A kind of simple separation of Human plactnta myofibroblast mother cell and authentication methodInvention field
The present invention relates to be separated into myofibroblast from Human plactnta decidua face and myofibroblast mother cell is identifiedMethod.
Background technology
Placenta is considered as most attractive " adult stem cell ", can be broken up under suitable inner or in vitro environmentFor various kinds of cell such as myocyte, liver cell, Gegenbaur's cell, cartilage cell, fat cells.Due to placenta stem-cell material source phaseTo convenient, it is easy to separation and purifying, still repeatedly there are stem cell properties after passage amplification, and the spy of immunological rejection is not presentProperty；Therefore in recent years turn into stem-cell research in focus, immunological diseases treatment, HSCT, organizational project,The fields such as genetic engineering have preferable application prospect.
Myofibroblast mother cell refers to the muscular tissue precursor containing myofilament in endochylema, from mesodermal stem cell,First differentiated in embryo development procedure by mesenchymal cell, then divide, be fused into multinuclear muscle fibre, form flesh smallPipe, is further divided into the Skeletal Muscle Cell of maturation.
A kind of Oliver C isolated cells from deciduate placenta, the cell can express smooth muscle actin and haveSimilar to the submicroscopic structure of myofibroblast；The myofibroblast that Zuzana Strakova are separated from placenta, in spyDetermine under culture medium induction, fat cell, Gegenbaur's cell can be divided into, it was demonstrated that the multipotency of placenta myofibroblast,Theories integration is provided for its research in regenerative medicine field；Ludovic Micallef research find myofibroblast byPlayed an important role in terms of the reparation of injured tissue scar and functional rehabilitation；And Ori Eyal in vitro placenta myofibroblasts inThe inducible factor co-incubation such as estrogen, it was demonstrated that prolactin can prevent the decidua of uterine cell by the mechanism self secretedChange.The characteristics of myofibroblast is exclusive and multipotency, and its material source are abundant, are that cell model is set up and regenerative medicineResearch provide good material.
However, the domestic separation for placenta fibroblast at present is rarely reported, it is also main to concentrate even if having been reported thatIn terms of the separation of MSC or stem cell, such as A of CN 102586184（Chinese Patent Application No. 201210044638.6, publication dateJuly 18 2012 phase）The invention of entitled " method for setting up placental mesenchyme stem cell library " is disclosed；CN 101395266 A（Chinese Patent Application No. 200680053575.3, March 25 2009 publication date）.
In the world in the report of placenta fibroblast, separation method all concentrates on enzyme digestion, but is digestingDuring have the participations of many enzymes：As University of Illinois of U.S. Zuzana Strakova separate placental fibroblasts when,Fiber finer is obtained into using clostridiopetidase A, deoxyribonuclease, hyaluronidase, four kinds of enzyme hybrid reaction digestion of pronaseBorn of the same parents, the participation of a variety of enzymes will certainly cause the unnecessary waste of complexity and resource in experimental procedure.
Myofibroblast mother cell is by Derived from Mesenchymal Stem Cells, then it should remain the one of mescenchymal stem cellA little characteristics, comprising adherent growth characteristic, the expression of specific cells surface antigen marker thing, into fat, the differentiation in skeletonization directionAbility etc..
Myofibroblast mother cell is one kind of adult stem cell, can self-renewing and can specialization formation composition suchThe cell of type tissue, it may express one or more kinds of multipotential stem cell factors.
Desmin（Desmin）It is a kind of intermediate filament protein, is widely present in the smooth muscle cell of big blood vessel, passes through identificationThe expression of desmin, can verify the myogen characteristic of cell.And have document report with the purifying of myofibroblast mother cell, tie eggWhite expression also progressively strengthens.
The content of the invention
It is an object of the invention to provide it is a kind of it is simple and practical from deciduate placenta layer be largely separated into myofibroblastThe method of method and the myofibroblast mother cell of identification separation.
The placenta fibroblast separation method of the present invention is that the blood transfusion group in decidua face is rejected using scalpel and tweezersThe tissue such as knit, only leave and take and sloughs off face close to fetus, simply use collagenase digesting tissue, its specific steps includes：
1st, fresh human placenta tissue is taken, the tissue of about 5 cm length and width is taken in placenta centre with scalpel；It is clear with physiological salineWash away except blood in tissue；
2nd, the vascular tissue on amnion and decidua basalis is divested with tweezers scalpel, fetus face decidua tissue is left and taken；
3rd, placenta baby face decidua tissue 75% alcohol quick wash 10 seconds；
4th, after 75% alcohol disinfecting is washed with brine twice；
5th, fetus face decidua tissue is cut into 0.5 cm or so fragment with scalpel；
6th, with 37 DEG C of digestion 16-18 h of DMEM digestive juices containing 1% clostridiopetidase A I with fragment of tissue in equal volume, digest moltenWithout stopping digestion after placenta tissue piece in liquid；
7th, mononuclearcell is collected by centrifugation, is trained with the dual anti-DMEM/F12 of 10% hyclone and 200 unit mycillinsSupport base and cell is resuspended；
8th, in 37 DEG C, 5% CO2, 48 h are cultivated under saturated humidity in incubator, induce adherent；
9th, after cell formation monoclonal, picking monoclonal cell culture, when cell fusion degree reaches 90%, 0.25% pancreatinDigestion, produces placenta myofibroblast mother cell.
The present invention also passes through flow cytometer detection cell surface antigen and cell cycle, cell multinomial differentiation potential experimental identification pointFrom the interstitial cell characteristic of gained cell after purification, by the dryness of the Testing and appraisal cell of the multipotent stem cells factor, pass throughThe myogenicity of the expression identification cell of morphological observation and cell desmin.
10th, under normal condition culture of isolated myofibroblast, observation cell growth feature and form in incubationLearn change；
11st, the third generation cell of culture, flow cytometer detection cell surface antigen CD90, CD44, CD29, CD45, HLA- are takenDR, while carrying out cell cycle detection；
12nd, the third generation cell of culture is taken to carry out the multipotential stem cell factor OCT4, SOX2, Nanog detection of expression；
13rd, the third generation cell of culture is taken to carry out the detection of expression of myogenicity desmin；
14th, the third generation cell of culture is taken to carry out to skeletonization, into the detection of Adipose Differentiation.
The present invention rejects the tissues such as the blood transfusion tissue in decidua face using scalpel and tweezers, and that only leaves and takes nearly fetus sloughs off face,Collagenase digesting tissue is simply used, unnecessary tissue is rejected, enzyme usage amount can not only be reduced, cost is reduced, when reducing digestionBetween, while avoiding the introducing of a large amount of heteroproteose cells, make cell purification more convenient.Gained primary cell can be made in shorter culturePurifying is purpose cell in time.
The present invention is using mechanical dispersion method and a kind of one-step method of enzymic digestion, and this method is simple to operate, and short time consumption is shorter, makesIt is the separating effect that can reach with a small amount of digestive ferment, is conducive to the scale of later stage such separation method to amplify.Use simultaneouslySpecial reagent pre-treatment tissue samples, in the case where samples sources do not confirm, substantially reduce the risk of pollution.
The authentication method combination for the separating obtained muscle fibril mother cell that the present invention is provided, the one-stop identification for solving cellProblem, and authentication method and credible result, effectively.
Brief description of the drawings
Fig. 1：Cellular morphology：
A：Individual cells photo
B:Cell monoclonal photo
C:Cell fusion degree reaches 90% photo
Fig. 2：Flow cytometry cell phenotype
Fig. 3：The Flow cytometry cell cycle
Fig. 4：The identification detection of cell Gegenbaur's cell
Fig. 5：Cell lipoblast is detected
Fig. 6：Cell multipotential stem cell factors check, be respectively from left to right：OCT4, SOX2, Nanog RT-PCR electrophoresisPhoto
Fig. 7：Cell desmin expression is detected
Embodiment
The invention discloses a kind of separation of Human plactnta myofibroblast mother cell and authentication method, those skilled in the artPresent disclosure can be used for reference, technological parameter realization is suitably modified.In particular, all similar replacements and change pairIt is it will be apparent that they are considered as being included in the invention for those skilled in the art.
In order that those skilled in the art are better understood from technical scheme, with reference to specific specific embodimentThe present invention will be further described in detail.
The placenta myofibroblast mother cell of embodiment one is separately cultured
The fresh human placenta tissue of natural labor baby is taken in sterile biological safety cabinet（Within 12 hours）, with scalpel in placentaCentre takes the tissue of about 5 cm length and width, and 250 mL brines 2 times extrude placenta tissue, make the blood in tissue with strengthLiquid is fully washed out, and the blood transfusion tissue on amnion and decidua basalis is divested with tweezers and scalpel；Leave and take fetus face decidua tissue, 250Fetus face decidua tissue, the wide size of 1.5 cm length, 0.5 cm is cut into scalpel by mL brines 1 time；75% wineSmart Rapid Cleaning is sterilized 10 seconds, then with 250 mL brines 2 times；With with fragment of tissue in equal volume contain 1% clostridiopetidase A I37 DEG C of DMEM digestive juices digestion 16-18 h, without stopping digestion after placenta tissue piece in digestion solution；By postdigestive tissueLiquid average mark is loaded in 2 50 mL centrifuge tubes, and addition physiological saline to 50 mL, 2300 rpm, 10 min of centrifugation are collected by centrifugationMononuclearcell；Brine 2 times, 2000 rpm centrifuge 10 min, with containing 10% hyclone and the blue or green chain of 200 unitsMononuclearcell is resuspended in the dual anti-DMEM/F12 culture mediums of mycin；In 37 DEG C, 5% CO2, cultivate under saturated humidity.
Primitive cell culture after 48 hours full dose change liquid, remove non-adherent cell, add fresh culture, wait to be dispersed in adherentAfter cell formation monoclonal, picking monoclonal cell is individually cultivated, and is obtained placenta fibroblast, is treated that cell fusion degree reachesWhen 90%, pancreatin had digestive transfer culture.
The placenta myofibroblast mother cell Morphological Identification of embodiment two
The myofibroblast separated according to the method for embodiment one is cultivated, it is visible under microscope to be dispersed in after inducing adherent 2 daysFusiformis attached cell, culture supernatant is all outwelled, while remove not adherent contaminating cell, after culture 5-7 days, it is seen thatFormed into radial monoclonal cell, cultivated with cell scraper scraping monoclonal cell in 12 orifice plates, treat that cell quantity reachesTo 5 × 106During left and right, for follow-up other identifications.
Micro- Microscopic observation is visible（Fig. 1）Cell is in fusiformis or spindle, polarity, small volume, with incubation timeIncrease, cell volume becomes big, and fusion growth, these features meet the growth characteristic and Morphological Characteristics of myofibroblast.
The placenta myofibroblast mother cell surface marker of embodiment three is identified
The 3rd generation cell for taking different placenta sources to be cultivated according to embodiment one respectively, Flow cytometry cell surfaceMark, observes the change of different placenta derived cell surface markers.Cell is collected in digestion, and 1 × 10 is taken after counting6It is individual, PBS washingsOnce, 1500 rpm, centrifuges 10 min；Supernatant is abandoned, 200 uL are remained, blows and beats and mixes cell, the CD90 of addition FITC marks,Each 10 uL of CD44 antibody of CD29, HLA-DR and PE mark of CD45 and APC marks, and 1 pipe is set as blank control, at 4 DEG CLower lucifuge reacts 30 min, and PBS washed once, 1500 rpm, centrifuges 10 min, abandons supernatant, remains 200 uL, upper machine testing.
Flow cytometer detection result figure 2 shows different placenta derived cell CD90（99% or so）、CD44（98% or so）、CD29（97% or so）The expression of three kinds of antigens is above more than 95%, CD45（0.7% or so）、HLA-DR（0.4% or so）Two kinds of antigensExpression be below 2%.Cell surface marker result shows that cell has interstitial fibers cell characteristics.
The example IV placenta myofibroblast mother cell cell cycle is detected
Cell is cultivated according to embodiment one, when cell growth degrees of fusion is 90%, cell cycle detection is carried out.Digestion is receivedCollect cell, 1 × 10 is taken after counting6Individual, PBS washed once, and abandon supernatant, and -20 DEG C of ethanol for adding 70% fixes 2 h, and centrifugation is goneIt except ethanol, washed once with PBS, add the uL of RNaseI 5 (20 mg/mL), 37 DEG C are reacted 30 min, and PBS washed once, plusEnter propidium iodide（PI, 50 ug/mL）0.5 uL, 4 DEG C of lucifuges react 30 min, upper machine testing DNA content.
Cell cycle analysis result figure 3 shows, in the G0/G1 phases, the S phases, G2M phase cell proportions average out to 93.72%,3.7%、2.57%.Prove that the placenta myofibroblast mother cell most cells in culture are in G0/G1 quiescent stages, it is only a small number of thinBorn of the same parents are in active S proliferation periods, with typical stem cell increasing feature.
The placenta myofibroblast mother cell Osteoblast Differentiation potential of embodiment five is identified
3 generation above cells are cultivated according to embodiment one, single cell suspension is made in digestion, according to 2 × 104Individual/cm2DensityUsing containing 10% hyclone and the dual anti-DMEM/F12 culture medium inoculateds of 200 unit mycillins in having overlay I type Collagen type-IsIn 6 orifice plates, in 37 DEG C, 5% CO2, cultivate under saturated humidity, after cell fusion degree reaches 50%, change and contain dexamethasone 0.1μM, ascorbyl phosphate 50 μ g/mL, β-mM of phosphoglycerol 10 fresh culture Fiber differentiation, change liquid once within 3 days, altogetherInduction 3 weeks, cell, Alizarin red staining, the formation of the micro- extracellular calcium Stromal Precipitation of Microscopic observation are fixed using 95% ethanol.
Observation Fig. 4 results show that cellular matrix substantially has calcium Stromal Precipitation, show that cell is converted to Gegenbaur's cell, haveThere is the characteristic of interstital stem cell.
The placenta myofibroblast mother cell of embodiment six is identified into fat differentiation potential
3 generation above cells are cultivated according to embodiment one, single cell suspension is made in digestion, according to 1 × 105Individual/cm2DensityUsing containing 10% hyclone and the dual anti-DMEM/F12 culture medium inoculateds of 200 unit mycillins in having overlay I type Collagen type-IsIn 6 orifice plates, in 37 DEG C, 5% CO2, cultivate under saturated humidity, after cell fusion degree reaches 80%, change the μ containing dexamethasone 1M, 0.5 mM of mM, IBMX of indocin 0.1, the μ g/ml of insulin 5 fresh culture Fiber differentiation, change liquid once in 3 days, lure altogetherLead 3 weeks, 10% paraformaldehyde fixes cell, oil red dyeing.
Observation Fig. 5 results show that the fat that cell is produced dyes red by specificity, show that cell turns to fat cellChange, the characteristic with interstital stem cell.
The placenta myofibroblast mother cell multipotential stem cell factor expression of embodiment seven is identified
The 3rd generation cell for taking different placenta sources to be cultivated according to embodiment one, cell concentration reaches 5 × 106It is individual, extract cellTotal serum IgE, reverse transcription obtain cDNA, using cDNA as template, using following primer carry out RT-PCR, detection OCT4, SOX2 andThe expression situation of Nanog genes, the operation is carried out with kit specification, and primer sequence is as shown in table 1.
Table 1：RT-PCR primer sequence and its characteristic
In Fig. 6, it is respectively from left to right：OCT4, SOX2, Nanog RT-PCR electrophoresis photographs, as a result show, different tiresDisk source myofibroblast mother cell expression two kinds of multipotential stem cell factors of OCT4, Nanog, do not express or low expression SOX2 becauseSon.This experiment absolutely proves the characteristics of placenta myofibroblast mother cell has stem cell.
The placenta myofibroblast mother cell desmin expression of embodiment eight is identified
The 3rd generation cell for taking different placenta sources to be cultivated according to embodiment one, cell concentration reaches 5 × 106It is individual, extract cellTotal serum IgE, reverse transcription obtains cDNA, using cDNA as template, carries out RT-PCR using following primer, the operation illustrates with kitBook is carried out, and primer sequence is as shown in table 2.
Table 2：RT-PCR primer sequence and its characteristic
Fig. 7 is Desmin electrophoresis pictures, as a result shows that placenta myofibroblast mother cell expresses desmin, it was demonstrated that it is derived fromMusculature.