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Human hematopoietic stem cell expressing human exogenous proinsulin and application thereof

A technology of human insulin and stem cells, applied in the field of medical molecular biology

Inactive Publication Date: 2015-01-28
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The invention patent with the application number 200710100326.1 discloses a method for preparing insulin-secreting stem cell lines using lentiviral vectors encoding various foreign genes. After transfection of 293T cells for packaging, virus particles were collected and concentrated to transfect human embryonic pancreatic stem cells. This lentiviral vector containing a variety of insulin regulatory genes can greatly improve the ability of stem cells to synthesize insulin after transfection of stem cells. And produce obvious insulin release when stimulated by glucose. The patent only introduces that in vitro culture can improve insulin secretion, but the effect of transplanting cell lines into animal models is unknown.

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  • Human hematopoietic stem cell expressing human exogenous proinsulin and application thereof
  • Human hematopoietic stem cell expressing human exogenous proinsulin and application thereof
  • Human hematopoietic stem cell expressing human exogenous proinsulin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of recombinant lentiviral expression vector

[0033] (1) Preparation of Insert DNA

[0034] According to the human proinsulin gene mRNA sequence (GenBank NO.NM_000207) provided by GenBank, the sequence (5'-GAATTC-3') of EcoR I was added at the 5' end of the sequence by TaKaRa Company, and the sequence of BamHI was added at the 3' end (5 '-GGATCC-3'), a total of 12 nucleotides, the artificially synthesized human proinsulin gene includes enzyme cleavage sites, a total of 345bp, the artificially synthesized human proinsulin gene is synthesized into the pMD18-T simple vector to obtain The pMD18-T simple plasmid containing the human proinsulin gene sequence, through DNA sequence analysis, confirmed that the sequence of the synthetic preproinsulin was completely correct.

[0035] The pMD18-T simple plasmid containing the human proinsulin gene sequence was digested with EcoRI / BamHI, and the steps were as follows:

[0036] 1) Prepare an enzyme digestion...

Embodiment 2

[0061] Example 2 Packaging of recombinant lentiviral particles

[0062] (1) 293FT cell culture

[0063] 1) Preparation of culture medium: After adding FBS with a final concentration of 10% and penicillin and streptomycin at a final concentration of 1% to the DMEM basal medium, and then preheating in a 37° C. water bath.

[0064] 2) Take out the cells when the cell abundance is 70-80%, discard the old medium gently, wash the cells with HBSS preheated at 37°C, add an appropriate amount of trypsin / EDTA to digest the cells.

[0065] 3) Observing under a microscope, if the cells are separated into a single state, immediately add twice the volume of medium, and gently blow and mix to terminate the digestion.

[0066] 4) Add the cell suspension into a 15ml centrifuge tube, centrifuge at 800rpm for 3min at room temperature, and discard the supernatant.

[0067] 5) Add an appropriate amount of culture medium and pipette gently to make a cell suspension.

[0068] 6) Take 1 / 3 of the c...

Embodiment 3

[0087] Example 3 Construction of human hematopoietic stem cells (proinsulin UCB CD34+ cells) expressing human exogenous proinsulin

[0088] (1) The umbilical cord blood (UCB) of newborns was collected according to standard procedures with the joint signature and consent of the hospital ethics committee and the puerpera. According to the Ficoll-Plaque method, umbilical cord blood mononuclear cells were isolated, and CD34+ cells (UCBCD34+) were isolated from the monocytes using the kit (CD34 microbeads kit) provided by Miltenyi according to the instructions, and cultured in vitro by standard human hematopoietic stem cells. , to culture UCBCD34+ cells.

[0089] (2) According to the standard method of lentivirus infection, the prepared pCDH-CMV-insulin-EF1-GFP-T2A-Puro recombinant lentiviral particles were used to infect UCBCD34+ cells. On the second day after infection, the green fluorescein was detected by flow cytometry The proportion of protein (GFP) positive cells, the resul...

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Abstract

The invention belongs to the field of medical molecular biology, and particularly relates to a human hematopoietic stem cell expressing a human exogenous proinsulin and a preparation method and an application thereof. The human hematopoietic stem cell expressing the human exogenous proinsulin is obtained by the steps: a DNA recombinant technology is adopted, a human proinsulin gene is cloned into a lentiviral expression vector, a lentiviral expression vector which can express the human proinsulin gene is obtained, the expression vector and a lentiviral packaging plasmid are co-transfected to a 293FT cell, a lentiviral particle is packaged, a cell culture solution supernatant containing a virus is collected, newly-isolated UCB HSCs are infected, and the human hematopoietic stem cell expressing the human exogenous proinsulin is obtained. The human hematopoietic stem cell can be amplified in vitro, and an amplified cell is transplanted by a cell and is capable of normal self renewal and multi-directional differentiation in vivo; the secreted proinsulin can activate an insulin receptor and a downstream target gene, is transplanted to an NOD / SCID / Diabetes I mouse, can significantly reduce blood glucose of the type I diabetic mouse, and improves the survival rate of the mouse.

Description

technical field [0001] The invention belongs to the field of medical molecular biology, and in particular relates to a human hematopoietic stem cell expressing human exogenous insulin proinsulin and its preparation method and application. Background technique [0002] Diabetes is one of the major diseases that seriously endanger human health, and has become the third major disease after cardiovascular diseases and tumors. In recent years, the incidence of diabetes has been increasing, and the onset is getting younger. [0003] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defective insulin secretion or impaired biological action, or both. Diabetic patients have disorders of glucose metabolism due to insulin production disorders or insulin failure to exert physiological functions, resulting in microvascular disease, causing a series of serious complications such as limb necrosis, blindness and renal failure. Currently ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N15/66C12N5/0789A61K35/28A61P3/10C12R1/93
Inventor 叶治家韩月雯汤玲
Owner ARMY MEDICAL UNIV
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