CN102706788B - System for detecting breast cancer - Google Patents
System for detecting breast cancer Download PDFInfo
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- CN102706788B CN102706788B CN201210164367.8A CN201210164367A CN102706788B CN 102706788 B CN102706788 B CN 102706788B CN 201210164367 A CN201210164367 A CN 201210164367A CN 102706788 B CN102706788 B CN 102706788B
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- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 33
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 33
- 150000001875 compounds Chemical class 0.000 claims abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 238000013016 damping Methods 0.000 claims description 27
- 239000012530 fluid Substances 0.000 claims description 27
- -1 halide anion Chemical group 0.000 claims description 20
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 claims description 13
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical group CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- 125000000217 alkyl group Chemical group 0.000 claims description 9
- 229960002319 barbital Drugs 0.000 claims description 6
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 claims description 6
- 125000001972 isopentyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 6
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 6
- 125000001280 n-hexyl group Chemical group C(CCCCC)* 0.000 claims description 6
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 claims description 6
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 6
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 5
- 239000011734 sodium Substances 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 239000001488 sodium phosphate Substances 0.000 claims description 5
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 5
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 5
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 claims description 4
- NPYPAHLBTDXSSS-UHFFFAOYSA-N Potassium ion Chemical compound [K+] NPYPAHLBTDXSSS-UHFFFAOYSA-N 0.000 claims description 3
- LMPDLIQFRXLCMO-UHFFFAOYSA-L dipotassium;hydrogen phosphate;phosphoric acid Chemical compound [K+].[K+].OP(O)(O)=O.OP([O-])([O-])=O LMPDLIQFRXLCMO-UHFFFAOYSA-L 0.000 claims description 3
- 150000001455 metallic ions Chemical class 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229910001414 potassium ion Inorganic materials 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 claims description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical group [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Chemical group BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 239000000460 chlorine Chemical group 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 229910052731 fluorine Inorganic materials 0.000 claims description 2
- 239000011737 fluorine Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 239000011630 iodine Chemical group 0.000 claims description 2
- 229910052740 iodine Inorganic materials 0.000 claims description 2
- 150000002500 ions Chemical class 0.000 claims description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- WVULZDFWPQCPPJ-UHFFFAOYSA-N potassium;hydrochloride Chemical compound Cl.[K] WVULZDFWPQCPPJ-UHFFFAOYSA-N 0.000 claims description 2
- 229910052711 selenium Inorganic materials 0.000 claims description 2
- 125000004434 sulfur atom Chemical group 0.000 claims description 2
- 229910052714 tellurium Inorganic materials 0.000 claims description 2
- SPRBJFWIUVWXBT-UHFFFAOYSA-K tripotassium 2-hydroxypropane-1,2,3-tricarboxylate 2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [K+].[K+].[K+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O SPRBJFWIUVWXBT-UHFFFAOYSA-K 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 2
- 125000001153 fluoro group Chemical group F* 0.000 claims 1
- 229910052717 sulfur Inorganic materials 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 30
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 47
- 238000004043 dyeing Methods 0.000 description 30
- 239000006285 cell suspension Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 10
- 206010028980 Neoplasm Diseases 0.000 description 8
- 210000000481 breast Anatomy 0.000 description 8
- 238000011580 nude mouse model Methods 0.000 description 7
- 241000699660 Mus musculus Species 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 210000004881 tumor cell Anatomy 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 150000001721 carbon Chemical group 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 208000030270 breast disease Diseases 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 2
- 0 CC[*@@]1c(cccc2)c2NC1=CC(C)=Cc1*(CC)c2ccccc2[n]1 Chemical compound CC[*@@]1c(cccc2)c2NC1=CC(C)=Cc1*(CC)c2ccccc2[n]1 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 208000035010 Term birth Diseases 0.000 description 1
- 208000024799 Thyroid disease Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960000796 barbital sodium Drugs 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 208000011803 breast fibrocystic disease Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
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- 238000003759 clinical diagnosis Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 102000043827 human Smooth muscle Human genes 0.000 description 1
- 108700038605 human Smooth muscle Proteins 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000009607 mammography Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009247 menarche Effects 0.000 description 1
- 230000004630 mental health Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 229940127234 oral contraceptive Drugs 0.000 description 1
- 239000003539 oral contraceptive agent Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000002559 palpation Methods 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- RGHFKWPGWBFQLN-UHFFFAOYSA-M sodium;5,5-diethylpyrimidin-3-ide-2,4,6-trione Chemical compound [Na+].CCC1(CC)C([O-])=NC(=O)NC1=O RGHFKWPGWBFQLN-UHFFFAOYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium atom Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 description 1
- 208000021510 thyroid gland disease Diseases 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/0008—Methine or polymethine dyes, e.g. cyanine dyes substituted on the polymethine chain
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1488—Methods for deciding
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- Biochemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The present invention provides a kind of system for Diagnosis of Breast cancer, and the system comprises reagent I and reagent II and flow cytometers, wherein the reagent I is the buffer of pH value 6~8, the reagent II is the compound with structure shown in following formula I:
Formulas I diagnostic system sensitivity of the invention is 100 times of traditional single molecule probe or more, and accuracy rate about 97~99% is easy to operate, and detection time is short and result is quantized values, without description, is not necessarily to experience, more objective and accurate.
Description
Technical field
The invention belongs to field of medicaments, in particular to a kind of supermolecular probe that can be used for diagnosing mammary cancer.
Background technology
Breast cancer is one of modal malignant tumour of women, is that one has a strong impact on the even life-threatening modal malignant tumour of women's physical and mental health.Up-to-date statistics shows that the incidence of disease of breast cancer is positioned at first of all kinds of cancers, and mortality ratio is also only second to lung cancer and occupies second.Death toll accounts for 14% of various cancers, is a kind of very serious tumor disease.
The cause of disease of breast cancer is also completely not clear and definite, but the factors such as sterile, fertility number of times is few, first tire term birth evening at age, age of menarche morning, benign breast disease history, family history of breast cancer, oral contraceptive, radiation exposure have been identified with breast cancer related.The long-term factor such as dietary structure, habits and customs causes the excessive acidifying of physique, the function of human body integral declines, cause and suffer from a deficiency of the kidney, The liver and the kidney have a common source, the liver of suffering from a deficiency of the kidney is also empty, and then causes that part of the body cavity above the diaphragm housing the heart and lungs metabolic cycles is slack-off, causes thyroid disease and endocrinopathy, immunologic function to decline, thereby develop into breast tissue paraplasm, whole carcinogenesis.
The methods for clinical diagnosis of breast cancer has a lot, as visual examination, palpation, mammography, ultrasound diagnosis, mammary gland light scattering imaging, latex dust introscope, breast MRI inspection and pathology biopsy etc., but these methods all need good operation skill and abundant clinical experience, it is a kind of more subjective determination methods, cannot produce the result of a quantification, this has just increased testing cost and detection difficulty.
Cyanine dyes is a kind of common dyes, has unique photaesthesia character, for centuries, is accompanied by its unique physics and chemistry, optical property is found, and is used as gradually developer, shadow agent, nonlinear optical material etc.
The inventor is by a large amount of research, finds unexpectedly, and cyanine dyes can be combined specifically with breast cancer tissue, thereby can be used in the detection of breast cancer.
Summary of the invention
One aspect of the present invention provides a kind of system for diagnosing mammary cancer, and described system comprises kit and flow cytometer.
According to system of the present invention, wherein said kit comprises reagent I and reagent II, and wherein reagent I is that pH value is 6～8 damping fluid, and reagent II is the compound shown in following formula I:
Formula I
Wherein: R
1for C
1-C
6the phenyl that replaces of alkyl, phenyl, alkyl; R
2, R
3, R
4and R
5independently selected from H or C
1-C
6alkyl, or R
2and R
3together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan, or R
4and R
5together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan; R
6and R
7for C
1-C
6the C that alkyl or sulfonic group replace
1-C
6alkyl; Y is gegenion, according to R
6and R
7institute charged difference and difference, if R
6and R
7for alkyl, Y is halide anion; If R
6and R
7only have one with sulfonate radical, without Y as gegenion; If R
6and R
7all, with sulfonate radical, Y is triethylamine kation; X
1, X
2independently selected from carbon (C), oxygen (O), sulphur (S), selenium (Se) or tellurium (Te).
According to system of the present invention, wherein, for the compound of formula I, its preparation method can be with reference to Leslie G.S., Brooker and Frank L.W., JACS, 1935, the synthetic route of recording in 547-551, also can prepare with additive method well known in the art.
According to system of the present invention, wherein reagent I is preferably the damping fluid that contains monovalent metallic ion of pH6～8, preferably contain the damping fluid of potassium ion or sodion, include but not limited to sodium phosphate-dibastic sodium phosphate damping fluid, potassium phosphate-potassium hydrogen phosphate damping fluid, barbital sodium-hydrochloride buffer, barbital potassium-hydrochloride buffer, citric acid-sodium citrate damping fluid or citric acid-potassium citrate damping fluid.
According to system of the present invention, wherein C
1-C
6alkyl be that carbon number is the alkyl of the straight or branched of 1-6, include but not limited to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl or isohesyl etc.
According to system of the present invention, wherein R
1be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl, isohesyl, phenyl, aminomethyl phenyl or 3,5-dimethylphenyl.
The method according to this invention, wherein R
2, R
3, R
4and R
5independently selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl or isohesyl.
According to system of the present invention, wherein R
2and R
3the carbon atom being connected with them can form saturated rings structure or the unsaturated ring structure of 5 yuan to 7 yuan, and described ring structure can contain or not contain nitrogen (N) or sulphur (S) atom.
According to system of the present invention, wherein R
4and R
5the carbon atom being connected with them can form saturated rings structure or the unsaturated ring structure of 5 yuan to 7 yuan, and described ring structure can contain or not contain N or S atom.
According to system of the present invention, wherein Y is preferably fluorine, chlorine, bromine, iodine negative ion or triethylamine kation.
One aspect of the present invention provides a kind of method of diagnosing mammary cancer, said method comprising the steps of:
(1) obtain experimenter's breast lesion tissue sample, and to prepare concentration with the damping fluid that pH value is 6～8 be 10
4～10
9the single cell suspension of individual cell/mL;
(2) in the middle single cell suspension obtaining of step (1), add the compound of appropriate formula I so that the compound concentration of single cell suspension Chinese style I is 0.5 μ M to 200 μ M, and the single cell suspension of acquisition is first hatched 10 minutes to 8 hours at 25-40 DEG C, then at 2-8 DEG C, hatch 10 minutes to 8 hours;
(3) collect the cell after hatching, the damping fluid that is 6～8 by pH value washing, and Eddy diffusion, obtaining concentration is 10
3～10
6the single cell suspension of individual cell/ml;
In 515nm～650nm or the above wavelength coverage of 670nm, the fluorescence of the cell suspension obtaining in step (3) is detected with flow cytometer, calculate the cell that has fluorescence signal to produce and account for the ratio of total cellular score, i.e. the rate of dyeing of cell;
By a large amount of experiments, the inventor finds that rate of dyeing when experimenter's mammary glandular cell is in the time that rate of dyeing is greater than 25%, while being preferably greater than 40%, more preferably greater than 60% time, can assert that experimenter suffers from breast cancer.
The method according to this invention, the compound of wherein said formula I is as defined above.
The method according to this invention, wherein said damping fluid is the damping fluid that contains monovalent metallic ion, be preferably the damping fluid that contains potassium ion or sodion, include but not limited to sodium phosphate-dibastic sodium phosphate damping fluid, potassium phosphate-potassium hydrogen phosphate damping fluid, barbital sodium-hydrochloride buffer or citric acid-sodium citrate damping fluid.
Compared with prior art, the advantage of the kit for diagnosing mammary cancer provided by the invention and method and diagnostic system is:
High sensitivity, the employing supermolecule fluorescence probe of novelty, sensitivity is the more than 100 times of traditional single molecule probe;
High specific, normal cell and cancer cell rate of dyeing difference are very large, accuracy rate approximately 97～99%.
Only need a step staining reaction, without making section etc., simple to operate, detection time is short;
Result is quantized values, without description, without experience, more objective and accurate.
Brief description of the drawings
Fig. 1 is the flow cytometer detection rate of dyeing statistical graph that represents the given the test agent obtaining according to one embodiment of the invention;
Fig. 2 is the flow cytometer detection rate of dyeing statistical graph that represents the given the test agent obtaining according to a further embodiment of the invention.
Embodiment
In the mode of specific embodiment, the present invention is described in more detail below with reference to accompanying drawings, but be to be understood that, the present invention can implement in a different manner, it is only in order to make this instructions fully with complete that these embodiment are provided, so that those skilled in the art can implement the present invention, scope of the present invention should not be defined as listed specific embodiment herein.
Embodiment 1
Raise 50 nude mices, 20 as blank, 30 inoculation breast cancer cells (MCF-7) are in nude mouse, after tumour grows up to, get tumor tissues from the nude mice of inoculation MCF-7, blank group is directly got mammary gland and near muscle cell, makes single cell suspension, sodium phosphate-dibastic sodium phosphate the damping fluid that is 6 by pH value washing, suspension, obtaining 20 parts of concentration is 10
4～10
9the normal cell suspension sample of individual cell/mL and 30 parts of concentration are 10
4～10
9the tumour cell suspension sample of individual cell/mL.
Making the concentration of this compound in every duplicate samples to the compound that adds following formula in every duplicate samples is 0.5 μ M
Every duplicate samples is hatched 1 hour in the incubator of 37 DEG C, then in 4 DEG C of refrigerators, continue to hatch 1 hour.
Hatch after end sample is centrifugal, remove dye solution, the sodium phosphate-dibastic sodium phosphate damping fluid washed cell that is 6 by pH value, and Eddy diffusion obtains 10
3～10
6the single cell suspension of individual cell/ml.
Detect cell at the fluorescence signal at 564-606nm place with flow cytometer, obtain the rate of dyeing of each sample.
Rate of dyeing to 50 parts of cell samples by obtaining with upper type carries out statistical study, and result as shown in Figure 1.
With reference to Fig. 1, can clearly be seen that all breast cancer sample rate of dyeing are greater than 95%, the rate of dyeing of all control samples is all less than 25%, thereby, adopt the diagnostic mode of the present embodiment easily the tumour cell of normal cell and breast cancer induction to be separated.
Embodiment 2
Adopt tumour cell and the normal cell of the step identical with embodiment 1 to above 50 nude mices to detect, difference is, the compound that uses is:
The concentration of this compound in sample is 50 μ M, and incubation temperature and time are at 37 DEG C 15 minutes, at 4 DEG C 15 minutes, then use fluorescence signal more than flow cytometer sample 670nm, and obtain the rate of dyeing of each sample.
Testing result: the rate of dyeing of all breast cancer samples is all more than 90%, and the rate of dyeing of all control samples is all below 20%.
Embodiment 3
Adopt tumour cell and the normal cell of the step identical with embodiment 1 to above 50 nude mices to detect, difference is, the compound using is:
Compound concentration is: 10 μ M, and incubation temperature and time are at 37 DEG C 7.5 hours, at 4 DEG C 0.5 hour, fluorescence signal more than flow cytometer detection 670nm, obtained the rate of dyeing of each sample.
Testing result: the rate of dyeing of all breast cancer samples is all more than 80%, and the rate of dyeing of all control samples is all below 25%.
Embodiment 4
Adopt tumour cell and the normal cell of the step identical with embodiment 1 to above 50 nude mices to detect, difference is, the compound using is:
Compound concentration is: 6 μ M, incubation temperature and time are at 37 DEG C 10 minutes, at 4 DEG C 8 hours, detect the fluorescence signal within the scope of 564-606nm with flow cytometer.
Testing result: the rate of dyeing of all breast cancer samples is all more than 70%, and the rate of dyeing of all control samples is all below 25%.
Adopt tumour cell and the normal cell of the step identical with embodiment 1 to above 50 nude mices to detect, difference is, the compound that uses is:
The final concentration of this compound in each sample is 2 μ M, and incubation temperature and time are at 37 DEG C 1 hour, at 4 DEG C 1 hour, detects fluorescence signal more than each sample 670nm with flow cytometer.
Testing result: the rate of dyeing of all breast cancer samples is all more than 80%, and the rate of dyeing of all control samples is all below 25%.
Embodiment 6
Cultivate human breast cancer cell (MCF-7).
Cultivation normal cell system, comprise hamster ovary cell (CHO), rat embryo fibroblast cell (NIH3T3), people's lung fibroblast (HLF), human smooth muscle cell (HUMC), human skin cell (HaCaT), with the washing of the barbital sodium of pH8-hydrochloride buffer, breast carcinoma cell strain and the normal cell system of cultivating that suspend make 10
4～10
9the single cell suspension of individual cell/mL, obtains sample of normal cells and breast cancer cell sample.
To the compound that adds following formula in every duplicate samples, so that the final concentration of this compound in each sample is 0.5 μ M,
Every duplicate samples is hatched 4 hours in 37 DEG C of incubators, then in 4 DEG C of refrigerators, continues to hatch 4 hours.
Hatch after end sample is centrifugal, remove dye solution, with barbital sodium-hydrochloride buffer washed cell of pH8, preparation 10
3～10
6the single cell suspension of individual cell/ml.
Fluorescence signal with flow cytometer detection cell at 564-606nm place, the ratio of acquisition stained positive cell mass, i.e. rate of dyeing.
To carrying out statistical study by the rate of dyeing that repeats 30 parts of cultured cell in vitro that obtain for 30 times with upper type, result as shown in Figure 2.
With reference to Fig. 2, can clearly be seen that all breast cancer cell sample rate of dyeing are greater than 40%, the rate of dyeing of all normal breast cell samples is all less than 25%, therefore, adopts the diagnostic mode of the present embodiment easily normal breast cell and breast cancer cell to be distinguished.
Embodiment 7
Adopt the step identical with embodiment 6 to detect breast cancer cell and normal breast cell line, difference is, the compound that uses is:
The final concentration of this compound in each sample is 200 μ M, and incubation temperature and time are at 37 DEG C 7.5 hours, at 4 DEG C 0.5 hour, detect fluorescence signal more than a sample 670nm with flow cytometer, and obtain the rate of dyeing of each sample.
Testing result: all breast cancer cell sample rate of dyeing are greater than 45%, and the rate of dyeing of all normal breast cell samples is all less than 25%.
Embodiment 8
Adopt the step identical with embodiment 6 to detect breast cancer cell and normal cell strain, difference is, the compound that uses is:
The final concentration of this compound in each sample is 1 μ M, and incubation temperature and time are at 37 DEG C 2 hours, at 4 DEG C 2 hours, detect the fluorescence signal at each sample 564-606nm place with flow cytometer, and obtain the rate of dyeing of each sample.
Testing result: all breast cancer cell sample rate of dyeing are greater than 35%, and the rate of dyeing of all normal breast cell samples is all less than 20%.
Embodiment by above indefiniteness can clearly be seen that, uses system and method for the present invention can distinguish easily breast cancer cell and normal mammary glandular cell, thereby can be greatly for the diagnosis of breast cancer facilitates.And the accuracy rate of method of the present invention is high, the reagent, the equipment that use also easily obtain, simple to operate.
It should be noted that, the flow cytometer using in the embodiment of the present invention is Becton, the flow cytometer of the FACSCalibur model that Dickinson and Company (BD company) produces, its fluorescence exciting wavelength is 488nm and 688nm, but the model of convection type cell instrument is not done any restriction in the present invention, as long as can implement method of the present invention; In the method for the invention to the concentration of each cell suspension sample do not do special restriction, as long as can meet the testing requirement of flow cytometer, those skilled in the art can carry out to determine the concentration of cell suspension as required.
Although described the present invention in the mode of specific embodiment, but be apparent that to those skilled in the art, in the case of not departing from the spirit and scope of the present invention that appended claims limits, can carry out variations and modifications to the present invention, these variations and amendment comprise within the scope of the invention equally.
Claims (9)
1. for detection of a system for breast cancer, described system comprises reagent I and reagent II and flow cytometer, and wherein said reagent I is the damping fluid of pH value 6～8, and described reagent II is the compound with structure shown in following formula I:
Wherein: R
1for C
1-C
6the phenyl that replaces of alkyl, phenyl, alkyl; R
2, R
3, R
4and R
5independently selected from H or C
1-C
6alkyl, or R
2and R
3together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan, and/or R
4and R
5together with the carbon atom connecting with them, form the ring structure of 5 yuan to 7 yuan; R
6and R
7for C
1-C
6the C that alkyl or sulfonic group replace
1-C
6alkyl; According to R
6and R
7charged different Y differences, if R
6and R
7for alkyl, Y is halide anion; If R
6and R
7in only have one with sulfonate radical, Y does not exist; If R
6and R
7all, with sulfonate radical, Y is triethylamine kation; X
1, X
2independently selected from C, O, S, Se or Te.
2. the system as claimed in claim 1, wherein said damping fluid is the damping fluid that contains monovalent metallic ion.
3. system as claimed in claim 2, wherein said damping fluid is the damping fluid that contains potassium ion or sodion.
4. system as claimed in claim 2, wherein said damping fluid is selected from sodium phosphate-dibastic sodium phosphate damping fluid, potassium phosphate-potassium hydrogen phosphate damping fluid, barbital sodium-hydrochloride buffer, barbital potassium-hydrochloride buffer, citric acid-sodium citrate damping fluid or citric acid-potassium citrate damping fluid.
5. the system as claimed in claim 1, wherein said C
1-C
6alkyl be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl or isohesyl.
6. the system as claimed in claim 1, wherein R
1be selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl, isohesyl, phenyl, aminomethyl phenyl or 3,5-dimethylphenyl.
7. the system as claimed in claim 1, wherein R
2, R
3, R
4and R
5independently selected from methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl, the tert-butyl group, amyl group, isopentyl, n-hexyl or isohesyl.
8. the system as claimed in claim 1, wherein said 5 yuan is the saturated or unsaturated ring structure that contains or do not contain N or S atom to 7 ring structures.
9. the system as claimed in claim 1, wherein Y is selected from fluorine, chlorine, bromine, iodine negative ion or triethylamine kation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210164367.8A CN102706788B (en) | 2012-05-24 | 2012-05-24 | System for detecting breast cancer |
| PCT/CN2013/076071 WO2013174262A1 (en) | 2012-05-24 | 2013-05-22 | Cancer diagnosing method, reagent kit, and system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210164367.8A CN102706788B (en) | 2012-05-24 | 2012-05-24 | System for detecting breast cancer |
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