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Application of peroxidase to preparation of drug for preventing and treating cerebrovascular diseases

A technology for peroxidase and cerebrovascular disease, which is applied in the field of biomedicine to achieve the effects of relieving nerve function damage, improving vascular ischemia damage, and preventing and treating cerebrovascular disease.

Inactive Publication Date: 2013-12-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no application report on how to use Prdx1 to reverse the oxidative stress damage of cerebrovascular endothelial cells and prevent ischemic stroke, vascular dementia and other brain injury diseases based on cerebrovascular lesions.

Method used

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  • Application of peroxidase to preparation of drug for preventing and treating cerebrovascular diseases
  • Application of peroxidase to preparation of drug for preventing and treating cerebrovascular diseases
  • Application of peroxidase to preparation of drug for preventing and treating cerebrovascular diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment one Construction of Prdx1 overexpression recombinant plasmid

[0035] 1.1 The first strand of cDNA was synthesized by reverse transcription using the total RNA of Hela cells as a template, and quantified.

[0036]Source of Hela cells: CCL-2 Hela cells from American type culture collection (ATCC).

[0037] Hela cell total RNA extraction experimental steps:

[0038] 1) First discard the DMEM medium in the six-well plate, and add 1ml TRIzol to make a cell suspension.

[0039] 2) Let stand at room temperature, transfer the cell suspension to an EP tube, add 0.2ml chloroform / phenol, shake by hand for 15 seconds, let stand for 2-3 minutes, and centrifuge at 12000g for 15 minutes (2°C-8°C).

[0040] 3) Divide three layers of RNA and dissolve in the upper aqueous phase, absorb the aqueous phase, add 0.5ml of isopropanol, and centrifuge at 12000g for 10 minutes (2°C-8°C).

[0041] 4) After centrifugation, discard the supernatant and add 1ml of 75% ethanol to wash...

Embodiment 2

[0069] Embodiment two Construction of Prdx1 overexpression lentivirus

[0070] 2.1 Prdx1 overexpression vector construction (the vector is pCDH-CMV-MCS-EF1-copGFP)

[0071] ① Obtain the target gene fragment by digesting the chemically synthesized Prdx1 gene.

[0072] The chemically synthesized target gene was digested with EcoR I and BamH I, and the 609bp target gene fragment was recovered from the gel.

[0073] ② Recover the linearized vector fragment after digestion of the expression vector.

[0074] Use EcoR I and BamH I to express the vector ( Figure 7 ) for double enzyme digestion, and the vector fragment of about 7.5 kb was recovered after electrophoresis of the digested product.

[0075] ③The target gene fragment is connected with the linearized vector fragment.

[0076] A. Reaction system (Table 1)

[0077]

[0078] Note: The positive control and the self-ligating control, the added vector and the connecting group are the same, but the target gene fragment a...

Embodiment 3

[0112] Embodiment three Protective effect of overexpression of Prdx1 on vascular endothelial cell injury under glucose and hypoxia oxidative stress injury

[0113] Vascular endothelial cells EA.hy 926 were inoculated in a 35mm diameter culture dish, cultured in the incubator to 80% confluence rate, and after 6 hours of transient transfection with pEGFP-Prdx1 plasmid, the transfection supernatant was removed and replaced with into normal DMEM medium (containing 10% FBS) to continue culturing for 48 hours. Then OGD modeling was carried out for 6 h, that is, the experimental group was preheated to 37 ° C with HBSS (NaCl 8.00, KCl 0.20, CaCl 2 0.14, MgSO 4 .7H 2 O 0.20, Na 2 HPO 4 .H 2 O 0.06, KH 2 PO 4 0.06, NaHCO 3 0.35) washed three times, and incubated cells with HBSS instead of high-sugar medium to simulate a sugar-deficient environment. Then the cells were placed in an anoxic airtight box (the product of Billups-Rothenberg, USA, MIC-101) and a mixed gas (cont...

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Abstract

The invention provides application of peroxidase Prdx1 (peroxiredoxin-1) to preparation of a drug for preventing and treating cerebral injury diseases based on cerebral arterial thrombosis, vascular dementia and other cerebrovascular diseases. According to the invention, a Prdx1 gene-carrying overexpression plasmid and a recombinant lentiviral vector are established, cell and whole animal level tests prove that the Prdx1 is capable of effectively reversing the ischemic injury of vascular endothelial cells, protecting a BBB (Blood Brain Barrier) and relieving nerve function deficits caused by cerebral microcirculatory disturbance, and therefore, the effect of preventing and treating the cerebrovascular diseases is achieved. The Prdx1 gene lentiviral vector provided by the invention is expected to become an important way for developing the drug for treating the cerebrovascular diseases, and experimental basis is provided to the application of the Prdx1 gene to gene therapy of the cerebrovascular diseases.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and relates to the medicinal use of peroxidase Prdx1 (peroxiredoxin-1), in particular to the application of Prdx1 in the treatment of brain injury diseases based on cerebrovascular lesions such as ischemic stroke and vascular dementia . Background technique [0002] Cerebrovascular disease is a common and frequently-occurring disease that endangers human life and health, and has the characteristics of high morbidity, disability, mortality and recurrence. With the acceleration of the population aging trend, the incidence of cerebrovascular diseases in my country continues to increase, which brings a heavy medical and economic burden to patients, their families and society. Statistics show that ischemic cerebrovascular disease is more common. Ischemic cerebrovascular disease is a complex pathological process with multiple factors and layers, and the functional state of cerebral microvessel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/44A61K48/00A61P9/10A61P25/28C12N15/53C12N15/867C12N7/01C12Q1/02
Inventor 韩峰陶蓉蓉陆楠楠王欢廖美华黄继云田允
Owner ZHEJIANG UNIV
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