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Neural stem cell carrying tumor-related gene and preparation method and application of neural stem cell

A tumor-related gene and neural stem cell technology, which is applied in the field of neural stem cells carrying tumor-related genes and the preparation thereof, can solve the problems of difficulty in elucidating tumor stem cells in detail, inability to study tumorigenesis mechanisms in depth, and inability to effectively simulate tumors, etc. The effect of important scientific significance and application value

Active Publication Date: 2020-05-05
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, similar to a large number of previous tumor-related research works, these works focus on primary glioma or tumor cell lines as the research object, and conduct research on the established tumor cell population, so they cannot effectively simulate the occurrence of tumors. However, it is difficult to clarify whether the tumor stem cells are the initial cells of the tumor or the cell populations that can continuously proliferate and form tumors during tumor progression, so that it is impossible to deeply study the mechanism of tumorigenesis

Method used

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  • Neural stem cell carrying tumor-related gene and preparation method and application of neural stem cell
  • Neural stem cell carrying tumor-related gene and preparation method and application of neural stem cell
  • Neural stem cell carrying tumor-related gene and preparation method and application of neural stem cell

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Embodiment 1

[0069] Example 1 Establishment and Identification of Pluripotent Stem Cell Lines Carrying Tumor-Related Genes

[0070] The present invention relates to activating the KRAS expression by inducing the mutation of the proto-oncogene KRAS, overexpressing the upstream gene EGFR and the downstream gene BRAF of the RAS signaling pathway, and knocking down the expression of the PTEN gene in human embryonic stem cells (hESCs). The present invention first designs and constructs lentiviral plasmid vectors containing human KRAS gene (including mutation site G13D), human BRAF gene and EGFR gene fragments respectively through molecular cloning methods, constructs shRNA lentiviral plasmid vectors for knockdown expression of PTEN gene, and recombines The vectors all contain the Puro resistance gene for positive clone screening, and then the plasmid is packaged as a lentivirus, and the exogenous gene KRAS is infected by the lentivirus method. G13D , BRAF, and EGFR were overexpressed, the PTEN ...

Embodiment 2

[0097] Example 2. ESCs carrying tumor-associated genes undergo in vitro directed differentiation to produce neural stem cells (NPCs)

[0098] With the H1-KRAS obtained in embodiment 1 G13D , H1-shPTEN, H1-BRAF-shPTEN, H1-shPTEN-KRAS G13D , H1-BRAF-EGFR-shPTEN cells, according to image 3 The flow chart of neural direction differentiation of induced pluripotent stem cells shown, further in vitro directional differentiation into neural stem cells, the specific method is as follows:

[0099] 1. Directed differentiation of neural stem cells

[0100] 1) Culture hESCs in a 6-well plate until the cell confluency reaches 90% or above. The day before cell subculture, the 12-well culture plate (500 μL / well) was coated with 100× Matrigel overnight, and the hESCs were passaged, and the cells in one well of the 6-well plate were transferred to one well of the 12-well plate to ensure the second passaging. The cell density reaches over 95% (preferably 100%) two days later, and the cell c...

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Abstract

The invention discloses a neural stem cell carrying a tumor-related gene and a preparation method and application of the neural stem cell. According to the neural stem cell, a KRAS (kirsten rat sarcoma viral oncogene) gene mutant as well as an upstream gene EGFR (epidermal growth factor receptor) and a downstream gene BRAF (V-raf murine sarcoma viral oncogene homolog B1) of a combined over-expression Ras / raf / MAPK (mitogen-activated protein kinase) signal channel are transferred into a human multipotential stem cell, and PTEN (phosphate and tension homology deleted on chromsome ten) gene expression is knocked off or reduced, so that a multipotential stem cell carrying the tumor-related gene is obtained; and the multipotential stem cell is further induced to differentiate to a nerve direction, then the neural stem cell is obtained, the dynamic transformation process of a tumor stem cell and occurrence of a tumor is effectively simulated, and a novel and reliable cell model is provided for research on pathogenesis of neural tumors such as gliomas. By adopting the technology, possibility is provided for dynamic observation on the occurrence and development process of the gliomas in multiple angles, novel research ideas are also brought to glioma treatment medicine screening and targeting treatment, and great scientific significances and application value can be made.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a neural stem cell carrying a tumor-related gene, a preparation method and application thereof. Background technique [0002] Nervous system tumors seriously endanger human health. Glioma occurs in the neuroectoderm and is the most common tumor of the central nervous system, accounting for 40-50% of all primary intracranial tumors. Because of its highly proliferative and invasive biological characteristics, surgical resection is difficult, and it is not sensitive to radiotherapy and chemotherapy, so tumor recurrence is common, and there is still a lack of effective treatment methods. Therefore, finding more effective treatment methods has become an urgent task at present, and in-depth elucidation of the pathogenesis of glioma is a key issue that must be resolved to determine key therapeutic targets and molecular markers for discovery, differential diagnosis, and prognosis ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12Q1/02
CPCC12N5/0623C12N15/86C12N5/0606C12N5/0696G01N33/5011G01N33/5058C12N2501/15C12N2501/727C12N2740/15043C12N2800/107C12N2506/02C12N2506/45C12N2501/115C12N2501/11C12N2503/02G01N2500/10
Inventor 王丽辉梁伟业
Owner JINAN UNIVERSITY
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