CN110161226A - A kind of antibody, kit and detection method detecting senile cell - Google Patents

Abstract

The present invention relates to Cell Measurement Technique field, a kind of antibody detecting senile cell, the antibody is at least one of the following: p16^INK4a, nucleotide sequence is as shown in SEQ ID No.1, p53, and nucleotide sequence is as shown in SEQ ID No.2, p21, and nucleotide sequence is as shown in SEQ ID No.3, pRB, and nucleotide sequence is as shown in SEQ ID No.4, p19^ARF, nucleotide sequence is as shown in SEQ ID No.5, p38^MAPK, nucleotide sequence is as shown in SEQ ID No.6, p14^ARF, nucleotide sequence is as shown in SEQ ID No.7, p15^INK4b, nucleotide sequence is as shown in SEQ ID No.8, p62, and nucleotide sequence is as shown in SEQ ID No.9, p65, and nucleotide sequence is as shown in SEQ ID No.10.

Description

A kind of antibody, kit and detection method detecting senile cell

Technical field

The present invention relates to Cell Measurement Technique fields, especially detect antibody, the senile cell marker of senile cell Detection method and kit and preparation method thereof.

Background technique

Chronic disease is to threaten the main hidden danger of health of people, according to the related data that health department is announced, in recent years Chronic disease illness trend, which is presented always, rises situation, and is not controlled effectively；The characteristics of chronic disease be organ not Reversible progressive structural disease becomes, once make a definite diagnosis the lifelong medication of needs.Therefore, prevention is carried out to chronic disease and illness is compared in intervention Treatment afterwards is more advantageous to the control state of an illness.

Aging is a complicated biological phenomena, often while people's aging, the risk of chronic diseases It is stepped up；The development of the chronic diseases such as aging and cancer, diabetes, cardiovascular disease and neurodegenerative disease has more Close relationship (Jan M.van Deursen .Nature.May 22；

509(7501):439–446,2014).Since the division number of culture cell is limited by Hai Fulike (Hayflick Et al., Exp.Cell Res.37:585-621,1961) since discovery, cell cycle research is always cell biology one Hot spot.In recent years research and propose telomere shorten, DNA damage reaction, epigenetic change, mitochondria dysfunction, cell generation Thank functional disturbance, extracellular matrix functional disturbance etc. be the most important factor of cell ageing (NainaBhatia-Deyetal., Front Gennet.,12February 2016.00013).Wherein telomere, which shortens, opens cell ageing program, and telomere shortens It is as caused by cellular replication aging and DNA damage, senile cell accumulates in vivo with rheological properties aging is increased.Cell ageing can To exhaust stem cell or progenitor cells, this is by damaging tissue's reparation, regeneration function, so as to cause the decaying of function of organization；Meanwhile it declining Uneducated person, which closes the cell factor that secretion phenotype generates, will affect and destroy institutional framework and function；And generate chronic low-level inflammation Immune surveillance function is set to weaken (Francis Rodier, Judith Campisi；J Cell Biol.2011Feb 21；192 (4):547–556).Effectively prove that senile cell has (Bennett G in a large amount of agings or pathological tissue Childs et al.,Nat Med.2015Dec；21 (12): 1424-1435), being found to be for senile cell is gradually disclosed It cracks various chronic diseases and more research directions is provided, to explain the nosogenesis of chronic disease.

Senile cell is usually expressed as following multiple characteristics: 1, aging division is permanent stagnates, and will not pass through known proliferation The stimulation of signal and take a turn for the worse.2, the appearance of senile cell becomes larger, twice of size of sometimes non-senile cell.3, aging is thin The relevant beta galactosidase of cellular expression aging (SA- β-gal) reflects the increase of cytase weight.4, most of agings Cell expresses p16^INK4a, static or terminal differentiation cell do not express.5, DNA damage reaction (DDR) signal of duration can lure It sends out nucleus and forms lesion, the DNA of referred to as reinforcement aging changes segment (DNA-SCARS).DNA-SCARS0 includes functional disturbance Telomere or telomere dysfunction induction lesion.6, senile cell expresses and can secrete molecule relevant to aging, claims For the relevant secretion phenotype (SASP) of aging.SASP phenotype is DNA damage, telomere dysfunction, epigenetics or has silk point Split signal, the result of oxidative stress and some other agings induction stimulation.7, the nucleus of senile cell loses structural proteins. (Freund et al.,Mol.Biol.Cell 23:2066-75(2012)；Davaloset al.,J.Cell Biol.201: 613-29(2013)；Ivanov et al.,J.Cell Biol.DOI:10.1083/jcb.201212110,1-15page； On July 1st, 2013 delivers online；Funayamaet al.,J.Cell Biol.175:869-80(2006)).

The program of cell ageing involves starting up various active protein ingredient, promotes cell to enter the process of aging, participates in it In have；p53,pRb,p16^INK4a、p21、p19^ARF、p38^MAPK、p14^Arf、p15^INK4b, the significant albumen such as p62, p65.Cell The process of aging be it is irreversible, if p53 or pRb protein inactivation, cell can reverse aging form to continue to divide.Aging is thin Born of the same parents also express betagalactosidase activity simultaneously, for increasing plasminogen activator inhibitor (PAI) level, contaminate at pH6 Color beta galactosidase (SA- β-Gal) shows activity (Sharpless et al., J.Clin.Invest.113:160- 168,2004).It is by making the cell cycle protein dependent kinase of Rb phosphorylation (CDK) compound that the irreversible G1 phase, which stagnates, It inactivates to mediate.P21 is accumulated in senile cell and is inhibited CDK4-CDK6.p16^INK4aInhibit CDK4-CDK6 and β galactolipin Glycosides enzyme activity and in proportion in cell volume accumulate (Stein et al., Mol.Cell.Biol.19:2109-2117, 1999).P21 expression when cell ageing starts, but not continue exist in cell；And p16^INK4aExpression can continue at carefully Whole process after born of the same parents' aging.

Evidence suggests there are relationships between replicative senescence and aging.Culture cell Proliferation from old donor compared with It is few, occur compared with the culture cell of young donor more senile cells (Martin et al., Lab.Invest.23: 86-92,1970；Schneider et al.,Proc.Nat.Acad.Sci.USA 73:3584-3588,1976).From short-lived Life species culture cells show gone out more less than the cell proliferation of long-life species and more senile cell phenomenons (Rohme, D.,Proc.Nat.Acad.Sci.USA 78:5009-3320,1981).And from have heredity early ageing syndrome (such as Werner syndrome) donor cell culture, show than not there is the cell of early ageing sign donor more senile cells occur (Goldstein, Genetics of Aging, 171-224,1978；Martin, Genetic Effects on Aging, 5- 39,1990)。

However, there are also other aging approach, these agings commonly known as induction property early ageing other than replicative senescence (SIPS).Response to oxidative stress may cause to telomere and shorten (von Zglinicki, Trends Biochem.Sci.27:339- 344,2002), and evidence show oxygen environments can Induction of Cellular senescence appearance.Human fibroblasts are in G1 phase early metaphase When by gamma-rays irradiate with activate p53 independent path cause aging (Di Leonardo et al., Genes Dev.8: 2540-2551,1994).Ultraviolet radiation also can Induction of Cellular senescence (Krtolica et al., Proc.Nat.Acad.Sci.USA 98:12072-12077,2001).Some other treatment and drug can induce cell ageing, It is such that cell ageing is led to by destruction cell DNA.Cell ageing be present in vivo it is verified that.In Dimri et al. in 1995 In the research delivered in year, senescent fibroblast shows beta galactosidase (SA- β-Gal) vital staining in pH6.This A little cells fail to mix tritium-labeled thymidine after renewed vaccination and retain SA- β-Gal activity, but not divide.It is static at Fibrocyte fails to detect SA- β-Gal activity.Horn cell, huve cell and galactophore epithelial cell can be shown SA- β-Gal activity increases out.SA- β-Gal dyeing is carried out in Skin biopsy to test whether aging, dermal cell With age-dependent sexual norm is observed in epidermal cell, wherein the biggish cell of donor age shows SA- β-Gal activity (Dimri et al.,Proc.Nat.Acad.Sci.USA 92:9363-9367,1995).Relative to the tested of lung health Person has suffered from and shows that senescent fibroblast exists and quantity increase (M ü ller et in the pneumonocyte of pulmonary emphysema subject al.,Resp.Res.7:32-41,2006)。

Replicative senescence is the Senescent type that Hayflick is initially observed, and the type is by the dye in fission process Colour solid telomere foreshortens to the cell ageing for being unable to effective protection DNA structure and causing.Replicability cell ageing can pass through expression Telomerase extends the length of telomere, so will not trigger cell ageing mechanism (Mathon and Lloyd, Nat.Rev.Cancer 3:203-213,2001；Martins,U.M.Exp Cell Res.256:291-299,2000)；When thin When cellular expression Telomerase, human fibroblasts can be replicated indefinitely.Most of cancer cell expression Telomerases are to maintain Telomere length and infinite copy.The a small number of cancers for not expressing Telomerase have lengthening of telomeres (ALT) mechanism of substitution.

The stroma cell of aging can promote tumour, while play neighbouring epithelial cell the tumour of paracrine action Inhibiting factor；These effects include mitogenesis and anti-apoptotic (Chang et al., Proc.Nat.Acad.Sci.USA 97 (8):4291-4296,2000).The epithelial cell of canceration before senescent fibroblast irritation cancer has been displayed, but cannot stimulate normal Epithelial cell tumour is formed in mouse；(the Krtolica in the fibroblast aging within 10% occurs for such case et al.,Proc.Nat.Acad.Sci.USA 98:12072-12077,2001).By senile cell secretion tumor promotion because Subdivision mediates (Roninson, Cancer Res.63:2705-2715,2003) by p21.The stroma cell of aging can provide Cancer anterior epithelium cornea cell survival, migration and the microenvironment of division (Campisi, Nat.Rev.Cancer 3:339- near allowing 349,2003)。

In short, cell ageing occurs in vivo really, and the microenvironment of cells survival can be damaged.And some groups The illness rate knitted can increase with the age and increase risk.Aging assigns the change of cellular functional, to a certain extent and respectively The age-related disease of kind has direct relation (Chang et al., Proc.Nat.Acad.Sci.USA 97 (8): 4291- 4296,2000)。

Summary of the invention

It is an object of the invention to pass through CBA (Cytometric Bead Array) Flow cytometry senile cell. Senile cell can gradually accumulation in vivo, will affect the function of histoorgan, which can carry out senile cell Detection, with understand the senile cell in histoorgan accumulation degree and caused by harm.

Technical purpose of the invention is achieved in that

A kind of antibody detecting senile cell, which is characterized in that the antibody is at least one of the following:

p16^INK4a, nucleotide sequence as shown in SEQ ID No.1,

P53, nucleotide sequence as shown in SEQ ID No.2,

P21, nucleotide sequence as shown in SEQ ID No.3,

PRB, nucleotide sequence as shown in SEQ ID No.4,

p19^ARF, nucleotide sequence as shown in SEQ ID No.5,

p38^MAPK, nucleotide sequence as shown in SEQ ID No.6,

p14^ARF, nucleotide sequence as shown in SEQ ID No.7,

p15^INK4b, nucleotide sequence as shown in SEQ ID No.8,

P62, nucleotide sequence as shown in SEQ ID No.9,

P65, nucleotide sequence is as shown in SEQ ID No.10.

A kind of kit detecting senile cell, it is characterised in that: the kit includes fluorescence viability dye, is directed to The antibody of the fluorescent marker of senile cell target or magnetic bead target, the antibody include p16^INK4a、p53、p21、pRB、p19^ARF、 p38^MAPK、p14^ARF、p15^INK4b, at least one of p62, p65 antibody.

A kind of preparation method for the kit detecting senile cell, it is characterised in that: prepare magnetic bead and antibody sample respectively Product carry out antibody coupling after removing unreacted component.

The magnetic bead for preparing sequentially includes the following steps:

The magnetic bead for taking out 65~85 μ l blank is washed with deionized twice, magnetic bead is vortexed and is ultrasonically treated to avoid poly- Collect and ensures that the bead in solution is uniformly distributed；It is suspended in 80 μ L later and contains 100mM sodium phosphate, 10 μ L N- hydroxy ambers In the activation buffer of amber acid imide and 10 μ L 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides, it is diluted to The concentration of 50mg/mL is spare；

1M DTT is configured with deionized water；2 μ L 1M DTT are taken to be added in the Ep pipe containing magnetic bead and be vortexed again for mixing It is even；After mixing, magnetic bead is protected from light to heat preservation 20 minutes at room temperature；It is washed three times with PBS buffer solution, at room temperature in orbital oscillation After being incubated for 2 hours on device, in the washing/store buffer liquid for being suspended in 1mL, it is kept in dark place at 2 DEG C to 8 DEG C.

The antibody samples that prepare sequentially include the following steps:

Magnetic bead is kept suspending by rotation, the conjugate solution of 100 μ l is added, under protein concentration maximum case, Each magnetic bead is in combination with about 5 × 10⁶A tested protein molecular；Mixture is warm in the dark at room temperature on orbit shaker It educates 1 hour.

The removal unreacted component sequentially includes the following steps:

Magnetic bead is washed three times to remove the target of non-specific binding with BCB buffer, and magnetic bead is resuspended in 2mL and contains phosphorus In the activation buffer of sour sodium, Sulfo-NHS and EDC and pass through the dispersion magnetic bead that is vortexed.

A kind of preparation method of kit, it is characterised in that: the antibody coupling sequentially includes the following steps:

Activation of protein in buffer is transferred in the Ep pipe containing the functional magnetic bead previously prepared and is vortexed mixed It is even, and room temperature is protected from light incubation 1 hour on the oscillator；2 μ l are contained into sodium phosphate, the activation buffer of Sulfo-NHS and EDC add Enter into the Ep pipe containing functional magnetic bead and activated protein, is vortexed and mixes and be incubated for 15 minutes on the oscillator；It, will after incubation 1ml store buffer liquid is added in pipe, centrifugation；It is washed in triplicate with store buffer liquid；Then, it is slow that 0.5ml storage will be resuspended in Magnetic bead particles in fliud flushing store at 4 DEG C, and concentration is 6 × 10⁶Magnetic bead/ml.

A kind of senile cell detection method, it is characterised in that: carried out using aging marker of the kit to senile cell Label reapplies fluidic cell statistics aging marker.

The invention has the advantages that: the present invention is by the conventional flow cytometry of kit cooperation to senile cell The case where can detecting to senile cell, know senile cell, testing result is accurate, easy to operate quick.

Detailed description of the invention

Fig. 1 a: by aging induction, aging marker increases display IMR90 cell comprehensively compared with the control group；

Fig. 1 b: detecting after inducing aging to mescenchymal stem cell, the data of acquisition aging marker compared with the control group It is to increase comprehensively, and otherness is obvious.

Fig. 2 a: for the histogram of flow cytometry analysis after IMR90 cell ageing induction；

Fig. 2 b: for the histogram through overflow-type Cytometric Analysis after mescenchymal stem cell aging induction.

Fig. 3 a: senile cell tests progress at any time in diabetic mouse model, and quantity gradually increases.

Fig. 3 b: the various markers of senile cell are also identical as the ratio of blood glucose rise, gradually increase, wherein p16 albumen It is promoted in the ratio of the second half expression very fast.

Fig. 4 a: senile cell quantity experimental group is being stepped up compared with the control group in atherosclerosis mouse model, Increase to the experiment obvious large scale of later period quantity.

Fig. 4 b: atherosclerosis mouse each aging marker levels in lysis are also increasing.

Fig. 5 a: senile cell quantity experimental group is gradually rising compared with the control group in idiopathic pulmonary fibrosis mouse model Height increases to the experiment obvious large scale of later period quantity.

Fig. 5 b: idiopathic pulmonary fibrosis mouse each aging marker levels in lysis are also increasing.

Fig. 6 a: senile cell quantity experimental group is gradually rising compared with the control group in chronic obstructive pulmonary disease mouse model Height increases to the experiment obvious large scale of later period quantity.

Fig. 6 b: chronic obstructive pulmonary disease mouse model each aging marker levels in lysis are also increasing.

Fig. 7: the dyeing of aging correlation beta galactosidase carries out fluorescence for aging marker p16, p53, p21, pRB albumen Labeled Antibodies Staining analysis, diabetes, atherosclerosis, chronic obstructive pulmonary disease, the experimental group of idiopathic pulmonary fibrosis with Control group senile cell map.

Fig. 8: immunoprecipitation carries out electrophoretic analysis, diabetes, artery for aging marker p16, p53, p21, pRB albumen Atherosis, chronic obstructive pulmonary disease, the experimental group of idiopathic pulmonary fibrosis and control group senile cell electrophoresis map analysis.

Fig. 9: flow cytometry carries out fluorescent labeled antibody dyeing point for aging marker p16, p53, p21, pRB albumen Analysis, diabetes, atherosclerosis, chronic obstructive pulmonary disease, the experimental group of idiopathic pulmonary fibrosis and control group senile cell Fluorescence level representative diagram spectrum analysis.

The analysis of the thermal map of Figure 10: SASP secretion experimental group and control group mice, the secretion of experimental group each component is obvious to be risen, And showing comprehensive red, i.e., the secretion of the initial SASP of disease shows a large amount of increase after a week.

Specific embodiment

With reference to the accompanying drawings of the specification, the invention will be further described.

P16 used in the present embodiment^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、p15^INK4b, p62, p65 egg The white specific protein to express in cellular senescence process, these albumen participate in cellular senescence process or cause cell autophagy program It opens.

It can be coloured in conjunction with a variety of fluorescent dyes in hydrophobic dye using polystyrene magnetic beads.And N- hydroxy Succinimide (Sulfo-NHS) and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and antigen molecule Covalent coupling, Sulfo-NHS contain n-hydroxysuccinimide and EDC carbodiimide group.NHS ester at pH7-9 with primary amine Reaction forms amido bond, and carbodiimide group reacts to form stable thioether bond at pH4.5-6.0 with sulfydryl.It realizes to declining The detection of old cell marker.

Usually and easily, antibody used in method of the invention and kit is for target cell (or magnetic bead) target The monoclonal or polyclonal antibody marked and be conjugated with fluorochrome label object.

Senile cell marker detection kit preparation method is as follows:

Magnetic bead preparation

The magnetic bead for taking out 65~85 μ l blank is washed with deionized twice, and magnetic bead is vortexed and is ultrasonically treated that (2 are followed Ring) to avoid assembling and ensure that the bead in solution is uniformly distributed.It is suspended in 80 μ l later and contains 100mM sodium phosphate, 10 μ l N- Hydroxy thiosuccinimide (Sulfo-NHS) and 10 μ l 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides (EDC) in activation buffer, the concentration for being diluted to 50mg/mL is spare.1M DTT (dithiothreitol (DTT)) is configured with deionized water； 2 μ l 1M DTT are taken to be added in the Ep pipe containing magnetic bead and be vortexed again for mixing.After mixing, magnetic bead is protected from light to guarantor at room temperature Temperature 20 minutes.It is washed three times with PBS (phosphate buffer), after being incubated for 2 hours on orbit shaker at room temperature, with suspension In washing/store buffer liquid [PBS, 1% bovine serum albumin(BSA), 0.02% polysorbas20,0.05% sodium azide] of 1mL, 2 DEG C to being kept in dark place at 8 DEG C.

Cell ageing marker antibody samples

Magnetic bead is kept suspending by rotation, the conjugate solution of 100 μ l is added, under protein concentration maximum case, Each magnetic bead is in combination with about 5 × 10⁶A tested protein molecular.Mixture is warm in the dark at room temperature on orbit shaker It educates 1 hour.

Remove unreacted component

With BCB [BCB: phosphate buffer, supplement 1% bovine serum albumin(BSA) (BSA)] buffer washing magnetic bead three times with Magnetic bead is resuspended in 2mL and contains sodium phosphate by the target for removing non-specific binding, in the activation buffer of Sulfo-NHS and EDC And pass through the dispersion magnetic bead that is vortexed.

Antibody coupling

Activation of protein in buffer is transferred in the Ep pipe containing the functional magnetic bead previously prepared and is vortexed mixed It is even, and room temperature is protected from light incubation 1 hour on the oscillator.2 μ l are contained into sodium phosphate, the activation buffer of Sulfo-NHS and EDC add Enter into the Ep pipe containing functional magnetic bead and activated protein, is vortexed and mixes and be incubated for 15 minutes on the oscillator.It, will after incubation 1ml store buffer liquid is added in pipe, centrifugation.It is washed in triplicate with store buffer liquid.Then, it is slow that 0.5ml storage will be resuspended in Magnetic bead particles in fliud flushing store at 4 DEG C, and concentration is 6 × 10⁶Magnetic bead/ml.

The detection of sample

Before the use, reagent is placed under room temperature, all operating process needs are carried out in the dark.

The magnetic bead of 1 μ l is taken to be used for the detection of every kind of senile cell marker.Magnetic bead is washed with 0.5ml buffer, with 200 × G centrifugation.Supernatant is removed, magnetic bead is resuspended in dilution, and volume is increased into 50 μ l, is vortexed and is incubated at room temperature 15 minutes.Prepare that 50 μ l are added in each test tube and dilute corresponding to 12 × 75mm test tube of experiment marker matching number Magnetic bead；20 μ l storage liquid is added into negative control test tube；The senile cell marker sample that 20 μ l have been diluted with 1:25 is added Product (being diluted with storage buffer) arrive test tubes；It is vortexed after mixing and is protected from light incubation 30 minutes in room temperature；Centrifugation, 1000 × g, 10 Minute, abandon supernatant；The antibody (being diluted with PBS with 1:200) of 150 μ l PE label is added, is vortexed after mixing and is protected from light incubation in room temperature 30 minutes；Centrifugation, 1000 × g 10 minutes, abandon supernatant, are resuspended in 150 μ l washing buffers；It is examined with flow cytometer It surveys.

The present invention provides it is a kind of detect senile cell kit, wherein comprising fluorescence viability dye and for aging it is thin The fluorescent labeled antibody of born of the same parents (or magnetic bead) target, includes p16^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、 p15^INK4b, a variety of or whole markers of at least one of p62, p65 albumen.

Senile cell detection kit includes at least one or more of marker antibody, that is, is used for while detection and label are given Multiple target albumen in random sample product, the albumen include p16^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、p15^INK4b、 p62,p65.According to advantageous embodiment, the method that 1~10 while label can be used can be detected highly accurately Senile cell (or magnetic bead) described herein.

Different (aniso-) fluorochrome label of different fluorescent labeled antibodies, makes it possible in list in the present invention The senile cell marker (such as passing through flow cytometry) combined by the antibody is separated in a detecting step.The kit can Optionally comprising that can identify the specific antibody of senile cell (or magnetic bead) pedigree, the senile cell usually by it is identical (or It is substantially equivalent) fluorochrome label, more generally with the fluorochrome label identical or substantially equivalent with fluorescence viability dye.

In embodiments, the antibody of label is selected from least 1~10 kind of different target, the cell target p16^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、p15^INK4b, p62, p65 albumen.Every kind of possibility represents the present invention Independent embodiment.In certain embodiments, kit includes multiple cells (or magnetic bead) counting group, and each group comprising living Power dyestuff and a variety of antibody for different cells (or magnetic bead) target.

In an example, the culture cell induced by aging is realized to p16^INK4a、p53、p21、pRB、p19^ARF、 p38^MAPK、p14^ARF、p15^INK4b, the corresponding albumen of p62, p65 detection, such detection can be a kind of albumen it is a variety of or Whole albumen.In embodiments, at least one aging biomarker of cell detection sample exists or its variant or segment In the presence of.Sample may include fibroblast, osteoblast, osteoclast, macrophage, mast cell, fat cell, acidophilia Granulocyte, neutrophil leucocyte, T lymphocyte, bone-marrow-derived lymphocyte, K lymphocyte, NK lymphocyte, fills basophilic granulocyte Matter stem cell, multipotential stem cell, embryonic stem cell, nerve cell, Deiter's cells, cardiac muscle cell, smooth muscle cell, bone Myocyte, epithelial cell, PECTORAL LIMB SKELETON, endothelial cell, cartilage cell.

In an example, aging marker p16 is detected in diabetic mice^INK4a, p53, p21, pRB egg White expression up-regulation, the being positively correlated property always in disease progression, when the fatty senile cell of mouse reaches 5% ratio When, diabetes enter the period of expansion of disease, and when senile cell reaches 19%, disease has entered the symptom phase, 5%~19% During ratio, the diabetes early stage disease asymptomatic formation phase can be considered as, such marker plays promotion to development such as diabetes Effect has clinical meaning.The icotype of the up-regulation of aging marker is related to the diabetes in mouse and the mankind.For Preventive test is carried out in health population, can effectively the metabolism such as pre- preventing obesity, diabetes, metabolic syndrome class disease be sent out It is raw.The present invention is not limited to above-mentioned experiments, and the preventive test of metabolism class chronic disease used can be used.

In an example, data show that atherosclerosis includes that macrophage, vascular smooth muscle with aging are thin Born of the same parents, while the activity and p16 of aging correlation beta galactosidase (SA- β-GAL)^Ink4a, pRB, p53 and p21 albumen expression, make Cell is easy aging.P16 is observed in human peripheral blood detection^INK4aProtein positive rate is higher, while p53, p21, pRB, p19^ArfAlbumen The trend gradually risen is showed, shows that senile cell is gradually formed in diseased region.It is carried out by the tissue to lesion patch Culture, isolate macrophage and arterial wall smooth muscle cell, and carry out SA- β-Gal dyeing observation, the macrophage of aging with The ratio of smooth muscle cell is gradually rising, ratio by 4.3% rise to 15.3% level, show its formation with disease Journey has direct relationship.After patch formation, the trend of aging tends towards stability, but marker index is in high-level shape always State.Data show, SA- the β-Gal, p16 of experimental mice^INK4a、p53、p21、p19^Arf, pRB protein level be apparently higher than control Group mouse.Vascular smooth muscle cells are also showed with progression of atherosclerosis and the gradually process of aging, to the tension of blood vessel It is gradually reduced with ductility.

Preferably, cardiovascular disease can be angina pectoris, arrhythmia cordis, atherosclerosis, cardiomyopathy, congested mental and physical efforts Failure, coronary artery disease (CAD), carotid disease, endocarditis, (coronary artery thrombosis is formed heart disease, myocardial infarction [MI]), any one or more of hypertension pressure/hypertension, aortic aneurysm, cerebral aneurysm, cardiac fibrosis, heart relax Dysfunction is opened, hypercholesterolemia/hyperlipidemia, mitral valve prolapse, peripheral artery disease is (for example, peripheral arterial disease (PAD)), cardiac stress resistance and apoplexy.

In an example, by the relationship of senile cell detection and related disease development to respiratory system, to aging Respiratory disease such as: idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, branch The detection of the diseases such as tracheaectasy and pulmonary emphysema.

Idiopathic pulmonary fibrosis (IPF) is a kind of progressive fibroproliferative tuberculosis that reason is unknown, is lung at fibre It ties up the formation of cell stove, the disorder of alveolar unit structure and lung and forms hardening and cicatricial tissue.It can lead to respiratory failure, lung cancer and Heart failure.Pulmonary fibrosis is related with the reparation of epithelial cell.The accumulation of fibroblast/myofibroblast and excessively generation The abnormal remodeling of the lung structure of matrix rich in I-type collagen.Myofibroblast is main cell type, is responsible for Excess generation extracellular matrix in fibroblast stove, this is the feature of fibrotic processes.The participation table of cell ageing in IPF Bright, the disease incidence of the disease increases with advancing age, and the lung tissue of IPF patient is rich in p16^INK4aProtein positive is thin Born of the same parents and contain high-caliber cell.The p16 of aging label^INK4a, p53, p21, pRB albumen be that IPF and cell ageing are common Risks and assumptions.

In another example, the cell ageing of smoke from cigarette induction participates in chronic obstructive pulmonary disease (COPD)/pulmonary emphysema Pathogenesis, the cell that lung airway Constituent cell (epithelium and fibroblast) shows that smoke from cigarette induces in vitro and in vivo decline It is old to increase.Cell ageing feature p16^INK4a, the expression of p53, p21 and pRB albumen, beta galactosidase (SA- relevant to aging β-GAL) activity increase.Smoke from cigarette generates inflammatory reaction to respiratory tract, and active oxygen promotes the aging of pneumonocyte, leads Cause senile cell factor release damage lung.Genetic predisposition may also lead to disease.

In embodiments, test sample can be in test subject's body, be also possible to experimental animal (such as Mouse or rat) or people, wherein this method is based on intracorporal test.Alternatively, sample can be vitro samples or external sample. Therefore, cell to be measured can be in tissue sample (for based on external test) or cell can grow (body in culture Outer sample).Preferably, biological sample is vitro samples.

In embodiments, at least one of test sample senile cell biomarker or its variant or segment in vitro Presence.Sample may include blood, blood plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate Liquid, sperm, vaginal secretion, excrement, cervical smear, cell, amniotic fluid, intraocular liquid, mucus, moisture, breathing, animal tissue, cell are split Solve object, tumor tissues, hair, skin, oral cavity scrapings, nail, marrow, cartilage, prion, bone meal, earwax or combinations thereof.

Preferably, at least one of test sample senile cell biomarker diseases associated with senescence or illness in vitro. Obstacle or disease include cognitive illnesses (for example, mild cognitive impairment (MCI), Alzheimer disease and other dementias；Huntingdon dance Step disease)；Cardiovascular disease (such as atherosclerosis, Diastolic dysfunction, aortic aneurysm, angina pectoris, arrhythmia cordis, the heart Myopathy, congestive heart failure, coronary artery disease, myocardial infarction, endocarditis, hypertension, carotid disease, peripheral blood vessel Disease, cardiac stress reaction, myocardial fibrosis)；Metabolic disease and disorder (as fat, diabetes, metabolic syndrome)；Move function Energy disease and obstacle are (for example, Parkinson's disease, motor neuron dysfunction (MND)；Huntington's chorea)；Cranial vascular disease； Apoplexy；Osteoarthritis；Benign prostatauxe；Pulmonary disease (such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), Pulmonary emphysema, bronchiolitis, asthma)；Inflammation/autoimmune disease and illness are (for example, osteoarthritis, eczema, ox-hide Tinea, osteoporosis, catarrh transplant related disease and illness)；Ophthalmology disease or illness (such as age-related macular degeneration, Cataract, glaucoma, hypopsia, presbyopia)；Diabetic ulcer；Side effects of chemotherapy, radiotherapy side effect；It is related to aging Disease and illness (such as humpback, renal insufficiency is weak, alopecia, hearing disability, muscular fatigue, skin, flesh Meat reduces disease and the protrusion of the intervertebral disc) and other disease agings related with the age (for example, radiation, chemotherapy, smoking, feed height Disease/disorder caused by fat/high-carbonhydrate diet and environmental factor)；Wound healing；Skin disease (such as: cutaneous nevus, with light sensitivity Or the related disease of light aging and illness, wrinkle, itch is insensitive, acidophilus dermatoses, reactive neutrophilic skin Skin disease, pemphigus, pemphigoid, immune dermatosis, skin fiber histiocytosis, skin lymphoma and cutaneous lupus)； Fibrotic disease and illness (for example, cystic fibrosis, kidney fibrosis, liver fibrosis, pulmonary fibrosis, oral submucosa fibrosis, Cardiac fibrosis and pancreatic fibrosis).Tumour (respiratory system: lung cancer, nasopharyngeal carcinoma, laryngocarcinoma, gland cancer；Digestive system: gastric cancer, liver Cancer, cancer of the esophagus, intestinal cancer, cancer of pancreas, gallbladder cancer, peritoneal cancer, cancer of anus, peri-ampullar carcinoma, carcinoma of mouth, carcinoma of cecum, carcinoma of parotid gland, print Guard against cell cancer, insulinoma；Urinary system: kidney, bladder cancer, prostate cancer, carcinoma of urethra, clear cell carcinoma；The circulatory system: white blood Disease, lymph cancer, hemangioma, aneurysm, hemangioblastoma；Kinematic system: osteocarcinoma, ganglion, liomyoma, neurinoma； Reproductive system: cervical carcinoma, uterine cancer, oophoroma, carcinoma of testis, choriocarcinoma, carcinoma of fallopian tube, carcinoma of vagina, carcinoma of penis and fat Tumor, breast cancer, brain tumor, squamous carcinoma, cutaneum carcinoma, thyroid cancer, lip cancer, hypophysoma, hamartoma, sebaceous cyst, glioma, syringocystadenoma, Matter tumor, cranium tumour, sarcoma, chondroma, head and neck cancer, fibroma, cancer eye, thoracic cavity cancer, gingival carcinoma, basal cell adenocarcinoma, wellability Duct carcinoma, cholesteatoma of external auditory meatus, acoustic neurinoma, pheochromocytoma, mediastinal tumor).

The above description is only a preferred embodiment of the present invention, and for those skilled in the art, the present invention can have Various modifications and variations.All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on, should all It is included within protection scope of the present invention.

Embodiment 1:

Detectable substance: senile cell culture and induction

1, IMR90 cell culture and induction

We simulate the process of cell ageing using IMR90 cell.Cell is grown in containing 10% fetal calf serum (FBS) In DMEM culture medium, 10%CO₂, grow and reach 1 × 10⁵Density when pass on.Before analysis keep static control cultures Converge or kept for 4 days in 0.1%FBS, and replaces culture medium daily.It induces aging: using Etoposide and H₂O₂Inducing cell Aging is handled 48 hours with 100 μM of Etoposides when IMR90 cell is merged there are about 60-70%, and uses H₂O₂It is trained in serum-free (400 μM) of base are supported to handle 1 hour.Then culture medium is replaced and in the 7th day harvest cell.It collects cell and is used for appraisal of aging.

2, mescenchymal stem cell culture and induction

With DMEM cultivate mescenchymal stem cell, add 10%FBS, 100ml units of Penicillin, 100 μ g/ml streptomysins and 2mM glutamine induces aging: cultivating the O 20% and 3%₂Under concentration, senile cell group and non-aging group are obtained respectively. Culture medium is replaced weekly twice, and passage in cell every 7 days is primary, up to the approximation for reaching 80% is converged.Monitor in-vitro cell growth And cell number is counted with hemacytometer.

Testing result

In order to verify senile cell marker kit, using the culture cell experiment of two independent aging inductions, survey The ability that two kinds of different cell lines induced and then formed aging marker detection by aging is tried；By flow cytometry analysis It is shown after statistics, the IMR90 cell and mescenchymal stem cell which can induce aging detect.It is therein p16^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、p15^INK4b, p62, p65 albumen have significance difference compared with the control group It is different.

Embodiment 2:

High fat diet or high fruit are given as animal for research using 6~8 week old C57BL/6 mouse in the present embodiment Sugared diet；Control group is using normal feeding.It weighs in weekly and extracts peripheric venous blood 1ml, for measuring senile cell mark Will object and blood glucose level；After measurement, using CO₂Mouse is put to death, fat cell is collected and pancreatic cell carries out aging point Analysis, and be compared with the aging marker in blood.

In feeding, blood glucose level gradually rises mouse after two weeks, carries out staining analysis to SA- β-Gal activity and shows that aging is thin Born of the same parents also rise to 5% by initial 3%, and accounting steps up in the process, when blood glucose level is stablized in 16mmol/L or so When, the ratio of senile cell is close to 19%, while rising scale eases up；And the symptom of diabetes typical case's three-many-one-little starts to show It is existing；P16 is carried out to senile cell^INK4a, p53, p21, pRB Protein Detection, display aging marker content also gradually rise therewith It is high.This shows during onset diabetes, senile cell always involved in wherein, while senile cell content 19% it Before be the disease pathogenetic quick phase, to this period carry out senile cell monitoring, can be very good prevention disease generation, hair Exhibition.

p16^INK4a, the various albumen of p53, p21, pRB be nearly no detectable at model foundation initial stage, as model mouse blood glucose contains Amount is stepped up, p16^INK4a, p53, p21, pRB albumen appearance also show its cellular senescence process participate in timeliness spy Point, detect at first content it is higher be p53, p21, pRB, p16, and p16 is increased with cell ageing ratio at direct after increasing Positive correlation, other albumen can also maintain one elevation.

The factors checks such as SASP secretion factor IL-6, IL-8, IL-12, MMP-1, MMP-2, TNF are shown, with aging degree Deng intensification, the content of such factor is also being stepped up, and can be stablized after increasing to a certain extent in a level, the SASP factor Secretion can gradually influence surrounding healthy cells, therefore, senile cell also can raising gradually.

Embodiment 3:

Cell ageing and the research of pathogenic process are carried out with the atherosclerosis-inducing model of experimental animal.Start in experiment Entire research during, contain the high fat diet of 42% calorie from fat to experimental group LDLR mouse (10 weeks) feeding.Control Group LDLR mouse (10 weeks) feeding normal diet.The incidence of experimental animal is detected weekly, when being observed continuously nine weeks Between, during which by periphery hematometry senile cell marker levels, physiology be dissected and observed atherosclerotic plaque formed with it is outer All blood senile cell marker detection results are verified each other.

5 experimental groups and control group mice are put to death weekly, observe formation and the macrophage, arterial wall of its arterial wall plaque The aging marker of smooth muscle cell, measurement record blood lipid level.In dissecting mouse aorta it is observed that patch with The progress of disease is incrementally increasing.The content of lipid is gradually increasing when just starting, and the toughness of blood is also increasing, from third Start intra-arterial in week and initially form patch, the volume of patch is also incrementally increasing increase with time.Human peripheral blood detection is observed p16^INK4aProtein positive rate is higher, while p53, p21, pRB, p19^ARFAlbumen shows the trend gradually risen, shows that aging is thin Born of the same parents gradually form in diseased region.It is cultivated by the tissue to lesion patch, it is smooth with arterial wall to isolate macrophage Myocyte, and SA- β-Gal dyeing observation is carried out, the macrophage of aging and the ratio of smooth muscle cell are gradually rising, ratio 15.3% level is risen to by 4.3%, shows that the forming process of itself and disease has direct relationship.Senile cell is trained simultaneously Nutrient secretion analysis shows that, the level and p53, p21, pRB, p19 of the macrophage of aging and smooth muscle cell secretion SASP^ARF The expression correlation of albumen.Wherein SASP factor M MP3, MMP13, PAIL p21's, IGFBP2, IL-1A and IL-1B Expression is obviously increased.After patch formation, the trend of aging tends towards stability, but albumen index is in high-level state always. Data show, SA- the β-Gal, p16 of experimental mice^INK4a、p53、p21、p19^Arf, pRB albumen and SASP factor level it is obvious Higher than control group mice.Vascular smooth muscle cells are also showed with progression of atherosclerosis and the gradually process of aging, to blood The tension of pipe is gradually reduced with ductility.

Embodiment 4:

The transgenosis C57BL6/J mouse species Research of Animal Model for Study with injury of lungs is induced to assess using bleomycin The effect and there is potential significance to IPF diagnosis that senile cell plays in idiopathic pulmonary fibrosis (IPF) forming process.? For in the bleomycin damage model of IPF, pulmonary fibrosis development can be completely presented in mouse after bleomycin processing in 2 months Process.Select feed to 6-8 week old, weight for 19-23g C57BL6/J mouse as experimental subjects.With bleomycin 2.5U/kg is dissolved in 100 μ L physiological saline, and intraperitoneal injection, the experiment mice of control group gives physiological saline, in bleomycin The 1st week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks, monitor mouse by using experimental animal pulse blood oxygen instrument after processing Oxygen saturation, for assessing experiment mice lung function.With isoflurane (1.5%) anesthetized animal, the 30 second time of mouse is monitored Periphery capillary is averaged oxygen saturation (SpO₂Calculate the measured value in the duration).Bleomycin is after one week of dosing just IPF symptom is showed, for the overall process data for obtaining disease development, which carried out for 8 weeks altogether.With each time point to reality Test mouse SpO₂After detection, using CO₂Mouse is put to death, sputum, lung cells, tracheal cell is collected and carries out aging analysis.

During lasting bleomycin administration, the lung function SpO of mouse₂Level significantly reduces, and mouse was at 2,4,6,8 weeks There is different degrees of alveolar inflammation, pulmonary fibrosis.Experimental group alveolar inflammation, pulmonary fibrosis started to gradually rise in 2 weeks, in It peaks within 6-8 weeks.Carry out SA- β-Gal expression to studies have shown that senile cell by 6.4% rise to 28.3% level, than Example extends with experimental period and gradually increases；p16^INK4a, p53, p21, pRB protein content is also being stepped up；In senile cell In culture solution pass through immunohistochemistry show SASP secretion the factor: the inflammatory factors such as IL-6, IL-8, TNF-α, chemotactic because Son, matrix metalloproteinase etc. significantly improve；

Pulmonary alveolitis, the feature of lung fibrosis are gradually shown over time, meanwhile, senile cell items mark Object numerical value shows the characteristic risen comprehensively with the intensification of experiment progress time, and after lung fibrosis molding, senile cell Also region is gentle for the trend that every numerical value rises, this illustrates that senile cell depth participates in IPF pathogenic process, while senile cell number The degree correlation of amount and progression of disease.

Designing in the studies above can also be in other mice study.

Chronic obstructive pulmonary disease (COPD) and senile cell are tested in Animal Model, and mouse is placed in smoke from cigarette In.The senile cell generated by the sucking to smoke from cigarette makes smoke from cigarette with lung function and pulmonary histopathology assessment At lung harm studied.

It selects the C57BL6 mouse of 6 week old to be placed in for a long time to make by oneself in the closed smoke from cigarette container that can be generated, lights 6 every time Zhi Xiangyan, mouse receive smoke from cigarette exposure in 6 hours in total daily, 5 days weekly, continue 12 weeks.In the bimestrial perfume (or spice) of exposure After cigarette smog, oxygen saturation is monitored by using experimental animal pulse blood oxygen instrument to assess lung function.With isoflurane (1.5%) fiber crops Liquor-saturated animal simultaneously applies toe clip.It monitors mouse 30 seconds and calculates the mean peripheral capillary oxygen saturation within the duration (SpO₂) measured value.

At the end of experiment periods, control group is in each time point lung tissue without significant change.Experimental mice is in smog exposure 4 Visible mouse lung tissue inflammatory cell invades profit when all, part alveolar space slightly expands；Occur obvious alveolar space when to the 12nd week to expand Greatly, alveolar wall is thinning, alveolar septum ruptures and fusion, pulmonary emphysema are formed.SA- β-Gal activity dye is carried out to the pneumonocyte of mouse Color shows, senile cell 5.8% rose to the 12nd week 20.5% ratio at the 2nd week, senile cell with disease into It opens up and shows and gradually increase.P16 in test sample^INK4a, p53, p21, pRB protein content is also being stepped up；In aging Cell SASP detection similarly shows the inflammatory factors such as IL-6, IL-8, TNF-α, chemotactic factor (CF), matrix metalloproteinase equal part Secretion is improving；

It is shown according to the data that experiment is completed, mouse COPD model gradually shows alveolar space expansion over time Greatly, alveolar wall is thinning and the feature of alveolar septum rupture and fusion, meanwhile, senile cell items marker numerical value with test into The intensification of exhibition time shows the characteristic risen comprehensively, and after chronic obstructive pulmonary disease molding, senile cell items are numerically Also region is gentle for the trend risen, this illustrates that senile cell depth participates in IPF pathogenic process, at the same senile cell quantity and disease into The degree correlation of exhibition.

Sequence table

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Ile Phe His Met Ser Leu Leu Ala Cys Ala Leu Glu Val Val Met Ala

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Ser Phe Pro Trp Ile Leu Asn Val Leu Asn Leu Lys Ala Phe Asp Phe

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Gly Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile Glu

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Gly Ser Asp Glu Ala Asp Gly Ser Lys His Leu Pro Gly Glu Ser Lys

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Phe Gln Gln Lys Leu Ala Glu Met Thr Ser Thr Arg Thr Arg Met Gln

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Asp Asp Asp Gly Gln Arg Ser Arg Ser Ser Ser Ser Ala Gln Leu Arg

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Val Arg Gly Ser Ala Gly Cys Ala Arg Cys Leu Gly Ser Pro Ala Ala

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Arg Leu Gly Pro Arg Ala Gly Thr Ser Arg His Arg Ala Ile Phe Ala

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Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His

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Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu

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Glu Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp

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Leu Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln

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Phe Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala

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Asp Ile Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu

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Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp

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Asp Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu

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Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser

195 200 205

Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro

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Gly Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu Arg Leu Val Gly

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Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser Ala Arg

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Val Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu Leu Glu Lys Met

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Leu Val Leu Asp Ser Asp Lys Arg Ile Thr Ala Ala Gln Ala Leu Ala

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His Ala Tyr Phe Ala Gln Tyr His Asp Pro Asp Asp Glu Pro Val Ala

305 310 315 320

Asp Pro Tyr Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu

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Trp Lys Ser Leu Thr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro

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Leu Asp Gln Glu Glu Met Glu Ser

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Glu Trp Ala Ala Pro Gly Ala Pro Ala Ala Val Ala Leu Val Leu Met

35 40 45

Leu Leu Arg Ser Gln Arg Leu Gly Gln Gln Pro Leu Pro Arg Arg Pro

50 55 60

Gly His Asp Asp Gly Gln Arg Pro Ser Gly Gly Ala Ala Ala Ala Pro

65 70 75 80

Arg Arg Gly Ala Gln Leu Arg Arg Pro Arg His Ser His Pro Thr Arg

85 90 95

Ala Arg Arg Cys Pro Gly Gly Leu Pro Gly His Ala Gly Gly Ala Ala

100 105 110

Pro Gly Arg Gly Ala Ala Gly Arg Ala Arg Cys Leu Gly Pro Ser Ala

115 120 125

Arg Gly Pro Gly

130

<210> 8

<211> 138

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 8

Met Arg Glu Glu Asn Lys Gly Met Pro Ser Gly Gly Gly Ser Asp Glu

1 5 10 15

Gly Leu Ala Ser Ala Ala Ala Arg Gly Leu Val Glu Lys Val Arg Gln

20 25 30

Leu Leu Glu Ala Gly Ala Asp Pro Asn Gly Val Asn Arg Phe Gly Arg

35 40 45

Arg Ala Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu

50 55 60

Leu Leu Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu

65 70 75 80

Thr Arg Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu

85 90 95

Val Val Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp

100 105 110

Gly Arg Leu Pro Val Asp Leu Ala Glu Glu Arg Gly His Arg Asp Val

115 120 125

Ala Gly Tyr Leu Arg Thr Ala Thr Gly Asp

130 135

<210> 9

<211> 548

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 9

Met Ala Thr Ser Ser Glu Glu Val Leu Leu Ile Val Lys Lys Val Arg

1 5 10 15

Gln Lys Lys Gln Asp Gly Ala Leu Tyr Leu Met Ala Glu Arg Ile Ala

20 25 30

Trp Ala Pro Glu Gly Lys Asp Arg Phe Thr Ile Ser His Met Tyr Ala

35 40 45

Asp Ile Lys Cys Gln Lys Ile Ser Pro Glu Gly Lys Ala Lys Ile Gln

50 55 60

Leu Gln Leu Val Leu His Ala Gly Asp Thr Thr Asn Phe His Phe Ser

65 70 75 80

Asn Glu Ser Thr Ala Val Lys Glu Arg Asp Ala Val Lys Asp Leu Leu

85 90 95

Gln Gln Leu Leu Pro Lys Phe Lys Arg Lys Ala Asn Lys Glu Leu Glu

100 105 110

Glu Lys Asn Arg Met Leu Gln Glu Asp Pro Val Leu Phe Gln Leu Tyr

115 120 125

Lys Asp Leu Val Val Ser Gln Val Ile Ser Ala Glu Glu Phe Trp Ala

130 135 140

Asn Arg Leu Asn Val Asn Ala Thr Asp Ser Ser Ser Thr Ser Asn His

145 150 155 160

Lys Gln Asp Val Gly Ile Ser Ala Ala Phe Leu Ala Asp Val Arg Pro

165 170 175

Gln Thr Asp Gly Cys Asn Gly Leu Arg Tyr Asn Leu Thr Ser Asp Ile

180 185 190

Ile Glu Ser Ile Phe Arg Thr Tyr Pro Ala Val Lys Met Lys Tyr Ala

195 200 205

Glu Asn Val Pro His Asn Met Thr Glu Lys Glu Phe Trp Thr Arg Phe

210 215 220

Phe Gln Ser His Tyr Phe His Arg Asp Arg Leu Asn Thr Gly Ser Lys

225 230 235 240

Asp Leu Phe Ala Glu Cys Ala Lys Ile Asp Glu Lys Gly Leu Lys Thr

245 250 255

Met Val Ser Leu Gly Val Lys Asn Pro Leu Leu Asp Leu Thr Ala Leu

260 265 270

Glu Asp Lys Pro Leu Asp Glu Gly Tyr Gly Ile Ser Ser Val Pro Ser

275 280 285

Ala Ser Asn Ser Lys Ser Ile Lys Glu Asn Ser Asn Ala Ala Ile Ile

290 295 300

Lys Arg Phe Asn His His Ser Ala Met Val Leu Ala Ala Gly Leu Arg

305 310 315 320

Lys Gln Glu Ala Gln Asn Glu Gln Thr Ser Glu Pro Ser Asn Met Asp

325 330 335

Gly Asn Ser Gly Asp Ala Asp Cys Phe Gln Pro Ala Val Lys Arg Ala

340 345 350

Lys Leu Gln Glu Ser Ile Glu Tyr Glu Asp Leu Gly Lys Asn Asn Ser

355 360 365

Val Lys Thr Ile Ala Leu Asn Leu Lys Lys Ser Asp Arg Tyr Tyr His

370 375 380

Gly Pro Thr Pro Ile Gln Ser Leu Gln Tyr Ala Thr Ser Gln Asp Ile

385 390 395 400

Ile Asn Ser Phe Gln Ser Ile Arg Gln Glu Met Glu Ala Tyr Thr Pro

405 410 415

Lys Leu Thr Gln Val Leu Ser Ser Ser Ala Ala Ser Ser Thr Ile Thr

420 425 430

Ala Leu Ser Pro Gly Gly Ala Leu Met Gln Gly Gly Thr Gln Gln Ala

435 440 445

Ile Asn Gln Met Val Pro Asn Asp Ile Gln Ser Glu Leu Lys His Leu

450 455 460

Tyr Val Ala Val Gly Glu Leu Leu Arg His Phe Trp Ser Cys Phe Pro

465 470 475 480

Val Asn Thr Pro Phe Leu Glu Glu Lys Val Val Lys Met Lys Ser Asn

485 490 495

Leu Glu Arg Phe Gln Val Thr Lys Leu Cys Pro Phe Gln Glu Lys Ile

500 505 510

Arg Arg Gln Tyr Leu Ser Thr Asn Leu Val Ser His Ile Glu Glu Met

515 520 525

Leu Gln Thr Ala Tyr Asn Lys Leu His Thr Trp Gln Ser Arg Arg Leu

530 535 540

Met Lys Lys Thr

545

<210> 10

<211> 551

<212> PRT

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 10

Met Asp Glu Leu Phe Pro Leu Ile Phe Pro Ala Glu Pro Ala Gln Ala

1 5 10 15

Ser Gly Pro Tyr Val Glu Ile Ile Glu Gln Pro Lys Gln Arg Gly Met

20 25 30

Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser Ala Gly Ser Ile Pro Gly

35 40 45

Glu Arg Ser Thr Asp Thr Thr Lys Thr His Pro Thr Ile Lys Ile Asn

50 55 60

Gly Tyr Thr Gly Pro Gly Thr Val Arg Ile Ser Leu Val Thr Lys Asp

65 70 75 80

Pro Pro His Arg Pro His Pro His Glu Leu Val Gly Lys Asp Cys Arg

85 90 95

Asp Gly Phe Tyr Glu Ala Glu Leu Cys Pro Asp Arg Cys Ile His Ser

100 105 110

Phe Gln Asn Leu Gly Ile Gln Cys Val Lys Lys Arg Asp Leu Glu Gln

115 120 125

Ala Ile Ser Gln Arg Ile Gln Thr Asn Asn Asn Pro Phe Gln Val Pro

130 135 140

Ile Glu Glu Gln Arg Gly Asp Tyr Asp Leu Asn Ala Val Arg Leu Cys

145 150 155 160

Phe Gln Val Thr Val Arg Asp Pro Ser Gly Arg Pro Leu Arg Leu Pro

165 170 175

Pro Val Leu Ser His Pro Ile Phe Asp Asn Arg Ala Pro Asn Thr Ala

180 185 190

Glu Leu Lys Ile Cys Arg Val Asn Arg Asn Ser Gly Ser Cys Leu Gly

195 200 205

Gly Asp Glu Ile Phe Leu Leu Cys Asp Lys Val Gln Lys Glu Asp Ile

210 215 220

Glu Val Tyr Phe Thr Gly Pro Gly Trp Glu Ala Arg Gly Ser Phe Ser

225 230 235 240

Gln Ala Asp Val His Arg Gln Val Ala Ile Val Phe Arg Thr Pro Pro

245 250 255

Tyr Ala Asp Pro Ser Leu Gln Ala Pro Val Arg Val Ser Met Gln Leu

260 265 270

Arg Arg Pro Ser Asp Arg Glu Leu Ser Glu Pro Met Glu Phe Gln Tyr

275 280 285

Leu Pro Asp Thr Asp Asp Arg His Arg Ile Glu Glu Lys Arg Lys Arg

290 295 300

Thr Tyr Glu Thr Phe Lys Ser Ile Met Lys Lys Ser Pro Phe Ser Gly

305 310 315 320

Pro Thr Asp Pro Arg Pro Pro Pro Arg Arg Ile Ala Val Pro Ser Arg

325 330 335

Ser Ser Ala Ser Val Pro Lys Pro Ala Pro Gln Pro Tyr Pro Phe Thr

340 345 350

Ser Ser Leu Ser Thr Ile Asn Tyr Asp Glu Phe Pro Thr Met Val Phe

355 360 365

Pro Ser Gly Gln Ile Ser Gln Ala Ser Ala Leu Ala Pro Ala Pro Pro

370 375 380

Gln Val Leu Pro Gln Ala Pro Ala Pro Ala Pro Ala Pro Ala Met Val

385 390 395 400

Ser Ala Leu Ala Gln Ala Pro Ala Pro Val Pro Val Leu Ala Pro Gly

405 410 415

Pro Pro Gln Ala Val Ala Pro Pro Ala Pro Lys Pro Thr Gln Ala Gly

420 425 430

Glu Gly Thr Leu Ser Glu Ala Leu Leu Gln Leu Gln Phe Asp Asp Glu

435 440 445

Asp Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Ala Val Phe Thr

450 455 460

Asp Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln

465 470 475 480

Gly Ile Pro Val Ala Pro His Thr Thr Glu Pro Met Leu Met Glu Tyr

485 490 495

Pro Glu Ala Ile Thr Arg Leu Val Thr Gly Ala Gln Arg Pro Pro Asp

500 505 510

Pro Ala Pro Ala Pro Leu Gly Ala Pro Gly Leu Pro Asn Gly Leu Leu

515 520 525

Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala

530 535 540

Leu Leu Ser Gln Ile Ser Ser

545 550

Claims (8)

1. a kind of antibody for detecting senile cell, which is characterized in that the antibody is at least one of the following:

p16^INK4a, nucleotide sequence as shown in SEQ ID No.1,

P53, nucleotide sequence as shown in SEQ ID No.2,

P21, nucleotide sequence as shown in SEQ ID No.3,

PRB, nucleotide sequence as shown in SEQ ID No.4,

p19^ARF, nucleotide sequence as shown in SEQ ID No.5,

p38^MAPK, nucleotide sequence as shown in SEQ ID No.6,

p14^ARF, nucleotide sequence as shown in SEQ ID No.7,

p15^INK4b, nucleotide sequence as shown in SEQ ID No.8,

P62, nucleotide sequence as shown in SEQ ID No.9,

P65, nucleotide sequence is as shown in SEQ ID No.10.

2. a kind of kit of the detection senile cell based on antibody described in claim 1, it is characterised in that: the kit Comprising fluorescence viability dye, for senile cell target or the antibody of the fluorescent marker of magnetic bead target, which includes p16^INK4a、p53、p21、pRB、p19^ARF、p38^MAPK、p14^ARF、p15^INK4b, at least one of p62, p65 antibody.

3. a kind of preparation method of the kit of the senile cell of detection as claimed in claim 2, it is characterised in that: prepare respectively Magnetic bead and antibody samples carry out antibody coupling after removing unreacted component.

4. the preparation method of the kit of detection senile cell as claimed in claim 3, it is characterised in that: described to prepare magnetic Pearl sequentially includes the following steps:

The magnetic bead for taking out 65~85 μ l blank is washed with deionized twice, magnetic bead is vortexed and is ultrasonically treated to avoid aggregation simultaneously Ensure that the bead in solution is uniformly distributed；It is suspended in 80 μ L later and contains 100mM sodium phosphate, 10 μ L N- hydroxy succinyls In the activation buffer of imines and 10 μ L 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides, it is diluted to 50mg/ The concentration of mL is spare；

1M DTT is configured with deionized water；2 μ l 1M DTT are taken to be added in the Ep pipe containing magnetic bead and be vortexed again for mixing；It is mixed After conjunction, magnetic bead is protected from light to heat preservation 20 minutes at room temperature；It is washed three times with PBS buffer solution, at room temperature on orbit shaker After being incubated for 2 hours, in the washing/store buffer liquid for being suspended in 1mL, it is kept in dark place at 2 DEG C to 8 DEG C.

5. the preparation method of the kit of detection senile cell as claimed in claim 4, it is characterised in that: the preparation is anti- Body sample sequentially includes the following steps:

Magnetic bead is kept suspending by rotation, the conjugate solution of 100 μ L is added, under protein concentration maximum case, each Magnetic bead is in combination with about 5 × 106 tested protein moleculars；Mixture is incubated 1 in the dark at room temperature on orbit shaker Hour.

6. the preparation method of the kit of detection senile cell as claimed in claim 5, it is characterised in that: the removal is not Reaction component sequentially includes the following steps:

Magnetic bead is washed three times to remove the target of non-specific binding with BCB buffer, and magnetic bead is resuspended in 2mL and contains phosphoric acid In the activation buffer of sodium, Sulfo-NHS and EDC and pass through the dispersion magnetic bead that is vortexed.

7. the preparation method of the kit of detection senile cell as claimed in claim 6, it is characterised in that: the antibody is even Connection sequentially includes the following steps:

Activation of protein in buffer is transferred in the Ep pipe containing the functional magnetic bead previously prepared and the mixing that is vortexed, and Room temperature is protected from light incubation 1 hour on the oscillator；2 μ l are contained into sodium phosphate, the activation buffer of Sulfo-NHS and EDC are added to In Ep pipe containing functional magnetic bead and activated protein, it is vortexed and mixes and be incubated for 15 minutes on the oscillator；After incubation, by 1ml Store buffer liquid is added in pipe, centrifugation；It is washed in triplicate with store buffer liquid；Then, 0.5ml store buffer liquid will be resuspended in In magnetic bead particles stored at 4 DEG C, concentration be 6 × 10⁶Magnetic bead/ml.

8. a kind of senile cell detection method based on kit described in claim 2, it is characterised in that: using kit to declining The aging marker of old cell is marked, and reapplies fluidic cell statistics aging marker.