Search

CN-122012375-A - Preparation method and application of functional islet beta cells for type 2 diabetes

CN122012375ACN 122012375 ACN122012375 ACN 122012375ACN-122012375-A

Abstract

The invention belongs to the technical field of cell biology, and discloses a preparation method and application of functional islet beta cells for type 2 diabetes. According to the method, the physical isolation co-culture of the mesenchymal stem cells and the primary islet cells is realized by the first semi-permeable membrane separation device, and the specific induction factor combination (FGF/EGF/BMP is taken as a core) is added to drive MSCs to be efficiently and directionally differentiated into functional islet beta cells, so that the insulin positive rate of the obtained cell population is greatly improved, and the insulin secretion amplification is greatly improved after glucose stimulation. The method solves the industrial problems of scarce stem cell sources, uncontrollable differentiation and unstable functions. The method has the advantages of realizing the effect of breaking through source limitation, replacing dilute shortage of staff islet cells by MSCs, realizing the effect of precise differentiation control, ensuring directional differentiation efficiency by semipermeable membrane isolation, ensuring that the glucose response capability reaches the physiological level, being superior to the prior art, being clinically applicable, facilitating separation and purification by physical isolation design and reducing the transplantation risk.

Inventors

  • LIU YANYOU
  • ZHANG QIAN
  • JIN PENG
  • TAN RUFU
  • ZOU LI
  • CAI BO

Assignees

  • 四川中天鑫源生命科技有限公司

Dates

Publication Date
20260512
Application Date
20260211

Claims (10)

  1. 1. A preparation method of functional islet beta cells for type 2 diabetes is characterized in that the preparation method is a physical isolation co-culture system directly aiming at mesenchymal stem cells and primary islet cells, and after a specific induction factor composition is added, the mesenchymal stem cells are driven to directionally differentiate into the functional islet beta cells; The physical isolation co-culture system is that mesenchymal stem cells and primary islet cells are co-cultured by utilizing a semipermeable membrane separation device; The insulin positive cell amount in the functional islet beta cells accounts for not less than 90 percent.
  2. 2. The method for producing functional pancreatic islet beta cells for type 2 diabetes according to claim 1, the semi-permeable membrane separation device is characterized by being a Transwell system; The membrane aperture of the semi-permeable membrane separation device is 0.4-1.0 mu m.
  3. 3. The method of claim 2, wherein the semi-permeable membrane separator has a membrane pore size of 0.6 μm.
  4. 4. The method for preparing functional islet beta cells for type 2 diabetes according to claim 1, comprising the specific steps of: s1, obtaining primary islet cells: Obtaining ex vivo pancreatic tissue from a mammal, digesting the pancreatic tissue by pancreatic duct perfusion collagenase; Monitoring the digestive process under a microscope, terminating the digestive process when more than 70% of the islet cells are dissociated from acinar cells; Purifying by adopting a density gradient centrifugation method to obtain primary islet cells; S2, a physical isolation co-culture system: selecting the obtained primary islet cells and mesenchymal stem cells, and performing co-culture under a physical isolation co-culture condition; s3, adding a specific induction factor composition consisting of basic factors and synergistic factors into the physical isolation co-culture system to continuously perform cell culture, and inducing the directional differentiation of the mesenchymal stem cells into functional islet beta cells.
  5. 5. The method for preparing functional pancreatic islet beta cells for type 2 diabetes according to claim 4, wherein in S1, pancreatic tissue is obtained by the specific steps of: Selecting 5 SD rats, performing anesthesia by injecting ketamine hydrochloride with the mass concentration of 80mg/kg into the abdominal cavity, ligating the opening of a biliary pancreatic duct, pouring normal saline through the pancreatic duct, and picking pancreatic tissues; The collagenase is V-type collagenase; the concentration of collagenase in the digestive juice is 1.0-2.0 mg/mL.
  6. 6. The method for preparing functional pancreatic islet beta cells for type 2 diabetes according to claim 4, wherein in S2, the specific step of physically isolating the co-culture system comprises: Selecting mesenchymal stem cells and primary islet cells, and placing the primary islet cells in the lower layer of the device by using a physical isolation device; And in the step S3, a specific induction factor composition is added, and the culture is continued for 7-14 days under the conditions of 35-37 ℃ and CO 2 with the volume fraction of 5%.
  7. 7. The method for preparing functional pancreatic islet beta cells for type 2 diabetes according to claim 4, wherein in S3, the basic factor is composed of 10-20 ng/mL fibroblast growth factor, 5-10 ng/mL epidermal growth factor and 50-100 ng/mL bone morphogenic protein; the synergistic factor consists of 0.1-1 mu M retinoic acid, 20-50 ng/mL beta-cytokine and 10-20 mM niacinamide; The mesenchymal stem cells are selected from umbilical cord blood, bone marrow or adipose tissue-derived mesenchymal stem cells.
  8. 8. A functional islet beta cell population obtained by the method of producing a functional islet beta cell for type 2 diabetes according to any one of claims 1 to 7.
  9. 9. Use of a functional islet beta cell population according to claim 8 for the preparation of a glucose concentration-dependent insulin-secreting cell formulation for the treatment of type 2 diabetes, wherein said functional islet beta cell population has a function responsive to a change in glucose concentration.
  10. 10. The use according to claim 9, wherein the insulin secretion amount of the functional islet β cell population is at a basal value of 5.6mM at a glucose concentration; at a glucose concentration of 16.7mM, the insulin secretion amount was increased by not less than 200%.

Description

Preparation method and application of functional islet beta cells for type 2 diabetes Technical Field The invention belongs to the technical field of cell biology, and particularly relates to a preparation method and application of functional islet beta cells for type 2 diabetes. Background Type 2 diabetes mellitus (Type 2 Diabetes Mellitus,T2DM) is one of the metabolic diseases with highest global morbidity, and has the key pathological characteristics that the insulin resistance of islet beta cells is caused by progressive decline of the functions of the islet beta cells and insulin resistance of peripheral tissues, the insulin resistance leads to the decrease of the sensitivity of organisms to insulin, the islet beta cells need to compensatory increase insulin secretion to maintain blood sugar homeostasis, long-term compensatory load causes the loss of proliferation capacity and increase of apoptosis of beta cells, finally leads to insufficient insulin secretion, can not effectively regulate blood sugar, and forms a persistent hyperglycemia state. 1. Limitations of conventional therapeutic techniques The current main clinical treatment scheme mainly comprises oral hypoglycemic agents (such as sulfonylurea, biguanide, DPP-4 inhibitor and the like) and exogenous insulin injection, and the scheme can only stimulate insulin secretion through chemical drugs, improve insulin sensitivity or directly supplement insulin to realize temporary control of blood sugar, can not reverse the functional damage of islet beta cells and can not fundamentally solve the pathological root cause of insulin resistance. After long-term application, oxidative stress, inflammatory reaction and the like caused by blood sugar fluctuation can further aggravate islet failure, and induce cardiovascular and cerebrovascular diseases (such as cerebral infarction and coronary heart disease), nephropathy (diabetic nephropathy), retinopathy (diabetic retinopathy), neuropathy and other serious complications, obviously reduce the life quality of patients and increase the death rate, so that development of a treatment technology capable of reversing islet beta cell function damage becomes a critical problem to be solved in clinical urgent need. 2. Application state and defect of stem cell technology in type 2 diabetes treatment The stem cell factor has multidirectional differentiation potential, self-renewing capacity and immunoregulation property, is considered as an ideal seed cell for regenerating islet beta cells and repairing islet functions, and provides a new direction for root treatment of type 2 diabetes. However, the related technology of the existing stem cell therapy has the following core defects, which severely limit the clinical transformation and the large-scale application: (1) Cell source scarcity and large-scale acquisition difficulty Seed cells for islet beta cell regeneration mainly comprise primary islet cells and stem cells (such as Induced Pluripotent Stem Cells (iPSCs), mesenchymal Stem Cells (MSCs), etc.). The primary islet cells are seriously donated by human pancreas organs, are influenced by the number of donors, ethical limitation, short organ preservation time, immune rejection risk and other factors, cannot realize large-scale acquisition, and are difficult to meet the mass requirements of clinical treatment, while stem cells can be theoretically amplified in vitro, iPSCs have ethical disputes and tumorigenic risks, the differentiation efficiency of adult stem cells such as MSCs is low, and the quality difference of stem cells from different donors is large, so that the standardized preparation of seed cells is further restricted. (2) Uncontrollable differentiation of Stem cells In the prior art, the induction and differentiation process of stem cells to islet beta cells has obvious non-target cell mixing problems that when iPSCs are taken as an example, growth factors (such as actin A, BMP4, GLP-1 and the like) are added for in vitro induction, the stem cells are easy to differentiate into non-islet beta cell types such as liver cells, nerve cells, fibroblasts and the like, so that the differentiation efficiency of islet beta cells is low (the differentiation efficiency in the prior art is generally lower than 30 percent), the heterogeneity of differentiated cell populations is high, and high-purity functional islet beta cells cannot be obtained. The core reason for the problem is that the induction system cannot accurately simulate microenvironment signals of islet development in vivo, so that the differentiation direction is out of control. (3) Functional stability of in vitro differentiated islet beta cells is poor Even if part of islet beta cell-like cells are obtained, the insulin secretion function is still immature, and the specific characteristics are that the basal insulin secretion amount is higher, the response capability to the change of glucose concentration is weak, normal islet beta cells can realiz