CN1059702C - Method for cerebral acetylcholinesterase obtained from cells of mamma animals - Google Patents

Abstract

The present invention provides a method for obtaining cerebral acetylcholinesterase from mammalia cells, which is on the basis that apoptosis mammalia cells are proved to express acetylcholinesterase for the first time in the world. All cells (besides nerve cells and erythrocytes) of mammalia animals are induced to be in apoptosis and to express the acetylcholinesterase; then, the cells are cracked, and the chromatography of tacrine affinity columns is carried out; the high-purity cerebral acetylcholinesterase of which the molecule weight is 66 KDa is obtained through separation and purification.

Description

From cells of mamma animals, obtain cerebral acetylcholinesteraseobtained method

The present invention relates to the method for separation and purification enzyme from biomass cells, especially about the cerebral acetylcholinesteraseobtained method of separation and purification from the cells of mamma animals of non-nerve, non-red blood cell source.

(EC 3.1.17, acetylcholinesterase AChE) are the lytic enzyme of neurotransmitter acetylcholine to acetylcholinesterase.Mainly be present in the electric organ of electric ray, mammiferous cholinergic nerve, neuromuscular junction, erythrocyte membrane (Zakut H, et al.J-Clin-Invest.1990,86 (3): (A.Shafferman and B.Velan 900-8) and in the rodentine megalokaryocyte thrombocyte, Multidisplinary approaches tocholinesterase functions.1992 Plenum Press, New York).Acetylcholinesterase can be divided into neuromuscular type and erythrocytic form with the position.Can be divided into ' different subunits poly build ' (heteromeric class) and ' identical subunit poly build ' (homomeric class) according to molecular structure.The catalytic subunit of different subunits poly build is connected or is connected with lipid with triple helical collagen subunit.Identical subunit poly build is divided into wetting ability (hydrophilic) and lipotropy hypotypes such as (glycophospholipid-linked) again.The about 260KDa of acetylcholinesterase tetramer molecular weight on the erythrocyte membrane.The function of acetylcholinesterase is the hydrolysis neurotransmitter acetylcholine at cholinergic nerve system, makes it to generate choline and acetate.Also there is report to think that acetylcholinesterase has the hydrolysis of protein activity of serine protease.In recent years, since the tumor cell line of vitro culture also detect AChE mRNA transcribe with chemotherapy after the serum of ovarian cancer patients detect acetylcholine esterase active, there is the author to think that acetylcholinesterase with tumour relevant (Lev-LehmanE takes place, et al.Blood.1997,89 (10): 3644-53).

Commercial in the world at present acetylcholinesterase, mainly from HRBC, ORBC, the electric organs of serum and discharge fish such as electric ray separates and obtains, and does not have the acetylcholinesterase of human brain, traces it to its cause, be exactly to be difficult to obtain human brain in order to the separation and purification acetylcholinesterase, therefore, with regard to human brain acetylcholinesterase, market supply is blank.

Acetylcholinesterase is an important enzyme in the mammalian body, except lytic enzyme as neurotransmitter acetylcholine, the function of the acetylcholinesterase of other cell expressing and meaning are still unclear, research people acetylcholinesteraseinhibitors, the enzyme that must produce with the human cell is an object, therefore, the requirement to this enzyme is very big.Be that to separate the approach that obtains acetylcholinesterase be limited in the source with the human brain.If there are wide material sources to be easy to get, simple and reliable method obtains this kind of enzyme, has just opened convenience for research.

In addition, studies have shown that senile dementia patient affected area has a large amount of acetylcholinesterases to exist, and, irrelevant with the neurotransmitter degradation function, what is its appearance meaning so? how to study them? also have, having proved that senile dementia patient affected area has apoptotic cell to exist, what relation does the two have so? these are all unclear.The acetylcholinesterase that obtains a large amount of human brains source is very difficult again.The inventor is devoted to the work that goes in for the study for many years always in this field, be separated to cerebral acetylcholinesteraseobtainedly from apoptotic cell, substitutes acetylcholinesterase from the human brain separation and purification with this kind of enzyme, and its meaning is very great.

The object of the invention provides from Mammals and comprises human cell strain, and except known nerve and red corpuscle, the cell of not expressing acetylcholinesterase under the standard state is through obtaining the method for acetylcholinesterase after apoptosis-induced.

The present invention is a kind of method that obtains acetylcholinesterase from cells of mamma animals, be to comprise that from Mammals human all cell strains (except neurocyte and the red corpuscle) are through cell death inducing, make it to express acetylcholinesterase, pass through lysing cell again, the separation and purification of tacrine affinity column chromatography obtains cerebral acetylcholinesteraseobtained method.

The present invention obtains the method for acetylcholinesterase from cells of mamma animals, be a brand-new invention in scientific research.Its principle is that the cell of not expressing acetylcholinesterase under the normal circumstances is expressed cerebral acetylcholinesteraseobtained in apoptosis process.Therefore, present method at first is to obtain apoptotic cell, collects then through apoptosis-induced cell, respectively extracting mRNA, DNA and protein can obtain a large amount of acetylcholinesterase AChE mRNA and reverse transcription RT-PCR product, trapezoidal dna segment and activated acetylcholinesterase respectively.

Present method has been used non-tumor cell strain and tumor cell line.The non-tumor cell strain comprises people's lung fibroblast (HLF), Human umbilical vein endothelial cells, mouse endotheliocyte (SIEC), l cell strain (NIH/3T3), rat hepatocytes strain (BRL), rat aorta smooth muscle cell (SMC).Tumor cell line comprises HELA and PC-3, HL-60, M07e, HEL, DAMI or the like.

Cell death inducing can adopt several different methods, and physics (radiation), chemistry (chemotherapeutics) and biological (cytokine adds deduct) method and naturally-aged method are wherein arranged.The naturally-aged method is the simplest and cheap.

The naturally-aged method: cultivate the cell of adherent growth, as HLF and PC-3 cell strain, do not change nutrient solution in 3-4 days, at this moment, the part cell loses adherent ability, has entered apoptotic process, and the suspension cell of collecting at this moment can obtain a large amount of acetylcholinesterases.If the suspension growth cell, under the situation of not changing nutrient solution, the part cell begins conglomerate as HL-60, a few cell has arrived the apoptosis later stage (referring to Fig. 4, dna break appears), present the flocculence bead, the cell of collecting at this moment also can obtain a large amount of acetylcholinesterases.

The induced by chemotherapeutic agents method: join in the HELA cell culture fluid with the antitumor drug daunorubicin, a large amount of apoptosis of cell behind the certain hour, the cell of collecting at this moment can obtain a large amount of acetylcholinesterases.

Go cytokine induction apoptosis method: for the cell strain of cytokine dependence, as the strain of people M07e megalokaryocyte, as long as in nutrient solution, do not add GM-CSF, after 2-3 days, a large amount of cell generation apoptosis, the cell of collecting at this moment can obtain a large amount of acetylcholinesterases.

Radiation-induced apoptosis method: 5 Gy gamma-radiations irradiation cultured cells, collecting cell can obtain a large amount of acetylcholinesterases after 4 hours.

For example from apoptosis HLF and apoptosis HL-60 cell difference extracting DNA, carry out agarose electrophoresis, as seen the dna segment (Fig. 4) of the above different sizes of 200bp proves that apoptosis has taken place these cells really, and trapezoidal dna segment electrophorogram is the most reliable evidence of present identification of cell apoptosis.Never detect in the apoptotic cells, and from apoptotic cell, detect the AChE activity, show that apoptotic cell can express acetylcholinesterase less than the AChE activity.

Obtained containing the apoptotic cell of acetylcholinesterase, carried out acetylcholinesterase then and separate and evaluation.Identify cerebral acetylcholinesteraseobtained can carrying out from two levels, the one, at transcriptional level, promptly use the method for RT-PCR, detect whether transcribing of AChE mRNA of cell.The 2nd, the protein level after the translation, i.e. activity by detecting this enzyme and be separated to this enzyme.

The complete sequence of people's acetylcholinesterase in genome is clear, and totally 10 sections, wherein contain 6 exons (Extron) and 4 introns (Intron), different shear-forms is arranged after transcribing.Common has three kinds, E1-E2-E3-E4-E6 type (hydrophilic also is brain type or " cynapse type "); E1-E2-E3-E4-E5-E6 type (PI connects hydrophobic type, also is erythrocytic form) and E1-E2-E3-E4-I4-E5-E6 type (PI is connected the type of reading over).

1. transcriptional level detects

PCR design of primers and RT-PCR step, PCR design of primers sequence is as follows:

(1)1522(+)5’-

CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3′

(2)2003(-)5′-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3′

(3)5’-CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3’

(4)5’-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3’

Primer 1 and primer 2 lay respectively in the E3 and E6 of AChE gene, in order to detect brain type AChEmRNA (E1-E2-E3-E4-E6); Primer 1 and primer 3 are readed over type in order to detection, i.e. E1-E2-E3-E4-I4-E5-E6 type (PI connects the type of reading over); Primer 1 and primer 4 are in order to detect erythrocytic form, i.e. E1-E2-E3-E4-E5-E6 type (PI connects hydrophobic type).Do not detect erythrocytic form in this experiment and read over type.

The RT-PCR step: carry out reverse transcription after total RNA extracting, step is according to the schedule of operation of this reagent.Pcr amplification product detects in 1.6% agarose gel electrophoresis.For example from losing the apoptosis PC-3 cell extracting mRNA of adherent ability, be traceable to transcribe (Fig. 1) of a large amount of AChE mRNA with the RT-PCR method, the PC-3 cell that adherent growth conditions is good then is difficult to detect transcribing of AChE mRNA, also detects the activity less than acetylcholinesterase.Primer as mentioned above, the result is detected to be brain type AChEmRNA (E1-E2-E3-E4-E6).(scope of this primer amplification is between AChE gene 1522 to 2003, the PCR product should be 482bp), rather than E1-E2-E3-E4-E5-E6 type (PI connects hydrophobic type, also is erythrocytic form) neither E1-E2-E3-E4-I4-E5-E6 type (PI connects the type of reading over).

2. protein level detects, and available the following step detects the feature of the acetylcholinesterase of protein level.

The first step lysing cell

With reference to " molecular cloning " book preparation lysate, lysate is added lysing cell in the cell, centrifugal 5 minutes of 1000g removes uncracked cell debris.

The second step tacrine affinity column separates the acetylcholinesterase of apoptotic cell

Reference literature (Richard T, et al.Protein Expression and Purification 6,389-393,1995).

The 3rd step acetylcholine esterase active is identified

Activity identification and active inhibition experiment can be carried out on cell levels and running gel.Preparation acetylcholinesterase substrate reactions liquid.Detect cell or gel acetylcholine esterase active, make cell room temperature hatching 4-12 hour in reaction solution, if having enzyme to exist then the positive reaction (see figure 2) of tawny occurs.Suppressing experiment is to add acetylcholinesterase specific inhibitor BW284c51 (Sigma company) in reaction solution.If active reaction is suppressed, prove that this activity is by acetylcholinesterase catalysis institute extremely.

SDS-PAGE carries out molecular weight identification: determining molecular weight uses international SDS-PAGE electrophoresis method at present.Fig. 3 is exactly the SDS-PAGE electrophorogram, the 5th swimming lane is the molecular weight protein marker thing among the figure, wherein a Zui Da band is a bovine serum albumin, the about 67KDa of molecular weight, the 4th swimming lane is bovine serum albumin (buying from Sigma company), the 3rd swimming lane is HRBC acetylcholinesterase (buying from Sigma company), the 2nd swimming lane be the inventor from the DAMI cell strain separate acetylcholinesterase and the 1st swimming lane be the inventor from the PC-3 cell strain separate acetylcholinesterase, molecular weight all is 66KDa, similar to the neural acetylcholinesterase of people, be cerebral acetylcholinesteraseobtained.

Comprehensive foregoing, proof nerve and the red corpuscle mammalian cell of not expressing acetylcholinesterase are at ordinary times in addition expressed the important phenomenon of acetylcholinesterase to the inventor when apoptosis takes place in the world first.Proof: 1. Mammals, as the people, mouse and rat cell in case apoptosis has taken place, are all expressed acetylcholinesterase, and all are the brain types, and the conservative property of its evolutionary process is described; 2. tumor cell line and the former foster normal diploid cell of being commissioned to train in case apoptosis has taken place, are just expressed acetylcholinesterase, illustrate that the expression of acetylcholinesterase and tumour are related not quite, and with apoptosis-related.There is the material of biopsy of report primary tumor not see expression (the Karpel R of acetylcholinesterase, et al.Experimental CellResearch, 210:268-277,1994) and detect in the serum before the primary tumor chemotherapy less than detecting acetylcholine esterase active in acetylcholine esterase active and the treatment back most of patients serum and increase (Zakut H, et al.Cancer 61:727-737,1988), this two example report proves a very strong support to of the present invention.

The present invention obtains the method for acetylcholinesterase from cells of mamma animals, can be used as to obtain acetylcholinesterase from apoptotic cell and prepare various antibody and be used for commerce.And on the research apoptosis basis relevant with the expression of acetylcholinesterase, with the method for anti-acetylcholinesterase, treat and carry out the sexual cell apoptosis in the senile dementia process, have certain meaning.

Advantage of the present invention: the present invention comprises the mankind from Mammals, and is cerebral acetylcholinesteraseobtained through obtaining after apoptosis-induced except known nerve and exo-erythrocytic all cell strains, is first in the world.There is acetylcholinesterase in human senile dementia patient's cerebral tissue, obtains acetylcholinesterase from human brain and be not easy very much.Use method of the present invention, any one can carry out the experiment condition of cell cultures, just has the ability also can be separated to this kind of enzyme easily.

The present invention is further elaborated by following drawings and Examples, but does not place restrictions on scope of the present invention.

Description of drawings

Fig. 1. be the agarose gel electrophoresis figure of the RT-PCR product of PC-3 cell.

1 PCR mark (bp): 1,543 994 695 515 377;

2 use the RT-PCR product electrophoretogram of primer 1 and primer 3, prove that product is not to read over type;

3 use the RT-PCR product electrophoretogram of primers 1 and primer 2, and the PCR product is 482bp, and that prove that PC-3 in the apoptotic process transcribes is cerebral acetylcholinesteraseobtained mRNA;

4 use the RT-PCR product electrophoretogram of primer 1 and primer 4, prove that product is not an erythrocytic form.

Fig. 2. from the PC-3 apoptotic cell, separate the non-sex change PAGE of the acetylcholine esterase active reaction zone electrophorogram that obtains.

Fig. 3. separate the SDS-PAGE electrophorogram of the acetylcholinesterase that obtains with the DAMI apoptotic cell from PC-3.

1 separates the acetylcholinesterase obtain, molecular weight 66KDa from the PC-3 apoptotic cell;

2 separate the acetylcholinesterase obtain, molecular weight 66KDa from the DAMI apoptotic cell;

3 HRBC acetylcholinesterases, molecular weight 66KDa;

4 bovine serum albumins;

5 molecular weight protein marker things, wherein a Zui Da band is a bovine serum albumin, molecular weight 67KDa.

The apoptotic dna ladder shape of Fig. 4 .HLF and HL-60 fragment agarose electrophoresis figure.

1 123 dna markers, (buying) from Sigma company;

2 people's lung fibroblast strains (HLF) through the apoptotic cell after inducing, show dna ladder shape fragment;

3 human leukemia cell lines (HL-60) through the apoptotic cell after inducing, show dna ladder shape fragment;

4 human leukemia cell lines (HL-60), well-grown, no dna ladder shape fragment proves apoptosis does not take place.

Embodiment 1 obtains acetylcholinesterase (characteristics: people, tumour, induced by chemotherapeutic agents apoptosis) from Human Prostate Cancer Cells strain (PC-3)

Cultivator PC-3 cell, adherent growth.Nutrient solution RPMI Medium 1640 (GibcoBRL) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Control group does not add induced drug, induce experimental group add 2 μ M daunorubicins (daunorubicin, Italy), collecting cell after 18-20 hour.

Identify and cerebral acetylcholinesteraseobtainedly carry out from two levels, the one, at transcriptional level, promptly use the method for RT-PCR, detect whether transcribing of AChE mRNA of cell.The 2nd, the protein level after the translation, i.e. activity by detecting this enzyme and be separated to this enzyme.

1. transcriptional level:

After the extracting of PC-3 nucleus, obtain mRNA, reverse transcription arrives cDNA, is that template is made PCR with this cDNA.PCR primer and RT-PCR step:

PCR design of primers sequence is as follows:

(1)1522(+)5’-

CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3′

(2)2003(-)5′-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3′

(3)5’-CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3’

(4)5’-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3’

Primer 1 and primer 2 lay respectively in the E3 and E6 of AChE gene, in order to detect brain type AChEmRNA (E1-E2-E3-E4-E6); Primer 1 and primer 3 are readed over type in order to detection, i.e. E1-E2-E3-E4-I4-E5-E6 type (PI connects the type of reading over); Primer 1 and primer 4 are in order to detect erythrocytic form, and promptly (PI connects hydrophobic type to the E1-E2-E3-E4-E5-E6 type.Do not detect erythrocytic form in this experiment and read over type.

The RT-PCR step: Boehringer, the Tripure of Mannheim company are used in total RNA extracting ^TMSeparation agent.Synthetic six primers at random that use Promega company of cDNA, reverse transcription uses Boehringer, the Expend of Mannheim company ^TMReversed transcriptive enzyme, method is according to the schedule of operation of this reagent.Pcr amplification uses 480 amplification instrument of Perkin-Elmer company, and condition is as follows: sex change, 94 ℃, 1 minute; Anneal 65 ℃ 1 minute; Extend, 72 ℃, 1 minute (last circulation 5 minutes); Cycle number, 39 times.Pcr amplification product detects in 1.6% agarose gel electrophoresis.

Detect a large amount of AChEmRNA are arranged in the apoptotic cell, its shear-form is that E1-E2-E3-E4-E6 type (hydrophilic also is the brain type) is seen Fig. 1, (scope of this primer amplification is between 1522 to 2003, the PCR product should be 482bp), rather than E1-E2-E3-E4-E5-E6 type (PI connects hydrophobic type, also is erythrocytic form) neither E1-E2-E3-E4-I4-E5-E6 type (PI connects the type of reading over).

2. protein level detects:

The first step lysing cell

With reference to " molecular cloning " book preparation lysate (50m mol/L Tris-HCl pH8.0,150mmol/l NaCl, 0.02 μ g sodium azide, 1 μ g/ml aprotinin (Aprotinin), 1% triton (Triton X-100).Lysate is added lysing cell in the cell, and centrifugal 5 minutes of 1000g removes uncracked cell debris.

The second step tacrine affinity column separates the acetylcholinesterase of apoptotic cell

Reference literature (Richard T, et al.Protein Expression and Purification6,389-393,1995), with epoxy activated agarose gel 6B (Epoxy-activated Sepharose6B) 10g, " Sigma company; E-6754 ", with 13g 9-aminoacridine HCl, " No.A3840-1 of Aldrich company " preparation Tacrine affinity column, at first use 0.2mol/L NaCl 50m mol/LTris-HCl pH8.0 damping fluid balance columns, sample is placed on and goes up sample, flow velocity 0.3ml/ minute in the balance liquid.Wash-out 10m mol/L Tacrine, 50m mol/L Tris-HCl pH8.0 elutriant, the acetylcholinesterase of wash-out with 10m mol/L Tris-HCl pH8.0 dialysis, changes dialyzate three times again, dialyses 24 hours.Lyophilize.

The 3rd step acetylcholine esterase active is identified

Reference literature (Richard T, et al.Protein Expression and Purification6,389-393,1995).

Activity identification and active inhibition experiment can be carried out on cell levels and running gel.Preparation acetylcholinesterase substrate reactions liquid (0.1mol/L phosphoric acid buffer pH6.0 150ml, acetyl thio choline 100mg/200ml, 0.1mol/L Trisodium Citrate 11ml, 30m mol/L copper sulfate 20ml, 5m mol/L Tripotassium iron hexacyanide 20ml).Used Bio-RAD mini-PROTEIN II electrophoresis apparatus in this experiment, the 6%PAGE gel, get cell pyrolysis liquid, add 0.2% triton x-100, carry out non-sex change electrophoresis, 20 volts of voltages, 12 hours, take out gel behind the electrophoresis, tawny These positive bands (Fig. 2) appears in the place that is placed on the vagusstoff ester of preparation, and other protein does not then have positive reaction.Suppressing experiment is to add 10 μ M BW284c51 (Sigma company) acetylcholinesterase specific inhibitors in reaction solution, and enzymic activity can be suppressed, and proves this proteic acetylcholinesterase really.

SDS-PAGE carries out molecular weight identification: using SDS-PAGE electrophoresis method determining molecular weight, prove that isolating activated protein is 66KDa (first swimming lane among Fig. 3) from the PC-3 cell of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 2 obtains acetylcholinesterase (characteristics: people, non-tumour, the factor is apoptosis-induced) from Human umbilical vein endothelial cells

The former foster Human umbilical vein endothelial cells of being commissioned to train, adherent growth.Nutrient solution M199 (Gibco) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Control group does not add the factor, induces experimental group to add 5 μ g/ml TGF-β, collecting cell after 18-20 hour.

Protein level detects, and institute is in steps with embodiment 1.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from people's umbilical cord endotheliocyte of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 3 obtains acetylcholinesterase (characteristics: people, non-tumour, naturally-aged apoptosis) from people's lung fibroblast

People's lung fibroblast of adherent growth, nutrient solution RPMI1640 (Gibco) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Do not add the factor, do not change nutrient solution, collecting cell after 4 days in 4 days.

1. nucleic acid level detects with embodiment 1, and apoptotic cell is traceable to brain type AChEmRNA with RT-PCR and expresses, and adherent normal cell then anencephaly type AChE mRNA is expressed.2. protein level detects, and institute is in steps with embodiment 1.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from people's lung fibroblast acquisition acetylcholinesterase of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 4 obtains acetylcholinesterase (characteristics: people, tumour, induced by chemotherapeutic agents apoptosis) from human leukemia cell line (HL-60)

Suspension culture human leukemia cell line (HL-60), nutrient solution RPMI Medium 1640 (GibcoBRL) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Control group does not add induced drug, induce experimental group add 2 μ M daunorubicins (daunorubicin, I-taly), collecting cell after 18-20 hour.

1. nucleic acid level detects with embodiment 1, and apoptotic cell is traceable to brain type AChE mRNA with RT-PCR and expresses, and normal cell then anencephaly type AChE mRNA is expressed.

2. protein level detects, and institute is in steps with embodiment 1.

The SDS-PAGE electrophoresis method carries out molecular weight identification, illustrates that isolating activated protein is 66KDa from the HL-60 cell of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 5 obtains acetylcholinesterase (characteristics: people, tumour cell removes the cytokine induction apoptosis) from people's megalokaryocyte strain (M07e)

Cultivator M07e cell, suspension growth.Nutrient solution α-Medium adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Because this cell strain is cytokine GM-CSF dependent form, so control group adds GM-CSF 2.5ng/ml, induces experimental group not add GM-CSF, collecting cell after 18-20 hour.

Protein level detects, and institute is in steps with embodiment 1.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from the M07e cell that accent is died, and is cerebral acetylcholinesteraseobtained.

Embodiment 6 obtains acetylcholinesterase (characteristics: people, tumour cell, naturally-aged apoptosis) from people's megalokaryocyte strain (DAMI)

Cultivator DAMI cell, suspension growth.Nutrient solution RPMI Medium 1640 adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Do not change nutrient solution in 4 days, collect apoptotic cell after 4 days.

Protein level detects, and institute is in steps with embodiment 1.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from the DAMI cell of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 7 obtains acetylcholinesterase (characteristics: mouse, non-tumour, naturally-aged apoptosis) from l cell strain (NIH/3T3)

The NIH/3T3 cell of adherent growth, nutrient solution RPMI Medium 1640 (GibcoBRL) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Do not add the factor, do not change nutrient solution, the 4th day collecting cell in 3-4 days.

Protein level detects, and institute is in steps with embodiment 1, apoptotic cells acetylcholine esterase active reacting positive.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from the NIH/3T3 cell of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 8 obtains acetylcholinesterase (characteristics: rat, non-tumour, the factor is apoptosis-induced) from the rat aorta smooth muscle cell strain

The adherent growth rat aorta smooth muscle cell, nutrient solution M199 (GibcoBRL) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO ₂Cultivate in the incubator.Control group does not add the factor, induces experimental group to add 5 μ g/ml TGF-β, collecting cell after 18-20 hour.

Protein level detects, and institute is in steps with executing example 1.Acetylcholine esterase active reacting positive in the rat aorta smooth muscle cell of apoptosis.

The SDS-PAGE electrophoresis method carries out molecular weight identification, proves that isolating activated protein is 66KDa from the rat aorta smooth muscle cell of apoptosis, is cerebral acetylcholinesteraseobtained.

Embodiment 9 detects the experiment of HLF apoptotic cell

The DNA method for extracting of HLF cell normally reaches old and feeble cell (2-3 * 10 referring to document (Jaffrezou JP, et al.The EMBO Journal, 15:2417-2424,1996) ⁶) centrifugal respectively, precipitation is washed with PBS, and centrifuged deposit is resuspended with 0.2mol/L Sodium phosphate dibasic/0.1mol/L citric acid (192: 8) of 40 μ l again, and room temperature was placed 60 minutes.2000g is centrifugal 30 minutes then, and supernatant changes 1.5ml Eppendorf pipe over to, adds 3 μ l 0.25%NP-40 and 3 μ l RNaseA (10mg/ml), and 37 ℃ of incubations 60 minutes add 3 μ l Proteinase Ks (10mg/ml) again, 50 ℃ of incubations 30 minutes.Reaction finishes and adds 5 μ l sample loading buffers, 1% agarose gel electrophoresis.See 2 among Fig. 4, show that dna fragmentation is trapezoidal, this is the most reliable evidence of apoptosis.

Embodiment 10 detects the experiment of HL-60 apoptotic cell

The DNA method for extracting of HL-60 cell normally reaches old and feeble cell (2-3 * 10 referring to document (Jaffrezou JP, et al.The EMBOJournal, 15:2417-2424,1996) ⁶) centrifugal respectively, precipitation is washed with PBS, and centrifuged deposit is resuspended with 0.2mol/L Sodium phosphate dibasic/0.1mol/L citric acid (192: 8) of 40 μ l again, and room temperature was placed 60 minutes.2000g is centrifugal 30 minutes then, and supernatant changes 1.5ml Eppendorf pipe over to, adds 3 μ l 0.25%NP-40 and 3 μ lRNaseA (10mg/ml), and 37 ℃ of incubations 60 minutes add 3 μ l Proteinase Ks (10mg/ml) again, 50 ℃ of incubations 30 minutes.Reaction finishes and adds 5 μ l sample loading buffers, 1% agarose gel electrophoresis.See 3 among Fig. 4, show that dna fragmentation is trapezoidal, this is the most reliable evidence of apoptosis.

Claims (3)

1. method that from cells of mamma animals, obtains acetylcholinesterase, it is characterized in that this method is the cells of mamma animals process cell death inducing with non-nerve, non-red blood cell source, make it to express acetylcholinesterase, pass through lysing cell again, the tacrine affinity column chromatography, separation and purification obtains acetylcholinesterase.

2. the method that obtains acetylcholinesterase from cells of mamma animals as claimed in claim 1 is characterized in that described cell death inducing can adopt naturally-aged method, induced by chemotherapeutic agents method, go cytokine induction method or radiation-induced method.

3. the method that obtains acetylcholinesterase from cells of mamma animals as claimed in claim 1 is characterized in that the acetylcholinesterase that obtains is the cerebral acetylcholinesteraseobtained of molecular weight 66KDa.

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