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Method for preparing insulin secretory cell

An insulin secretion and cell technology, applied in the field of biochemistry, can solve the problems of complex methods, poor repeatability, and the induction efficiency needs to be further improved, achieve high transfection efficiency, and overcome the effect of complex construction

Inactive Publication Date: 2008-03-05
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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Problems solved by technology

Studies have shown that bone marrow mesenchymal stem cells may differentiate into insulin-secreting cells. Wang Qiwei et al. found that the differentiated bone marrow mesenchymal stem cells had the mRNA expression of insulin 1 after being induced by stimulation factors such as high glucose, activin A, retinoic acid, and β-mercaptoethanol. (Wang Qiwei et al., Activin A and retinoic acid induced bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells in vitro. China Clinical Rehabilitation. Volume 10, Issue 17, 2006), Tang et al. Induced IPCs (Tang DQ, Cao LZ, Brant R et al. Invivo and in vitro characterization of insulin in-producing cells obtain from murinebone marrow. Diabetes. 2004, 53: 1721-1732.), but these methods are complex and poorly reproducible , and the induction efficiency needs to be further improved

Method used

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  • Method for preparing insulin secretory cell

Examples

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Effect test

example 1

[0057] 1. Obtaining methods of nestin positive cells:

[0058] Bone marrow tissue was taken in serum-free L-DMEM medium (containing heparin 100u / ml), washed repeatedly, blown evenly, and made into a single cell suspension. Centrifuge at 1000rpm for 8min, discard the supernatant and fat. Resuspend in serum-free L-DMEM, gently superimpose the cell suspension on the wall into a 15ml plastic centrifuge tube with 1 / 2 volume of lymphocyte separation medium (specific gravity 1.077), make the suspension in the upper layer, centrifuge at 2500rpm, 25℃ After 20 minutes, collect the mononuclear cell layer like white film in the middle into another centrifuge tube, wash twice with PBS (1000 rpm, centrifuge for 8 minutes), and discard the supernatant. Cells were resuspended in neural stem cell culture medium containing 10% fetal bovine serum (FBS) at 1 × 10 7 Inoculate the cells at a density of 1 / ml in a disposable plastic culture bottle in a constant temperature incubator at 37°C and 5% ...

example 2

[0132] 1. Preparation of nestin positive cells: same as example 1;

[0133] 2. Preparation of recombinant plasmid pEGFP-C2-PDX-1: same as Example 1;

[0134] 3. Recombinant plasmid pEGFP-C2-PDX-1 transfected nestin-positive cells: same as example 1;

[0135] 4. Induced differentiation after transfer: transfer the above nestin + The cells were inoculated in L-DMEM containing 100 nM glucagon-like peptide-1 and 20% FBS at a ratio of 1:2 and cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity.

example 3

[0137] 1. Preparation of nestin positive cells: same as example 1;

[0138] 2. Preparation of recombinant plasmid pEGFP-C2-PDX-1: same as Example 1;

[0139] 3. Recombinant plasmid pEGFP-C2-PDX-1 transfected nestin-positive cells: same as example 1;

[0140]4. Induced differentiation after transfer: transfer the above nestin + The cells were inoculated in L-DMEM with a final concentration of 100 nM of glucagon-like peptide-1, glucose at a final concentration of 4.Sg / L, and 20% FBS at 37°C and 5% CO at a ratio of 1:2. 2 , Cultivated in a saturated humidity constant temperature incubator.

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Abstract

The present invention provides one kind of insulin secreting cell, which is prepared through pre-inducing marrow stem cell in nerve stem cell culturing medium into nestin+ cell, introducing PDX-1 gene-containing eukaryon expressing vector to the nestin+ cell by means of cell nucleus transfection, and final culture in low sugar DMEM containing cell factor and 20 % concentration fetal calf serum for induction differentiating into insulin secreting cell. The cell factor is glucose and / or glicetin-1. The insulin secreting cell of the present invention can generate and secrete insulin and C peptide, and may be used as substitute of beta-cell for the cell treatment or gene treatment of diabetes.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for preparing undifferentiated mammalian cells, especially marrow-derived stem cells modified by introducing foreign genetic material. Background technique [0002] In 2000, there were 150 million people suffering from diabetes in the whole world. According to relevant research, this number will double in 2025. There are two main types of diabetes; type 1 diabetes and type 2 diabetes. For diabetic patients (type 1 and some type 2) with impaired islet cell function, implantation of islet β cells or their substitutes is an ideal treatment. In recent years, portal vein islet cell transplantation technology has been developed clinically, but the lack of cell sources and severe immune rejection have greatly limited the wide application of this therapy. The use of artificial, non-β-cell-derived β-cells for gene or cell therapy to treat diabetes has attracted widespread attention....

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
Inventor 王海澜高毅蒋泽生
Owner ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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