GAG (glycosaminoglycan) analogue realizing cell membrane modification, synthetic method of GAG analogue and application method of GAG analogue in in-vitro induced stem cell directional differentiation

A technology of glycosaminoglycans and synthesis methods, which is applied in the field of glycosaminoglycans labeled with green fluorescein and can solve the problems of uncontrollable sulfonation degree, limited wide application, complex synthesis and purification process, etc., and achieves The synthesis method and purification process are simple, the degree of sulfonation is easy to control, and the effect of strong universality

Active Publication Date: 2016-07-13
SUZHOU UNIV
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Problems solved by technology

However, whether it is natural GAG molecules or chemically synthesized GAG analogs, although they can obtain certain biological activities in the process of interacting with cells, these polymer molecules will be internalized by cells in a short time, making functional Polymer molecules cannot directly interact with specific growth factors and corresponding receptors on the cell surface. Therefore, a phospholipid group-anchored GAG analog is prepared by cell membrane surface engineering technology, and GAG The analogs are directly introduced into the cell membrane to induce the differentiation of ESCs, and its application in tissue engineering has important scientific value and social significance
[0003] At present, the main method for preparing GAG analogs anchored by phospholipid groups is as follows: first, synthesize a RAFT reagent containing phospho

Method used

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  • GAG (glycosaminoglycan) analogue realizing cell membrane modification, synthetic method of GAG analogue and application method of GAG analogue in in-vitro induced stem cell directional differentiation
  • GAG (glycosaminoglycan) analogue realizing cell membrane modification, synthetic method of GAG analogue and application method of GAG analogue in in-vitro induced stem cell directional differentiation
  • GAG (glycosaminoglycan) analogue realizing cell membrane modification, synthetic method of GAG analogue and application method of GAG analogue in in-vitro induced stem cell directional differentiation

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preparation example Construction

[0044] A kind of synthetic method of the sugar amine group analog of phospholipid group anchoring, comprises the following steps:

[0045] (1) Using RAFT technology to prepare a copolymer of SS, MAG and FluMA three monomers (molecular weight is in the range of 6000-10000), and using the chemical reaction between active groups to synthesize MAL-DPPE and DPPE-pSMF;

[0046](2) Dissolving the prepared DPPE-pSMF in a serum-free culture solution at a certain concentration, and incubating the Hela cells for a certain period of time in a 37°C constant temperature incubator, and then studying the modification of the cell membrane surface;

[0047] (3) Dissolve DPPE-pSMF in serum-free culture medium, incubate ESCs in a constant temperature incubator at 37°C for a certain period of time, add fresh neural differentiation induction medium after washing, and study how it promotes the directional differentiation of ESCs into neurons after several days Case.

[0048] The principle of the pr...

Embodiment 1

[0054] (1) Select SS, MAG, FluMA as monomers, in DMF / H 2 RAFT polymerization is carried out in a mixed solvent of O (V / V=1:1). After a certain period of reaction, the reaction solution is dialyzed and purified to obtain a terpolymer pSMF, which is added to MAL-DPPE to obtain a phospholipid group. Anchored glycosaminoglycan analog DPPE-pSMF;

[0055] (2) Characterize the obtained substance by proton nuclear magnetic resonance spectrum and infrared spectrum.

[0056] The result is as figure 1 As shown, the phospholipid-anchored glycosaminoglycan analog DPPE-pSMF 1 H-NMR spectrum.

[0057] The phospholipid-anchored glycosaminoglycan analogs are used to modify the cell membrane of the model cells, including the following steps: (1) the model cells Hela are planted on the cell climbing plate, and a certain concentration of phospholipid-anchored glycosaminoglycans is prepared. Serum-free medium of analogs, incubate Hela cells at 37°C for a certain period of time;

[0058] (2) W...

Embodiment 2

[0060] (1) Plant Hela cells on the cell slide plate, add serum-free high-glucose DMEM culture medium containing 3.4 μM terpolymer pSMF and phospholipid group-anchored polymer DPPE-PSMF respectively, and incubate at 37°C Incubate in the box for 1 h, and the cells incubated in serum-free high-glucose DMEM without any polymer were used as the control group;

[0061] (2) Discard the above-mentioned culture solution for incubating cells, wash the cells with sterile PBS 2 to 3 times to remove excess polymers, fix the cells on the slide plate with 4% paraformaldehyde solution, and use DAPI reagent to carry out cell nuclei After staining, the modification of the phospholipid-anchored polymer on the surface of the cell membrane was observed under a confocal fluorescence microscope.

[0062] The result is as figure 2 Shown, Hela cells were incubated with pSMF and DPPE-pSMF in the culture solution environment without adding polymer for 1 h, the situation of polymer modification on the ...

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Abstract

The invention discloses a GAG (glycosaminoglycan) analogue realizing cell membrane modification, a synthetic method of the GAG analogue and an application method of the GAG analogue in in-vitro induced stem cell directional differentiation. The GAG analogue can be inserted into the surface of a cell membrane and can prompt a stem cell to be directionally and efficiently differentiated into a mature neuronal cell in a shorter cycle. The phospholipid anchored GAG analogue is prepared firstly through RAFT (reversible addition-fragmentation chain transfer) polymerization, and then phospholipidation of the GAG analogue is realized through maleimidized lipid molecules; the phospholipidized GAG analogue is introduced to the surface of the stem cell through affinity interaction between phospholipid groups and the cell membrane, so that the stem cell can be better prompted to be efficiently differentiated into the mature neuronal cell. A synthetic process is simple, and a powerful means capable of realizing phospholipidation of almost every RAFT polymer is provided.

Description

technical field [0001] The invention belongs to the fields of small molecules, polymer synthesis and biomedical applications, and in particular relates to a maleimidated phospholipid molecule, a green fluorescein-labeled glycosaminoglycan analog, and a phospholipid-anchored glycosaminoglycan analog The synthesis method of the substance uses the reaction between chemically active groups and RAFT technology to obtain a polymer substance that can be inserted into the cell membrane and can efficiently promote the directional differentiation of embryonic stem cells into mature neurons in a short period of time. Background technique [0002] Embryonic stem cells (ESCs) are a type of totipotent cells isolated from the inner cell mass of the blastocyst stage, which can self-renew and differentiate into almost all somatic cells under certain conditions. Promising cell source for cell therapy and tissue transplantation. Natural glycosaminoglycan (GAG) is a highly structurally heterog...

Claims

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Application Information

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IPC IPC(8): C08F212/14C08F220/58C08F220/32C08F8/40C12N5/0793C12N5/0735C12N5/0775C12N5/0797
CPCC08F8/40C08F2438/03C12N5/0619C12N2501/90C12N2506/02C12N2506/08C12N2506/1353C08F212/14C08F220/58C08F220/32
Inventor 陈红陈高健袁琳刘琦曹丽敏吕仲林
Owner SUZHOU UNIV
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