CN103173405A - Simple separation and identification method of human placenta myofibroblasts - Google Patents
Simple separation and identification method of human placenta myofibroblasts Download PDFInfo
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Abstract
The invention relates to a separation and identification method of human placenta myofibroblasts. The separation and identification method comprises the following steps of: mechanically crushing the decidual tissue of the placenta fetal surface, digesting the decidual tissue by DMEM digestive juice containing collagenase I and thereby separating the decidual tissue into individual karyocytes; cultivating the karyocytes by using a DMEM/F12 culture medium containing 10% of fetal calf serum under the conditions of 37 DEG C, 5% of CO2 and saturated humidity; inducing adherence to wall and completely replacing the liquid in 48 hours, removing cells not adhering to the wall, adding fresh culture medium, and after the cells adhering to the wall is mono-cloned, cultivating each monoclonal cell separately; and when the cells are fused by about 90%, performing pancreatin passage, thereby obtaining the myofibroblasts. The method further identifies the obtained myofibroblasts by the combined method of cell morphology, cell cycle, cell surface marker detection, cell adipogenesis and osteogenesis differentiative capacity detection, cell multipotential stem cell factor detection and cell desmin expression detection.
Description
Invention field
The present invention relates to the method that is separated into myofibroblast and the myofibroblast parent cell is identified from people's deciduate placenta face.
Background technology
Placenta is considered to the most attractive " adult stem cell ", can be divided into the various kinds of cell such as myocyte, liver cell, scleroblast, chondrocyte, adipocyte in suitable body or under external environment.Because the placenta stem-cell material source is relatively convenient, be easy to separate and purifying, still have the stem cell characteristic after the amplification of repeatedly going down to posterity, and do not have the characteristic of immunological rejection; Therefore become in recent years the focus in stem-cell research, had application prospect preferably in fields such as Immunological diseases treatment, hematopoietic stem cell transplantation, organizational project, genetically engineereds.
The myofibroblast parent cell refers to contain in endochylema the muscular tissue precursor cell of myofilament, derive from mesodermal stem cell, first differentiated by mesenchymal cell in embryo development procedure, then divide, be fused into the multinuclear myofiber, form sarcotubules, more further be divided into ripe Skeletal Muscle Cell.
Oliver C separates from deciduate placenta and obtains a kind of cell, and this cell can be expressed smooth muscle actin and the submicroscopic structure that is similar to myofibroblast is arranged; The myofibroblast that Zuzana Strakova separates from placenta, under defined medium is induced, can be divided into adipocyte, scleroblast, prove the multipotency of placenta myofibroblast, support for its research in the regenerative medicine field provides theoretical; Ludovic Micallef research finds that myofibroblast is playing an important role aspect the reparation of wounded tissue scar and functional rehabilitation; And Ori Eyal external placenta myofiber cell in inducible factor co-cultivation such as oestrogenic hormon, proved that prolactin can stop by the mechanism that the oneself secretes the decidua of uterine cell.The characteristics that myofibroblast is exclusive and multipotency, and material source is abundant are for cell model is set up and the research of regenerative medicine provides good material.
Yet, domestic separation for the placenta fibroblast at present rarely has report, even report is arranged, also mainly concentrate on the separation aspect of MSC or stem cell, as CN 102586184 A(Chinese patent application numbers 201210044638.6, open July 18 2012 date) openly be entitled as the invention of " method of setting up placental mesenchyme stem cell library "; CN 101395266 A(Chinese patent application numbers 200680053575.3, open March 25 2009 date).
In the world in the report about the placenta fibroblast, separation method all concentrates on enzyme digestion, but the participation of a lot of enzymes is arranged in digestive process: as the Zuzana Strakova of U.S. University of Illinois when separating the placenta inoblast, use collagenase, deoxyribonuclease, Unidasa, four kinds of enzyme hybrid reactions digestion of PRONASE A to obtain inoblasts, the participation of plurality of enzymes will certainly cause complexity on experimental procedure and the unnecessary waste of resource.
The myofibroblast parent cell is by Derived from Mesenchymal Stem Cells, and it should keep some characteristics of mescenchymal stem cell so, comprises the adherent growth characteristic, and the expression of specific cells surface antigen mark is to becoming fat, the ability of the differentiation of skeletonization direction etc.
The myofibroblast parent cell is a kind of of adult stem cell, can self and can specialization form the cell that forms the type tissue, and it may express one or more multipotential stem cell factors.
Desmin (Desmin) is a kind of intermediate filament protein, extensively is present in the smooth muscle cell of great vessels, is tested and appraised the expression of desmin, can verify the myogen characteristic of cell.And bibliographical information is arranged along with the purifying of myofibroblast parent cell, the expression of desmin also progressively strengthens.
Summary of the invention
The purpose of this invention is to provide and a kind ofly simple and practical be separated in a large number the method for myofibroblast and identify the method for the myofibroblast parent cell that separates from the deciduate placenta layer.
Placenta fibroblast separation method of the present invention is, utilizes scalpel and tweezers to reject the tissues such as blood transfusion tissue of decidua face, only leaves and takes the face of sloughing off near fetus, and just with the collagenase digesting tissue, its concrete steps comprise:
1, get the fresh human placenta tissue, get the approximately tissue of 5 cm length and width with scalpel in the placenta centre; Clean with physiological saline and remove blood in tissue;
2, divest vascular tissue on amnion and decidua basalis with the tweezers scalpel, leave and take fetus face decidua tissue;
3, placenta baby face decidua tissue washed 10 seconds fast with 75% alcohol;
4,75% alcohol disinfecting washing is rear with the physiological saline washed twice;
5, with scalpel, fetus face decidua tissue is cut into 0.5 cm left and right fragment;
6, with 37 ℃ of digestion 16-18 h of the isopyknic DMEM Digestive system that contains 1% collagenase I of fragment of tissue, in digestion solution without stopping digestion after the placenta tissue sheet;
7, centrifugal collection mononuclearcell is with 10% foetal calf serum and the two anti-DMEM/F12 substratum re-suspended cells of 200 unit mycillins;
8, at 37 ℃, 5% CO
2, cultivate 48 h under saturated humidity in incubator, induce adherent;
9, after cell formed mono-clonal, the picking monoclonal cell was cultivated, and when the cytogamy degree reached 90%, 0.25% trysinization namely got placenta myofibroblast parent cell.
The present invention is the mesenchymal cell characteristic by gained cell after flow cytometer detection cell-surface antigens and cell cycle, the separation and purification of the multinomial differentiation potential experimental identification of cell also, the dryness of the detection identification of cell by the multipotent stem cells factor, the myogenicity of the expression identification cell by morphological observation and cell desmin.
10, the myofibroblast of culture of isolated under normal condition, observation of cell growth characteristic and morphological change in culturing process;
11, get the third generation cell of cultivation, flow cytometer detection cell-surface antigens CD90, CD44, CD29, CD45, HLA-DR carry out simultaneously the cell cycle and detect;
12, the third generation cell of getting cultivation carries out the detection of expression of multipotential stem cell factor OCT4, SOX2, Nanog;
13, the third generation cell of getting cultivation carries out the detection of expression of myogenicity desmin;
14, the third generation cell of getting cultivation carries out to skeletonization, becomes the detection of fat differentiation.
The present invention utilizes scalpel and tweezers to reject the tissues such as blood transfusion tissue of decidua face, only leave and take the face of sloughing off of nearly fetus, just organize with collagenase digesting, reject unnecessary tissue, can not only reduce the enzyme usage quantity, reduce costs, reduce digestion time, avoided simultaneously the introducing of a large amount of heteroproteose cells, make cell purification more convenient.Can make gained primary cell purifying in shorter incubation time is the purpose cell.
The present invention adopts the single stage method of mechanical dispersion method and a kind of enzymic digestion, and this method is simple to operate, and the time consuming time is shorter, uses a small amount of digestive ferment can reach good separating effect, and the scale that is conducive to this type of separation method of later stage is amplified.Adopt simultaneously special reagent pre-treatment tissue samples, do not confirm in the situation that sample is originated, greatly fall oligosaprobic risk.
The authentication method combination of separating obtained sarcostyle parent cell provided by the invention, the evaluation problem of one-stop solution cell, and authentication method and credible result, effective.
Description of drawings
Fig. 1: cellular form:
A: individual cells photo
B: cell monoclonal photo
C: the cytogamy degree reaches 90% photo
Fig. 2: Flow cytometry cell phenotype
Fig. 3: Flow cytometry cell cycle
Fig. 4: cell scleroblast identification and detection
Fig. 5: the cell lipoblast detects
Fig. 6: cell multipotential stem cell factors check is respectively from left to right: the RT-PCR electrophoresis photo of OCT4, SOX2, Nanog
Fig. 7: the cell desmin expression detects
Embodiment
The invention discloses a kind of separation and authentication method of people's placenta myofibroblast parent cell, those skilled in the art can use for reference this paper content, suitably improves processing parameter and realizes.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.
In order to make those skilled in the art better understand technical scheme of the present invention, the present invention will be further described in detail below in conjunction with concrete specific embodiment.
Embodiment one placenta myofibroblast parent cell separation and Culture
Get natural labor baby's fresh human placenta tissue (in 12 hours) in the sterilized bio safety cabinet, get the approximately tissue of 5 cm length and width with scalpel in the placenta centre, 250 mL physiological saline washings 2 times, push with strength placenta tissue, blood in tissue is fully washed out, divest blood transfusion tissue on amnion and decidua basalis with tweezers and scalpel; Leave and take fetus face decidua tissue, 250 mL physiological saline washings 1 time cut into 1.5 cm length, the wide size of 0.5 cm to fetus face decidua tissue with scalpel; 75% alcohol Rapid Cleaning sterilization 10 seconds, then with 250 mL physiological saline washing 2 times; With with 37 ℃ of digestion 16-18 h of the isopyknic DMEM Digestive system that contains 1% collagenase I of fragment of tissue, in digestion solution without stopping digestion after the placenta tissue sheet; Postdigestive tissue juice average mark is loaded in 2 50 mL centrifuge tubes, adds physiological saline to 50 mL, 2300 rpm, the centrifugal 10 centrifugal collection mononuclearcells of min; Physiological saline washing 2 times, 2000 rpm, centrifugal 10 min are with containing resuspended mononuclearcell in the two anti-DMEM/F12 substratum of 10% foetal calf serum and 200 unit mycillins; At 37 ℃, 5% CO
2, cultivate under saturated humidity.
Primary cell culture after 48 hours full dose change liquid, remove non-adherent cell, add fresh culture, after being dispersed in attached cell formation mono-clonal, picking monoclonal cell single culture obtains the placenta fibroblast, when the cytogamy degree reached 90%, trysinization was gone down to posterity.
Embodiment two placenta myofibroblast parent cell Morphological Identifications
The myofibroblast that cultivation separates according to embodiment one method, after inducing adherent 2 days, the fusiformis attached cell that as seen microscopically is dispersed in, culture supernatant is all outwelled, remove simultaneously not adherent impurity cell, cultivate after 5-7 days, as seen become radial monoclonal cell to form, cultivate in 12 orifice plates with cell scraper scraping monoclonal cell, treat that cell quantity reaches 5 * 10
6During the left and right, be used for follow-up other evaluation.
Microscopically is observed visible (Fig. 1) cell and is fusiformis or fusiform, polarity, and volume is little, and along with the increase of incubation time, it is large that cell volume becomes, and fusion growth, these characteristics meet growth characteristic and the Morphological Characteristics of myofibroblast.
Embodiment three placenta myofibroblast parent cell surface markers are identified
Get respectively different placentas sources according to embodiment one cultivate the 3rd generation cell, the Flow cytometry cell surface marker is observed the variation of different placenta derived cell surface markers.The digestion collecting cell gets 1 * 10 after counting
6Individual, PBS washs once, 1500 rpm, centrifugal 10 min; Abandon supernatant, residual 200 uL blow and beat the mixing cell, add CD90, CD45 and CD29, the HLA-DR of APC mark and each 10 uL of CD44 antibody of PE mark of FITC mark, and establish 1 pipe for blank, at 4 ℃ of lower lucifuge reaction 30 min, PBS washs once, 1500 rpm, centrifugal 10 min, abandon supernatant, residual 200 uL, upper machine testing.
Flow cytometer detection Fig. 2 as a result shows different placenta derived cell CD90(99% left and right), the CD44(98% left and right), the CD29(97% left and right) expression of three kinds of antigens is all higher than more than 95%, the CD45(0.7% left and right), the HLA-DR(0.4% left and right) expression of two kinds of antigens is all lower than 2%.The cell surface marker result shows that cell has the interstitial fibers cell characteristics.
The four placenta myofibroblast parent cell cell cycles of embodiment are detected
According to embodiment one culturing cell, when the Growth of Cells degrees of fusion is 90%, carries out the cell cycle and detect.The digestion collecting cell gets 1 * 10 after counting
6Individual, PBS washs once, abandons supernatant, add 70% ethanol-20 ℃ to fix 2 h, centrifugal removal ethanol with the PBS washing once, adds RNaseI 5 uL (20 mg/mL), 37 ℃ of reaction 30 min, the PBS washing once adds propidium iodide (PI, 50 ug/mL) 0.5 uL, 4 ℃ of lucifuge reaction 30 min, upper machine testing DNA content.
Cell cycle analysis Fig. 3 as a result shows, is in G0/G1 phase, S phase, G2M phase cell proportion average out to 93.72%, 3.7%, 2.57%.Placenta myofibroblast parent cell most cells during proof is cultivated is in the G0/G1 quiescent stage, only has a few cell to be in active S proliferation period, has typical stem cell increasing feature.
Embodiment five placenta myofibroblast parent cell Osteoblast Differentiation potential are identified
According to embodiment one cultivation above cell of 3 generation, single cell suspension is made in digestion, according to 2 * 10
4Individual/cm
2Density use and to contain 10% foetal calf serum and the two anti-DMEM/F12 culture medium inoculateds of 200 unit mycillins in having overlay I type mouse tail collagen 6 orifice plates, at 37 ℃, 5% CO
2, cultivate under saturated humidity, after the cytogamy degree reaches 50%, replacing contains the fresh culture inducing culture of dexamethasone 0.1 μ M, ascorbyl phosphate 50 μ g/mL, β-phospho-glycerol 10 mM, changed liquid once in 3 days, 3 weeks of coinduction, use 95% ethanol fixed cell, Alizarin red staining, microscopically are observed the formation of the outer calcium matrix precipitation of born of the same parents.
Observe Fig. 4 result and show, cell matrix obviously has calcium matrix precipitation, shows that cell transforms to scleroblast, has the characteristic of interstital stem cell.
Embodiment six placenta myofibroblast parent cells become the fat differentiation potential to identify
According to embodiment one cultivation above cell of 3 generation, single cell suspension is made in digestion, according to 1 * 10
5Individual/cm
2Density use and to contain 10% foetal calf serum and the two anti-DMEM/F12 culture medium inoculateds of 200 unit mycillins in having overlay I type mouse tail collagen 6 orifice plates, at 37 ℃, 5% CO
2, cultivate under saturated humidity, after the cytogamy degree reaches 80%, change the fresh culture inducing culture that contains dexamethasone 1 μ M, INDOMETHACIN 0.1 mM, IBMX 0.5 mM, Regular Insulin 5 μ g/ml, changed liquid once in 3 days, 3 weeks of coinduction, 10% paraformaldehyde fixed cell, oil red dyeing.
Observe Fig. 5 result and show, the fat that cell produces is dyed redness by specificity, shows that cell transforms to adipocyte, has the characteristic of interstital stem cell.
Embodiment seven placenta myofibroblast parent cell multipotential stem cell factor expressions are identified
Get different placentas sources according to embodiment one cultivate the 3rd generation cell, cell concentration reaches 5 * 10
6Individual, total RNA of extraction cell, reverse transcription obtains cDNA, take cDNA as template, uses following primer to carry out RT-PCR, detects the expression situation of OCT4, SOX2 and Nanog gene, and this operation is carried out with the test kit specification sheets, and primer sequence is as shown in table 1.
Table 1:RT-PCR primer sequence and characteristic thereof
In Fig. 6, be respectively from left to right: the RT-PCR electrophoresis photo of OCT4, SOX2, Nanog, result show that myofibroblast parent cell expression OCT4, two kinds of multipotential stem cell factors of Nanog that different placentas are originated are not expressed or the low SOX2 of the expression factor.This experiment proves absolutely that placenta myofibroblast parent cell has the characteristics of stem cell.
Embodiment eight placenta myofibroblast parent cell desmin expression are identified
Get different placentas sources according to embodiment one cultivate the 3rd generation cell, cell concentration reaches 5 * 10
6Individual, total RNA of extraction cell, reverse transcription obtains cDNA, take cDNA as template, uses following primer to carry out RT-PCR, and this operation is carried out with the test kit specification sheets, and primer sequence is as shown in table 2.
Table 2:RT-PCR primer sequence and characteristic thereof
Fig. 7 is Desmin electrophoresis picture, and result shows placenta myofibroblast parent cell expression desmin, proves that it derives from muscle tissue.
Claims (13)
1. the easy separation method of people's placenta myofibroblast parent cell, comprise and get placenta, divest vascular tissue on amnion and decidua basalis with the tweezers scalpel, leave and take fetus face decidua tissue, used 75% alcohol Quick disinfection washing tissue 10 seconds, then with physiological saline washing 2 times, with scalpel, fetus face decidua tissue is slit into 1.5 cm length, the wide large small shreds of 0.5 cm; Add and the isopyknic DMEM Digestive system that contains 1% collagenase I of fragment of tissue, 37 ℃ of digestion 16-18 h digest centrifugal collection mononuclearcell without stopping after the placenta tissue sheet in digestion solution; With containing the two anti-DMEM/F12 substratum of 10% foetal calf serum and 200 unit mycillins at 37 ℃, 5%CO
2, cultivate 48h under saturated humidity, induce adherent, after cell forms mono-clonal, picking monoclonal cell single culture, when the cytogamy degree reaches 90%, trysinization.
2. according to claim 1, placenta myofibroblast parent cell comes from placenta baby face decidua tissue.
3. according to claim 1, washed fast placenta tissue 10 seconds with 75% alcohol.
4. according to claim 1, with scalpel, fetus face decidua tissue is slit into 1.5 cm length, the wide big or small fragment of 0.5 cm.
5. according to claim 1, Digestive system is the DMEM solution that contains collagenase I.
6. according to claim 1, digestion time is 16-18 h.
7. according to claim 1, cell cultures is used and is contained 10% foetal calf serum and the two anti-DMEM/F12 substratum of 200 unit mycillins.
8. according to claim 1, after forming mono-clonal, picking monoclonal cell single culture is come purifying cells.
9. the authentication method combination of identifier's placenta source fibroblast, comprise: morphocytology, the cell cycle, cell surface marker detects, the cytodifferentiation ability detects, and cell multipotential stem cell factors check and cell desmin expression detect totally 6 kinds of methods.
10. according to claim 9, cell surface marker detects and comprises surface antigen CD90, CD44, CD29, CD45, HLA-DR.
11. according to claim 9, adopt to becoming fat, the method for Osteoblast Differentiation detects the differentiation capability of cell.
12. according to claim 9, adopt detection OCT4, SOX2,3 kinds of factors of Nanog to carry out the evaluation of cell versatility.
13. adopt according to claim 9, the myogenicity of desmin expression identification of cell.
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