VEGF plasmid-liposome complex and its usage

A growth factor and vascular endothelial technology, applied in the field of vascular endothelial growth factor plasmid-liposome complex and its preparation of pharmaceutical compositions, can solve the problems of immune rejection that cannot be ignored, infection, local infection, etc.

Inactive Publication Date: 2005-01-26
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the rapid metabolism of protein in the body, exogenous injection of VEGF must be perfused for a long time, which can easily cause systemic or local infection in the brain
However, the use of AAV-VEGF transfection has solved the problem of infection caused by long-term perfusion, but it is still a problem that cannot be ignored for the cytotoxicity mediated by the virus or the induction of the body's immune rejection to the virus.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Preparation of VEGF plasmid-liposome complex, and its intracerebral injection experiment and expression identification

[0014] 1. Preparation of VEGF plasmid-liposome complex

[0015] VEGF-EGFP-N 1 Plasmid and liposome are mixed to form a complex. VEGF is the full-length sequence of human VEGF165 DNA (gene bank number: AB021221), inserted into pEGFP-N 1 Between the plasmid promoter pCMV and EGFP sequence. Lipofectamine using LipofectAMINE TM 2000 or DOTAP. The plasmid vector and liposome were purchased from Roche, Germany. The mixing ratio of the liposome and the plasmid is: plasmid DNAlug-10ug, liposome 2ul-20ul.

[0016] 2. Preparation of experimental animal models of focal cerebral ischemia and intracerebral VEGF plasmid-liposome complex injection

[0017] Clean-grade male Sprague-Dawley rats weighing 220-250 grams. The rats were randomly divided into VEGF plasmid injection group and control plasmid injection group. The local cerebral ischemia model adopts the...

Embodiment 2

[0029] Example 2 Detection of the protective effect of VEGF plasmid protein expression on the brain

[0030] 1. Detection of neurological score and physiological indexes in rats after cerebral ischemia

[0031] Within 1-14 days of ischemia-reperfusion, the rat's nerve function was measured every day. The scoring of neurological function is carried out according to the method of Longa et al. The function scoring index is divided into six grades.

[0032] 2. Preparation of frozen tissue sections

[0033] After 1-4 weeks of ischemia-reperfusion, the left ventricle of the rat was perfused with normal saline and 4% paraformaldehyde, and then the whole brain was taken for frozen coronal section with a thickness of 30 μm. The brain slice was stored at -20°C.

[0034] 3. Detect the volume of cerebral infarction

[0035] A brain slice was taken every 240 μm and stained with tar violet to identify viable cells. Normal brain tissue is dark blue, and the infarcted area is lightly stained. Obt...

Embodiment 3

[0040] Example 3 Detecting the neuron regeneration effect of VEGF plasmid protein expression

[0041] 1. BrdU labeling and detection:

[0042] On days 0-12 of cerebral ischemia-reperfusion, BrdU (30 mg / kg body weight) was injected intraperitoneally every day to mark newly proliferated cells in the brain. Mouse anti-BrdU monoclonal antibody combined with immunohistochemical staining was used to observe the incorporation of BrdU into cells.

[0043] 2. BrdU and MAP-2, CRMP-4 immunohistochemical double label detection:

[0044] BrdU and MAP-2, CRMP-4 immunohistochemical double labeling were used to detect neuronal regeneration. The brain slices were incubated with mouse anti-BrdU monoclonal antibody (1∶100), developed by Vector Blue, and incubated with polyclonal rabbit anti-MAP-2 (1∶200) and polyclonal rabbit anti-CRMP-4 (1∶200) , Perform DAB color development, mount the slides with lemon oleoresin, observe the labeling signal under an optical microscope, and carry out the counting ...

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Abstract

The invention belongs to biological medical and biological technology field. It relates to a vascular endothelia growth factor(VEGF) plasmid-liposome complex and its usage in the preparation of the pharmaceutical composition, in particular to the usage of VEGF plasmid-liposome complex in preparing pharmaceutical composition for treating cerebral ischemia injury, promoting differentiation of the adult cranial nerve stem cells and regenerating of the nerve cells. The complex of the VEGF and liposome is proved by the local cerebral ischemia animal model that said complex expresses highly of VEGF with only once injection, having no need of infusing for long period of time, having biological effects of brain protection and adult cerebral nerve stem cell differentiation promotion and regeneration of the nerve cell without the virus infection.

Description

Technical field [0001] The invention belongs to the field of biomedicine biotechnology, and specifically relates to a vascular endothelial growth factor (VEGF) plasmid-liposome complex and its use in preparing pharmaceutical compositions. Background technique: [0002] Stroke is the third leading cause of death in humans, and the first leading cause of disability. Therefore, it is the common goal of neuroscience and pharmacologists to find effective drugs to promote the repair of damaged brain after stroke. [0003] The pathological mechanism of neuron ischemia injury is more complicated, therefore, the angle of research and treatment is quite different. Including studies on the effects of neurotransmitter transmission, anti-oxidative damage, anti-apoptosis, or exogenous treatment with growth factors. [0004] Vascular endothelial growth factor (VEGF) is derived from vascular endothelial cells. Recent studies have shown that it also exists in neurons, and has the functions of qui...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/18A61K48/00A61P25/00A61P25/16A61P25/28
Inventor 孙凤艳张玲妹陆世铎
Owner FUDAN UNIV
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