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A kind of brain target stem cell and its preparation method and application

A stem cell and brain-targeting technology, applied in the field of stem cell therapy, can solve problems such as insufficient targeting of stem cells, and achieve the effect of protecting its own activity, reducing safety hazards, and reducing aggregation

Active Publication Date: 2015-09-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention provides a brain-targeted stem cell, which solves the problem of insufficient targeting of stem cells in the prior art

Method used

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  • A kind of brain target stem cell and its preparation method and application
  • A kind of brain target stem cell and its preparation method and application
  • A kind of brain target stem cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of brain-targeted stem cells

[0040] 1. Preparation of brain-targeted stem cells

[0041] (1) Rat bone marrow mesenchymal stem cells were extracted and expanded in vitro. The specific steps were as follows: take the tibia and femur of 3-week-old male SD rats, wash the bone marrow with a 1mL syringe into DMEM low-sugar culture medium, and pass through 200 mesh cells Sieve, and centrifuge the sieved culture solution at 1200rpm for 5min, discard the supernatant, resuspend the cells in DMEM low-sugar medium containing 15% fetal bovine serum and double antibody, and place at 37°C, 5%CO 2 cultured in an environment, digested and passaged when the cells grew to 80% full of the culture dish, the second to fifth generation cells were used for experiments;

[0042] (2) Take the above-mentioned primary cultured MSCs, digest them with EDTA-containing trypsin and centrifuge, and then resuspend them in serum-free DMEM low-sugar medium to prepare a cell suspens...

Embodiment 2

[0060] Embodiment 2 brain targeting experiment

[0061] 1. Establishment of middle cerebral artery occlusion (MCAO) model

[0062] SD rats were anesthetized with 10% chloral hydrate, and the common carotid artery, internal carotid artery and external carotid artery on the right side of the rat were bluntly dissected, and then a nylon thread with a diameter of 0.26 mm was inserted from the external carotid artery and passed through the internal carotid The artery reaches the beginning of the middle cerebral artery (MCA), and the depth of the thread is about 17-18mm. The blood supply of the MCA is blocked for 1.5 hours, and then the nylon thread is pulled out to restore the recanalization.

[0063] 2. Brain targeting experiments

[0064] (1) MSCs were resuspended in serum-free DMEM low-glucose medium, and Vybrant nuclear dye (Invitrogen, USA, 2 μL / 10 6 cells), incubated at 37°C for 30 minutes, centrifuged at 1500rpm for 5 minutes, washed twice with phosphate buffer, and then a...

Embodiment 3

[0069] Example 3 Brain Targeted Nucleic Acid Drug Delivery Experiment

[0070] 1. Preparation of drugs for treating ischemic stroke

[0071] (1) Synthesis of Spermine-Pullulan (SP)

[0072] Dissolve 50 mg of pullulan in 5 mL of DMSO, add 225 mg of CDI for activation for 5 minutes, add dropwise into 45 mL of DMSO containing 1873.5 mg of spermine, stir at room temperature for 18 hours, and dialyze with three-distilled water for 48 hours (dialysis bag molecular weight cut-off is 12,000-14,000) , freeze-dried.

[0073] (2) Preparation of transfection system

[0074] SP was prepared into a solution with a concentration of 4 mg / mL with ultrapure water, and miRNA-133b was prepared with an appropriate amount of DEPC water to a solution with a final concentration of 30 μmol / L. After diluting miRNA-133b and SP with Opti-MEM respectively, press The ratios of nitrogen and phosphorus were 5:1, 10:1, and 20:1, respectively, and incubated at room temperature for 30 minutes to prepare the ...

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Abstract

The invention discloses a brain-targeted stem cell as well as a preparation method and application thereof. The brain-targeted stem cell is prepared by modifying brain-targeted peptide on the cell membrane surface of a stem cell, wherein the stem cell is a mesenchymal stem cell (MSC), and an amino acid sequence of the targeted peptide is represented as CLEVSRKNC. The application refers to an application of the brain-targeted stem cell as a nucleic acid delivery carrier. According to the brain-targeted stem cell disclosed by the invention, the mesenchymal stem cell is modified through the targeted peptide with palmitic acid, thus enhancing targeting of the MSCs on the part of ischemic cerebral apoplexy, relieving aggregation of the MSCs in a non-targeted tissue and reducing potential safety hazard; a cerebral arterial thrombosis treating medicine prepared from the brain-targeted stem cell is a multifunctional complex, and the brain-targeted stem cell not only serves as a nucleic acid delivery carrier but also develops therapeutic action thereof; the cerebral arterial thrombosis treating medicine has a therapeutic action on cerebral ischemia from three aspects, namely stem steel, targeted peptide and nucleic acid; therefore, the medicine is more excellent in curative effect.

Description

technical field [0001] The invention belongs to the field of stem cell therapy, and in particular relates to a brain-targeted stem cell and its preparation method and application. Background technique [0002] With the aging of human society and changes in the global ecological environment, brain diseases have increasingly become major diseases that endanger human life and health. In the study of brain diseases, cerebrovascular injury diseases are a major problem affecting human health. Cerebrovascular disease is a disease that occurs in the blood vessels of the brain and causes brain tissue damage due to intracranial blood circulation disorders. Because of its sudden onset, it is also called stroke or cerebrovascular accident. Stroke is divided into two types: hemorrhagic stroke and ischemic stroke, and the latter accounts for about 80% of all cerebrovascular diseases. According to WHO statistics, stroke has become the second killer of human death, and there are more tha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K48/00A61P9/10
Inventor 高建青黄冰
Owner ZHEJIANG UNIV
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