Stem cell preparation for treating diabetes mellitus and preparation method of preparation

Abstract

The invention provides a stem cell preparation for treating diabetes mellitus and a preparation method of the preparation. The stem cell preparation consists of bone marrow pluripotent stem cells, bone marrow pluripotent stem cell source islet-like cells and adipose-derived stem cells which are evenly mixed in normal saline in a ratio of 1:1:1. The cells adopted by the stem cell preparation have the multiple differentiation potential and the high proliferation characteristic and are abundant in source, convenient and easy to take and small in damage to organisms, in vitro separation, culture and induction operations can be conducted easily, abundant cell sources can be provided for treatment of the diabetes mellitus through the cells, and the preparation can be clinically used on a large scale. The stem cell preparation is free of immunological rejection when being implanted into a body. A specific induction culture system is used in the preparation method of the stem cell preparation, and the bone marrow pluripotent stem cells are directionally differentiated into the islet-like cells in vitro, so that the problem that cells implanted under the continuous high blood glucose condition of the diabetes mellitus are difficult to differentiate into cells capable of secreting insulin is solved.

Description

technical field

[0001] The invention relates to a stem cell preparation for treating diabetes and a preparation method thereof, belonging to the technical field of stem cell regenerative medicine. 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 hyperglycemic state caused by the joint action of genetic factors and environmental factors. It is due to the absolute and relative insufficiency of insulin secretion, which leads to the disorder of glucose metabolism, which makes blood sugar too high and diabetes occurs. The main 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. Epidemiological studies in recent years have shown tha...

Examples

specific Embodiment 1

[0041] Specific embodiment 1: a kind of preparation method of the stem cell preparation for treating diabetes comprises the following steps:

[0042] Step 1) Preparation of normal saline: first prepare normal saline, then add oligosaccharides, albumin, cholesterol, sodium chloride, propylene glycol and ammonium bicarbonate to make the concentrations 50g / L, 50g / L, 0.3g / L respectively , 9g / L, 5%, 20g / L;

[0043] Step 2) Preparation of bone marrow pluripotent stem cell suspension: including the separation and extraction of bone marrow pluripotent stem cells, collecting bone marrow pluripotent stem cells and mixing with the normal saline prepared in step 1) to prepare a concentration of 1×10 7 ~1×10 8 Individual / ml bone marrow pluripotent stem cell suspension;

[0044] Step 3) Preparation of bone marrow pluripotent stem cell-derived islet-like cell suspension: including collecting the bone marrow pluripotent stem cells prepared in step 2), inducing differentiation of bone marr...

specific Embodiment 2

[0053] Specific Example 2: Flow cytometric detection and identification of bone marrow pluripotent stem cells

[0054] Flow cytometry identification steps are as follows: collect the third to fifth generation bone marrow pluripotent stem cells, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000r / min for 5min. Discard the supernatant, add pre-cooled PBS, resuspend the cells, mix well, and centrifuge again. Repeat this 3 times. Then add PBS solution to mix the cells well to make 1×10 6 / ml of the cell suspension was added to the special test tube for flow cytometry. CD29, CD34, CD44, CD45 monoclonal antibodies were added sequentially, and incubated at 4°C for 60 min. Then rinse with PBS three times, add fluorescein isothiocyanate (FITC)-labeled secondary antibody working solution 1:50, and incubate at 4°C for 60 min. Rinse with PBS solution 3 times. Perform flow cytometry detection, and use PBS as the primary antibody to set up a negative control. ...

specific Embodiment 3

[0056] Specific Example 3: Identification of Islet-like Cells by Dithizone Staining

[0057] After the induced cells were washed with buffer, 1 mg / L dithizone solution was added, incubated in a 37°C incubator for 20 min, and the cells were observed with an inverted microscope. The induced cells were stained with dithizone, and observed under an inverted microscope, most of the cell clusters were bright red, such as image 3 As shown, it indicates that the cytoplasm of the induced cells contains zinc ions, and the dithizone staining of the uninduced bone marrow pluripotent stem cells is negative.

technical field

[0001] The invention relates to a stem cell preparation for treating diabetes and a preparation method thereof, belonging to the technical field of stem cell regenerative medicine. 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 hyperglycemic state caused by the joint action of genetic factors and environmental factors. It is due to the absolute and relative insufficiency of insulin secretion, which leads to the disorder of glucose metabolism, which makes blood sugar too high and diabetes occurs. The main 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. Epidemiological studies in recent years have shown tha...

Examples

specific Embodiment 1

[0041] Specific embodiment 1: a kind of preparation method of the stem cell preparation for treating diabetes comprises the following steps:

[0042] Step 1) Preparation of normal saline: first prepare normal saline, then add oligosaccharides, albumin, cholesterol, sodium chloride, propylene glycol and ammonium bicarbonate to make the concentrations 50g / L, 50g / L, 0.3g / L respectively , 9g / L, 5%, 20g / L;

[0043] Step 2) Preparation of bone marrow pluripotent stem cell suspension: including the separation and extraction of bone marrow pluripotent stem cells, collecting bone marrow pluripotent stem cells and mixing with the normal saline prepared in step 1) to prepare a concentration of 1×10 7 ~1×10 8 Individual / ml bone marrow pluripotent stem cell suspension;

[0044] Step 3) Preparation of bone marrow pluripotent stem cell-derived islet-like cell suspension: including collecting the bone marrow pluripotent stem cells prepared in step 2), inducing differentiation of bone marr...

specific Embodiment 2

[0053] Specific Example 2: Flow cytometric detection and identification of bone marrow pluripotent stem cells

[0054] Flow cytometry identification steps are as follows: collect the third to fifth generation bone marrow pluripotent stem cells, transfer the cell suspension to a centrifuge tube, and centrifuge at 1000r / min for 5min. Discard the supernatant, add pre-cooled PBS, resuspend the cells, mix well, and centrifuge again. Repeat this 3 times. Then add PBS solution to mix the cells well to make 1×10 6 / ml of the cell suspension was added to the special test tube for flow cytometry. CD29, CD34, CD44, CD45 monoclonal antibodies were added sequentially, and incubated at 4°C for 60 min. Then rinse with PBS three times, add fluorescein isothiocyanate (FITC)-labeled secondary antibody working solution 1:50, and incubate at 4°C for 60 min. Rinse with PBS solution 3 times. Perform flow cytometry detection, and use PBS as the primary antibody to set up a negative control. ...

specific Embodiment 3

[0056] Specific Example 3: Identification of Islet-like Cells by Dithizone Staining

[0057] After the induced cells were washed with buffer, 1 mg / L dithizone solution was added, incubated in a 37°C incubator for 20 min, and the cells were observed with an inverted microscope. The induced cells were stained with dithizone, and observed under an inverted microscope, most of the cell clusters were bright red, such as image 3 As shown, it indicates that the cytoplasm of the induced cells contains zinc ions, and the dithizone staining of the uninduced bone marrow pluripotent stem cells is negative.

Images