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A three-dimensional compound cell aggregate model and its preparation method and application

A three-dimensional composite and aggregate technology, applied in the field of three-dimensional composite cell aggregate model and its preparation, can solve the problems of rare reports of human-derived cadherin protein, and achieve the promotion of stem cell proliferation and differentiation function expression, good stability, immobilization The effect of simple method

Active Publication Date: 2018-10-12
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few reports on the related research on human cadherin protein

Method used

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  • A three-dimensional compound cell aggregate model and its preparation method and application
  • A three-dimensional compound cell aggregate model and its preparation method and application
  • A three-dimensional compound cell aggregate model and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1 obtains pcDNA3.1-hE-cad-Fc gene plasmid

[0058] Human epithelial cell adhesion factor (hE-cadherin) ectodomain (hE-cad) and human immunoglobulin (IgG 1) Fc fragment (Fc) were fused by gene recombination technology to obtain a plasmid containing the target gene hE-cad and Fc pcDNA3.1-hE-cad-Fc. That is: using the full length of human E-cadherin mRNA (Gene bank: NM 004360.3) as a template, the synthetic primers were used to specifically amplify the hE-cadherin ectodomain fragment. The primer sequences are:

[0059] 5'-CGCAAGCTTATGGGCCCTTG-GAGCCGCAGC-3';

[0060] 5'-TTGCGGCCGCAGGCAGGAATTTGCAATCCTGC-3'.

[0061] like figure 1 As shown, the PCR product was analyzed by 1% agarose gel electrophoresis, and the gel-recovered PCR product was double-digested with Hind III and Not I, and inserted between the Hind III and Not I sites of pcDNA3.1-Fc. Plasmid pcDNA3.1-Fc was donated by Professor Toshihiro Akaike of Tokyo Institute of Technology.

[0062] like fig...

Embodiment 2

[0064] Embodiment 2 obtains hE-cad-Fc fusion protein

[0065] The plasmid pcDNA3.1-hE-cad-Fc containing the target gene constructed in Example 1 was transfected into 293F cells, cultured in suspension for 72 hours, the cell culture supernatant was collected, and the hE-cad-Fc fusion protein was purified by rProtein AFF column . The purified fusion protein was detected by immunoblotting using an anti-E-cadherin antibody, as image 3 As shown, there are no other non-specific exposure bands in the figure, and the band with a molecular weight of about 120KD is reduced E-cadherin ( image 3 Middle 1), the band at 240KD is non-reduced E-cadherin ( image 3 Medium 2). This result shows that the hE-cad-Fc fusion protein prepared by the present invention has a base sequence as shown in 1 in the sequence table, and the fusion protein can form a stable dimer structure in a non-reduced state.

Embodiment 3

[0066] Example 3 Preparation of hE-cad-Fc fusion protein matrix-modified surface-modified PLGA microspheres

[0067] First, microspheres with a particle size range of 15±5 μm were prepared by chemically synthesizing the polymer PLGA and placed in an EP tube; then the above hE-cad-Fc fusion protein solution was diluted to 10 μg / ml, soaked into the microspheres, and incubated at 37°C for 2 hours; the supernatant was discarded by centrifugation, rinsed with PBS three times to remove the unimmobilized fusion protein hE-cad-Fc, and obtained hE-cad-Fc matrix-modified PLGA microspheres, which were named hE-cad- Fc-PLGA.

[0068] The invention detects the effect of the protein concentration of the hE-cad-Fc solution on the surface modification of the matrix of PLGA microspheres by an Elisa method. The hE-cad-Fc substrate method is the same as above. Namely: after obtaining hE-cad-Fc-PLGA, add 1% BSA to block at room temperature for 1 hour, add anti-E-cadherin antibody and incubate a...

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Abstract

Provided is a three-dimensional composite cell pellet model, a method of preparing same, and an application of same. The model is prepared by using the following manner: using a natural or chemical synthetic polymer as a matrix material to prepare submicron-level microspheres, and immobilizing, on the surface of the microspheres, a fusion protein formed of an extracellular domain of a cadherin protein and an FC domain, and forming microspheres / cell composite pellets after mixing with stem cells.

Description

technical field [0001] The invention relates to the technical field of in vitro cell culture, in particular to a three-dimensional composite cell aggregate model and its preparation method and application. Background technique [0002] In the body, cells are in a highly informational microenvironment, which includes various physical and chemical signals that change dynamically in time / space, and cells are regulated by various signal stimuli to achieve their specific biological functions. This interaction between cells and the microenvironment includes a variety of complex biochemical, biomechanical, and bioelectrical signal stimuli responses from surrounding cells, extracellular matrix, and soluble factors. Among them, the interaction between cells occurs through direct contact or paracrine soluble factors. This intercellular communication is an important link in maintaining the structure and function of cells, tissues and organs, and is also a key factor in regulating tiss...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/0775
Inventor 杨军张妍李素华徐建斌
Owner NANKAI UNIV
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