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Application of amsc-malat1-exo in the preparation of medicines for treating liver diseases and its preparation method

A liver disease and drug technology, which is applied in the preparation of liver disease treatment biological preparations, the construction of adipose-derived mesenchymal stem cells and the preparation of exosomes, and the application fields of liver macrophage targeting biological preparations, which can solve the problem of difficult mass preparation. , the limitation of preparation technology, etc., to achieve the effect of reducing the required amount, reducing the effective dose, and improving the treatment effect of liver diseases

Active Publication Date: 2021-06-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limitations of the current MSC-Exo preparation technology, it is still difficult to prepare a large amount of MSC-Exo that meets the clinical therapeutic dose.
Therefore, the promotion of its clinical application still depends on improving its efficacy through modification.

Method used

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  • Application of amsc-malat1-exo in the preparation of medicines for treating liver diseases and its preparation method
  • Application of amsc-malat1-exo in the preparation of medicines for treating liver diseases and its preparation method
  • Application of amsc-malat1-exo in the preparation of medicines for treating liver diseases and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 AMSC-MALAT1-Exo can mediate the transfer of MALAT1 to macrophages

[0071] qRT-PCR analysis of MALAT1 was performed as described above. Such as figure 1 As shown on the left, AMSC-MALAT1-Exo contains high levels of MALAT1.

[0072] AMSC-MALAT1-Exo, AMSC-Ctrl-Exo, and native AMSC-Exo were added to macrophage RAW264.7, and the cells were collected after 24 hours for qPCR analysis of MALAT1 levels.

[0073] qPCR results showed that AMSC-MALAT1-Exo could significantly up-regulate the MALAT1 level of macrophages compared with AMSC-Ctrl-Exo and native AMSC-Exo ( figure 1 right).

Embodiment 2

[0074] Example 2 AMSC-MALAT1-Exo regulates macrophage phenotype

[0075] Add gradient concentrations of AMSC-MALAT1-Exo, AMSC-Ctrl-Exo and native AMSC-Exo (0.2 μg / mL, 4 μg / mL, 80 μg / mL) to macrophage RAW264.7, and add corresponding volume of PBS as In the control group (Vehicle), the cells were collected after 24 hours for qPCR analysis of macrophage phenotype-related molecules.

[0076] Compared with AMSC-Ctrl-Exo and natural AMSC-Exo, AMSC-MALAT1-Exo can more significantly up-regulate the expression of molecules related to M2 phenotype of macrophages Arg1 and Fizz-1, while down-regulate the expression of M1 phenotype under the same concentration conditions Expression of related molecules CD86 and iNOS. AMSC-MALAT1-Exo can significantly affect the expression of M1 / 2 phenotype-related molecules at a concentration of 0.2 μg / mL, while AMSC-Ctrl-Exo and native AMSC-Exo can significantly regulate the M1 / 2 phenotype at a concentration of 80 μg / mL Expression of related molecules (...

Embodiment 3

[0077] Example 3 AMSC-MALAT1-Exo regulates the secretion of macrophage inflammatory factors

[0078] Macrophages RAW264.7 were treated with gradient concentrations of AMSC-MALAT1-Exo, AMSC-Ctrl-Exo and natural AMSC-Exo (0.2 μg / mL, 4 μg / mL, 80 μg / mL) for 24 hours, and corresponding volumes of PBS was used as the control group (Vehicle); and then stimulated with LPS (100ng / mL) for 12h, and then ELISA was used to detect the levels of IL-1β, IL-33 and HMGB1 in the cell supernatant.

[0079] Under the same concentration conditions, AMSC-MALAT1-Exo can more significantly reduce the secretion of LPS-induced macrophage inflammatory factors IL-1β, IL-33 and HMGB1 than AMSC-Ctrl-Exo and natural AMSC-Exo. AMSC-MALAT1-Exo can significantly inhibit the secretion of macrophage inflammatory factors at a concentration of 0.2 μg / mL, and its inhibitory effect is comparable to that of AMSC-Ctrl-Exo and natural AMSC-Exo at a concentration of 80 μg / mL ( image 3 ).

[0080] Moreover, the AMSC-Ex...

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Abstract

The invention belongs to the field of biotechnology, and relates to the new application of exosomes derived from mesenchymal stem cells, in particular to the construction of adipose-derived mesenchymal stem cells modified by lncRNA MALAT1 and the preparation of exosomes, and its application in the preparation of biological preparations for liver disease treatment and Application of liver macrophage targeting biologics. The idea of ​​the present invention is to construct MALAT1-modified AMSC, prepare its exosomes, and clarify the regulatory advantages of AMSC-MALAT1-Exo on the functional phenotype of macrophages at the cellular level through ELISA, WB, flow cytometry and other experiments, and through in vivo The experiment clarified the curative effect of MALAT1-modified AMSC-derived exosomes on liver diseases and its role in regulating the functional phenotype of liver macrophages. And further prepare liver macrophage targeting biological preparations or drugs with improved curative effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the new application of exosomes derived from mesenchymal stem cells, in particular to the construction of adipose-derived mesenchymal stem cells modified by lncRNA MALAT1 and the preparation of exosomes, and its application in the preparation of biological preparations for liver disease treatment and Application of liver macrophage targeting biologics. Background technique [0002] The mechanism of liver inflammation is very complex, involving many factors such as etiology, host genetics, body immunity, and clinical intervention, among which the body's immune response plays a decisive role in the occurrence and development of liver inflammation. Various innate immune cells, such as monocytes / macrophages, natural killer cells, and dendritic cells, are recruited, activated, and proliferated in the inflamed liver, suggesting that the innate immune system plays a very important role in the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10
CPCA61K35/28A61P1/16C12N5/0667C12N15/86C12N2510/00C12N2740/15043
Inventor 楼国华刘艳宁陈智
Owner ZHEJIANG UNIV
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