CN108841786A - The amnion-derived mescenchymal stem cell preprocess method of people and its application - Google Patents

Abstract

The invention discloses the amnion-derived mescenchymal stem cell preprocess methods of people, including：Secondary culture is separated to AMSC and AEC respectively, collects the AEC in the AMSC and P5 generation in P10 generation；AEC in AMSC and P5 generation in P10 generation is subjected to Indirect co-culture processing；Or the AEC in P3 generation is used to prepare the AMSC being used in P10 generation after conditioned medium and carries out CMC model；Collect treated AMSC, obtain pretreated AMSC, method of the invention is able to maintain the stable cytologic characteristic of AMSC and good proliferation activity, so as to ensure the quality and quantity of MSC needed for clinical application, increase AMSC to go back to the nest and intracorporal implantation quantity, treats required cell quantity so as to meet；Have the characteristics that simplicity and due to cheap, effectively reduces use cost, meanwhile, this method safety is good, no obvious toxic-side effects, clinical application to AMSC and improves HSCT success rate and curative effect has important practical significance.

Description

The amnion-derived mescenchymal stem cell preprocess method of people and its application

Technical field

The invention belongs to medicine technology fields, are related to hematology technical field, in particular between a kind of people is amnion-derived Mesenchymal stem cells preprocess method and its application.

Background technique

Hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) is blood system System malignant tumour, aplastic anaemia, severe immunologic deficiency disease and the most effective treatment means of part genetic disease One of.Currently, only just newly-increased 40,000 people, positive waiting hematopoietic stem cell transplantation save the patient of life to leukaemic every year in China Nearly million people.And after HSCT, stem cell survive, break up, being proliferated and the recovery of hematopoiesis and immune function, directly affect treatment Effect.Problems can be inevitably encountered in migration process, such as the destruction of hematopoieticmicroenviron-ment, stem cell homing and the anti-place of graft Main reaction (graft versus host reaction, GVHR) influences to transplant success or failure.Therefore, how HSCT is preferably improved Success rate is the key subjects that hematology worker faces.

Research discovery mescenchymal stem cell (mesenchymal stem cells, MSC) in recent years is hematopoieticmicroenviron-ment Important composition ingredient, combined clinical transplanting MSC and HSC be improve hematopoietic reconstitution ability, reduction or mitigate graft-versus-host The effective means that sick (GVHD) occurs.But it is micro- to rebuild marrow for many research reports MSC or stroma cell after venoclysis at present The ability of environment is very limited, and the MSC quantity that increase moves to marrow has been found preferably improve clinical treatment Effect.Therefore, for MSC transplanting obtains good therapeutic effect, it is desirable to which we find the effective raising of one kind, and it is gone back to the nest With the method for implantation.

People's amnion MSC (amniotic MSC, AMSC) be a kind of abundance, without invasive operation, materials hardly by Limitation, and the MSC new sources of isolated culture method simplicity, can not only break up to mesoblastemas such as skeletonization, fat, endotheliums, It can also break up under optimum conditions to the ectoderms such as nerve, liver cell and endoderm cell, have in vitro similar with BMSC Hematopoiesis support function, it might even be possible to expression hematopoietic cytokine more more abundant than BMSC, while immunogenicity is low, can significantly press down Lymphopoiesis processed is likely to become the source of a kind of more ideal MSC clinical research and application.SDF1/CXCR4 axis exists It plays a significant role in MSC transition process, as point of penetration, its ability of going back to the nest is improved by the external pretreatment to AMSC, It not only will be applied to clinic for AMSC and theoretical foundation is provided, and also open up new way more effectively to improve transplanting curative effect.Meanwhile by Not only to marrow after MSC intravenous administration, additionally it is possible to specifically be migrated to other damage locations, participate in local damage and repair It is multiple, therefore improve its transfer ability and be also possible to improve MSC and treating bone and joint diseases, inflammatory bowel disease, cirrhosis, nervous system The curative effect of disease etc. provides new approaches.

The MSC clinical test carried out at present mostly uses the method through venoclysis, and required MSC quantity is larger, from 0.4 ×10⁶/ kg to 10 × 10⁶/ kg weight differs.Preferable clinical efficacy is achieved although with such large dosage, but MSC is in body The quantity being implanted into is but still very limited.Such as the report such as Bartsch, after BMSC and HSC combined transplantation, the periphery of receptor The complete donor that hematopoietic cell in blood and marrow has all reached 100% is chimeric, and its MSC still remains receptor source, not Detect being chronically implanted for donor source MSC.Recent research confirms that MSC, which is directly injected into ossis, preferably to promote Radiation in jury and mitigation GVHD,

Many researchs confirm, the migration of stem cell and the process specifically gone back to the nest to tissue be mainly by chemotactic factor (CF) and its Receptor is come what is adjusted, and wherein SDF-1 and its receptor CXCR 4 play very important effect.Just whether the function of SDF-1/CXCR4 Often, it is played a very important role during marrow is implanted into after hematopoietic stem cell transplantation.SDF-1 can be by the more of body The expression of kind histoorgan or secretion, especially marrow.In the case where tissue/organ damage, it can promote stem cell, inflammation Disease recruiting cells are to damage location, and the key cytokines for being both adjusting tissue/organ injury repair and an adjusting are locally The important chemotactic factor (CF) of inflammation.The functional expression of CXCR4 has been had been found that in many stem cell surfaces at present, wherein also wrapping Include MSC.

Can up-regulation CXCR4 expression enhance the ability of going back to the nest of other tissue-derived MSC (such as AMSC) other than marrow？Before Phase, inventor are improved using the method that the Presence of Several Cytokines (Flt-3ligand, SCF, IL-6, HGF, IL-3) stimulates Expression of the BMSC to CXCR4, and these cell factors are not necessarily all suitable for clinic, and due to the pleiotropism of cell factor, in this way Combination of cytokines may cause unforeseen serious side reaction, therefore this method is although effectively, be not suitable for facing Bed is promoted.Easier, safe and effective promotion MSC can be found to go back to the nest method？This in prior art is still to have Problem to be solved.

It is more and more studies have shown that MSC can selectively go back to the nest in the damage location of Various Tissues, due to quantity pole of going back to the nest Less, MSC plays a role limited, therefore the efficiency for improving stem cell homing seems quite urgent.It is improved using various measures dry thin The efficiency that born of the same parents go back to the nest, it will improve the effect of stem cell transplantation.MSC pretreatment be improve its efficiency of going back to the nest main path it One.

There is the primary MSC of studies have shown that ability of going back to the nest stronger.But the MSC clinical test carried out at present mostly uses defeated through vein The method of note, required MSC quantity is larger, is difficult to meet using primary MSC transplanting and treats required cell quantity.

It is transfected using gene and increases the molecule that MSC expression promotes it to go back to the nest, and improve the strategy of its efficiency of going back to the nest at present One of.Currently used Gene transfer techniques are mainly virus carrier system and non-viral carrier systems, they respectively have excellent lack Point.Viral vectors transfection efficiency is relatively high, but its immunogenicity is big, targeting is poor, and has potential oncogenicity.Non-viral load Body safety and targeting are preferable, but transfection efficiency is not good enough always.And Gene transfer techniques take a long time in vitro.

Early period, inventor is using the Presence of Several Cytokines (Flt-3ligand, SCF, IL-6, HGF, IL-3) stimulation Method is not only with high costs come the method that improves expression of the BMSC to CXCR4, but also safety can not ensure.These cells because Son is not all to go through to be applied to clinic, and due to the pleiotropism of cell factor, such combination of cytokines is used for people Body is possible to induce the appearance of unpredictable serious side reaction.

Summary of the invention

The object of the present invention is to provide a kind of preprocess methods for AMSC, are able to maintain the stable cytologic characteristic of AMSC And good proliferation activity, so as to ensure the quality and quantity of MSC needed for clinical application, while more using easy and price Cheap method effectively improves implantation capability after AMSC migration and transplanting, specifically, the invention is realized in this way：

The first aspect of the present invention provides the amnion-derived mescenchymal stem cell preprocess method of people, including：It is right respectively AMSC and AEC separates secondary culture, collects the AEC in the AMSC and P5 generation in P10 generation, will be in the AMSC and P5 generation in P10 generation AEC carry out Indirect co-culture processing；Or the AEC in P3 generation is used to prepare to the AMSC being used in P10 generation after conditioned medium CMC model is carried out, treated AMSC is collected, obtains pretreated AMSC.

Further, the AMSC and AEC separation secondary culture further includes purifying and amplification processing.

Further, the separation secondary culture of the AMSC, purifying and amplification, which are handled, includes：

It draws materials and is inoculated with, after inoculation, complete DMEM/F12 culture medium is added, be placed in humidified incubator and stand training It supports；It is slightly dry to tissue block, when can securely be attached in bottle wall, culture solution is made slowly to infiltrate tissue block, stationary culture；

Supply complete medium afterwards for 24 hours, liquid is changed after 3~5 days and remove the tissue block of not adherent floating, when after cell it is raw Long density carries out secondary culture when reaching 80~90% fusion；

Remove tissue block, suck tissue block and outmoded culture solution, with D-Hank ' s liquid, it is possible to use PBS or Hanks etc. its After his balanced salt solution cleaning, 0.125% trypsin solution is added into culture bottle and digests at room temperature；

It to cell body retraction, is rounded, space between cells becomes larger but when without departing from bottle wall, and it is whole that newborn bovine serum stoste is added Only digest；

The cell formation cell suspension to fall off in bottle wall, centrifugal treating,

Supernatant is abandoned, full DMEM/F12 culture medium is added and cell is resuspended, seed cells into culture bottle, then is complete to adding DMEM/F12 culture medium is labeled as P1 generation, sets in incubator and cultivate；

When attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3 carry out passage inoculation；

Collect AMSC within P10 generation.

Further, the separation secondary culture of the AEC, purifying and amplification, which are handled, includes：

Block amnion tissue fetch into centrifuge tube, 0.125% pancreatin is added, is placed in 37 DEG C of incubators and digests 3 times, with serum Digestion is terminated, the liquid of 3 digestion, 200 mesh net filtrations, centrifugal treating are collected；

Cell is resuspended with complete DMEM/F12 culture medium, is inoculated in culture bottle, and add complete DMEM/F12 culture medium； It is fused to each other to attached cell up to 80~90%, outwells outmoded culture solution, after being cleaned with D-Hank ' s liquid, be added into culture bottle Trypsin solution digests at room temperature；

It to cell body retraction, is rounded, space between cells becomes larger but when without departing from bottle wall, and it is whole that newborn bovine serum stoste is added It only digests, the cell in bottle wall that falls off makes to form cell suspension centrifugal treating；Supernatant is abandoned, full DMEM/F12 culture medium is added and is resuspended Cell seeds cells into culture bottle, then to complete DMEM/F12 culture medium is added, is labeled as P1 generation, sets in incubator and train It supports；

When attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3 carry out passage inoculation；

Collect AEC within P5 generation.

Further, the AEC progress Indirect co-culture in the AMSC and P5 generation by P10 generation, which is handled, includes：

The AMSC within the P10 generation and AEC within P5 generation is taken respectively, and outmoded culture medium in respective culture bottle is sucked out respectively, And washed 2 times with D-Hank ' s liquid, trypsin digestion is added, after the respective cell body retraction of AMSC and AEC, being rounded, Newborn bovine serum stoste is added immediately and terminates digestion, will cell shift centrifuge tube centrifugal treating after discard supernatant liquid, with being free of Cell is resuspended in the DMEM/F12 culture medium of serum；After the AMSC handled well is put into suspension type culture dish, the AEC that will handle well It is seeded in suspension type culture dish and obtains carrying out Indirect co-culture in small indoor placement to incubator；

It discards suspension type culture dish after 48h to take out AMSC, digestion is collected, and pretreated AMSC is obtained.

Further, the AEC by P3 generation is used to prepare the AMSC being used in P10 generation after conditioned medium and carries out item Part culture includes：

(1) preparation of conditioned medium：The AEC within P3 generation is taken, outmoded culture medium in culture bottle is sucked out, and use D- Hank ' s liquid is washed 2 times, and the DMEM/F12 culture medium for being free of serum is added, and places to culture 48h in incubator, culture is sucked out Base filters culture medium into sterile centrifugation tube with the Millex syringe filters that aperture is 0.42umPVDF film, freezes after sealing up for safekeeping It deposits spare, obtains conditioned medium；

(2) AMSC within P10 generation is taken, outmoded culture medium in culture bottle is sucked out, and washed 2 times with D-Hank ' s liquid, is added Trypsin digestion, after cell body retraction, be rounded after, immediately be added newborn bovine serum terminate digestion, by cell be transferred to from Centrifugation in heart pipe；Discard supernatant liquid；It is added into thawing, carrying out CMC model in the conditioned medium resuspension after rewarming, through condition Pretreated AMSC is obtained after cultivating 48h.

It is a further object of the invention to provide the amnion-derived mescenchymal stem cell preprocess methods of people to fill between raising Application in matter stem cell homing ability and et al. Ke quantity.

It is a further object of the invention to provide the amnion-derived mescenchymal stem cell preprocess methods of people to improve hematopoiesis Application in stem cell transplantation success rate.

Above-mentioned technical proposal of the invention has following beneficial technical effect：

1. preprocess method through the invention, after AMSC and AEC co-culture pretreatment, due to AEC can secrete it is many thin Intracellular cytokine co-cultures under conditions of serum-free with AMSC, can maintain the good growth activity of AMSC, while AEC by dividing certainly It secretes or paracrine, generation can promote the cell factor of AMSC surface expression CXCR4；Under conditions of lacking nutrition, AMSC is Maintenance own growth needs, and can also increase the table of cell CXCR4 with autocrine Some cytokines, these cell factors It reaches, so as to make the CXCR4 of AMSC express obvious up-regulation, enhances the SDF-1/CXCR4 axis effect of AMSC, while the work of AMSC Power is not significantly affected；Early-stage study shows that enhancing the effect of SDF-1/CXCR4 axis increases going back to the nest and intracorporal implantation for AMSC Quantity, clinical application and raising HSCT success rate and curative effect to AMSC have important practical significance；2. through the invention Preprocess method, after the pretreatment of AEC conditioned medium, the cytokine profiles secreted in conditioned medium containing AEC, and Conditioned medium is free of serum, under CMC model state, AMSC in order to maintain own growth to need, autocrine part cell because Son, these cell factors increase the expression of cell CXCR4 jointly.

3. conditioned medium preprocess method is advanced optimizing and improveing to Indirect co-culture preprocess method, utilize Soluble cytokine caused by AEC, carries out growth support to AMSC, while improving it and going back to the nest and plant under serum-free condition Enter ability, conditioned medium preparation is easier, has many advantages, such as that at low cost, method is easy, it is spare to freeze.

4. Indirect co-culture since AEC and AMSC is in a common cultivating system, has, there are two kinds of iuntercellulars The possibility of the Inter Modulation (cross-talk) of signal is carried out by soluble factor, mechanism of action is complex.And AMSC Under serum-free culturing conditions, CXCR4 is also increased, it was demonstrated that its own cell factor generated is wherein rising centainly Effect, but cell Proliferation and activity are decreased obviously.Conditioned medium pretreatment both saved AEC generation soluble cell because Son, supports the growth of AMSC, at the same these cell factors can cooperate with AMSC in the case where lacking nutritional status autocrine it is solvable Sex factor, collective effect raises the CXCR4 expression of AMSC, and is obviously promoted its migration and implantation.

5. preprocess method through the invention increases AMSC and goes back to the nest and intracorporal implantation quantity, so as to meet Cell quantity required for treating；

It is easy and due to cheap 6. preprocess method of the invention, use cost is effectively reduced, meanwhile, this Method security is good, no obvious toxic-side effects.

Detailed description of the invention

Fig. 1 is the form of AMSC and growth pattern, growth curve display figure in embodiment 1；

The immunophenotype that Fig. 2 is AMSC in embodiment 1 shows figure；

Fig. 3 is AMSC in embodiment 3 in vitro at rouge and osteoblast differentiation photo；

Fig. 4 is the form of AEC and phenotypic evaluation photo and schematic diagram in embodiment 4；

Fig. 5 is to be co-cultured pretreated AMSC in embodiment 7 to enhance schematic diagram along SDF-1 concentration gradient transfer ability；

Fig. 6 is the schematic diagram that 7 conditional culture group AMSC of embodiment shows migration advantage；

Fig. 7 is PKH26 in embodiment 7⁺AMSC goes back to the nest the schematic diagram of capacity variation in short term；

Fig. 8 is that the processing of AEC conditioned medium increases AMSC and goes back to the nest in short term the schematic diagram of quantity in embodiment 7；

Fig. 9 is the schematic diagram that 250cGy irradiates Radiation in jury situation after Recipient mice transplanting in embodiment 7；

Figure 10 is the schematic diagram that real-time quantitative PCR detects the implantation of human archeocyte in Recipient mice marrow in embodiment 7；

Figure 11 is the people in embodiment 7 in immunohistochemical analysis receptor NOD/SCID mouse marrow long-term cultivation stromal cells layers The schematic diagram of source cell.

Wherein：

There is cell to climb out of (× 100) around visible tissue block within 7 days after the inoculation of A. amnion in Fig. 1.B.AMSC l is for cell master It to be fibroblast sample, a small amount of epithelial cell like cell is mingled with (× 100) therebetween.C.3 uniform for cellular morphology, it is radial It grows (× 100).D.6 still it is in spindle shape for cell, there are good proliferative capacity (× 100).E.AMSC growth curve.

In Fig. 3, A. is at rouge blank control；B. oil red O dye is positive after adipogenic induction；C. skeletonization blank control；D. skeletonization Von Kossa dyes visible a large amount of calcium depositions after induction.

In Fig. 4, A：HE dyes (200 ×)；B：Pan-CK immunohistochemical staining (200 ×)；C：Flow cytometry analysis AEC The expression of upper pan-CK.

In Fig. 5, in three time point A, B, C (for 24 hours, 48h, 72h), co-cultivation group and free serum culture group AMSC migration Ability is significantly higher than serum free culture system group (P < 0.05).Co-culture the AMSC transfer ability no significant difference (P with free serum culture ≥0.05).AMSC all shows dose dependent to the migration of SDF-1.

In Fig. 7, A. control group (injecting normal saline group)；B. CMC model group；C. serum free culture system group；D.CXCR4 is neutralized Antibody group.

In Fig. 9, A. serum IgG concentation：Transplanting is through the pretreated AMSC group of conditioned medium (i.e. condition group) peripheral blood Cell resume speed is accelerated compared with serum group, then reduces this effect through the incubation of CXCR4 neutralizing antibody.B. after Recipient mice transplanting Sections of Bone Marrow detects marrow hemopoiesis hyperplasia degree：A. pretreated group, b. serum group, c. neutralizing antibody group, d. is blank control group (100×)。

In Figure 10, A. standard curve：Genome is extracted after being mixed with AMSC gradient dilution with NIH 3 T 3 cells in vitro system DNA carries out PCR reaction, to establish standard curve.B. the serum group receptor using routine culture system culture AMSC transplanting is small It is not detected the implantation of human archeocyte in mouse marrow, and AEC conditioned medium treated AMSC transplantation group (condition group, i.e., in advance Processing group) human archeocyte implantation can be measured in mouse bone marrow cells, and if pre-process AMSC with the neutralizing antibody of CXCR4 (ɑ-CXCR4), And when being therewith injected into Mice Body, human archeocyte implantation level is decreased obviously (P<0.01, n=3)

In Figure 11, A. human specific HLA-I antibody dyes (100 ×)：A. serum group；B. pretreated group；C. pretreated group+ α-CXCR4.B. human specific 5B5 antibody dyeing (fibroblast) (200 ×):A. serum group；B. pretreated group.C. people is special Property vWF antibody dyeing (400 ×)：Pretreated group.

Specific embodiment

In order to make the objectives, technical solutions and advantages of the present invention clearer, With reference to embodiment and join According to attached drawing, the present invention is described in more detail.It should be understood that these descriptions are merely illustrative, and it is not intended to limit this hair Bright range.In addition, in the following description, descriptions of well-known structures and technologies are omitted, to avoid this is unnecessarily obscured The concept of invention.

Technical terms according to the present invention：Hematopoietic stem cell transplantation (hematopoietic stem cell transplantation,HSCT)；Mescenchymal stem cell (mesenchymal stem cells, MSC)；Medulla mesenchyma is dry thin Born of the same parents (bone marrow MSC, BMSC)；Human amnion mesenchymal stem cell (amniotic MSC, AMSC)；Amniotic epithelial cells (epithelial cells, AEC), Chemokine receptor CXCR4 are chemotactic factor (CF) stromal cell derived factor-1 (SDF1) Specific receptors, SDF1/CXCR4 axis play a significant role in MSC transition process；

Embodiment 1AMSC's is separately cultured, purifies and expands

(1) it draws materials：Placenta (the serum such as a hepatitis full set, HIV, syphilis of health full term Cesarean esction fetus are taken under aseptic condition Learn reaction and show feminine gender, multipara's informed consent), the amnion of blunt separation placental fetal surface about 10cm × 10cm floats repeatedly It washes, removes blood clot.

(2) it shears：Amnion is shredded into about 1mm³Fritter, in rotten shape.

(3) it is inoculated with：Several tissue fritters are drawn with suction pipe, are placed in culture bottle, the dispersion of tissue fritter is inoculated into culture In bottom of bottle, mutual distance about 0.5cm~1cm is advisable between fritter, every 75cm²Culture bottle can be inoculated with 45~60 pieces.

(4) it cultivates：After inoculation, the complete DMEM/F12 culture medium of about 3ml is added, lightly culture bottle is overturn, is made The bottom of bottle for being inoculated with tissue block upward, marks, is placed in 37 DEG C, 5%CO₂Stationary culture in saturated humidity incubator.2~4h Afterwards, slightly dry to tissue block, when can securely be attached in bottle wall, then culture bottle is slowly overturn, culture solution is made slowly to infiltrate tissue block, Stationary culture.It supplies complete medium afterwards for 24 hours, liquid is changed after 3~5 days and removes the tissue block of not adherent floating, about 1 week or so Cell can be observed to climb out of from tissue block edge, form outgrowth.Thereafter according to culture medium color and cell growth status, every 4 ~5d full dose is changed liquid 1 time.About two weeks or so cell densities reach 80~90% fusions, can carry out secondary culture at this time.This The primary cell of kind method culture is the mixture of the various kinds of cell such as MSC, fibroblast and epithelial cell, in the training of cell During supporting, liquid is changed by full dose and removes not adherent cell.Since AMSC has extremely strong adherent Adhering capacity, adherent mistake Journey is fast compared with other cells, therefore, in secondary culture can by control pancreatin digestion time, adherent method gradually removes epithelium repeatedly And fibroblast, realize the purifying of AMSC.Cell quality is provided for the clinical application curative effect and raising HSCT success rate of AMSC It ensures.

(5) it passes on for the first time：Tissue block is gently picked out with suction pipe, sucks tissue block and outmoded culture solution, with D-Hank ' s liquid After cleaning 2 times, 0.125% trypsin solution about 4ml is added into culture bottle, digests at room temperature.It is returned to cell major part cell space It contracts, be rounded, space between cells becomes larger but when without departing from bottle wall, and the newborn bovine serum stoste that about 1ml is added terminates digestion, when digestion Between be usually no more than 2min.Cell in piping and druming bottle wall makes it fall off, and forms cell suspension, is transferred in centrifuge tube, 1000r/ Min is centrifuged 5min.Supernatant is abandoned, full DMEM/F12 culture medium is added and cell is resuspended, count.Cell presses 3~6 × 10⁵/cm²It is close Degree is inoculated into 75cm²In culture bottle, then complete DMEM/F12 culture medium is added to each bottle and sets culture labeled as P1 generation to 15ml It is cultivated in case.Liquid is changed in time thereafter according to culture medium color change, is generally advisable with 3~4d.

(6) continue to pass on：When attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3 Passage inoculation is carried out, algebra is successively marked.

(7) it collects AMSC within P10 generation and is used as pretreatment object.

Through isolating and purifying, cellular morphology is uniform, maintains spindle shape, keeps good proliferative capacity (Fig. 1).Flow cytometry inspection It surveys AMSC high as the result is shown and expresses CD29 (97.69% ± 0.18%), CD44 (98.70% ± 0.25%), CD105 (99.20% ± 0.20%), the negative expression (< 1.5%) of repeated detection CD31, CD34, CD45, Pan-CK display, as shown in Figure 2.

Embodiment 2.

AEC's is separately cultured, purifies and expands

(1) amnion method of drawing material is the same.

(2) 0.125% pancreatin 5ml is added, is placed in 37 DEG C of incubators into centrifuge tube with tweezers clamping several piece amnion tissue During digestion, it is primary to rock centrifuge tube every 20min oscillation for digestion 3 times, each 40min or so.It is terminated and is digested with serum.It receives Collect the liquid of 3 digestion, 200 mesh net filtrations, 1000r/min is centrifuged 5min.

(3) cell is resuspended with complete DMEM/F12 culture medium, with 6 × 10⁵/cm²It is inoculated in 75cm²In culture bottle, add Full DMEM/F12 culture medium is to 15ml.

(4) liquid is changed after 48h for the first time, hereafter every 2d changes liquid, and attached cell is fused to each other up to 80~90% within about 5~7 days, outwells Outmoded culture solution after cleaning 2 times with D-Hank ' s liquid, 0.25% trypsin solution about 3ml is added into culture bottle, disappears at room temperature Change.It to cell major part cell space retraction, is rounded, space between cells becomes larger but when without departing from bottle wall, and the newborn ox blood of about 1ml is added Clear stoste terminates digestion, digestion time 5min or so.Cell in piping and druming bottle wall makes it fall off, and forms cell suspension, is transferred to In centrifuge tube, 1000r/min is centrifuged 5min.Supernatant is abandoned, full DMEM/F12 culture medium is added and cell is resuspended, count.Cell presses 6 ×10⁵/cm²Density be inoculated into 75cm²In culture bottle, then to each bottle complete DMEM/F12 culture medium is added to 15ml, be labeled as It in P1 generation, sets in incubator and cultivates.

(5) when attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3, which carry out passage, connects Kind, passage label is followed successively by P2, P3 generation.

(6) it collects AEC within P5 generation and is used for subsequent experimental.

Using pancreatin substep, repeatedly digestion method can obtain a large amount of cells in a short time, while cell being kept well to live Property.The assurance to digestion time is especially paid attention in digestion process, the time, too short digestion was insufficient, so that the cell concentration of harvest is few； Overlong time is easy digestion excessively, and cell viability is caused to decline.Can get purer AEC after passage, for building AEC in next step with AMSC is co-cultured and CMC model system provides abundant cell origin.

Embodiment 3 identifies the AMSC that embodiment 1 obtains

The identification of AMSC depends on comprehensive to the progress such as its morphology, cell surface molecule mark and differentiation capability Close identification.The AMSC for obtaining high quality is the premise for obtaining good clinical efficacy, ensureing patient safety.

The immunophenotype of 3.1 Flow cytometry AMSC

(1) take P3 for 0.125% trypsin digestion of cell after, washed 1 time with the PBS containing 0.5%BSA, abandon supernatant, It is resuspended with the PBS containing 0.5%BSA, is separately added into following primary antibody：Monoclonal antibody CD11a, CD11b of mouse anti human, CD29, CD31, CD34, CD44, CD45, CD105, HLA-DR, Pan-CK set 4 DEG C of incubation 30min.

(2) it is washed 2 times with the PBS containing 0.5%BSA, the sheep anti mouse secondary antibody of FITC label is added, sets 4 DEG C of incubation 30min.

(3) it is washed 2 times, cell is suspended in the PBS of 300 μ l with the PBS containing 0.5%BSA, carry out flow cytometer point Analysis.Primary antibody is replaced with the PBS containing 0.5%BSA, FITC secondary antibody is added, as blank control.As a result 2.9 software of WinMDI is used Analysis.

3.2 directional induction AMSC are to Adipocyte Differentiation

(1) take the good P3 of growth conditions for AMSC by 3 × 10⁴/ ml density is inoculated in 25cm²It is raw to cell in culture bottle After 60% fusion, lipoblast induction differentiation liquid (10 is added^-6Mol/L dexamethasone+0.5mol/L 1- methyl -3- is different Butyl-xanthine+0.1mol/L Vit C+1% blueness-streptomysin+10%FBS IMDM) it is used as experimental group, control group, which is not added, to lure Lead differentiation liquid.

(2) every 3d is changed the liquid once in half, and the formation of fat drop is observed under inverted microscope.Rouge is detected using oil red O stain Fat drop：Cell 10min is fixed with 10% formaldehyde, distilled water is cleaned, oil red O stain 5min, then redyes cell with alum hematoxylin Core, distilled water are cleaned, glycerin gelatine mounting.

(3) it microscopically observation and takes a picture.

3.3 directional induction AMSC are to osteoblast differentiation

(1) take the good P3 of growth conditions for AMSC by 2 × 10⁴/ ml density is inoculated in 25cm²It is raw to cell in culture bottle After 60% fusion, addition osteoblast induction differentiation liquid (10-7mol/L dexamethasone+10mol/L β phosphoglycerol+ 0.05mol/L Vit C+1% blueness-streptomysin+10%FBS IMDM) it is used as experimental group, induction differentiation liquid is not added in control group.

(2) every 3d is changed the liquid once in half.Detection calcified matrix precipitating is dyed using Von Kossa：It is fixed with 10% formaldehyde After cell 1h, deionized water is cleaned, and 2% silver nitrate solution is added, and 37 DEG C are protected from light 10min, after being cleaned with deionized water, day 15min is exposed under light lamp.

(3) light microscopic observation calcified matrix is precipitated and is taken a picture.Identification the result shows that, such as Fig. 2, above-mentioned separation shown in 3 is pure The cell of change is AMSC.

Embodiment 4 identifies the AEC that embodiment 2 obtains

It is current studies have shown that amnion is not only that fetus provides good growing environment, also deeply grinding for stem cell Study carefully and provide new sources, the development and utilization to amnion cell function is the hot fields of medical experiment and clinical research in recent years One of.AEC can secrete many cell factors, while maintenance AMSC good growth activity, can raise the CXCR4 of AMSC Expression, to enhance its migration and implantation capability.Therefore, the purifying AEC for obtaining sufficient amount of high quality is to ensure the present invention The precondition of pretreating effect.

4.1 AEC HE dyeing

(1) cell climbing sheet is prepared：Creep plate is placed in the concentrated sulfuric acid and impregnates and stays overnight, is rushed first with tap water after next day taking-up Wash 20 times, then be placed in absolute alcohol and impregnate 6h, later use distilled water flushing 3 times, creep plate is placed in culture dish dry it is laggard Horizontal high voltage disinfection.It is put into oven later to dry, is put into spare in superclean bench.

(2) it takes P3~P5 for cell, after digestion, centrifugation, cell is resuspended with complete DMEM/F12, single cell suspension is made. Slide is put into culture dish, cell suspension is slowly dropped into slide center (2~3 drop).It is observed under inverted microscope thin Culture dish is put into 37 DEG C, stationary culture in 5%CO2 saturated humidity incubator after adherent satisfaction by the adherent situation of born of the same parents.

(3) cell climbing sheet is taken out, PBS is gently washed 3 times, and 4% neutral formalin is fixed, after room temperature fixes 15~30min, by table Face liquid air-dries.

(4) slide prepared is washed 2 times, each 1min with PBS, distillation washing 1min；Hematoxylin disseminates 10min；From Water rinses 20min；1% hydrochloride alcohol breaks up 0.5~1min；Again with distilled water flushing a moment to a few hours；1% ammonium hydroxide returns indigo plant 1min；Yihong aqueous solution disseminates 30s；Distilled water is developed a film quarter；75% alcohol I is dehydrated 3~5inin；95% alcohol II dehydration 3~ 5min；Absolute alcohol is dehydrated 3~5min of I；Absolute alcohol is dehydrated 3~5min of II；Transparent liquid I, transparent 5~10min；Transparent liquid II, transparent 5~10min；Neutral gum mounting is used after drying.Microscopically observation.

The identification of 4.2pan-CK immunofluorescence dyeing

(1) digestion collects P3 for cell, is washed 1 time, 3000r/min with PBS, is centrifuged 5min.Supernatant is abandoned, 500 μ are added in every pipe Cell, room temperature is resuspended in l Fix/Perm, and darkroom is incubated for 20min.3000r/min is centrifuged 5min, abandons supernatant, and 1ml is added in every pipe Cell, room temperature is resuspended in Perm/Wash, and darkroom is incubated for 10min.

(2) after being incubated for, 3000r/min is centrifuged 5min, abandons supernatant, and cell is resuspended with 50 μ l Perm/Wash in every pipe, The pan-CK antibody of 4 μ l PE label, room temperature is added, darkroom is incubated for 40min.Add 1ml Perm/Wash, 3000r/min, is centrifuged 5min.Supernatant is abandoned, flow cytometer row detects after cell is resuspended in 300 μ l PBS.

(3) replace the pan-CK antibody of PE label as blank control using the PBS of equivalent in step (2).As a result it uses The analysis of 2.9 software of WinMDI.

The identification of 4.3pan-CK immunohistochemistry

(1) cell climbing sheet production is the same.

(2) it takes P3~P5 for cell, after digestion, centrifugation, cell is resuspended with complete DMEM/F12, single cell suspension is made. Slide is put into culture dish, cell suspension is slowly dropped into slide center (2~3 drop).It is observed under inverted microscope thin Culture dish is put into 37 DEG C, stationary culture in 5%CO2 saturated humidity incubator after adherent satisfaction by the adherent situation of born of the same parents.

(3) cell climbing sheet is taken out, PBS is gently washed 3 times, and 4% neutral formalin is fixed, after room temperature fixes 15~30min, by table Face liquid air-dries Hou Song pathology department row pan-CK immunohistochemical staining.

Histochemical staining confirms pan-CK high expression, as shown in figure 4, showing that the method for our above-mentioned isolated AEC can obtain The AEC of high-purity provides the source AEC of high quality for subsequent co-cultivation and preparation condition culture medium.

The preprocess method of 5 Indirect co-culture system of embodiment

(1) it takes P3 for AMSC, outmoded culture medium in culture bottle is sucked out, D-Hank ' s liquid is washed 2 times, and 0.125% pancreas egg is added White enzyme 1.5ml digests, and observes under inverted phase contrast microscope, and after cell major part cell space retraction, being rounded, 0.5ml is added immediately Newborn bovine serum stoste terminate digestion, digestion time be no more than 2min.It is blown and beaten with elbow straw, cell is fully transferred to In the disposable sterilized centrifuge tube of 15ml, 1000r/min is centrifuged 5min.Liquid is discarded supernatant, with the single DMEM/F12 for being free of serum Cell is resuspended in culture medium, counts, with 1 × 10⁵A/hole is seeded to 6 orifice plates, adjusts every hole culture medium to 2.5ml.

(2) it is put into Millicell suspension type culture dish (0.4 μm of aperture).

(3) it takes P3~P5 for AEC, outmoded culture medium in culture bottle is sucked out, D-Hank ' s liquid is washed 2 times, and 0.25% pancreas is added Protease 1ml digests, and observes under inverted phase contrast microscope, and after cell major part cell space retraction, being rounded, 0.5ml is added immediately Newborn bovine serum stoste terminate digestion, digestion time 5min or so.It is blown and beaten with elbow straw, cell is fully transferred to 15ml Disposable sterilized centrifuge tube in, 1000r/min, be centrifuged 5min.Liquid is discarded supernatant, is cultivated with single DMEM/F12 without serum Base weight hangs cell, counts, with 1 × 10⁴A/hole is seeded to the small interior Millicell, adjusts every hole culture medium to 1.5ml.Label For co-cultivation group, place to cultivating in incubator.

(4) in order to observe with after AEC co-cultivation, AMSC is in cellular morphology, cell activity, CXCR4 expression and migration energy Free serum culture and serum free culture system (i.e. Nostoc commune Vanch mode) is arranged as control in variation in terms of the biological characteristics such as power.It takes Same batch P3 is resuspended respectively with single DMEM/F12 culture medium, complete DMEM/F12 culture medium, by above-mentioned side for AMSC after digestion Method and concentration are seeded to 6 orifice plates, are added without AEC, as a control group, are respectively labeled as free serum culture group, serum free culture system group, and It is cultivated in placement to incubator.

(5) 6 orifice plates are taken out after 48h, discards the cell Millicell, it is stand-by that AMSC is collected in digestion.

Preprocess method of the embodiment 6 based on conditioned medium

(1) preparation of conditioned medium：The AEC within P3 generation is taken, outmoded culture medium in culture bottle, D-Hank ' s liquid is sucked out It washes 2 times, the DMEM/F12 culture medium 15ml for being free of serum is added.It places to culture 48h in incubator, culture medium is sucked out, with Aperture be 0.42umPVDF film Millex syringe filters (Millipore company of the U.S.) filter culture medium to 15ml it is sterile from In heart pipe, be put into after sealing up for safekeeping in -80 DEG C of refrigerators freeze it is spare.Culture medium culture without serum can promote AEC lacking battalion More cell factors are secreted in the case where supporting, to maintain own growth to need, these cell factors can both be tieed up in pretreatment AMSC growth activity is held, while can effectively raise its CXCR4 expression.Use aperture for the filtering of the filter of 0.42 μm of pvdf membrane, Not only the soluble cytokine in conditioned medium can be allowed to pass through, but also cell fragment, impurity and possibility therein can be removed Mixed floating AEC etc..- 80 DEG C freeze can soluble cytokine effectively in preservation condition culture medium, avoid its degradation.

(2) AMSC within P10 generation is taken, outmoded culture medium in culture bottle is sucked out, D-Hank ' s liquid is washed 2 times, is added 0.125% trypsase 4ml digests, and observes under inverted phase contrast microscope, after cell major part cell space retraction, being rounded, immediately The newborn bovine serum that 1ml is added terminates digestion, and digestion time is no more than 2min.It is blown and beaten with elbow straw, cell is all shifted In to disposable sterilized centrifuge tube, 1000r/min is centrifuged 5min.Discard supernatant liquid.Wherein the AMSC in CMC model group is added It thaws, the conditioned medium after rewarming is resuspended, supplementary condition culture medium to 15ml, labeled as CMC model group.Blood is set simultaneously Clear culture (i.e. Nostoc commune Vanch mode) group and free serum culture group are as control；Take P10 generation within AMSC, after digestion respectively with Complete DMEM/F12 culture medium (containing 10%FBS), DMEM/F12 culture medium are resuspended, and are respectively complemented to corresponding culture medium 15ml.It is cultivated in placement to incubator.

(3) each group is taken out after cultivating 48h, carries out subsequent detection.

The biological nature of the pretreated AMSC of embodiment 7 detects

1, CCK-8 method detection co-cultures AMSC cell-proliferation activity

(1) inoculating cell：Single cell culture liquid is made in the AMSC of each time point of each group, in addition to blank group other Every hole inoculation about 10⁴A cell is to 96 orifice plates, every 100 μ l of pore volume, 4 groups of every plate point, i.e. blank group, co-cultivation group, serum-free Culture group, serum free culture system group test divide equally 4 groups of progress below, 6 multiple holes of every group of setting, totally 3 plate.It is put into incubator and trains It supports overnight.

(2) 10 μ l of CCK-8 solution (being careful not to inject air formation bubble in a liquid) is added in every hole, and culture plate is set 3~4h is incubated in incubator.

(3) colorimetric：490nm is selected to measure wavelength, is returned to zero with blank well, measures each hole absorbance value (i.e. with microplate reader OD value), the results are shown in Table 1 for record, and experiment is repeated 3 times.

The detection of 1 CCK-8 method of table co-cultures AMSC cell-proliferation activity (indicating with OD value)

* it is higher than serum-free group, P < 0.05 in 48h, 72h, co-cultivation group and the AMSC absorbance value of serum free culture system group；And Co-culture the no significant difference between serum free culture system group

2, mtt assay testing conditions culture AMSC cell-proliferation activity

(1) inoculating cell：Single cell culture liquid is made in the pretreated AMSC of CMC model, in addition to blank group its He is inoculated with about 10 in every hole³A cell is to 96 orifice plates, every 100 μ l of pore volume, 4 groups of every plate point, i.e. blank group, CMC model group, blood Clear culture group, free serum culture group, test divide equally 4 groups of progress below, 6 multiple holes of every group of setting, totally 3 plate.It is put into incubator Middle overnight incubation.

(2) colour generation：10 μ l of MTT solution (being careful not to inject air formation bubble in a liquid), culture is added in every hole Case continues to be incubated for 4h.

(3) colorimetric：The supernatant in each hole is gently removed with micro sample adding appliance, (is paid attention to as far as possible not by the purple crystal in hole It removes in order to avoid influencing the measurement of absorbance value).100 μ lDMSO (dimethyl sulfoxide) are added in every hole, vibrate 10min, make to have crystallized Fully dissolved.Microplate reader measures absorbance value (OD492nm) at 492nm wavelength.Record is as a result, experiment is repeated 3 times.As a result such as table 2 It is shown.

2 mtt assay testing conditions culture AMSC cell-proliferation activity (being indicated with OD value) of table

* the AMSC absorbance value of CMC model group and serum free culture system group is higher than serum-free group, P < 0.05；And CMC model The no significant difference between serum free culture system group.

3, trypan blue staining detects AMSC cell viability

(1) Single cell culture liquid is made in the AMSC of each time point of each group, inhales 5 drop cell suspensions to Ep pipe with suction pipe In, 5 0.4% trypan blue dye liquors of drop are added, uniformly, living cells has an achromatophilia to trypan blue for piping and druming, and being added after dye liquor can be Living cells and dead cell are distinguished under microscope.

(2) 10 μ l cell suspensions drop is drawn in cell counting board, under the microscope.It counts thin in the block plaid at four angles Born of the same parents' (under not several on number, number is left not to count the right side), calculation formula：Cell viability=(total number of cells-indigo plant dye cell number)/total number of cells × 100%, experiment is repeated 3 times.As a result as shown in Table 3, 4.

The detection of 3 Trypan Blue of table co-cultures AMSC survival rate

* for 24 hours, 48h, 72h, the AMSC survival rate of co-cultivation group and serum free culture system group is above serum-free group, P < 0.05；And co-culture the no significant difference between serum free culture system

4 Trypan Blue testing conditions culture AMSC survival rate of table

* the AMSC survival rate of CMC model group and serum free culture system group is above serum-free group, P < 0.05；And CMC model The no significant difference between serum free culture system.

4, the expression of direct immuno-fluorescent antibody assay AMSC surface C XCR4

(1) cell is collected in digestion, is washed 2 times, 3000r/min with PBS, and 5min is centrifuged.

(2) supernatant is abandoned, cell is resuspended with 50 μ L PBS, the mouse anti human CXCR4 monoclonal antibody of 4 μ L PE label is added, Room temperature, darkroom are incubated for 40min.

(3) it after being incubated for, is washed twice with PBS, up flow type machine testing.

(4) labelled antibody is replaced with the PBS of equivalent in step (2), as blank control.As a result WinMDI 2.9 is used Software analysis, as shown in Table 5,6.

5 flow cytomery AMSC surface C XCR4 of table expression

* for 24 hours, 48h, 72h, the AMSC surface C XCR4 expression of co-cultivation group and free serum culture group is apparently higher than serum Culture group, P < 0.01；And co-culture the no significant difference between serum free culture system.

The expression of 6 flow cytomery each group AMSC surface C XCR4 of table

After * grouping culture 48h, the AMSC surface C XCR4 expression of CMC model group and free serum culture group is apparently higher than blood Clear culture group, P < 0.01；And no significant difference between CMC model group and serum free culture system group.

5, AMSC migration experiment is co-cultured

Migration test carries out in the cell Millicell (Millipore company), and detailed process is as follows：

(1) C-Buffer (DMEM/F12 culture medium+0.5%BSA) is prepared, chemotactic liquid and cell suspension use C-Buffer It prepares, using preceding pre-balance to 37 DEG C.

(2) upper chamber film (12 μm of aperture) is coated with FN：10ng/ml, 37 DEG C of 1h, is washed twice with PBS.

(3) chemotactic liquid is prepared：SDF-1 is diluted to 0,100,200,300ng/ml with C-Buffer.Add in 24 orifice plates Enter the hole chemotactic liquid 1ml/, is then put into upper chamber, 37 DEG C of balance 1h.

(4) each group AMSC cell dissociation is collected, is resuspended with C-Buffer, single cell suspension is made, adjust cell concentration It is 2~4 × 10⁵A/ml.150 μ l cell suspensions are added in each Millicell upper chamber.To verify the specificity migrated, In part Experiment, the neutralizing monoclonal antibody (10 μ g/ml, Ebioscience company) that anti-CXCR4 is added is incubated for.

(5) 37 DEG C, cultivate for 24 hours in 5%CO2 saturated humidity incubator.

(6) cell Millicell is carefully taken out with tweezers, blots upper chamber liquid, is transferred to 800 μ l methanol being previously added Hole in, the fixed 30min of room temperature.

(7) cell Millicell is taken out, upper chamber fixer is blotted, is transferred to 800 μ l crystal violet dye liquors being previously added Kong Zhong, room temperature dye 15~30min.

(8) cell Millicell is put into clear water to rinse and is impregnated for several times, taken out, blot upper chamber liquid, it is small with wet swab stick The heart wipes the cell of upper chamber bottom filter membrane surface.

(9) filter membrane is carefully taken off with tweezers, bottom surface is dried upward, is transferred on glass slide, with neutral gum mounting.

(10) it counts and is migrated to the cell number of filter membrane outer surface under microscope, every filter membrane takes 5 visuals field to count at random, real It tests and is repeated 3 times.

As a result see Fig. 5,6.

6, the detection for ability of going back to the nest in AMSC body after pretreatment

(1) prepared by Recipient mice model：NOD/SCID mouse (6-8 week old, female) is through gamma-rays sublethal dose 250cGy irradiation；

(2) AMSC is transplanted：In NOD/SCID mouse 4h after irradiation, pretreated AMSC (6 × 10 will be passed through⁶/ only) warp Tail vein injection enters in Mice Body, and the AMSC group (6 × 10 of serum free culture system is arranged⁶/ only), blank control group injects isometric physiology Salt water.In the transplantation group of part, preceding neutralizing monoclonal antibody (10 μ g/ are only) the preparatory incubated cell with anti-CXCR4 of transplanting, and By cell together with excessive neutralizing antibody intravenous administration mouse.

(3) AMSC dyes (carrying out according to the specification of Sigma company) with PKH26.

(4) AMSC through PKH26 dyeing is transplanted

By receptor NOD/SCID mouse (6-8 week old/female) in advance after caesium source full-body exposure 250cGy in 4h from tail Vein inputs the AMSC (6 × 10 dyed through PKH26⁶/ only), control group mice then injects isometric physiological saline.In some realities In testing, with neutralizing monoclonal antibody (the 10 μ g/10 of anti-CXCR4 before transplanting⁶Cell) preparatory incubated cell, and by cell and mistake The neutralizing antibody of amount enters mouse through tail vein injection together.

All mouse raise in an aseptic environment to transplant after for 24 hours, with cervical dislocation put to death mouse.Take Recipient mice Marrow, with flow cytometry analysis wherein PKH26⁺The quantity of donorcells, and to make without the marrow of cell transplantation mouse For control, false positive is removed.As a result as shown in Fig. 7,8.

7, the ability detection of the promotion Radiation in jury of AMSC

(1) Radiation in jury

After the transfer the 1st, 3,6,9,12,15,22,29,36,43 day, blood is taken from the tail vein of Recipient mice, analysis is outer The quantity variation of leucocyte, red blood cell and blood platelet in all blood.As shown in Figure 9.

(2) marrow hemopoiesis restores detection

The 14th day after transplanting, partial receptor mouse is put to death, prepares Sections of Bone Marrow, HE dyeing observation hematopoietic cell restores feelings Condition, as shown in Figure 9.

8, AMSC is chronically implanted and breaks up

The 4-6 month after transplanting Recipient mice is put to death, marrow is taken, detects human archeocyte using the method for real-time quantitative PCR It is implanted into situation.In order to further appreciate that the differentiation situation of implantation cell by flow cytometry, is exempted from using human-specific antibody The method of epidemic disease group and RT-PCR, detect Recipient mice in specific human hematopoietic cell surface marker (CD34, CD45, CD2, CD7, CD10, CD13, CD14, CD19, CD33, CD38 etc.), the expression feelings of endothelial cell marker vWF and HLAclass-I antigen The staining conditions of condition and human specific fibroblast monoclonal antibody 5B5.

To understand implantation of the human archeocyte in Recipient mice bone marrow matrix, Dexter marrow long-term cultivation body is used System, changes the liquid once in half weekly, after 37 DEG C, 5%CO2 culture 2-3 weeks, discards supernatant, suspension cell is removed, paraformaldehyde is solid Determine stromal cells layers, the implantation of human archeocyte is detected with anti-human HLAclass-I monoclonal antibody, with specific C D45, vWF and 5B5 antibody It detects donorcells and breaks up situation.

8.1 real-time quantitative PCR

With the content of the human specific DNA in the method detection Recipient mice marrow of TaqMan.Specific method is expanded with PCR Increase the specific fragment of one section of long 480bp on No. 17 chromosome of people.Using QLAamp DNA mini kit (QIAamp DNA Mini Kit, Qiagen company) extracting bone marrow cell genomic DNA.Primer is：5 '-GGG ATA ATT TCA of upstream GCT TAA ACA G-3 ', 5 '-AAA CGT CCA CTT GCA GAT TCT AG-3 ' of downstream.Probe is：5'FAM CAC GTT TGA AAC ACT CTT XT TTG CAG GATC p (X=TAMRA).Reaction uses 50uL system, containing AmpliTaq gold Board enzyme (AmpliTaq Gold enzyme, Applied Biosystems), 200uM dNTP, 2mM MgCl2,250nM primer, The genomic DNA template of 10nM TaqMan probe and 250ng is analyzed, reaction condition with ABI7500 real-time PCR For 94 DEG C of initial denaturations 10min, 94 DEG C of denaturation 15s, 60 DEG C of annealing 50s, totally 45 circulations, each sample set 3 multiple holes, use AMSC Genomic DNA is extracted after mixing with NIH 3 T 3 cells in vitro system, PCR reaction is carried out, to establish standard curve.Such as Figure 10 institute Show.

8.2 flow cytometry

Marrow single cell suspension is prepared, with erythrocyte cracked liquid lysed erythrocyte, after employment AB serum block Fc receptors, is added Enter human specific monoclonal antibody HLA class-I, CD45, CD2, CD7, CD10, CD13, CD14, the CD19 of FITC or PE label, CD33, CD34, CD38 or rat anti-mouse CD45 monoclonal antibodies are incubated for, and with the addition of the mouse bone marrow cells of non-acceptor's stem cell transplantation Allo-antibody dyeing is as control.The isotype control Ab marked with FITC or PE is dyed to remove non-specific fluorescence background. Flow cytomery, analysis, as shown in table 7.

Human archeocyte after table 7. is transplanted in 6 months flow cytometry receptor NOD/SCID mouse marrow

8.3 immunohistochemistry

1. with medulla biopsy slice is prepared.

2. 3% hydrogen peroxide H2O2 is incubated for 5min, to close endogenic peroxidase, 0.1MPBS rinses 2 times, often Secondary 3min.

3. Normal Goat Serum working solution is closed, why it is incubated for 10min.

4. being added dropwise, through appropriate diluted human specific primary antibody, (CD45, human HLA class-I, vWF and human specific are fine Tie up mother cell monoclonal antibody 5B5), it is incubated at room temperature 40min, 0.1M PBS is rinsed 2 times, each 3min.

5. universal biotinylation IgG antibody is added dropwise, it is incubated at room temperature 40min, 0.1M PBS is rinsed 2 times, each 3min.

6. horseradish enzyme label strepto- avidin working solution is added dropwise, incubation at room temperature 40min, 0.1M PBS flushing 2 times, every time 3min。

7. DAB solution develops the color.Distilled water flushing, haematoxylin redye, be dehydrated, mounting.

8. observing, take a picture.

As a result such as table 8, shown in Figure 11.

8. immunohistochemical staining of table analyzes the human archeocyte in receptor NOD/SCID mouse marrow

It should be understood that above-mentioned specific embodiment of the invention is used only for exemplary illustration or explains of the invention Principle, but not to limit the present invention.Therefore, that is done without departing from the spirit and scope of the present invention is any Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.In addition, appended claims purport of the present invention Covering the whole variations fallen into attached claim scope and boundary or this range and the equivalent form on boundary and is repairing Change example.

Claims (10)

1. the amnion-derived mescenchymal stem cell preprocess method of people, it is characterised in that including：

Secondary culture is separated to AMSC and AEC respectively, collects the AEC in the AMSC and P5 generation in P10 generation；

AEC in AMSC and P5 generation in P10 generation is subjected to Indirect co-culture processing；Or the AEC in P3 generation is used to prepare item CMC model is carried out for the AMSC in P10 generation after part culture medium；

Treated AMSC is collected, pretreated AMSC is obtained.

2. the amnion-derived mescenchymal stem cell preprocess method of people as described in claim 1, which is characterized in that the AMSC It further includes purifying and amplification processing that secondary culture is separated with AEC.

3. the amnion-derived mescenchymal stem cell preprocess method of people as claimed in claim 2, which is characterized in that the AMSC Separation secondary culture, purifying and amplification processing include：

It draws materials and is inoculated with, after inoculation, complete DMEM/F12 culture medium is added, is placed in stationary culture in humidified incubator；To Tissue block is slightly dry, when can securely be attached in bottle wall, culture solution is made slowly to infiltrate tissue block, stationary culture；

Supply complete medium afterwards for 24 hours, liquid is changed after 3~5 days and remove the tissue block of not adherent floating, when after cell growth it is close Degree carries out secondary culture when reaching 80~90% fusion；

Remove tissue block, suck tissue block and outmoded culture solution, after being cleaned with balanced salt solution, is added into culture bottle 0.125% trypsin solution digests at room temperature；

It to cell body retraction, is rounded, space between cells becomes larger but when without departing from bottle wall, and the termination of newborn bovine serum stoste is added and disappears Change；

The cell formation cell suspension to fall off in bottle wall, centrifugal treating,

Supernatant is abandoned, full DMEM/F12 culture medium is added and cell is resuspended, seed cells into culture bottle, then add complete DMEM/ F12 culture medium is labeled as P1 generation, sets in incubator and cultivate；

When attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3 carry out passage inoculation；

Collect AMSC within P10 generation.

4. the amnion-derived mescenchymal stem cell preprocess method of people as claimed in claim 2, which is characterized in that the AEC Separation secondary culture, purifying and amplification processing include：

Block amnion tissue fetch into centrifuge tube, 0.125% pancreatin is added, is placed in 37 DEG C of incubators and digests, is disappeared with serum termination Change, collects the liquid of 3 digestion, filter centrifugation processing；

Cell is resuspended with complete DMEM/F12 culture medium, is inoculated in culture bottle, and add complete DMEM/F12 culture medium；Wait paste Parietal cell is fused to each other up to 80~90%, outwells outmoded culture solution, and after being cleaned with balanced salt solution, pancreas egg is added into culture bottle White enzyme solution, digests at room temperature；

It to cell body retraction, is rounded, space between cells becomes larger but when without departing from bottle wall, and the termination of newborn bovine serum stoste is added and disappears Change, the cell in bottle wall that falls off makes to form cell suspension centrifugal treating；Supernatant is abandoned, full DMEM/F12 culture medium is added and is resuspended carefully Born of the same parents seed cells into culture bottle, then to complete DMEM/F12 culture medium is added, are labeled as P1 generation, set in incubator and cultivate；

When attached cell is fused to each other up to 80~90%, aforesaid operations are repeated, by 1:2 or 1:3 carry out passage inoculation；

Collect AEC within P5 generation.

5. the amnion-derived mescenchymal stem cell preprocess method of people as described in claim 1, which is characterized in that described to incite somebody to action The AEC in AMSC and P5 generation in P10 generation carries out Indirect co-culture processing and includes：

The AEC in the AMSC and P5 generation in P10 generation is taken respectively, outmoded culture medium in respective culture bottle is sucked out respectively, and with flat Weighing apparatus salting liquid liquid digests after washing, and after the respective cell body retraction of AMSC and AEC, being rounded, digestion is terminated immediately, by cell Liquid is discarded supernatant after transfer centrifuge tube centrifugal treating, cell is resuspended with single DMEM/F12 culture medium without serum；

The AMSC handled well is put into suspension type culture dish, the AEC handled well is seeded in the suspension type culture dish simultaneously Indirect co-culture is carried out in placement to incubator；

In for 24 hours~72h after discard suspension type culture dish and take out AMSC, digestion is collected, and pretreated AMSC is obtained.

6. the amnion-derived mescenchymal stem cell preprocess method of people as described in claim 1, which is characterized in that described by P3 AEC in generation is used to prepare after conditioned medium：

(1) preparation of conditioned medium：The AEC within P3 generation is taken, outmoded culture medium in culture bottle is sucked out, and use balanced salt solution Liquid is washed, and the DMEM/F12 culture medium for being free of serum is added, and places to culture in incubator, culture medium is sucked out, filter culture medium It into sterile centrifugation tube, freezes spare after sealing up for safekeeping, obtains conditioned medium；

(2) AMSC within P10 generation is taken, outmoded culture medium in culture bottle is sucked out, and digested after being washed with balanced salt solution liquid, After cell body retraction, being rounded, digestion is terminated immediately, cell is transferred in centrifuge tube and is centrifuged；Discard supernatant liquid；Be added into Thaw, carry out CMC model during the conditioned medium after rewarming is resuspended, after pretreated AMSC.

7. the amnion-derived mescenchymal stem cell preprocess method of people as claimed in claim 6, which is characterized in that the filtering It is that 0.42umPVDF membrane filter is filtered that culture, which is using aperture,.

8. the amnion-derived mescenchymal stem cell preprocess method of people as claimed in claim 4, which is characterized in that the filtering Centrifugal treating is with centrifugal treating after 200 mesh net filtrations.

9. the amnion-derived mescenchymal stem cell preprocess method of people described in above-mentioned any one claim fills between raising Application in matter stem cell homing ability and et al. Ke quantity.

10. the amnion-derived mescenchymal stem cell preprocess method of people described in any one of claim 1~8 is made in raising Hemocytoblast transplants the application in success rate.