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Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof

An ischemic stroke, stem cell technology, applied in the field of stem cell therapy, can solve the problems of low transfection efficiency of non-viral vectors, achieve high safety, low genotoxicity, and improve therapeutic effects.

Active Publication Date: 2019-03-08
广州赛隽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The overexpression of chemokine receptors plays a central role in improving the targeted chemotaxis of cells. However, the viral vectors currently used for gene therapy have potential safety risks; on the other hand, the transfection efficiency of non-viral vectors is relatively low. low, which makes adopting a safer and more efficient transport carrier

Method used

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  • Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof
  • Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof
  • Mesenchymal stem cells overexpressing CCR2 for treating acute ischemic stroke and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Establishment of mesenchymal stem cells (MSCs) overexpressing CCR2 CCR2 )

[0045] 1. MSC culture and phenotypic identification:

[0046] Take 20ml of bone marrow from healthy volunteers, dilute it with 1×PBS at a ratio of 1:1, and use Ficoll-Paque lymph separation medium to separate mononuclear cells from the bone marrow by density gradient centrifugation (2000rpm, 30min). 1×10 5 / cm2 density was inoculated into 75cm2 culture flasks for cultivation. After culturing with L-DMEM medium for 3 days at 37°C and 5% CO2, the suspended cells were removed, and the medium was changed to continue culturing. After the cells grew to 80% density, the medium was sucked off, washed twice with PBS, digested with 0.125% trypsin for 1-ni72min, and the passage ratio was 1:3. MSCs are isolated from bone marrow donated by healthy donors. The isolation, expansion, cryopreservation, and recovery of MSCs for clinical use are all carried out under conditions that comply with GMP...

Embodiment 2

[0096] Example 2 Identification of MSC Biological Properties

[0097] 1. Identification of MSC Phenotypes

[0098] The MSCs cultured in vitro were digested into a single cell suspension, washed once with PBS (pH 7.4) containing 0.1% BSA+0.05% NaN3, the supernatant was discarded, and the cell density was adjusted to 106 / ml in a flow tube. Antibody CD29, CD34, CD44, CD45, CD73, CD90, CD105, CD166 labeled MSCs, fully oscillated and mixed, incubated at 4°C in the dark for 30min, and then washed two times with PBS (pH 7.4) containing 0.1%BSA+0.05%NaN3 to remove excess antibody; discard the supernatant, resuspend the cells with 200ul 1% PFA, and detect the phenotype of MSCs (CD29+, CD34-, CD44+, CD45-, CD73+, CD90+, CD105+, CD166+) by flow cytometry, proving that in vitro culture No effect on MSC cell phenotype, the results are as follows figure 2 As shown in A. 2. Identification of multilineage differentiation ability of MSC

[0099] (1) Osteogenic differentiation: the modif...

Embodiment 3

[0101] Example 3 MCAO model construction

[0102] (1) MCAO model construction

[0103]① Rats were fasted for 12 hours before operation, and had free access to water. Induce anesthesia by intraperitoneal injection of 10% chloral hydrate 350mg / kg, fix in supine position, take median neck incision, separate right common carotid artery, external jugular vein and internal jugular vein, electrocoagulate and burn off the branches of external carotid artery, ligate and free the carotid For the main trunk of the external artery, cut a small opening on the free section, insert the 4-0 nylon thread with a rounded end into the external carotid artery, pass through the bifurcation of the common carotid artery such as the internal carotid artery, and the depth is calculated from the bifurcation of the common carotid artery From about 18 to 20mm, until there is a slight sense of resistance. After 1.5 hours of cerebral ischemia in rats, the thread plug was retracted to the stump of the ex...

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Abstract

The invention discloses MSCs (mesenchymal stem cells) overexpressing CCR2 and a preparation method thereof, and further discloses use of the MSCs overexpressing the CCR2 in a medicament for treating AIS (Acute Ischemic Stroke). The invention optimizes the treatment method of the AIS, which is higher in targeting property and effectiveness for treatment with the CCR2 gene modified MSCs, so that thetreatment effect of the MSCs can be significantly enhanced. According to the preparation method of the MSCs overexpressing the CCR2, the MSCs can be enabled to overexpress the receptor CCR2 by constructing plasmids which express the CCR2 and using an mRNA transfection method, and the method does not affect the phenotype, the differentiation capacity and the immune adjustment capacity of the MSCs;due to relatively low genotoxicity, the MSCCCR2 can be directionally migrated to a lesion part in the body, so as to achieve the targeted treatment aim more effectively.

Description

technical field [0001] The invention relates to the technical field of stem cell therapy, in particular to a CCR2-overexpressed mesenchymal stem cell (Mesenchymal stromal cells, MSC) and a preparation method and use thereof. Background technique [0002] Acute ischemic stroke (AIS) is a sudden local brain dysfunction disease caused by cerebral blood circulation disorder, clinically manifested as symptoms and signs of transient or permanent brain dysfunction , is a common and frequently-occurring disease in neurology, accounting for about 60% to 80% of all strokes. AIS mainly includes two types of thrombosis and embolism, occasionally seen in vasospasm or brain tumor compressing blood vessels. Thrombosis is that the cerebrovascular wall becomes smaller and smaller due to atherosclerosis, or even completely blocked; embolism is the sudden appearance of an embolus in a blood vessel, which is stuck in a blood vessel with a smaller diameter along with the blood flow, hindering b...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/11A61K35/28A61P9/10
CPCA61K35/28A61P9/10C07K14/7158C12N5/0662C12N15/85C12N2510/00
Inventor 项鹏汪建成黄一浓李启豪宋武
Owner 广州赛隽生物科技有限公司
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