CN103446184A - Application of amniotic mesenchymal stem cells in preparation of medicine for prolonging life, health product or cosmetic - Google Patents

Abstract

The invention belongs to the field of bioengineering, and discloses application of amniotic mesenchymal stem cells in preparation of a medicine for prolonging life, a health product or a cosmetic. The series study shows that the amniotic mesenchymal stem cells and/ or secretory products of the amniotic mesenchymal stem cells have anti-aging effect and can be used for preparing the medicine for prolonging the life, the health product or the cosmetic.

Description

The application of amnion mesenchymal stem cell in preparing the medicine of life-saving, health product or cosmetics

Technical field

The invention belongs to bioengineering field, relate to the application of amnion mesenchymal stem cell in preparing the medicine of life-saving, health product or cosmetics.

Background technology

Along with the arrival of global aging society, the control of the elder's health and Senile disease has become the important public hygiene problem that international community faces.China is the country that world population is maximum, is also the country that aging population are maximum.The 6th national census key data that State Statistics Bureau announces show, during with on November 1st, 2,010 zero, is as the criterion, and China's total population is 13.4 hundred million people, and wherein 60 years old and above population accounting have reached 13.26%.This means, China will march toward " aging society " very soon.The puzzlement brought in order to solve aging society, defying age medical science starts to rise.Defying age also can life lengthening.Explore Aging mechanism, find antidotal new target drone and new therapy, become the key subjects of life sciences.

In recent decades, along with interdisciplinary develop rapidlys such as modern genetics, molecular biology, cytobiology and molecular immunologies, people have had profound understanding to old and feeble mechanism, some causes of senescence with scientific value have been proposed: procedural causes of senescence, the replicative senescence theory, free radical theory, DNA damage is repaired theory etc.

Old and feeble free radical theory (free radical theory) is always by the representative causes of senescence of people's common concern.At first this theory is proposed in 1956 by British scholar Harman, thinks that aging is because the radical pair cell component is attacked the result caused.Maintain antioxidant and the free radical scavenger level of proper level in body, can play the effect [1] of life-saving and slow down aging.Although this theory can't be explained the very complicated process of old and feeble this body fully, between free radical and aging, close ties have obtained the extensive approval of Chinese scholars.

Approximately more than 95%, belong to oxygen-derived free radicals (oxygen free radicals in total free radical in human body, OFR) [2], claim again active oxygen (reactive oxygen species, ROS), oxygen-derived free radicals is mainly derived from the mitochondrial respiratory chain reaction, the oxygen that enters cell more than 90% by mitochondrion for biological oxidation, in oxidative phosphorylation, consume, generate ATP when metabolite is oxidized.At Mitochondria, generate in the process of ATP, nearly 1～4% oxygen of taking in is converted into oxygen-derived free radicals.Due to the existence of body antioxidant system, in the normal condition lower body, the generation of free radical in dynamic equilibrium, can not produce injury to human body with elimination.In body, the performance antioxidation mainly contains antioxidant reductase (as superoxide dismutase (superoxide dismutase, SOD), catalase (catalase, CAT) and glutathion peroxidase (glutathione peroxidase,) and non-enzyme antioxidant (as vitamin C, vitamin E and melatonin (melatonin, MT) etc.) GSH-px) etc.Along with the increase at age, in human body, Free Radical Level shows a rising trend, and free radical scavenging mechanism but is degradation trend simultaneously, result causes oxygen-derived free radicals to accumulate in vivo, and acting on the biomacromolecule of body cell, the base generation chemical reaction as with nucleic acid, cause DNA damage; Make biomembranous polyunsaturated fatty acid generation lipid peroxidation, generate lipid peroxide (lipid peroxidation, LPO), malonaldehyde (the malonaldehyde produced when LPO decomposes, MDA) can make DNA, protein occur crosslinked etc., thereby cause the body different physiological roles and obstacle occurs, promote generation, the development of multiple infirmities of age (as atherosclerosis, cardiovascular and cerebrovascular disease, cranial nerve cell degeneration, diabetes and tumor etc.), cause the aging of body.

The expression product of Bmi-1 (B cell specific moloney murine leukemia virus insertion site 1) gene is that the muroid lymphoma filters virus (Bmi-1) oncogene autoploid (GenBank:L13689.1).Bmi-1 belongs to Polycomb (PcG) gene family, and the Space-time speciality that the PcG gene is considered to maintain the HOX house-keeping gene usually in the biological development process has played transcripting suppressioning action [3] in expressing.1994, Nathalie etc. in depth study the Bmi-1 gene in the body function, use homologous recombination technique to knock out this gene at embryonic stem cell, set up Bmi-1 gene knockout (Bmi-1 ^-/-) mouse model, find Bmi-1 ^-/-mice is old and feeble phenotype [4] before neural and hemopoietic system have obvious developmental defect and maturation.Further Bmi-1 is found in research ^-/-the ability of the nerve of mice and the self renewal of hematopoietic stem cell is impaired, and the stable of stem cell pool is broken, and stem cell constantly decays, major injury hemopoietic and neural growth and function [5-7].Separately there are some researches show Bmi-1 ^-/-the ROS level of the many histiocytes of mice whole body (thymus, spleen, bone marrow) obviously increases.Antioxidant N-acetyl-cysteine (N-acetylcysteine, NAC) can make the ROS level obviously reduce and approach WT mice level, and partly corrects Bmi-1 ^-/-the old and feeble phenotype [8] of mice.This results suggest Bmi-1 may bring into play antidotal effect by antioxidation.

Body aging is the final result of cell ageing, and aging simultaneously is also the performance of stem cell and body cell lazy weight.Stem cell, as the source of all cells, has reproducibility, becomes thus the new fulcrum of defying age medical research, and increasing people expects that defying age medical science takes the lead in making a breakthrough in the stem cell field.

Stem cell is of a great variety, for using the stem-cell research defying age, at first, people pay close attention to embryonic stem cell (embryonic stem cells, ES), because it has infinite multiplication and to the ability [9] of three differentiation of germinal layers, but ES transplant have ethics dispute [10] and transplant after tumor and rejection [11] are arranged into.Recently research is found, adult stem cell also has to the ability of three differentiation of germinal layers, wherein that most study is mescenchymal stem cell (the bone marrow-mesenchymal stem cells that derives from bone marrow, BM-MSCs) [12], but BM-MSCs must obtain by the wound approach is arranged, and with advancing age, the quantity of cell significantly descend [13], differentiation capability reduces, so the stem cell of these types is all because of more difficult the promoting the use of of problem [14] of existence separately.

Studies have found that recently, amnion mesenchymal stem cell (amnion mesenchymal stem cells, AMSCs) there is the phenotype similar to BM-MSCs, the potential that there is self renewal and Multidirectional Differentiation, express Interstitial cell mark CD44, CD90, CD105, CD166[15], do not express hematopoietic stem cell mark CD34, CD45 and mononuclear cell mark CD14.AMSCs may be a middle-bracket stem cell between embryonic stem cell and adult stem cell, so multiplication capacity is than BM-MSCs stronger [16].The low HLA-ABC that expresses of AMSCs, and expression of HLA-DR not, thereby illustrate that AMSCs has reduced immunogenicity [15], after allogeneic, Xenogeneic, immunological rejection can not occur all, and AMSCs is without oncogenicity [17,18], this cell replacement therapy that is various diseases provides new selection.Amniotic membrane, be the garbage after delivery of fetus, be a kind ofly be easy to obtain, output is high, without the regenerative medicine cell derived of ethics restriction.These advantages have caused the extensive concern of people to AMSCs, have been applied at present research and the diseases such as treatment liver cirrhosis, myocardial infarction, nerve retrograde affection [19].

The current report for the preparation of the medicine of life-saving by amnion mesenchymal stem cell not still.

Summary of the invention

The purpose of this invention is to provide the application of amnion mesenchymal stem cell in preparing the medicine of life-saving, health product or cosmetics.

The objective of the invention is to realize by following technical proposal:

Amnion mesenchymal stem cell and/or the application of its secretory product in preparing the medicine of life-saving, health product or cosmetics.

Described application, wherein the medicine of life-saving, health product or cosmetics refer to antiaging agent, health product or cosmetics.

Described application, wherein antiaging agent, health product or cosmetics refer to medicine, health product or the cosmetics of anti-immunocyte aging, removing free radical or enhancing activities of antioxidant enzymes.

Described application, wherein antiaging agent, health product or cosmetics refer to the inhibitor of Wnt16 protein expression inhibitor or cyclin dependent kinase inhibitive factor p16, p19 or p27 albumen.

Described application, wherein the medicine of life-saving, health product or cosmetics refer to medicine, health product or the cosmetics that improve hematological function.

Described application, wherein improve hematological function and refer to and improve hematopoietic disorder, especially improves the lymphocyte series hematopoietic disorder.

Described application, wherein the medicine of life-saving, health product or cosmetics refer to and promote bone growth medicine, health product or cosmetics.

Described application, wherein promote bone growth to refer to promoting bone growing and/or improve osteoporosis.

Described application, wherein the medicine of life-saving, health product or cosmetics refer to medicine, health product or the cosmetics that improve the Bmi-1 disappearance.

Beneficial effect of the present invention:

The present invention uses Bmi-1 in embodiment 1 ^-/-mice, as aging model, will arrive Bmi-1 through lumbar injection from the 2nd generation AMSCs of pregnant middle and advanced stage β-gal transgenic mice ^-/-in Mice Body, observation Bmi-1 ^-/-the variation of survival time of mice and body weight, utilized the Method Comparisons such as histopathology, immunohistochemistry, molecular biology brood WT mice in 4 week age, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the phenotypic difference of different tissues of mice organ.Result shows that AMSCs is as a kind of pluripotent stem cell, can move to the different histoorgan of whole body by it, be divided into the cell of Different Organs, again can be by raising the activity of antioxidase, remove oxygen-derived free radicals, suppress SAG, the performance part is corrected the effect that the Bmi-1 disappearance causes senilism.It acts on the direct effect of existing AMSCs, and the effect of AMSCs secretory product is also arranged.Therefore, AMSCs can be used for preparing the Novel anti-aging medicine, and in addition, AMSCs also has following advantage for the preparation of antiaging agent: do not belong to embryonic stem cell, limit without ethics; Easily obtain; Non-invasive; Twice laid; Rejection is very little; Application is wide etc.

The accompanying drawing explanation

The genotype of Fig. 1: WT and Bmi-1KO mice

+ /+is an only aobvious 687bp band of WT mice;-/-is an only aobvious 399bp band of Bmi-1 homozygote mice; +/-is aobvious 2 bands of Bmi-1 heterozygote mice, and wherein one is 687bp, and one is 399bp.Neo: meaning neomycin opposing gene (neomycin-resistance gene), is the marker gene the most frequently used for the identification of knock out mice.

The preparation of Fig. 2: AMSCs and dryness feature

(A) morphological characteristic of 2nd generation AMSCs (400x); (B) the 2nd generation amnion mesenchymal stem cell in β-gal transgenic mice source presents Lac Z stained positive (400x); (C) express alkali phosphatase (400x) after the differentiation of AMSCs skeletonization direction induction; (D) become the rear positive adipose cell (400x) of oil red that shows of fat direction induction differentiation; (E-L) flow cytometry analysis mescenchymal stem cell labelled molecule CD29, CD44, CD73, CD90, CD105, embryonic stem cell labelled molecule SSEA-4 and hematopoietic stem cell labelled molecule CD34, CD45 are in the expression of AMSCs; 2nd generation amnion mesenchymal stem cell (M) the Bmi-1 gene PCR figure in β-gal transgenic mice source and (N) Bmi-1Western blot figure; (O) RT-PCR analysis embryonic stem cell labelled molecule Oct4 and CXCR4 are in the expression of AMSCs; (P-R) immuning fluorescent dyeing analysis embryonic stem cell labelled molecule CXCR4 is in the expression (100x) of AMSCs. ^*: p<0.05, ^*: p<0.01, with matched group, compare; Negtive: negative control.

The amplification of Fig. 3: AMSCs

With matched group (A), control compares, respectively through somatomedin (B) epidermal growth factor (epidermal growth factor, EGF), (C) basic fibroblast growth factor (basic fibroblast growth factor, bFGF), (D) EGF and bFGF and (E) activated vitamin D (1,25-dihydroxyvitamin D, 1,25 (OH) ₂d ₃) intervene, 2nd generation AMSCs is processed to the area (methylene blue staining) that obviously increases afterwards methyl blue stained positive AMSC in 8 days; (F) 2nd generation AMSCs quantitative result of methyl blue stained positive AMSC area after 8 days after A-E processes.

Fig. 4: AMSCs transplants Bmi-1 ^-/-the impact of mice existence and growth

(A) brood WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-mouse survival rate cartogram; (B) 4 week age the Mouse Weight change curve; (C) 4 week age mice substantially according to; (D) 4 week age mouse thymus the form size; (E) 4 week age mouse spleen the form size. ^*: p<0.01, ^{* *}: p<0.001, with WT mice on the same group, compare; #:p<0.05, ##:p<0.01, with matched group Bmi-1 on the same group ^-/-mice is compared.

Fig. 5: AMSCs transplants Bmi-1 ^-/-the impact that mouse thymus and spleen t-cell are grown

Brood 4 week age WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-mice (A) thymus and (B) splenic T lymphocyte flow cytometer showed figure as a result; In thymus, (C) CD4 and CD8 jack to jack adapter (DN), (D) CD4 and CD8 two positive (DP), (E) CD8 reach (F) the mono-positive cell ratio of CD4 cartogram; In spleen, (G) DP, (H) CD8 reach (I) the mono-positive cell ratio of CD4 cartogram. ^{* *}: p<0.001, compare ##:p<0.01 with brood WT mice, ###:p<0.001, with littermate control group Bmi-1 ^-/-mice is compared.

Fig. 6: AMSCs transplants Bmi-1 ^-/-mouse bone marrow cells and the cytocerastic impact of spleen B

Brood 4 week age WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-mice (A) bone marrow and (B) spleen bone-marrow-derived lymphocyte streaming interpretation of result figure; (C) pro-B cell in bone marrow, (D) pre-B cell, (E) immature B cell and (F) mature cell proportion cartogram; (G) T1 phase B cell proportion cartogram in spleen. ^*: p<0.05, ^*: p<0.01, ^{* *}: p<0.001, with brood WT mice, compare; #:p<0.05, ##:p<0.01, with littermate control group Bmi-1 ^-/-mice is compared.

Fig. 7: AMSCs transplants Bmi-1 ^-/-the impact of mice skeleton aging

Brood 4 week age WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-the total collagen staining section in mice (A) mouse tibia upper end micro-image (50x); (B) tibial metaphysis district and (C) the key district HE stained micro-image (400x) of tibia; (D) Cbfa1, IGF-1 and PPAR γ Western blot figure; (E) bone volume (BV/TV, %) cartogram; (F) osteoblast number (N.Ob/T Area, #/mm ²) cartogram; (G) adipose cell number (N.Ad/T Area, #/mm ²) cartogram; (H) Cbfa1, (I) IGF-1 and (J) the horizontal cartogram of PPAR γ albumen relative expression. ^*: p<0.01, ^{* *}: p<0.001, with brood WT mice, compare; #:p<0.05, ##:p<0.01, ###:p<0.001, with littermate control group Bmi-1 ^-/-mice is compared.

Fig. 8: the distribution of donorcells in host's organ

Brood 4 week age matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-(A) Bmi-1 gene PCR figure and (B) Bmi-1Western blot figure in mouse core, liver, spleen, lung, kidney, bone marrow and thymic tissue; (C) the positive AMSCs of Lac Z in β-gal transgenic mice source is at matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-distribution in mouse lung, spleen and kidney (400x).

Fig. 9: AMSCs transplants Bmi-1 ^-/-the impact of mice Different Organs oxidative stress index of correlation

Brood 4W WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-each histoorgan of mice whole body (A) ROS relative level, (B) H ₂o ₂relative amount, (C) catalase (catalase, CAT) relative activity and (D) cartogram of total superoxide dismutase (total-superoxide dismutase, T-SOD) relative activity. ^*: p<0.05, ^*: p<0.01, ^{* *}: p<0.001, with brood WT mice, compare; #:p<0.05, ##:p<0.01, ###:p<0.001, with littermate control group Bmi-1 ^-/-mice is compared.

Figure 10: AMSCs transplants Bmi-1 ^-/-the impact of the old and feeble protein expression of mice

Brood 4 week age WT, matched group Bmi-1 ^-/-with transplantation group Bmi-1 ^-/-mouse Bone tissue (A) Wnt16, p16, p19 and p27Western blot figure; (B) Wnt16, (C) p16, (D) p19 and (E) the horizontal cartogram of p27 albumen relative expression. ^*: p<0.05, ^*: p<0.01, ^{* *}: p<0.001, with brood WT mice, compare; #:p<0.05, ##:p<0.01, ###:p<0.001, with littermate control group Bmi-1 ^-/-mice is compared.

The specific embodiment

The invention will be further elaborated by the following examples.

Embodiment 1: amnion mesenchymal stem cell is implanted in the effects anb Mechanism research in the aging of anti-Bmi-1 disappearance mice

1. laboratory animal

In this research, all experiment mices are raised, are bred by the SPF of Nanjing Medical University level zoopery center, and animal is raised at barrier environment, 22～26 ℃ of temperature, humidity 45%～75%.Adopt Bmi-1 Gene Knock-Out Animal Model heterozygote mice (purchased from Dutch institute of oncology) in embodiment 1, carry out the male and female pairing and mate, obtain wild type (WT) and Bmi-1 homozygote (Bmi-1 ^-/-) mice.β-gal transgenic mice (purchased from Canadian McGill university animal center), cut after tail 1cm (method see under) the identified gene type that dyes by LacZ.Cell experiment partly adopts pregnant middle and advanced stage β-gal transgenic mice; Due to Bmi-1 ^-/-mice is most of dead in latter one month of birth, so latter 2 days brood WT of birth and Bmi-1 are partly selected in zoopery ^-/-mice is as the receptor Mus, and β-gal transgenic mice is as donor Mus (pregnant middle and advanced stage).

2. the animal gene type is identified

2.1 the evaluation of β-gal transgenic mice:

2.1.1 with LacZ buffer (2mM MgCl ₂, 0.01% NaTDC, 0.02%Nonidet-P40 (NP-40, Ameresco, USA) in0.01M PBS, PH7.3) and clean the Mus tail 3 times, each 5min.

2.1.2 by the chloro-3-indole-β of the bromo-4-of 0.5mg/ml5--D-galactoside (X-galactoside, X-gal) be dissolved in 50 μ l/ml dimethyl sulfoxide (galactoside, DMSO), 5mM potassium ferrocyanide and the 5mM potassium ferricyanide are dissolved in to the LacZ buffer simultaneously, then the two are mixed.

2.1.3 this dyeing liquor is added in the 1.5mlEP pipe that fills the Mus tail to every pipe 500 μ l.

2.1.4 be placed in the constant-temperature incubation case, 37 ℃, the lucifuge overnight incubation.

2.1.5 observe the painted situation of Mus tail inferior morning, is colored as β-gal transgenic mice, is not colored as wild-type mice.

2.2.2Bmi-1 ^-/-the genotypic evaluation of mice:

2.2.2.1 dissolve the Mus tail: add 350 μ l Mus tail lysates (to contain 100mM Tris-HCl, pH8.5,5mM EDTA, 0.2%SDS, 200mM NaCl in the 1000ml lysate in 1.5ml EP pipe, room temperature preservation) and 10mg/ml E.C. 3.4.21.64 10 μ l, 55 ℃, shaking table rocks and spends the night with 170revolutions per minute (rpm).

2.2.2.2 add 350 μ l phenol in each pipe: chloroform: isoamyl alcohol (25:24:1) solution, thermal agitation 15 seconds, the standing 3min of room temperature.

2.2.2.3 centrifugal 15min (12000rpm), make liquid be divided into three layers.

2.2.2.4 get the colourless aqueous phase liquid in upper strata, approximately 200 μ l are in new 1.5ml EP pipe, and middle level liquid is not drawn in attention.

2.2.2.5, after adding 400 μ l dehydrated alcohol in new EP pipe, put upside down for several times to producing flocculent deposit.

2.2.2.6 centrifugal 5min (12000rpm).

2.2.2.7 abandon liquid, be inverted the EP pipe in absorbent paper, drying at room temperature 10min.

2.2.2.8, after adding 100 μ l sterile distilled waters in each pipe, 55 ℃ of water-baths 1 hour are with dissolving DNA.

2.2.2.9Bmi-1PCR reaction system

The forward primer sequence of Bmi-1 is: 5 '-CAGTTAGGCAGTATGTAGTTTTC-3 ', and reverse primer is 5 '-GTTGTGGTGGAGTGTAAGAGTGT-3 ';

The forward primer of NEO gene is: 5 '-AAGATGTTGGCGACCTCGTATTGG-3 ', reverse primer is: 5 '-GCAAGACCTGCCTGAAACCGAACT-3 '.

Primer is synthetic by Shanghai Invitrogen company.

2.2.2.10Bmi-1PCR reaction condition

The same Bmi-1 of NEO PCR reaction system and reaction condition.The Bmi-1 amplified production is 687bp, NEO amplified production 399bp.

2.2.2.11PCR product electrophoresis

Prepare 2% agarose gel: take the 0.5g agarose, add 50ml TAE buffer, mix, microwave heating 5～7min, melt fully to agarose, taking-up is put in fume hood, be cooled to 65 ℃, add 2 μ l ethidium bromide (EB) liquid, mix, pour in preprepared electrophoresis tank, insert immediately comb.Complete when cooling until it, extract comb, adjust the gel direction and gel is placed in to the TAE electrophoretic buffer.

The PCR product is added to 6 * sample-loading buffer, 4 μ l, mix, centrifugal, get 12 μ l and add in the loading hole of 2% prefabricated agarose gel, 120V, electrophoresis 10min.Gel after electrophoresis is taken pictures, filed, record genotype (Fig. 1).

Seed cell preparation

3.1 the primary and cultivation of going down to posterity of mice AMSCs

3.1.1 getting pregnant middle and advanced stage β-gal transgenic mice is studied.

3.1.2 mice is through etherization, the cervical vertebra dislocation is put to death, and immerses 75% ethanol.

3.1.35min after, mice is taken out from 75% ethanol, move on in super-clean bench.

3.1.4 amniotic membrane is mechanically peeled off, with PBS, repeatedly rinse, remove bloodstain, shred amniotic membrane.

3.1.5 add 0.05%trypsin+0.02%EDTA, in 37 ℃, 200r/min, digestion, 55min.

3.1.6 centrifugal, 1500r/min, 5min, discard pancreatin.

3.1.7 with PBS, rinse, centrifugal, 1000r, 5min, supernatant discarded.

3.1.8 the resuspended fragment of tissue of α MEM standard medium (α MEM+15%FBS+2mM L-glutaminate+100U/ml mycillin+50 μ g/ml ascorbic acid), plant in culture dish, in 37 ℃, and 5%CO ₂middle cultivation.Every 3 days, half amount was changed liquid once, and when growing into 80%～90% integration, adherent cell collecting is as primary AMSCs.

3.1.9 when growing into 80%～90% fusion, with the pancreatin of 2.5g/L and the ethylenediaminetetraacetic acid of 0.02M (ethylenediamine tetraacetic acid, EDTA), in 37 ℃, digest 3～5min, stop digestion according to the cell shrinkage degree of observing under microscope, piping and druming mixes, low-speed centrifugal 1000r/min, centrifugal 5min, add standard medium to go down to posterity and continue to cultivate by 1:2, obtain passage cell.

4. zoopery

4.1 the evaluation of seed cell

Use LacZ cytochemical staining method to determine seed cell expression beta galactosidase.

4.1.1 after cell culture fluid is toppled over, with 0.01M PBS, rinse 3 times each 3min.

4.1.2 add fixedly 45min of periodate-lysine-paraformaldehyde (periodate-lysine-paraformaldehyde, PLP), then with LacZ buffer solution for cleaning 3 times, each 5min.

4.1.3 0.5mg/ml X-gal is dissolved in to 50 μ l/ml DMSO, 5mM potassium ferrocyanide and the 5mM potassium ferricyanide are dissolved in to the LacZ buffer simultaneously, then the two is mixed.

4.1.4 this dyeing liquor is added in culture dish, be placed in 37 ℃ of incubator lucifuge overnight incubation, under washing, microscope, take pictures.

4.2AMSCs intraperitoneal injection

With No. 31 microsyringes by 0.1ml cell suspension (5 * 10 ⁶aMSCs/50 μ l) lumbar injection is to the birth Bmi-1 of latter 2 days ^-/-in Mice Body, PBS studies in contrast, 1 time weekly, continues 3 times.

4.3Lac-Z histochemical stain

4.3.1 experimental procedure

4.3.1.1 for following three groups of mice: WT, Bmi-1 ^-/-and Bmi-1 ^-/-+ AMSCs(Bmi-1 ^-/-mice is transplanted the AMSCs group) mice, studied every group of 5 mices, totally 15 during 4 week age mice.

4.3.1.22% chloral hydrate lumbar injection (0.2ml/10g) anesthesia.

4.3.1.3 cut off chest and abdominal part along anterior midline, catheter needle is inserted to apex, cut off liver simultaneously, carry out the normal saline perfusion.

4.3.1.4 be rinsed when clean until blood circulation, then pour into PLP100ml.

4.3.1.5 carefully take off each histoorgan specimen of whole body, heart preparation, along coronalplane, on average is cut into upper and lower two parts by it; The kidney specimen tears kidney peplos off, along sagittal plane, it is divided into to left and right two parts.Liver, lungs specimen are cut off.

4.3.1.6 use PBS to clean PLP.

4.3.1.7 for above three treated animal models, respectively get the whole body specimen of three animals and carry out the Lac-Z histochemical stain,

Colouring method is with above genotype identification.

4.4 gross examination of skeletal muscle and HE dyeing

4.4.1 experimental procedure

4.4.1.1, after carrying out LacZ histochemical stain 24h, under stereomicroscope, observe and take pictures.

4.4.1.2 after taking pictures, carry out PLP4 ℃ after fixing 72h.

4.4.1.3 de-cured aquation, section.

4.4.1.4 get the section cut, be placed in dimethylbenzene and dewax, each 5min, totally three times.100% ethanol twice, each 3min, 95%, 80%, 70% gradient ethanol aquation, each 3min, flowing water rinses 5min.

4.4.1.5 haematoxylin dyeing 15s, flowing water rinses 5min, and 1% hydrochloride alcohol differentiation 20 seconds, till flowing water rinses and is blueness to observation of cell core under optical microscope.

4.4.1.6 pure molten Yihong dyeing 1 minute, 80%, 95%, 100% gradient alcohol dehydration, each 2min.

4.4.1.7 dimethylbenzene is transparent twice, each 2min.

4.4.1.8 neutral gum mounting.

4.5 total collagen tissue chemical staining

4.5.1 the preparation of the straight red colouring liquid of supersaturation picric acid

Get picric acid 0.2 gram, add the 150ml sterilized distilled water, fully stir, get supernatant 100ml after standing, add 0.1 gram Picro-Sirius red (Sirius Red) in supernatant, fully stir, keep in Dark Place.

4.5.2 experimental procedure

4.5.2.1 get the section that each group has cut, de-cured aquation.

4.5.2.2 the straight red colouring liquid of supersaturation picric acid prepared is hatched on specimen to incubated at room 1 hour.

4.5.2.3 flowing water rinses 5 minutes, haematoxylin is redyed 1 minute, and flowing water rinses 3 minutes, and 1% hydrochloride alcohol differentiation 10 seconds, till flowing water rinses and is blueness to observation of cell core under optical microscope.

4.5.2.4 gradient alcohol dehydration, dimethylbenzene is transparent, the neutral gum mounting.

4.5.2.5 just putting, fluorescence microscope (Japanese Olympus company) is lower to be observed, and with DP70 type CCD (Japanese Olympus company), gathers image.

4.5.2.6 carry out image quantitative analysis with Northern Eclipse software (U.S. Empix Imaging company), quantitative target comprises: BV/TV: i.e. the percentage ratio of the area in total collagen positive area in bone trabecula zone and bone trabecula zone.With the SPSS13.0 statistical software, analyzed.

5. flow cytometer is surveyed active oxygen (ROS) level

5.1 experimental procedure

5.1.1 the de-cervical vertebra of experiment mice is got each histoorgan of whole body after putting to death, the tibia tissue is gone out medullary cell with the 5ml syringe and piping and druming repeatedly, with syringe, from syringe needle, releases, and makes single cell suspension.Its hetero-organization, shred, and through 200 eye mesh screens, filters and make single cell suspension.1500rpm, 4 ℃ of centrifugal 5min, abandon supernatant.

5.1.2 add the PBS of 1ml pre-cooling to make single cell suspension, the concentration of adjusting cell to be measured is 1 * 10 ⁶～10 ⁷individual/ml.1500rpm, 4 ℃ of centrifugal 10min, abandon supernatant.

5.1.3 add prefabricated fluorescent probe 2 ', 7 '-dichloro-dihydro fluorescein diethylester (2,7-dichlorofluorescin dictate, DCF-DA) working solution, mix gently latter 37 ℃ and hatch 30min.1500rpm, 4 ℃ of centrifugal 10min, abandon supernatant.

Hatch 20min 5.1.4 add 37 ℃ of 10% hyclones.1500rpm, 4 ℃ of centrifugal 10min, abandon supernatant.

5.1.5 add appropriate PBS, flow cytometer detects the average fluorescent strength of fluorescent probe in cell.

5.1.6 analyzed with the SPSS13.0 statistical software.

6. organize total superoxide dismutase (total superoxide dismutase, T-SOD), hydrogen peroxide (hydrogen dioxide, H ₂o ₂) and the detection of catalase (catalase, CAT) content

6.1 experimental procedure

6.1.1 the preparation of tissue homogenate

6.1.1.1 get each histoorgan piece (0.2g-1g) rinsing in the normal saline of pre-cooling of whole body, remove blood, filter paper is wiped away dry, weighs, and puts into the test tube of 4ml.

6.1.1.2 measure 0.86% cold saline with pipet, the volume total amount of normal saline should be 9 times of piece of tissue weight, and normal saline is moved in test tube, piece of tissue is shredded with eye scissors as far as possible.

6.1.1.3 machine homogenate: with tissue refiner, with 15000r/min, 10 second/times of homogenate times, 30 seconds, interval, 3-5 time continuously, carry out on ice.

6.1.1.4 10% tissue homogenate that will prepare with low speed centrifuge at 4 ℃, with 2500r/min centrifugal 10 minutes, centrifugal good homogenate leave and take supernatant abandon following precipitation need not.

6.1.2 Coomassie brilliant blue determining the protein quantity

Prepare 1% tissue homogenate with the normal saline dilution supernatant, measurement range is to be advisable in the 1mg/ml left and right.By specification behaviour

Making step carries out:

Pressing formula calculates:

6.1.3T-SOD active, detect

Measuring principle: by xanthine and xanthine oxidase response system, produce O ₂ ^-, latter's oxidation azanol forms nitrite, under the developer effect, presents aubergine, while in sample, containing SOD, superoxide anion is had to narrow spectrum inhibitory action, and the nitrite formed is reduced.Calculate the activity of T-SOD according to the height of suppression ratio.

Assay method, get and organize supernatant, then be diluted to 1% homogenate with cold saline 1:9.By specification operation step is carried out:

Calculate:

A, definition: every milligram of histone when in the 1ml reactant liquor, the SOD suppression ratio reaches 50% corresponding SOD amount be a SOD unit of activity (U).

B, formula:

6.1.4H ₂o ₂content detection

Measuring principle: hydrogen peroxide H ₂o ₂can generate a kind of complex with the molybdic acid effect and can calculate H at 405nm place its growing amount of mensuration ₂o ₂amount.

Assay method, get and organize supernatant, according to different histone content, then becomes suitable ratio with normal saline dilution, and the by specification operating procedure is carried out:

Computing formula:

6.1.5CAT vigor detects

Measuring principle: catalase (CAT) decomposing H ₂o ₂reaction can be by ammonium molybdate and end rapidly remaining H ₂o ₂produce a kind of flaxen complex with the ammonium molybdate effect, measure its growing amount at the 405nm place, can calculate the vigor of CAT.

Assay method: get and organize supernatant, then become best sampling concentration with normal saline dilution, to be measured.

Calculate

A. definition: every milligram of histone decomposes the H of 1 μ mol each second ₂o ₂amount be a unit of activity.

B. formula:

6.1.6 analyzed with the SPSS13.0 statistical software.

7. Western blot (Western Blot) detects

7.1 experimental procedure

7.1.1 reagent preparation

7.1.1.1RIPA agent prescription:

Annotate: precipitation occurs after each composition mixes, need dissolve with NaOH, then add the Cocktail1 sheet after adjusting pH to 7.2 with concentrated hydrochloric acid, 500 μ l are distributed into 4ml EP pipe.

7.1.1.22 * sample loading buffer formula:

7.1.1.3 separation gel and concentrated glue formula:

7.1.1.430% acrylamide (AA) formula of liquid:

Annotate: mix rear 37 ℃ of dissolvings, filter paper filtering, brown bottle is stored in room temperature.Must again prepare (pH value is no more than 7.0 energy use, otherwise must again prepare) every the several months.

7.1.1.510% dodecyl sodium sulfate (SDS) is filled a prescription:

Reagent name dosage

Electrophoresis level SDS 10.0g

DdH ₂o 100ml (standardize solution)

Annotate: be heated to 68 ℃ and also use the magnetic stirrer hydrotropy, concentrated hydrochloric acid is adjusted pH7.2, is settled to 100ml, room temperature storage.7.3.1.610% Ammonium persulfate. (APS) is filled a prescription:

Reagent name dosage

Ammonium persulfate. (APS) 0.1g

ddH ₂O 1ml

Annotate: 4 ℃ of preservations, Ammonium persulfate. is slowly decay in solution, therefore fresh configuration every other week.

7.1.1.7 protein electrophoresis liquid (10 *) formula:

7.1.1.8 Protein transfer buffer formulation:

7.1.1.90.05%PBST (cleaning mixture) fills a prescription:

Reagent name dosage

1 * phosphate buffer (PBS) 1000ml

Tween-20 500μl

7.1.1.105% defatted milk powder-PBST fills a prescription:

Reagent name dosage

Defatted milk powder 5g

PBST 100ml

7.1.2, for above three treated animal models, after transplanting 4 weeks, get the whole body Different Organs and studied, every group of 5 mices, totally 15.

Add RIPA in sample cell 7.1.3 be about the 1:25 ratio in mass/volume, homogenizer is homogenate on ice, puts in ice chest and shake 30min on shaking table.

7.1.413000rpm4 ℃ centrifugal 15min.

7.1.5 the supernatant under the absorption fat deposit (about 100-200 μ l) is in new EP pipe.

Add in new EP pipe 7.1.6 separately get 10 μ l, utilize the coomassie brilliant blue staining method, on the nucleic acid-protein analyzer, survey protein concentration.

7.1.7 add equal-volume 2 * sample loading buffer (loading buffer) in EP pipe, boiling water boiling 5min ,-40 ℃ of preservations.

7.1.8 get two blocks of glass plates different in size, absorbent cotton (or ethanol) wiping with being stained with methanol, evaporate into dry.The glass plate is sealed to guarantee not leak glue.

7.1.9 record separation gel: according to the separation gel of detected destination protein molecular weight preparation debita spissitudo, gel solution is injected to two-layer glass plate (injecting from the centre position of glass plate with steady flow velocity) reposefully, slowly continue to be injected into liquid level apart from broach 1.5cm place (or comb tooth length+1cm), carefully inject again one deck water (or 10%SDS on liquid level, about 2-3cm is high), to completely cut off air, standing to gel formation.

7.1.10 the concentrated glue of preparation: after the complete polymerization of glue to be separated (glue face and top water have obvious interface, about 30min), incline and cover layer liquid, with the filter paper supernatant liquid that exhausts.Mix concentrated glue, inject on separation gel between concentrated glue to two glass plate with the flow velocity of continuous and stable, then insert immediately comb.After the complete polymerization of glue to be concentrated, carefully pull up comb, note not changing the relative position of glass plate.

7.1.11 gel slab is placed in to electrophoresis tank, at electrophoresis tank, be to inject electrophoretic buffer in the slot electrode of both sides up and down.

7.1.12 application of sample: inhale sample with microsyringe and be close to the glass plate application of sample, inhale the fresh protein electrophoresis liquid after each application of sample and clean injector.

7.1.13 electrophoresis: constant voltage electrophoresis 110V2h.

7.3.14 taking-up gel: unload the glass plate from electrophoretic apparatus, with skid plate, pry open the glass plate.Excise one jiao of orientation with the mark gel on the gel top near Far Left one hole.

7.1.15 transferring film: 1 * protein delivery buffer of packing into enough in container, by gel, two, sponge, four of filter paper, 1 of nitrocellulose filter (must on angle, with pencil, mark to distinguish positive and negative), bubble balance 10～15min in 1 * protein delivery buffer.By from the blackboard to the blank, fill successively lastblock sponge, two filter paper, gel, nitrocellulose filter, two filter paper, a sponge, use the roller roll extrusion, keep whole device interior moistening and there is no a bubble.

7.1.16 this electrotransfer device is put into to electrophoresis tank, and gel is towards negative electrode, the face anode, install.Fill transfering buffering liquid (need flood gel).

7.1.17 constant current 0.3A shifts 60-120min.

7.1.18 after transferring film finishes, take out film and gel, after film takes out, use 3 times * 10min of PBST rinsing.

7.1.19 sealing: nitrocellulose filter is put into plate, adds the 5%PBSTM confining liquid, 37 ℃ of sealing 1h * 2 time.

7.1.20 primary antibodie is hatched: film is put into to hybridization bag, and the sealing surrounding, cut an osculum at one jiao, adds primary antibodie (shown in the working concentration by specification, diluting with 1%PBST), around film, do not stay as far as possible bubble, and 4 ℃ are spent the night.

7.1.21 wash film: film is washed 3 times with 5%PBST, each 5min.

7.1.22 two anti-hatching: film is put into to new hybridization bag, add two anti-(shown in the working concentration by specification, diluting with 1%PBST), put room temperature reaction 2h on shaking table.

7.1.23 film is washed 5 times with PBST, each 15min.

7.1.24 develop: film lies on card-protecting film and (faces up), Chemiluminescent Substrate kit (German Roche Cat No.11500708001) A liquid and each 300 μ l1:1 of B liquid are mixed to (according to film size adjustment consumption), evenly drip full whole face, reaction 1min.Blot film residual liquid on every side with absorbent paper, with card-protecting film, film is sealed, note not staying bubble, put into the tabletting box, close daylight lamp, give the red light to, cut and the film of the similar size of film, be pressed on film, shut the tabletting box and exposed and (be generally 5min the 1st time, fully exposure, can select time of exposure according to primary result for the second time).Be ready to three pallets, from left to right successively: developer solution, tap water, fixative solution.Take out film and drop in developer solution, rinse in developer solution to till occurring that band or film are transparent on film, after removing developer solution with water rinse, drop into and get final product (seeing that the film bleach gets final product) in fixative solution tens of seconds, turn on light, water washes down film, dries.

7.1.25 film is put on scanner and is gathered positive band, analyze experimental result.

8. mice AMSCs cultivation, proliferation and differentiation and coherent detection

8.1 mice AMSCs cultivates

Use β-gal transgenic mice amniotic membrane to prepare primary AMSCs and 2nd generation and 3 generation AMSCs, method is the same.

8.2 the detection of the 3rd generation AMSCs multiplication capacity

8.2.1 the 3rd generation AMSCs cell suspension is divided into 5 parts, wherein 4 parts to add respectively final concentration be 10 ^-8mM activated vitamin D (1,25-dihydroxyvitamin D, 1,25 (OH) ₂d ₃), 10 ^-7mM prostaglandin 2 (prostaglandin2, PEG2), 10ng/ml epidermal growth factor (epidermal growth factor, EGF), 10ng/ml fibroblast growth factor (basic fibroblast growth factor, bFGF), in the culture dish that every group is planted respectively three diameter 60mm, carry out labelling, in 37 ℃, 5%CO ₂, saturated humidity incubator in cultivate, within every 3 days, change culture fluid one time, after cultivating 7 days, go culture fluid stop to cultivate, carry out methylene blue staining.

8.2.2 methylene blue staining: after cell rinses with PBS, dehydrated alcohol is 30s fixedly, distilled water flushing, the borate buffer solution of 10mM is washed 3mins, under 1% methylene blue room temperature, hatches 30mins, after dyeing finishes, rinse well with borate buffer solution, in air, dry and take pictures.

8.2.3IPP image processing software calculates the percentage ratio that cell accounts for the culture dish floor space, and mean with mean ± standard deviation (mean ± SEM), row statistical analysis while adopting SPSS13.0 software, relatively adopt one factor analysis of variance between group, relatively adopt the t check between two groups of means, P<0.05 shows to have significant difference.

8.3 the 3rd generation AMSCs differentiation capability detects

8.3.1 induce the 3rd generation AMSCs to Osteoblast Differentiation and detection

8.3.1.1AMSCs be inoculated in culture dish, discard culture medium after 24h, change osteoblast induced liquid (α MEM culture fluid, 15% hyclone, 100U/ml penicillin, 100g/mL streptomycin, 10 into ^-8m dexamethasone, 50 μ g/ml ascorbic acid), in 37 ℃, 5%CO ₂, saturated humidity incubator in cultivate, within every 3 days, change one time culture fluid, within 14 days, stop afterwards cultivating and carrying out alkaline phosphatase staining.

8.3.1.2 alkali phosphatase (alkaline phosphatase, ALP) staining: cell cleans 1 time with PBS, dehydrated alcohol is fixed, and outwell a moment, with tap water, rinses gently, with borate buffer solution, clean 1 time again, add ALP substrate solution (after naphthols 1mg is dissolved in 50 microlitre ethylene glycol phenyl ethers, with the fast red 5ml PH9.2Tris-HCL that is dissolved in of 2mg simultaneously), room temperature, lucifuge, 15min.Tap water rinses, and dries rear Microscopic observation and takes pictures.

8.3.2 induce the 3rd generation AMSCs to the fat differentiation and detect

8.3.2.1AMSCs after being inoculated in culture dish, discard culture medium after 24h, change into fat inducing culture liquid into (containing 15% hyclone, 2mM L-glutamine, 100U/ml penicillin, 100U/ml streptomycin, 50 μ g/ml ascorbic acid, 100 μ g/ml insulins, 10 ^-8the M dexamethasone), in 37 ℃, 5%CO ₂, saturated humidity incubator in cultivate.Every 3 days, change liquid 1 time.Observe growing state, within the 14th day, stop cultivating and carrying out oil red dyeing.

8.3.2.2 oil red staining: after cell rinses gently with PBS, 4 ℃, PLP is 30min fixedly, and PBS rinses, and adds oil red dye liquor 15min.60% isopropyl alcohol, vibration gently, remove excess dyestuff, and tap water rinses, and adds a small amount of phosphate buffer, micro-Microscopic observation film making.

9.RT-PCR

9.1 experimental procedure

9.1.1 after cell culture fluid is toppled over, with 0.01M PBS, rinse 3 times each 3min.

9.1.2 add 1ml TRIzol in the 100mm culture dish, with the 1ml rifle head blunt end without RNase, cell is scraped from the culture dish bottom, then together with TRIzol, together be transferred to the Eppendorf pipe of 1.5ml without RNase.

9.1.3 the homogenate sample is placed to 5min in room temperature, the nucleic acid-protein complex is separated fully.

9.1.4 every use 1ml TRIzol adds the 0.2ml chloroform, thermal agitation 15s, and room temperature is placed 3min.

9.1.52-8 the centrifugal 15min of ℃ 10000 * g.Sample is divided into three layers: bottom is yellow organic facies, and upper strata is colourless water and an intermediate layer.RNA is mainly in water, and the water volume is about 60% of TRIzol reagent used.

9.1.6 water is transferred in new pipe, and with the RNA in the isopropanol precipitating water, every use 1ml TRIzol adds the 0.5ml isopropyl alcohol, vibration mixes, and room temperature is placed 10min.

9.1.72-8 the centrifugal 10min of ℃ 10000 * g, do not see the RNA precipitation before centrifugal, gelatinous precipitate occurs in pipe side and the pipe end after centrifugal, abandons supernatant.

9.1.8 by 75 ℅ washing with alcohol RNA precipitations.Every use 1ml TRIzol at least adds 1ml75 ℅ ethanol.Be no more than 7500 * g, 2-8 ℃ of centrifugal 5min, abandon supernatant.

9.1.9 room temperature is placed dry RNA precipitation, 5-10min approximately dries in the air.Add the water of 25-200 μ l without RNA enzyme (RNAase), with the rifle head, inhale and beat several times, 55-60 ℃ of water-bath 10min dissolves RNA.

9.1.10 getting the total RNA of 1 μ l is diluted to 100 μ l measures RNA concentration on the nucleic acid determination instrument.

Carry out reverse transcription 9.1.11 get the total RNA of 2 μ g (X μ l): get the total RNA of X μ l and add sterilizing 0.1%DEPC water to 10 μ l+50pmol/ μ l Oligo (d) T151 μ l+10mM dNTP Mix1 μ l and mix, put on ice fast after 65 ℃ of water-bath 5min.

9.1.12 add following reaction system:

9.1.13 pipettor blow and beat gently mix centrifugal, 37 ℃ of water-bath 55min.

9.1.1470 ℃ water-bath 15min.Collect reverse transcription product (cDNA) in-86 ℃ of Refrigerator stores.

9.1.15PCR reaction primer:

The forward primer sequence of Bmi-1 is: 5 '-ATTGATGCCACTACCATAAT-3 ', and reverse primer is 5 '-CCTGGACATCACAAATAGGAC-3 ';

The forward primer sequence of Oct4 is: 5 '-CGCCCGCATACGAGTTCT-3 ', and reverse primer is 5 '-CTTCTCCAACTTCACGGCATT-3 ';

The forward primer sequence of CXCR4 is: 5 '-GACGGACAAGTACCGGCTGC-3 ', and reverse primer is 5 '-GACAGCTTAGAGATGATGAT-3 ';

The forward primer of GAPDH gene (internal reference) is: 5 '-CATTTCACTCAAGGTTGTCAGC-3 ', reverse primer is: 5 '-ATCATACTTGGCAGGTTTCTCC-3 '.

Primer is synthetic by Shanghai Invitrogen company.

9.1.16PCR reaction system

9.1.17PCR condition:

The PCR reaction system of GAPDH and condition are with gene to be checked.The Bmi-1 amplified production is 103bp, and the Oct4 amplified production is 188bp, and the CXCR4 amplified production is 480bp, GAPDH amplified production 346bp.

9.1.18 collecting reaction product, the molecular weight that carries out agarose gel electrophoresis checking fluorescence quantitative RT-RCR product is little and clear and definite without non-specific amplification.

9.1.19 use ultraviolet photometry (ultraviolet photometry, UVP) the computer picture scanning analysis system measure the gene Messenger RNA that detects (messenger ribonucleic acid, mRNA) level, with glyceraldehyde phosphate dehydrogenase (glyceraldehyde phosphate dehydrogenase, GAPDH) be internal reference, calculate the relative amount of the gene mRNA that detects, the percentage rate (%) that result is accounted for the GAPDH ribbon density with detected gene mRNA means.

10. low cytometric analysis

10.1 anesthesia: lumbar injection 1% pentobarbital 6-10 μ l/g body weight.Separate thymus, spleen, bilateral femur and tibia, be placed on ice.

10.2 screen cloth is placed in to culture dish on ice, grind thymus, spleen, obtain cell suspension, standing on ice; With PBS, long bone bone marrow is gone out, obtained cell suspension, standing on ice.

10.32000rpm centrifugal 5min, remove supernatant, adds PBS100 μ l re-suspended cell.

In this step, cell quantity need to generally be adjusted, to be no less than 1x10 ⁷/ part cell is good, with the negative contrast of wild type control mice (WT) cell suspension.

10.4 traget antibody:

10.5 myeloid element: WT stays 3 parts of cells, and 2 parts compare

10.6 lucifuge adds antibody: (c-kit is the surface markers of hematopoietic stem cell (haemopoietic stem cell, HSC) for Sca-1, Lin, wherein, and sca-1 _-PEthe anti-mice sca-1 antibody that refers to second channel PE red fluorescence labelling, Lin _{-Alexa flour488}the anti-mice Lin antibody that refers to first passage Alexa Flour488 green fluorescence labelling, c-kit _-PE-CY5.5the anti-mice c-kit antibody that refers to third channel PE-CY5.5 purple fluorescence labelling);

Negative control: only add the PE homotype and contrast 1.25 μ l;

The 2nd part of contrast: add sca-1 _-PE1.25 μ l; C-kit _-PE-CY5.50.3 μ l;

Sample: add sca-1 _-PE1.25 μ l, c-kit _-PE-CY5.50.3 μ l, Lin _{-Alexa flour488}5 μ l.

10.7 room temperature, lucifuge 20min.Add PBS to 1ml resuspended, 2000rpm, 5min, abandon cleaning.

10.8.200 μ l PBS is resuspended, adds 200 μ l4% paraformaldehydes and mixes, 4 ℃ of lucifuges are treated machine testing.

10.9 bone marrow B cell:

Lucifuge adds antibody:

Negative control: only add homotype contrast-FITC0.25 μ l;

Sample: add B220 _-PE2.5 μ l, CD43 _-FITC0.25 μ l, IgM _-PECy5.51.25 μ l.

10.10 same myeloid element.

10.11 spleen B cell:

Lucifuge adds antibody:

Negative control: do not add;

Sample: add B220 _-PE2.5 μ l, IgM _-PECy5.51.25 μ l, IgD _{-Alex flour647}0.625 μ l.

10.12 same myeloid element.

10.13 spleen t-cell:

Lucifuge adds antibody

Negative control: do not add;

Sample: add: CD4 _-FITC0.5 μ l, CD8 _-PE1.25 μ l.

10.14 same myeloid element.

10.15 thymus T cell

Lucifuge adds antibody:

Negative control inspection: do not add;

Sample: add CD4 _-FITC0.5 μ l, CD8 _-PE1.25 μ l, CD25 _-APC0.625 μ l, CD44 _--PECy5.51.25 μ l.

(APC, Allophycocyanin, allophycocyanin, blue-fluorescence)

10.16 same myeloid element.

10.17 upper machine analysis.

11. cytometry

11.1 get WT in brood 4 week age, matched group Bmi-1 ^-/-with processed group Bmi-1 ^-/-mice, each 6.

11.2 each treated animal etherization, adopt the heart blood collection method, gets peripheral blood 20 μ l, adds in 180 μ l diluents, mixes rear upper machine testing.Every mice detects 3 parts.

12. statistical analysis

Data mean by mean ± standard deviation, with SPSS13.0 software, carry out one factor analysis of variance or t check, and p<0.05 is considered to statistical significance.

Result:

1.1AMSCs preparation and dryness feature

Use the primary AMSCs of trypsin digestion separation and Culture, cultivate after 3d days, attached cell is increased rapidly, and the visible cell adherent growth is fusiformis, polygon etc.The spindle shape (Fig. 2 A) that is homogeneous by the 2nd generation AMSCs of the amplification of going down to posterity.Through LacZ cytochemical staining, polymerase chain reaction,PCR (polymerase chain reaction, PCR) and western blotting (Western blot) confirm that respectively the 2nd generation AMSCs in β-gal transgenic mice source presents beta galactosidase activity (Fig. 2 B, the LacZ positive), express Bmi-1 gene and albumen (Fig. 2 M and N), be used as donorcells.

For the multi-lineage potential of clear and definite AMSCs, we to osteoblast and adipose cell direction induction, detect the ability of AMSCs to osteoblast, the differentiation of adipose cell direction by alkaline phosphatase staining, oil red colouring method by the 3rd generation AMSCs respectively.Result shows: to the osteoblast inducing culture, after 14 days, alkaline phosphatase staining is positive, i.e. alkali phosphatase positive expression (Fig. 2 C); To the adipose cell inducing culture, after 14 days, oil red dyeing shows that the part cell has been divided into adipose cell (Fig. 2 D); Thereby illustrated that AMSCs has to the potential of osteoblast and Adipocyte Differentiation.

For the versatility characteristic that confirms AMSCs and the feature of Interstitial cell thereof, detected embryonic stem cell labelled molecule CXCR4, Oct-4, SSEA4 by RT-PCR, immunofluorescence, flow cytometer, hematopoietic stem cell labelled molecule CD34, CD45 and mescenchymal stem cell labelled molecule CD29, CD44, CD73, CD90, CD105 express at 2nd generation AMSCs.Found that: 2nd generation AMSCs can high expressed Interstitial cell labelled molecule CD29, CD44, CD73, CD90, CD105 (Fig. 2 E-I), do not express hematopoietic stem cell label CD34, CD45 (figure K-L), and express embryonic stem cell labelled molecule CXCR4, Oct4, SSEA4 (Fig. 2 J, O-R).These presentation of results AMSCs has the phenotypic characteristic of mescenchymal stem cell.

1.2AMSCs amplification

For the proliferation potential of clear and definite AMSCs and whether be subject to the adjusting of somatomedin or hormone, 2nd generation AMSCs is through 10ng/ml EGF, 10ng/ml bFGF or 10 ^-8m1,25 (OH) ₂d ₃process after 8 days, detected the propagation situation of 2nd generation AMSCs through methylene blue staining.Result shows: somatomedin EGF and bFGF processing or Combined Treatment and 1,25 (OH) ₂d ₃after processing, can obviously increase the area (Fig. 3 A-F) of methyl blue dyeing AMSC.These presentation of results: somatomedin EGF and bFGF and 1,25 (OH) ₂d ₃all there is the effect that promotes AMSC propagation, can be used for stimulating the AMSC amplification in vitro.

Extend Bmi-1 1.3AMSCs transplant ^-/-life-span of mice and improve its growth promoter

For clear and definite AMSCs transplants Bmi-1 ^-/-the impact of old and feeble phenotype before the mice maturation, we are by lumbar injection 10 ⁶individual AMSCs/50 μ lPBS is to Bmi-1 ^-/-mice, 2 days from being born start, and 1 time weekly, inject 3 times, matched group lumbar injection PBS, then respectively to brood WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the mouse survival rate is monitored.The normal WT mice 2-3 of generally surviving, Bmi-1 ^-/-the mean survival time (MST) of mice is only about 4 weeks, AMSCs transplantation group Bmi-1 ^-/-obviously extend the life cycle of mice, within about 14 weeks, still has mouse survival (Fig. 4 A).The result shows that the AMSCs transplanting can extend Bmi-1 significantly ^-/-the life cycle of mice.

Compare Bmi-1 with brood WT mice ^-/-it is smaller that mice gives birth to latter 1 day physique, with age, engenders bow-backed grade for performance.4 week age Bmi-1 ^-/-mice shows as small, and body weight is light, and Thymus and spleen size, weight are markedly inferior to the WT mice.Carrying out property retarded growth after presentation of results Bmi-1 gene knockout causes mice to be born.

For clear and definite AMSCs transplants Bmi-1 ^-/-the impact of Mouse Weight, thymus and spleen, our comparative analysis brood WT in 4 week age, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the difference of the build of mice, body weight and Thymus and spleen size.Result shows: with littermate control Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-the mice build obviously increases (Fig. 4 C), and from the 3rd week, body weight obviously raise (Fig. 4 B), but still was starkly lower than brood WT mice; The more brood Bmi-1 of the size of Thymus and spleen ^-/-mice obviously increases, and still is less than brood WT mice (Fig. 4 D and E).These presentation of results AMSCs can improve the retarded growth of Bmi-1 disappearance senescence accelerated mouse.

Improve Bmi-1 1.4AMSCs transplant ^-/-the hematopoietic disorders of mice

For clear and definite AMSCs transplants Bmi-1 ^-/-the impact of Mouse Blood cell parameters, we get brood WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the peripheral blood of mice carries out the detection of blood routine parameter.Result shows: with matched group Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-it is that cell proportion is significantly corrected (table 1) that mice is drenched.The transplanting of these presentation of results AMSCs has improved the hematopoietic disorders of disappearance senescence accelerated mouse.

Table 1.AMSCs transplants Bmi-1 ^-/-the impact of mouse peripheral blood cell parameters

^*: p<0.05, ^*: p<0.01, ^{* *}: p<0.001, with brood WT mice, compare; #:p<0.05, with littermate control group Bmi-1 ^-/-mice is compared.

Improve Bmi-1 1.5AMSCs transplant ^-/-the mouse lymphocyte dysplasia

For after further observing AMSCs and transplanting to Bmi-1 ^-/-mice T system and B are the impact that lymphocyte is grown, and we are by immunofluorescence dyeing, and it is that in lymphocyte and bone marrow and spleen, B is the variation of each stage ratio of lymphocyte that flow cytometer has detected Thymus and spleen T.

Thymus is the place of T development and cell differentiation, and the size variation of thymus has reflected the change of T cell development state.We observe Bmi-1 in 4 week age ^-/-the thymus of mice is significantly less than brood WT mice.Flow cytometry is found to compare with brood WT mice, Bmi-1 ^-/-cD4 in mouse thymus ⁺cD8 ⁺the ratio of two positive cells is starkly lower than WT mice, CD4 ^-cD8 ^-jack to jack adapter sexual cell ratio obviously increases.AMSCs transplants Bmi-1 after 4 weeks ^-/-mice CD4 ⁺cD8 ⁺the ratio of two positive cells obviously increases (Fig. 5 A and 5D), CD4 ^-cD8 ^-jack to jack adapter sexual cell ratio obviously reduces (Fig. 5 A and 5C).These presentation of results AMSCs transplants and obviously to have improved thymocyte proliferation that the Bmi-1 disappearance causes and ripe obstacle.

Ripe T cell can be settled down in peripheral lymphoid organs, and we have further detected the ratio of mature T cells in the spleen and have changed.Flow cytometry is found WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the CD4 of maturation in mouse spleen ⁺and CD8 ⁺the equal no significant difference of T cell proportion (Fig. 5 B and 5G).

In bone marrow, the growth of B cell probably is divided into pro-B cell (B220+IgM ^-cD43 ⁺), pre-B cell(B220 ⁺igM ^-cD43 ^-), immature B cell(B220 ⁺igM ⁺cD43 ^-) three phases.From bone marrow, immature B cell out can enter spleen and settles down, through T1 (IgM ⁺igD ^-) and T2 (IgM ⁺igD ⁺) two stages finally become ripe B cell (B220 ⁺igM ⁺cD43 ^-).We are by immunofluorescence dyeing, flow cytometry analysis AMSCs transplant after on the cytocerastic impact of different phase B in Bmi-1 disappearance mouse bone marrow cells and spleen.Result shows: compare Bmi-1 with brood WT mice ^-/-in mouse bone marrow cells, pro-B cell, pre-B cell and immature B cell subsets ratio significantly descend, mature B cell no significant difference (Fig. 6 A, C-F), T1 (IgM in spleen ⁺igD ^-) cell proportion significantly descend (Fig. 6 B, G).These results have confirmed that the disappearance of Bmi-1 causes the change of bone marrow and spleen B cell subsets ratio.With matched group Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-mice pro-B cell proportion increases significantly in bone marrow, and in spleen, the T1 cell also significantly increases, and reaches the level (Fig. 6 C) of brood WT mice.These presentation of results AMSCs transplant the disappearance can partly correct Bmi-1 and cause the abnormal of part B cell subsets ratio in spleen and bone marrow.

Improve the aging of Bmi-1 disappearance senescence accelerated mouse skeleton 1.6AMSCs transplant

For clear and definite AMSCs transplants Bmi-1 ^-/-the impact of mice skeleton aging, we utilize the method for histology, histochemistry and Western blot, have observed AMSCs and have transplanted Bmi-1 ^-/-the impact of adipose cell and correlation molecule protein expression level in Mouse Bone amount, osteoblast, bone marrow.Compare matched group Bmi-1 with the WT mice ^-/-mouse Bone capacity and osteoblast number all obviously reduce, and in osseous tissue, core connects factor a-1 (Cbfa-1) and all obviously downwards of insulin-like growth factor-i (insulin-like growth factor, IGF-1) protein expression level; But, in bone marrow, the adipose cell number obviously increases, in osseous tissue, gene outcome peroxisome proliferator-activated receptor γ (peroxisome Proliferator-activated receptor γ, the PPAR γ) expression of leading Adipocyte Differentiation obviously raises (Fig. 7).These results have confirmed Bmi-1 ^-/-there is the skeleton senilism phenotype that ripe osteoid is loose in mice.With matched group Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-mouse Bone capacity and osteoblast number all obviously raise, and Cbfa-1 and IGF-1 protein expression level all obviously raise; In bone marrow, the adipose cell number obviously reduces, and in osseous tissue, PPAR γ expression is obviously lowered, yet these indexs all do not reach the level of normal WT.These presentation of results AMSCs transplants and can suppress adipose cell in marrow and form by increasing the osteoblast bone formation, and it is loose to improve the ripe osteoid that the Bmi-1 disappearance causes.

1.7 the AMSCs transplanted can move in host's Different Organs

For the AMSCs that clearly transplants at Bmi-1 ^-/-the distribution of Different Organs in Mice Body, we have detected Bmi-1 gene and albumen at matched group and transplantation group Bmi-1 by PCR and Westernblot and LacZ histochemical stain ^-/-expression in the mice Different Organs and donorcells are at transplantation group Bmi-1 ^-/-the distribution of mice Different Organs.Result shows: at matched group Bmi-1 ^-/-can't detect the expression of Bmi-1 gene and albumen in mouse core, liver, spleen, lung, kidney, bone marrow and thymic tissue, and at transplantation group Bmi-1 ^-/-in these organ-tissues of mice, the expression (Fig. 8 A and B) of Bmi-1 gene and albumen can be detected.Observe the LacZ positive cell be dispersed in transplantation group mouse lung, spleen and renal tissue, control group mice can't detect LacZ positive cell (Fig. 8 C).These presentation of results AMSCs is transplanted to Bmi-1 ^-/-in Mice Body, can, by migration, distribute and arrive whole body different tissues organ.

Suppress 1.8AMSCs transplant the oxidative stress increase that the Bmi-1 disappearance causes

For transplanting, clear and definite AMSCs extends Bmi-1 ^-/-life-span of mice and whether improve its senilism phenotype relevant with its anti-oxidative stress, we have detected brood WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-mice whole body different tissues reactive oxygen species (reactive oxygen species, ROS) level, hydrogen peroxide (hydrogen peroxide, H ₂o ₂) content, total superoxide dismutase (total-superoxide dismutase, T-SOD) is active and the variation of catalase (catalase, CAT) activity.Found that: compare matched group Bmi-1 with brood WT mice ^-/-in Mouse Liver, spleen, lung, kidney, bone marrow, thymic tissue cell, the ROS level obviously raises, transplantation group Bmi-1 ^-/-in Mouse Liver, spleen, lung, bone marrow, thymic tissue cell, the ROS level is improved (Fig. 9 A) significantly.Compare matched group Bmi-1 with brood WT mice ^-/-h in mouse core, liver, spleen, lung, kidney, bone marrow, thymic tissue ₂o ₂content significantly increases, H in the heart, spleen, bone marrow, thymus after AMSCs transplants ₂o ₂content obviously reduces (Fig. 9 B).Simultaneously, matched group Bmi-1 ^-/-in mouse core, liver,spleen,kidney, bone marrow, thymic tissue, CAT is active significantly reduces, active obviously correct (Fig. 9 C) of CAT in the heart, spleen, kidney, bone marrow, thymic tissue after AMSCs transplants.Matched group Bmi-1 ^-/-in mouse core, liver, spleen, lung, kidney, bone marrow, thymus, antioxidase T-SOD is active significantly reduces, and after AMSCs transplants, in the heart, spleen, bone marrow, thymus, (Fig. 9 D) appears improving significantly in the activity of T-SOD.These presentation of results AMSCs transplants can reduce Bmi-1 ^-/-the Oxygen Free Radical In Mouse level, the activity of enhancing antioxidase, reach and suppress Bmi-1 ^-/-the oxidative stress of mice, thus performance extends Bmi-1 ^-/-the life-span of mice is also improved the effect of its old and feeble phenotype.

Suppress 1.9AMSCs transplant the expression that Bmi-1 lacks the old and feeble correlation molecule caused

Whether for clear and definite AMSCs transplants, to improve the skeleton senilism that the Bmi-1 disappearance causes relevant with the expression that suppresses SAG, and we have detected the change of Wnt16 and cyclin dependent kinase inhibitive factor p16, p19 and p27 expression in the osseous tissue by Western blot.Result shows: compare matched group Bmi-1 with brood WT mice ^-/-the expression of mice Wnt16, p16, p19 and p27 albumen all significantly raises, and AMSCs transplants all significantly downwards (Figure 10) of expression of rear these albumen.These presentation of results AMSCs transplant improve skeleton senilism that the Bmi-1 disappearance causes may with lower regulating senescence molecule Wnt16, p16, p19 are relevant with the p27 protein expression level.AMSCs and secretory product thereof can be used for preparing the inhibitor of Wnt16, p16, p19 and p27 albumen.

Discuss:

Amnion tissue mainly is comprised of the amniotic mesenchymal cell (amnion mesenchymal stem cells, AMSCs) in mesoderm source and amniotic epithelial cells (amnion epithelial cells, AECs) the two class cells in ectoderm source.According to the literature, AECs does not express the mesenchymal cell labelled molecule, is distinctive paving stone sample outward appearance during In vitro culture, and AMSCs high expressed mesenchymal cell labelled molecule, adherent growth during In vitro culture, be spindle shape, fibroblast sample morphological characteristic [17,20-23].Our result of study has also confirmed that AMSCs is fusiformis, and spindle goes down to posterity and cultivates the fibroblast sample morphological characteristic that is uniformity, expresses CD29, CD44, CD73, CD90, CD105, thereby illustrates that the cell that we separate is AMSCs.We further confirm that AMSCs can express embryonic stem cell labelled molecule CXCR4, Oct4 and SSEA-4, do not express hematopoietic stem cell labelled molecule CD34, CD45, can be divided into osteoblast or adipose cell through external evoked cultivation, this and previous AMSCs result of study consistent [17,18,23].Simultaneously, we also find that the propagation of AMSCs is subject to somatomedin bFGF and EGF, 1,25 (OH) ₂d ₃adjusting.BFGF, EGF and 1,25 (OH) ₂d ₃can promote significantly AMSCs propagation, bFGF and EGF Combined Treatment can promote AMSCs propagation more significantly.The mechanism of action of these molecules in promoting AMSCs propagation still requires study.Our result supports AMSCs to have the conclusion of multipotency mescenchymal stem cell feature.

Stem cell defying age therapy also claims " regenerative medicine technology ", is described as at present the most effective antidotal therapy means in the world.Its possible mechanism of action is as follows: 1. direct effect: stem cell is a kind of cell with multi-lineage potential as emerging seed cell.Transplant stem cell energy self renewal and can be divided into various kinds of cell, tissue is repaired.2. paracrine action: stem cell can secrete a large amount of cytokines, take mescenchymal stem cell as example, and its excreted factor mainly can be divided into immunomodulating, anti-apoptotic, blood vessel generation, the propagation of supporting ancestral cells and differentiation, chemical chemotactic, the large class of anti-cicatrix six on function.They,, by the mode of autocrine or paracrine, improve the body local microenvironment, promote migration and the differentiation of endogenous retinal stem cells, save and are on the verge of the cell of apoptosis, thereby reach the purpose [24-25] of antidotal therapy.

The stem cell that Recent study is maximum is mesenchymal cell (the bone marrow-mesenchymal stem cells that derives from bone marrow, BM-MSCs), but BM-MSCs must obtain by the wound approach is arranged, and with advancing age, its quantity and stem cell potential significantly descend.Because AMSCs system separates and obtains from the amnion tissue of garbage in puerperal, not only source is abundant, the amplification ability is strong, reduced immunogenicity, and therefore its use, is reproduced medical domain and is considered as a kind of important donorcells [16] without ethnics Problem.Previous existing research shows: after AMSCs is transplanted to allogeneic or heterogenous allosome animal, can be by migration, the pathological tissues organ of going back to the nest, and then be divided into the cell of relevant histoorgan, as myocardial cell [17], liver cell [26], neurocyte etc., reach the different disease for the treatment of, as liver cirrhosis [26], parkinsonism [27] etc.

The Bmi-1 gene is one of Polycomb Group gene family member, has suppressed the Space-time speciality of Hox gene and express in growth and development process.Bmi-1 disappearance mice show as ripe before senilism, significantly shorten life cycle, only can survive about 4 weeks, the self-renewal capacity decline relevant [4-7,28] of main and neural stem cell, hematopoietic stem cell and mesenchymal stem cells MSCs.In this research, we will be transplanted to Bmi-1 as donorcells by lumbar injection from the 2nd generation AMSCs that β-the gal transgenic mice obtains ^-/-in Mice Body, to inquire into AMSCs, be implanted in the effects anb Mechanism in defying age.Found that the AMSCs transplanting can obviously extend Bmi-1 ^-/-the life cycle of mice, and then we are to 4 week age brood WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-the phenotype of mice compares analysis.

Our result of study is found, is compared matched group Bmi-1 with the WT mice ^-/-mice build, Thymus and spleen significantly reduce, and body weight obviously reduces, these consistent with result in the past [28].With matched group Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-mouse Weight obviously increases, and build, Thymus and spleen all obviously increase.Bmi-1 has significantly been corrected in the transplanting of the above results explanation AMSCs ^-/-the retarded growth of mice part.

When Bmi-1 lacks, mice shows the abnormal phenotype [29] of blood system.Our result of study has also confirmed Bmi-1 in 4 week age ^-/-in mouse peripheral blood, leukocyte, platelet significantly reduce, and pouring is that the myeloid cell ratio is inverted, and other blood parameters (RBC number, hemoglobin concentration, packed cell volume, mean corpuscular volume (MCV)) does not change.After AMSCs transplants, pouring is that cell proportion is corrected significantly, and other parameter is unchanged.Bmi-1 can have partly been corrected in the transplanting of this explanation AMSCs ^-/-mouse peripheral blood cell parameters abnormal.

Thymus is the place of T development and cell differentiation, and the size variation of thymus has reflected the change of T cell development state.T cell development, function and survival obstacle will cause serious cellular immunity deficiency.Cellular immunity deficiency comprises heritability immunodeficiency and acquired immunodeficiency.And propose the generation of doing T cell in thymus, it is the physiological approach that conforms with most that recovers the T cellular immunity.But difficulty is that the amount and the function that increase thymus with the age are more and more poor.Consistent with experimental result in the past, we find equally: compare Bmi-1 with the WT mice ^-/-mouse thymus obviously reduces, Bmi-1 ^-/-cD4 in mouse thymus ⁺cD8 ⁺the ratio of two positive cells is starkly lower than WT mice, CD4 ^-cD8 ^-jack to jack adapter sexual cell ratio obviously increases [11].So Bmi-1 ^-/-may there be the heritability immunodeficiency in mice.We find: after AMSCs transplants, and Bmi-1 ^-/-cD4 in mouse thymus ⁺cD8 ⁺the ratio of two positive cells obviously increases, CD4 ^-cD8 ^-jack to jack adapter sexual cell ratio obviously reduces, but does not return to the WT level.Therefore, our result of study is provable: AMSCs transplants and can partly correct the cellular immunity deficiency that the Bmi-1 disappearance causes.

In bone marrow, the growth of B cell probably is divided into pro-B cell, pre-B cell, immature B cell several stages [30,31].We found that, the disappearance of Bmi-1 causes the change of bone marrow and spleen B cell subsets ratio.With matched group Bmi-1 ^-/-mice is compared, transplantation group Bmi-1 ^-/-mice pro-B cell proportion increases significantly in bone marrow, and in spleen, the T1 cell also significantly increases, and reaches the level of brood WT mice.The increase of spleen volume may be relevant with the increase of mature B cell ratio.The transplanting of these presentation of results AMSCs may promote the maturation of B cell in the growth of pro-B cell in bone marrow and spleen simultaneously, thereby part is corrected bone marrow and the abnormal phenotype of spleen.Therefore, our result of study has proved: AMSCs transplants and can partly correct the hematopoietic disorders that the Bmi-1 disappearance causes.

Previous result of study shows, 4 week age Bmi-1 ^-/-mice shows as ripe front osteoporosis [28].We found that, compare matched group Bmi-1 with the WT mice ^-/-mouse Bone capacity and osteoblast number all obviously reduce, and in osseous tissue, Cbfa-1 and IGF-1 protein expression level are all obviously lowered; But in bone marrow, the adipose cell number obviously increases, in osseous tissue, PPAR γ expression obviously raises, this with in the past to Bmi-1 ^-/-the result of study of mice consistent [28].After AMSCs transplants, although These parameters does not return to WT mice level, the protein expression level of bone volume and osteoblast digital display work increase, Cbfa1 and IGF-1 significantly raises, and adipose cell digital display work reduces and Ppar γ protein expression level is lowered.These presentation of results AMSCs transplants and can form by promoting osteoblast bone formation and inhibition adipose cell in marrow, not only partly correct the loose senilism phenotype of ripe osteoid that the Bmi-1 disappearance causes, and promoted the rectification of the hematopoietic disorders that the Bmi-1 disappearance causes by improving hematopoieticmicroenviron-ment.

AMSCs transplants not only can improve hematopoieticmicroenviron-ment, and can further maintain the skeletonization microenvironment by the rectification of hemopoietic system, improves its oxidative stress level, promotes skeletonization, brings into play preferably the osteoporotic effect of control.

Beta galactosidase is a very effective molecular marked compound, and the spike donorcells moves in vivo, distributes, breeds and breaks up effectively, with clear and definite its effect in participating in normal structure self renewal and damaged tissue reparation.In this research, we use and are transplanted to Bmi-1 from the AMSCs that β-the gal transgenic mice obtains as donorcells ^-/-in Mice Body, by the LacZ histochemical stain, come the spike donorcells at Bmi-1 ^-/-the distribution of mice whole body different tissues organ.We found that at transplantation group Bmi-1 ^-/-mouse lung, spleen and kidney detect some LacZ positive cells, in control group mice, can't detect.Simultaneously, we utilize the method for PCR and Western blot at Bmi-1 ^-/-mice whole body Different Organs detects the expression of Bmi-1 gene and albumen.Our another research passes through to transplant the AMSCs in β-gal transgenic mice source to the WT mice, utilize the two marks of Neuron-specific labelled molecule and β-gal immunofluorescence, proved that the AMSCs transplanted can be divided into neurogliocyte and oligodendrocyte [19].The AMSCs that these presentation of results are transplanted can move to Bmi-1 by blood circulation ^-/-the different tissues of mice organ, be divided into the cell of different tissues, and bring into play the direct effect that rectification Bmi-1 disappearance causes senilism.

Recent research shows in the AMSCs culture supernatant to contain a large amount of cytokines [46].The experimental result of the holostrome skin burn rat of Franz etc. [47] injection AMSCs supernatant treatment coli-infection shows, after treatment the 25th day, the supernatant treatment group infects matched group wound healing degree every other week 15% raising, and increase the i.e. injection every other day of the injection frequency, more can improve 20% wound healing degree.Illustrated that thus AMSCs has effective paracrine action.

When various factors has been broken the dynamic equilibrium of interior free yl generation and elimination, thereby will make free radical surpass the physiology limit, cause tissue injury.Old and feeble Free radicals thinks with advancing age, and active oxygen constantly produces accumulation at human body, causes the aging [48] of cell, tissue and organ.The somatic biomacromolecule in ROS attack plane (lipid, protein, nucleic acid) of accumulation and cause tissue oxygen voltinism damage to cause the generation of old and feeble and relevant disease.SOD is the important metalloenzyme of defence oxidative damage in organism, its catalysis superoxide anion O ₂ ^-dismutation reaction occurs, thereby removes O ₂ ^-; CAT be take the desmoenzyme that iron porphyrin is prothetic group.It can impel H ₂o ₂be decomposed into molecular oxygen and water, remove the body hydrogen peroxide, thereby make cell avoid suffering H ₂o ₂murder by poisoning, be the key enzyme of the imperial system of biological biological and ecological methods to prevent plant disease, pests, and erosion.Previous research [8] shows: antioxidant removes to correct Bmi-1 by the level that reduces oxidative stress ^-/-the old and feeble phenotype of mice.Have recently research [49] to show: stem cell transplantation also can be used as a kind for the treatment of means, go to treat many diseases relevant with oxidative stress, as acute myocardial infarction [50], cerebral ischemia [51] and diabetes [52,53], but the effect of stem cell transplantation in anti-oxidation stress there is not yet report.Rat MSCs and human bone marrow substrate cell are cultivated under the condition of ascorbic acid can expression activity SOD1, SOD2, CAT, GSH-px and sulfur oxygen albumen reductase are proved [54,55].We found that: AMSCs transplants can extend Bmi-1 ^-/-in the life-span of mice, it is loose that part is corrected hematopoietic disorders and ripe osteoid that Bmi-1 disappearance causes.For clear and definite AMSCs is implanted in the mechanism of action in defying age, we have detected brood WT mice, matched group Bmi-1 ^-/-mice and transplantation group Bmi-1 ^-/-rOS level, H in mice whole body different tissues cell ₂o ₂the variation of content, T-SOD and CAT activity.Found that: AMSCs transplants can make Bmi-1 ^-/-different tissues of mice organ ROS and H ₂o ₂content reduces, and the good CAT of T-SOD is active to raise.These presentation of results ASMCs transplants and can increase activities of antioxidant enzymes by paracrine action, removes free radical and brings into play and correct the old and feeble phenotype that the Bmi-1 disappearance causes.

1987, Rijsewijk etc. [56], by the contrast of the gene order to the aptery gene Wg of fruit bat and mice oncogene int-1, found that the two has the sequence homology of height, therefore is collectively referred to as Wnt by the two.The common feature of Wnt is the one group of Wnt albumen (Wnt1～Wnt16) [39,57] containing 23～24 cysteine conserved sequences of can encoding.The Wnt signal path is comprised of extracellular signaling molecule (Wnt albumen), transmembrane receptor and the complicated interior cascade albumen of born of the same parents, can be divided into two kinds of classical pathway and non-classical approach.Study the most at present, be clear that Wnt/ β-catenin signal path is also referred to as classical Wnt signal path most.Wnt/ β-catenin signal path participates in the processes [58] such as cell proliferation, differentiation, apoptosis and celluar localization control.The research of Binet etc. [59] shows, no matter at replicative senescence, still oncogene, induce in old and feeble MEFs, the Wnt16 albumen of high expressed all can be detected, in culture fluid, add Wnt16 albumen can promote that MEFs has advanced aging, and after suppressing the Wnt16 protein expression with siRNA, in MEFs, p53 and p21 protein expression level are lowered, and cell ageing is suppressed.Our result of study is also found, compares Bmi-1 with the WT mice ^-/-in the Mouse Bone tissue, the Wnt16 protein expression level obviously raises, and illustrates that the Wnt16 up-regulated is at Bmi-1 ^-/-in the mice aging, also play an important role.Our AMSCs transplanting that found that has obviously suppressed Wnt16 at Bmi-1 ^-/-expression in the Mouse Bone tissue.It may be one of mechanism of its performance anti-aging effects that these presentation of results AMSCs transplanting suppresses the Wnt16 protein expression by paracrine action.

Previous research report Bmi-1 can regulate by p16 and p19 path the proliferation and apoptosis of neural stem cell [6,54] and hematopoietic stem cell [5,55].P27 has a very important role [60] as one of Cyclin kinase inhibitor in the cell cycle regulation process, is another negative regulate molecule of Bmi-1 cell cycle regulation.For clear and definite AMSCs is transplanted to Bmi-1 ^-/-whether the bone amount caused in Mice Body increases relevant with rise CDKI with the adipose cell minimizing, and we have detected p16 by Western-blot, p19, and p27 is in the expression of osseous tissue.Result shows and brood Bmi-1 ^-/-mice is compared, and p16, p19 and the p27 expression in receptor Mouse Bone tissue obviously reduces.These presentation of results AMSCs transplants also the expression that can suppress p16, p19 and p27 by paracrine action and brings into play its anti-aging effects.

This research shows that pluripotent stem cell AMSCs transplanting can move to the different histoorgan of whole body, is divided into the cell of Different Organs, and the performance part is corrected the direct effect that the Bmi-1 disappearance causes senilism; Can pass through paracrine action, raise the activity of antioxidase, remove oxygen-derived free radicals, suppress Aging-associated gene expression, the performance part is corrected the indirect action that the Bmi-1 disappearance causes senilism simultaneously.This research provides theory and experimental basis for AMSCs transplants slow down aging.

Brief summary:

The present invention, in embodiment 1, is implanted in the effects anb Mechanism in defying age in order to study amnion mesenchymal stem cell, and the 2nd generation AMSCs that we will derive from β-gal transgenic mice is transplanted to Bmi-1 ^-/-in Mice Body, comparative analysis transplantation group Bmi-1 in 4 week age ^-/-mice and matched group Bmi-1 ^-/-the phenotypic difference of mice Different Organs, brief summary is as following table 2:

Table 2:

Annotate: ↓ ↓ ↓: significantly reduce ↓: reduce (statistical significance is arranged)

↑ ↑ ↑: significantly increase (statistical significance is arranged) ↑ ↑: obviously increase

↑: increase (statistical significance is arranged)-: negative+: the positive

Conclusion:

This result of study shows that the AMSCs transplanting can move to the different histoorgan of whole body by it, be divided into the cell of Different Organs, can raise the activity of antioxidase through paracrine action by the different factors of secretion again, remove oxygen-derived free radicals, suppress SAG, the performance part is corrected the effect that the Bmi-1 disappearance causes senilism.

Initialism

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Claims (9)

1. amnion mesenchymal stem cell and/or the application of its secretory product in preparing the medicine of life-saving, health product or cosmetics.

2. application according to claim 1, is characterized in that medicine, health product or the cosmetics of life-saving refer to antiaging agent, health product or cosmetics.

3. application according to claim 2, is characterized in that antiaging agent, health product or cosmetics refer to medicine, health product or the cosmetics of anti-immunocyte aging, removing free radical or enhancing activities of antioxidant enzymes.

4. application according to claim 2, is characterized in that antiaging agent, health product or cosmetics refer to the inhibitor of Wnt16 protein expression inhibitor or cyclin dependent kinase inhibitive factor p16, p19 or p27 albumen.

5. application according to claim 1, is characterized in that medicine, health product or the cosmetics of life-saving refer to medicine, health product or the cosmetics that improve hematological function.

6. application according to claim 5, is characterized in that improving hematological function and refer to and improve hematopoietic disorder, especially improves the lymphocyte series hematopoietic disorder.

7. application according to claim 1, is characterized in that medicine, health product or the cosmetics of life-saving refer to promotion bone growth medicine, health product or cosmetics.

8. application according to claim 7, is characterized in that promoting bone growth to refer to promoting bone growing and/or improve osteoporosis.

9. application according to claim 1, is characterized in that medicine, health product or the cosmetics of life-saving refer to medicine, health product or the cosmetics that improve the Bmi-1 disappearance.

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