CN104483472B - A kind of diagnostic reagent for cervical carcinogenesis cell detection and preparation method thereof - Google Patents
A kind of diagnostic reagent for cervical carcinogenesis cell detection and preparation method thereof Download PDFInfo
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- 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
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- 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 kind of diagnostic reagent for cervical carcinogenesis cell detection and preparation method thereof.This diagnostic reagent is made up of following content of component: glucose 10 parts, 0.5 part, folic acid, methylene blue 0.4 part, Lyeopene 0.8 part, 3mol/L aqueous sodium hydroxide 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: glucose and Lyeopene, acetic acid to be dissolved in dimethyl sulfoxide (DMSO) respectively; Folic acid joins the dimethyl sulfoxide (DMSO) being dissolved with glucose and Lyeopene after joining and dissolving in aqueous sodium hydroxide solution again, add methylene blue stirring and dissolving again, add the dimethyl sulfoxide (DMSO) being dissolved with acetic acid again, after mixing, add dithiothreitol (DTT) and Sodium Methyl Hydroxybenzoate.Diagnostic reagent of the present invention is easy to use, picks during use with cotton swab, its sensitivity and stability high, diagnosis recall rate reach 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 cancer 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 cancer new cases.But cervical cancer 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 cancer.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 disturbance 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 differential diagnosis 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 precision 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 cancer mainly carries out detecting based on large-scale instrument and equipment mainly contains: uterine neck ThinPrep cytologic test, HPV Viral diagnosis, vaginoscope detect.These detection technique sensitivity and specificity higher, but plant and instrument is expensive, needs veteran operator and reviewer, and there is certain false positive and false negative, examination period 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 cells.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 expression level and cell response to oxidative stress, the color reaction by methylene blue realizes examination cervical cancer 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 application, 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 epithelium sick cell and preparation technology and cotton swab " of 103808551A discloses a kind of compound acetic acid white solution diagnostic reagent detected for epithelium 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 epithelium 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 cancer 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, Lyeopene 0.8 part, 3mol/L aqueous sodium hydroxide 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 study 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, in fact folacin coupled mixture 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.Stablizer add the stability considerably increasing this diagnostic reagent, the normal temperature shelf time reaches 2 years.
Because the compositions such as the methylene blue in this diagnostic reagent, glucose, Lyeopene 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, Lyeopene are added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L aqueous sodium hydroxide solution, afterwards folic acid is joined in aqueous sodium hydroxide 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 resistance of oxidation after 34 days for 45 DEG C to reduce less than 10%), far away higher than other analogous 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 shelf 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 distinguishes the colour-change after scraping HaCaT cell and Hela cell with the cotton swab infiltrating diagnostic reagent of the present invention.
Fig. 2 is the relation of cervical cell in various degree between pathology and diagnostic reagent colour-change of the present invention.
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, Lyeopene 0.8 part, 3mol/L aqueous sodium hydroxide 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, Lyeopene 0.8 part are added in 70 parts of dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L aqueous sodium hydroxide solution 0.5 part, afterwards 0.5 part of folic acid is joined in aqueous sodium hydroxide solution, stirring and dissolving;
(3) 4 parts of acetic acid are added in 20 parts of dimethyl sulfoxide (DMSO), 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 standardized solution (0.025mol/L immediately, diluted by 0.1mol/LI2 standardized solution) be titrated to light yellow, colour-fastly in 30s be terminal, the I2 standardized solution volume that record consumes, by the resistance of oxidation relatively carrying out calculation sample of the I2 standardized 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 epithelium 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 staging life, the oxidation-resistance of each sample is measured by iodimetric method, result shows: reagent disclosed in 103808551A, the decline of resistance of oxidation is there is after 2 to 3 days, more than 90% is reduced in one week, diagnostic reagent prepared by embodiment 1 is deposited resistance of oxidation 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 grownup'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 coeruleum have high affinity, and the oxidation-reduction quality of methylenum coeruleum makes methylenum coeruleum 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 colour-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 colour-change in different time, the results are shown in Table 1.
103808551A: in " diagnostic reagent detected for epithelium 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), Lyeopene is 1.4 parts;
Reagent 4: prepare same embodiment 1, not containing dithiothreitol (DTT), Lyeopene is 2 parts;
Reagent 5: prepare same embodiment 1, not containing dithiothreitol (DTT), Lyeopene 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 color 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 application 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, observe the colour-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-greenish colour, tissue is without acetic acid albefaction district simultaneously, then show there is inflammation; If cotton swab becomes green or blue-greenish colour, tissue has acetic acid albefaction district simultaneously, be then indicated as CIN-1 or pointed condyloma; 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), vaginoscope, multiple punch biopsies simultaneously.Result is as follows: meet 18 examples, 2 examples are false positive, accuracy 90%, susceptibility 90%; Innocent 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-greenish colour 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-greenish colour, 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 gold standard evaluating this detection method, wherein, in cervicitis and CIN-1 pathology, this reagent should present green or blue-greenish colour, 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, Lyeopene 0.8 part, 3mol/L aqueous sodium hydroxide 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, Lyeopene are added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L aqueous sodium hydroxide solution, afterwards folic acid is joined in aqueous sodium hydroxide solution, stirring and dissolving;
(3) acetic acid is added in appropriate dimethyl sulfoxide (DMSO), stirring and dissolving;
(4) joined by the liquid that step (2) obtains in the liquid that step (1) obtains, limit edged stirs, then adds methylene blue, and Keep agitation makes it mix and reacts completely;
(5) joined by the liquid that step (3) obtains in the liquid that step (4) obtains, 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, Lyeopene 0.8 part are added in 70 parts of dimethyl sulfoxide (DMSO), stirring and dissolving;
(2) prepare 3mol/L aqueous sodium hydroxide solution 0.5 part, afterwards 0.5 part of folic acid is joined in aqueous sodium hydroxide solution, stirring and dissolving;
(3) 4 parts of acetic acid are added in 20 parts of dimethyl sulfoxide (DMSO), stirring and dissolving;
(4) joined by the liquid that step (2) obtains in the liquid that step (1) obtains, limit edged stirs, then adds 0.4 part of methylene blue, Keep agitation 5min, mixing;
(5) joined by the liquid that step (3) obtains in the liquid that step (4) obtains, 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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| CN103808551A (en) * | 2013-10-15 | 2014-05-21 | 深圳市华中生物药械有限公司 | Diagnosis reagent for detecting pathological cells of epithelial tissue and preparation technology thereof as well as cotton swab |
| 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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| NZ540800A (en) * | 2000-06-30 | 2007-06-29 | Zila Inc | Methylene blue diagnostic agent and in vivo diagnostic methods for detection of epithelial cancer |
| JP4004385B2 (en) * | 2002-11-07 | 2007-11-07 | 独立行政法人科学技術振興機構 | Cancer detection method by double staining |
| KR20110027823A (en) * | 2004-03-24 | 2011-03-16 | 트리패스 이미징, 인코포레이티드 | Methods and compositions for the detection of cervical disease |
| CN101608228A (en) * | 2008-06-20 | 2009-12-23 | 上海主健生物工程有限公司 | Kit for genetic detection of cervical cancer |
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| CN105510321A (en) * | 2011-12-29 | 2016-04-20 | 闫文广 | Detection agent composition for epithelial tissue tumor cells and preparing method thereof |
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