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Method for inducing transdifferentiation of fibroblasts into neuronal cells and application thereof

A technology of neuron cells and fibroblasts, applied in the fields of biotechnology and neurodevelopment, can solve problems such as cancer risk, low success rate, and time-consuming

Active Publication Date: 2014-06-11
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the limitations of cloning and embryonic stem cells, induced pluripotent stem cells are undoubtedly a step forward, but its shortcomings are also obvious
It is not easy to obtain, and the success rate is extremely low; it takes a long time and is relatively lacking in stability, and has a risk of cancer

Method used

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  • Method for inducing transdifferentiation of fibroblasts into neuronal cells and application thereof
  • Method for inducing transdifferentiation of fibroblasts into neuronal cells and application thereof
  • Method for inducing transdifferentiation of fibroblasts into neuronal cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Obtaining and culturing primary skin fibroblasts

[0129] Under aseptic conditions in the operating room, fresh human skin tissue was cut, the epidermis and subcutaneous tissue were removed, washed repeatedly with saline, placed in DMEM / F12 culture medium containing double antibodies, and brought back to the intercellular space. Place the tissue block in a petri dish, rinse with PBS three times, cut the tissue block repeatedly with ophthalmic scissors to a suitable size, take the tissue block with tweezers and inoculate it on a 6 cm petri dish, leaving a distance of about 0.3-0.5 cm between the tissue blocks. Place in a 37°C incubator for 30 minutes to dry the cultured tissue pieces slightly. Take out the culture bottle from the incubator, gently inject 3ml of fibroblast culture medium (90%DMEM / F12, 10%FBS) into the bottom of the dish, let the culture medium slowly cover the small tissue pieces on the wall of the attached bottle, be careful not to Let the cell block fl...

Embodiment 2

[0132] Construction of miRNA-302 / 367 cluster expression vector and preparation of retrovirus

[0133] According to the sequence of miRNA-302 / 367 cluster, design two primers:

[0134] Forward primer 5'GCTCCCTTCAACTTTAACA3' (SEQ ID NO: 11)

[0135] Reverse primer 5'-CCATCACCATTGCTAAAGT 3' (SEQ ID NO: 12)

[0136] The above primer sequences were artificially synthesized by Yingwei Jieji Company.

[0137] Digest the skin fibroblasts obtained in Example 1 with 1ml trizol, add 0.2ml chloroform, mix well, centrifuge at 10000g for 15min, discard the water phase, add 0.3ml absolute ethanol, mix well, centrifuge at 2,000×g for 5min, discard Wash the DNA pellet with 0.1M sodium citrate containing 10% ethanol. Mix and let stand for 30min, centrifuge at 2,000×g for 5min, discard supernatant, wash twice with 75% ethanol, centrifuge at 2,000×g for 5min, discard supernatant, add 10μl 8mM NaOH to dissolve after drying, and then dilute with 90μl TE buffer.

[0138] The genomic DNA thus obta...

Embodiment 3

[0142] Construction of miRNA-9 and miRNA-124 expression vectors and preparation of retroviruses

[0143] The precursor sequence of miRNA-9 is synthesized, and the precursor sequence of miRNA-9 is shown as SEQ ID NO:2.

[0144] The precursor sequence of miRNA-124 was synthesized, and the precursor sequence of miRNA-124 is shown in SEQ ID NO:3.

[0145] Insert the precursor sequences of miRNA-9 and miRNA-124 into the lentivirus3 plasmid (using the H1 promoter and carrying the GFP reporter gene), and co-transfect the 293T cells with the expression plasmid lentivirus3 and the packaging plasmid (env vector and pacvector), and collect in 48 hours Cleared, concentrated by ultrafiltration for later use.

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Abstract

The invention relates to a method for inducing transdifferentiation of fibroblasts into neuronal cells and application thereof. Specifically, miRNA-302 / 367 clusters, miRNA-9 and miRNA-124 are indispensable for the transdifferentiation of the fibroblasts into the neuronal cells; no obvious stem cell cloning stage takes place in the transformation process, and a retrovirus system is adopted to stably and efficiently express the miRNA-302 / 367 clusters, the miRNA-9 and the miRNA-124 in human fibroblasts, and therefore, a series of biochemical reactions of cells are adjusted and the fibroblasts are trans-differentiated into the neuronal cells. The invention also provides application of a miRNA combination (the miRNA-302 / 367 clusters, the miRNA-9 and the miRNA-124).

Description

technical field [0001] The invention belongs to the fields of biotechnology and neurodevelopment, in particular, the invention relates to a method for inducing fibroblasts to differentiate into neuron cells and its application. Background technique [0002] Nervous system diseases, especially central nervous system degenerative diseases are common and frequently-occurring diseases in clinical practice, which seriously endanger human health. According to a report released by the World Health Organization in February 2007, more than 1 billion people worldwide suffer from neurodegenerative diseases. In 2005, more than 24 million people worldwide suffered from Alzheimer's disease; other degenerative diseases of the central nervous system, Such as multiple sclerosis, Huntington's disease and Parkinson's syndrome, also bring great suffering to patients. [0003] Because some nerve cells in these patients are irreversibly damaged, and it is difficult to be supplemented by autologo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/0793C12N15/113C12N15/63A61K35/30A61P25/00
Inventor 顾红峰王蓓周晨楼觉人
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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