CN106854638B - Method for inducing mesenchymal stem cells to differentiate into islet-like cells - Google Patents
Method for inducing mesenchymal stem cells to differentiate into islet-like cells Download PDFInfo
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Abstract
A method for inducing mesenchymal stem cells to differentiate into islet-like cells relates to a method for inducing mesenchymal stem cells to differentiate into islet-like cells. The invention aims to provide a method for differentiating mesenchymal stem cells from different sources into islet-like cells so as to obtain a large amount of islet-like cells. In order to achieve the above object, the specific method is as follows: 1) separating and primary culturing umbilical cord, placenta or adipose-derived mesenchymal stem cells; 2) the preparation of the differentiation medium comprises: preparing a Brain Tissue Conditioned Medium (BTCM), a cell differentiation inducing solution and a stem cell conditioned medium (SCM); 3) inducing the mesenchymal stem cells to differentiate into islet-like cells in three stages; 4) identification of the islet-like cells obtained. The islet-like cells obtained by the method have high maturity, can achieve the effect of quickly reducing blood sugar by a small number of cells, have wide application prospect, and are applied to the field of biomedicine.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a method for inducing stem cells to differentiate directionally into islet-like cells in vitro and application of the islet-like cells.
Background
Diabetes is a chronic metabolic and multifactorial disease, hyperglycemia is caused by absolute (type I diabetes) and relative (type II diabetes) insulin deficiency, the two diseases have the common point that the function of islet beta cells is insufficient, no obvious symptoms exist in the early clinical stage, more than three or less phenomena can occur in the symptom period, the damage of multiple systems of the body is often accompanied, the body is seriously injured, and even the life is threatened. The main cause of type I diabetes is the destruction of islet beta cells by an autoimmune response, which fails to maintain stable glucose levels. Islet transplantation, particularly for severe patients, is a more direct treatment method, but cannot be widely applied clinically due to the problems of donor deficiency, high pancreas/islet separation cost, multiple transplantations of inflammatory reactions generated by transplantation, long-term taking of immunological rejection medicines and the like. The improvement of the pancreatic islet function of the diabetic is particularly important for delaying the occurrence of complications, and an endogenous insulin secretion system is reconstructed in the body of the diabetic by means of a stem cell technology, so that the improvement becomes a research direction which is concerned about in the field of regenerative medicine. The stem cells have multipotential differentiation potential, wherein Mesenchymal Stem Cells (MSCs) are multipotential stem cells with high self-renewal and multipotential differentiation potential in mesoderm, widely exist in various tissues throughout the body, can be cultured and expanded in vitro, and can be differentiated into various tissue cells such as nerve, sweat gland, bone, cartilage, fat, liver and the like under specific conditions. The MSC from fat, umbilical cord and placenta has the advantages of convenient material acquisition, strong passability, low immunoreaction, small probability of pollution, no ethical limitation and the like, can be differentiated into islet beta cells or insulin-producing cells (IPCs) under certain conditions, and becomes an important research way for replacing the existing short islet cells and realizing clinical diabetes treatment. Studies have shown that pancreatic endocrine cells are derived from endoderm and express neuronal markers, while insulin gene transcripts are found in vertebrate brain, and these results all show that islet cells share similarities with neurons and are therefore likely to be more beneficial to the growth of islet-like cells under the action of neuronal conditioned media.
Disclosure of Invention
The invention aims to provide a method for inducing mesenchymal stem cells to be differentiated into islet-like cells, which comprises the steps of preparing a Brain Tissue Conditioned Medium (BTCM) by using a newborn rat brain, inducing the mesenchymal stem cells to be differentiated into the islet-like cells by using cytarabine, nicotinamide, B27, Conophylline and the like, discussing the differentiation potential of the mesenchymal stem cells through further identification, and simultaneously searching a high-efficiency differentiation system of the islet-like cells to lay a foundation for the clinical application of differentiating the mesenchymal stem cells into mature islet-like cells.
The invention relates to a method for inducing mesenchymal stem cells to differentiate into islet-like cells, which comprises the following steps:
firstly, separating and obtaining mesenchymal stem cells from umbilical cord, placenta or adipose tissues, and subculturing;
preparation of differentiation Medium
The differentiation culture medium comprises a brain tissue conditioned medium, a cell differentiation inducing solution and a stem cell conditioned medium;
the preparation process of the brain tissue conditioned medium is as follows: taking out the brains of rats growing for 6-7 days under an aseptic condition, washing the brains with PBS or saline, centrifuging the brains for 6min at 1000rpm, discarding the supernatant, resuspending the brains in a 10% FBS DMEM high-sugar culture medium for precipitation, mashing the brain tissues into single cells, collecting the brain tissue cells, culturing the brain tissue cells for 1d in 10% FBS DMEM high-sugar, adding 1-2 mu mol/L cytarabine for culture, and collecting a culture solution at the 5 th d of culture to obtain BTCM;
the cell differentiation-inducing solution comprises the following components: DMEM/F12 is taken as a basic culture medium and contains 2% of FBS, 80-100 mug/L of vinblastine derivatives and 2% of B27;
the preparation process of the stem cell conditioned medium comprises the following steps: culturing MSCs in DMEM/F12 culture medium containing 2% FBS and 10mmol/L nicotinamide, and collecting culture solution after 3 days to obtain SCM;
and thirdly, differentiating the umbilical cord, placenta or adipose tissue mesenchymal stem cells subjected to subculture in the first step into islet-like cells, and identifying to finish the differentiation of the induced mesenchymal stem cells into the islet-like cells.
The vinblastine derivative is Conophylline.
The invention has the following beneficial effects:
1) the induction system provided by the invention is safe and has no side effect, and the BTCM is prepared by adopting rat brain, wherein the BTCM contains a plurality of natural induction factors and efficiently induces the generation of insulin secreting cells by combining the synergistic effect of cytokines such as nicotinamide, Conophylline and B27. Compared with the traditional method, the induction system uses less kinds of cytokines and has less dosage, thereby reducing the clinical use risk. SCM in the final stage provides protection for insulin-like cell clusters, and overcomes the defects of easy apoptosis, immature differentiation, glucose-induced toxicity and the like of cells.
2) The islet-like cells obtained by the invention have strong secretory capacity. The reported data show that the insulin secretion amount of the islet-like cell mass is about 50-70 mU/L under high glucose stimulation, but the islet-like cell mass induced by the invention has higher and faster insulin secretion capacity, reaches (33.88 +/-4.05 mU/L) under low glucose stimulation and reaches (105.65 +/-7.43 mU/L) under high glucose action, the insulin secretion capacity of the islet-like cell is improved to a great extent, and the rapid response to the glucose stimulation is realized, so that the function of rapidly reducing the blood glucose is achieved.
Compared with the traditional multi-cytokine combined induction, the induction method of the invention can obtain more and mature islet-like cells, obviously improves the induction differentiation efficiency, has long survival time and high proliferation activity of the differentiated islet-like cells, can make quick response in a short time under the stimulation of glucose, and achieves the effect of quickly reducing blood sugar. Meanwhile, the method does not need gene transfection, avoids gene change and tumorigenic risks, and has wide application prospect.
Drawings
FIG. 1 is a graph of dithizone staining of islet-like cells;
FIG. 2 is a diagram showing gene expression;
FIG. 3 is a graph showing the results of regulation of insulin secretion by external environmental sugars;
FIG. 4 is a graph showing blood glucose levels of rats on days 3 and 10 after islet-like cell transplantation, respectively.
Detailed Description
The first embodiment is as follows: the method for inducing the mesenchymal stem cells to differentiate into the islet-like cells of the embodiment comprises the following steps:
firstly, separating and obtaining mesenchymal stem cells from umbilical cord, placenta or adipose tissues, and subculturing;
preparation of differentiation Medium
The differentiation culture medium is a brain tissue conditioned medium, a cell differentiation inducing solution and a stem cell conditioned medium;
the preparation process of the brain tissue conditioned medium is as follows: taking out the brains of rats growing for 6-7 days under an aseptic condition, washing the brains with PBS or saline, centrifuging the brains for 6min at 1000rpm, discarding the supernatant, resuspending the brains in a 10% FBS DMEM high-sugar culture medium for precipitation, mashing the brain tissues into single cells, collecting the brain tissue cells, culturing the brain tissue cells for 1d in 10% FBS DMEM high-sugar, adding 1-2 mu mol/L cytarabine for culture, and collecting a culture solution at the 5 th d of culture to obtain BTCM;
the cell differentiation-inducing solution comprises the following components: DMEM/F12 is taken as a basic culture medium and contains 2% of FBS, 80-100 mug/L of vinblastine derivatives and 2% of B27;
the preparation process of the stem cell conditioned medium comprises the following steps: culturing MSCs in DMEM/F12 culture medium containing 2% FBS and 10mmol/L nicotinamide, and collecting culture solution after 3 days to obtain SCM;
and thirdly, differentiating the umbilical cord, placenta or adipose tissue mesenchymal stem cells subjected to subculture in the first step into islet-like cells, and identifying to finish the differentiation of the induced mesenchymal stem cells into the islet-like cells.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the specific process for differentiating the umbilical cord, placenta or adipose tissue mesenchymal stem cells subjected to subculture in the step one into the islet-like cells is as follows:
subculturing the mesenchymal stem cells of umbilical cord, placenta or adipose tissue of step one to P3 generation at 4X 104Inoculating each/mL of the cells into a 6-well plate, culturing in a 10% FBS-containing DMEM/F12 medium for 24h, replacing BTCM for 7 days, replacing BTCM once every 2 days, culturing for 7 days, and culturing in a cell differentiation-inducing solution for 7 daysChanging the inducing liquid every 2 days, and then jointly culturing the cell differentiation inducing liquid and SCM for 14 days according to the volume ratio of 1:1 to obtain mature islet-like cells. The rest is the same as the first embodiment.
The third concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the umbilical cord mesenchymal stem cell separation and subculture process comprises the following steps:
taking 15-20 cm of umbilical cord of a full-term fetus, cleaning the umbilical cord with normal saline and alcohol with the volume percentage of 75%, shearing the umbilical cord into pieces with the length of 2-3 cm, respectively stripping off arteries and veins, separating out the Wharton jelly in the middle, and shearing the umbilical cord into pieces with the length of 0.5-2 mm under the aseptic condition3Inoculating tissue blocks according to a bottle of 1g/T75, culturing with 10% FBS culture medium, changing the culture solution after 5d, digesting with pancreatin with volume percentage concentration of 0.125% after the cell fusion degree reaches above 80%, obtaining single cells, and inoculating with 8 × 105Inoculating the individual cells/T75 in a bottle, culturing by adopting a 10% FBS culture medium, placing in a carbon dioxide incubator, carrying out passage after the cell fusion degree reaches more than 90%, simultaneously detecting the immune markers of the MSCs in the umbilical cord, and detecting qualified retention, namely completing the separation and passage culture of the umbilical cord mesenchymal stem cells.
The rest is the same as the first embodiment.
The detection qualification in the embodiment of this market is as follows: after detection, the surface antigens of the representative MSCs are expressed: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45 and endothelial cell surface antigen CD31, which accord with the characteristics of stem cells, namely the detection is qualified.
The fourth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the placenta mesenchymal stem cell separation and subculture process comprises the following steps:
under the aseptic condition, firstly, the placenta collecting bag is disinfected by alcohol with the volume percentage of 75 percent, then the placenta is washed for 2 times by normal saline, after the amnion is stripped, the chorion tissue of the placenta is taken, the normal saline is added for washing the bloodstain, and the chorion tissue is placed in a 50mL clean centrifugal tube after being washedInside, cut into 0.2-1 mm3Adding collagenase type I with the volume 3 times of the tissue block, digesting for 30min in a shaking table at 37 ℃, adding 10mL of serum-free culture medium to stop digestion, filtering in a 50mL clean centrifugal tube, centrifuging for 6min at 1300r/min, discarding the supernatant, taking the precipitate, and centrifuging according to the proportion of 1 multiplied by 106Inoculating each cell/T75 bottle, culturing by adopting 10% FBS culture medium, placing in a carbon dioxide incubator, carrying out passage after the cell fusion degree reaches more than 90%, detecting the immune marker of the placenta MSCs, and detecting qualified retention, namely completing the separation and passage culture of the placenta mesenchymal stem cells.
The rest is the same as the first embodiment.
The detection qualification in the embodiment of this market is as follows: after detection, the surface antigens of the representative MSCs are expressed: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45 and endothelial cell surface antigen CD31, which accord with the characteristics of stem cells, namely the detection is qualified.
The fifth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the adipose-derived mesenchymal stem cell separation and subculture process comprises the following steps:
under the aseptic condition, firstly, treating a fat collection bottle with 75% alcohol by volume percentage, then cleaning fat for 2-3 times by using normal saline, transferring adipose tissues to a 50mL centrifuge tube, adding type I collagenase with the volume 3 times of that of tissue blocks, digesting for 35min in a shaking table at 37 ℃, adding about 15mL serum-free medium to stop digestion, filtering to a 50mL centrifuge tube, centrifuging for 6min at 1300r/min, discarding supernatant, taking precipitate, and centrifuging according to the proportion of 1 multiplied by 106～1.5×106Inoculating the individual cells/T75 in a bottle, culturing by adopting a 10% FBS culture medium, placing in a carbon dioxide incubator, carrying out passage after the cell fusion degree reaches more than 90%, simultaneously detecting the immune markers of the adipose MSCs, and detecting qualified retention, namely completing the isolation and passage culture of the adipose mesenchymal stem cells.
The rest is the same as the first embodiment.
The detection qualification in the embodiment of this market is as follows: after detection, the surface antigens of the representative MSCs are expressed: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45 and endothelial cell surface antigen CD31, which accord with the characteristics of stem cells, namely the detection is qualified.
The invention is not limited to the above embodiments, and one or a combination of several embodiments may also achieve the object of the invention.
The beneficial effects of the present invention are demonstrated by the following examples:
example 1:
preparation of mesenchymal Stem cells
1.1 preparation of umbilical cord mesenchymal Stem cells
Taking 15-20 cm of umbilical cord of a full-term fetus under aseptic condition, cleaning the umbilical cord with normal saline and alcohol with the volume percentage of 75%, cutting the umbilical cord into small sections with the length of 2-3 cm, respectively peeling off artery and vein, separating out the Wharton's jelly in the middle, and dissecting the umbilical cord into sections with the length of 0.5-2 mm under aseptic condition3Inoculating small tissue blocks according to a bottle of 1g/T75, culturing with 10% FBS culture medium, changing the culture solution after 5d, digesting with pancreatin with volume percentage concentration of 0.125% after the cell fusion degree reaches above 80%, obtaining single cells, and inoculating to a culture medium with a concentration of 8 × 105Inoculating each cell/T75 bottle, culturing in 10% FBS culture medium, placing in a carbon dioxide incubator, carrying out passage after the cell fusion degree reaches above 90%, and detecting the immune marker of umbilical cord MSCs to find that the cells all express typical MSCs surface antigen: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45, and the endothelial cell surface antigen CD31, consistent with the characteristics of stem cells.
1.2 preparation of placental mesenchymal Stem cells
Under the aseptic condition, firstly, disinfecting a placenta collecting bag by using 75% alcohol, then, cleaning the placenta for 2 times by using normal saline, stripping amnion, taking placenta chorion tissue, adding the normal saline to clean bloodstain, placing the chorion tissue into a 50mL clean centrifugal tube after cleaning, and cutting into pieces with the thickness of 0.2-1 mm3Adding collagenase type I3 times the volume of the tissue blocks, and shaking at 37 deg.CDigesting in bed for 30min, adding 10mL serum-free culture medium to stop digestion, filtering in 50mL clean centrifugal tube, centrifuging at 1300r/min for 6min, discarding supernatant, collecting precipitate, and concentrating at 1 × 106Inoculating each cell/T75 bottle, culturing in 10% FBS culture medium, placing in carbon dioxide incubator, subculturing after cell fusion degree reaches above 90%, and detecting immune marker of placenta MSCs to find that they all express typical MSCs surface antigen: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45, and the endothelial cell surface antigen CD31, consistent with the characteristics of stem cells.
1.3 preparation of adipose-derived mesenchymal Stem cells
Under the aseptic condition, firstly treating a fat collection bottle with 75% alcohol, then cleaning fat for 2-3 times with normal saline, transferring adipose tissues to a 50mL centrifuge tube, adding type I collagenase with the volume being 3 times of that of tissue blocks, digesting for 35min in a shaking table at 37 ℃, adding about 15mL serum-free medium to stop digestion, filtering to the inside of the 50mL centrifuge tube, centrifuging for 6min at 1300r/min, discarding supernatant, taking precipitate, and carrying out centrifugation according to the proportion of 1 × 106～1.5×106Inoculating each cell/T75 bottle, culturing in 10% FBS culture medium, placing in a carbon dioxide incubator, subculturing after the cell fusion degree reaches above 90%, and detecting the immune marker of the adipose MSCs to find that the cells all express typical MSCs surface antigen: CD13, CD29, CD44, CD54, CD73, CD105, does not express hematopoietic stem cell surface antigen: CD3, CD14, CD34, CD45, and the endothelial cell surface antigen CD31, consistent with the characteristics of stem cells.
Example 2:
preparation of differentiation Medium
2.1 Brain Tissue Conditioned Medium (BTCM) preparation
Taking out the brains of rats growing for 6-7 days under the aseptic condition, washing the brains with PBS or saline water, centrifuging the brains for 6min at 1000rpm, discarding the supernatant, carrying out heavy suspension precipitation (brain tissue) with 10% FBS-DMEM, mashing the brain tissue into single cells, collecting the brain tissue cells, culturing the brain tissue cells with 10% FBS-DMEM, adding 2 mu mol/L cytarabine on the second day, and collecting a culture solution which is BTCM on the fifth day of culture.
2.2 preparation of cell differentiation-inducing solution
DMEM/F12 is used as a basic culture medium and contains 2% FBS, 10mmol/L nicotinamide, beta-cell regulator and Conophylline100 mu g/L.
2.3 Stem cell conditioned Medium (SCM) preparation
MSCs are cultured in DMEM/F12 medium containing 2% FBS, 10mmol/L nicotinamide and 2% B27, and the culture solution is collected after 3 days to obtain the SCM.
Example 3:
in vitro induction of mesenchymal stem cells to differentiate into islet-like cells
3.1 in vitro induced differentiation into islet-like cells
The P3 generation umbilical cord, placenta or adipose derived mesenchymal stem cells are cultured at 4X 104Each/ml was inoculated in a 6-well plate in DMEM/F12 containing 10% FBS, and the BTCM was replaced after 24 hours for 7 days and every 2 days. Culturing the cell differentiation inducing solution for 7 days, changing the solution every 2 days, and co-culturing the cell differentiation inducing solution and SCM for 14 days to obtain mature pancreatic islet-like cells.
3.2 identification of in vitro induced islet-like cells
(1) Dithizone dyeing identification
And (3) carrying out dithizone staining on the induced islet-like cells, removing a cell culture medium, washing the islet-like cells for 2 times by using PBS, adding 1ml of PBS and 50 mu l of dithizone working solution, incubating the islet-like cells for 20min at 37 ℃, removing the dithizone staining solution, washing the islet-like cells for 2 times by using PBS, observing the cell staining condition under an inverted microscope, and taking a picture for recording. The result shows that the induced cells are changed into round shape from the long spindle shape of the mesenchymal stem cells, and the cells are stained into brick red, and are positive islet-like cells, and the result is shown in figure 1.
(2) RT-PCR specific gene detection
Extracting total RNA of the mesenchymal stem cells and the induced islet-like cells by using a Trizol reagent, detecting the expression of human Insulin (Insulin) and Glucagon (Glucagon) genes by using RT-PCR, taking the beta-actin genes as a reference, and showing the primers in a table 1 and the RT-PCR result in a table 2.
Table 1: RT-PCR gene primer
| Gene | Upstream primer | Downstream primer |
| Insulin | 5’-GCCTTTGTGAACCAACACCTG-3’ | 5’-GTTGCAGTAGTTCTCCAGCTG-3’ |
| Glucagon | 5’-AGGCAGACCCACTCAGTGA-3’ | 5’-AACAATGGCGACCTCTTCTG-3’ |
| β-actin | 5’-TGGCACCCAGCACAATGAA-3’ | 5’-TAAGTCATAGTCCGCCTAGAAGCA-3’ |
The results show that the mesenchymal stem cells and the islet-like cells both express the internal reference genes, the mesenchymal stem cells do not express the insulin and glucagon genes, and the islet-like cells generated after induction express the insulin and glucagon genes, which indicates that the mesenchymal stem cells are induced to differentiate into the islet-like cells.
(3) Glucose-stimulated insulin secretion test
Grouping experiments: the control group is non-induced mesenchymal stem cells; in the experiment 1 group, the induced islet-like cells are cultured in 5.8mmol/L glucose DMEM; experiment 2 group was cultured in 18.7mmol/L glucose DMEM for islet-like cells after induction.
About 50 induced cell clusters (50-150 mu m) are picked into a 1.5ml centrifuge tube, washed for 2 times by PBS and added with 1ml sugar-free DMEM for pre-culture for 3-6 h. Then, the cells were cultured in 300. mu.l of DMEM containing 5.8mmol/L glucose and 25mmol/L glucose at 37 ℃ for 2 hours in this order. Collecting supernatant, and detecting the insulin secretion amount of the supernatant under the stimulation of different concentrations of glucose by an ELISA method. The results show that almost no insulin can be detected in the cell supernatant of the control group, while the insulin secretion of the induced cell mass under the stimulation of 5.8mmol/L glucose is (33.88 +/-4.05 mU/L), the insulin secretion after the cell mass is incubated for 2h with 25mmol/L glucose is (105.65 +/-7.43 mU/L), and the difference is very obvious along with the increase of the glucose concentration. It is thus clear that after induction, islet-like cell masses are sensitive to glucose challenge, and insulin secretion is regulated by the external environmental sugars (see FIG. 3).
Example 4: in vivo transplantation test
The diabetic rat model is made by adopting SD rats with half male and female bodies, the weight of the SD rats is about 250g, blood is taken from tail veins after fasting for 12h (fasting without water), a glucometer is used for measuring the blood sugar, and patients with fasting blood sugar of more than 11.1mmol/L are removed, so that the hyperglycemia before model making is eliminated. Rats were injected intraperitoneally with a single injection of streptozotocin in 0.1M sodium citrate buffer (pH 4.5) at a dose of 50 mg/kg. The induction of diabetes was confirmed by evaluation of blood glucose 4 days after streptozotocin injection, and rats with blood glucose higher than 16.7mmol/L and showing polydipsia, polyphagia, polyuria were considered to be successfully modelled. Rats successfully modeled after diabetes mellitus were randomly selected and divided into three groups, namely a normal group, a diabetic control group and an islet-like cell transplantation group. Blood glucose levels were measured in both groups on days 3 and 10 after islet-like cell transplantation, and the results are shown in table 2 and fig. 4.
Specific experimental animal groups were as follows:
normal group: 5 healthy rats
Diabetes control group: 5 rats receiving streptozotocin-induced type I diabetes were not treated;
islet-like cell transplantation group: 5 diabetic rats received islet-like cell transplantation (10)2-103Cell intravenous injection once)
TABLE 2 blood glucose levels (mmol/L) (poor (x. + -. s,%)
As shown by the results in Table 2 and FIG. 4, the blood sugar of rats in the diabetic group was generally high, and the blood sugar of rats after islet-like cell transplantation was decreased and significantly different. The blood sugar of the islet-like cell transplantation group is further reduced along with the prolonging of the time, and the difference is obvious, and the results show that the differentiated islet-like cells have a certain blood sugar reducing effect on diabetic rats.
Claims (5)
1. A method for inducing mesenchymal stem cells to differentiate into islet-like cells is characterized by comprising the following steps:
firstly, separating and obtaining mesenchymal stem cells from umbilical cord, placenta or adipose tissues, and subculturing;
preparation of differentiation Medium
The differentiation culture medium comprises a brain tissue conditioned medium, a cell differentiation inducing solution and a stem cell conditioned medium;
the preparation process of the brain tissue conditioned medium is as follows: taking out the brains of rats growing for 6-7 days under an aseptic condition, washing the brains with PBS or saline, centrifuging the brains for 6min at 1000rpm, discarding the supernatant, resuspending the brains in a 10% FBS DMEM high-sugar culture medium for precipitation, mashing the brain tissues into single cells, collecting the brain tissue cells, culturing the brain tissue cells for 1d in 10% FBS DMEM high-sugar, adding 1-2 mu mol/L cytarabine for culture, and collecting a culture solution at the 5 th d of culture to obtain BTCM;
the cell differentiation-inducing solution comprises the following components: DMEM/F12 is taken as a basic culture medium and contains 2% FBS, 10mmol/L nicotinamide, beta-cell regulator and Conophylline100 mu g/L;
the preparation process of the stem cell conditioned medium comprises the following steps: culturing MSCs in DMEM/F12 culture medium containing 2% FBS, 10mmol/L nicotinamide and 2% B27, and collecting culture solution after 3 days to obtain SCM;
thirdly, replacing the P3 umbilical cord, placenta or adipose-derived mesenchymal stem cells by 4 x 104One/ml of the culture medium was inoculated into 6-well platesThe mature islet-like cells can be obtained by culturing 24h after replacing BTCM in DMEM/F12 containing 10% FBS for 7 days, replacing BTCM every 2 days, culturing the cell differentiation inducing solution for 7 days, replacing the cell differentiation inducing solution every 2 days, and culturing the cell differentiation inducing solution and SCM for 14 days.
2. The method for inducing the differentiation of mesenchymal stem cells into islet-like cells according to claim 1, wherein the specific process of differentiating the umbilical cord, placenta or adipose tissue mesenchymal stem cells after the first subculture into islet-like cells is as follows:
subculturing the mesenchymal stem cells of umbilical cord, placenta or adipose tissue of step one to P3 generation at 4X 104Inoculating each/mL of the cultured cells into a 6-well plate, culturing in 10% FBS-containing DMEM/F12, replacing BTCM after 24h of culture, replacing BTCM once every 2 days, culturing for 7 days, then culturing for 7 days with a cell differentiation inducing solution, replacing the inducing solution once every 2 days, and then jointly culturing for 14 days by using the cell differentiation inducing solution and SCM according to the volume ratio of 1:1 to obtain mature islet-like cells.
3. The method for inducing mesenchymal stem cells to differentiate into islet-like cells according to claim 1, wherein the umbilical cord mesenchymal stem cells are isolated and subcultured as follows:
taking 15-20 cm of umbilical cord of a full-term fetus, cleaning the umbilical cord with normal saline and alcohol with the volume percentage of 75%, shearing the umbilical cord into pieces with the length of 2-3 cm, respectively stripping off arteries and veins, separating out the Wharton jelly in the middle, and shearing the umbilical cord into pieces with the length of 0.5-2 mm under the aseptic condition3Inoculating tissue blocks according to a bottle of 1g/T75, culturing with 10% FBS culture medium, changing the culture solution after 5d, digesting with pancreatin with volume percentage concentration of 0.125% after the cell fusion degree reaches above 80%, obtaining single cells, and inoculating with 8 × 105Inoculating individual cells/T75 in bottle, culturing with 10% FBS culture medium, placing in carbon dioxide incubator, subculturing after cell fusion degree reaches above 90%, detecting the immune marker of MSCs in umbilical cord, and detecting qualified retention, i.e.And completing the separation and subculture of the umbilical cord mesenchymal stem cells.
4. The method for inducing differentiation of mesenchymal stem cells into islet-like cells according to claim 1, wherein said placental mesenchymal stem cells are isolated and subcultured as follows:
under the aseptic condition, firstly, 75% alcohol by volume percentage is used for disinfecting a placenta collecting bag, then the placenta is washed for 2 times by normal saline, after the amnion is stripped, the chorion tissue of the placenta is taken, the normal saline is added for washing blood stain, the chorion tissue is placed in a 50mL clean centrifugal tube after being washed, and the tube is cut into 0.2-1 mm3Adding collagenase type I with the volume 3 times of the tissue block, digesting for 30min in a shaking table at 37 ℃, adding 10mL of serum-free culture medium to stop digestion, filtering in a 50mL clean centrifugal tube, centrifuging for 6min at 1300r/min, discarding the supernatant, taking the precipitate, and centrifuging according to the proportion of 1 multiplied by 106Inoculating each cell/T75 bottle, culturing by adopting 10% FBS culture medium, placing in a carbon dioxide incubator, carrying out passage after the cell fusion degree reaches more than 90%, detecting the immune marker of the placenta MSCs, and detecting qualified retention, namely completing the separation and passage culture of the placenta mesenchymal stem cells.
5. The method for inducing differentiation of mesenchymal stem cells into islet-like cells according to claim 1, wherein said adipose mesenchymal stem cells are isolated and subcultured:
under the aseptic condition, firstly, treating a fat collection bottle with 75% alcohol by volume percentage, then cleaning fat for 2-3 times by using normal saline, transferring adipose tissues to a 50mL centrifuge tube, adding type I collagenase with the volume 3 times of that of tissue blocks, digesting for 35min in a shaking table at 37 ℃, adding about 15mL serum-free medium to stop digestion, filtering to a 50mL centrifuge tube, centrifuging for 6min at 1300r/min, discarding supernatant, taking precipitate, and centrifuging according to the proportion of 1 multiplied by 106～1.5×106Inoculating individual cells/T75 in bottle, culturing in 10% FBS culture medium, placing in carbon dioxide incubator, passaging after cell fusion degree reaches above 90%, and immunizing fat MSCsAnd (4) performing label detection, and detecting qualified retention, namely completing the separation and subculture of the adipose tissue-derived mesenchymal stem cells.
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| CN111454880B (en) * | 2020-04-09 | 2022-04-19 | 厦门大学 | Method for differentiating human umbilical cord mesenchymal stem cells into insulin-like masses with insulin secretion function through in-vitro induction |
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