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A method for inducing insulin-secreting cells based on human skin cells and its application

A technology of insulin secretion and skin cells, applied in the field of molecular cytology, can solve the problems of single cells, cumbersome directed differentiation steps, and lack of complete function of pancreatic islet beta cells

Active Publication Date: 2020-07-31
INST OF ADVANCED TECH UNIV OF SCI & TECH OF CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, when fibroblasts are directly transformed into insulin-producing or secreting cells through the directed differentiation of stem cells or the use of small molecule drugs, objectively there are cumbersome directed differentiation steps, and the induced cells do not have the full function of pancreatic beta cells or formed cells. Not single enough, the formed cells have multiple cellular functions, such as simultaneous production of insulin, glucagon, etc.
Although there are reports in the prior art on the induction of other human cells or stem cells into insulin-secreting cells, there is no report on the induction of human skin cells into insulin-secreting cells

Method used

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  • A method for inducing insulin-secreting cells based on human skin cells and its application
  • A method for inducing insulin-secreting cells based on human skin cells and its application
  • A method for inducing insulin-secreting cells based on human skin cells and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Induction of Exogenous Transcription Factor Infection of Human Foreskin Fibroblast Insulin-secreting Cells

[0034] In this example, the inventor first constructed the gene into the lentiviral vector Psin, and transfected it with PMD2G and PSPAX2 packaging plasmids in 293T cells, with a transfection ratio of 2:2:1. Viruses were harvested 48 hours after virus transfection. The virus was infected into human fibroblasts, and the medium was changed after 24 hours. Two days after the cells were infected with the virus, they were cultured using a specific medium (IMDM+1×ITS+10ng / ml EGF+10ng / ml bFGF).

Embodiment 2

[0035] Example 2 Determination of insulin secretion

[0036] RT-PCR experiment: When the virus infected human fibroblasts for 20 days, the collected cells were lysed with Trizol, RNA was extracted, RNA was reverse-transcribed using a reverse transcription kit, PCR was used to perform PCR on related genes, and electrophoresis was used to detect , to observe the expression levels of related genes. Such as figure 1 shown.

[0037] Cellular immunofluorescence: when the virus infected human fibroblasts for 20 days, the cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes, Triton-100 was perforated for 15 minutes, 2% BSA was blocked and incubated for 1 hour, and the primary antibody (buffer was 2% BSA) for 2 hours, ALEXA-secondary antibody (secondary antibody with fluorescence) for 1 hour and then mounted. Insulin production was observed under a fluorescent microscope, as figure 2 shown.

Embodiment 3

[0038] Example 3 Cellular glucose response experiment

[0039] The RT-PCR experimental steps are as implemented in 2, and the results are as follows image 3 shown.

[0040] Cell immunofluorescence experimental steps are as in implementation 2, and the results are as follows Figure 4 shown.

[0041] Insulin secretion experiment in vitro: the cultured cells were firstly washed with KRB buffer several times. Then pre-incubate KRB buffer containing 2 mM glucose for 2 hours to remove insulin remaining in the medium. The cells were washed twice again with KRB buffer, then incubated with KRB buffer containing 2mM glucose for 30min, and the supernatant was collected. Then the cells were washed twice with KRB buffer again, then incubated with KRB buffer containing 20 mM glucose for 30 min, and the supernatant was collected. Cells were quantified by BCA method for protein standard; human insulin detection kit was used to detect the level of insulin released by cells under 2mM glu...

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Abstract

The invention discloses an induction method for insulin secretion cells based on human skin cells and application thereof, belonging to the technical field of molecular cytology. The method comprises the following steps: constructing a lentiviral vector Psin loaded with obtained exogenous transcription factors consisting of PDX1, Mafa, Ngn3, Hnf6 and NeuroD1; then transfecting virus incubation cells with the lentiviral vector and packaging plasmids and carrying out culture; after culture, transfecting human skin cells with a virus obtained in the previous step; and carrying out culture so as to obtain the insulin secretion cells. According to the invention, it is determined for the first time that five transcription factors consisting of PDX1, Mafa, Ngn3, Hnf6 and NeuroD1 can be used for induced differentiation of human fibroblasts into islet beta cells with complete functions; and the cells formed after induction do not form tumors in the body, has the only capability of secretion of insulin, and can lower the level of blood sugar in vivo.

Description

technical field [0001] The invention relates to an induction method and application of insulin-secreting cells based on human skin cells, and belongs to the technical field of molecular cytology. Background technique [0002] Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia caused by defects in insulin secretion and / or insulin action. Sustained high blood sugar and long-term metabolic disorders can lead to damage and dysfunction of tissues and organs throughout the body, especially the eyes, kidneys, cardiovascular and nervous systems, and even failure. Insulin plays an important role in the treatment of diabetes, especially for patients with type I diabetes. [0003] At present, when fibroblasts are directly transformed into insulin-producing or secreting cells through the directed differentiation of stem cells or the use of small molecule drugs, objectively there are cumbersome directed differentiation steps, and the induced cells do not h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 吴缅梅一德初波李启东汪国兴
Owner INST OF ADVANCED TECH UNIV OF SCI & TECH OF CHINA
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