CN1299876A - Cell structure dectecting method - Google Patents
Cell structure dectecting method Download PDFInfo
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- CN1299876A CN1299876A CN 00129316 CN00129316A CN1299876A CN 1299876 A CN1299876 A CN 1299876A CN 00129316 CN00129316 CN 00129316 CN 00129316 A CN00129316 A CN 00129316A CN 1299876 A CN1299876 A CN 1299876A
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Abstract
The persent invention relates to a method for detecting cell structure by using organelle chromogenic agent. The organelle chromogenic agent of said invented methoc an be reduced into crystal by means of succinate dehydrogenase to make the organelle richly containing said dehydrogenase and microtubule system implement crystallization, so that it is favourable for observation under the microscope. Said invented method can simply, quickly and effectively detect cell structure, and possesses high application value.
Description
The present invention relates to the organoid developer and detect cyto-architectural method.
In recent years, cyto-architectural research has obtained remarkable progress, yet existing method is many based on immune response, and the wherein used antigen-antibody and the preparation of fluorescent marker are all complicated, and conditional request is very high, needs expensive equipment and consuming time longer.Therefore, inquire into a kind of new saving trouble simply fast and reliably method just become current important topic.
One of purpose of the present invention is to provide a kind of method of utilizing the organoid developer to detect cellularstructure, information transmission, cell cycle and small-molecule substance running.
Two of purpose of the present invention is to provide a kind of method of utilizing organoid developer diagnosing malignant tumor.
The present invention utilizes the organoid developer to detect cyto-architectural method to comprise that step is as follows: 1) culturing cell; 2) add the organoid developer; With 3) the observation of cell structure.
Available organoid developer comprises tetrazolium and mixture thereof among the present invention.
Above-mentioned tetrazolium and mixture thereof comprise:
3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl Thiazolyl blue tetrazolium bromide (MTT claims Thiazolyl blue tetrazolium bromide again, and is English by name: and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide);
Thiazolyl blue (TB), English by name: thiazolyl blue, 2-(2 Benzothiazolyl)-5-styryl-3-(4phthalhydrazidyl) tetrazolium chloride;
The tetrazolium orchid claims dioxy 3 again, 3 '-[3,3 '-dimethoxy (1,1 '-biphenyl)-4,4 '-two bases]-two [2,5 biphenyl-2H-tetrazolium], English by name: tetrazolium blue:3,3 '-[3,3 '-dimethoxy (1,1 '-biphenyl)-4,4 '-diyl]-bis[2,5-diphenyl-2H-tetrazolium] dichloride;
(1H)-and tetrazolium, English by name: 1H-tetrazole;
Vitastain (1,3,5-Triphenyl Tetrazolium Chloride, or chlorination 2,3,5-Triphenyl Tetrazolium Chloride), English by name: tetrazolium red:1,3,5-triphenyltetrazolium or 2,3,5-triphenyltetrazolium chloride:
Chlorination (2,3,5-triphen-2-H-tetrazolium, English by name: 2,3,5-triphenyl-2-H-tetrazolium;
Chlorination 5-cyano group-2,3-two ditolyl tetrazoliums (CTC), English by name: 5-cyano-2,3-ditolyltetrazolium chloride;
P-iodonitrotetrazolium violet, claim again (2-[4-allusion quotation benzene]-3-[4-oil of mirbane]-5-phenyltetrazole chlorine) (INT), English by name: p-iodonitrotetrazolium violet or (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride;
The nitro tetrazole violet;
The chlorination iodonitrotetrazolium;
Tetrazole violet;
The violet tetrazolium claims chlorination (2,5-biphenyl-3-[α-Nai Ji]-tetrazolium) again, and is English by name: tetrazoliumviolet:(2,5-diphenyl-3-[α-naphthyl]-tetrazolium chloride;
Chlorination 2 (P-iodobenzene)-P-oil of mirbane-5-benzene tetrazolium (INPT), English by name: 2-(p-iodophenyl)-p-itrophenyl-5-phenyltetrazolium chloride;
The blue tetrazolium of chlorination P-nitro;
The blue monotetrazolium of chlorination nitro;
Oxidation nitro neotetrazolum;
The blue tetrazolium of chlorination m-nitro;
The blue tetrazolium of chlorination;
PIPERONYL TETRAZOLIUM BLUE, English by name: piperonyl tetrazolium blue;
TOLYL TETRAZOLIUM RED (oTTR or p-TTR), English by name: o-Tolyl tetrazolium red, or p-Tolyl tetrazolium red;
Neotetrazolium chloride claims chlorination 2,2 ', 5 again, 5 '-four benzene-3,3 '-[p-two inferior benzene] two tetrazoliums), English by name: neotetrazolium chloride:(NT:2,2 ', 5,5 '-tetraphenyl-3,3 '-[p-diphenylene] ditetrazolium chlorid);
The blue tetrazolium of oxidation nitro also claims chlorination P-nitro blue tetrazolium, or chlorination 2,2 '-two-oil of mirbane-5,5 '-phenylbenzene-3,3 ' [3,3 '-dimethoxy-4,4 two phenylene]-two tetrazoliums (NBT), English by name: nitro blue tetrazoliumor p-nitrotetrazolium blue or 2,2 ' di-p-nitrophenyl-5,5 '-diphenyl-3,3 ' [3,3 '-dimethoxy-4,4 ' diphenylene]-tetrazolium chloride;
Tetranitro blue tetrazolium (TNBT), English by name: tetranitro blue tetrazolium;
The blue tetrazolium (NBT) of nitro, English by name: nitro blue tetrazolium
Chlorination 2,2 '-two [p-nitre benzene]-5,5 '-two [p-thiocarbamyl benzene]-3,3 '-[3,3 '-dimethoxy-4,4 '-two inferior benzene] two tetrazoliums (TC-NBT), English by name: 2,2 '-di[p-nitrophenyl]-5,5 ' di[p-thiocarbamylphenyl]-3,3 '-[3,3 '-dimethoxy-4,4 '-biphenylene] ditetrazolium chloride, claim Thiocarbamyl nitro-BT again, English by name: thiocarbamyl nitro blue tetrazolium;
Tetrazotized o-dianisidine (TTD), English by name: tetrazotized 0-dianisidine;
3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl methoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium saltses (MTS), English by name: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt;
4-(3-iodophenyl)-2-(4-oil of mirbane)-2H-5-tetrazolium]-1,3-benzene disulfonate (WST-1), English by name: 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5tetrazolio]-1,3-benzene disulfonate;
2, two (2-methoxyl group-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyl anilids (XTT) of 2-, English by name: 2,2-bis (2-methoxyl-4-notro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide;
1-[4,5-dimethylthiazole-2-yl]-2,5-hexichol Thiazolyl blue tetrazolium bromide (1-DDTT), English by name: 1-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide;
Thiazolyl blue tetrazolium bromide claims 3-[4 again, 5-dimethylthiazole-2-yl]-2,5-hexichol Thiazolyl blue tetrazolium bromide (3-DDTT), English by name: 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide;
3 '-[the 1-[(phenyl amino)-phosphinylidyne]-3, the 4-tetrazolium]-two (4-methoxyl group-6-nitro) benzene-sulfo group acid sodium hydroxide (PCTT), English by name: sodium 3 '-[1-[phenylamino)-carbonyl]-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene-sulfonic acid hydrate);
P-Anisyl Blue Tetrazolium Chloride (pABT or pApNBT), English by name: p-Anisyl blue tetrzolium chloride, or p-Anisyl-p-nitro blue tetrazolium chloride;
Lamb's-quarters is basic tetrazolium orchid recklessly.
Above organoid developer (organoid crystal material) can singly select or multiselect.
Multiselect more singly is elected to be with effective.Can select different organoid developers for use according to various objectives.As, Vitastain can be used for the dyeing of centrosome, and MTT then is used for the dyeing of microfilament microtubule, interior knitmesh, nucleus, nuclear membrane, centriole, centrosome, spindle body etc.
Major ingredient among the present invention can be made various formulations because of different needs, also can make various concentration because of different needs.
The concentration of organoid developer of the present invention is decided because of particular case, can be good with 5 mcg/ml-500 mcg/ml from 0.1 mcg/ml-800 mcg/ml, is the best with 20 mcg/ml-200 mcg/ml.
Clear-cells device developer of the present invention can be made into multiple formulation.Solid-state as, injection, muddy suspension, ointment machin as particle and powder.But be not limited thereto.
Organoid among the present invention comprises that all cells structure and microfilament microtubule system comprise all subcellular structures, as, but be not limited to interior knitmesh, nucleus, nuclear membrane, nuclear plate, caryoplasm, nuclear pore complex, nuclear cover, karyomit(e), chromatin, core tube, kinetochore, telomere, serous coat, golgi body, centriole, lysosome, centrosome, other centriole material, shrunk ring, rrna, endosome, phagosome, proteasome, peroxisome, spindle body, microvillus, flagellum, cilium, and other microfilament microtubule etc.
Organoid among the present invention also comprises the albumen that is positioned on the said structure and small-molecular peptides etc.
As the key enzyme of tricarboxylic acid cycle, succinodehydrogenase also is distributed widely in other organoid except being present in plastosome, and normal cell obviously increases in tumour cell.When the mixture that contains tetrazolium existed, succinodehydrogenase can be converted into it corresponding crystal product.Because succinodehydrogenase borrows its film incarceration albumen mortise on the membranous structure of organoid, formed crystal is not to be free on endochylema and extracellular fluid, but concentrates on the organoid that is rich in this enzyme.The cellularstructure of crystalization is high-visible at microscopically.
The normal cell of succinodehydrogenase in the cancer cells obviously increases, and the normal cell of the cellularstructure in the cancer cells is obviously unusual.Wherein, quantity showed increased such as centrosome and microfilament microtubule.When the mixture that contains tetrazolium exists,, succinodehydrogenase makes the cellularstructure crystalization that is rich in succinodehydrogenase thereby can being converted into it corresponding crystal product.Because succinodehydrogenase borrows its film incarceration albumen mortise on the membranous structure of organoid, formed crystal is not to be free on endochylema and extracellular fluid, but concentrates on the organoid that is rich in this enzyme.The cellularstructure of crystalization is high-visible at microscopically.Relatively the degree of cell structure crystalization helps the good virulent discriminating of cell.Cyto-architectural crystalization not only causes the mechanicalness loss of cell, also can cause the function stagnation, and the information transmission is obstructed, cell division arrest.
The basis that the present invention detects cyto-architectural method is the application of new organoid developer.Succinodehydrogenase also is distributed widely in other organoid except being present in plastosome, obviously increases at the normal cell of tumour cell.Succinodehydrogenase is the basic substance that tumour is depended on for existence, and its effect is a unlimited hyperplasia of keeping tumour cell and blood vessel thereof: thereby except that removing the cancer-resisting substance in the oncocyte or reducing the active and expression that tumour cell reduces the antitumor action of many chemotherapeutics to the picked-up of cancer-resisting substance and causes the generation of cross resistance and adjustable protective system and function system.Therefore, not only can be used as cyto-architectural detection based on the organoid developer of succinodehydrogenase, and can be used for the discriminating of cellularity.Has application foundation widely.
The present invention has determined that also many medicines, particularly anti-mitosis medicine all act on succinodehydrogenase.Therefore, this is found to be the result of treatment of estimating this type of medicine a kind of new method and theoretical direction is provided.
The present invention uses a kind of organoid developer and detects cyto-architectural method and can further be illustrated by following examples, but is not limited thereto.
Embodiment 1: observe the brain tumor cell centrosome.
With 9L glioma sarcomatosum cell cultures 24 hours, add tetrazole violet 20 mcg/ml then, to handle 5-10 hour, the observation of cell centrosome is as Fig. 1 (amplifying 200 times).The visible star-shaped structure of oncocyte centrosome among the figure.
Embodiment 2: observe the lung tumor cell centrosome.
The SHP77 lung carcinoma cell was cultivated 24 hours, added Vitastain 70 mcg/ml then and handled 12 hours, the observation of cell centrosome is as Fig. 2 (amplifying 200 times).The visible star-shaped structure of oncocyte centrosome among the figure.
Embodiment 3: observe the brain tumor cell micro-tubular structure.
With 9L glioma sarcomatosum cell cultures 24 hours, add the MTT30 mcg/ml then and handled 4-8 hour, the observation of cell micro-tubular structure is as Fig. 3 (amplifying 400 times).Visible oncocyte is done the radial stretching, extension of tubular construction among the figure.
Embodiment 4: observe the lung tumor cell micro-tubular structure.
SHP77 lung carcinoma cell 4000 was cultivated 24 hours, added the MTT50 mcg/ml then and handled 12 hours, observation of cell is done tubular construction, as Fig. 4 (amplifying 400 times).The visible radial stretching, extension of oncocyte micro-tubular structure among the figure.
Embodiment 5: observe brain tumor cell lysosome structure.
With 9L glioma sarcomatosum cell cultures 24 hours, add the MTT30 mcg/ml then and handled 12-18 hour, observe the brain tumor cell structure of mitochondria.As Fig. 5 (amplifying 200 times).Visible tumour cell plastosome is pearl shape encirclement nucleus among the figure.
Embodiment 6: observe brain tumor cell nuclear membrane and/or interior web structure.
With 9L glioma sarcomatosum cell cultures 24 hours, add the MTT30 mcg/ml then and handled 12-24 hour, observe brain tumor cell nuclear membrane and/or interior web structure.As Fig. 6 (amplifying 200 times).Visible tumour cell, brain tumor cell nuclear membrane and/or interior web structure circular layer shape surround nucleus among the figure.
Application cell device developer of the present invention detects cyto-architectural method and also can be applicable to the diagnosis of tumour cell and differential diagnosis, cyto-architectural dynamic observing except that above-mentioned application.The monitoring cancer therapy drug is the curative effect of anti-cell division medicine particularly, and the research in fields such as the running of small-molecule substance, the transmission of cell information and cell cycle.Therefore, the present invention produces great pushing effect the research of pair cell biology and oncology, has very application prospects and remarkable economical and social value.
The foregoing description is intended to illustrate but not only is confined to application cell device developer and detects cyto-architectural method.
Claims (8)
1. cyto-architectural detection method comprises using a kind ofly can being reduced into crystalline organoid developer through succinodehydrogenase that concrete steps are as follows: 1) culturing cell; 2) add the organoid developer; With 3) the observation of cell structure.
2. cyto-architectural detection method according to claim 1 is characterized in that, described organoid developer comprises tetrazolium and mixture thereof.
3. according to the described cyto-architectural detection method of claim 2; it is characterized in that; described organoid developer tetrazolium and mixture thereof comprise 3-(4; 5-dimethylthiazole-2-yl)-2; 5-biphenyl Thiazolyl blue tetrazolium bromide; Thiazolyl blue; the tetrazolium orchid; (1H)-tetrazolium; Vitastain; chlorination 2; 3; 5-triphen-2-H-tetrazolium; chlorination 5-cyano group-2; 3-two ditolyl tetrazoliums; P-iodonitrotetrazolium violet; the nitro tetrazole violet; the chlorination iodonitrotetrazolium; tetrazole violet; the violet tetrazolium; chlorination 2 (P-iodobenzene)-P-oil of mirbane-5-benzene tetrazolium; the blue tetrazolium of chlorination P-nitro; the blue monotetrazolium of chlorination nitro; chlorination nitro neotetrazolum; the blue tetrazolium of chlorination m-nitro; the blue tetrazolium of chlorination; PIPERONYL TETRAZOLIUM BLUE; TOLYL TETRAZOLIUM RED; neotetrazolium chloride; Tetrazolium Nitro BT; tetranitro blue tetrazolium; the blue tetrazolium of nitro; chlorination 2; 2 '-two [p-nitre benzene]-5; 5 '-two [p-thiocarbamyl benzene]-3; 3 ' [3; 3 '-dimethoxy-4; 4 '-two inferior benzene] two tetrazoliums; tetrazotized o-dianisidine; 3-(4; 5-dimethylthiazole-2-yl)-5-(3-carboxyl methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salts; 4-[3-(iodophenyl)-2-(4-oil of mirbane)-2H-5-tetrazolium]-1; the 3-benzene disulfonate; 2; two (2-methoxyl group-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyl anilids of 2-; 1-[4; 5-dimethylthiazole-2-yl]-2; 5-hexichol Thiazolyl blue tetrazolium bromide; Thiazolyl blue tetrazolium bromide; 3 '-[the 1-[(phenyl amino)-phosphinylidyne]-3; the 4-tetrazolium]-two (4-methoxyl group-6-nitro) benzene-sulfo group acid sodium hydroxide; p-Anisyl Blue Tetrazolium Chloride; lamb's-quarters is basic tetrazolium orchid recklessly, and above organoid crystal material can singly select or multiselect.
4. according to the described cyto-architectural detection method of claim 1, it is characterized in that, described organoid developer comprises all subcellular structures, as, interior knitmesh, nucleus, nuclear membrane, nuclear plate, caryoplasm, nuclear pore complex, nuclear cover, karyomit(e), chromatin, core tube, kinetochore, telomere, serous coat, golgi body, centriole, lysosome, centrosome, other centriole material, shrunk ring, rrna, endosome, phagosome, proteasome, peroxisome, spindle body, microvillus, flagellum, cilium, and other microfilament microtubule.
5. according to the described cyto-architectural detection method of claim 1, it is characterized in that, described organoid developer can be good with 5 mcg/ml～500 mcg/ml from 0.1 mcg/ml～800 mcg/ml, is the best with 20 mcg/ml～200 mcg/ml.
6. according to the described cyto-architectural detection method of claim 1, it is characterized in that described cell comprises normal and tumour cell, the inflammatory cell that comes from people and animal, has the microorganism cells of subcellular structure, various hybridoma.
7. according to the described cyto-architectural detection method of claim 1, it is characterized in that described organoid developer can be made into various formulations and medicine closes, solid-state as injection, muddy suspension, ointment machin as particle and powder.
8. the method for a diagnosing malignant tumor comprises and uses the described cyto-architectural detection method of claim 1-7.
Priority Applications (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00129316 CN1112447C (en) | 2000-11-13 | 2000-11-13 | Cell structure dectecting method |
| EP01930891A EP1294922B1 (en) | 2000-04-29 | 2001-04-27 | Treating cancer using organelle crystallizing agents |
| IL15248401A IL152484A0 (en) | 2000-04-29 | 2001-04-27 | Method for treating cancer, visualizing cell structures, and isolating organelles using organelle crystallizing agents |
| AT01930891T ATE464046T1 (en) | 2000-04-29 | 2001-04-27 | TREATMENT OF CANCER USING ORGANELLIC CRYSTALLIZING COMPOUNDS |
| AU5738301A AU5738301A (en) | 2000-04-29 | 2001-04-27 | Method for treating cancer, visualizing cell structures, and isolating organelles using organelle crystallizing agents |
| DE60141829T DE60141829D1 (en) | 2000-04-29 | 2001-04-27 | TREATMENT OF CANCER USING ORGANIC CRYSTALLIZING COMPOUNDS |
| CA2407112A CA2407112C (en) | 2000-04-29 | 2001-04-27 | Method for treating cancer, visualizing cell structures, and isolating organelles using organelle crystallizing agents |
| AU2001257383A AU2001257383B2 (en) | 2000-04-29 | 2001-04-27 | Method for treating cancer, visualizing cell structures, and isolating organelles using organelle crystallizing agents |
| PCT/US2001/013730 WO2001082780A2 (en) | 2000-04-29 | 2001-04-27 | Method for treating cancer, visualizing cell structures, and isolating organelles using organelle crystallizing agents |
| IL152484A IL152484A (en) | 2000-04-29 | 2002-10-24 | Salts of tetrazolium derivatives as organelle and cytoskeleton crystallizing agents and compositions containing them for killing tumor cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00129316 CN1112447C (en) | 2000-11-13 | 2000-11-13 | Cell structure dectecting method |
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| Publication Number | Publication Date |
|---|---|
| CN1299876A true CN1299876A (en) | 2001-06-20 |
| CN1112447C CN1112447C (en) | 2003-06-25 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00129316 Expired - Fee Related CN1112447C (en) | 2000-04-29 | 2000-11-13 | Cell structure dectecting method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285828B (en) * | 2008-05-30 | 2012-09-26 | 中国农业大学 | Rapid quantitative analysis method for animals skeletal muscle fiber |
-
2000
- 2000-11-13 CN CN 00129316 patent/CN1112447C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101285828B (en) * | 2008-05-30 | 2012-09-26 | 中国农业大学 | Rapid quantitative analysis method for animals skeletal muscle fiber |
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| CN1112447C (en) | 2003-06-25 |
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