CN1245437A - Method for detecting abnormal epithelial cell shedding - Google Patents
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- CN1245437A CN1245437A CN 97181675 CN97181675A CN1245437A CN 1245437 A CN1245437 A CN 1245437A CN 97181675 CN97181675 CN 97181675 CN 97181675 A CN97181675 A CN 97181675A CN 1245437 A CN1245437 A CN 1245437A
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Abstract
A method for detecting abnormal cell shedding rates of mature epithelial cells of a warm-blooded animal, such as the epithelial cells of the gastric or colonic glands. A labeling composition containing, e. g., a cyanine dye is applied to epithelial cells, following which the site is observed over time. Abnormal cell shedding rates are indicative of disease states such as carcinoma.
Description
Invention field
The present invention relates to the method that live body detects abnormal epithelial cell shedding speed, it can be used as diagnostic method.
Description of related art
The variation of known epithelial cell kinetics (phenomenon that is called cell proliferation, migration, differentiation, aging and forfeiture) is relevant with different morbid states, comprises inflammation and cancer (epithelial pernicious cancer).Therefore, setting up the cytokinetic exact method of mensuration is very important for medical science and association area.Before the radioautography of the thymidine (3H-thymidine) that uses tritiate, cyto-dynamics typically uses the whole bag of tricks and learns and measure, and comprises detecting cell mitogen, measure the size (for example by microscopic count or measurement) of gastrointestinal body of gland and carry out simple cell counting in the cell in vitro culture medium.
Dynamic (dynamical) conventional method use of analysis of cells and the synthetic relevant label of the DNA in the proliferative cell.Proliferative cell can come labelling with 3H-thymidine or thymidine analog-bromodeoxyuridine (BrdU), and they can be inserted in the cell DNA between synthesis stage apace at DNA.But this method is only measured the synthetic speed of DNA, rather than directly measures the cell growth in vivo, moves or come off.
Because use the method for the label synthetic relevant with DNA easy to implement, they are suspected to have unusual dynamic (dynamical) epithelial prefered method as assessment usually.The use of these methods is limited by following factor: the time that must cultivate 3-H thymidine and BrdU regular length with proliferative cell.In addition, can not in human body, carry out with 3H-thymidine and BrdU labelling.Must extract HEP's sample on one's body from the patient, in the culture medium that comprises 3H-thymidine or BrdU, carry out tissue culture then.
Measure the level of some enzyme,, also be used to measure cyto-dynamics as proliferating cell nuclear antigen (PCNA) and thymidine kinase.Though these methods do not need tissue culture, they can not be as method in the body of assessment cyto-dynamics (comprise cell migration or come off).
Cyanine dye has been used for multiple biologic applications.The dioxy carbonyl cyanine dye has been used to carry out leukocyte differentiation counting.Gunter Valet, Max Plank Ges Wissensch; Patent Accession No.84-102307/17 is by selectively staining and measurement volumes and fluorescence while quantitative assay leukocyte (Simultaneous Quantitative Determination of Blood Cells by SelectiveStaining and Measuring volume and Fluorescence).The dyestuff that is used for these researchs is short chain carbonyl cyanine dye (being lower than 10 carbon atoms), and the variation of membrane potential is produced reaction.The short chain carbonyl cyanine dye enters in the cell mitochondrial, and is Cytotoxic.When washed cell, no matter whether the membrane potential of cell changes, and these dyestuffs all leak out from cell easily.Tricarbocyanine dye (Fox, people such as I.J., Proc.May Clinic, 32:478-484,1957) and Evans blue dyestuff (Schad, people such as H., Pfluegers Arch.Eur.J.Physiol., 370 (2): 139-144,1977) be used for by dilution method in the output of the live body assessment heart.Dow (Dow, P., Physiol.Rev., 36:77-102,1956) these methods have been described, it comprises: the blood vessel internal indicator of injecting known quantity in the venous side of lung, measure indicator tremulous pulse concentration in time then, to measure the volume between injection point and the sample point.But these dyestuffs are not used in staining cell.
The 4th, 762, No. 701 U.S. Patent Publications be used to the cell of the cyanine dye labelling following the trail of the cell of cyanine labelling and deliver medicine to the host by measurement rate of disappearance measure method in the body of cell survival, the full content of the document is incorporated at this.
The 4th, 783, No. 401 U.S. Patent Publications with the method for cyanine dye labelling survivaling cell, one of its purpose is to measure the growth rate of the cell of being cultivated.The full content of the document is incorporated at this.
The 4th, 859, No. 584 U.S. Patent Publications measure the method for the growth rate of the cell of the cyanine labelling of growth in the external and body, the full content of the document is incorporated at this.
Before the present invention, still have no talent and study the kinetics of cell by the exfoliation rates of the ripe superficial epithelium cell of inspection in the body.Therefore, disclosed document comprises the healthy or unusual ripe epithelial exfoliation rates on considerably less relevant for example gastrointestinal tract or other mucomembranous surface.The time of washing stomach lining fixed length, measure loss cell by the cell DNA of measuring in the wash solution then, can assess the cell detachment in the stomach thus.It is coarse that this method has been proved to be, and this is because of its program complexity and is difficult to standardization that the cell that obtains during the washing is not enough to carry out DNA analysis, and might obtain the cell of non-definite target location during washing because of carelessness.
The invention summary
Therefore, the purpose of this invention is to provide the method that detects abnormal epithelial cell shedding speed in a kind of body.
It is the method for the morbid state of feature with abnormal cell exfoliation rates in the ripe epithelial cell that a further object of the present invention provides a kind of in-vivo diagnostic, and it comprises that the exfoliation rates of the superficial epithelium cell of the target location of measuring homoiothermic animal in the body is normally or unusual.
Another purpose of the present invention provides improving one's methods of a kind of cancer diagnosis, and described cancer for example is that gastric cancer and colon cancer and other are the disease of feature with abnormal epithelial cell shedding speed.
For realizing above-mentioned and other purpose, the invention provides the method for abnormal cell exfoliation rates in the epithelial cell of the ripe epithelial cell that detects homoiothermic animal in a kind of body such as mucomembranous surface, it may further comprise the steps: the ripe superficial epithelium cell of target-marking position, and monitor this position then and whether have label.In the preferred embodiment of this method, the cell of labelling remains on the mucomembranous surface, and wherein the gastrointestinal mucomembranous surface provides particularly preferred target.
It is the method for the morbid state of feature with abnormal cell exfoliation rates in the ripe epithelial cell of homoiothermic animal that the present invention also provides a kind of diagnosis, it comprises: the sophisticated epithelial cell of labelling, measure the exfoliation rates of labeled cell, then the exfoliation rates of contrast marker cell and the healthy epithelial known exfoliation rates that is in similar position.
The accompanying drawing summary
Figure l is the part sketch map of gastric mucosa.
Fig. 2 is the part sketch map of mucous membrane of colon.
The description of preferred embodiment
Below will describe the preferred embodiments of the invention in detail. The invention provides and detect temperature in the body Improving one's methods of the ripe epithelial abnormal cell exfoliation rates of blood animal, described homoiothermy is moving Thing comprises the people. The ripe superficial epithelium cell marking composition at target location such as mucomembranous surface place Carry out mark. It is known being suitable for the epithelial marking composition of body internal labeling, and it comprises Cyanine dye, other chemical dye such as acetic acid crystal violet, Hoechsdye H33342, eosin and Floxyn, and antibody base label (namely, can detect and covalent bond is connected section on the antibody Divide, such as fluorescence molecule, radio isotope, roentgenopaque compound). Preferably wrap at this The marking composition that contains the cyanine dye part. The cyanine dye part can play in marking composition The effect of detectable independent material. In addition, other detectable material such as other chemical dye Perhaps roentgenopaque compound (X ray developer) also can be present in the marking composition. But this other detectable material chemical coupling perhaps also can be simply on the cyanine dye part Be present in the mixture with cyanine dye or other labeled compound.
According to the present invention the cell mass of target mark preferred main (if not words completely) by Ripe epithelial cell forms. Ripe epithelial cell is that those mitosis are finished and lost Remove the cell of multiplication capacity. Therefore, target location lose detectable label be because The cell of mark has been from exuviation, and this is different from, and cell division between the labeled cell causes The label dilution.
The speed that labeled cell comes off from target location is with what lose in the time of pre-fixed length Detectable represents that available several different methods is measured. At present preferably directly visual by the doctor Detect the target location, but also can use other method that whether mensuration exist label (for example, Use X ray or be used in combination other radioactivity imaging method of X ray developer label).
Refer to compared with normal cell detachment speed more at this used term " abnormal cell exfoliation rates " Height or lower exfoliation rates. Unique distinction of the present invention is, ripe superficial epithelium cell Exfoliation rates can directly be assessed in vivo, to estimate cyto-dynamics and to help to determine the disease shape The existence of attitude.
There is not cell turnover just can not keep function of organization and morphologic integrality. People's gastric mucosa With mucous membrane of colon be carry out continuously quick cell loss (by the tube chamber endepidermis come off and cell dead Die) and the example of the tissue that upgrades. Carrying out constant cell turnover to keep the thin of specific quantity In born of the same parents' the organ, it almost is with identical speed in the tissue of health that cell produces with cell loss Rate is carried out. In gastric mucosa, has the gastric epithelial cell in fixing life-span at the bottom of being positioned at the stomach nest The propagation region generating of the office, continuous top of section and body of gland. These cells migrate upwardly to The surface of skin, with additional mucomembranous cell, and differentiation and ripe in this process, substitute downwards Gastric gland cell. Because gastric epithelial cell has advanced junction complexes, so The order of cell does not change in the transition process. Consequently, old epithelial cell at first takes off Drop in the tube chamber. This is according to the cell age and the phenomenon that comes off in tube chamber of order is called " pipeline System (pipe line system) ". In the epithelium of colon, the propagation zone is positioned at lower three of glandular tube In/two parts. These cells also show " pipeline system " that order comes off, but and stomach The epithelial cell difference, it is upwards migration only. These processes are logical in healthy and abnormal epithelial tissue The normal feature that all has cell increase and cell loss in this collectively is " cell detachment ".
Have now found that,, can assess cyto-dynamics from the exfoliation rates on the mucomembranous surface by observing labeled cell when labelling for example during the ripe epithelial cell on the mucomembranous surface of internal organs.Observed exfoliation rates can compare with the ripe epithelial exfoliation rates of other position of (1) this organ and/or the standard exfoliation rates of (2) particular type of being assessed and the cell of position.Therefore, opposite with the cytokinetic method of current measurement, the present invention measures the ripe epithelial exfoliation rates of having broken up, rather than measures the growth rate of proliferative cell.But the exfoliation rates of assessing these ripe superficial epithelium cells is provided for diagnosing and/or monitoring the important information of epithelial cell group's health status Xiang the doctor.Particularly, these information help to diagnose and monitor high proliferation and low propagation pathological changes and be the morbid state of feature with abnormal cell kinetics, comprise gastric cancer and colorectal cancer.
According to the present invention, for detecting the unusual exfoliation rates of superficial epithelium cell, with detectable material labeled cell, this material is preferably cyanine dye.Method of the present invention is not limited to any concrete Target organ or any concrete marking composition, and is applicable to the cell detachment speed research that any epithelization surface (epithelium) of health is carried out usually.These surfaces comprise stomach, bile duct, colon, urethra, blood vessel, the respiratory tract that comprises nasal cavity, cornea, esophagus, ductus pancreaticus, small intestinal, the genitals who comprises vagina and ovary and prostate.The last leather lining (gastric mucosa) of stomach and the last leather lining (mucous membrane of colon) of large intestine are the positions that is easy to form cancer especially, and account for the major part of the dead population in the world.Therefore, below will at length illustrate the present invention at these two target locations.
When enforcement was of the present invention, the selection of cell marker should make and be easy to detect its existence when it is incorporated into the target epithelial surface.Label should be connected on the epithelium, but can change epithelial essence sharply.Label also should keep connecting the sufficiently long time, and non-degradable in the time of diagnosing.
The preferred marking composition that is used for the inventive method comprises the cyanine dye of following formula:
Wherein:
Y is the methylene that oxygen, sulfur, methylene or alkyl replace;
M is 0-3; And
N is 12-22.Be meant single or dibasic methylene at this used " methylene that alkyl replaces ", substituent group is methyl, ethyl or propyl group.The also available following simple general formula of the chemical compound of said structure is represented (Sims, people such as P.J., Biochem., 13:3315 (1974)):
DiYC
n(2m+1) therefore, for example wherein Y be that the chemical compound of sulfur and carbon atom with three bridge joint rings and two 14 carbon aliphatic chains is called DiSC
14(3).Similarly, DiIC
14(5) represent that wherein Y is the chemical compound of isopropyl and carbon with five bridge joint rings and two 14 carbon aliphatic chains.
Following formula chemical compound with one or more replacement is also included within the above chemical compound that is called cyanine dye, its condition is that these chemical compounds that are substituted may be dissolved in is in the required cell marking medium of labelling at least, and has sufficiently high film partition coefficient to keep the association with cell.These chemical compounds also must be when the required concentration of labelling not the viability of pair cell produce any negative effect.Except that iodine salt, also can use the cyanine dye of the form of can accepting on the materia medica, comprise upward acceptable salt of other medicines.
More preferably, carry out labelling with the compositions of the cyanine dye that comprises following formula:
Or
The dyestuff of formula I (" PKH2 ", iodate 1-docosyl-1 '-propyl group oxygen carbocyanine) and the dyestuff (" PKH26 " of formula II, iodate 1-docosyl-1 '-myristyl-3,3,3 ', 3 '-tetramethyl indole carbocyanine) all can buy from Sigma BioSciences, and by Phanos Technologies, Inc. makes.PKH2 and PKH26 be specificity and optionally staining cell film in vivo, and the character of pair cell does not have a negative impact.These two kinds of dyestuffs can easily be differentiated by fluorescence.These chemical compounds also can be used as the bonding agent with lipophilic affinity, and other diagnostic compounds or other treatment chemical compound can connect thereon.
Except that these advantages, PKH2 and PKH26 are nonantigenic, non-cell toxicities, have the long half-life, and have the ability of stable and lasting staining cell.This dyestuff only needs to be applied in a few minutes on the superficial epithelium cell, just can realize enough dyeing.
Other cyanine dye can be buied from various sources, perhaps also can use known synthetic method to make from existing starting material.Referring to Hamer, F.M., cyanine dye and related compound (TheCynine Dyes and Related Compounds), Interscience Publishers (1964).In the time of in being used for human body, the dyestuff of effective dose is dissolved in the aqueous sucrose solution, can prepares the solution of PKH2 or PKH26 thus.PKH2 and PKH26 can be from Phanos Technologies, obtain in the dye reagent box that Inc. sells, and they provide the dye material liquid of 500 μ l and the diluent of 60ml.Therefore, when using wherein a kind of dye reagent box to prepare dye solution, dyestuff is 1: 120 to the concentration of diluent.
PKH2 and PKH26 be known concentration level when the concentration level that is used for compositions of the present invention is similar to these dyestuffs at external staining cell.Accurate concentration to be used is variable, and easy optimization.The volume of dye composite to be used depends on the size of the concentration of cyanine dye in the said composition and target location and changes.Applied volume for example can change between 1-100ml, and can use the dye composite of about 10ml in many cases.Accurate applied volume is variable, and easy optimization.
Marking composition is included in the cyanine dye in the medium (diluent), and this medium is safe when using, and repeatably cell marking is provided.Usually use osmotic pressure regulator, make cyanine dye need to form stable solution in the time of labelling at least.Acceptable osmotic pressure regulator comprises sugar, comprises monosaccharide such as glucose, fructose, sorbose, xylose, ribose, and disaccharide such as sucrose; Sugar alcohol comprises mannitol, glycerol, inositol, xylitol and ribitol; Aminoacid comprises glycine and arginine; And some Good ' s buffer agent, as N-three (methylol)-methyl-3-aminopropanesulfonicacid acid.Can in the labelling medium, add a spot of buffer agent, to regulate hydrogen ion concentration (pH).Also can use other conventional reagent, as antibiotic and antiseptic.
Preparing the experimenter when implementing method of the present invention, can use and carry out the required conventional formulation of splanchnoscopy.Before carrying out method of the present invention, should follow conventional readiness program, for example go on a diet.For increasing cyanine dye such as PKH2 and PKH26 and last cutaneomucosal cohesive force, can use mucus to remove method, this method is utilized protease (pronase is made by Kaken Seiyaku).This isorheic removal method is described in " Endoscopic Diagnosis of GastricCancer with Dye Scattering " (Amer.J.Gastroenterology, the 63rd volume, No. 4,316-320 page or leaf, in April, 1975) of people such as K.Ida.The content of the document is incorporated into fully at this.In brief, at preceding 20 minutes administration anticonvulsants of inspection, the about 80ml of oral administration is through dimethyl polysiloxane (Gasoon) solution of ten times of dilutions then, and this solution is mixed with the sodium bicarbonate of 1g and the protease of 20000p.u. (pronase).
Can use the superficial epithelium cell administration marking composition of various approach to target location.Can use sustained-release oral dosage forms to come the administration marking composition.Preferably, directly the compositions of solution form is being used (spraying) on last cutaneomucosal surface under the observation of endoscope, perhaps the form with potus delivers medicine to the experimenter with solution.Though expectation directly can increase solution spraying on the skin cohesive force with mucosa from the teeth outwards, oral administration solution also is preferred, and this is because can alleviate experimenter's burden.
According to one embodiment of the invention, marking composition is applied in the concrete zone on epithelization surface.By splanchnoscopy, the doctor can determine easily whether this regional area is unusual, and whether establishment is the good candidate of the present invention's exfoliation rates appraisal procedure thus.The doctor also can be applied in marking composition contiguous normal position, to compare between these two positions.
The advantage of only using marking composition on the regional area of cell rather than the major part at whole mucomembranous surface is to save dyestuff and reduce administration and Diagnostic Time.In a preferred method, also can use the major part of marking composition certainly with the whole mucomembranous surface of covering internal organs Target organ.In the time of for example on the major part that cyanine dye is applied in whole stomach lining, the normal exfoliation rates that can observe mature endothelial cell by the fluorescence intensity of checking dyeing epithelial cell position.
According to the present invention during the labelling gastric epithelial cell, different with the epithelial cell of other organ, the time span of administration marking composition when observing depends on the following fact on average: the life-span of stomach surface mucomembranous cell is about 2 days.Therefore, when the exfoliation rates (wherein cancer slows down normal exfoliation rates) of assessment gastric epithelial cell, administration was at least once checked after 2 days.On the contrary, whether there is the disease that makes the gastric mucosal cell hyper-proliferative, at least once checks in 2 days after administration for measuring.During this period, the experimenter can keep its normal life and diet style, but material such as aspirin and the ethanol that should avoid quickening stomach and other epithelial cell shedding speed.
As viewed in animal and human's experiment, the life cycle of whole body of gland is estimated to be about 4-7 days in the colon.Generally, should behind the administration dye solution, carry out the diagnosis of colon cancer in 4-7 days again.
When enforcement was of the present invention, directly perusal was through the superficial epithelium cell of labelling.According to the preferred method of the present invention, directly whether perusal exists ripe epithelial cell through labelling at medicine-feeding part, the exfoliation rates of these cells can be evaluated as normal or unusual thus.The observed fluorescence intensity of target site comes off and reduces with labeled cell.If still there is label in the time that comes off above labelling position normal epithelium cell, then show hyper-proliferative.Equally, label disappears before normal epithelium cell comes off, and shows that then propagation is low excessively.Advantageously, only whether the preselected time place qualitative determination after markers step exists label.Though can carry out quantitatively label, this is not necessary for implementing diagnosis algorithm of the present invention.
According to the preferred method of the present invention, the position of labeled cell is exposed to exciting light, observe and/or measure fluorescence intensity then, can detect the cyanine dye labelling thing thus.For example, PKH2 and PKH26 have fixed absorbing wavelength and wavelength of fluorescence.When observing maximum fluorescence, the exciting light that PKH2 need about 490-504nm, and the exciting light that PKH26 need about 551-567nm.Use for example suitable filters and photodetector, can observe fluorescence by endoscope.
When enforcement is of the present invention, can use common fibrescope.Particularly, optionally will go up the light that mucocutaneous membrane is exposed to specific wavelength, wherein use the fibrescope that has wave filter, and described filter configuration will be for can only transmit desired portion in total spectrum.Preferably, be about the light of 490-504nm from light source (as halogen lamp) transmission wavelength for the PKH2 wave filter, for PKH26 then transmission wavelength be about the light of 55l-567nm.The light of launching from labeled cell is passed through from suitable narrow band filter, and randomly from image intensifier, pass through, arrive the fluorescence detector that produces corresponding to the signal of detected light intensity, can detect fluorescence thus.Also can use other device during the object observing position, as the endoscope that high-resolution CCD detector is housed.
The washing gastral cavity is measured the DNA in the wash solution, measures the quantity of exfoliative cyte in the gastric juice then, can obtain the existing public data of relevant ripe epithelial cell from the gastrointestinal tract mucous speed that comes off thus.According to these data, per minute has 500000 cells to split away off from gastric mucosa approximately.Have recognized that this cell loss increases when atrophic gastritis.
Though the data of relevant exfoliation rates are limited, the cyto-dynamics of tumor is known.Generally, when in a certain organ canceration taking place, and the kinetics of healthy cell turnover in this organ is in health tissues when slack-off, and the cyto-dynamics of cancerous cell increases.On the contrary, if the epithelial cell kinetics of healthy organ is high and when canceration took place, cyto-dynamics then reduced.For example, the healthy liver cell experiences cell division hardly.Cyto-dynamics then increases in hepatoma carcinoma cell.In gastrointestinal tract mucous healthy cell, cyto-dynamics under normal circumstances is exceedingly fast.When cancer, cyto-dynamics sharply descends.
In healthy people gastrointestinal tract mucous, the length of S phase (DNA synthesis stage) is between 10-11 hour.The gastric mucosa circulation time (T) that is known that the people is about 24-48 hour, and the circulation time that intestinal tissue transforms is about 37 hours, and the circulation time of mucous membrane of colon is about 40 hours, and rectal mucosal circulation time is about 24-48 hour.Comparatively speaking, people's gastric cancer circulation time is about 2.5-13 days, and the circulation time of colon cancer is about 4.2-7.0 days.Therefore, can find out clearly that cell proliferation obviously reduces when cancer.In addition, in people's cancer, though the time that tumor cell doubles (D) (promptly, number of cells double the needed time) usually between about 30-120 days, for gastrointestinal cancer be the utmost point slowly; Early gastric cancer is about 555-3076 days, and late gastric cancer is about 105-305 days, and colon cancer is about 636 days.
Cell cycle time (T) difference between health and the cancerous cell is about 10 times.On the exuviation speed that the difference of this growth rate also is reflected in normally and the carcinomatous mucosa epithelial cell is showed.The ordinary life cycle of stomach surface mucomembranous cell is about 1-2 days.The epithelial ordinary life cycle of mucous membrane of colon is about 4-7 days.If the exfoliation rates of cancerous cell equals this normal cycle period, show that then the exfoliation rates of cancerous cell is faster than its T, mean that basically cancerous cell has disappeared from health.Therefore, can think that the exfoliation rates of cancerous cell must be slower than ten times of Normocellular exfoliation rates (cancerous cell and Normocellular T difference), so that cancerous cell remains in the health.So be apparent that, between the exfoliation rates of normal and carcinomatous mucosa, exist evident difference.Peculiar methods of the present invention has been utilized the difference of this exfoliation rates.
When enforcement was of the present invention, the precision of the concrete instrument that observation post uses (promptly, distinguishing normal cell and cancer) was very important.Two kinds the endoscope of frequent use be fibrescope and fujinon electronic video endoscope.Though the fibrescope of each manufacturer and the resolution differences of fujinon electronic video endoscope are bigger, the resolution of fibrescope is generally 600 μ m, and the resolution of fujinon electronic video endoscope is generally about 100 μ m.As object of the present invention, diameter is that the epithelial exfoliation rates in the 5-10mm damage can be differentiated for normal or unusual person.Therefore, should select suitable instrument.
Because the exfoliation rates of normal stomach surface mucomembranous cell is about 2 days, cyanine dyeing remarkable explanation that disappears before this time has the normal person of exfoliation rates disease existence faster.If cyanine dyeing still can obviously be observed, show that then the disease (as gastric cancer) that has exfoliation rates to slow down exists after 2 days.
Atrophic gastritis takes place in gastric mucosa with age ageing usually.Because known cell turnover increase when atrophic gastritis is so think that exfoliation rates also increases.
The turnover of cell during gastric ulcer is usually above normal gastric mucosa.The data that do not have relevant exfoliation rates, but for compensating the loss of mucosa, grow normally.Therefore, can think that cell loss is not delayed.Also can think, have evident difference between this disease and gastric cancer, the cell turnover reduces among the latter.
Below will further specify the present invention by embodiment, these embodiment only are used to illustrate the present invention, rather than limitation of the present invention.Embodiment 1
For whether investigation can come analysis of cells kinetics by the epithelial exfoliation rates of check mark, with the epithelial cell in PKH2 and the PKH26 dyeing Mus gastric mucosa.The purpose of this research is to assess cyto-dynamics with respect to the synthetic method of known investigation DNA on the basis of epithelial migration of maturation and exfoliation rates in proliferative cell.Carry out following experiment in 2 ages in week on (160-200g) male Wistar rats.
For reducing under one's belt residual, feed with 7% sucrose, 0.5% sodium chloride solution 2 days to rat.Be living again property (generative) cell compartment of labelling gastric mucosa, with the BrdU (5-bromo-2-deoxyuridine (sigma Chemical Co.)) of 6 hours interval intraperitoneal administration 50mg/kg (every about 10mg of animal) 4 times.Behind the 4th administration BrdU immediately the through port cannula cavity to the pronase solution (pronase 20000 units and 1g sodium bicarbonate are in 80ml water) of rat administration 3ml, to remove attached to the surperficial mucus on the gastric mucosa.30 minutes the time, the through port cannula cavity is the PKH2 of administration 3ml or PKH26 in stomach in an identical manner behind oral administration pronase solution.Killed rat in 1,8,16,24,32,40,48,60 and 72 hour behind final administration BrdU, and the excision stomach, freezing in liquid nitrogen immediately then.
, prepare 4 and be respectively the thick sample of 5 μ m through refrigerated tissue with the cryostat cutting.
Existence with first freezing sample proof PKH2 or PKH26.Use cyanoacrylate resin (Bond Aron Alpha is made by Toa Gousei Kagaku Co.) to fix this sample immediately, make the sample of no dyeing.With all the other 3 freezing sample stuck-at-s 0% in buffered formalin 3 minutes.A sample dyes with haematoxylin-eosin.All the other 2 samples dye with anti--BrdU monoclonal antibody as described below by enzyme-antibody act.
With 0.1MPBS (phosphate buffered saline (PBS), Experimental Bio Medical ResearchInc.) flushing sample, then in 2N hydrochloric acid in 37 ℃ of following incubations 30 minutes, with dna breakage.The flushing sample is 3 times in 0.1MPBS.For blocking-up endogenous peroxidase, at room temperature handled sample 10 minutes, and in 0.1M PBS, wash 3 times with 0.3% hydrogenperoxide steam generator.Then, be the blocking-up nonspecific reaction, (Snow Brand ProductsCo., Ltd.) middle incubation is 10 minutes, washs 3 times in 0.1M PBS then at the Block-ACE of double dilution.Afterwards, sample is with original antibody (anti--BrdU, Mus monoclonal, Becton Dickinson) incubation 1 hour at room temperature, and described original antibody is with 50 times of the 0.1MPBS dilutions that comprises 1%BSA.As negative contrast, all the other samples were at room temperature used the normal mouse serum incubation 1 hour, and described Mus serum dilutes 10000 times in comprising the 0.1M PBS of 1%BSA.Sample washes 3 times with 0.1M PBS then, and at room temperature uses biotinylated Mus IgG incubation 30 minutes.After 0.1M PBS flushing 3 times, sample and ABC reagent (Vector) at room temperature react 30 minutes, and reuse 0.1M PBS washes 3 times, then incubation 1 minute in DAB solution (Dojindo).After the washing, sample carried out nuclear staining 1 minute with Mayer haematoxylin stain (Merck) in flowing water, and then washed 10 minutes with flowing water.Make the sample dehydration with ethanol, in toluene, clean, embed HSR then.
Each several part perpendicular to the mucomembranous surface excision is carried out following research.Fig. 1 has shown the various zones of the gastric mucosa of a part of excision.Live again sexual cell zone (G) be defined as behind the 4th administration BrdU 1 hour in the proliferative zone from the painted cell of the BrdU of the superiors to the distance between the painted cell of orlop BrdU.Be called surperficial mucomembranous cell zone (S) in the painted cell of the BrdU of the superiors to the zone of mucomembranous surface.The ratio of BrdU positive cell and total cellular score is called breeding blanket field mark index in the sexual cell zone of living again at this moment.Measure epithelial cell and migrate to the needed time of mucomembranous surface from the BrdU positive cell.The BrdU positive cell arrives the used time of mucomembranous surface and is called surperficial mucomembranous cell update time.As shown in Figure 1, distance (A) is the distance that is equivalent to the painted orlop cell from mucomembranous surface to PKH2, and distance (B) is equivalent to the distance of the painted orlop cell from mucomembranous surface to PKH26.
In last stained specimens, there are PKH2 and PKH26 with fluorescence microscopy.PKH2 is used for the wave filter inspection of FITC, and PKH6 is used for the wave filter inspection of rhodamine (rhodamine).Measurement is by the distance of PKH2 and the painted mucomembranous surface of PKH26.Losing of staining cell is called turnover.Use eyepiece micrometer to measure histology's measuring distance of each sample.
Behind the 4th administration 50mg/kg BrdU, the sample in contrast at 6 hours at interval the 1st hour.The BrdU positive cell mainly is confined to the top of body of gland.In the cell in the sexual cell zone of living again, the 98.2%th, the BrdU positive cell.Therefore, the most cells of living again in the sexual cell zone has absorbed BrdU.
The migration of continuous review BrdU positive cell can be observed the migration of surperficial mucomembranous cell thus.In first sample that 1 hour obtains after administration, can see the BrdU positive cell (promptly, living again to see the superficial cell zone of 110 μ on the sexual cell zone) of the superiors at about 110 μ places.Therefore, can clearly draw, the BrdU positive cell is upwards migration in time, and in 60 hours, can see the BrdU positive cell at the mucomembranous surface place after administration.So the life cycle that can measure the surperficial mucomembranous cell of rat is about 60 hours.
Existing reporting, the life cycle of people surface mucomembranous cell is about 72 hours.By relating to data that this research that the BrdU labeled cell arrives the needed time of mucomembranous surface obtains very near the numerical value of bibliographical information.
Calculate from mucomembranous surface, dyestuff PKH2 can dye and be about the surperficial mucomembranous cell at 75 μ places in the degree of depth.The painted degree of depth of PKH2 passs in time and becomes more and more shallow, and disappears after 40 hours.PKH26 dyeing is more shallow than PKH2, calculates about 45 μ of as many as that can dye from mucomembranous surface.In the case, the dyed cell degree of depth also passs in time and becomes more and more shallow, and disappears after 24 hours.Measure as sentencing BrdU at about 110 μ, surperficial mucomembranous cell needed time that comes off is 60 hours, is 40 hours when dyeing with PKH2 when mucomembranous surface calculates about 75 μ, is 24 hours when dyeing with PKH26 when about 45 μ.It is believed that not being both of exfoliation rates because the dye level difference of each label.The result of this research is can obviously draw PKH2 and PKH26 and can be used as the label that the surperficial mucomembranous cell of analysis comes off.Embodiment 2
For whether investigation can come analysis of cells kinetics by the epithelial exfoliation rates of check mark, with the epithelial cell in PKH2 and the PKH26 dyeing Mus mucous membrane of colon.Carry out following experiment in 2 ages in week on (160-200g) male Wistar rats.
For being reduced in residual in the colon, feed with 7% sucrose, 0.5% sodium chloride solution 2 days to rat.Be the cell compartment of living again of labelling mucous membrane of colon, with BrdU4 time of 6 hours interval intraperitoneal administration 50mg/kg (every about 10mg of animal).Behind the 4th administration BrdU immediately by the pronase solution (pronase 20000 units and 1g sodium bicarbonate in 80ml water) of anus, to remove attached to the surperficial mucus on the mucous membrane of colon to rat administration 3ml.Behind oral administration pronase solution 30 minutes the time, also PKH2 or the PKH26 by anus administration 3ml in colon.Killed rat in 1,8,16,24,32,40,48,60 and 72 hour behind final administration BrdU, and the excision colon, freezing in liquid nitrogen immediately then.
, prepare 4 and be respectively the thick sample of 5 μ m through refrigerated tissue with the cryostat cutting.
Existence with first freezing sample proof PKH2 or PKH26.Use cyanoacrylate resin (Bond Aron Alpha is made by Toa Gousei Kagaku Co.) to fix this sample immediately, make the sample of no dyeing.With all the other 3 freezing sample stuck-at-s 0% in buffered formalin 3 minutes.A sample dyes with haematoxylin-eosin.All the other 2 samples dye with anti--BrdU monoclonal antibody as described below by enzyme-antibody act.
With 0.1M PBS (phosphate buffered saline (PBS), Experimental Bio Medical ResearchInc.) flushing sample, then in 2N hydrochloric acid in 37 ℃ of following incubations 30 minutes, with dna breakage.The flushing sample is 3 times in 0.1M PBS.For blocking-up endogenous peroxidase, at room temperature handled sample 10 minutes, and in 0.1M PBS, wash 3 times with 0.3% hydrogenperoxide steam generator.Then, be the blocking-up nonspecific reaction, (Snow Brand ProductsCo., Ltd.) middle incubation is 10 minutes, washs 3 times in 0.1M PBS then at the Block-ACE of double dilution.Afterwards, sample is with original antibody (anti--BrdU, Mus monoclonal, Becton Dickinson) incubation 1 hour at room temperature, and described original antibody is with 50 times of the 0.1M PBS dilutions that comprises 1%BSA.As negative contrast, all the other samples were at room temperature used the normal mouse serum incubation 1 hour, and described Mus serum dilutes 10000 times in comprising the 0.1M PBS of 1%BSA.Sample washes 3 times with 0.1M PBS then, and at room temperature uses biotinylated Mus IgG incubation 30 minutes.After 0.1M PBS flushing 3 times, sample and ABC reagent (Vector) at room temperature react 30 minutes, and reuse 0.1M PBS washes 3 times, then incubation 1 minute in DAB solution (Dojindo).After the washing, sample carried out nuclear staining 1 minute with Mayer haematoxylin stain (Merck) in flowing water, and then washed 10 minutes with flowing water.Make the sample dehydration with ethanol, in toluene, clean, embed HSR then.
Each several part perpendicular to the mucomembranous surface excision is carried out following research.Fig. 2 has shown the various zones of the mucous membrane of colon of a part of excision.Live again sexual cell zone (G) be defined as behind the 4th administration BrdU 1 hour in the proliferative zone from the painted cell of the BrdU of the superiors to the body of gland distance between the painted cell of orlop BrdU.Be called table in the painted cell of the BrdU of the superiors to the zone of mucomembranous surface and mucomembranous cell zone (S).The ratio of BrdU positive cell and total cellular score is called breeding blanket field mark index in the sexual cell zone of living again at this moment.Measure epithelial cell and migrate to the needed time of mucomembranous surface from the BrdU positive cell.The BrdU positive cell arrives the used time of mucomembranous surface and is called surperficial mucomembranous cell update time.As shown in Figure 2, distance (A) is the distance that is equivalent to the painted orlop cell from mucomembranous surface to PKH2, and distance (B) is equivalent to the distance of the painted orlop cell from mucomembranous surface to PKH26.
In the sample that is unstained, there are PKH2 and PKH26 with fluorescence microscopy.PKH2 is used for the wave filter inspection of FITC, and PKH6 is used for the wave filter inspection of rhodamine.Measurement is by the distance of PKH2 and the painted mucomembranous surface of PKH26.Losing of staining cell is called turnover.Use eyepiece micrometer to measure histology's measuring distance of each sample.
Behind the 4th administration 50mg/kg BrdU (about 10mg/ animal), time in contrast at 6 hours at interval the 1st hour.The BrdU positive cell mainly is confined in the bottom 7/10 of body of gland.Last 3/10 is the superficial cell zone, fathom to be about in the cell of 60 μ in the sexual cell zone of living again, and the 98.2%th, the BrdU positive cell.Therefore, the most cells of living again in the sexual cell zone has absorbed BrdU.
The migration of continuous review BrdU positive cell can be observed the migration of surperficial mucomembranous cell thus.After administration in 1 hour, can see the highest BrdU positive cell (promptly, living again to see the superficial cell zone of 60 μ on the sexual cell zone) at about 60 μ places.Can clearly draw, the BrdU positive cell is upwards migration in time, and in 32 hours, can locate to see the BrdU positive cell at the mucosa table after administration.So the life cycle that can measure the surperficial mucomembranous cell of rat is about 32 hours.Because the life-span of the positive colon surface mucomembranous cell of Mus BrdU is about 32 hours, and this part cell accounts for the last 3/10 of body of gland, is about 107 hours or 4.5 days so estimate the life-span of whole body of gland.
Existing reporting, the life cycle of people surface mucomembranous cell is about 4-7 days.By relating to data that this research that the BrdU labeled cell arrives the needed time of mucomembranous surface obtains very near the numerical value of bibliographical information.
Calculate from mucomembranous surface, dyestuff PKH2 can dye and be about the surperficial mucomembranous cell at 40 μ places in the degree of depth.The painted degree of depth of PKH2 passs in time and becomes more and more shallow, and disappears after 16 hours.PKH26 dyeing is darker than PKH2, calculates about 50 μ of as many as that can dye from mucomembranous surface.The degree of depth of this label also passs in time and becomes more and more shallow, and disappears after 32 hours.Measure as sentencing BrdU at about 60 μ, surperficial mucomembranous cell needed time that comes off is 32 hours, is 16 hours when about 40 μ sentence PKH2 dyeing, and is 32 hours when about 50 μ sentence PKH26 dyeing.It is believed that not being both of exfoliation rates because the dye level difference of each label.Embodiment 3
Carry out vitro examination, different with the exfoliation rates of normal people's colon cell and gastric cells to determine whether passing through to differentiate human colon cancer cell with stomach cancer cell, come from normal mucosa, to distinguish the cancer damage.The gained result shows that human colon carcinoma and gastric cancer can diagnose by measuring exfoliation rates.
Be sprayed on the target site by the cell marking compositions that direct observation will comprise PKH2 and PKH26 by means of endoscope, excise disease injury and this damage normal structure on every side then.Through refrigerated sample,, and measure exfoliation rates with fluorescence microscope with existing of discriminating PKH2 or PKH26.For preparation is used for the stomach of this method, at first use to comprise pronase 20000 units and the solution of 1g sodium bicarbonate in 80ml water is eliminated gastric mucus.After the removing, carry out normal colonoscopy, wherein by means of endoscope by direct observation with PKH2 and PKH26 solution spraying on damage location and peripheral region.
In colon, carry out colonoscopy, and prepare the affected areas of after normal colonoscopy, excising, the wherein water that uses about 137g Polyethylene Glycol and 2-4 to rise.The patient is forbidden food, medicine, other might influence cytokinetic medicament such as steroid drugs, aspirin and ethanol, checks first after 4 days, and then carries out following inspection.
Because the case of being studied relates to early-stage cancer, thus carry out endoscope's excision (biopsy), with the excision damage location.Part through excision is kept in the liquid nitrogen immediately.With cryostat tissue is cut into the sample of 5 μ m, forms two samples thus.Behind the freezing sample of preparation, fix one of them sample immediately, and form the sample of no dyeing, whether there are PKH2 or PKH26 to differentiate.Second sample stuck-at-0% in buffered formalin 3 minutes used haematoxylin-eosin dyeing then.On the painted sample of haematoxylin-eosin, carry out damage.Use fluorescence microscope and the wave filter that is used for FITC to differentiate the existence of PKH2, and differentiate the existence of PKH26 with fluorescence microscope and the wave filter that is used for rhodamine.The patient 1
Patient 1 is 60 years old man, suffers from early gastric cancer (grand swollen type, IIa type) at pyloric antrum, and diameter is 20mm.After using pronase 20000 units and the solution of 1g sodium bicarbonate in 80ml water to carry out the gastric mucus removing, carry out normal splanchnoscopy.After using endoscope's naked eyes to confirm that damage exists, spray the PKH2 solution of 10ml immediately, with the dyeing damage.Behind splanchnoscopy, 4 days the time, use to divest biopsy method excision IIa type early gastric cancer.Use said method freezing tissue sample, and check with microscope.From the image of H-E dyeing tissue, distinguishing the residing mucosa in tangible adenocarcinoma zone is normal mucosa on every side.With fluorescence microscope and after using the FITC wave filter to observe cancerous area, find that the surface of cancerous issue is 30 μ by the painted degree of depth of PKH2.
On the contrary, though identify intestinal tissue distortion and pyloric gland in the normal cell around, do not detect PKH2 fluorescence.Therefore, lost owing to coming off by the painted normal mucosa epithelial cell of PKH2, and do not had by the painted cancerous cell of PKH2.The patient 2
Patient 2 is male of 63 years old, suffers from rectal cancer (grand swollen I type rectal cancer), and diameter is 10mm.After knowing feces with Polyethylene Glycol, carry out colonoscopy.After using endoscope's naked eyes to confirm that damage exists, spray the PKH26 solution of 10ml immediately, with dyeing damage and peripheral region.After colonoscopy 4 days the time, excision damage under endoscope's direct observation.Use the method identical to prepare resection organization with above-mentioned patient 1.The painted tissue of H-E demonstrates the tangible adenocarcinoma of difference and is confined on the mucosa, and fluorescence microscopy finds that the surface of cancerous issue is by the degree of depth of PKH26 dyeing to 60 μ.Also find to be come off by the painted epithelial cell of PKH26 in the mucosa around.Therefore, come off by the painted normal mucosa of PKH26, and still existed by the painted cancer mucosa of PKH26.So, can find out significantly, between the exfoliation rates of cancer mucosa and normal mucous membrane of colon, there are differences.
In mucous membrane of colon, the zone of proliferative cell comprises following 2/3rds of body of gland.When external the measurement, the persistent period in S stage is 9-20 hour, and total cycle time is between 24-48 hour.According to the data of experiment in vitro, the cell quantity in the large intestine in S stage (perhaps label index LI.) changes between 12-25%.Go out from these numerical computations, the replacement time of this tissue is 4-8 days.Research provides usually than short S persistent period in stage (7.2-11.2 hour) and lower label index numerical value (1.5-17%) in the body.Therefore, the estimated value of total cell time can provide than the bigger numerical value that obtains in the body (external is 77.2-129.9 hour, is 24-48 hour in the body).
Foregoing description and accompanying drawing only are to be used to the preferred embodiment that realizes the object of the invention, feature and advantage is described, and do not mean that scope of the present invention just is limited to this.In marrow of the present invention and scope, also can carry out various improvement.
Claims (17)
1, the method for the abnormal cell exfoliation rates of the ripe superficial epithelium cell of detection homoiothermic animal in the body, it may further comprise the steps:
The ripe superficial epithelium cell of the target site of the described animal of labelling, then
Whether this position of monitoring exists label after above-mentioned markers step.
2, the method for claim 1, wherein described cell cyanine dye labelling.
3, method as claimed in claim 2, wherein, described cyanine dye is the following formula person:
Wherein:
Y is the methylene that oxygen, sulfur, methylene or alkyl replace;
M is 0-3; And
N is 12-22.
4, method as claimed in claim 2, wherein, described cyanine dye is following formula person:
Or
5, the method for claim 1, wherein described position is a mucomembranous surface.
6, the method for claim 1, wherein described position mucomembranous surface that is stomach.
7, the method for claim 1, wherein described position mucomembranous surface that is colon.
8, the method for claim 1, wherein after described markers step, observe the marker concentrations variation at described position, detect cell detachment thus at the time place of preliminary election.
9, method as claimed in claim 8, wherein, described label is a cyanine dye, and described position is exposed to exciting light, detects the fluorescence intensity that obtains thus then, observes the variation of marker concentrations thus.
10, method as claimed in claim 9, wherein, the described light wavelength that excites is about 490-504nm or for about 551-567nm.
11, the method for claim 1, wherein directly marking composition is applied on the described position, comes the described epithelial cell of labelling thus.
12, the method for claim 1, wherein marking composition is administered orally in described homoiothermic animal, comes the described epithelial cell of labelling thus.
13, the method for claim 1, wherein with the marking composition rectally in described homoiothermic animal, come the described epithelial cell of labelling thus.
14, the method for the exfoliation rates of ripe superficial epithelium cell on a kind of mucomembranous surface of assessing homoiothermic animal, it comprises: the marking composition that will comprise the label that can detect is applied on the ripe superficial epithelium cell of target site, observes the speed that label is lost from described target site then.
15, method as claimed in claim 14, it removes mucous step from target site before being included in and using marking composition.
16, method as claimed in claim 14, wherein, described label is a cyanine dye.
17, method as claimed in claim 16, wherein, described cyanine dye is the following formula person:
Wherein:
Y is the methylene that oxygen, sulfur, methylene or alkyl replace;
M is 0-3; And
N is 12-22.
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