CN104483472A - Diagnostic reagent for cervical cancer cell detection and preparation method thereof - Google Patents
Diagnostic reagent for cervical cancer cell detection and preparation method thereof Download PDFInfo
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- CN104483472A CN104483472A CN201410804606.0A CN201410804606A CN104483472A CN 104483472 A CN104483472 A CN 104483472A CN 201410804606 A CN201410804606 A CN 201410804606A CN 104483472 A CN104483472 A CN 104483472A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention discloses a diagnostic reagent for cervical cancer cell detection and a preparation method thereof. The diagnostic reagent comprises components in parts by mass as follows: 10 parts of glucose, 0.5 parts of folic acid, 0.4 parts of methylene blue, 0.8 parts of lycopene, 0.5 parts of a 3mol/L sodium hydroxide aqueous solution, 90 parts of dimethyl sulfoxide, 4 parts of acetic acid, 0.01 parts of dithiothreitol and 0.05 parts of sodium methyl parahydroxybenzoate. The preparation method comprises steps as follows: the glucose, the lycopene and acetic acid are dissolved in dimethyl sulfoxiderespectively; the folic acid is added to the sodium hydroxide aqueous solution for dissolution and then added to the dimethyl sulfoxide with theglucose and the lycopene dissolved, then methylene blue is added, stirred and dissolved, the dimethyl sulfoxide with theacetic acid dissolved is added, and the dithiothreitol and the sodium methyl parahydroxybenzoate are added after even mixing. The diagnostic reagent is convenient to use and high in sensitivity and stability, cotton swabs are dipped in the diagnostic reagent during usage, and the diagnostic detection rate is up to 90%.
Description
Technical field
The present invention relates to cancerous tumor cell diagnostic reagent, be specifically related to a kind of diagnostic reagent for cervical carcinogenesis cell detection and preparation method thereof.
Background technology
Cervical carcinoma is one of women's common cancer, is worldwide number two in woman cancer, occupies China's female reproductive system malignant tumour the first.Domestic annual newly-increased cases of cervical cancer about 13.5 ten thousand people, accounts for 1/5 of world's cervical carcinoma new cases.But cervical carcinoma is again the clear and definite and cancer that can prevent and treat of unique cause of disease in all cancers of the current mankind, by the anti-cancer examination of specification, Timeliness coverage and treatment precancerous lesion, just can block it and develop into cervical carcinoma.Therefore how can improve sensitivity, the specificity of cervical carcinoma screening, can meet again simple, the economic requirement of patient, be the very necessary and work of arduous task of gynaecology medical personnel one.
In the world, similar tumor screening technology, as tumor marker check up and appraisal etc., is main flow examination means.Tumor marker check up and appraisal refers to biochemical property by tumour cell and metabolic disorder thereof, namely tumour patient body fluid, get rid of thing and tissue in occur matter or amount on change material to carry out tumor screening, as carcinomebryonic antigen, tumour antigen, sugar antigen etc.Tumor marker is mainly used in the prediction etc. of discovery to primary tumo(u)r, the screening of tumor high-risk, the antidiastole of optimum and malignant tumour, the judgement of tumor development degree, the O&A of oncotherapy effect and tumor recurrence and prognosis clinically.But, similar tumor screening technology (as tumor marker check up and appraisal etc.) sense cycle is long, needs exact instrument and special operative technique, especially needs operator to have rich experience, cost is high, is 100 to 1000 times of diagnostic reagent of the present invention.
The technology that domestic current diagnosis cervical carcinoma mainly carries out detecting based on large-scale instrument and equipment mainly contains: uterine neck ThinPrep cytologic test, HPV Viral diagnosis, gynecatoptron detect.These detection technique sensitivity and specificity higher, but instrument and equipment is expensive, needs veteran operating personnel and reviewer, and there is certain false positive and false negative, proof cycle is long, and cost is high.
At present clinically a simple chromoscopy cancerous tissue cell be the method become based on acetic acid albefaction reaction diagnosing cervical, by certain density acetic acid solution is applied in cervical epithelial tissue, institute's spreader portion bit organization is observed after about 3-5 minute, if occur, acetic acid albefaction is reacted, then may have cancer cell.Show that the sensitivity of observation method of naked eye diagnosing cervical is 53.8% according to the study, specificity is 92%, positive predictive value 38.9%, negative predictive value 95.4%, coincidence rate 88.7%.The method is simple, without any need for specific apparatus and special operative technique, cheap, and result meets the requirement of current low cost, the wide cervical carcinoma screening strategy covered, and has higher practical value in economically less developed region.
There is a kind of technology---folacin receptor mediated uterine neck specific stain of diagnosis cervical lesions newly in the market.This technology make use of two features of tumour cell, and namely strong in cell surface folacin receptor high expressed and cell response to oxidative stress, the chromogenic reaction by methylene blue realizes examination cervical carcinoma fast and accurately and precancerous lesion.Because this is emerging technology, technical know-how is very high, so the producer of domestic production similar-type products is little.Existing market is not very stable based on the product result of use of this technology and product stability, limit its market development and clinical practice, this is because this acetic acid white solution composition is more, high technology is needed to its composition, content, preparation, the sensitivity of these factors and this product, stability is directly related, and the cell category that the solution that different proportionings obtains is suitable for is also different.
Publication number is that the Chinese patent application " diagnostic reagent detected for epithelial tissue sick cell and preparation technology and cotton swab " of 103808551A discloses a kind of compound acetic acid white solution diagnostic reagent detected for epithelial tissue sick cell, containing 0.03-4% methylene blue, the fructose of 1-20%, the glucose of 1-20%, the Vc of 0.5-5%, the folic acid of 0.1-5%, the distilled water of 5-90%, the dimethyl sulfoxide (DMSO) of 5-90%, 1-7% acetic acid in this solution reagent of 100mL.But it is not strong that the epithelial tissue sick cell of this diagnostic reagent to privileged sites detects specificity, also there is the problem of product stability difference.
Summary of the invention
The object of the present invention is to provide a kind of diagnostic reagent for cervical carcinogenesis cell detection, this diagnostic reagent is based on redox reaction, and the colour developing by methylene blue realizes examination cervical carcinoma fast and accurately and precancerous lesion.Thus doctor can be made to obtain the firsthand information of pathology, help doctor's clinical decision, as intended examining and diagnosis and treatment scheme.
The present invention also aims to the preparation method that the above-mentioned diagnostic reagent for cervical carcinogenesis cell detection is provided.
Object of the present invention is achieved through the following technical solutions:
A kind of diagnostic reagent for cervical carcinogenesis cell detection, be made up of following content of component: glucose 10 parts, 0.5 part, folic acid, methylene blue 0.4 part, lycopene 0.8 part, 3mol/L sodium hydrate aqueous solution 0.5 part, dimethyl sulfoxide (DMSO) 90 parts, acetic acid 4 parts, dithiothreitol (DTT) 0.01 part, Sodium Methyl Hydroxybenzoate 0.05 part.
Clinical research proves, folacin receptor is in most of tumor cell surface overexpression, and existence in normal cell is little, so folic acid composite can as tumour-specific target reporter molecule, for the Diagnosis and Treat of tumour.And diagnostic reagent of the present invention mainly utilizes folic acid as the instrumentality of color, because only have folic acid composite could as tumour-specific target reporter molecule, and this needs folic acid to be effectively connected with effector molecules, in fact folacin coupled compound is prepared very difficult, successfully few at present, some are also only only limitted to laboratory stage, and entering clinically also needs the time.
Glucose in diagnostic reagent of the present invention, as carbohydrate reductive agent, has the advantages such as cheap, convenience of drawing materials, reductibility are strong.Acetic acid in reagent provides acid environment; In addition, the use of acetic acid contributes to methylene blue quick penetration cell, thus with the high oxygen pressure reaction solution of cell.Adding of methylene blue, make traditional acetic acid white solution can locate abnormal epithelial tissue pathology better.Stabilizing agent add the stability considerably increasing this diagnostic reagent, the normal temperature holding time reaches 2 years.
Because the compositions such as the methylene blue in this diagnostic reagent, glucose, lycopene relate to redox reaction, in the process of actual production, the oxygen easily and in air reacts, and therefore inserts order to the priority of composition and there are certain requirements.The preparation method of diagnostic reagent of the present invention, comprises the steps:
(1) glucose, lycopene are added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L sodium hydrate aqueous solution, afterwards folic acid is joined in sodium hydrate aqueous solution, stirring and dissolving;
(3) acetic acid is added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(4) joined by the liquid in (2) in (1) liquid, limit edged stirs, then adds methylene blue, and Keep agitation makes it mix and reacts completely;
(5) joined by the liquid in (3) in the liquid that (4) obtain, limit edged stirs, and Keep agitation makes it mix and reacts completely;
(6) after above-mentioned solution mixes, dithiothreitol (DTT) and Sodium Methyl Hydroxybenzoate is added.
Each step of the diagnostic reagent described in preparation is carried out all at normal temperatures and pressures.
Diagnostic reagent normal temperature for cervical carcinogenesis cell detection sealing of the present invention is stored in brown bottle, picks during use with cotton swab.
The present invention has the following advantages and effect relative to prior art tool:
(1) the present invention is used for the diagnostic reagent sensitivity (94.43%) of cervical carcinogenesis cell detection and stability (depositing oxidation resistance after 34 days for 45 DEG C to reduce less than 10%), far away higher than other similar products, diagnoses recall rate to reach 90%.
(2) stability of diagnostic reagent of the present invention is good, and sealing is stored in the brown bottle of 5mL, and the normal temperature holding time reaches 2 years.
(3) diagnostic reagent of the present invention is easy to use, picks during use with cotton swab.
Diagnostic reagent for cervical carcinogenesis cell detection of the present invention overcomes the deficiency of like product in the past, decreases working doctor intensity, easy and simple to handle, has good potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is that the color after using the cotton swab difference scraping HaCaT cell of infiltration diagnostic reagent of the present invention and Hela cell changes.
Fig. 2 be cervical cell in various degree pathology and diagnostic reagent color of the present invention change between relation.
Embodiment
Below in conjunction with specific embodiment, further detailed description is done to the present invention.Should be understood that the following examples are only not used in the scope limiting this technology for illustration of the present invention.
Embodiment 1 is for the preparation of the diagnostic reagent of cervical carcinogenesis cell detection
The diagnostic reagent that the present invention is used for cervical carcinogenesis cell detection is made up of following content of component: glucose 10 parts, 0.5 part, folic acid, methylene blue 0.4 part, lycopene 0.8 part, 3mol/L sodium hydrate aqueous solution 0.5 part, dimethyl sulfoxide (DMSO) 90 parts, acetic acid 4 parts, dithiothreitol (DTT) 0.01 part, Sodium Methyl Hydroxybenzoate 0.05 part.Its preparation method comprises the steps:
(1) glucose 10 parts, lycopene 0.8 part are added in 70 parts of dimethyl sulfoxide (DMSO)s, stirring and dissolving;
(2) prepare 3mol/L sodium hydrate aqueous solution 0.5 part, afterwards 0.5 part of folic acid is joined in sodium hydrate aqueous solution, stirring and dissolving;
(3) 4 parts of acetic acid are added in 20 parts of dimethyl sulfoxide (DMSO)s, stirring and dissolving;
(4) slowly at the uniform velocity joined by the liquid in (2) in (1) liquid, limit edged stirs, then adds 0.4 part of methylene blue, Keep agitation 5min, mixing;
(5) slowly at the uniform velocity joined by the liquid in (3) in the liquid that (4) obtain, limit edged stirs, Keep agitation 5min, and mixing, until this diagnostic reagent is light yellowish brown.
(6) in (5) solution, add 0.01 part of dithiothreitol (DTT), 0.05 part of Sodium Methyl Hydroxybenzoate, obtains the diagnostic reagent for cervical carcinogenesis cell detection, seals stand-by.
Embodiment 2 is for the mensuration of the stability of diagnostic reagent of cervical carcinogenesis cell detection
Iodine direct titrimetric method: preparation accurately draws 3mL testing sample solution, fully mix with 3mL indicator amidin (10g/L), use I2 standard solution (0.025mol/L immediately, by 0.1mol/L I2 standard solution dilute) be titrated to light yellow, colour-fastly in 30s be terminal, the I2 standard solution volume that record consumes, by the oxidation resistance relatively carrying out calculation sample of the I2 standard solution volume of consumption.
The diagnostic reagent being used for cervical carcinogenesis cell detection prepared by embodiment 1, reagent sample in " diagnostic reagent detected for epithelial tissue sick cell and preparation technology and cotton swab (103808551A) " patent is (containing methylene blue 0.37% in this solution reagent of 100mL, fructose 4.69%, glucose 9.38%, Vc 1.00%, folic acid 0.43%, distilled water 5.00%, dimethyl sulfoxide (DMSO) 76.33%, acetic acid 2.80%) deposit in 45 DEG C, between storage period, the inoxidizability of each sample is measured by iodimetric method, result shows: reagent disclosed in 103808551A, the decline of oxidation resistance is there is after 2 to 3 days, more than 90% is reduced in one week, diagnostic reagent prepared by embodiment 1 is deposited oxidation resistance after 34 days and is reduced less than 10%.
Embodiment 3 for the diagnostic reagent of cervical carcinogenesis cell detection to the detection of cell carcinogenesis
Hela clone comes from into human cervical cancer 1, be a kind of can the cancerous cell line of infinite multiplication, be the model study clone of the tumour cell in biology and medical research.HaCaT clone is the immortalized cell line coming from adult's skins, shows normal differentiation state, for the regulation and control studying human epithelial cells provide a kind of desirable model.Although HaCaT clone presents obvious immortalization, but still do not possess tumour cell characteristic, can as Normocellular model.
Tumour cell and methylenum careuleum have high affinity, and the oxidation-reduction quality of methylenum careuleum makes methylenum careuleum in the redox system of tumour cell, have different metachromasia spectrum.
(1) specificity is good
With cotton swab scraping HaCaT cell and the Hela cell respectively infiltrating diagnostic reagent prepared by embodiment 1, observe color after 5min, the cotton swab of scraping HaCaT cell is almost without color change, and the cotton swab of scraping Hela cell is in green (Fig. 1).Illustrate that this diagnostic reagent is highly sensitive, specificity is good.
(2) detection time is short
With cotton swab scraping Hela cell (A) and the HaCaT cell (B) respectively infiltrating following different reagent, observe the color change in different time, the results are shown in Table 1.
103808551A: in " diagnostic reagent detected for epithelial tissue sick cell and preparation technology and cotton swab " patent in this solution reagent of reagent: 100mL containing methylene blue 0.37%, fructose 4.69%, glucose 9.38%, Vc 1.00%, folic acid 0.43%, distilled water 5.00%, dimethyl sulfoxide (DMSO) 76.33%, acetic acid 2.80%.
Reagent 1: diagnostic reagent prepared by embodiment 1;
Reagent 2: prepare same embodiment 1, not containing dithiothreitol (DTT);
Reagent 3: prepare same embodiment 1, not containing dithiothreitol (DTT), lycopene is 1.4 parts;
Reagent 4: prepare same embodiment 1, not containing dithiothreitol (DTT), lycopene is 2 parts;
Reagent 5: prepare same embodiment 1, not containing dithiothreitol (DTT), lycopene is 2.6 parts.
Table 1
The wherein quantity representative color reaction depth degree of "+", the quantity representative of "-" does not have color reaction to occur.
As can be seen from the above results, within 15min, diagnostic reagent chromogenic reaction prepared by embodiment 1, close to completing, forming notable difference with other reagent, illustrating that this diagnostic reagent is short for detection time.
Embodiment 4 is for the clinical practice of the diagnostic reagent of cervical carcinogenesis cell detection
The using method of diagnostic reagent prepared by embodiment 1 is as follows: pick this diagnostic reagent with cotton swab, then cotton swab is stretched into vagina, makes cotton swab head be coated with 3 to 5 circles at cervical surface, can exert oneself a little, take out cotton swab, after 3 to 5 minutes, observes the color change of smearing position.If cotton swab is without variable color, spreader portion bit organization is without acetic acid albefaction reaction zone simultaneously, then show that tissue is without pathology; If cotton swab is without variable color, spreader portion hyte is woven with acetic acid white reaction zone simultaneously, then show not get rid of tissue without pathology; If cotton swab becomes green or blue-green, tissue is without acetic acid albefaction district simultaneously, then show there is inflammation; If cotton swab becomes green or blue-green, tissue has acetic acid albefaction district simultaneously, be then indicated as CIN-1 or condyloma acuminatum; If cotton swab becomes black, be then indicated as CIN-2 or more pathology (Fig. 2).Under the prerequisite soliciting patient's agreement, get living tissue in the region of tissue generation acetic acid albefaction reaction and send pathologic finding analysis.
(1) this diagnostic reagent is carried out Clinical practice in Shenzhen hospital, People's Hospital, Hubei Province, Central-South hospital of Hubei Province, verify in conjunction with the accuracy of means to this diagnostic reagent such as Liquid-based thin-layer cytology test (TCT), gynecatoptron, multiple punch biopsies simultaneously.Result is as follows: meet 18 examples, 2 examples are false positive, accuracy 90%, susceptibility 90%; Benign tumour 30 example: 2 examples are carcinoma in situ, accuracy 93.3%, susceptibility 93.3%; Inflammation 10 example: accuracy 100%, susceptibility 100%.
(2) choose the women that 10000, Wufeng County meets inclusion criteria sign Informed Consent Form and participate in examination examination, first phase detects 795 routine patient's sample altogether, the results are shown in Table 2.
Table 2
Check result shows, and there is a large amount of Cervicitis and above patient in this population sample, wherein yellowish-brown and green proportion are 36.10%, and blue-green accounts for 40.62%, and black accounts for 23.27%.In green, what ratio was the highest is Cervicitis (79.45%), and all the other are CIN-1, CIN-2, CIN-3; What in blue-green, ratio was the highest is CIN-1 (67.49%), and what in black, ratio was the highest is CIN-2 (57.84%).Pathological examination is the goldstandard evaluating this detection method, wherein, in cervicitis and CIN-1 pathology, this reagent should present green or blue-green, is 98.19%, with pathological examination matching degree in CIN-2 or more pathology, this reagent should present black, is 98.72% with pathological examination matching degree.
Claims (3)
1. the diagnostic reagent for cervical carcinogenesis cell detection, it is characterized in that: be made up of following content of component: glucose 10 parts, 0.5 part, folic acid, methylene blue 0.4 part, lycopene 0.8 part, 3mol/L sodium hydrate aqueous solution 0.5 part, dimethyl sulfoxide (DMSO) 90 parts, acetic acid 4 parts, dithiothreitol (DTT) 0.01 part, Sodium Methyl Hydroxybenzoate 0.05 part.
2. the preparation method of the diagnostic reagent for cervical carcinogenesis cell detection according to claim 1, is characterized in that comprising the steps:
(1) glucose, lycopene are added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L sodium hydrate aqueous solution, afterwards folic acid is joined in sodium hydrate aqueous solution, stirring and dissolving;
(3) acetic acid is added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(4) joined by the liquid in (2) in (1) liquid, limit edged stirs, then adds methylene blue, and Keep agitation makes it mix and reacts completely;
(5) joined by the liquid in (3) in the liquid that (4) obtain, limit edged stirs, and Keep agitation makes it mix and reacts completely;
(6) after above-mentioned solution mixes, dithiothreitol (DTT) and Sodium Methyl Hydroxybenzoate is added.
3. the preparation method of the diagnostic reagent for cervical carcinogenesis cell detection according to claim 2, is characterized in that comprising the steps:
(1) glucose 10 parts, lycopene 0.8 part are added in 70 parts of dimethyl sulfoxide (DMSO)s, stirring and dissolving;
(2) prepare 3mol/L sodium hydrate aqueous solution 0.5 part, afterwards 0.5 part of folic acid is joined in sodium hydrate aqueous solution, stirring and dissolving;
(3) 4 parts of acetic acid are added in 20 parts of dimethyl sulfoxide (DMSO)s, stirring and dissolving;
(4) joined by the liquid in (2) in (1) liquid, limit edged stirs, then adds 0.4 part of methylene blue, Keep agitation 5min, mixing;
(5) joined by the liquid in (3) in the liquid that (4) obtain, limit edged stirs, Keep agitation 5min, mixing, then adds 0.01 part of dithiothreitol (DTT) and 0.05 part of Sodium Methyl Hydroxybenzoate.
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| CN108318478A (en) * | 2017-01-18 | 2018-07-24 | 四川天莆高科医疗器械有限公司 | A kind of detection reagent and its preparation method and application of human epithelia's cancer cell |
| CN108760733A (en) * | 2018-06-13 | 2018-11-06 | 迦娜生物科技（武汉）有限公司 | A kind of cervical carcinoma urine detection reagent and its application |
| CN109187145A (en) * | 2018-08-23 | 2019-01-11 | 杭州元研细胞生物科技有限公司 | A kind of diagnosis dyeing liquor and preparation method thereof early sieved for cervical carcinoma |
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| CN109187145A (en) * | 2018-08-23 | 2019-01-11 | 杭州元研细胞生物科技有限公司 | A kind of diagnosis dyeing liquor and preparation method thereof early sieved for cervical carcinoma |
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