CN102199573B - Method for inducing and differentiating functional cardiocytes by utilizing endometrial stem cells - Google Patents
Method for inducing and differentiating functional cardiocytes by utilizing endometrial stem cells Download PDFInfo
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Abstract
The invention discloses a method for inducing and differentiating functional cardiocytes by utilizing endometrial stem cells. The method comprises the following steps of: 1) performing separation culture on the endometrial stem cells and amplifying; and 2) inducing and differentiating in vitro, namely digesting 4th to 6th generation of cells in good growth condition, which is obtained in step 1) by using pancreatin, terminating the digestion by using a Dulbecco modified eagle medium (DMEM) containing 13 to 17 volume percent fetal calf serum when the cells are changed into single circular shapes under a microscope, centrifuging, washing an obtained cell precipitate by using phosphate buffer solution (PBS), adding an induction culture medium into the cells washed by the PBS, putting into anincubator which contains 4 to 6 percent of CO2 and has the saturated humidity of between 94 and 96 percent at the temperature of between 36 and 38 DEG C, and culturing to obtain the functional cardiocytes, wherein the culture time is 68 to 76 hours. The obtained functional cardiocytes can be used for treating myocardial infarction.
Description
Technical field
The present invention relates to the in utero film stem cell method of inducing the differentiation function myocardial cell of a kind of utilization.
Background technology
At present the congestive heart failure sickness rate raises year by year, and there are 1,500 ten thousand patients in the whole world, and annual new cases are 46.5 ten thousand, for the over-65s crowd reason of primarily being in hospital, also is the main reason of cardiovascular death.With the stem cell be the Transplanted cells therapy of seed resource can promote the infarct local vascular newborn with cardiac muscle regeneration, and then improve Heart Function After Myocardial Infarction, become the research focus of present cardiovascular field.Yet limited, the inconvenience of drawing materials of the embryonic myocardium ability of cell proliferation that can supply transplant, skeletal myoblast can not form that the slit is connected with the myocardial cell again and can proarrhythmia, cause this treatment means to be gone no further.Therefore the cell therapy that with the stem cell is seed resource becomes damaged tissue reparation and alternate important means.Wherein, human embryo stem cell receives the restriction of himself some factor, like tumorigenicity, immunogenicity and ethics problem etc., and still can not be as ideal transplanted cells source.
(mesenchymal stem cell MSC) is the adult stem cell of many differentiation directions of tool ability to mescenchymal stem cell, because of it carries out the restriction that autotransplantation does not have rejection and source, is ideal transplanted cells source.Wherein, mesenchymal stem cells MSCs is present main source of human stem cell.Reduce although its ethics problem is compared with embryonic stem cell greatly, medullary cell must obtain through the approach of invading, and with advancing age, stem cell population significantly reduces.Because of the restriction of acquisition mode and self content, mesenchymal stem cells MSCs is faced with the bottleneck that is difficult to break through in clinical application.Mainly there is following problem in the mescenchymal stem cell of derived from bone marrow:
1. the potential that is divided into functioning cells such as myocardial cell, neuronal cell is not enough;
2. transplant the most of necrocytosis in back;
3. it is undesirable to transplant curative effect;
4. cell quantity restriction.
The endometrial cell cording has very strong self ability.In every month all can be in a organized way and the dramatic growth of blood vessel through the cycle, this propagation process can stop along with the end of menstrual cycle.The medium multinational scientist's of America and Japan current research is presented in the endometrial tissue that comes off with women's menses, has a considerable amount of mescenchymal stem cells (stroma stem cell)-in utero film stem cell, and quantity is 30 times of derived from bone marrow; These stem cell vigor are stronger, and stronger self and multiplication capacity, differentiation potential are more near embryonic stem cell.Have and discover that in utero the film stem cell not only has the surprising speed of duplicating once in almost per 24 hours, and they produce the speed of unique growth factor, than big last 100,000 times of the stem cell that comes from Cord blood.
Summary of the invention
The technical problem that the present invention will solve provides the in utero film stem cell method of inducing the differentiation function myocardial cell of a kind of utilization, and the function myocardial cell of gained can be used to treat myocardial infarction.
In order to solve the problems of the technologies described above, the present invention provides a kind of method of utilizing the uterine endometrium stem cell to induce the differentiation function myocardial cell, may further comprise the steps:
1), in utero separation and Culture of film stem cell and amplification:
Menses are after the germicidal treatment that contains antibiotic disinfecting liquid, centrifugal, remove supernatant; Add the ChangShi collective media then and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate;
Cultivate and change liquid after 5～7 days and remove not attached cell; In case when cell grows to 75～85% degree of converging, promptly adopt trysinization to go down to posterity;
Generally, can be whenever change liquid once at a distance from 2～4, per 3～4 days passage cells once;
2), external evoked differentiation:
With above-mentioned steps 1) gained the 4th～6 generation the upgrowth situation good cell with trysinization; When microscopically sees that it is that the DMEM substratum of 13～17% foetal calf serums stops digestion to contain volumetric concentration when circular that cell becomes single; Centrifugal, the cell precipitation of gained cleans with PBS;
Add inducing culture in the cell after above-mentioned PBS cleans and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate; Per 1 * 10
5Add 0.8～1.2ml inducing culture in the individual cell, said inducing culture is: the U-18496 that in serum-free DMEM substratum, contains 1～10 μ M; Incubation time is 68～76 hours, gets functional myocardial cell.
As the improvement that utilizes the uterine endometrium stem cell to induce differentiation function myocardial cell's method of the present invention: step 1) is followed successively by:
1., menses are collected:
Hank ' s the balanced salt solution (HBSS) of 20ml is set in collection tube, and adds following composition to following concentration: vancomyein (Vancomycin) 60～100 μ g/mL, Cephalexin Monohydrate Micro/Compacted (Claforan) 150～350 μ g/mL, kantlex (Kanamycin A Sulfate) 50～150 μ g/mL, qingfengmeisu qiong (Gentamycin) 80～160 μ g/mL, amphotericin B (Amphotericin B) 2～3 μ g/mL and 300～500 units of heparin sodium; With this as containing antibiotic disinfecting liquid; The menses of 15～20ml are put into above-mentioned collection tube, preserved 24～48 hours down in 0～4 ℃ low temperature; Centrifugal then, remove supernatant;
2., former be commissioned to train foster:
A), the ChangShi collective media of getting 6～10ml joins step 1. in the gains;
B), with steps A) gains put into culturing bottle and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate; Cultivate after 5～7 days full dose and change liquid; Again with upright 3～8 minutes of above-mentioned culturing bottle, thereby make not adherent cell landing to culturing bottle bottom, remove the substratum in the culturing bottle with transfer pipet;
C), at step B) the ChangShi collective media that adds 2～4mL in the culturing bottle of gained carries out rinse (rinse cellular layer), removes the substratum in the culturing bottle with transfer pipet then;
D), at step C) add the ChangShi collective media of 6～10mL in the culturing bottle of gained, place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate;
The compound method of ChangShi collective media is following: under aseptic condition; In sterile chamber, add 650mL MEM-alpha substratum (MEM alpha; Invitrogen company), 160～200mL Chang B base fluid (Irvine Scientific company), 10～30mL Chang C base fluid (Irvine Scientific company), 5～15mL penicillin/streptomycin two anti-(10,000U/mL sodium benzylpenicillin, 10; 000 μ g/mL Streptomycin sulphate); The concentration of 5～15mL is the L-glutaminate (L-glutamine, Invitrogen company) of 200mM, the foetal calf serum (ES-FBS, Invitrogen company) of 130～170mL; Fully mixing is put into 0～4 ℃ of refrigerator and is preserved for use behind the high pressure steam sterilization;
3., passage is cultivated:
In case step when 2. former generation, cultured cells grew to 75～85% degree of converging, promptly adopts trysinization to go down to posterity; (general per 3～4 days passage cells once); Specific as follows:
A) in case step when 2. former generation, cultured cells grew to 75～85% degree of converging, is removed nutrient solution, the PBS washings with no calcium ions and magnesium ions washs then;
B), add 2～4ml pancreatin, place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in hatched 4～6 minutes;
C), the ChangShi collective media of adding 4～6ml makes the pancreatin inactivation;
D), soft piping and druming, attached cell come off and become unicellular state;
E), go down to posterity by 1: 8 ratio;
F), the ChangShi collective media that in the cell suspension of every 1ml, adds 5～7ml; Place 4～6%CO then
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate.
In the present invention, hatching and cultivate all is at 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in carry out.
In the present invention, the pancreatin that is used to digest is meant the pancreatin (trypsinase) of conventional 0.25% or 0.2% (mass volume ratio).
The present invention relates generally to following content:
1. the in utero evaluation of cultivation of film stem cell and molecular marked compound.
2. experiment in vitro: setting up U-18496 directional induction menses differentiation of stem cells is functional myocardial cell's external model.
3. the structure of congested back animal model for heart failure after the myocardial infarction.
4. experiment in the body: will not induce with the functional myocardial cell who successfully induces differentiation and carry out transplanting in the body, in vivo differentiation situation and heart function improved situation after assessment MSCs transplanted.
The present invention filters out stable in utero film stem cell line through external cultivation and evaluation through hemocytoblast; Through U-18496 directional induction in vitro model, successfully differentiate functional myocardial cell; Utilize the Transplanted cells art; To not induce and the infarct kitchen range periphery of directed differentiation, study the effect of in utero film stem cell transplantation congested back animal model for heart failure treatment after myocardial infarction for myocardial cell's animal model for heart failure after the hemocytoblast orthotopic transplantation is congested after myocardial infarction; Intravital differentiation situation of postoperative track cells and heart function improve situation; Utilization Kaplan-Meier survival curve is analyzed the in utero curative effect of film stem cell transplantation art congested back animal model for heart failure after the treatment myocardial infarction, confirms the feasibility of its treatment myocardial infarction.
In utero film (menses) stem cell is adult stem cell a kind of of recent findings, belongs to mescenchymal stem cell (MenSC).Finding in the endometrial tissue to have abundant mescenchymal stem cell, excrete with menses at intermenstrual period, is a kind of source of human stem cell mode of not having wound, safety.On cell marking developed by molecule and cell proliferation, differentiation capability, more similar with embryonic stem cell through hemocytoblast.The present invention induces film stem cell directional in utero to be divided into functional myocardial cell's external model through U-18496; Employing is without inducing the method for transplanting in the in utero film stem cell body that breaks up with induced orientation; Observe the effect that stem cell breaks up in vivo, for congested back treatment in heart failure after the myocardial infarction provide a kind of efficient, use solution widely.
Utilization of the present invention is the film stem cell method of inducing the differentiation function myocardial cell in utero, has the following advantages:
1, in utero the film stem cell to express myocardial cell's specific gene GREM1 be 10 times of bone marrow stem cell.
2, external evoked differentiation rate is high, can be up to 60%.
3, in utero the film stem cell can reach more than February at petty action object internal memory live time.
In sum, the present invention sets up directional induction in vitro myocardial cell model, utilizes the stem cell transplantation technology, studies the effect of in utero film stem cell transplantation congested back animal model for heart failure treatment after myocardial infarction; Confirm the feasibility of its treatment myocardial infarction, lay the foundation for improving the stem cell clinical therapeutic efficacy.
Description of drawings
Do further explain below in conjunction with the accompanying drawing specific embodiments of the invention.
Fig. 1 is immunohistochemical experiment figure as a result, and the U-18496 of 5 μ M was induced back 72 hours, β-Actin coloration result;
Fig. 2 is the RT-PCR experimental result picture, and the U-18496 of 5 μ M was induced back 72 hours, and three bands are respectively β-MHC, cTNT and GAPDH from top to bottom;
Fig. 3 is the interior transplantation experiments of body figure as a result;
Fig. 4 is the interior transplantation experiments of body figure as a result;
Fig. 5 is for transplanting seraglio inner membrance stem cell viability evaluation map.
Embodiment
One, in utero separation and Culture of film stem cell and amplification:
Recruit the menstrual woman of 10 different ages sections, under voluntary fully prerequisite, contribute the menstrual blood sample.
The concrete operations rules are following:
1, menses sample collection:
With the menses collection tube, menses cup put into the menses collecting cassette, deliver in the menses donor hand.Collection tube is equipped with 20mlHBSS (Hank ' s balanced salt solution) in advance gathers liquid, wherein contains Vancomycin (vancomyein) 80 μ g/mL, Claforan (Cephalexin Monohydrate Micro/Compacted) 250 μ g/mL, Kanamycin A Sulfate (kantlex) 100 μ g/mL, Gentamycin (qingfengmeisu qiong) 120 μ g/mL, Amphotericin B (amphotericin B) 2.7 μ g/mL and 400 units of heparin sodium.With this as containing antibiotic disinfecting liquid.
The preparation method who is above-mentioned HBSS (Hank ' s balanced salt solution) collection liquid is following:
In the Hank ' of 20ml s balanced salt solution, add following composition to following concentration: vancomyein (Vancomycin) 80 μ g/mL, Cephalexin Monohydrate Micro/Compacted (Claforan) 250 μ g/mL, kantlex (Kanamycin A Sulfate) 100 μ g/mL, qingfengmeisu qiong (Gentamycin) 120 μ g/mL, amphotericin B (Amphotericin B) 2.7 μ g/mL and 400 units of heparin sodium; And then under HTHP, sterilize according to conventional procedure.
Donor is in a few days ago (the 1st to 3 day) that menstruation begins, utilizes the menses cup to obtain through blood sample.The menses (belonging to same donor) of per 15～20ml are put into above-mentioned 1 collection tube.Pair cell is cultivated (the essential approval that obtains to formulate evaluation committee of this informed consent) under the situation that obtains cell contributor informed consent.Must they be preserved (preservation period was generally 24 to 48 hours) under low temperature (0～4 ℃) condition before after obtaining sample, being sent to treating lab.
1) preserves after 24 hours, observe the sample in the menses collection tube,, can sample be filtered via the 100micron filtering net if any deposition;
2) prepare centrifugal before, carefully with the periphery wiped clean of sample collection tube; Guarantee the balance of centrifuge tube on the whizzer;
3) centrifugal sample 7 minutes under 840g, 4.0 ℃ condition;
4) carefully take out sample collection tube, do not upset the cell layering;
5) with the sample collection tube periphery with after the alcohol swab wiping sterilization, move into Biohazard Safety Equipment and carry out next step operation;
6) remove supernatant; Cellular layer is not upset in careful imbibition, causes extra cell loss.
Above-mentioned supernatant can be used for carrying out Bacteria Detection, is about to this supernatant and carries out the detection of anerobes and aerophil, fungi according to the hemoculture method of routine, and identify according to ordinary method.If positive findings then finishes separation and Culture and the amplification of whole in utero film stem cell.
Above-mentioned HBSS (Hank ' s balanced salt solution) is gathered the menses that have been processed in the liquid after 24 hours, carry out microorganism detection and infectious disease pathogens safety detection in a conventional manner, generally common virus such as HIV, HBV, HCV, CMV and syphilis are detected.If positive findings then finishes separation and Culture and the amplification of whole in utero film stem cell.
Above-mentioned 2 kinds of testing goals are the safety in utilization for the function myocardial cell who guarantees final gained.
2, former be commissioned to train foster:
The substratum preparation---the composition of ChangShi collective media is following:
1) 650mL MEM alpha substratum
2) 180mL ChangShi B liquid (basic medium) (18%v/v)
3) 20mL ChangShi C liquid (2%v/v)
4) the 10mL penicillin/streptomycin is two resists (10,000U/mL sodium benzylpenicillin, 10,000 μ g/mL Streptomycin sulphates; Be to contain 10, the Streptomycin sulphate of the sodium benzylpenicillin of 000U and 10,000 μ g in the two anti-solution of every ml penicillin/streptomycin)
5) the L-glutaminate 200mM (100x) of 10mL
6) foetal calf serum of 150mL (15%v/v)
The compound method of ChangShi collective media is following: under aseptic condition; In sterile chamber, add 650mLMEM-alpha substratum (MEM alpha; Invitrogen company), 180mL Chang B base fluid (Irvine Scientific company), 20mL Chang C base fluid (Irvine Scientific company), 10mL penicillin/streptomycin two anti-(10,000U/mL sodium benzylpenicillin, 10; 000 μ g/mL Streptomycin sulphate); The concentration of 10mL is the L-glutaminate (L-glutamine, Invitrogen company) of 200mM, the foetal calf serum (ES-FBS, Invitrogen company) of 150mL; Fully mixing is put into 0～4 ℃ of refrigerator in a conventional manner and is preserved for use behind the high pressure steam sterilization.
1) get the 7mLChangShi collective media, join remove supernatant in blood sample (being the gains of step 1), softly blow and beat mixing; Get cell suspension;
2) get a T-25 culturing bottle, whole cell suspensions of step 1) gained are pipetted in this culturing bottle;
3) put into 36.0-38.0 ℃ incubator (5%CO
2, 95% saturated humidity) cultivate;
4) cultivated 5-7 days, full dose is changed liquid (promptly changing the 7mLChangShi collective media).In Biohazard Safety Equipment,, treat that not adherent cell major part with substratum landing to culturing bottle bottom, removes substratum with transfer pipet earlier with upright 5 minutes of culturing bottle.
5) get the 3mLChangShi collective media, slowly splash at the culturing bottle sidewall.Slowly culturing bottle is set level, softly rocked, the rinse cellular layer with upright 5 minutes of culturing bottle, is removed substratum with transfer pipet then.
6) get the 7mLChangShi collective media, slowly splash at the culturing bottle sidewall.
7) mirror is observed down, and the attached cell that is scattered is arranged.
8) put into 36.0-38.0 ℃ of CO
2Incubator (5%CO
2, 95% saturated humidity) and interior continuation cultivation.In culturing process, wait the stem cell cell attachment after, remove not adherent cell, general every liquid that changed at a distance from 2～4 days once continues cultivation.
The back observation of one week, the cell growth has agglomerating cell proliferation, and this moment, cell grew to 80% degree of converging.
3, passage is cultivated
1) 8 of the above-mentioned former foster step of being commissioned to train of the removal) nutrient solution in the gains;
2) the PBS washings with no calcium ions and magnesium ions washs;
The preparation of the PBS washings of above-mentioned no calcium ions and magnesium ions is following:
Behind high pressure steam sterilization (conventional procedure), putting into 4 ℃ of refrigerators preserves for use.
3) add the 3ml pancreatin, hatch (5%CO for 36-38 ℃
2, 95% saturated humidity) and 5 minutes;
4) the ChangShi collective media of adding 5ml makes the pancreatin inactivation;
5) soft piping and druming comes off attached cell and becomes unicellular state;
6) go down to posterity by 1: 8 ratio: in the cell suspension of 8ml, get in the new T-25 culturing bottle of 1ml to, add the ChangShi collective media of 6ml again, mixing;
7) put into 36.0-38.0 ℃ of CO
2Incubator (5%CO
2, 95% saturated humidity) and interior the cultivation;
8) every 3-4 days passage cell once (promptly begins to go down to posterity one in every 3-4 days till the step 7) from step 1)
Inferior), the cell of hypertrophy can reduce the natural differentiation rate of cell.
4, the molecular phenotype of menses mescenchymal stem cell is identified:
The Flow cytometry cell-surface antigens
1) cell reach 2-4 for the time collecting cell, 0.25% trypsin digestion cell is processed cell suspension, cell concentration is 1 * 10
6
2) the 0.5ml EP that gets desired data respectively manages, and adds mouse anti human monoclonal antibody (CD34, CD44, CD45, CD73, CD90, CD105, CD29, SSEA-4, OCT-4, and homotype contrast) 20 μ l respectively;
3), the pipe in add cell sample (step 1) gained) 50 μ l respectively, lucifuge is hatched 30min for 4 ℃;
4), 300g is centrifugal 5 minutes;
5), abandon supernatant, the PBS 1ml of adding 1% (mass concentration) human serum, abundant mixing, centrifugal 5 minutes of 300g;
6), abandon supernatant, add PBS adjustment sample size to 100 μ l, the upflowing cell instrument is analyzed;
The gained result is: CD44, and CD73, CD90, CD105, CD29, SSEA-4, OCT-4 is positive, CD34, CD45 is negative; Thereby confirm that gained really is film stem cell in utero.
Two, set up the external model that film stem cell directional in utero is divided into functional myocardial cell
Get the in utero film stem cell of cultivating 4-6 generation and carry out experiment in vitro, adopt 1 μ M, 5 μ M, the U-18496 of 10 μ M different concns, and combine different induction times, in utero the film stem cell directional is induced into functional myocardial cell.Differentiation gained cell is carried out myocardial myosin heavy chain (MHC); Cardiac muscle myosin light chain (MLC-2v); The immunocytochemical stain of sign such as cardiac troponin (Troponin), and relevant myocardium specific molecular sign such as Nkx2.5, ANF; GATA4, the RT-PCR evaluation of Na+-Ca2+ exchanging pump and phosphoric acid protein receptor etc. etc.
The concrete operations rules are following:
1, directional induction in vitro in utero the film differentiation of stem cells be cardiac-like muscle cell
Get 4-6 for the upgrowth situation good cell; Show as the cell that growth is vigorous, cell space is big, karyon is clear, endochylema is abundant, the microscopically refractivity is strong; With pancreatin (for example being 0.2% trypsinase) digestion, microscopically sees that cell becomes and singlely stops digestion with the DMEM that contains 15% foetal calf serum when circular that soft piping and druming back is centrifugal; Obtain cell precipitation, PBS washes (purpose is for materials such as flush away trypsinase, foetal calf serums) 2 times.Inducing culture dispels cell precipitation, is inoculated in then to carry out inducing culture in 6 orifice plates, and inoculating cell density is 1 * 10
5/ hole, every hole add 1ml inducing culture (being settled to 1ml).Place 5%CO
2, cultivate in 36～38 ℃ of incubators of 95% saturated humidity.
In order to confirm best inducing culture; Be provided with following 3 kinds of inducing cultures respectively: the serum-free DMEM substratum that contains 1 μ M concentration U-18496; The serum-free DMEM substratum that contains 5 μ M concentration U-18496s contains the serum-free DMEM substratum of 10 μ M concentration U-18496s.With the serum-free DMEM substratum that do not contain U-18496 as contrast.
The preparation method of the above-mentioned serum-free DMEM substratum that contains 1 μ M concentration U-18496 is following: in serum-free DMEM substratum, adding U-18496, is 1 μ M until the concentration of U-18496.Behind high pressure steam sterilization (usual manner), putting into 4 ℃ of refrigerators preserves for use.Change the consumption of U-18496, thereby obtain the serum-free DMEM substratum that contains the serum-free DMEM substratum of 5 μ M concentration U-18496s and contain 10 μ M concentration U-18496s accordingly.
After hatching 72h, what in above-mentioned inducing culture, hatch all can obtain functional myocardial cell; Wherein, it is best to contain the pairing effect of serum-free DMEM substratum of 5 μ M concentration U-18496s.And the serum-free DMEM substratum that does not contain U-18496 can not obtain functional myocardial cell.
With above-mentioned functions property myocardial cell with containing 10%FBS (foetal calf serum; Volume content) DMEM substratum changes liquid; Every change liquid once, continued to be cultured to the 8th week at a distance from 2-3 days full doses, every group comprise 20 cell climbing sheets (be every group be provided with respectively induce before with induced back 72 hours and the 2nd, 3,4 weeks).U-18496 is induced after 72 hours and is accomplished, and continues in the DMEM culture medium culturing that contains 10%FBS (foetal calf serum), and purpose is the variation of inducing the back myocardial cell in order to observe.
2, immunohistochemical methods is identified
1) U-18496 of getting 5 μ M is induced 72 hours cell climbing sheet, and 4% Paraformaldehyde 96 room temperature is fixed.
2) add 3%H
2O
2Block endogenous property px.
3) add β-Actin one to be measured and resist, 4 ℃ are spent the night.
4) adding biotin labeled two resists.Normal temperature is hatched 10min.
5) mirror is observed down.
The gained result is as shown in Figure 1: Fig. 1 is the immunohistochemical experiment result, and the U-18496 of 5 μ M was induced back 72 hours, β-Actin coloration result.β-Actin is a kind of specific proteins of myocardial cell, learns from Fig. 1, and in utero the film stem cell has been induced to differentiate into cardiac-like muscle cell.
3, RT-PCR detects the expression of myocardial cell's specific gene (β-MHC, cTNT)
Got for the 2nd generation and induced back 72 hours and the 1st, 2,3,4,6,8 pericytes, count about 1 * 10
6Individual, the Trizol method is extracted total RNA.Rt also carries out amplification in vitro.The required primer of PCR sees the following form 1:
Table 1
The gained result is as shown in Figure 2.Fig. 2 is the RT-PCR experimental result, and the U-18496 of 5 μ M was induced back 72 hours, and three bands are respectively β-MHC, cTNT and GAPDH from top to bottom.According to Fig. 2, we learn: after the U-18496 of 5 μ M was induced 72 hours, the inductive cardiac-like muscle cell was expressed myocardial cell's specific gene β-MHC and cTNT.
4. the structure of congested back animal model for heart failure after the myocardial infarction
15 of 8 monthly age healthy male SD rats, physique amount (500 ± 25) g is divided into 10 of experimental group, 5 of control groups.Experimental group rat ligation LADCA causes left ventricle antetheca myocardial infarction; MAIN OUTCOME MEASURES: record rat electrocardiogram(ECG.Postoperative 4 all ultrasonic cardiography graph evaluation ligation effects, RCCA intubate measure blood flow kinetic parameter is put to death the back from left ventricle root intubate injection blue ink, estimates the scope of myocardial infarction, the thickness of frozen section examination left ventricular wall.The result: the rat postoperative clear and definite myocardial infarction occurs and changes, and comprises that Electrocardiographic change, ultrasonic cardiogram are measured LVEF decline, haemodynamics is measured ventricular end diastolic pressure and raise, dP/dt40.Descend with-dP/dt, the big area myocardial infarction and the frozen section of blue ink prompting confirm the obvious attenuation of left ventricle antetheca thickness.
5, experiment in the body: will induce the functional myocardial cell of differentiation in animal model, to transplant in the body
Animal divides into groups: a. control group (above-mentioned myocardial infarction and ischemia model experimental group+DMEM injection); B. experimental group (the film stem cell of above-mentioned myocardial infarction and ischemia model experimental group+in utero-DMEM suspension injection).Every group each 5, be the injection volume of every rat 2ml.
1) transplants in the body:, get the functional myocardial cell (U-18496 of 5 μ M is induced back 72 hours gained) that the present invention induces the differentiation gained at heart stalk back 30min; In-situ injection is divided 5 injection cells in the myocardial infarction focus periphery of experimental mice through animalcule ultrasonic developing-out unit location, and every mouse injected cells quantity is controlled at 10
7About.Control group mice is injected DMEM at corresponding site.
2) heart function evaluation after the transplanting: respectively group is transplanted warp, cardiac ejection fraction in the blind method use of animal cardiac ultrasonic evaluate left ventricular contraction in back 30 days and the diastole end.And put through carotid artery with the Muller conduit and to manage left ventricle, assessment left indoor pressure, dp/dt.MicroPET observes the myocardial blood flow perfusion: with 18F-FDG is pet imaging agent;, transplanting back 30 day animal preceding to transplanting carries out FDG-PET and detects; Observe the FDG metabolism aggregation extent of each wall of ventricle; And set the local region of interest (ROI) of each wall of cardiac muscle, the local radiation of quantitatively determined ROI aggregation extent, the FDG metabotic change before and after relatively transplanting.Following Fig. 3 of result and shown in Figure 4.
Fig. 3 is transplantation experiments result in the body; The U-18496 of 5 μ M is induced back 72 hours in utero film stem cell (being the functional myocardial cell that the present invention induces the differentiation gained) treatment heart stalk mouse; The ultra detected result of 30 right overheads is as shown in Figure 3; Experimental group each item parameters of left ventricular function has in various degree than control group to be improved, the most remarkable with IVSTd.The IVSi-IVSTd, LVPWi-LPWT, LVIDi-left ventricular interior diameter, s-systole, d-diastole.
Fig. 4 is transplantation experiments result in the body; The U-18496 of 5 μ M is induced back 72 hours in utero film stem cell (being the functional myocardial cell that the present invention induces the differentiation gained) treatment heart stalk mouse; The ultra detected result of 30 right overheads is as shown in Figure 4, and experimental group each item parameters of left ventricular function has in various degree than control group to be improved.Chamber, FS-left side major axis LVFS, the EF-LVEF.
3), transplanting back stem cell viability estimates: transplant the back 60 days active strong positives (of Fig. 5) that show of animal immune group detection human mitochondrion.Among Fig. 5: A: control group, B: experimental group.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (1)
1. the method for utilizing film stem cell in utero to induce the differentiation function myocardial cell is characterized in that may further comprise the steps:
1), in utero separation and Culture of film stem cell and amplification:
Menses are after the germicidal treatment that contains antibiotic disinfecting liquid, centrifugal, remove supernatant; Add the ChangShi collective media then and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate; Cultivate and change liquid after 5～7 days and remove not attached cell; In case when cell grows to 75～85% degree of converging, promptly adopt trysinization to go down to posterity;
Be followed successively by:
1., menses are collected:
Hank ' s the balanced salt solution of 20ml is set in collection tube, and adds following composition to following concentration: vancomyein 60～100 μ g/mL, Cephalexin Monohydrate Micro/Compacted 150～350 μ g/mL, kantlex 50～150 μ g/mL, qingfengmeisu qiong 80～160 μ g/mL, amphotericin B 2～3 μ g/mL and 300～500 units of heparin sodium; With this as containing antibiotic disinfecting liquid;
The menses of 15～20ml are put into above-mentioned collection tube, preserved 24～48 hours down in 0～4 ℃ low temperature; Centrifugal then, remove supernatant;
2., former be commissioned to train foster:
A), the ChangShi collective media of getting 6～10ml joins step 1. in the gains;
B), with steps A) gains put into culturing bottle and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate; Cultivate after 5～7 days full dose and change liquid; Again with upright 3～8 minutes of above-mentioned culturing bottle, thereby make not adherent cell landing to culturing bottle bottom, remove the substratum in the culturing bottle with transfer pipet;
C), at step B) the ChangShi collective media that adds 2～4mL in the culturing bottle of gained carries out rinse, removes the substratum in the culturing bottle with transfer pipet then;
D), at step C) add the ChangShi collective media of 6～10mL in the culturing bottle of gained, place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate;
The compound method of said ChangShi collective media is following: under aseptic condition; The Chang B base fluid, the Chang C base fluid of 10～30mL, the 5～15mL penicillin/streptomycin that in sterile chamber, add 650mLMEM-alpha substratum, 160～200mL are two anti-; Contain 10 in the two anti-solution of every ml penicillin/streptomycin; The Streptomycin sulphate of the sodium benzylpenicillin of 000U and 10,000 μ g, the concentration of 5～15mL are the L-glutaminate of 200mM, the foetal calf serum of 130～170mL; Fully mixing is put into 0～4 ℃ of refrigerator and is preserved for use behind the high pressure steam sterilization;
3., passage is cultivated:
In case step when 2. former generation, cultured cells grew to 75～85% degree of converging, promptly adopts trysinization to go down to posterity; Specific as follows:
A) in case step when 2. former generation, cultured cells grew to 75～85% degree of converging, is removed nutrient solution, the PBS washings with no calcium ions and magnesium ions washs then;
B), add 2～4ml pancreatin, place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in hatched 4～6 minutes;
C), the ChangShi collective media of adding 4～6ml makes the pancreatin inactivation;
D), soft piping and druming, attached cell come off and become unicellular state;
E), go down to posterity by 1: 8 ratio;
F), the ChangShi collective media that in the cell suspension of every 1ml, adds 5～7ml; Place 4～6%CO then
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate;
2), external evoked differentiation:
With above-mentioned steps 1) gained the 4th～6 generation the upgrowth situation good cell with trysinization; When microscopically sees that it is that the DMEM substratum of 13～17% foetal calf serums stops digestion to contain volumetric concentration when circular that cell becomes single; Centrifugal, the cell precipitation of gained cleans with PBS;
Add inducing culture in the cell after above-mentioned PBS cleans and place 4～6%CO
2, 94～96% saturated humidities 36～38 ℃ of incubators in cultivate; Per 1 * 10
5Add 0.8～1.2ml inducing culture in the individual cell, said inducing culture is: the U-18496 that in serum-free DMEM substratum, contains 1～10 μ M; Incubation time is 68～76 hours, gets the function myocardial cell.
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| CN1397646A (en) * | 2001-07-13 | 2003-02-19 | 上海杰隆生物工程股份有限公司 | Process for cloning myocardial cell, heart tissue and heart |
| CA2461121A1 (en) * | 2001-09-20 | 2003-04-03 | Kyowa Hakko Kogyo Co., Ltd. | Multipotent stem cell in the interstitial tissues of skeletal muscle |
| EP1876233A1 (en) * | 2005-01-24 | 2008-01-09 | Japan Health Sciences Foundation | Cells capable of differentiating into cardiac muscle cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1397646A (en) * | 2001-07-13 | 2003-02-19 | 上海杰隆生物工程股份有限公司 | Process for cloning myocardial cell, heart tissue and heart |
| CA2461121A1 (en) * | 2001-09-20 | 2003-04-03 | Kyowa Hakko Kogyo Co., Ltd. | Multipotent stem cell in the interstitial tissues of skeletal muscle |
| EP1876233A1 (en) * | 2005-01-24 | 2008-01-09 | Japan Health Sciences Foundation | Cells capable of differentiating into cardiac muscle cells |
Non-Patent Citations (2)
| Title |
|---|
| NAOKO HIDA et al.Novel Cardiac Precursor-Like Cells from Human Menstrual.《STEM CELLS》.2008,第26卷1695-1704. * |
| 王昕荣等.子宫内膜干细胞研究进展.《中国优生与遗传杂志》.2008,第16卷(第1期),1-3. * |
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