Non-integrative reprogramming method of peripheral blood mononuclear cell for MERRF patient

Abstract

The invention discloses a non-integrative reprogramming method of the peripheral blood mononuclear cell for an MERRF patient. The non-integrative reprogramming method comprises the following steps: resuscitating the cryopreserved peripheral blood mononuclear cell (PBMC), inoculating the resuscitated peripheral blood mononuclear cell to a 24-hole cell culture plate, and carrying out continuous culture for 4 days under the saturated humidity condition with the temperature of 37 DEG C and with 5% CO2, wherein liquid is exchanged each day; carrying out vital cell staining counting by adopting trypan blue, adjusting the cell density, abandoning a culture solution containing viruses, carrying out further culture for three days by adopting PBMC complete culture solution, and transferring the infected cell to a 6-hole cell culture plate covered by Matrigel basement-membrane matrix; carrying out median liquid exchanging by adopting StemPro-34 containing no cell factors, after three days, taking the PSC culture solution, and after cloning with suitable size is obtained, carrying out passage, carrying out enlarged culture, and carrying out cryopreservation. The detection experiment proves that the obtained cell line has totipotency and normal karyotype, and completely contains no exogenous genes, meanwhile, the mutation existing in the mitochondria genetic material of the MERRF patient can be simulated, and various demands for MERRF disease research can be adapted.

Description

technical field [0001] The present invention relates to the technical field of cell biology and biomedicine, in particular to a method for non-integrated reprogramming of peripheral blood mononuclear cells of MERRF patients. Background technique [0002] Clonic epilepsy with ragged-red fibers syndrome (myoclonic epilepsy with ragged-red fibers, MERRF) is a hereditary multiple systemic disease and one of the common types of mitochondrial muscle encephalopathy. The main clinical symptoms are myoclonic seizures, tonic-clonic seizures, limb weakness, cerebellar ataxia, mental decline or dementia, and mental abnormalities. MERRF syndrome is mainly caused by the mitochondrial DNA (mtDNA) 8344th A to G mutation (mtDNA 8344A→G) (80%-90%) causing respiratory chain damage, and then corresponding clinical symptoms [1]. The disease can be clearly diagnosed by checking the mutations of the corresponding mitochondrial DNA loci using mtDNA first-generation or second-generation sequencing ...

AI Technical Summary

Problems solved by technology

[0004] A foreign laboratory has reprogrammed the cells of another common mitochondrial genetic disease, mitochondrial encephalopathy with lactic acidosis and stroke-like attack syndrome (MELAS), and due to the different pathogenic sites, the induced pluripotent stem cells of MELAS are not the same. Cannot be fully used for MERRF research

At the same time, due to the use of integrative induction technology based on lentiviral or retroviral vectors, there are potential genomic changes in these cells, which cannot fully simulate the patient's situation

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