CN104001216B - The CFU-GM of skin-derived is utilized to prepare the method for organization engineering skin - Google Patents
The CFU-GM of skin-derived is utilized to prepare the method for organization engineering skin Download PDFInfo
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- CN104001216B CN104001216B CN201410244591.7A CN201410244591A CN104001216B CN 104001216 B CN104001216 B CN 104001216B CN 201410244591 A CN201410244591 A CN 201410244591A CN 104001216 B CN104001216 B CN 104001216B
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Abstract
The invention discloses a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin, the method comprises the following steps: 1) be separated SKPs; 2) separation is obtained? SKPs moves in amplification culture medium and increases; 3) the DiI fluorochrome label of SKPs; 4) prepare organization engineering skin, collagen sponge card punch is made diameter 8mm circular collagen sponge Co60 radiation sterilization, SKPs cell 1 × 105/20-30uL culture medium is added on sponge exasperate, cultivates.SKPs in organization engineering skin of the present invention can be divided into smooth muscle cell, vascular endothelial cell, nervous tissue, the smooth muscle cell differentiated, vascular endothelial cell can participate in forming blood vessel structure, promote that wound new vessels is formed, thus improve skin healing speed and healing quality.
Description
Technical field
The present invention relates to organization engineering skin preparing technical field, especially relate to a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin.
Background technology
Refractory Diabetic skin injury (as: diabetic foot) is one of common chronic complicating diseases of diabetes, is that diabetics is disabled, lethal main cause.Its high incidence, high disability rate, difficult therapeutic, high medical expense etc., make the quality of life degradation of diabetics, bring heavy financial burden to patient and society.The routine clinical treatment means of Refractory Diabetic skin injury comprises control blood glucose, improve the nutriture of patient, improve local blood supply, infection, promotes peripheroneural functional rehabilitation, intensive care, debridement, skin grafting etc., but therapeutic effect is usually not good enough, and current treatment emphasis is still avoids amputation.Therefore more efficiently Therapeutic Method must be found.
At present, tissue engineering skin has become a kind of more perfect engineered tissue, and Preliminary Applications is in clinical, reparation for skin injury provides new method, but still there are problems, as: there is not the component of organization such as blood vessel, melanocyte, skin appendages structure can not be rebuild, mechanical performance with normally have larger gap etc. at body skin.
Stem cell is the cell that a class has self replication and multi-lineage potential, has the potential being divided into Various Tissues type.In recent years, stem cell has become new study hotspot as seed cell treatment Refractory Diabetic skin lesion.Research proves, ESC and MSC all can produce the composed component of skin histology, thus participates in the repair process of skin trauma, and this feature can make up the defect of engineered skin substitute, improves repair ability and the healing quality of skin.But the existence of the problems such as the carcinogenic possibility existed after the relevant ethics morals problem caused owing to using above-mentioned cell, transplanting, cell derived is not enough, make clinical practice greatly limited, we need to find new seed cell to avoid the problems referred to above.
Calendar year 2001, Tomaetal., from the dermal layer of the skin taking from rodent newborn individual and adult, is separated the CFU-GM (skin-derivedprecursors, SKPs) obtaining skin-derived.Multinomial research in recent years shows, SKPs is a kind of multipotency CFU-GM of novel, neural crest, is present in mammal skin corium, in vitro in experiment, finally can produce nerveous system filial generation and mesoderm filial generation.At present, the cell with SKPs character has also been separated and has obtained from all ages and classes and the human skin of origin, routine can also obtain from human foreskin.Because SKPs has multipotency, no matter when normal physiological renewal or injury repairing, the mesoderm composition participating in forming dermal layer of the skin all may be broken up, comprises dermal fibroblast, vascular smooth muscle cell etc.There are some researches show, SKPs can be divided into vascular smooth muscle cell, also may have the ability forming endotheliocyte.
After using existing SKPs to be applied to skin wound, vascular smooth muscle and endotheliocyte can be divided into, thus promote that wound new vessels is formed, wound healing.But its healing rate is still comparatively slow, effect is still not ideal enough.How to find one and can promote that wound new vessels is formed in a large number fast, promote that the method for wound surface quickly-healing has become current problem demanding prompt solution, wound surface quickly-healing damages the clinical practice for the treatment of to diabetic skin and has great significance, and the clinical treatment that also can be the chronic wound surface in refractory to treatment of DM brings very big help.
Summary of the invention
Instant invention overcomes shortcoming of the prior art, provide one and can promote that wound new vessels is formed in a large number fast, promote that the CFU-GM of skin-derived that utilizes of wound surface quickly-healing prepares the method for organization engineering skin.
In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions: a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin, comprises the following steps:
1), SKPs is separated
Skin samples is put into aseptic PB, is separated and discards subcutaneous tissue under microscope, epidermis and dermal tissue are cut into small pieces and put into dispase enzyme, 4 DEG C of digestion are spent the night; Within second day, take out, be separated and discard skin portion, retain skin corium tissue; Add collagenase 37 DEG C digestion 1h; Piping and druming is organized until unicellular, with 40um aperture cell screen filtration cell; Unicellular with complete culture medium culturing, changed liquid once every seven days;
2), SKPs rapid amplifying
Carry out passage when cell forms clone ball and shade in centre, go down to posterity in the ratio of 1:2, amplification cultivation in amplification culture medium;
3), the DiI fluorochrome label of SKPs
After hatching 5min in cell suspension 37 DEG C of incubators, be that 3ug/mlDiI fluorescent dye hatches 8min with middle concentration; Wash 3 times with PBS afterwards, remove excess dyestuff, be resuspended in normal saline, cell counting is for subsequent use, 4 DEG C, cell preservation before transplanting;
4), organization engineering skin is prepared
The circular collagen sponge of diameter 8mm made by collagen sponge pretreatment card punch, puts into o.o1MPBS, aerofluxus; Co60 radiation sterilization, irradiation dose 20K; Bed board in super-clean bench, ultra-vioket radiation 0.5h; Every hole 300ul culture medium, spends the night in 37 DEG C of incubators; Sponge is transferred in another orifice plate; Cell 1 × 105/20-30uL culture medium is added on sponge exasperate; 4h is hatched, cell attachment in 37 DEG C of incubators; After 4h, culture medium 200-300uL/ hole adds, and hatches in 37 DEG C of incubators; After 1 day, change liquid once, from the imbibition of sponge edge, inoculating cell was transplanted after 3 days.
Preferably, described skin samples is epidermis and dermal tissue.
Preferably, described skin samples is newborn C57 neonatal rat skin of back.
Preferably, wherein said step 1) condition of culture be cultivate in 37 DEG C of incubators.
Preferably, wherein said step 2) in, described amplification culture medium is complete medium; Condition of culture is 37 DEG C of incubators.
Preferably, wherein said step 4) in, described bed board mode is 48 orifice plates, exasperate upwards, exasperate upper berth cell.
Compared with prior art, tool of the present invention has the following advantages: organization engineering skin of the present invention promotes that the quantity of angiogenic growth is 2.95 times of control group, be 1.68 times of collagen group, it can promote that wound new vessels is formed in a large number fast, promotes wound surface quickly-healing.It is the effective means that diabetic skin damage is carried out treating, and is the optimum selection of the clinical treatment of the chronic wound surface in refractory to treatment of DM.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the organization engineering skin that the present invention prepares;
Fig. 2 is the comparison chart that the present invention tests 1 each experimental group wound healing;
Fig. 3 is the shrinkage factor figure of each test group wound adopting IPP6.0 image analysis software to calculate:
Wound healing figure when Fig. 4 is each test group of Microscopic observation 7 days;
Wound healing figure when Fig. 5 is each test group of Microscopic observation 14 days;
Fig. 6 is skps+collagen group Nestin immunofluorescence SKPs survival colored graph;
Fig. 7 is skps+collagen group vWF new vessels colored graph;
Fig. 8 is skps+collagen group SMA smooth muscle cell colored graph;
Fig. 9 is skps+collagen group tubulin neurocyte colored graph;
Figure 10 is skps+collagen group SMA+nestin new vessels and smooth muscle cell colored graph;
Figure 11 is skps+collagen group vWF+nestin new vessels colored graph;
SMA new vessels colored graph when Figure 12 is each processed group 14 days.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not paying the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1:
1, isolation identification, the labelling of SKPs
The newborn C57 neonatal rat neck that breaks is put to death, and back unhairing, cuts skin of back, puts into aseptic PBS.
Be separated under Stereo microscope and discard subcutaneous tissue, epidermis and dermal tissue being cut into small pieces and putting into dispase enzyme, 4 DEG C of digestion are spent the night; Within second day, take out, be separated and discard skin portion, retain skin corium tissue; Add collagenase 37 DEG C digestion 1h; Piping and druming is organized until unicellular, with 40um aperture cell screen filtration cell; Unicellular with complete culture medium culturing, changed liquid once every seven days, the condition of culture of this step is cultivate in 37 DEG C of incubators.
2, SKPs rapid amplifying
Carry out passage when cell forms clone ball and shade in centre, go down to posterity in the ratio of 1:2, carry out in complete medium; Condition of culture is 37 DEG C of incubators.
3, the labelling of SKPs
After hatching 5min in cell suspension 37 DEG C of incubators, be that 3ug/mlDiI fluorescent dye hatches 8min with final concentration.Wash 3 times with PBS afterwards, remove excess dyestuff, be resuspended in normal saline, cell counting is for subsequent use, 4 DEG C, cell preservation before transplanting.
4, organization engineering skin is prepared
(1) collagen sponge pretreatment
A, size: the circular collagen sponge of diameter 8mm made by card punch, puts into o.o1MPBS, aerofluxus;
B, Co60 radiation sterilization, irradiation dose 20K, (Tianjin physics Institute).
(2) collagen sponge bed board
Bed board in a, super-clean bench (48 orifice plates, exasperate upwards, exasperate upper berth cell) ultra-vioket radiation 0.5h;
B, every hole 300ul culture medium, spend the night in 37 DEG C of incubators.
(3) collagen sponge paving cell
A, sponge is transferred in another 48 orifice plate;
B, cell 1 × 105/20-30uL culture medium are added on sponge exasperate;
4h is hatched, cell attachment in c, 37 DEG C of incubators;
After d, 4h, culture medium 200-300uL/ hole adds, and hatches in 37 DEG C of incubators;
E, after 1 day, change liquid once, from the imbibition of sponge edge, interference cell is not adherent as far as possible;
F, inoculating cell were transplanted after 3 days, and organization engineering skin is shown in Fig. 1.
The making process of composition engineering skin sample
1, light rinsing: soft rinsing Combined culture thing 2--3min × 3 time in the PBS of 37 DEG C of preheatings.
2, fixing
2h in (1) 2.5% glutaraldehyde 37 DEG C incubator.
(2), after changing 2.5% glutaraldehyde, in 2.5% glutaraldehyde, 4 DEG C are spent the night.
3, gradient alcohol dehydration
4, after naturally drying 4h, evacuation 2h.For subsequent use.
To quantity and the active observation (mtt assay) of survivaling cell in the organization engineering skin of embodiment 1 preparation
1,5mg/mlMTT25ul adds in Combined culture thing respectively, and 37 DEG C of incubators hatch 4h.
2, develop the color: pipettor siphons away supernatant, and being cut by sponge with sterilizing scissors is 4 lobes, and every hole lucifuge adds 300ulDMSO, beats gently with pipettor.Agitator shakes 15min, omnidistance attention lucifuge.
3, colorimetric: every hole is got 50ul and is transferred in 96 new orifice plates, surveys OD value (wavelength: 490nm).
4, each time point (after transplanting 1,3,5 day) is surveyed 3 times respectively, often organizes survey three samples at every turn, averages.
Expert's conclusion
1, the separation of cell.The method of Tomaetal. has been used for reference in this experiment, skin is digested to unicellular after, use serum-free medium cultivate.Separation efficiency is very high, maintains the CFU-GM state of cell.By document comparison, can prove, great majority plant the isolated cell of method therewith about the isolated nestin positive cell of hair follicle stem cells document is instantly homologous cell, all from neural ridge.
2, cell amplification in vitro and go down to posterity.This cell increasess slowly under the condition of serum-free, fully maintains its CFU-GM feature.After cell adds the culture medium of variable concentrations serum, its corresponding increase of rising in value, differentiation also corresponding beginning.
3, the differentiation qualification of cell.In the growth course of cell, remove somatomedin, and add serum, cell can carry out multidirectional differentiation, as nerve, fat, smooth muscle etc., differentiation efficiency is different according to different differentiation directions from the time, and the easiest differentiation direction of hair follicle stem cells is smooth muscle cell and neurocyte.
4, the qualification work of cell progenitors and paracrine ability has been carried out.This cellular expression nestin, this is the labelling of neural progenitor cell, and nanog and OCT3/4 is the labelling of embryonic stem cell, and the expression of SDF-1 and bFGF proves that it has the ability of paracrine.
The foundation of test 1:C57 mice T2DM skin injury model
(1) anaesthetize: 4% chloral hydrate 350mg/kg lumbar injection is in C57 mice T2DM model.
(2) preserved skin, fixing, sterilization: remove mouse back Mus hair, the outside of belly is fixed on downwards in surgical console, and skin of back is sterilized.
(3) set up skin lesion model: on mouse back median line, cut with operating scissors the circular skin injury that a diameter is 8mm, lesion thickness reaches skin holostrome, front and back each.
(4) transplant: arrange three groups of migration process groups, each processed group gives different disposal.Control group: normal healing; Collagen group: transplant collagen sponge; Skps-collagen group: transplant organization engineering skin of the present invention.
(5) fixing sponge: with 7/0 suturing with thread management fixing glue olynthus in mouse skin wound place.Notice that sponge and wound expose organize well involutory.
(6) postoperative: Sterilized application covers.The single cage of all mices is fed, and ad lib high-sugar-fat-diet, freely drinks water.
Test the result of 1 wound repair
1, wound healing general appearance
Whole wound all without infection, wound is shunk to wound center gradually by wound edge, Fig. 2 illustrate control group, collagen group and collagen+skps group 0 day, 7 days and 14 days time wound healing state.
1) control group: wound surface contraction of skin is slow, crust is belittled, granulation tissue poor growth under crust, and color and luster is dark red, and matter is crisp, and very easily damaged under external force, healing quality is poor.Hinder latter 14 days, wound surface heals substantially, and comparatively obvious by skin healing cicatrix, crust obviously remains.
2) collagen group: wound surface contraction of skin is fast, and visible thin layer granulation tissue under crust, color and luster is dark red.Hinder latter 14 days, wound surface heals substantially, leaves a small amount of crust.
3) collagen+skps group: wound surface contraction of skin is fast, under crust, granulation tissue growth is comparatively firm, and color and luster is more ruddy, and wound surface is dry, and neoplastic skin is smooth, and skin healing cicatrix is obviously less.Hinder latter 14 days, wound healing, part crust comes off completely.
2, speed of wound healing
Perusal: transplant in 7 days, collagen, skps-collagen group wound contraction is all faster than control group.
(Fig. 3) is calculated through IPP6.0 image analysis software, when transplanting latter 7 days, each group of wound contraction rate has notable difference (p < 0.01), wound contraction rate when 7 days: skps-collagen group > collagen group > control group.When 14 days, each group wound contraction rate zero difference, wound is all substantially closed.
3, Microscopic observation wound healing quality (× 100 times of visual field picture mosaics)
When 7 days (Fig. 4), wound granulation tissue is formed, epithelium by wound edge to wound central authorities hypertrophy, bottom wound and a large amount of new vessels of wound edge formed; The visible granulation tissue of control group is formed poor, and bottom wound, new vessels is formed.Compared with control group, collagen group and collagen+skps form mouth place cambium and are formed more plentiful, and granulation tissue is formed good, bottom wound and the visible a large amount of new vessels of window edge formed, inflammatory cell invades profit, and organizational structure is more complete.
When 14 days (Fig. 5), respectively form a mouthful cambium structural integrity.By contrast, control group granulation is formed slowly, and structure is belittled, poor structural integrity.
4, organization engineering skin of the present invention is to smooth muscle, endothelium, Neural Differentiation, promotes that new vessels is formed and analyzes;
Skps+collagen group Nestin immunofluorescence SKPs survival colored graph (Fig. 6): skps+collagen group immunofluorescence dyeing when 7 days, bottom wound and the visible DiI positive cell of wound edge overlap with nestin positive cell, the SKPs survival that there is transplanted cells source is described in tissue, and maintains the labelling of neural progenitor cell.
Skps+collagen group vWF new vessels colored graph (Fig. 7): bottom wound and wound edge DiI positive cell overlap with vWF positive cell.The cell survival existing in tissue and transplant SKPs source is described, and expresses the labelling of endotheliocyte.The SKPs of provable transplanting is divided into endotheliocyte, take part in the formation of new vessels, participates in tissue repair.
Skps+collagen group SMA smooth muscle cell colored graph (Fig. 8): bottom wound and wound edge DiI positive cell SMA positive cell overlap.The cell survival existing in tissue and transplant SKPs source is described, and expresses the labelling of smooth muscle cell, participate in forming tubular structure.The SKPs of provable transplanting is divided into smooth muscle cell, take part in the formation of new vessels, participates in tissue repair.
Skps+collagen group tubulin neurocyte colored graph (Fig. 9): wound edge is easily shown in that DiI positive cell overlaps with tubulin positive cell.The cell survival existing in tissue and transplant SKPs source is described, and expresses the labelling of neurocyte.The SKPs of provable transplanting is divided into nervous tissue, take part in the formation of newborn nervous tissue.
Skps+collagen group SMA+nestin new vessels and smooth muscle cell colored graph (Figure 10): bottom wound and wound edge easily see that nestin positive cell overlaps with SMA positive cell, nestin is one of SKPs labelling, nestin positive cell expresses smooth muscle cell labelling, take part in the formation of new vessels.
Skps+collagen group vWF+nestin new vessels colored graph (Figure 11): bottom wound and wound edge easily see that nestin positive cell overlaps with vWF positive cell, nestin is one of SKPs labelling, nestin positive cell expresses endothelial cell marker, take part in the formation of new vessels.
5, damage zone blood vessel number calculates
According to SMA coloration result (Figure 12), counting is transplanted after latter 14 days, blood vessel number in wound area.Result: skps-collagen group blood vessel number, more than control group, has statistical significance (p < 0.05).Blood vessel number in wound area: skps-collagen group > collagen group > control group.Skps-collagen combined transplantation facilitates new vessels and is formed, thus promotes skin wounds healing, and each test group blood vessel number sees the following form.
| Grouping | control | collagen | skps-collagen |
| 14 days blood vessel number | 21.07±4.99 | 39.82±0.14 | 62.07±0.11 |
Testing surface, organization engineering skin of the present invention promotes that the quantity of angiogenic growth is 2.95 times of control group, and be 1.68 times of collagen group, it can promote that wound new vessels is formed in a large number fast, promotes wound surface quickly-healing.It is the effective means that diabetic skin damage is carried out treating, and is the optimum selection of the clinical treatment of the chronic wound surface in refractory to treatment of DM.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (5)
1. utilize the CFU-GM of skin-derived to prepare a method for organization engineering skin, comprise the following steps:
1), SKPs is separated
Skin samples is put into aseptic PBS, is separated and discards subcutaneous tissue under microscope, epidermis and dermal tissue are cut into small pieces and put into dispase enzyme, 4 DEG C of digestion are spent the night; Within second day, take out, be separated and discard skin portion, retain skin corium tissue; Add collagenase 37 DEG C digestion 1h; Piping and druming is organized until unicellular, with 40um aperture cell screen filtration cell; Unicellular with complete culture medium culturing, changed liquid once every seven days;
2), SKPs rapid amplifying
Carry out passage when cell forms clone ball and shade in centre, go down to posterity in the ratio of 1:2, amplification cultivation in amplification culture medium;
3), the DiI fluorochrome label of SKPs
After hatching 5min in cell suspension 37 DEG C of incubators, be that 3ug/mlDiI fluorescent dye hatches 8min with middle concentration; Wash 3 times with PBS afterwards, remove excess dyestuff, be resuspended in normal saline, cell counting is for subsequent use, 4 DEG C, cell preservation before transplanting;
4), organization engineering skin is prepared
The circular collagen sponge of diameter 8mm made by collagen sponge pretreatment card punch, puts into 0.01MPBS, aerofluxus; Co60 radiation sterilization, irradiation dose 20K; Bed board in super-clean bench, ultra-vioket radiation 0.5h; Every hole 300ul culture medium, spends the night in 37 DEG C of incubators; Sponge is transferred in another orifice plate; Cell 1 × 105/20-30uL culture medium is added on sponge exasperate; 4h is hatched, cell attachment in 37 DEG C of incubators; After 4h, culture medium 200-300uL/ hole adds, and hatches in 37 DEG C of incubators; After 1 day, change liquid once, from the imbibition of sponge edge, inoculating cell was transplanted after 3 days;
Step 1) described in skin samples be epidermis and dermal tissue.
2. a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin as claimed in claim 1, is characterized in that described skin samples is newborn C57 neonatal rat skin of back.
3. a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin as claimed in claim 1, is characterized in that step 1) described in condition of culture be cultivate in 37 DEG C of incubators.
4. a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin as claimed in claim 1, is characterized in that step 2) in, described amplification culture medium is complete medium; Condition of culture is 37 DEG C of incubators.
5. a kind of method utilizing the CFU-GM of skin-derived to prepare organization engineering skin as claimed in claim 1, is characterized in that step 4) in, described bed board mode is 48 orifice plates, exasperate upwards, exasperate upper berth cell.
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| CN1563366A (en) * | 2004-04-09 | 2005-01-12 | 西北农林科技大学 | Method for preparing corneal epithelium form epidermis stem cell through fabrication of tissue engineering, and application |
| DE102008017990A1 (en) * | 2007-02-07 | 2009-10-08 | Dagmar Briechle | Method for producing dendritic cell-like cells and use of these cells in in-vitro test methods for determining the influence of exogenous substances |
| CN102462864A (en) * | 2010-11-12 | 2012-05-23 | 中国辐射防护研究院 | Novel method for constructing tissue engineering skin |
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| CN1563366A (en) * | 2004-04-09 | 2005-01-12 | 西北农林科技大学 | Method for preparing corneal epithelium form epidermis stem cell through fabrication of tissue engineering, and application |
| DE102008017990A1 (en) * | 2007-02-07 | 2009-10-08 | Dagmar Briechle | Method for producing dendritic cell-like cells and use of these cells in in-vitro test methods for determining the influence of exogenous substances |
| CN102462864A (en) * | 2010-11-12 | 2012-05-23 | 中国辐射防护研究院 | Novel method for constructing tissue engineering skin |
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