Method for immortalizing myoblasts

Abstract

The invention discloses a method for immortalizing myoblasts. According to the method, low-density culture is adopted, retrovirus infection under the centrifugal condition is combined, improvement of repeated adherent enrichment immortal myoblasts is combined, operation conditions are optimized and controlled, the cell infection efficiency of the retroviruses is greatly improved, and the yield of the immortal myoblasts is greatly increased. According to the method, as for samples with very small initial sample size, particularly very low myoblast proportion (lower than 10% or even only 1%) in initial samples, immortalized myoblasts still can be successfully and permanently formed, and most samples can ensure that immortalized myoblasts with the purity of 70% or above can be finally obtained. According to the scheme, all muscular dystrophy patients can be benefited, and good application value and prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. More specifically, it relates to a method for immortalizing myoblasts, and the resulting immortalized myoblasts can be used for research on pathophysiology and therapeutic targets of muscular dystrophy and development of related drugs. Background technique [0002] Muscular dystrophies are a group of genetic disorders characterized by progressive muscle weakness and degeneration of muscles that govern movement. Muscular dystrophies include congenital muscular dystrophy, Becker muscular dystrophy and other types. Some forms of muscular dystrophy can result in impaired movement or even paralysis. Clinically, it mainly manifests as progressively aggravated skeletal muscle atrophy and weakness in varying degrees and distributions, and may also involve the myocardium. The study of the pathophysiology and therapeutic targets of muscular dystrophy has been limited by the limited proliferative cap...

AI Technical Summary

Problems solved by technology

But the technical flaw: Before immortalizing cells, that is, before viral transduction, it is necessary to use flow cytometry to purify myogenic CD56-positive myoblasts

However, the defect of this technology: the same as the above-mentioned prior art, retrovirus is directly added to the culture dish covered with cells to achieve the purpose of introducing CDK4 and hTERT target genes into cells. Current research data show that this method reverse transcription The effect of virus-infected cells is not significant, and the possibility of successful selection of immortalized myogenic CD56-positive myoblasts is low

The initial human muscle biopsy sample used to isolate cells in this technology is 500 to 700 mg, and the myogenic CD56-positive myoblasts in the final immortalized cells are 14%. In the absence of enrichment of CD56-positive cells, the cloning ring Screening of immortalized muscle-derived CD56-positive monoclonal cells, the screening efficiency is low

[0006] In addition, the problem of poor samples is often encountered in clinical practice. For example, some patients are old, muscle diseases lead to muscle atrophy, and the initial sample size is very small (less than 30mg). Therefore, after separation of some samples, the number of myoblasts in them The absolute number and proportion are very low, among which the proportion of myoblasts is basically less than 10%, or even only 1%

For such samples, it is currently difficult to successfully perform efficient immortalization and enrichment, and it is difficult to obtain high-purity immortalized myoblasts

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