CN103756954A - Inducer for inducing human adipose derived stromal cells into insulin-secretion cells, induction medium and method - Google Patents
Inducer for inducing human adipose derived stromal cells into insulin-secretion cells, induction medium and method Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and relates to an inducer for inducing human adipose derived stromal cells into insulin-secretion cells, an induction medium and a method. The inducer for inducing human adipose derived stromal cells into insulin-secretion cells consists of the following components: a rhodiola rosea injection, nicotinamide, taurine and GLP-1. Every 1,000ml of the induction medium containing the inducer comprises 20-40g of the rhodiola rosea injection, 0.97-1.47g of the nicotinamide, 0.25-0.50g of the taurine, 26.84-40.27mg of the GLP-1 and the balance being a human adipose derived stromal cell serum-free medium, which are mixed uniformly, filtered and degermed. According to the inducer for inducing human adipose derived stromal cells into insulin-secretion cells, the induction medium and the method, differentiation of the human adipose derived stromal cells into the insulin-secretion cells is induced by using the injection of a traditional Chinese medicine, namely, rhodiola rosea, and each component is safe and nontoxic. The method has few steps and is short in time and high in the inducing efficiency.
Description
Technical field
The invention belongs to biomedical sector, relate to inductor, inducing culture and method that induced dry-cell is divided into insulin secretory cell.
Background technology
Diabetes (diabetes mellitus, DM) are a kind of endocrine metabolism diseases that glycolipid metabolism disorder is principal character of take.According to the prediction of the World Health Organization and IDF's announcement, to the year two thousand thirty, global diabetic subject will reach 3.7 hundred million.China DM patient surpasses 4,500 ten thousand at present, is only second to India and occupies second.Although its cause of disease is different, all show as the defect of islet cells quantity and function, finally all need to use Regular Insulin to treat.Although the application due to Regular Insulin, especially in recent years at aspects such as Regular Insulin formulation and route of administration, all have made some progress, but facts have proved that exogenous insulin can not ideally be controlled blood sugar as human body self excreting insulin and constant to maintain blood sugar normal.Cell replacement therapy is the important method for the treatment of diabetes, is a kind of effective ways that more approach physiology mode, can realize blood sugar is continued to monitor and meticulous adjusting.In recent years, THPV was implanted islet cells treatment diabetes and had also been obtained some curative effects, but islet cell transplantation is faced with two difficult problems: donor source is not enough and serious immunological rejection.
Stem cell, as a kind of cell with self and Multidirectional Differentiation, can be divided under certain condition and have functional islet cells, therefore can be used as the brand-new source of islet cells.By differentiation of stem cells, obtain islet cells at present and have three kinds of approach: 1, ES cell differentiation; 2, pancreatic stem cell or the differentiation of pancreas islet precursor cell; 3, adult stem cell differentiation.Because embryonic stem cell has dispute of ethic, and be difficult to obtain pancreatic stem cell, form soma cell derived extensive, without dispute of ethic, can be used as the source of islet cells.
In fatty tissue, contain the cell that a class has Multidirectional Differentiation potentiality, fat mesenchymal stem cell, is called for short fat stem cell.Its biological property and mesenchymal stem cells MSCs are similar, and can be to various kinds of cell direction differentiation such as fat, bone, cartilage, muscle, endothelium, liver, pancreas islet and nerves.Because fatty tissue reserves in human body are abundant, obtain easy wound little, at aspects such as organizational project, organ reparation, gene therapies, all have broad application prospects, so fat mesenchymal stem cell has become another focus receiving much concern of stem cell field after mesenchymal stem cells MSCs.
That current stem cell induction mode mainly contains is external evoked, genetic modification, protein transduction and tissue microenvironment induction, and wherein external evoked is to adopt different stimulated combinations of factors, and stem cell is induced to differentiate into object cell.Each laboratory-induced differentiation condition is different, and induction differentiation mechanism is still not clear, and induction differentiation efficiency is low, and islet secretion ability is only normal pancreas islet 1% left and right, and Induction Process is complicated, and induction time is long, and gained cell quantity is few, and function is low.
Summary of the invention
The object of the invention is, in order to solve the above-mentioned problems in the prior art, provides a kind of inductor of human adipose mesenchymal stem cells being induced into insulin secretory cell.This inductor composition safety non-toxic, induction success ratio is high.
For achieving the above object, the technical solution used in the present invention is: a kind of inductor of human adipose mesenchymal stem cells being induced into insulin secretory cell, and composed of the following components: rhodiola kirilowii Regel injection liquid, niacinamide, taurine and GLP-1.
The mass concentration proportioning of described each component of inductor is: rhodiola kirilowii Regel injection liquid 20～40 g/L, niacinamide 0.97～1.47g/L, taurine 0.25～0.50 g/L, GLP-1 26.84～40.27mg/L.
Second object of the present invention is to provide a kind of inducing culture that contains above-mentioned inductor; described inducing culture is prepared from by the following method: described in every 1000ml, in inducing culture, contain rhodiola kirilowii Regel injection liquid 20～40 g, niacinamide 0.97～1.47g, taurine 0.25～0.50 g, GLP-1 26.84～40.27mg; surplus behaviour mesenchymal stem cell serum-free culture medium, mixes filtration sterilization.
Another object of the present invention is to provide a kind of method of human adipose mesenchymal stem cells being induced into insulin secretory cell, and the method comprises the following steps:
1, the preparation of fat mesenchymal stem cell: by after fatty tissue digestion, carry out primary cell culture, the cultivation of going down to posterity;
2, the evaluation of fat mesenchymal stem cell: get P3 fat subsitutes mescenchymal stem cell, flow cytometer detection cell surface marker;
3, induction human adipose mesenchymal stem cells is divided into islet cells: the fat mesenchymal stem cell that passed for 3 generations, 0.125%～0.01%Trypsin-EDTA solution digestion collecting cell, prepare cell suspension, cell counting count board living cell counting density, and adjust density 1 * 10
4/ cm
2, be inoculated in 24 orifice plates that are placed with in advance the disinfection cap slide of processing through poly-lysine, prepare cell climbing sheet.Treating that cell reaches approaches 80% fusion, grows when vigorous and induces differentiation with inductor;
4, after induction, islet cells is identified: (1) dithizone staining reaction; (2) chemiluminescence immunoassay detects insulin level; (3) ELISA test kit detects and induces the insulin-like cell obtaining whether to have insulin secretion function; (4) glucose stimulation test; (5) transplantation experiments in animal model.
In recent years, traditional Chinese medicine research constantly makes progress, and rhodiola kirilowii Regel injection liquid is the new Chinese medicine injection liquid by rhodiola kirilowii Regel is separated through microwave assisted extraction, refining, membrane filtration is made.Rhodiola kirilowii Regel injection liquid contains rhodioside (Salidroside), tyrosol (Tyrosol), rosavin (Rosavin), rosarin (Rozarin), rhodiola essence (Rosin), polysaccharide; there is anti-hypoxia, anti-oxidant, radioprotective, antitumor action; can Cell protection film; Promote cell's growth; changing the character of cell coat etc., is the Chinese medicine that is worth very much research and development.
Niacinamide is a kind of form of vitamin B3, it is a kind of ADP-ribose synthetase inhibitors, by its main metabolites Reduced nicotinamide-adenine dinucleotide (NAD), participate in vital movement widely, comprise Nutrition and Metabolism, signal transduction, remain genomic complete etc., niacinamide can be induced differentiation and the maturation of human embryos pancreatic islet endocrine.
Taurine is a kind of non-protein amino acid of sulfur-bearing, exists in vivo with unbound state, does not participate in the biosynthesizing of albumen in body.Can be combined with insulin receptor, promote cellular uptake and utilize glucose, accelerate glycolysis-, reduce blood sugar concentration.
GLP-1 (glucagon-like peptide 1) can stimulate insulinoma cell proliferation, differentiation, suppresses Intra-islet Apoptosis; And can promote insulin gene to transcribe, promote emiocytosis Regular Insulin.
Inductor, inducing culture and method of human adipose mesenchymal stem cells being induced into insulin secretory cell of the present invention, application Chinese medicine rhodiola kirilowii Regel injection liquid induced lipolysis Derived from Mesenchymal Stem Cells is insulin secreting cells, has following advantage:
1, without gene transfection, therefore without gene alteration and risk of cancer;
2, induction step is few: the method for current bibliographical information be take two-step approach or three-step approach as main, adopts inductor of the present invention, inducing culture and method only to need a step;
3, induction time is short: the general derived need of current document more than 10 days, adopts inductor of the present invention, inducing culture and method only to need 7 days;
4, adopting inductor of the present invention, inducing culture and method that fat mesenchymal stem cell is induced into insulin secretory cell induces differentiation efficiency high;
5, inductor of the present invention, the equal safety non-toxic of each composition, and performance TCM Features;
6, after autologous fat mesenchyma stem cell differentiation induction is insulin secretory cell, without repelling, without ethics problem, safe after autotransplantation, there is wide potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is fat mesenchymal stem cell, p3,200 *;
Fig. 2 takes on a red color after the dyeing of induction group dithizone is shown in dithizone staining reaction.
Embodiment
embodiment 1the present embodiment human adipose mesenchymal stem cells is induced into the inductor of insulin secretory cell, by the component of following mass concentration proportioning, formed: rhodiola kirilowii Regel injection liquid 20 g/L, niacinamide 1.2g/L, taurine 0.50 g/L, GLP-1 30.5mg/L.
The composition and ratio of the inducing culture that contains above-mentioned inductor is: rhodiola kirilowii Regel injection liquid 20 g, niacinamide 1.2 g, taurine 0.5 g in inducing culture described in every 1000ml; GLP-1 30.5 mg; surplus behaviour mesenchymal stem cell serum-free culture medium, above-mentioned each composition mixes rear filtration sterilization and can be made into inductor substratum of the present invention.
Each composition of inductor of the present invention and inducing culture is commercially available prod: human mesenchymal stem cell serum free medium, LONZA, 00190632; Rhodiola kirilowii Regel injection liquid, the beautiful panacea industry in Tonghua; Niacinamide, Sigma, N0636; Taurine, Sigma, T8691; GLP-1 (glucagon-like peptide 1), Sigma, scp0153.
embodiment 2the present embodiment is the method for insulin secretory cell that human adipose mesenchymal stem cells is induced into, comprises the following steps:
One, fat mesenchymal stem cell preparation:
1, receive fatty tissue, the container outer wall of the alcohol wipe dress fatty tissue with 75%.
2, packing fatty tissue, each T175 culturing bottle packing fatty tissue is 50ml.10ml transfer pipet, removes suction nozzle, first draws lower floor's red liquid discard in fatty collecting bottle, after residue upper strata fat mixes, carries out packing.
3, washing fatty tissue, removes hemocyte.In T175 culturing bottle, add 100ml sodium chloride injection, tighten lid, acutely rock 3 minutes fully to wash fatty tissue, follow static 3-5 minute, difference is separated, suck lower floor's water; Repeat above operation three times, until subnatant is comparatively limpid.
4, collagenase I digestion: collagenase I solution (the 0.1% collagenase I compound method: take 0.1g collagenase I powder dissolution and do not add in the substratum of any factor in 100ml that adds the preheating (half an hour is in the gas bath shaking table preheating of 37 ℃ in advance) of the new preparation of equivalent, with 37 ℃ of preheatings before), sealed membrane sealing, acutely rock culturing bottle 5 ~ 10 seconds, be placed in vibration gas bath pot, 37 ℃, 200rpm, digest 60 minutes, every 15 minutes, acutely rock culturing bottle 5 ~ 10 seconds, until seem comparatively level and smooth.
5, isolation medium blood vessel component (SVF): postdigestive tissue is divided in the core barrel that installs to 50ml with aseptic 40 mesh filter screens, centrifugal 10 minutes of room temperature 400g, the precipitation obtaining is SVF.
6, purify precipitation: after centrifugal, SVF is deposited on centrifuge tube bottom, with transfer pipet, carefully removes the collagenase solution of upper strata grease and lower floor from top to bottom.Attention: leave a small amount of solution above SVF precipitation, in order to avoid disturbance sedimentation cell.Appropriate physiological saline re-suspended cell, dispels, room temperature 400g, and 10 minutes are centrifugal.Centrifugal complete, carefully suck supernatant liquor, can not directly outwell.During absorption, head of pipette should be placed in the top of centrifuge tube so that go out to deoil thoroughly.10ml substratum suspension cell, is then aggregated into cell in 50ml centrifuge tube, crosses 100 mesh sieves, room temperature 300g again, and 10 minutes are centrifugal.
7, cell seeding: add 20ml substratum after centrifugal and fully mix.Based on tissue block method: carry out cell seeding according to the area of culturing bottle.The fat quantity obtaining according to every square centimeter of inoculation 0.16ml liposuction is inoculated, and in each T75 culturing bottle, inoculates 12ml liposuction fat quantity.Be every 100ml fatty tissue, finally can inoculate 8 T75 culturing bottles.The cell suspension that carries out untreated fat quantity and finally obtain converts, and then inoculating cell.
8, primary cell culture: horizontal culturing bottle, is positioned over carbonic acid gas fixed temperature and humidity incubator by culturing bottle.Culture condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is 5 ± 0.2%.Substratum: human mesenchymal stem cell serum free medium (LONZA, 00190632).
9, change liquid: former culture the 24th hour, carries out full dose and changes liquid.After this every 3 days full doses, change liquid, place carbonic acid gas fixed temperature and humidity incubator and cultivate.
10, primary cell results: about 7 days, when the area percentage of primary cultured cells cloning cluster arrives 70%～80%, digestion results.
11, primary cell results: (digestive ferment is 0.125%Trypsin～0.01% EDTA solution, and before using, room temperature (20～25 ℃) is placed 15～25min, every 75 cm to add digestive ferment in culturing bottle
2add 2ml digestive ferment solution), digestion time is 1.5～2.5min, at the bottom of adding substratum 2～3ml repeatedly to blow and beat bottle, come off to cell major part, move in 50ml centrifuge tube, in former culturing bottle, add 4～5ml chloride injection-liquid washing bottle wall, add and in centrifuge tube, be settled to 50ml, after transfer pipet piping and druming suspends, the aseptic strainer filtering of 100 order, filtered liquid is collected in 50ml centrifuge tube, 1000rpm, 10min centrifuge washing.
12, primary cell goes down to posterity: observe remaining cell precipitation amount in single centrifuge tube, suitably merge in several centrifuge tubes in cell precipitation to 1 centrifuge tube, add appropriate substratum, blow and beat gently resuspension cell, be settled to 30ml, piping and druming mixes, sampling counting.1000rpm after counting, 10min secondary centrifuging.Remove supernatant liquor, add substratum appropriate in centrifuge tube, blow and beat gently resuspension cell, be seeded in new culture vessel after constant volume, passage cell density is 5000～6000/cm
2, i.e. (3.75 ~ 4.5) * 10
5individual cells/T75, according to 4.5 * 10
5individual cells/T75 goes down to posterity.On culture vessel, indicate the information such as cell algebraically and incubation time.Culture vessel is positioned over to carbonic acid gas fixed temperature and humidity incubator to start to cultivate.Culture condition: carbonic acid gas fixed temperature and humidity incubator.Condition: 37 ± 0.5 ℃, carbonic acid gas volume fraction is 5 ± 0.2%.Be cultured to cytogamy and reach 85%～90%.
Two, the evaluation of fat mesenchymal stem cell
Get P3 fat subsitutes mescenchymal stem cell, flow cytometer detection cell surface marker, streaming the results are shown in Table 1.
Table 1 flow cytometer detection cell surface marker
Wherein, positive mark's thing CD29, CD73, CD90, CD49d express and are greater than 95%, and negative marker thing CD14, CD34, CD45, HLA-DR express lower than 2%, and proving this cell is fat mesenchymal stem cell.
Three, induction human adipose mesenchymal stem cells is divided into islet cells: the fat mesenchymal stem cell that passed for 3 generations, 0.125%～0.01%Trypsin-EDTA solution digestion collecting cell, prepare cell suspension, cell counting count board living cell counting density, and adjust density 1 * 10
4/ cm
2, be inoculated in 24 orifice plates that are placed with in advance the disinfection cap slide of processing through poly-lysine, prepare cell climbing sheet.Treating that cell reaches approaches 80% fusion, grows when vigorous and induces differentiation again, and grouping is in Table 2.
Table 2 induction grouping
Four, after induction, islet cells is identified
1, dithizone staining reaction
Get respectively above three groups of insulin-like cell that induction obtains for 7 days afterwards, shift out former substratum, PBS washes 2 times, respectively adds 2ml PBS and 50ul dithizone working fluid, hatches 10min for 37 ℃, shifts out staining fluid, PBS washed twice, and the painted situation of observation of cell is also taken pictures.Result as shown in the figure, takes on a red color (as shown in Figure 1) after two induction group dithizone dyeing, positive reaction, and control group is negative.
2, chemiluminescence immunoassay detects insulin level
Get respectively above three groups and induce cells and supernatant after 7 days, detect insulin content, the emiocytosis insulin concentration of induction group 2 is 408mU/L, emiocytosis insulin concentration 226.0mU/L far above induction group 1, and blank group detection insulin concentration is zero, illustrate that inductor of the present invention and inducing culture can significantly improve induction efficiency.
3, ELISA test kit detects with the insulin-like cell that inductor of the present invention and inducing culture induction obtain whether have islet cell function
" C-peptide ELISA assays " standard procedure operation that this testing process directly provides according to Mercodia company.Net result detects C-peptide, illustrates that the islet cells of inductor of the present invention and inducing culture induction can excreting insulin.
4, glucose stimulation test
100 inductor induction differentiation of the present invention of picking islet cells group (50 ~ 150um) of 7 days is to 1.5ml centrifuge tube, with PBS, clean 2 times, add 1ml sugar-free DMEM preculture 3 ~ 6h, then with 300ul, contain 5.6mmol/L glucose, the DMEM of 16.7mmol/L glucose cultivates 2h successively, collect supernatant liquor, by ELISA method, detect the secretory volume of the lower Regular Insulin of different glucose stimulation in supernatant, in cellular control unit supernatant, almost can't detect Regular Insulin, and under 5.6mmol/L glucose stimulates, have a small amount of secretion through the islet cells group of induction, after 16.7mmol/L glucose incubation 2h, amount of insulin secretion obviously raise (P<0.001), be about 2 times under low sugar condition, result is known thus, after induction, islet cells group stimulates responsive to glucose, the secretion of its Regular Insulin is subject to the regulation and control of external environment.
5, transplantation experiments in body
First make diabetes rat model.Get adult Wistar rats, male and female are not limit, and body weight is 180 ~ 200g approximately.According to 70mg/kg dosage, give every rats by intraperitoneal injection U-9889.0.1M citrate buffer solution for U-9889 pulvis (PH=4.5) is made into liquid and uses, now with the current.When rat blood sugar raises (>=16.7mmol/L) and stablizes one week, show that diabetes model builds up.Under aseptic condition, under diabetes rat kidney packing or hepatic portal arteries and veins subbranch inject the insulin-like cell group (50 ~ 150um) that 500 inductors inductions of the present invention obtain.Postoperative, periodic observation blood sugar situation.Result: diabetes rat is being implanted cell blood sugar 6.2mmol/L that on average declines after 5 days.Show to adopt the islet cells group that inductor of the present invention and inducing culture obtain to there is significant hypoglycemic activity.
Claims (9)
1. human adipose mesenchymal stem cells is induced into an inductor for insulin secretory cell, it is characterized in that: composed of the following components: rhodiola kirilowii Regel injection liquid, niacinamide, taurine and GLP-1.
2. inductor of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 1; it is characterized in that: the mass concentration proportioning of each component is: rhodiola kirilowii Regel injection liquid 20～40 g/L, niacinamide 0.97～1.47g/L, taurine 0.25～0.50 g/L, GLP-1 26.84～40.27mg/L.
3. an inducing culture that contains inductor as claimed in claim 1 or 2; it is characterized in that: described inducing culture is prepared from by the following method: described in every 1000ml, in inducing culture, contain rhodiola kirilowii Regel injection liquid 20～40 g, niacinamide 0.97～1.47g, taurine 0.25～0.50 g, GLP-1 26.84～40.27mg; surplus behaviour mesenchymal stem cell serum-free culture medium, mixes filtration sterilization.
4. human adipose mesenchymal stem cells is induced into a method for insulin secretory cell, the method comprises the following steps:
(1), the preparation of fat mesenchymal stem cell: by after fatty tissue digestion, carry out primary cell culture, the cultivation of going down to posterity;
(2), the evaluation of fat mesenchymal stem cell: get P3 fat subsitutes mescenchymal stem cell, flow cytometer detection cell surface marker;
(3), induction human adipose mesenchymal stem cells is divided into islet cells: the fat mesenchymal stem cell that passed for 3 generations, 0.125%～0.01%Trypsin-EDTA solution digestion collecting cell, prepare cell suspension, cell counting count board living cell counting density, and adjust density 1 * 10
4/ cm
2, be inoculated in 24 orifice plates that are placed with in advance the disinfection cap slide of processing through poly-lysine, prepare cell climbing sheet; Treating that cell reaches approaches 80% fusion, grows when vigorous and induces differentiation with inductor;
(4), after induction, islet cells is identified.
5. method of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 4, is characterized in that: after the induction described in step (4), islet cells authentication method is: dithizone staining reaction.
6. method of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 4, is characterized in that: after the induction described in step (4), islet cells authentication method is: chemiluminescence immunoassay detects insulin level.
7. method of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 4, is characterized in that: after the induction described in step (4), islet cells authentication method is: whether the insulin-like cell that adopts ELISA test kit to detect induction acquisition has insulin secretion function.
8. method of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 4, is characterized in that: after the induction described in step (4), islet cells authentication method is: glucose stimulation test.
9. method of human adipose mesenchymal stem cells being induced into insulin secretory cell according to claim 4, is characterized in that: after the induction described in step (4), islet cells authentication method is: transplantation experiments in animal model.
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| CN113943699B (en) * | 2020-07-17 | 2023-10-27 | 北京中卫医正科技有限公司 | Umbilical cord mesenchymal stem cell induction liquid for resisting high sugar injury, method and application |
| CN113943699A (en) * | 2020-07-17 | 2022-01-18 | 北京中卫医正科技有限公司 | Umbilical cord mesenchymal stem cell induction liquid for resisting high-sugar damage, method and application |
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| CN112251397A (en) * | 2020-10-30 | 2021-01-22 | 湖南赛诺生物科技股份有限公司 | Culture method of newborn pig islet cells |
| CN113181217A (en) * | 2021-04-28 | 2021-07-30 | 吉林大学 | Cell scaffold, islet cell carrying compound thereof, preparation method and application |
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