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Treatment of Pulmonary Fibrosis by Carrying Epo Using Directed Chemotaxis Properties of MSCs

A technology for pulmonary fibrosis and purpose, applied in the field of biopharmaceuticals, can solve the problems of uncertainty of treatment effect and unclear mechanism of action

Active Publication Date: 2021-02-26
ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although MSCs have shown certain therapeutic effects in animal and human tissue experimental models of fibrosis, most of the treatment methods are direct injection of cells, because the mechanism of action is not clear, and the therapeutic effect is uncertain.

Method used

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  • Treatment of Pulmonary Fibrosis by Carrying Epo Using Directed Chemotaxis Properties of MSCs
  • Treatment of Pulmonary Fibrosis by Carrying Epo Using Directed Chemotaxis Properties of MSCs
  • Treatment of Pulmonary Fibrosis by Carrying Epo Using Directed Chemotaxis Properties of MSCs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: Construction of EPO overexpression vector

[0045] Search the EPO coding sequence on the NCBI website, and design a pair of primers according to the nucleotide sequence NM 000799.2, wherein the forward primer: 5'GCCCGCTCTGCTCCGACAC 3'; the reverse primer: 5'TGCCCATGCCCGTGAGACC 3'. The complete cDNA sequence of EPO was cloned, and the length of the product was 1,168bp. The PCR product of EPO was inserted into the plasmid, and then ligated into the lentiviral vector.

Embodiment 2

[0046] Example 2: Acquisition of menstrual blood stem cells

[0047] 1. Sample collection

[0048] Volunteers for sample collection are 20-30-year-old women. After passing the ethical review, sign the informed consent form. In the middle of the menstrual cycle, use the method described in the example in 201510657971.8 to collect menstrual blood and transfer it to a storage tube containing collection fluid , sent to the laboratory within 24 hours.

[0049] 2. Sample processing

[0050] After the sample package is sterilized, it is processed on a 100-level operating table; the menstrual blood is diluted with PBS, filtered through a 120-mesh cell sieve, and the filtrate is centrifuged; the collected precipitate is resuspended in PBS, and mononuclear cells are collected by density gradient centrifugation; configured according to the method described in 201510657971.8 Medium, that is, added 4mmol / L glutamine, 5mg / mL human serum substitute (purchased from Wuhan Vinosai Biotechnolo...

Embodiment 3

[0053] Example 3: Construction of EPO overexpression vector to transfect uterine blood stem cells

[0054] The uterine blood stem cells (MenSCs) of passage 3-6 in Example 2 were selected and inoculated into a 10 cm culture dish. When the cells were 80% confluent, 2 ml of the concentrated virus solution obtained in Example 1 and 6 mg / L polybrene were added overnight. Replace with fresh medium containing 2 μg / ml puromycin for screening until most of the cells die, and live cells appear green (the carrier contains green fluorescent protein fragments) under a fluorescent microscope. The expression of EPO mRNA was verified by qPCR ( figure 1 A), collect total protein simultaneously, detect EPO protein expression situation with Western Blot method ( figure 1 B). The results showed that after MenSCs were transfected with EPO gene, the expression levels of EPO mRNA and protein were significantly increased. ** indicates P<0.01 compared with the control.

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Abstract

The invention relates to the use of MSCs directional chemotactic properties to carry EPO to treat pulmonary fibrosis. Using MSCs as a carrier, the EPO gene is introduced by means of genetic engineering, so that EPO is secreted in the lung organs and forms a local microenvironment, which promotes the secretion of MVs carrying anti-inflammatory and anti-fibrosis molecules, enters the lung cells through cell entry, and intervenes fibrosis. In this way, MSCs carrying and expressing EPO can directly target pulmonary fibrosis organs, improve the binding efficiency, and achieve twice the result with half the effort. At the same time, it can effectively prolong the biological half-life, promote the secretion of anti-inflammatory and anti-fibrosis molecules by MSCs, and improve the therapeutic effect.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to the treatment or prevention of pulmonary fibrosis. The invention realizes the purpose of treating pulmonary fibrosis by utilizing the directional chemotaxis property of MSCs to carry EPO. Background technique [0002] Fibrosis refers to the pathological process in which parenchymal cells of an organ undergo necrosis due to inflammation, and the extracellular matrix in the tissue increases abnormally and excessively deposits. In severe cases, tissue structure damage and organ sclerosis occur. Fibrosis can occur in various organs, such as lung, heart, liver, pancreas, kidney, etc. Continuous progress can lead to organ structural damage, functional decline, and even failure, which seriously threatens human health and life. Worldwide, tissue fibrosis is a major cause of disability and death in many diseases. According to relevant statistics in the United States, nearly 45% of the...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCA61K35/28A61K38/22A61K47/46
Inventor 袁巍陈露罗清清邢丽苹项春生
Owner ZHEJIANG SHENGCHUANG PRECISION MEDICAL TECH CO LTD