CN101914486A - Novel cervical exfoliated cell separation method - Google Patents
Novel cervical exfoliated cell separation method Download PDFInfo
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- CN101914486A CN101914486A CN2010102768633A CN201010276863A CN101914486A CN 101914486 A CN101914486 A CN 101914486A CN 2010102768633 A CN2010102768633 A CN 2010102768633A CN 201010276863 A CN201010276863 A CN 201010276863A CN 101914486 A CN101914486 A CN 101914486A
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Abstract
The invention discloses a novel cervical exfoliated cell separation method, which is characterized by: placing cervical exfoliated cells in phosphate buffer solution (PBS) and uniformly mixing by whirling to obtain cell suspension; adding the cell suspension into mucilage separating solution; centrifuging, removing supernate, adding the phosphate buffer solution (PBS) again and re-suspending sediment to obtain re-suspended cell suspension; and adding the re-suspended cell suspension into neutral granular cell separating solution Ficoll, centrifuging, removing supernate and obtaining the settlement which is pure cells after centrifugation. The method of the invention greatly purify clinically extracted cervical exfoliated cell specimens, adopts simple and convenient operation steps, is suitable for large-scale cell separation with stable and reliable effect, can obtain high-purity target cells, has high actual use value and a promising market prospect.
Description
Technical field
The present invention relates to a kind of method of cervical exfoliated cell separation.
Background technology
Cervical cancer is one of gynaecology's common cancer, and sickness rate is only second to mammary cancer, occupies second of women's malignant tumour, and its mortality ratio occupies the first place of gynecologic malignant tumor.In recent years, cervical cancer patient is with the speed increment in every year 2%～4%, meanwhile, with respect to developed country, in developing country, because uterine neck screening imperfection, the sickness rate of cervical cancer is 6 times of developed country, and be infiltrating carcinoma when wherein 80% patient makes a definite diagnosis, and cervical cancer exist long reversed a precancerous lesion phase, is the key of preventing and treating cervical cancer so the early discovery precancerous lesion is carried out therapeutic intervention.
The main method of present stage examination cervical cancer comprises the inspection of cervical smear exfoliative cytology, the inspection of epithelium of cervix uteri liquid based cytology etc., but also there is certain limitation, in above-mentioned two kinds of cytolgical examinations, being seen precancerous lesion is the comprehensive sign from optimum to a transitional stage of virulent in the smear, comprise: 1, the keratinocyte number in the cervical smear continues to increase, the cell of smear is the phenomenon of oestrogenic hormon long lasting effect, and the angling degree is equivalent to preovulatory highest level in mid-term in cycle; 2, have special-shaped or the precocious keratinocyte; 3, cell is huge, or cell is tiny; 4, dyskaryosis is huge, and is active and immature, and nuclear chromatin is dense to be dyed, and particle is often arranged in the endochylema; Karyolobism and multinuclear that 5, each layer squamous cell arranged; 6, bottom hyperplasia, nuclear often has dizzy in week; 7, serious reserve cell hyperplasia.
The liquid based cytology of epithelium of cervix uteri comparatively widely that adopts is clinically checked in the treating processes of sample sequencing at present, cell is only handled through flash liberation and is just directly entered the film-making step, though also separated mucus, hemocyte, inflammatory cell etc. to a certain extent, but after the film-making, visible part mucus still under the mirror, especially the neutrophil leucocyte of inflammatory, influence paracytic recall rate, false negative, false positive and dissatisfied smear rate are higher, and the clinical auxiliary diagnosis information comparatively accurately that provides can not be provided.
Summary of the invention
The present invention is for avoiding above-mentioned existing in prior technology weak point, a kind of method of novel cervical exfoliated cell separation is provided, and makes the quality of smear in the hope of raising, helps laboratory physician to draw accurate conclusion, and auxiliary clinicist's diagnosis.
Technical solution problem of the present invention adopts following technical scheme:
The characteristics of the method for cervical exfoliated cell separation of the present invention are to operate as follows:
A, get cervical exfoliated cell in the PBS phosphoric acid buffer, the vortex mixing gets cell suspension;
B, step a gained cell suspension is added in the mucus parting liquid; Through centrifugal, add the PBS phosphoric acid buffer once more after abandoning supernatant liquor, resuspended throw out obtains resuspended cell suspension;
C, cell suspension that step b gained is resuspended add among the neutrophil leucocyte parting liquid Ficoll, through centrifugal, abandon supernatant liquor, the gained throw out is pure cell.
The characteristics of the method for cervical exfoliated cell separation of the present invention also are:
The concentration of volume percent of the PBS phosphoric acid buffer among the described step a is 10%, and consumption is 5ml;
Mucus parting liquid consumption among the described step b is 3ml; Be used for resuspended sedimentary PBS phosphoric acid buffer by volume the concentration of per-cent be 10%, consumption is 3ml;
The consumption of neutrophil leucocyte parting liquid Ficoll among the described step c is 3ml.
The characteristics of the method for cervical exfoliated cell separation of the present invention also are:
The centrifugal condition is among the described step b: 100-4000g, and the time is 10 seconds-10 minutes; Centrifugal condition among the described step c is: 200-10000g, the time is 10-30 minute.
The centrifugal condition is among the described step b: 200g, and the time is 2 minutes; Centrifugal condition among the described step c is 2000g, and the time is 20 minutes.
Described each step is all carried out at normal temperatures.
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, the present invention has improved the quality of making smear greatly by two-step approach separation and purification cell, improves the isolating sharpness of clinical cervical exfoliated cell sample;
2, the inventive method is suitable for large-scale cellular segregation, its effect stability, reliable;
3, can be directly used in researchs such as genome extraction, karyotyping, cell cultures, cell transgenosis, cell induction differentiation, cell therapy with the inventive method isolated cells, also can be used for the purposes such as separation of the preparation of density gradient separation liquid, different mass molecule or compound simultaneously;
4, the inventive method has higher actual application value in fields such as cytobiology, functional genomics, clinical treatment, and market outlook are wide; Especially primary dcreening operation and the control for cervical cancer precancerosis provides strong directive significance, and clinical value is far-reaching.
Description of drawings
Fig. 1 a, Fig. 1 b are for using the learn a skill cervical exfoliated cell morphology sample of LCT preparation of conventional liquid basal cell;
Fig. 2 a, Fig. 2 b are the cervical exfoliated cell morphology sample with the inventive method preparation;
Fig. 3 utilizes the picture of the isolated human cervical cancer 1 cast-off cells of the inventive method when vitro culture.
Embodiment
The method of the cervical exfoliated cell separation in the present embodiment is operated as follows:
1, use the cervical exfoliated cell specific brush, brush gently is pressed on the uterine neck mouth, and good luck direction rotation 2-3 circle is brushed with this and to be got cervical exfoliated cell, and it is 10% PBS phosphoric acid buffer that brush is got concentration of volume percent that thing places 5ml, and the vortex mixing gets cell suspension;
2, add step 1 gained cell suspension in the mucus parting liquid of 3ml along tube wall; Through centrifugal, abandon supernatant liquor after, add the 3ml concentration of volume percent in the throw out after centrifugal and be 10% PBS phosphoric acid buffer, resuspended throw out obtains resuspended cell suspension;
3, cell suspension that step 2 gained is resuspended adds among the 3ml neutrophil leucocyte parting liquid Ficoll along tube wall, through centrifugal, abandon supernatant liquor, the throw out after centrifugal is the pure cell of gained.
More than each step all carry out at normal temperatures; The centrifugal condition is in the step 2: 100-4000g is preferably 200g; Centrifugation time is 10 seconds-10 minutes, and preferred centrifugation time is 2 minutes; Centrifugal condition in the step 3 is: 200-10000g, be preferably 2000g, and centrifugation time is 10-30 minute, and preferred centrifugation time is 20 minutes, and used mucus parting liquid and Ficoll liquid are commercially available cellular segregation liquid.
Use the present embodiment method, finish in seven days time the lock out operation of 210 pairs of cervical exfoliated cell samples nearly, the mucus 95% or more, blood and neutrophil leucocyte etc. mix composition can separatedly be removed, and has obtained the effect of being satisfied with.
Fig. 1 a, 1b are for using the learn a skill cervical exfoliated cell morphology sample of LCT preparation of conventional liquid basal cell, and cast-off cells are covered by more neutrophil leucocyte, make to read the sheet sharpness and descend; As seen compare with Fig. 1 b two figure by Fig. 1 a, by the conventional liquid basal cell sample effect instability that LCT makes that learns a skill.
Fig. 2 a, Fig. 2 b are that the separation clearance rate of mucus, blood and neutrophil leucocyte reaches more than 95%, and effect stability is reliable with the cervical exfoliated cell morphology sample of the inventive method preparation.
To the separating obtained uterine cervix squamous cell of present embodiment with conventional method (being BSA-DMEM nutrient solution+penicillin, Streptomycin sulphate 100,000 U/ml+2.5 μ g/ml amphotericin Bs) carry out former be commissioned to train foster, use microscopic examination one day after at the cell bed board, observations as shown in Figure 3.Observations showed cell growth conditions after separation is good, has integrity on the morphology, the mucus, blood and the neutrophil leucocyte that exist in the cervical exfoliated cell sample are effectively removed, increased the former foster purity of being commissioned to train, improve the quality of subsequent experimental.
Claims (5)
1. the method for a novel cervical exfoliated cell separation is characterized in that operating as follows:
A, get cervical exfoliated cell in the PBS phosphoric acid buffer, the vortex mixing gets cell suspension;
B, step a gained cell suspension is added in the mucus parting liquid; Through centrifugal, add the PBS phosphoric acid buffer once more after abandoning supernatant liquor, resuspended throw out obtains resuspended cell suspension;
C, cell suspension that step b gained is resuspended add among the neutrophil leucocyte parting liquid Ficoll, through centrifugal, abandon supernatant liquor, the gained throw out is pure cell.
2. the method for cervical exfoliated cell separation according to claim 1 is characterized in that:
The concentration of volume percent of the PBS phosphoric acid buffer among the described step a is 10%, and consumption is 5ml;
Mucus parting liquid consumption among the described step b is 3ml; Be used for resuspended sedimentary PBS phosphoric acid buffer by volume the concentration of per-cent be 10%, consumption is 3ml;
The consumption of neutrophil leucocyte parting liquid Ficoll among the described step c is 3ml.
3. the method for cervical exfoliated cell separation according to claim 1 is characterized in that the centrifugal condition is among the described step b: 100-4000g, and the time is 10 seconds-10 minutes; Centrifugal condition among the described step c is: 200-10000g, the time is 10-30 minute.
4. the method for cervical exfoliated cell separation according to claim 3 is characterized in that the centrifugal condition is among the described step b: 200g, and the time is 2 minutes; Centrifugal condition among the described step c is 2000g, and the time is 20 minutes.
5. the method for cervical exfoliated cell separation according to claim 1 is characterized in that described each step all carries out at normal temperatures.
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| CN2010102768633A CN101914486A (en) | 2010-09-09 | 2010-09-09 | Novel cervical exfoliated cell separation method |
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| CN2010102768633A CN101914486A (en) | 2010-09-09 | 2010-09-09 | Novel cervical exfoliated cell separation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN112378837A (en) * | 2020-09-15 | 2021-02-19 | 深圳市华中生物药械有限公司 | Cervical exfoliated cell detection method and related device |
-
2010
- 2010-09-09 CN CN2010102768633A patent/CN101914486A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN106047795B (en) * | 2016-06-22 | 2019-06-14 | 杭州海世嘉生物科技有限公司 | A kind of sample rate separating liquid and preparation method thereof |
| CN112378837A (en) * | 2020-09-15 | 2021-02-19 | 深圳市华中生物药械有限公司 | Cervical exfoliated cell detection method and related device |
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Application publication date: 20101215 |