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Method and application of directly reprogramming mouse liver cells into pancreatic beta cells

A β-cell and reprogramming technology, applied in the direction of insulin, animal cells, vertebrate cells, etc., can solve the problems of poor maturity, low reprogramming efficiency, safety, etc., and achieve improved maturity, high transfection efficiency, and improved safety effect

Active Publication Date: 2019-04-05
深圳市诺亚起航生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that Pdx1 can effectively drive the formation of pancreatic buds, but only Pdx1 cannot directionally drive the further development of cells, and requires the interaction of Ngn3, Neurod1, Mafa, Pax4, etc.
In 2008, the best combination of transcription factors Pdx1, Ngn3, and Mafa (PNM) screened out by Zhou et al. successfully reprogrammed mouse pancreatic exocrine cells directly into insulin-secreting cells, achieving a reprogramming efficiency of 20%. Islet-like structure, the secretion of insulin is far less than normal islet β cells
The direct reprogramming technology not only avoids the limitations of insufficient islet transplantation donors and immune rejection, but also avoids the low transdifferentiation efficiency of insulin cells differentiated from stem cells and safety issues
[0005] In recent years, the application of direct cell reprogramming technology to find the source of islet β cells has become a new hotspot, but this technology still has the problems of low reprogramming efficiency and poor maturity, and there are also safety and efficiency in the transfection method low problem

Method used

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  • Method and application of directly reprogramming mouse liver cells into pancreatic beta cells
  • Method and application of directly reprogramming mouse liver cells into pancreatic beta cells
  • Method and application of directly reprogramming mouse liver cells into pancreatic beta cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1, Construction, Identification and Efficiency Evaluation of Transcription Factor Plasmid Expression Vector

[0079] 1. Construction of transcription factor plasmid expression vector

[0080] The following genes Pdx1 (SEQ ID NO: 2), Ngn3 (SEQ ID NO: 1), Mafa (SEQ ID NO: 3), Neurod1 (SEQ ID NO: 4) and Pax4 (SEQ ID NO: 5) were artificially synthesized.

[0081] The amino acid sequence of protein Pdx1 encoded by gene Pdx1 is sequence 6;

[0082] The amino acid sequence of protein Pdx4 encoded by gene Pdx4 is sequence 7;

[0083] The amino acid sequence of the protein Neurod1 encoded by the gene Neurod1 is SEQ ID NO:8.

[0084] 1. Construction of tandem vectors pcDNA3.1(+)-Pdx1+Ngn3+Mafa—GFP and pcDNA3.1(+)-Pdx1, Ngn3, Mafa, Neurod1, Pax4

[0085] 1) Construction of tandem vector pcDNA3.1-Pdx1-2A-Ngn3-2A-Mafa-EGFP

[0086] The pcDNA3.1-Pdx1-2A-Ngn3-2A-Mafa-EGFP vector is a DNA fragment between the BamHI and HindIII restriction sites of the pCDNA3.1-EGFP vector r...

Embodiment 2

[0130] Example 2. Screening and identification of optimal transcription factor combinations and transfection time for direct cell reprogramming

[0131] In this experiment, by studying the order in which different transcription factors play a role and screening the best combination and time of reprogramming efficiency and maturity at different transfection periods, the reprogrammed β cells are identified from the aspects of genes and proteins, which will provide future reference. The clinical application lays the foundation, as follows:

[0132] The five recombinant vectors prepared in Example 1 were transfected into the NCTC-1469 mouse liver cell line according to the following grouping:

[0133] (1) Combination 1 Pdx1+Ngn3+Mafa: the recombinant vector pcDNA3.1(+)-Ngn3(N-Ngn3), pcDNA3.1(+)-Mafa(N-Mafa), pcDNA3.1(+)-Pdx1 (N-Pdx1) was mixed and co-transfected into the NCTC-1469 mouse liver cell line according to the mass ratio of 1:1:1;

[0134] (2) Combination 2 Pdx1+Ngn3+Neur...

Embodiment 3

[0237] Example 3. Research on the treatment of diabetes by directly reprogramming mouse liver cells into β cells

[0238] In this study, by constructing a mouse model of diabetes, the optimal combination and reprogrammed pancreatic islet β cells screened in the previous stage were transplanted under the left renal capsule of diabetic mice, and the blood glucose changes of the mice were monitored. Monitor the release of insulin and C-peptide, evaluate the hypoglycemic effect on diabetic model mice, and provide a certain basis for clinical treatment of diabetes.

[0239] 1. Construction of diabetes model

[0240] (1) After feeding SCID-Beige mice for 1 week, the body weight is about 20-25g.

[0241] (2) The mice were fasted for 14 hours without water, and the concentration of the prepared STZ solution was 20mg / ml; that is, 5-6ul / g mouse body weight.

[0242] (3) After the mice were injected with STZ, they were fed normally, blood was collected through the tail vein every morni...

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Abstract

The invention discloses a method for directly reprogramming mouse hepatocyte into an islet beta cell, and an application thereof. The method for reprogramming mouse hepatocyte into the islet beta cell is characterize in that Pdx1 protein encoding gene, Pax4 protein encoding gene and Neurod1 protein encoding gene are transferred into an in vitro liver cell to obtain the islet beta cell. The method has the following advantages: 1, a non-viral Entranster TM-D transfection reagent is adopted, so high transfection efficiency is realized, and the transfection safety in the direct reprogramming process is greatly improved; 2, a new optimum transfection combination Pdx1, Pax4 and Neurod1 suitable for being used in direct reprogramming to form the islet beta cell is screened according to the literature; and 3, an optimum transfection period is screened according to the action sequence of all transcription factors, so the maturity of the directly reprogrammed islet cell is improved, and the reprogramming efficiency in the researches in the invention reaches 23%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method and application for directly reprogramming mouse liver cells into pancreatic islet β cells. Background technique [0002] At present, diabetes is still a serious social health problem plaguing countries all over the world, and the prevalence of diabetes is on the rise. Type 1 diabetes is an autoimmune disease, which is caused by the selective destruction of islet β cells involving T cells. The application of islet cell transplantation technology is limited due to the lack of donors and immunosuppression. Therefore, more and more studies focus on the source of insulin-secreting cells. [0003] Direct cell reprogramming technology is a new technology after induced pluripotent stem cells (induced pluripotent stem cells, iPSCs) discovered by Takahashi and Yamanaka in 2006. This technology is different from induced pluripotent stem cells, which refers to the direct transformati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N5/071C07K14/62A61K38/28A61K35/39A61P3/10
Inventor 李富荣齐晖邓春艳张田田
Owner 深圳市诺亚起航生物科技有限公司
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