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Preparation method and application of stem cells capable of inductively secreting insulin

An insulin-inducible technology, applied in the field of cell biology, can solve the problems of MSC transplantation failure, difficult clinical application transformation, and the efficiency of differentiation cannot meet the requirements of clinical needs, so as to improve islet function and strong immune regulation. effect of function

Active Publication Date: 2022-05-17
深圳汉腾生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the technology of inducing MSCs to differentiate into islet β-like cells in vitro is relatively mature, and the differentiated islet β-like cells can theoretically secrete insulin for the treatment of T1D, but With the differentiation of MSCs, various cell differentiation markers, especially MHC molecules, are also up-regulated, which are easily recognized and eliminated by the human immune system after reinfusion, resulting in failure of MSC transplantation (Swijnenburg RJ, et al.2005) ; Drukker M, et al.(2002); Beatriz Suárez-Alvarez, et al.(2010)), another important issue is that the efficiency of UC-MSCs differentiation into islet β-like cells is far below the clinical requirement Quantity requirements, the clinical application of this technology is difficult

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  • Preparation method and application of stem cells capable of inductively secreting insulin
  • Preparation method and application of stem cells capable of inductively secreting insulin
  • Preparation method and application of stem cells capable of inductively secreting insulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Inserting the inducible insulin promoter and insulin gene fragment into the lentiviral vector pCDH-CMV-MCS-EF1-Puro

[0025] Entrust Beijing Huada Gene Technology Co., Ltd. to synthesize the inducible promoter (glucose responsiveInsulin Promoter) and human insulin gene (including signal peptide) (Insulin) sequence, and connect it to the PUC57 cloning vector, design primers (Table 1), so that The front and rear ends of the gene fragment sequence were respectively equipped with snaBI and BamHI restriction sites. After PCR amplification and double digestion, the pCDH-CMV-MCS-EF1-Puro vector was inserted into the pCDH-CMV-MCS-EF1-Puro vector after snaBI and BamHI double digestion, and transformed into E.coli ( stbl3), use the plasmid purification kit of OMEGA company to extract and purify plasmid, obtain such as figure 1 The indicated recombinant lentiviral vector plasmids. The target fragment of the plasmid is added with double restriction sites, linearized, amp...

Embodiment 2

[0028] Example 2 Preparation of induced insulin-secreting UC-MSCs

[0029] (1) Preparation of UC-MSCs

[0030] The umbilical cords of healthy newborns were aseptically transported to the GMP cell preparation laboratory within 3 hours after the delivery of the placenta. After blunt separation of the Wahrenheit jelly tissue, the UC-MSCs were isolated and cultured by the adherent method, and after expanding to the third generation (P3) UC-MSCs, the flow cytometric detection of cell surface markers and cell viability were performed. Rate, bacteria, fungi, endotoxin, mycoplasma, etc.

[0031] (2) Lentiviral packaging

[0032] 1. Take out the frozen 293T cells from the liquid nitrogen tank, and quickly put them into a 37°C water bath to dissolve the ice cubes of the cell suspension. After the ice cubes melted, slowly drop the cell suspension into 5 mL of preheated fresh complete medium (DMEM / 10% FBS), centrifuge at 1000 rpm for 5 min. Discard the supernatant after centrifugation...

Embodiment 3

[0071] Example 3 Therapeutic effect of induced insulin-secreting UC-MSCs cells on diabetic model mice

[0072] (1) Experimental animals: 6-week-old Balb / c mice, half male and half female, weighing 15-20 g, were purchased from the Animal Experiment Center of Guangdong Province. After intraperitoneal injection of 250 mg / kg body weight of streptozotocin (STZ), freshly dissolved in 114 mmol / L, pH=4.5 sodium citrate, hyperglycemia usually occurs after 3-5 days. The mice whose random blood glucose > 300 mg / dL was measured twice in a row and accompanied by symptoms of polydipsia, polyphagia, and polyuria were the successful model animals; 21 model mice were randomly divided into PBS control group, UC- MSCs group, insulin-secreting UC-MSCs group, 7 rats in each group, 3 groups in total.

[0073] (2) Preparation of inducible insulin-secreting UC-MSCs as shown in Example 2;

[0074] (3) The 5th passage UC-MSCs / induced insulin-secreting UC-MSCs cells were prepared into a density of 1×1...

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Abstract

The invention discloses a preparation method and application of stem cells capable of inductively secreting insulin. At present, recombinant insulin is mainly clinically used for treating insulin-dependent diabetes mellitus. Mesenchymal stem cells are pluripotent stem cells, have all generality of stem cells, namely self-renewal and multidirectional differentiation capabilities, have a relatively strong immune regulation function, and have a treatment effect on pancreas islet function reduction caused by autoimmune injury. The induced insulin secretion type umbilical cord mesenchymal stem cell capable of responding to the change of glucose concentration has a wide application prospect in treating diabetes mellitus.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a preparation method and application of induced insulin-secreting stem cells. Background technique [0002] Diabetes mellitus (DM) is a group of metabolic diseases characterized by long-term elevated blood glucose (referred to as blood sugar) levels, and is a chronic state of hyperglycemia caused by the joint action of genetic factors and environmental factors. It is due to the absolute and relative deficiency of insulin secretion, which leads to the disorder of glucose metabolism, high blood sugar, and sugar in urine. The main clinical manifestations are polydipsia, polyphagia, polyuria, weight loss and other symptoms. With the acceleration of the population aging process, the improvement of people's living standards, and the change of lifestyle, the incidence of diabetes has increased rapidly. At present, DM patients in the world have reached 4 billion. WHO predicts that by 2030, D...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/67C12N15/62C12N5/10A61K35/28A61P3/10
CPCC12N15/86C07K14/62C12N5/0668A61K35/28A61P3/10C12N2740/15043C12N2800/107C12N2830/002C07K2319/02C12N2510/00
Inventor 王冰李艳群张伟涛
Owner 深圳汉腾生物科技有限公司
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