effectively inhibit fibrosis of the detrusor smooth muscle cells or the urinary bladder, and has a treatment effect on fibrosis of neurogenic bladder.

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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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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