Inducer for inducing a mesenchymal stem cell to differentiate into an islet cell

Abstract

The present invention belongs to the field of biomedicine, and relates to an inducer for inducing a mesenchymal stem cell to differentiate into an islet cell. An inducer for inducing a mesenchymal stem cell to differentiate into an islet cell consisted of the following components: GLP-1, parathyroid hormone, paracetamol, rapamycin, icariin, trametinib, EPO and VEGF. Each component in a inducer for inducing a mesenchymal stem cell to differentiate into an islet cell of the present invention is safe and non-toxic, requiring fewer steps and short time to induce differentiation, with high induction efficiency.

Claims

1. An inducer for inducing a human umbilical cord mesenchymal stem cell to differentiate into an islet cell, wherein the inducer is a liquid consisting of the following components: GLP-1, parathyroid hormone, paracetamol, rapamycin, icariin, trametinib, EPO, and VEGF; wherein the mass concentration of each component is as follows: GLP-1 20-40 mg/L, parathyroid hormone 6-12 mg/L, paracetamol 2-8 mg/L, rapamycin 2-8 mg/L, icariin 2-8 mg/L, trametinib 0.3-0.6 mg/L, EPO 2-4 ?g/L, and VEGF 2-4 ?g/L.

2. The inducer for inducing a mesenchymal stem cell to differentiate into an islet cell according to claim 1, wherein the mass concentration ratio of each component is as follows: GLP-1 30 mg/L, parathyroid hormone 9 mg/L, paracetamol 5 mg/L, rapamycin 5 mg/L, icariin 5 mg/L, trametinib 0.4 mg/mL, EPO 3 ?g/L, and VEGF 3 ?g/L.

3. A method for preparing an inducer, wherein the inducer is for inducing a human umbilical cord mesenchymal stern cell to differentiate into an islet cell, the method comprising: adding GLP-1 20-40 mg/L, parathyroid hormone 6-12 mg/L, paracetamol 2-8 mg/L, rapamycin 2-8 mg/L, icariin 2-8 mg/L, trametinib 0.3-0.6 mg/L, EPO 2-4 ?g/L, and VEGF 2-4 ?g/L into a serum-free medium for human mesenchymal stem cells, mixing the medium, filtering the medium, and sterilizing the medium to obtain the inducer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The sole FIGURE is the reaction of dithizone staining, showing that the induced group of the present invention is red after dithizone staining.

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1

(2) The inducer of this example for inducing a mesenchymal stem cell into an islet cell was consisted of the following components with a mass concentration ratio: GLP-1 30 mg/L, parathyroid hormone 9 mg/L, paracetamol 5 mg/L, rapamycin 5 mg/L, icariin 5 mg/L, trametinib 0.4 mg/L, EPO 3 ?g/L, VEGF 3 ?g/L. The above components was added to serum-free medium for human mesenchymal stem cells (or DMEM+10% FBS or other commercially available medium for mesenchymal stem cells) in turn according to the mass concentration ratio, followed by mixing well, filtering and sterilizing.

(3) The components of the inducer of the present invention were all commercially available products: serum-free medium for human mesenchymal stem cells, brand LONZA, catalog number 00190632; GLP-1 (glucagon-like peptide 1), brand Sigma, catalog number scp0153; parathyroid hormone, brand Sigma, catalog number P7036; paracetamol, Shanghai Yiji Industrial Co., Ltd., catalog number Y903952; rapamycin, brand TargetMol, catalog number T1537; icariin, Shanghai Microcrystalline Bio, catalog No. 489-32-7; trametinib, brand Meilun Bio, catalog number MB5401; EPO, brand PeproTech, catalog number CYT-201; VEGF, brand PeproTech, catalog number 96-100-20-2.

Example 2

(4) The inducer of this example for inducing a mesenchymal stem cell into an islet cell was consisted of the following components with a mass concentration ratio: GLP-1 20 mg/L, parathyroid hormone 6 mg/L, paracetamol 2 mg/L, rapamycin 2 mg/L, icariin 2 mg/L, trametinib 0.3 mg/L, EPO 2 ?g/L, VEGF 2 ?g/L. The above components was added to serum-free medium for human mesenchymal stem cells (or DMEM+10% FBS) in turn according to the mass concentration ratio, followed by mixing well, filtering and sterilizing.

Example 3

(5) The inducer of this example for inducing a mesenchymal stem cell into an islet cell was consisted of the following components with a mass concentration ratio: GLP-1 40 mg/L, parathyroid hormone 12 mg/L, paracetamol 8 mg/L, rapamycin 8 mg/L, icariin 8 mg/L, trametinib 0.6 mg/L, EPO 4 ?g/L, VEGF 4 ?g/L. The above components was added to serum-free medium for human mesenchymal stem cells (or DMEM+10% FBS) in turn according to the mass concentration ratio, followed by mixing well, filtering and sterilizing.

Example 4

(6) The effect of the inducer was illustrated with human umbilical cord mesenchymal stem cells as an example

(7) I. Induction of Human Umbilical Cord Mesenchymal Stem Cells to Differentiate Into Islet Cells

(8) Human umbilical cord mesenchymal stem cells of passage 3 were digested with 0.125%?0.01% Trypsin-EDTA solution for cells collection, to prepare a cell suspension. The density of living cells counted by a cytometer was adjusted to 1?10.sup.4/cm.sup.2, and cells were inoculated in a 24-well plate with a polylysine-treated sterile cover glass in advance to prepare a cell slide. When cells reached close to 80% confluence, they were induced to differentiate when they grow vigorously, see Table 1 for grouping.

(9) TABLE-US-00001 TABLE 1 Induction grouping Groups Induction conditions Control group (blank Serum-free medium for human mesenchymal control) stem cells. Induction group 1 Conventional differentiation medium: (conventional method) niacinamide 1.2 g/L, taurine 0.50 g/L, GLP-1 30.5 mg/L; serum-free medium for human mesenchymal stem cells. Induction group 2 The differentiation medium of the (present invention) present invention in Example 1
II. Identification of Islet Cells After Induction

(10) (I) Dithizone staining reaction: the islet cells obtained after 5 days of induction in the above three groups (induction group 1 needed to induce differentiation for 10 days) were removed from the original medium, and washed twice with PBS, each with addition of 2 ml PBS and 50 ?l dithizone working solution, incubating at 37? C. for 10 min, followed by removal of the staining solution, washing twice with PBS, and then the staining of cells was observed and took pictures The results were shown in the FIGURE. The dithizone staining results in two induction groups were red (as shown in the sole FIGURE) indicating positive reaction, and there was negative reaction in the control group.

(11) (II) Chemiluminescence immunoassay to detect insulin level: the cell culture supernatants of the above three groups 5 days after induction (induction group 1 needed to be induced to differentiate for 10 days) were detected the insulin content. The concentration of insulin secreted by cells in induction group 2 was 518.7 mU/L, which was much higher than the concentration of insulin secreted by cells in induction group 1 of 212.1 mU/L, while the insulin concentration in the blank control group was zero, indicating that the inducer of the present invention could significantly improve the induction efficiency.

(12) (III) Detection of the function of islet cells for the islet-like cells induced by the inducer of the present invention by ELISA kit: This detection procedure directly followed the standard procedure of C-peptide ELISA assays provided by Mercodia. In the final result, C-peptide was detected, indicating that the islet-like cells obtained by differentiation induced by the inducer of the present invention could secrete insulin.

(13) (IV) Glucose stimulation test: 100 islet cell clusters (50?150 ?m) induced to differentiate for 5 days by the inducer of the present invention were picked into a 1.5 ml centrifuge tube, and washed twice with PBS, with addition of 1 ml sugar-free DMEM to pre-culture for 3-6 h, followed by culturing in 300 ?l DMEM containing 5.6 mmol/L glucose and 16.7 mmol/L glucose for 2 h in sequence, and the supernatants were collected. The secretion of insulin under the stimulation of different concentrations of glucose in the supernatant was detected by ELISA. Insulin was almost undetectable in the cell supernatant of the control group, and the induced islet cell group secreted a small amount under stimulation of 5.6 mmol/L glucose, but after 2 h incubation with 16.7 mmol/L glucose, insulin secretion increased significantly (P<0.001), which was about twice as high as under low-sugar conditions. The results showed that the cell mass was sensitive to glucose stimulation after induction, and its insulin secretion was regulated by the external environment.

(14) (V) Transplantation in vivo experiment: a diabetic rat model was created firstly. Adult Wistar rats (both sexes) with about 180-200 g weight were selected. Each rat was intraperitoneally injected with streptozotocin at a dose of 70 mg/kg. Streptozotocin powder was prepared into liquid with 0.1 M citric acid buffer (PH=4.5), ready for use freshly. When the blood glucose of the rat rose (?16.7 mmol/L) and kept stable for one week, it indicated that the diabete model had been established. Under aseptic conditions, 300 islet-like cell clusters (50-150 ?m) induced by the inducer of the present invention were injected into the kidney cyst or small branch of the hepatic portal vein of diabetic rats. After the operation, the blood glucose was observed regularly. Results: The blood glucose of diabetic rats dropped by 7.3 mmol/L on average 3 days after cell implantation. This showed that the islet cell cluster obtained by using the inducer of the present invention to induce differentiation had a significant hypoglycemic effect.