CN105755098A - Combined identification method for induced differentiation of hair follicle stem cells into vascular endothelial cells - Google Patents

Combined identification method for induced differentiation of hair follicle stem cells into vascular endothelial cells Download PDF

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CN105755098A
CN105755098A CN201610160138.7A CN201610160138A CN105755098A CN 105755098 A CN105755098 A CN 105755098A CN 201610160138 A CN201610160138 A CN 201610160138A CN 105755098 A CN105755098 A CN 105755098A
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vascular endothelial
endothelial cell
hair follicle
cell
follicle stem
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全仁夫
杜伟斌
郑宣
李强
曹国平
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HANGZHOU CITY XIAOSHAN DISTRICT TRADITIONAL CHINESE MEDICAL HOSPITAL
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Abstract

The invention provides a combined identification method for induced differentiation of hair follicle stem cells into vascular endothelial cells. The hair follicle stem cells are rat hair follicle stem cells, and the combined identification method comprises immunofluorescence detection of vascular endothelial cell markers, flow detection of the vascular endothelial cell markers, ultra-micro form detection of vascular endothelial cells and in-vitro function detection of the vascular endothelial cells. The four identification methods are combined, the effectiveness of induction of the hair follicle stem cells into the vascular endothelial cells is reflected from different perspectives, and the rough position, the existence number, the existence morphology and functional characteristics of the formed vascular endothelial cells are represented systematically and comprehensively.

Description

A kind of hair follicle stem cells is induced to differentiate into the associating authentication method of vascular endothelial cell
Technical field
The present invention relates to the biological detecting method for skin tissue engineering field, especially relate to a kind of hair follicle stem cells and be induced to differentiate into the associating authentication method of vascular endothelial cell.
Background field
Skin is as the organ of human body maximum, there is sensation, regulation body temperature, secrete and drain, prevent the multiple effects such as moisture evaporation, the most topmost function is barrier the stablizing with the interior environment of maintenance as human body with external environment, and it is also immune important component part simultaneously.Along with socioeconomic development, especially as China's manufacturing industry and the prosperity and development of handicraft of " world's factory ", the various wounds relating to defect of skin are particularly burnt, crush injury, incised injury etc. also get more and more.Wherein, the widespread skin defect that wound causes frequently results in the most serious physical disabilities, even dead.The standard treatments of clinical treatment defect of skin is auto-skin grafting at present, and owing to having without immunological rejection, survival rate high, it applies extremely wide clinically, and achieves good curative effect.But the shortcoming of auto-skin grafting is the most clearly, owing to being autologous drawing materials, itself it is exactly the damage again to patient, patient suffering has been significantly greatly increased, and has had generation skin donor site to infect, the risk of the complication such as disunion.More seriously, for above-mentioned large skin defect, often cause wound repair difficulty owing to lacking the autologous skin being enough available for transplanting, had a strong impact on treatment process, even resulted in death.
Although the most existing several artificial skin products above are available for selection of clinical, the most really promote the clinical treatment to large skin defect patient.But, according to the problem run into during our clinical application for many years, report in conjunction with document, it is considered that artificial skin transplanting at present there is also many problems, the most even include may causing treating " hard defects " proved an abortion: 1. owing to above artificial skin product does not all have Angiogenesis ability, cause the artificial skin transplanted not have vascular system to supply nutrition, so that artificial skin is susceptible to necrosis, make graft failure.2. contained in artificial skin product various variant cells easily cause immunological rejection, often occur the cutaneous necrosis transplanted clinically, come off, and serious the severe complications such as systemic immune response even occurs, and it is possible to spread disease.The most current all of artificial skin products all can only recover part anatomical structure and the physiological function of normal skin, and cannot regenerate the cutaneous appendages structure with critical function, such as: blood vessel, hair, sweat gland etc..
1997, (Asahara T, the et such as Asahara Al. 1997) there is the precursor that can be divided into ECs, by its named EPCs in finder's peripheral body first.EPCs is not only involved in brephic blood vessel and occurs, and plays a significant role in the angiogenic growth of adult is grown.But EPCs is primarily present in marrow and peripheral blood, its source and quantity are very limited, and the cell purity of acquisition is relatively low, multiplication capacity is poor, it is possible to the efficiency being successfully divided into ECs is lower, big (Gehling UM, the et al. 2000 of the damage to patient and the puncture of multiple positions is drawn materials；Casamassimi A, et al. 2007).The very important role additionally, ECs also deduces in the generation and forming process of blood vessel.But, the autologous source of drawing material of ECs is limited, easily causes the narrow, inaccessible of bridge blood vessel after drawing materials, and easily increases the weight of the damage to patient, and the most aging through the primary vascular endothelial cell being separately cultured, and the cell cycle is short, and multiplication capacity limits to very much.These all limit directly obtaining and application of ECs.Then the method considering to utilize stem cell to induce.
Research shows, hair follicle stem cells (Hair Follicle Stem Cells, HFSCs) is the stem cell that a class is present in raw coal bunker knuckle portion, have the features such as undifferentiated property, self and in-vitro multiplication ability are strong (Cotsarelis G, et al. 1990, Cotsarelis G. 2006).The HFSCs of in vitro culture shows high clonality, has the highest regeneration potential (Rochat A, et al.1994).It derives from skin, hair, and quantity is the most considerable, and does not has serious complication, non-immunogenicity, is available for autotransplatntation, is one of source of human stem cell being easiest to acquisition.The current research result that Stelios etc. deliver proves, the most successfully hair follicle stem cells differentiation and development is generated new vascular system.And vascular endothelial growth factor 165(VEGF165) it is one of 5 kinds of hypotypes of vascular endothelial growth factor, its activity is the strongest, and distribution is the widest, is the Main Subtype played a role in VEGF body.Study around the revascularization gene therapy centered by VEGF165 in recent years and become domestic and international study hotspot.
Summary of the invention
The associating authentication method being to provide a kind of hair follicle stem cells to be induced to differentiate into vascular endothelial cell is solved the technical problem that, to this end, the present invention provides following technical scheme required for the present invention:
A kind of hair follicle stem cells is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterized in that, described hair follicle stem cells is rat hair follicle stem cell, and described associating authentication method includes the Immunofluorescence test of vascular endothelial cell label, the flow cytometer detection of vascular endothelial cell label, the ultra micro Morphology observation of vascular endothelial cell and the external Function detection of vascular endothelial cell；
First, carry out the Immunofluorescence test of vascular endothelial cell label, for representing the expression position of endothelial cell marker thing qualitatively, be induced to differentiate into the situation of vascular endothelial cell for substantially expressing hair follicle stem cells；
Secondly, carry out the flow cytometer detection of vascular endothelial cell label, for detecting the positive rate of endothelial cell marker thing quantitatively；
Again, carrying out the ultra micro Morphology observation of vascular endothelial cell or external Function detection, described ultra micro Morphology observation is for proving the existence of endothelial cell distinctive W-P corpusculum, and described external Function detection is for detecting the function of endothelial cell.
Further, described vascular endothelial cell label is CD31 and VE-cadherin.
Further, the Immunofluorescence test of vascular endothelial cell label comprises the steps:
(1) cell after induction is with common pancreatin-PBS(1:3) dilution rinses 3 times, then is centrifuged with TrypLE Select (1X) trypsase replacement enzymic digestion 5 min, 1200 turns/5 min；
(2) it is inoculated in 1 × 105/ hole concentration and is coated on 37 DEG C, 5% CO with Matrix glue in advance2Incubator in 30 On the slide of min, adhere-wall culture 1 d；
(3) suck inducing culture, wash 3 times with 1XPBS；
(4) 4% PFA-PBS fixes 10 min at room temperature；
(5) 1XPBS washes 3 times, and 5 Min/ time；
(6) 1% BSA-PBS closes 30 min at room temperature；
(7) 1XPBS washes 2 times, and 5 Min/ time；
(8) hatching one resists, and the every anti-dilution of 15-25UL mono-, 4 DEG C overnight；
(9) 1XPBS washes 3 times, and 5 Min/ time；
(10) hatching two resists, lucifuge 1 h；
(11) 1XPBS washes 3 times, and 5 Min/ time；
(12) DAPI contaminates core, 10 min under room temperature；
(13) 1XPBS washes 2 times, and 5 Min/ time；
(14) mounting, takes pictures under laser co-focusing.
Further, the flow cytometer detection of vascular endothelial cell label comprises the steps:
(1) cell after induction rinses 3 times with common pancreatin-PBS dilution, then it is centrifugal to substitute enzymic digestion 5 min, 1200 turns/5 min with TrypLE Select (1X) trypsase, and 1XPBS washes 2 times afterwards；
Cell is blown and beaten into single suspension by (2) 80% methyl alcohol gently, and room temperature fixes 5min, and 1200 turns/3min is centrifugal abandons supernatant；
Cell is blown and beaten into single suspension by (3) 0.1% PBST gently, and room temperature stands 20 Min, 1200 turns/3 Min is centrifugal abandons supernatant；
(4) 5% Cell is blown and beaten into single suspension by BSA-PBS gently, and upper shaking table closes that 30 min, 1200 turns/3 min are centrifugal abandons supernatant；
(5) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(6) every fluid-guiding type pipe 100UL(1X Annexin-binding buffer), 1 × 106cell；
(7) adding one anti-, lucifuge stands 30 min；
(8) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(9) adding two anti-, lucifuge stands 30 min；
(10) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(11) 1XPBS every fluid-guiding type pipe 500UL, upper machine flow cytometer detection.
Further, the transmission electron microscope that ultra micro Morphology observation the is vascular endothelial cell detection of vascular endothelial cell, the detection of described transmission electron microscope comprises the steps:
(1) 2.5% The cell of centrifugal for digestion gained is fixed 4 hours or overnight by glutaric acid-PBS solution；
(2) 0.1 MPBS rinses 2 times/10-15 min；
(3) 1% osmic acids are fixed on 1 h at 4 DEG C；
(4) DdH2O rinses 2 times/10-15 min；
30 min are fixed/dyeed to (5) 2% acetic acid uranium；
(6) 50%, 70%, 90%, 100% Gradient elution using ethanol 1 time/10-15 min；
(7) 100% 2 times/10-20 of acetone serial dehydration min；
(8) again through permeating, embed, being polymerized three processes；
(9) ultramicrotome section, acetic acid uranium-lead citrate dyeing, observe W-P corpusculum under transmission electron microscope.
Further, the external Function detection of vascular endothelial cell is that the external official jargon of vascular endothelial cell forms experiment, and described external official jargon forms experiment and comprises the steps:
(1) from-20 DEG C of refrigerators, take out Matrigel matrigel and be placed on 4 DEG C of refrigerator overnight, make liquid；
(2) Micro pipette tip and 24 orifice plates are in precooling on ice；
(3) in 24 orifice plates, 100 μ L Matrigel matrigels are injected in every hole, and careful shake is allowed to be uniformly distributed in each position in hole, and avoids producing bubble, and all operations must be carried out on ice；
(4) then 24 orifice plates are put into 37 DEG C, 5% CO2In incubator 30 min；
(5) cell after induction through over cleaning, digest, collect, by every hole 2 × 105/500 μ L is inoculated in covering In 24 holes of Matrigel matrigel, set 3 multiple holes altogether；
(6) continuing to cultivate, respectively after 2 h, 4 h, 6 h, under 100 times of fluorescence microscopes, every hole randomly selects 3 visuals field and takes pictures.
Being explained as follows of CD31, VE-cadherin and W-P corpusculum involved in the present invention:
CD31
CD31 is endothelial specific marker's thing of discovered in recent years, it is also called blood platelet/endothelial cell adhesion molecule (Platelet/Endothelial Cell Adhesion Molecule-1, PECAM-1), it is expressed in blood platelet, monocyte, especially endothelial cell junction, is primarily present in endothelial cell cell membrane and endochylema.Low cytometric analysis or Immunohistochemical Method detection CD31 can be used to sort purifying and identify endothelial cell.
VE-cadherin, has another name called CD144, is that the specific calcium of blood vessel endothelium sticks albumen, exclusively expresses the surface at vascular endothelial cell, and concentration is distributed in endothelial cell junction, is the main adhesion molecule of adhesion junction.Numerous studies show, it is requisite that blood vessel endothelium calcium sticks the albumen integrality to maintaining vascular endothelial cell polarity and blood vessel.
Corpusculum
1964, Weibel and Palade was found that a kind of rod-shaped cells device being about 1-6um, the most named Weibel-Palade corpusculum (Weibel-Palade body) in vascular endothelial cell, is called for short W-P corpusculum, was the distinctive a kind of organelle of endothelial cell.Nineteen eighty-two, Wagner et al. has arrived the W-P corpusculum club shaped structure with longer axis parallel in rule by transmission electron microscope observing, and this structure is wide by about 0.1 ~ 0.3um, is about 0.5 ~ 5um, and the factor Ⅷ related antigen vWF factor is i.e. produced by the secretion of this corpusculum.Owing to existing only in endothelial cell, discovery rate can only achieve 30 %, therefore it is considered to be the most special morphological indexes of endothelial cell.
A kind of hair follicle stem cells provided by the present invention is induced to differentiate into the associating authentication method of vascular endothelial cell, described associating authentication method includes the Immunofluorescence test of vascular endothelial cell label, the flow cytometer detection of vascular endothelial cell label, the ultra micro Morphology observation of vascular endothelial cell and the external Function detection of vascular endothelial cell, the Immunofluorescence test of vascular endothelial cell label expresses the formation of vascular endothelial cell qualitatively, the flow cytometer detection of vascular endothelial cell label expresses the formation of vascular endothelial cell quantitatively, the ultra micro Morphology observation of vascular endothelial cell shows the morphological indexes-W-P corpusculum that vascular endothelial cell is the most special；The external Function detection of vascular endothelial cell reflects the functional character of vascular endothelial cell；By conjunctive use these four authentication method, reflecting hair follicle stem cells induction from different angles is the validity of vascular endothelial cell, system and characterize in all directions formed vascular endothelial cell approximate location, there is quantity, the pattern that exists and the functional character being had.
Accompanying drawing explanation
Fig. 1 is that Primary rat hair follicle stem cells provided by the present invention climbs out of from Hair Follicle Bulge portion, in nest like, epithelioid cell, arrange tight 100 ×；
Fig. 2 is IV Collagen Type VI provided by the present invention screening P3 after purification for rat hair follicle stem cell, in typical paving stone shape 100 ×；
Fig. 3 is the P3 provided by the present invention flow cytomery for rat hair follicle stem cell, respectively integrin β_1 (A), integrin a6(B), the positive rate of keratin 15 (C)；
Fig. 4 is P3 after purification provided by the present invention for rat hair follicle stem cell after induction 7 days, vascular endothelial cell label CD31(A) and immunofluorescence dyeing result VE-cadherin(B)；
Fig. 5 is P3 after purification provided by the present invention for rat hair follicle stem cell after induction 7 days, vascular endothelial cell label CD31(A) and streaming positive rate testing result VE-cadherin(B)；
Fig. 6 is P3 after purification provided by the present invention for rat hair follicle stem cell after induction 7 days, the vascular endothelial cell peculiar structure W-P corpusculum figure under transmission electron microscope；
Fig. 7 is the P3 after purification provided by the present invention external official jargon formation experimental result picture for rat hair follicle stem cell after induction 7 days, under fluorescence microscope.
Detailed description of the invention
With specific embodiment, the present invention is described in further details below in conjunction with the accompanying drawings.
1. rat hair follicle stem cell separation, cultivate, purify and identify
The separation of 1.1 rat hair follicle stem cells
Take newborn 1 week old SD rat, put into after the beaker equipped with 75% ethanol is sterilized and take out at cervical dislocation after death.In super-clean bench, cutting short antenna with eye scissors and cut antenna portion skin, 75% ethanol rinses 1 time again, rinses 3 times with PBS afterwards.Then, after digesting 90min with 1% type Ⅳ collagenase and 1%Dispase enzyme mixation 37 DEG C, twice is washed with PBS.With 1ml syringe needle, hair follicle is taken off sheath under stereomicroscope, cut two ends, leave knuckle portion, Hair Follicle Bulge portion is inoculated in the most coated culture dish of paving, adds 1 ml complete medium.
Described complete medium composition is: 44 ml DMEM/F12 nutrient solutions, 5 ml KSR serum substitute, 500 μ l mycillin mixed liquors, 500 μ l Glus, 500 μ l nonessential amino acid, 20 ng/ml recombinant human epidermal growth factors, 10 ng/ml recombination human basic fibroblast growth factors, 50 μ l hydroxyl ethanols, 10 ng/ml hydrocortisones.
The cultivation of 1.2 rat hair follicle stem cells
At 37 DEG C, 5% CO2Cultivate 1 h under condition of culture, then be slowly added into 2 ml complete mediums and continue to cultivate 3 h, wait organize the most adherent after continue to be slowly added to 3 ml complete mediums, every 2-3 d changes liquid afterwards.Fig. 1
The purifying of 1.3 rat hair follicle stem cells
By 100 μ g/ml IV Collagen Type VI is coated in culture dish according to the amount of 3 ml/100 mm dish, and room temperature stands 1 h；The primary cell of 100 mm culture dishes TrypLE Select (1X) trypsase is substituted enzymic digestion, after centrifugal collecting cell, blows and beats into single cell suspension and be inoculated in culture dish, after 20 min, by the most adherent cell together with nutrient solution sucking-off；The complete medium culture of adherent cell, changes liquid in every 3 days；P2 is for repurity once.Fig. 2
The qualification of 1.4 rat hair follicle stem cells
Using flow cytometry: collect the 3rd generation cell, adjusting cell density is 1.0x106/ml, loads the EP pipe of 1.5 ml, and 1200 turns/3min is centrifugal abandons supernatant.Cell room temperature is fixed 5 min by 80% methyl alcohol；0.1% PBST puts 20 min by gentle and quiet for Cytology Lab；On 5% BSA-PBS, 30 min closed by shaking table；PBS washes 1 time, and 1200 turns/3min is centrifugal abandons supernatant.Every fluid-guiding type pipe 100UL(1X annexin-binding buffer), 1 × 106 cell.It is separately added into Integrin β 1-PE；Integrin α 6, CK15, antibody lucifuge hatch 30 min.Add fluorescein to mark two anti-lucifuges and hatch 30 min.PBS washes 1 time, and 1200 turns/3min is centrifugal abandons supernatant.PBS every fluid-guiding type pipe 500UL, upper machine flow cytometer detection.Fig. 3
2. rat hair follicle stem cell is induced to differentiate into vascular endothelial cell
The preparation of 2.1 inducing cultures
10ml FBS hyclone, 1000 μ l mycillin mixed liquors, 1000 μ l Glus, 1000 μ l nonessential amino acid, 10 ng/ml vascular endothelial growth factor 165,10 ng/ml recombination human basic fibroblast growth factors, 100 μ l hydroxyl ethanols, 10 ng/ml hydrocortisones are dissolved in 88 ml DMEM/F12 nutrient solutions, fixed the most molten after be 0.22 membrane filtration by specification, the inducing culture finally given is standby.
The induction differentiation of 2.2 vascular endothelial cells is cultivated
After hair follicle stem cells after to be purified reaches 60% ~ 70% growth fusion in culture dish, change above-mentioned complete medium into inducing culture and induce, within every 2 days, change a not good liquor.At 37 DEG C, 5% CO2Continue under condition of culture to cultivate.
The qualification of the vascular endothelial cell after 2.3 induction differentiation
Using morphological observation record, immunofluorescence staining, flow cytometer detection method, transmission electron microscope carry out index of correlation qualification to the cell after induction, have finally given surveyed index and have reached optimal when 7 days.
The Immunofluorescence test of vascular endothelial cell label
(1) cell after induction 7 days is with common pancreatin-PBS(1:3) dilution rinses 3 times, then is centrifuged with TrypLE Select (1X) trypsase replacement enzymic digestion 5 min, 1200 turns/5 min；
(2) it is inoculated in 1 × 105/ hole concentration and is coated on 37 DEG C, 5% CO with Matrix glue in advance2Incubator in 30 On the slide of min, adhere-wall culture 1 d；
(3) suck inducing culture, wash 3 times with 1XPBS；
(4) 4% PFA-PBS fixes 10 min(room temperatures)；
(5) 1XPBS washes 3 times, and 5 Min/ time；
(6) 1% BSA-PBS closes 30 min(room temperatures)；
(7) 1XPBS washes 2 times, and 5 Min/ time；
(8) hatching one anti-(1:100), the every anti-dilution of 15-25UL mono-, 4 DEG C overnight；
(9) 1XPBS washes 3 times, and 5 Min/ time；
(10) hatching two anti-(1:500), lucifuge 1 h；
(11) 1XPBS washes 3 times, and 5 Min/ time；
(12) DAPI dye core (1:2000), room temperature 10 min；
(13) 1XPBS washes 2 times, and 5 Min/ time；
(14) mounting, takes pictures under laser co-focusing.
Result shows,Hair follicle stem cells is after induction 7 days, and vascular endothelial cell characteristic markers CD31 and VE-cadherin is at intracellular positive expression.
The flow cytometer detection of vascular endothelial cell label
(1) cell after induction 7 days is with common pancreatin-PBS(1:3) dilution rinses 3 times, then is centrifuged with TrypLE Select (1X) trypsase replacement enzymic digestion 5 min, 1200 turns/5 min, and 1XPBS washes 2 times afterwards；
Cell is blown and beaten into single suspension by (2) 80% methyl alcohol gently, and room temperature fixes 5min, and 1200 turns/3min is centrifugal abandons supernatant；
(3) 0.1% Cell is blown and beaten into single suspension by PBST gently, and room temperature stands that 20 min, 1200 turns/3 min are centrifugal abandons supernatant；
(4) 5% Cell is blown and beaten into single suspension by BSA-PBS gently, and upper shaking table closes that 30 min, 1200 turns/3 min are centrifugal abandons supernatant；
(5) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(6) every fluid-guiding type pipe 100UL(1X Annexin-binding buffer), 1 × 106cell；
(7) adding one anti-(ratio is with reference to antibody specification), lucifuge stands 30 min；
(8) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(9) adding two anti-(ratio is with reference to antibody specification), lucifuge stands 30 min；
(10) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(11) 1XPBS every fluid-guiding type pipe 500UL, upper machine flow cytometer detection.
Result shows,Hair follicle stem cells is after induction 7 days, and the positive rate of endothelial cell characteristics label CD31 and VE-cadherin is respectively 65.1% and 95.9%.
The transmission electron microscope detection of vascular endothelial cell
(1) 2.5% Glutaric acid-PBS rushes liquid and the cell of centrifugal for digestion gained is fixed 4 hours or overnight；
(2) 0.1 MPBS rinses 2 times/10-15 min；
(3) 1% osmic acids are fixed on 1 h at 4 DEG C；
(4) DdH2O rinses 2 times/10-15 min；
30 min are fixed/dyeed to (5) 2% acetic acid uranium；
(6) 50%, 70%, 90%, 100% Gradient elution using ethanol 1 time/10-15 min；
(7) 100% 2 times/10-20 of acetone serial dehydration min；
(8) again through permeating, embed, being polymerized three processes；
(9) ultramicrotome section, acetic acid uranium-lead citrate dyeing, observe W-P corpusculum under transmission electron microscope.
Result shows,Hair follicle stem cells, after induction 7 days, under 17000X transmission electron microscope, can observe the club shaped structure i.e. endothelial cell distinctive structure W-P corpusculum of black, this corpusculum typically width about 0.1-0.3 in endochylema Um, is about 0.5-5 um.
The external official jargon of vascular endothelial cell forms experiment
(1) from-20 DEG C of refrigerators, take out Matrigel matrigel and be placed on 4 DEG C of refrigerator overnight, make liquid；
(2) Micro pipette tip and 24 orifice plates are in precooling on ice；
(3) in 24 orifice plates, 100 μ L Matrigel matrigels are injected in every hole, and careful shake is allowed to be uniformly distributed in each position in hole, and avoids producing bubble, and all operations must be carried out on ice；
(4) then 24 orifice plates are put into 37 DEG C, 5% CO2In incubator 30 min；
(5) cell after induction through over cleaning, digest, collect, by every hole 2 × 105/500 μ L is inoculated in covering In 24 holes of Matrigel matrigel, set 3 multiple holes altogether；
(6) continuing to cultivate, respectively after 2 h, 4 h, 6 h, under 100 times of fluorescence microscopes, every hole randomly selects 3 visuals field and takes pictures.
Result shows,Hair follicle stem cells is after induction 7 days, and external official jargon forms experiment display: intracellular after 2h initially form simple line；The line of intracellular formation after 4h increases, and preliminarily forms circulus；After 6h, cell forms more, complete official jargon spline structure.
Above-mentioned detailed description of the invention is used for illustrating the present invention; it is only the preferred embodiments of the present invention; rather than limit the invention; in the protection domain of spirit and claims of the present invention; the any modification, equivalent substitution and improvement etc. making the present invention, both fall within protection scope of the present invention.

Claims (6)

1. a hair follicle stem cells is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterized in that, described hair follicle stem cells is rat hair follicle stem cell, and described associating authentication method includes the Immunofluorescence test of vascular endothelial cell label, the flow cytometer detection of vascular endothelial cell label, the ultra micro Morphology observation of vascular endothelial cell and the external Function detection of vascular endothelial cell；
First, carry out the Immunofluorescence test of vascular endothelial cell label, for representing the expression position of endothelial cell marker thing qualitatively, be induced to differentiate into the situation of vascular endothelial cell for substantially expressing hair follicle stem cells；
Secondly, carry out the flow cytometer detection of vascular endothelial cell label, for detecting the positive rate of endothelial cell marker thing quantitatively；
Again, carrying out the ultra micro Morphology observation of vascular endothelial cell or external Function detection, described ultra micro Morphology observation is for proving the existence of endothelial cell distinctive W-P corpusculum, and described external Function detection is for detecting the function of endothelial cell.
A kind of hair follicle stem cells the most according to claim 1 is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterised in that described vascular endothelial cell label is CD31 and VE-cadherin.
A kind of hair follicle stem cells the most according to claim 1 is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterised in that the Immunofluorescence test of vascular endothelial cell label comprises the steps:
(1) cell after induction is with common pancreatin-PBS(1:3) dilution rinses 3 times, then is centrifuged with TrypLE Select (1X) trypsase replacement enzymic digestion 5 min, 1200 turns/5 min；
(2) it is inoculated in 1 × 105/ hole concentration and is coated on 37 DEG C, 5% CO with Matrix glue in advance2Incubator in 30 min slide on, adhere-wall culture 1 d；
(3) suck inducing culture, wash 3 times with 1XPBS；
(4) 4% PFA-PBS fix 10 min at room temperature；
(5) 1XPBS washes 3 times, 5 min/ time；
(6) 1% BSA-PBS close 30 min at room temperature；
(7) 1XPBS washes 2 times, 5 min/ time；
(8) hatching one resists, and the every anti-dilution of 15-25UL mono-, 4 DEG C overnight；
(9) 1XPBS washes 3 times, 5 min/ time；
(10) hatching two resists, lucifuge 1 h；
(11) 1XPBS washes 3 times, 5 min/ time；
(12) DAPI contaminates core, 10 min under room temperature；
(13) 1XPBS washes 2 times, 5 min/ time；
(14) mounting, takes pictures under laser co-focusing.
A kind of hair follicle stem cells the most according to claim 1 is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterised in that the flow cytometer detection of vascular endothelial cell label comprises the steps:
(1) cell after induction rinses 3 times with common pancreatin-PBS dilution, then it is centrifugal to substitute enzymic digestion 5 min, 1200 turns/5 min with TrypLE Select (1X) trypsase, and 1XPBS washes 2 times afterwards；
Cell is blown and beaten into single suspension by (2) 80% methyl alcohol gently, and room temperature fixes 5min, and 1200 turns/3min is centrifugal abandons supernatant；
Cell is blown and beaten into single suspension by (3) 0.1% PBST gently, and room temperature stands that 20 min, 1200 turns/3 min are centrifugal abandons supernatant；
Cell is blown and beaten into single suspension by (4) 5% BSA-PBS gently, and upper shaking table closes that 30 min, 1200 turns/3 min are centrifugal abandons supernatant；
(5) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(6) every fluid-guiding type pipe 100UL(1X annexin-binding buffer), 1 × 106cell；
(7) adding one anti-, lucifuge stands 30 min；
(8) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(9) adding two anti-, lucifuge stands 30 min；
(10) 1XPBS washes 1 time, and 1200 turns/3 min are centrifugal abandons supernatant；
(11) 1XPBS every fluid-guiding type pipe 500UL, upper machine flow cytometer detection.
A kind of hair follicle stem cells the most according to claim 1 is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterized in that, the transmission electron microscope that ultra micro Morphology observation the is vascular endothelial cell detection of vascular endothelial cell, the detection of described transmission electron microscope comprises the steps:
The cell of centrifugal for digestion gained is fixed 4 hours or overnight by (1) 2.5% glutaric acid-PBS solution；
(2) 0.1 MPBS rinse 2 times/10-15 min；
(3) 1% osmic acids are fixed on 1 h at 4 DEG C；
(4) ddH2O rinses 2 times/10-15 min；
30 min are fixed/dyeed to (5) 2% acetic acid uranium；
(6) 50%, 70%, 90%, 100% Gradient elution using ethanol 1 time/10-15 min；
(7) 100% acetone serial dehydration 2 times/10-20 min；
(8) again through permeating, embed, being polymerized three processes；
(9) ultramicrotome section, acetic acid uranium-lead citrate dyeing, observe W-P corpusculum under transmission electron microscope.
A kind of hair follicle stem cells the most according to claim 1 is induced to differentiate into the associating authentication method of vascular endothelial cell, it is characterized in that, the external Function detection of vascular endothelial cell is that the external official jargon of vascular endothelial cell forms experiment, and described external official jargon forms experiment and comprises the steps:
(1) from-20 DEG C of refrigerators, take out Matrigel matrigel and be placed on 4 DEG C of refrigerator overnight, make liquid；
(2) Micro pipette tip and 24 orifice plates are in precooling on ice；
(3) in 24 orifice plates, 100 μ L Matrigel matrigels are injected in every hole, and careful shake is allowed to be uniformly distributed in each position in hole, and avoids producing bubble, and all operations must be carried out on ice；
(4) then 24 orifice plates are put into 37 DEG C, 5% CO230 min in incubator；
(5) cell after induction through over cleaning, digest, collect, be inoculated in by every hole 2 × 105/500 μ L in 24 holes covering Matrigel matrigel, set 3 altogether and answer holes；
(6) continuing to cultivate, respectively after 2 h, 4 h, 6 h, under 100 times of fluorescence microscopes, every hole randomly selects 3 visuals field and takes pictures.
CN201610160138.7A 2016-03-21 2016-03-21 Combined identification method for induced differentiation of hair follicle stem cells into vascular endothelial cells Pending CN105755098A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109141959A (en) * 2018-11-15 2019-01-04 甘肃农业大学 A kind of potato tubers ultra-thin section production method for transmission electron microscope observing
CN113030046A (en) * 2021-03-08 2021-06-25 苏州大学附属第一医院 Method for dynamically monitoring nitric oxide release of endothelial cells in real time by using total internal reflection fluorescence microscopic imaging system
CN113376186A (en) * 2021-05-12 2021-09-10 赵静 High-precision intelligent erythrocyte folic acid detector and detection method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
何威 等: "微环境诱导骨髓间充质干细胞分化为内皮样细胞", 《医学研究杂志》 *
许志成 等: "毛囊干细胞体外诱导分化为血管内皮细胞的实验研究", 《组织工程与重建外科杂志》 *
高瑞金 等: "大鼠血管内皮细胞的分离、鉴定及传代培养", 《山东畜牧兽医》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109141959A (en) * 2018-11-15 2019-01-04 甘肃农业大学 A kind of potato tubers ultra-thin section production method for transmission electron microscope observing
CN113030046A (en) * 2021-03-08 2021-06-25 苏州大学附属第一医院 Method for dynamically monitoring nitric oxide release of endothelial cells in real time by using total internal reflection fluorescence microscopic imaging system
CN113376186A (en) * 2021-05-12 2021-09-10 赵静 High-precision intelligent erythrocyte folic acid detector and detection method thereof
CN113376186B (en) * 2021-05-12 2022-12-23 无锡科金生物科技有限公司 High-precision intelligent erythrocyte folic acid detector and detection method thereof

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