CN1186859A - Method of screening anti-tumour medicine and determining clinical chemotherapy effect - Google Patents
Method of screening anti-tumour medicine and determining clinical chemotherapy effect Download PDFInfo
- Publication number
- CN1186859A CN1186859A CN97125220A CN97125220A CN1186859A CN 1186859 A CN1186859 A CN 1186859A CN 97125220 A CN97125220 A CN 97125220A CN 97125220 A CN97125220 A CN 97125220A CN 1186859 A CN1186859 A CN 1186859A
- Authority
- CN
- China
- Prior art keywords
- cell
- acetylcholinesterase
- apoptotic
- apoptosis
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The said medicine is based on cerebral acetyl choline esterase expressing mammual death cell, and the said choline esterase is proved first time by the present inventor, has a molecular weight of 66 KDa and is similar to acetyl choline esterase in nerve. Based on the principle and through detecting acetyl choline esterase activity in the normal cells unexpressing acetyl choline esterase, whether to produce cell death is determined. The said method is used in screeding new anti-tumor medicine and determining clinical chemotherapy effect for finding out optimal medicine.
Description
The present invention relates to antitumor drug effect determination methods, especially about by detecting the acetylcholine esterase active in the cells of mamma animals of normally not expressing acetylcholinesterase, identify whether apoptosis takes place, be used for the method that screening anti-tumor medicine and clinical chemotherapy effect are judged.
Known the clinical chemotherapeutics that is used for the treatment of tumour, all played a role by inducing apoptosis of tumour cell.The effect of cancer drug therapy tumour how so, and inducing apoptosis of tumour cell does not have a kind of means of identifying the cells in vivo apoptosis so far.People can not be for the effect that detects antitumor drug take out tissue at every turn in the human body and carry out electrophoretic analysis whether apoptosis has taken place.And, different tumours, different patients is different to the reaction of various chemotherapeutics, curative effect is fine on one's body this patient for the antitumor drug that has, and just bad to another patient's curative effect.If a kind of means of new identification of cell apoptosis are arranged, as drug sensitive experiment to bacterium, for clinical guidance select cancer therapy drug in tumor chemotherapeutic drug and the pharmacology research screening that will shoot the arrow at the target.Bring glad tidings to the patient.
The reliable means that experiment in vitro identification of cell apoptosis is commonly used is that agarose electrophoresis shows dna fragmentation trapezoidal (Wylie AH, et al.Am J Pathol 109:78-87,1982; Peitsch MC, et al.The EMBOJournal 12:371-377,1993; ), smear or tissue slice original position mark dna breakage are as TUNEL method (Stammler G and M Volm, Apoptosis 1:95-99,1996) and FACS show apoptotic peak (Bergamaschi G et al.Haematologica 79:86-93,1994).Yet some apoptotic cell had both been followed the trail of less than dna fragmentation is trapezoidal and has not been found also that FACS shows and this shows apoptotic peak (Di-Pietro R, et al.Cytokine9:463-470,1997), and the means of exploring a kind of new evaluation apoptotic cell are necessary.
The object of the invention provides the method for a kind of screening anti-tumor medicine and clinical chemotherapy effect judgement, this method is by detecting by the antitumor drug interaction in vitro behind the cell of not expressing acetylcholinesterase, the acetylcholine esterase active of cell expressing, or measure the serum acetylcholine esterase active and come identification of cell whether apoptosis takes place, take this screening antineoplastic drugs and the tumour patient clinical chemotherapy effect is judged.
Through experimental demonstration repeatedly, the inventor proves mammalian cell (except that neurocyte and red corpuscle) first in the world when apoptosis takes place, and apoptotic cell is expressed acetylcholinesterase.Be summarized as follows: 1. Mammals, as the people, mouse and rat cell (except that neurocyte and red corpuscle) in case apoptosis has taken place, are all expressed acetylcholinesterase, and the conservative property of its evolutionary process is described.2. no matter be tumor cell line or the former foster normal diploid cell of being commissioned to train, in case apoptosis has taken place, just express acetylcholinesterase, expression and tumour that acetylcholinesterase is described are related not quite, and it is closely related with apoptosis, by embodiment 3, embodiment 7 and embodiment 8, when being provided, the non-tumor cell apoptosis expresses the direct evidence of acetylcholinesterase.Author (Karpel R, et al such as the not high and Karpel of serum acetylcholine esterase active before the tumour patient treatment of author (Zakut H, et al.Cancer 61:727-737,1988) such as Zakut investigation in addition.ExperimentalCell Research 210:268-277,1994) be reported in the expression of following the trail of in the biopsy of primary tumor, all effectively support our argument less than any acetylcholinesterase mRNA.
The present invention around this principle, whether to have expressed acetylcholinesterase be foundation to detect the mammalian cell (except that neurocyte and red corpuscle) of normally not expressing acetylcholinesterase, judges whether this cell apoptosis has taken place.By detecting by the antitumor drug interaction in vitro behind the cell of not expressing acetylcholinesterase, the acetylcholine esterase active of cell expressing, or mensuration serum acetylcholine esterase active, as judging apoptotic means, take this screening antineoplastic drugs, help the doctor to select responsive especially chemotherapeutics, to improve result of treatment at patient.
1. experiment in vitro screening antineoplastic drugs: the research anti-cancer agent, in numerous drug candidates, screen, can inducing apoptosis of tumour cell be an important indicator.The inventive method can be screened anti-cancer agent in order to experiment in vitro.
The human tumor cell line of vitro culture, the antitumor drug candidate of the various dose that adding will detect after 18-20 hour, adopts " plasma clot method " or " polyacrylamide gel method ", and the cell of suspension is in the semi-solid grumeleuse.1% Paraformaldehyde 96 is fixed 10 minutes, uses the filter paper suck dry moisture, remakes acetylcholine esterase active dyeing.Preparation acetylcholinesterase substrate reactions liquid (0.1M phosphoric acid buffer Ph 6.0150ml, acetyl thio choline 100mg/200ml, 0.1M Trisodium Citrate 11ml, 30mM copper sulfate 20ml, 5mM Tripotassium iron hexacyanide 20ml) carries out enzyme reaction.When detecting the cell acetylcholine esterase active, cell room temperature in reaction solution was hatched 6-12 hour, be the positive reaction of tawny.Suppressing experiment is to add 10 μ M BW284c51 (Sigma company) acetylcholinesterase specific inhibitors in reaction solution.If active reaction is suppressed, prove that this activity is by acetylcholinesterase catalysis institute extremely.The albumen of non-sex change electrophoretic separation in this experiment, the acetylcholine esterase active reacting positive, this activity can be suppressed by BW284c51, proves this proteic acetylcholinesterase really.Above-mentioned positive reaction illustrates that this medicine induced apoptosis, is the effective antitumour medicine.
2. clinical tumor patient chemotherapy effect is judged
With the bacterial disease of antibiotic therapy the time, because antibiotic is widely-used, Resistant strain constantly occurs, to some people's infectious diseases, and the antibiotic poor effect that has.At this moment, the doctor often takes out patient's focus portion bacterium and carries out drug sensitivity assay, so that find the specific aim antibiotic therapy that the patient of Resistant strain is arranged, obtains best effect.
For different tumours, different patients is different to the reaction of various chemotherapeutics, and curative effect is fine on one's body this patient for the antitumor drug that has, and just bad to another patient's curative effect.The inventive method as above-mentioned drug sensitive experiment to bacterium, can help the clinician to select tumor chemotherapeutic drug targetedly, shoots the arrow at the target, and finds optimal drug in the shortest time, brings glad tidings to the patient.
Chemotherapeutics is at tumour cell, makes it to take place apoptosis, thereby reaches the purpose of treatment.The present invention is based upon on the newfound basis of scientific research.Its principle is cell great expression acetylcholinesterase in apoptosis process of not expressing acetylcholinesterase under the normal circumstances, mainly focuses in the nucleus, and along with nucleus is formed apoptotic body, acetylcholinesterase just is present in the apoptotic body.Normal people or tumour patient are under the situation that does not have treatment, though in the body apoptosis is arranged constantly, quantity is few, and the acetylcholinesterase in the macrophage phagocytic around the very fast quilt of apoptotic cell, apoptotic cell is not discharged in the blood.But, if effective chemotherapeutics, make a large amount of apoptosis of tumour cell, scavenger cell can't be engulfed all apoptotic cells very soon, perhaps be subjected to drug influence, the macrophage phagocytic ability reduces, and can not engulf apoptotic cell very soon, and at this moment a large amount of acetylcholinesterases enters in the blood with apoptotic cell and apoptotic body.Get the patients serum measure the serum acetylcholine esterase active this moment, will find it specific activity normal people or treatment before exceed 3-6 doubly.Therefore, measure patient treatment front and back serum acetylcholine esterase active, just how know chemotherapy effect, help the clinician in the shortest time, to find the tumor chemotherapeutic drug of targeted the best, shoot the arrow at the target,, strive for valuable time for saving patient's life.
If the patient is a leukemia, can directly get patient blood, separate with FICOLL 400 (Gibco BRL company), after removing red corpuscle, be divided into some branches, cultivate in the culture dish of 35mm, add different chemotherapeutics, after 18-20 hour, adopt " plasma clot method " or " polyacrylamide grumeleuse method ", the cell of suspension is in the semi-solid grumeleuse.1% Paraformaldehyde 96 is fixed 10 minutes, uses the filter paper suck dry moisture, remakes acetylcholine esterase active dyeing.Preparation acetylcholinesterase substrate reactions liquid (0.1M phosphoric acid buffer Ph 6.0 150ml, acetyl thio choline 100mg/200ml, 0.1M Trisodium Citrate 11ml, 30mM copper sulfate 20ml, 5mM Tripotassium iron hexacyanide 20ml) carries out enzyme reaction.When detecting the cell acetylcholine esterase active, cell room temperature in reaction solution was hatched 6-12 hour, be the positive reaction of tawny.Suppressing experiment is to add 10 μ M BW284c51 (Sigma company) acetylcholinesterase specific inhibitors in reaction solution.If active reaction is suppressed, prove that this activity is by acetylcholinesterase catalysis institute extremely.The albumen of non-sex change electrophoretic separation in this experiment, the acetylcholine esterase active reacting positive, this activity can be suppressed by BW284c51, proves this proteic acetylcholinesterase really, the apoptosis that shown induced by chemotherapeutic agents that the patient uses is effective chemotherapeutics.
If noumenal tumour then can be judged by the serum acetylcholine esterase active of measuring the treatment front and back, if activity exceeded 3-6 doubly before treatment back activity ratio treated, this shows that apoptosis has taken place tumour cell, illustrates that chemotherapy effect is good, can help clinician's specific aim to select chemotherapeutics.Measure the method for serum acetylcholinesterase, referring to Uete T, et al:Clin.Biochem.J 327-333,1970.
Also has a kind of method that the serum acetylcholinesterase is suppressed the back remaining activity of measuring, the inventor is by Zakut H et al:Cancer 61:727-737,1988 authors such as method .Zakut have measured the remaining activity after patient and 21 routine normal human serum acetylcholinesterase BW suppress before 77 example treatment back patients, the 11 example treatments, and the result is through 1 * 10
-5The acetylcholinesterase that BW suppresses to measure the back finds that treatment back enzymic activity obviously raises, but proof is not relevant with apoptosis of tumor cells for they, does not more propose as the effect of judging apoptosis and result of treatment.Prove that according to the inventor apoptotic cell expresses the result of acetylcholinesterase, propose to measure the method for serum acetylcholinesterase, identify chemotherapy effect by them.
Advantage of the present invention: the inventive method is by detecting by the antitumor drug interaction in vitro behind the cell of not expressing acetylcholinesterase, the cell expressing acetylcholine esterase active, or measure the serum acetylcholine esterase active and identify whether apoptosis takes place, apoptotic cell acetylcholinesterase reacting positive, illustrate that this medicine induced apoptosis, it is the effective antitumour medicine, take this screening antineoplastic drugs easily and effectively, also can help the doctor to select responsive especially chemotherapeutics, to improve result of treatment at patient.Detect acetylcholine esterase active with the inventive method and identify that the result of the apoptotic cell of vitro culture is consistent with classical way.And accurate positioning, method is simple, does not need expensive equipment, also too not expensive reagent, can be qualitative also can quantitative analysis.
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
The apoptotic dna ladder shape of Fig. 1 .HLF and HL-60 fragment agarose electrophoresis figure.
(1) 123 dna marker, (buying) from Sigma company;
(2) people's lung fibroblast strain (HLF) through the apoptotic cell after inducing, shows dna ladder shape fragment;
(3) human leukemia cell line (HL-60) through the apoptotic cell after inducing, shows dna ladder shape fragment;
(4) human leukemia cell line (HL-60), well-grown, no dna ladder shape fragment proves apoptosis does not take place.
Fig. 2 .FACS shows the apoptotic apoptotic peak figure of HL-60.Wherein A is an apoptotic peak, and B is the G0-G1 phase, and C is the G2-M phase.
Fig. 3. opticmicroscope shows the HL-60 cell, garden, left side and the smooth cell that there is no the substrate positive reaction is a normal cell, the right side form is irregular, and the cell of brown reaction is an apoptotic cell.×300。
Fig. 4. opticmicroscope shows the HL-60 cell, garden, left side and the smooth cell that there is no the substrate positive reaction is a normal cell, the right side form is irregular, and the cell of brown reaction is an apoptotic cell.×300。
Fig. 5. opticmicroscope shows the HL-60 cell, garden, left side and the smooth cell that there is no the substrate positive reaction is a normal cell, the right side form is irregular, has formed apoptotic body, and the cell of brown reaction is an apoptotic cell.×300。
Fig. 6. opticmicroscope shows the HL-60 cell, and cellular form is irregular, has formed apoptotic body, and the cell of brown reaction is an apoptotic cell.×300。
The agarose gel electrophoresis figure of the RT-PCR product of Fig. 7 .HL-60 cell.
(1) PCR mark (bp): 1,543 994 695 515 377;
(2) use the RT-PCR product electrophoretogram of primer 1 and primer 2, the PCR product is 482bp, and that prove HL-60 cell transcription in the apoptotic process is cerebral acetylcholinesteraseobtained mRNA;
(3) the RT-PCR product electrophoretogram of use primer 1 and primer 3 proves that product is not to read over type;
(4) the RT-PCR product electrophoretogram of use primer 1 and primer 4 proves that product is not an erythrocytic form.
Fig. 8. detect the HLF cell with polyacrylamide gel piece method, brown reaction be apoptotic cell, * 500.
Fig. 9. directly detect adherent HLF cell, brown reaction be apoptotic cell, apoptotic body is wherein arranged, * 300.
Figure 10. detect the PC-3 cell with the plasma clot method, brown reaction be apoptotic cell.×300。
Figure 11. detect the M07e cell with the plasma clot method, brown reaction be apoptotic cell, apoptotic body is especially obvious.×400
Figure 12. detect the HELA cell with the plasma clot method, brown reaction be the later stage apoptotic cell, and the blister projection appears.×400。
Figure 13. detect the HELA cell with the plasma clot method, brown reaction be viable apoptotic cell, enzyme is distributed in whole cell.×400。
Figure 14. detect the HELA cell with the plasma clot method, brown reaction be the apoptotic cell in mid-term, enzyme concentrates in the nucleus.×500。
Figure 15. detect the HIH/3T3 cell with the plasma clot method, brown reaction be apoptotic cell, * 300.
Figure 16. rat aorta unstriated muscle apoptotic cell transmission electron microscope figure, the acetylcholinesterase positive in the nuclear, nuclear begins to form apoptotic body, * 11500.
Embodiment 1 antitumor drug acts on the evaluation that the HL-60 cell causes apoptosis
Suspension culture human leukemia cell line (HL-60), nutrient solution RPMI Medium 1640 (GibcoBRL company) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO
2Cultivate in the incubator.Control group does not add antitumor drug, and experimental group adds 1 μ M antitumor drug daunorubicin, and (daunorubicin Italy), collects apoptotic cell after 18-20 hour.In order to confirm that cell is in apoptotic state, the inventor at first uses classic methods identification of cell apoptosis, then, identifies apoptotic cell with method of the present invention, and compares.
Classic methods identification of cell apoptosis
(1) agarose gel electrophoresis detects the apoptotic cell dna fragmentation
The DNA method for extracting of HL-60 cell normally reaches apoptotic cells (2-3 * 10 referring to document (Jaffrezou JP, et al The EMBOJournal 15:2417-2424,1996)
6) centrifugal respectively, throw out washs with PBS, and the centrifuged deposit thing is resuspended with 0.2M Sodium phosphate dibasic/0.1M 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 RNase A (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. 1, show that dna fragmentation is trapezoidal, normal group does not then have trapezoidal variation (4 among Fig. 1), and this is the most reliable evidence of apoptosis.
(2) flow cytometer detects apoptotic peak (referring to Bedi A, et a1.Blood 83:2-38-2-44,1994) the fixing inductive HL-60 cell of 50% ethanol, 0.1% triton (Triton X-100) room temperature treatment cell 5 minutes, hatched 15 minutes at 37 ℃ with 5 μ g/ml RNAse again, 50 μ g/ml PI (Propidium iodide) were 4 ℃ of dyeing 60 minutes, use flow cytometer (FACscan then, USA) apoptotic peak and the G0/G1 of the formation of the DNA fragment of tracking feature, the cell (see figure 2) of S/G2 and M phase.
(3) apoptotic morphologic: visible part karyopyknosis among Fig. 3-Fig. 6, cytolemma is balloon-shaped structure, apoptotic body, these are typical apoptotic cell (Kerr JFR, et al.26:239-257,1972; Plunkett W.Apoptosis-the story of suicide in the cell, CBC Oxford, UK, 1995).
The HL-60 cell that the present invention cultivates body carries out acetylcholine esterase active and detects
The cell of vitro culture is carried out acetylcholine esterase active to be identified and can implement at cell levels.Cell can carry out the enzymic activity reaction at suspended state, also can carry out the enzymic activity reaction at adherent state, adopt the plasma clot method, purpose is that cell is in the semisolid network, to avoid free resultant after the enzyme reaction, cause false positive attached to cell surface.Whole steps is: plasma clot method (cell is in the semisolid network)-fix-blot-enzyme reaction-microscopic examination.
Operating process:
Plasma clot method: get above-mentionedly, and prove same a collection of cell suspension (apoptotic cell and the original fluid) 0.8ml of apoptosis, 3.4%CaCl with classical way through after the antitumor drug effect
20.1ml ox true plasma (Gibco company) 0.1ml behind three's mixing, joins in the plastic culture dish of diameter 35mm immediately, is placed on 37 ℃ of hatchings 15 minutes, solidifies the back and fixes.
Fixing: as to fix 10 minutes in the 1% Paraformaldehyde 96 1ml adding plastic culture dish, outwell stationary liquid.
Blot: 5 of the filter paper of usefulness diameter 34mm, blot remaining moisture.Add substrate and carry out enzyme reaction.
Enzyme reaction: preparation acetylcholinesterase substrate reactions liquid (0.1M phosphoric acid buffer Ph 6.0 150ml, acetyl thio choline 100mg/200ml, 0.1M Trisodium Citrate 11ml, 30mM copper sulfate 20ml, 5mM Tripotassium iron hexacyanide 20ml).At room temperature reacted dereaction liquid, observations under opticmicroscope 6-12 hour.
Opticmicroscope is observed down: the cell of acetylcholinesterase occur the tawny reaction (Fig. 3, Fig. 4, Fig. 5, Fig. 6), and normal cell does not have positive reaction.Among Fig. 3-5, the cell in left side is normal, and cell surface is level and smooth, no enzyme positive reaction in the cell; The cell on right side is an apoptotic cell, the cell pyknosis, and there is vesicle shape projection on the surface, has occurred apoptotic body (Fig. 6 is fairly obvious) in some cell, so just apoptotic cell, acetylcholinesterase is positive.This result judges that with classical way apoptosis is consistent.And, show that apoptosis very particularly, but the ratio of each stage apoptosis number of quantitative analysis and apoptotic cell and normal cell number also, what do not resemble that electrophoresis and FACS show is that apoptotic cell is arranged in the general cell.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.About the type certification of acetylcholinesterase, see embodiment 2.
From this result as seen, HL-60 apoptotic cell acetylcholinesterase reacting positive, normal cell is negative reaction then, the apoptotic cell form of cellular form and bibliographical information is compared, the result matches fully, shows that this tumour medicine induced the HL-60 apoptosis, is the effective antitumour medicine.
The evaluation of embodiment 2 acetylcholinesterase types
Is proof HL-60 apoptotic cell expressed acetylcholinesterase among the embodiment 1, is the AChE of which type so? here made further proof.Identify that acetylcholinesterase carries out from two levels, the one, at transcriptional level, promptly use the method for RT-PCR, whether detect cell has transcribing of AChE mRNA.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 normal and inductive HL-60 nucleus is distinguished extracting, obtain mRNA, reverse transcription obtains 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, 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: 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 (scope of this primer amplification is between 1522 to 2003 to see Fig. 7 for E1-E2-E3-E4-E6 type (hydrophilic also is the brain type), 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 is with reference to " molecular cloning " book preparation lysate (50mmol/Tris.Cl Ph8.0,150mmol/LNaCl, 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.
Second step, the tacrine affinity column separates the acetylcholinesterase of apoptotic cell, reference literature (RichardTet al.Protein Expression and Purification 6,389-393,1995), with epoxy activated agarose gel 6B (Epoxy-activated Sepharose 6B) 10g, ＂ Sigma company, E-6754 ＂, with 13g 9-aminoacridine HCl, the No.A3840-1 ＂ of ＂ Aldrich company prepares the Tacrine affinity column, at first uses 0.2M NaCl 50mMTris-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 10mM Tacrine, 50 mM Tris-HCl Ph8.0 elutriants, the acetylcholinesterase of wash-out with 10mM Tris-HCl Ph8.0 dialysis, changes dialyzate three times again, dialyses 24 hours.Lyophilize.
In the 3rd step, acetylcholine esterase active is identified (reference literature Richard T et al.ProteinExpression and Purification 6,389-393,1995)
Activity identification and active inhibition experiment can be carried out on cell levels and running gel.Preparation acetylcholinesterase substrate reactions liquid (0.1M phosphoric acid buffer Ph 6.0 150ml, acetyl thio choline 100mg/200ml, 0.1M Trisodium Citrate 11ml, 30mM copper sulfate 20ml, 5mM 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, be placed in the acetylcholinesterase substrate reactions liquid of preparation reaction 4 hours.The result has the place of acetylcholinesterase the tawny These positive bands to occur, 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 from the HL-60 cell of apoptosis, is cerebral acetylcholinesteraseobtained.
The non-tumor cell people lung fibroblast (HLF) of embodiment 3 apoptosis can be expressed the experiment of acetylcholinesterase
People's lung fibroblast of adherent growth, nutrient solution RPMI 1640 (Gibco company) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO
2Cultivate in the incubator, do not change nutrient solution in 4 days, allow its naturally-aged apoptosis, collecting cell after 4 days.
With classic methods identification of cell apoptosis (seeing 2 among Fig. 1) with embodiment 1.Polyacrylamide gel piece method is adopted in this experiment, uses this ratio juris identical with " plasma clot method ", also is for fear of false positive reaction occurring.Step is cell+polyacrylamide gel piece (cell is in the semisolid network)-fixing-enzyme reaction-dried glue-microscopic examination.
The preparation of polyacrylamide gel is prepared 8% polyacrylamide gel 5ml with reference to " molecular cloning " method, and each component is as follows:
The apoptotic cell and the original fluid 3.65ml that suspend
30% acrylamide soln 1.3ml
10% Ammonium Persulfate 98.5 0.05ml
TEMED?0.003ml
Mix between the gel glass plate that adds Bio-RAD mini-PROTEIN II, room temperature is after 20 minutes, and grumeleuse forms, and takes out gel, and is fixing.
Fixing: as gel piece to be placed in the Flat bottom container that fills 1% Paraformaldehyde 96 20ml room temperature and to fix 10 minutes, outwell stationary liquid.With 0.1M phosphoric acid buffer Ph6.0 washing three times.
Carry out enzyme-substrate reactions then.Reaction conditions is with embodiment 1.
Dried glue: be placed on Model 583 Gel Dryer (Bio-RAD company), blotted in 2 hours.
Observations under opticmicroscope: apoptotic cell has acetylcholinesterase tawny reaction (Fig. 8), and does not normally have positive reaction.Apoptotic cell, the cell pyknosis, there is vesicle shape projection on the surface, has to have occurred apoptotic body in the cell.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.Further prove cerebral acetylcholinesteraseobtained, see embodiment 2.
If directly in attached cell, add stationary liquid, carry out substrate reactions then, also can show brown apoptotic cell, apoptotic body (Fig. 9) is wherein arranged.
Embodiment 4 antitumor drugs act on the evaluation that Human Prostate Cancer Cells (PC-3) causes apoptosis
Cultivator PC-3 cell, adherent growth.Nutrient solution RPMI Medium 1640 (Gibco BRL company) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO
2Cultivate in the incubator.Control group does not add induced drug, and experimental group adding antitumor drug 2 μ M daunorubicins (daunorubicin, Italy), collecting cell after 18-20 hour.
With classic methods identification of cell apoptosis and the present invention the cell of vitro culture being carried out acetylcholine esterase active detects with embodiment 1.Two kinds are detected apoptotic result is consistent.And the present invention can show concrete apoptosis (see Figure 10, brown cell is an apoptotic cell), and what do not resemble electrophoresis and FACS demonstration is that apoptotic cell is arranged in the general cell.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.Further prove cerebral acetylcholinesteraseobtained, with embodiment 2.This experiment shows that this antitumor drug induced the PC-3 apoptosis, is the effective antitumour medicine.
Embodiment 5 antitumor drugs act on the evaluation that people's megalokaryocyte (M07e) causes apoptosis
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, and experimental group does not add GM-CSF, collecting cell after 18-20 hour.
With classic methods identification of cell apoptosis and the present invention external evoked cultured cells being carried out acetylcholine esterase active detects.Two kinds are detected apoptotic result is consistent.And the present invention can show concrete apoptosis (seeing Figure 11), and brown demonstration is an apoptotic cell, and apoptotic body is especially obvious).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.Further prove cerebral acetylcholinesteraseobtained with embodiment 2, this experiment shows that this antitumor drug induced the M07e apoptosis, is the effective antitumour medicine.
Embodiment 6 antitumor drugs act on the evaluation that people HELA cell causes apoptosis
Cultivator HELA cell, adherent growth.Nutrient solution RPMI Medium 1640 (Gibco BRL company) 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.
With classic methods identification of cell apoptosis and the present invention the cell of vitro culture being carried out acetylcholine esterase active detects.Two kinds are detected apoptotic result is consistent.And the present invention can show concrete apoptosis (see Figure 12, Figure 13, Figure 14 show that brown is apoptotic cell), and Figure 12 shows the later stage apoptotic cell, and Figure 13 shows the apoptosis commitment, and Figure 14 shows the apoptosis mid-term stage.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.Further prove cerebral acetylcholinesteraseobtained, see embodiment 2, this experiment shows that this antitumor drug induced the HELA apoptosis, is the effective antitumour medicine.
The non-tumor cell l cell (NIH/3T3) of embodiment 7 apoptosis can be expressed the experiment of acetylcholinesterase
The NIH/3T3 cell of adherent growth, nutrient solution RPMI Medium 1640 (Gibco BRL company) 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 in 3-4 days, allow its naturally-aged, the 4th day collecting cell.
With classic methods identification of cell apoptosis and the present invention the cell of vitro culture being carried out acetylcholine esterase active detects.Two kinds are detected apoptotic result is consistent.And the present invention can show concrete apoptosis (seeing Figure 15, brown demonstration apoptotic cell).Suppressing experiment is to add 10 μ MBW284c51 (Sigma company) acetylcholinesterase specific inhibitor in reaction solution, and enzymic activity can be suppressed, and proves this proteic acetylcholinesterase really.
The non-tumor cell rat aorta smooth muscle cell of embodiment 8 apoptosis can be expressed the experiment of acetylcholinesterase
The adherent growth rat aorta smooth muscle cell, nutrient solution M199 (Gibco BRL company) adds 10% foetal calf serum, is placed on 37 ℃, 5%CO
2Cultivate in the incubator.Control group does not add the factor, and experimental group adds 5 μ g/ml TGF-β, collecting cell after 18-20 hour.Fixing immediately-enzyme substrates reaction (method is with embodiment 1), after 6 hours 1000 rev/mins centrifugal 5 minutes, wash 3 times with phosphoric acid buffer (seeing embodiment 1), fix 30 minutes after using 4 ℃ of 1% perosmic anhydrides (Osmium tetroxide) again.Distilled water wash 3 times, the dehydration of ethanol gradient, acetone treatment three times, the Epon-812 resin embedding, ultrathin section(ing) (LKB company slicing machine) without electron stain, is directly observed under Philips CM 10 type transmission electron microscopes.Because the resultant of acetylcholinesterase reaction contains metal, the electron density height, position that can clear demonstration resultant mainly concentrates in the nuclear (Figure 16), and the position of prompting enzyme is mainly in nucleus.Infer that thus this cell is in the mid-term of apoptosis, because, examine obvious pyknosis, there is nucleosome to occur, be the typical apoptotic morphologic phenotype.
Embodiment 9 noumenal tumours are judged chemotherapy effect by the serum acetylcholine esterase active of measuring the chemotherapy front and back
One prostate cancer patient, chemotherapy is adopted in not operation, for selecting suitable chemotherapeutics, collects before the chemotherapy respectively and chemotherapeutics treatment 20-48 hour the serum 0.04ml in back, measures the serum acetylcholine esterase active and judges chemotherapeutic efficacy.Measure the method for serum acetylcholinesterase, with reference to Uete T, et al:Clin.Biochem.J 327-333,1970. are summarized as follows:
Reagent:
1.Tris damping fluid, 0.2 mol/l, pH 7.4 and 8.0,
2. acetyl thio choline (Acetylthiocholine), 0.026 mol/l is dissolved in H
2O
3.HPO
3，25％W/V
4.O-phthalaldehyde (OPT), 0.1%, be dissolved in methyl alcohol
5. thiocholine (Thiocholine)
6. reduced glutathione (Reduced glutathione GSH)
7. blood sample: serum 0.04ml compares before getting patient's chemotherapy, gets the 20-48 hour serum in treatment back again as experimental group.
Operating process:
With 0.026 0.026 mol/l acetyl thio choline and the 0.2mol/l Tris damping fluid of serum sample 0.03ml adding 0.5ml, in pH 7.4 mixed solutions, be placed on 37 ℃ of hatchings 3 minutes.Add HPO
3, 25%W/V 0.6ml termination reaction.Abundant mixing, centrifugal 10 minutes precipitating proteins.Get in O-phthalaldehyde (OPT) mixed solution that supernatant 0.1ml joins the Tris damping fluid of 2ml pH 8.0 and 0.1ml 0.1%d, spectrophotometer detects after 15 minutes, and excitation wavelength 350nm under the room temperature detects wavelength of fluorescence 420nm.In contrast, reaction mixture and 0.6ml 25%HPO
3Mixing is hatched.The thiocholine that discharges after the serum effect, with the assessment of standard thiocholine, the thiocholine of normal content is handled with OPT equally.Unit of enzyme activity is 37 ℃ and discharges l micromole (micromole) thiocholine in following 1 minute.Acetylcholine esterase active is represented to discharge how many micromoles (micromole) thiocholine in 1 milliliter of serum 1 minute in the serum.Reduced glutathione is used as judges the sulfydryl that discharges after the effect of serum acetylcholinesterase.The fluorescence intensity of thiocholine-OPT approximately is 12% of a GSH-OPT fluorescence intensity.
The result judges: the amount that the preceding every milliliter of serum per minute of chemotherapy makes the acetyl thio choline be hydrolyzed into thiocholine is 11.24 μ mol after the 2.81 μ mol. chemotherapy, (exceeding 3-6 doubly is that chemotherapy is effective).
Embodiment 10 noumenal tumours are suppressed the remaining active chemotherapy effect of judging in back by the serum acetylcholinesterase of measuring the chemotherapy front and back
One patients with lung cancer, not operation, the employing chemotherapy for selecting suitable chemotherapeutics, is collected before the chemotherapy respectively and chemotherapeutics treatment 20-48 hour the serum 0.04ml in back, and mensuration serum acetylcholinesterase is suppressed the remaining activity in back and judges chemotherapeutic efficacy.Measure the method for serum acetylcholine esterase active,
Referring to Zakut H et al:Cancer 61:727-737,1988.
Present method is used reagent
1.[3H]-A?cetylcholine
2.Tetraisopropyl?pyrophosphoram?ide(iso-OMPA).
3.1，5-bis-(4-ally?ld?im?ethylam?m?onium?pheny1)-pentan-3-one?dibrom?ide(BW?284C?51).
Serum adds acetylcholinesterase specific inhibitor BW284C51 1 * 10 before the treatment
-5Behind the M, activity is suppressed substantially, and active 9pmol/ μ gP/hr (being the per hour pmol number of hydrolysis vagusstoff of every microgram serum protein) is only arranged, and the treatment back suppresses activity and rises to 29pmol/ μ gP/hr, and the preceding enzymic activity of activity ratio's chemotherapy exceeds 3.2 times after the chemotherapy.Result according to the present invention knows, this be since cells in vivo particularly tumour cell taken place apoptosis so, illustrate that chemotherapy effect is good.
Claims (1)
1. the method judged of screening anti-tumor medicine and clinical chemotherapy effect, it is characterized in that this method is by detecting by the antitumor drug interaction in vitro behind the cell of not expressing acetylcholinesterase, the acetylcholine esterase active of cell expressing, or measure the serum acetylcholine esterase active and come identification of cell whether apoptosis takes place, take this screening antineoplastic drugs and the tumour patient clinical chemotherapy effect is judged.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97125220A CN1072722C (en) | 1997-12-30 | 1997-12-30 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN97125220A CN1072722C (en) | 1997-12-30 | 1997-12-30 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1186859A true CN1186859A (en) | 1998-07-08 |
| CN1072722C CN1072722C (en) | 2001-10-10 |
Family
ID=5177198
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97125220A Expired - Fee Related CN1072722C (en) | 1997-12-30 | 1997-12-30 | Method of screening anti-tumour medicine and determining clinical chemotherapy effect |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1072722C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050667A1 (en) * | 2004-11-12 | 2006-05-18 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF |
| CN1808106B (en) * | 2005-01-21 | 2010-11-24 | 中国科学院上海药物研究所 | Method for screening acetylcholine esterase inhibitor by nuclear magnetic resonance |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101050473B (en) * | 2007-02-09 | 2010-08-18 | 北京大学 | Kit for sieving medication of anti cell proliferation or anti tumor, sieving method and usage |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IL102565A0 (en) * | 1991-07-22 | 1993-01-14 | Bio Technology General Corp | Expression of enzymatically active recombinant human acetylcholinesterase and uses thereof |
-
1997
- 1997-12-30 CN CN97125220A patent/CN1072722C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006050667A1 (en) * | 2004-11-12 | 2006-05-18 | Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences | MONOCLONAL ANTIBODIES AGAINST APOPTOSIS-RELATED ACETYLCHOLINESTERASE (AR-AChE) AND USE THEREOF |
| CN1808106B (en) * | 2005-01-21 | 2010-11-24 | 中国科学院上海药物研究所 | Method for screening acetylcholine esterase inhibitor by nuclear magnetic resonance |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1072722C (en) | 2001-10-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Hepatitis B virus pre‐S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes | |
| SEILHAMER et al. | Pancreatic phospholipase A2: isolation of the human gene and cDNAs from porcine pancreas and human lung | |
| Decker et al. | EH3 (ABHD9): the first member of a new epoxide hydrolase family with high activity for fatty acid epoxides | |
| Mencía Castaño et al. | Harnessing an inhibitory role of miR-16 in osteogenesis by human mesenchymal stem cells for advanced scaffold-based bone tissue engineering | |
| WO2008064519A1 (en) | Methods and compositions for diagnosis of esophageal cancer and prognosis and improvement of patient survival | |
| CN101031658A (en) | Viral and viral associated mirnas and uses thereof | |
| Yang et al. | miR-29b targets LPL and TDG genes and regulates apoptosis and triglyceride production in MECs | |
| Kawaguchi et al. | Development of an ENPP1 fluorescence probe for inhibitor screening, cellular imaging, and prognostic assessment of malignant breast cancer | |
| Perkins et al. | Indications for distinct pathogenic mechanisms of asbestos and silica through gene expression profiling of the response of lung epithelial cells | |
| CN1072722C (en) | Method of screening anti-tumour medicine and determining clinical chemotherapy effect | |
| Mechoulam et al. | Expression and activation of STAT3 in ischemia-induced retinopathy | |
| Bhat et al. | Interleukin-18-induced cell adhesion molecule expression is associated with feedback regulation by PPAR-γ and NF-κB in Apo E−/− mice | |
| Lu et al. | Synergistic inflammatory signaling by cGAS may be involved in the development of atherosclerosis | |
| Pan et al. | Cabozantinib Reverses Renal Cell Carcinoma–mediated Osteoblast Inhibition in Three-dimensional Coculture In Vitro and Reduces Bone Osteolysis In Vivo | |
| US20060188885A1 (en) | High throughput functional genomic screening methods for osteoarthritis | |
| Bandyopadhyay et al. | Development and gene expression profiling of a metastatic variant | |
| Zhang et al. | High expression of USP18 is associated with the growth of colorectal carcinoma | |
| CN1059702C (en) | Method for cerebral acetylcholinesterase obtained from cells of mamma animals | |
| CN102443630B (en) | Human PPP2R3A genetic mutation and application thereof | |
| CN1675374A (en) | Treatment of liver diseases | |
| Jia et al. | Transcriptome alterations in HepG2 cells induced by shRNA knockdown and overexpression of TMEM2 gene | |
| CN1844387A (en) | Antisense oligonucleotide structure inhibiting Sirt1 gene expression and use thereof | |
| CN1552876A (en) | Targetted liver cancer AFP gene siRNAs expression carrier and its constructing method and use | |
| Wang et al. | Correlation between miRNA-196a2 and miRNA-499 polymorphisms and bladder cancer | |
| Tang et al. | Post-transcriptional regulation of aortic calcification in KLOTHO deficient mice: Impact of miR-145 and miR-378 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SHANGHAI INST. OF LIFE SCIENCE, CAS Free format text: FORMER NAME OR ADDRESS: SHANGHAI INST. OF CYTOBIOLOGY, CHINESE ACADEMY OF SCIENCES |
|
| CP03 | Change of name, title or address |
Address after: 200031 No. 320, Yueyang Road, Shanghai Patentee after: Shanghai Institute of life Sciences, Chinese Academy of Sciences Address before: 200031 No. 320, Yueyang Road, Shanghai Patentee before: Shanghai Inst. of Cell Biology, Chinese Academy of Sciences |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20011010 Termination date: 20101230 |