CN106635995A - Circulating tumor cell negative enrichment method - Google Patents
Circulating tumor cell negative enrichment method Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, and in particular relates to a circulating tumor cell negative enrichment method. The method comprises the following steps that pretreatment is carried out on a blood sample by adopting density gradient centrifugation on the basis of a lymphocyte separating tube, so that the operating step of separating leukocytes is simplified, the enrichment time of the circulating tumor cell is shortened, and the whole operation is competed within 2.5h; the number of the left leukocytes is 30-50 % of that of original leukocytes of the blood sample, self-made negative enrichment immunomagnetic beads CD45 are adopted for marking the leukocytes and removed, so that the used number of the immunomagnetic beads is reduced, and the cost for negative enrichment of the circulating tumor cells is greatly saved. The lymphocyte separating tube is adopted for carrying out pretreatment on the blood sample, the negative enrichment method of the circulating tumor cell is improved, the stability of the enrichment operation is effectively improved, and the recovery ratio of the circulating tumor cell achieves 85% or above. The circulating tumor cell negative enrichment method further has the advantage of high sensitivity, and the circulating tumor cells can be stably detected within a few samples.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of circulating tumor cell feminine gender enrichment method.
Background technology
Circulating tumor cell (circulating tumor cells, CTC) refers to spontaneous or because operation of diagnosis and treatment is by solid tumor
It is the main cause that DISTANT METASTASES IN occurs in malignant tumor patient or MET discharges into the tumour cell of Peripheral Circulation.According to
Statistics, transfer is the cause of death of the tumor patient more than 90%.In recent years, it is continuous with some basic subject correlation techniques
Improve, CTC detect as a kind of new non-invasive diagnostic instrument, compare tradition tissue biopsy have it is noninvasive, can repeatedly obtain
Take, can the advantage such as real-time monitoring, therefore detect that CTC is visually referred to as " liquid biopsy ", with important clinical research and should
With value, it has also become the focus of clinical research.Existing a large amount of clinical researches have confirmed：By the inspection to circulating tumor cell
Survey, can effectively carry out early diagnosing kinds of tumors, Index for diagnosis, monitoring postoperative recurrence, individualized treatment, chemotherapeutic
The rapid evaluation of thing, monitoring of drug resistance etc..
A large amount of clinical researches of nearly 10 years show that detection CTC quantity contributes to colon cancer, liver cancer, breast cancer, prostate cancer
The prognosis evaluation of patient；It is past when Epithelial and stromal conversion (EMT), overexpression epithelial cell adhesion molecule (EpCAM) occurs in CTC
It is good toward prompting tumor patient prognosis,
Clinical research discovery, the cancer patient of early detection, 90% can cure.But, due to conventional detection technology
Limitation, the crowd of early detection cancer only occupies the minority, and China's early stage (I phase) cancer patient accounts for overall patient's number 5%-
10%, 70% patient belongs to late period discovery, 5 years survival rates only 10%～30% of this kind of patient.The World Health Organization early in
Just point out within 2006 the principle of preventing and treating three of cancer：Early discovery, early diagnosis, early treatment, it is seen then that be the discovery that the pass of prevention and control of cancer as early as possible
Key.But in the conventional detection means of tumour, such as CT or nuclear magnetic resonance check compare the diagnosing tumor less than 5 millimeters
Difficulty, is all middle and advanced stage after often finding to make a definite diagnosis such as cancer of pancreas, lung cancer.It has now been found that many situations of the tumour at 1 millimeter
Under circulating tumor cell (circulating tumor cells, CTC) can have been found in blood.Therefore, early detection
Judgement of the micrometastasis not only to tumor recurrence and prognosis is significant, and also has very big value to guiding clinical treatment.And
The discovery of clinical tumor at present and diagnosis are still highly dependent on the inspection of iconography and blood serum tumor markers, it is difficult to early detection
The transfer or recurrence of tumour, it is also difficult to reflect curative effect in time.Therefore, existing tumour routine screening, diagnostic method can not meet and face
Bed demand, it is clinical in the urgent need to high, the easy to operate screening for cancer of safe and accurate property, diagnosis new method.
Circulating tumor cell is present in blood with relatively low quantity in blood, and generally requiring could effective detection after enrichment
And analysis.Cell category in blood is various, absolutely accurately to identify the tumour cell in blood, is not easy to.CTC is swollen
It is extremely rare in knurl peripheral blood in patients, about 1～10/mL, and all hematocrite concentrations are 10 in adult normal's peripheral blood9/ mL,
The concentration of leucocyte is 4～10 × 106/mL.Far below the scarcity of other types cell quantity in blood CTC is caused in blood
It is extremely challenging in detection.
CTC detections generally comprise CTC enrichments and CTC identifies two parts, generally will be by big to remove the step of enrichment
Part normal cell, then identifies again the really CTC of the cell for obtaining.The enrichment method of CTC is broadly divided into physical method and life
The big class of thing chemical method two.The identification of CTC is mainly analyzed identification by cytomorphology or nucleic acid molecules level.
The enrichment method of CTC is broadly divided into physical method and the big class of biochemical method two.Physical is based on tumour cell
With normal cell physical property, the such as difference of size, morphotropism, density, dielectricity, using external force field such as magnetic field, fluid
, the effect of electric field etc. carries out separating trap.Biochemical process often relies on the antigen of surface of cell membrane and separates with being coupled at
Antibody on medium combines, and reaches the purpose of separating trap.
Being applied to the beneficiation technologies of CTC at this stage mainly includes：Based on the centrifugal process of cell density, based on cell volume
Filter membrane filtration method (isolation by size of epithelial tumour cells, ISET), immunomagnetic beads method
(magnetic activated cell sorting, MACS), CTC-Chip microfluidic chip technologies etc..
Existing density-gradient centrifugation method specificity is low, sensitiveness is low, the rate of recovery is low, stability is low, not only easily causes swollen
Oncocyte is lost, and depends on many factors such as the characteristic of tumour cell, time, the temperature of centrifugation, separates as single CTC
Means future development potentiality are extremely limited.ISET methods are although easy to operate, cheap, flux is high, can be while mass disposal
Sample, take it is shorter, but exist lack specificity, tumour cell be missed (≤8um) possibility, be also impossible to become future
Development trend and trend.
Most widely used of CTC previous cycle tumor cell enrichments be positive enrichment method, including immunomagnetic beads method sun
Property enrichment, the positive enrichment of micro-fluidic chip method.
Micro-fluidic chip is applied to the deficiency of rare cell sorting and is mainly manifested in that time-consuming, effect is poor, cytoactive is received
Damage three aspects.Additionally, the two indices that flux and purity often condition each other in most of chips, flux rises then anticipates
Taste purity decline, and vice versa, how to take into account both be the development of micro-fluidic sorting technology another challenge.Finally, it is contemplated that
The cell for capturing may contain different hypotypes, or the different growth phases of cell lineage, follow-up phenotype analytical pair
Clinical diagnosis is significant, and this proposes higher requirement to the activity for capturing cell.But, micro-structural in microchannel
Extruding, the flow field of high shear stream, additional electric field, sound field etc. all can cause certain damage to the activity of cell, to follow-up point
Analysis causes puzzlement.
The Chinese patent of Application No. 201110161224.7 discloses a kind of reagent of quantitative analysis circulating tumor cell
And kit, the probe of circulating tumor cell is detected by appropriate design, so as to Flow Cytometry can be coordinated, obtain circulation swollen
The measurement result of oncocyte.
Immunomagnetic beads method is the CTC separation methods being most widely used at present, positive concentration method is primarily referred to as, with simplicity
The high advantage of easy, separation purity, but the rate of recovery is low, there is the defect of missing inspection also clearly.At present, substantial amounts of immunomagnetic beads
The research of method CTC is separated according to the epithelium antigen of tumour, and thinks that EpCAM is current optimum tumor biomarker,
But there are some researches show the CTC of most of malignant tumours can lose EpCAM antigens.This means that this method is possible to meeting missing inspection
CTC with invasive.In fact, research finds that 134 kinds of different tumour cells of organization type only have 70% expression EpCAM,
But there is the otherness that expression intensity differs.At present, still without a kind of tumor biomarker of 100% specificity.
But circulating tumor cell positive enrichment method has CTC Specific markers shortage, monoclonal antibody screening and is stranded
It is difficult, the rate of recovery is low, the shortcomings of there is missing inspection (tumour cell of antibody None- identified is ignored), affect testing result.Due to CTC
Lack the mark of specificity, EpCAM (epithelial cell adhesion molecule) marked by magnetic bead is generally adopted at present and CTC is separated, be based on
The instruments such as the CellSearch that this principle grows up have been put into the market promotion.However, the method cannot detect low expression
The CTC of EpCAM is not expressed.Study and pointed out, separating CTC based on this principle will underestimate its quantity and lose with high turn
Shifting and the CTC subgroups of multiplication potentiality.One typical example is exactly, occur epithelial-mesenchymal conversion (EMT) CTC low expressions or
It is considered as one of the main reason for causing to shift that EpCAM subgroups are not expressed because having high migration force.
Compared with immunomagnetic beads positive enrichment method, CTC immunomagnetic beadses feminine gender enrichment method has more advantages.The skill
Art in conjunction with have CD45 monoclonal antibodies immunomagnetic beads specific binding leukocyte surface CD45 antigens (leucocyte is common
Antigen), magnetic mark is carried out to leucocyte, leucocyte is removed by externally-applied magnetic field, such that it is able to be independent of CTC surface markers
Thing, is enriched with out all subgroups of CTC, all types.CTC immunomagnetic beadses feminine gender beneficiation technologies are isolating all types
There is technical advantage, simultaneously because not relying on CTC surface markers, it is contemplated that can be used for the CTC of all types solid tumor on CTC
Enrichment.
Therefore, the separation method that exploitation does not rely on CTC surface markers is a technique direction with huge prospect.
On the other hand, the immunomagnetic beads method of negative enrichment strategy by effectively remove the leucocyte of CD45+ or the macrophage of CD61+,
Other cells of the peripheral bloods such as blood platelet and the rare epithelial cell of indirect enrichment, overcome in a way EpCAM caused by EMT positive
The defect of property enrichment strategy.Additionally, the circulating tumor cell obtained by negative enrichment strategy is also had not through any mark
The advantages such as note, high cytoactive.
In sum, although various CTC separation methods of existing Jing reports are each with unique detection advantage, but still exposure
Go out some problems, such as the circulating tumor cell rate of recovery not high (70%-80%), activated CTC, biomarker can not be obtained
Select difficulty, isolation technics complexity etc..
The content of the invention
The technical problem to be solved is：A kind of circulating tumor cell feminine gender enrichment method is provided, is solved existing
The circulating tumor cell rate of recovery that circulating tumor cell separation method is present is not high, can not to obtain activated circulating tumor thin
Born of the same parents, biomarker selects the complicated problem of difficult, isolation technics.
In order to solve above-mentioned technical problem, the technical solution used in the present invention is：A kind of negative enrichment of circulating tumor cell
Method, comprises the following steps：
S1：By density gradient separation liquid (Ficoll pague plus, GE Health) and tumor patient blood sample in lymph
Mix in cell Separating tube, add lavation buffer solution, carry out first time centrifugation；
S2：The baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred in the first centrifuge tube,
Second centrifugation is carried out, supernatant is sucked, erythrocyte cracked liquid is added, is stood；
S3：The first centrifuge tube after standing is carried out into third time centrifugation, supernatant is sucked, magnetic bead incubation buffering is added
Liquid, adds negative enrichment immunomagnetic beads, mixes, and is incubated 20-40min, and it is white that the negative enrichment immunomagnetic beads is combined with targeting
Cell surface broad-spectrum marker CD45；
S4：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and is stood, supernatant is fully transferred to
In second centrifuge tube, with lavation buffer solution the second centrifuge tube is rinsed；
S5：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, the second centrifuge tube supernatant is sucked, in the second centrifuge tube
It is applied on slide after adding cell fixer resuspended, dried overnight obtains dry circulating tumor cell.
The beneficial effects of the present invention is：Circulating tumor cell feminine gender enrichment method disclosed by the invention is thin using lymph
Density gradient centrifugation on the basis of born of the same parents' separating pipe carries out pre-treatment to blood sample, simplifies leucocyte separation application, shortens
The enrichment time of circulating tumor cell, whole operation can be completed in 2.5h；The quantity of leucocyte stayed Jing after processing is suitable
In the 30-50% of blood sample leucoblast quantity, the leucocyte to staying is marked using the negative enrichment immunomagnetic beads CD45 of self-control
Leucocyte is simultaneously removed, and reduces the consumption of immunomagnetic beads, greatlys save the cost of the negative enrichment of circulating tumor cell；The present invention
Pre-treatment is carried out to blood sample using separation of lymphocytes pipe, and circulating tumor cell feminine gender enrichment method is improved, effectively
Enrichment procedure stability is improve, the rate of recovery of circulating tumor cell reaches more than 85%；The present invention also has highly sensitive
Advantage, can stably detect circulating tumor cell in the few sample of quantity.
Description of the drawings
Fig. 1 for the specific embodiment of the invention a kind of circulating tumor cell feminine gender enrichment method and traditional density gradient from
The enriching and recovering rate block diagram of circulating tumor cell obtained in heart method；
Fig. 2 is thin to different type tumour for a kind of circulating tumor cell feminine gender enrichment method of the specific embodiment of the invention
The enriching and recovering rate block diagram of born of the same parents；
The SKOV3 cells that Fig. 3 is enriched with for a kind of circulating tumor cell feminine gender enrichment method of the specific embodiment of the invention
Identified by immunofluorescence result figure afterwards；
Fig. 4 is a kind of circulating tumor cell feminine gender enrichment method of the specific embodiment of the invention to varying number NCI-
The average recovery rate block diagram of H226 circulating tumor cells；
Fig. 5 is a kind of circulating tumor cell feminine gender enrichment method of the specific embodiment of the invention to varying number NCI-
The rate of recovery linear regression graph of H226 circulating tumor cells.
Specific embodiment
To describe the technology contents of the present invention in detail, being realized purpose and effect, below in conjunction with embodiment and coordinate attached
Figure is explained.
Abbreviation according to the present invention and Key Term are defined：
CTC：Circulating tumor cell；
CD45：LCA；
CK-FITC：Fluoresceinisothiocyanate anti-cytokeratin Ab；
CD45-PE：Phycoerythrin albumen coupling CD45 antibody；
DAPI：4', 6- diamidino -2-phenylindone.
The design of most critical of the present invention is：Blood sample is entered using the density gradient centrifugation on the basis of separation of lymphocytes pipe
Row pre-treatment, simplifies leucocyte separation application, shortens the enrichment time of circulating tumor cell, to the leucocyte for staying
Mark leucocyte and remove using the negative enrichment immunomagnetic beads CD45 of self-control, reduce the consumption of immunomagnetic beads, greatly save
The cost of the negative enrichment of circulating tumor cell.
A kind of circulating tumor cell feminine gender enrichment method, comprises the following steps：
S1：By density gradient separation liquid (Ficoll pague plus, GE Health) and tumor patient blood sample in lymph
Mix in cell Separating tube, add lavation buffer solution, carry out first time centrifugation；
S2：The baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred in the first centrifuge tube,
Second centrifugation is carried out, supernatant is sucked, erythrocyte cracked liquid is added, is stood；
S3：The first centrifuge tube after standing is carried out into third time centrifugation, supernatant is sucked, magnetic bead incubation buffering is added
Liquid, adds negative enrichment immunomagnetic beads, mixes, and is incubated 20-40min, and it is white that the negative enrichment immunomagnetic beads is combined with targeting
Cell surface broad-spectrum marker CD45；
S4：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and is stood, supernatant is fully transferred to
In second centrifuge tube, with lavation buffer solution the second centrifuge tube is rinsed；
S5：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, the second centrifuge tube supernatant is sucked, in the second centrifuge tube
It is applied on slide after adding cell fixer resuspended, dried overnight obtains dry circulating tumor cell.
The negative enrichment principle of the circulating tumor cell of the present invention：The invention discloses a kind of detection side of circulating tumor cell
Method, key step includes removing red blood cell and a large amount of neutrophil leucocytes using density gradient centrifugation, by erythrocyte splitting, enters one
Step removes residual red blood cells, by the negative enrichment immunomagnetic beads of the leucocyte for obtaining and self-control (targeting leukocyte surface wide spectrum mark
Thing CD45) it is incubated, Jing magnetic fields Adsorption leucocyte, the supernatant fraction not combined with magnetic bead is collected, smear after centrifugation is empty
Gas is dried film-making, and dry circulating tumor cell is obtained.
Above-mentioned circulating tumor cell feminine gender enrichment method, using the density gradient centrifugation pair on the basis of separation of lymphocytes pipe
Blood sample carries out pre-treatment, simplifies leucocyte separation application, shortens the enrichment time of circulating tumor cell, whole operation
Can complete in 2.5h；The quantity of leucocyte for staying equivalent to blood sample leucoblast quantity 30-50%, it is white to what is stayed
Cell is using the negative enrichment immunomagnetic beads CD45 mark leucocytes of self-control and removes, and reduces the consumption of immunomagnetic beads, saves significantly
The about cost of the negative enrichment of circulating tumor cell；The present invention carries out pre-treatment using separation of lymphocytes pipe to blood sample, and right
Circulating tumor cell feminine gender enrichment method is improved, and effectively increases enrichment procedure stability, the recovery of circulating tumor cell
Rate reaches more than 85%；The present invention also has highly sensitive advantage, and circulation can be stably detected in the few sample of quantity
Tumour cell.
Further, the S1 is specially：Density gradient separation liquid, brief centrifugation is added to make in separation of lymphocytes pipe
All ttom of pipe is arrived in centrifugation to density gradient separation liquid；Add 3mL's in the separation of lymphocytes pipe equipped with density gradient separation liquid
Tumor patient blood sample, and the lavation buffer solution more than 3mL is added, first time centrifugation is carried out, the condition of the first time centrifugation is
25℃、800g/15min。
Further, the S2 is specially：By the above-mentioned baffle plate upper strata in the separation of lymphocytes pipe of centrifugation for the first time
Solution is transferred in the first centrifuge tube, carries out second centrifugation, and the condition of second centrifugation is 25 DEG C, 300g/10min,
Supernatant is sucked, the erythrocyte cracked liquid of 5mL is added, 5min is stood.
Further, the S3 is specially：The first centrifuge tube after standing is carried out into third time centrifugation, the described 3rd
The condition of secondary centrifugation is 25 DEG C, 300g/10min, sucks supernatant, adds 5mL magnetic bead incubation buffers, adds 200uL negative
Enrichment immunomagnetic beads, piping and druming is mixed, and is mixed in 4 DEG C of slow speed verticals, is incubated 30min, and the negative enrichment immunomagnetic beads is combined with
Targeting leukocyte surface broad-spectrum marker CD45.
Further, the S4 is specially：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and is stood
5min, supernatant is fully transferred in the second centrifuge tube, and with lavation buffer solution the second centrifuge tube 2 times is rinsed.
Further, the S5 is specially：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, supernatant is sucked to residue
Volume is 100uL, in the second centrifuge tube add 100uL cell fixers, it is resuspended after be applied on slide, 25 DEG C are overnight done
It is dry, dry circulating tumor cell is obtained, the condition of the 4th centrifugation is 25 DEG C, 600g/5min.
Further, described circulating tumor cell feminine gender enrichment method also includes step：
S6：Using multiple-color immunofluorescence technology dry circulating tumor cell is carried out identifying and fluorescence is counted.
Further, the dyestuff that the multiple-color immunofluorescence technology is adopted is for CK-FITC, CD45-PE or DAPI.
Comparative example 1
A number of SiHa cells are mixed in healthy human peripheral blood, to contain circulating tumor thin to simulate cervical cancer patient
Born of the same parents' blood sample, in proportion dilution obtains 2 × 104Cell suspension, and under fluorescence microscope accurate metering and adjust to 100/
5uL, by cell suspension after counting 3mL peripheral bloods are mixed, and the peripheral blood sample containing 100 SiHa cells/3mL is obtained respectively, with
Above-mentioned blood sample as tumor patient blood sample, respectively according to following three kinds of blood pre-treatment scheme (A, erythrocyte splitting；B, density level bands
Degree centrifugation；C, erythrocyte splitting+density gradient centrifugation) process after, respectively according to embodiment 1 the step of be circulated tumour cell
Feminine gender enrichment；Red blood cell removal efficiency, leucocyte removal efficiency are evaluated.
Different blood pre-treatment scheme red blood cell removal efficiency, leucocyte removal efficiency are as shown in table 1.
Red blood cell removal efficiency, the leucocyte removal efficiency of the different blood pre-treatment schemes of table 1.
| Blood pre-treatment scheme | Red blood cell removal efficiency | Leucocyte removal efficiency |
| Erythrocyte splitting | ～98～99.5% | >99.5% |
| Density gradient centrifugation | >99.5% | ～99.7-99.9% |
| Erythrocyte splitting+density gradient centrifugation | >99.99% | ～99.7-99.9% |
Found out by table 1, using the pretreatment mode of erythrocyte splitting+density gradient centrifugation, red blood cell removal efficiency and white
Cell removal efficiency highest.
Embodiment 1
A kind of circulating tumor cell feminine gender enrichment method, comprises the following steps：
S1：Density gradient separation liquid, brief centrifugation is added to make density gradient separation liquid whole in separation of lymphocytes pipe
Ttom of pipe is centrifuged；The tumor patient blood sample of 3mL is added in the separation of lymphocytes pipe equipped with density gradient separation liquid, and is added
Lavation buffer solution more than 3mL, carries out first time centrifugation, and the condition of the first time centrifugation is 25 DEG C, 800g/15min；
S2：The above-mentioned baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred to into the first centrifuge tube
In, second centrifugation is carried out, the condition of second centrifugation is 25 DEG C, 300g/10min, sucks supernatant, and addition 5mL's is red
Cell pyrolysis liquid, stands 5min；
S3：The first centrifuge tube after standing is carried out into third time centrifugation, the condition of the third time centrifugation is 25 DEG C,
300g/10min, sucks supernatant, adds 5mL magnetic bead incubation buffers, adds the negative enrichment immunomagnetic beadses of 200uL, and piping and druming is mixed
It is even, mix in 4 DEG C of slow speed verticals, 30min is incubated, the negative enrichment immunomagnetic beads is combined with targeting leukocyte surface wide spectrum mark
Note thing CD45；
S4：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and stands 5min, supernatant is all turned
In moving to the second centrifuge tube, with lavation buffer solution the second centrifuge tube 2 times is rinsed；
S5：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, it is 100uL to suck supernatant to residual volume, second
It is applied on slide after adding 100uL cell fixers resuspended in centrifuge tube, 25 DEG C of dried overnights obtain dry circulating tumor
Cell, the condition of the 4th centrifugation is 25 DEG C, 600g/5min；
S6：Using multiple-color immunofluorescence technology dry circulating tumor cell is carried out identifying and fluorescence is counted, it is described many
The dyestuff that color immunofluorescence technique is adopted is for CK-FITC, CD45-PE or DAPI.
Embodiment 2
Fig. 1 is refer to, a number of SiHa cells are mixed in healthy human peripheral blood, contained with simulating cervical cancer patient
Circulating tumor cell blood sample, and evaluate enrichment effect of the set up circulating tumor cell feminine gender enrichment method to circulating tumor cell
Rate, concrete monitoring step is as follows：
1st, will cultivate and digest the SiHa cells of results carries out green fluorescence and marks in advance with Celltracker Green,
Dilution in proportion obtains 2 × 104Cell suspension, and accurate metering and adjust to 100/5uL under fluorescence microscope；
2nd, cell suspension mixes 3mL peripheral bloods after counting, and the peripheral blood containing 100 SiHa cells/3mL is obtained respectively
Sample；
3rd, the peripheral blood sample containing 100 SiHa cells/3mL is circulated respectively respectively according to following two schemes
The negative enrichment of tumour cell；
A, traditional density gradient centrifugal process；B, separation of lymphocytes pipe density gradient centrifugation (improvement project)
4th, peripheral blood sample is enriched with and detects using the circulating tumor feminine gender enrichment method of embodiment 1, and to circulating tumor
The bioaccumulation efficiency of cell feminine gender enrichment method is evaluated.
As can be seen from Figure 1, obtained with reference to circulating tumor feminine gender enrichment method using separation of lymphocytes pipe density gradient centrifugation
The rate of recovery of SiHa cells reaches 89%, is significantly higher than the rate of recovery 76% that traditional density gradient centrifugal process obtains SiHa cells.
Embodiment 3
Fig. 2 and Fig. 3 is refer to, a number of oophoroma SKOV-3, cervical carcinoma SiHa are mixed in healthy human peripheral blood
Cell, breast cancer SKBR3 cell, it is different swollen whether the circulating tumor cell feminine gender enrichment method that evaluation this programme is related to is applied to
The circulating tumor cell detection of knurl type, concrete detecting step is as follows：
1st, in the present embodiment with 100 oophoromas SKOV-3, Intrauterine device bleeding, the incorporation of breast cancer SKBR3 cell
The method of healthy human peripheral blood, to simulate different tumor patients circulating tumor cell blood sample is contained, and will cultivate and digest results
Different tumor type cells Celltracker Green carry out green fluorescence and mark in advance, and dilution in proportion obtains different dense
The cell suspension of degree, and the accurate metering under fluorescence microscope.
2nd, cell suspension mixes 3mL peripheral bloods after counting, and the peripheral blood of 100 different type tumour cell/3mL is obtained
Sample.
3rd, it is enriched with and detects peripheral blood sample using the circulating tumor feminine gender enrichment method of embodiment 1.
From figure 2, it is seen that the richness using circulating tumor according to the present invention feminine gender enrichment method to different type tumour cell
The collection rate of recovery is more than 85%.
Embodiment 4
Fig. 4 is refer to, varying number NCI-H226 cells is mixed in healthy human peripheral blood, with it as blood sample, using reality
The circulating tumor cell enrichment method for applying example 1 evaluates circulating tumor cell bioaccumulation efficiency, and concrete detecting step is as follows：
1st, the method in the present embodiment healthy human peripheral blood being mixed with non-small cell lung cancer NCI-H226 cells, to simulate
Patients with lung cancer contains circulating tumor cell blood sample.To cultivate and digest the NCI-H226 cell Celltracker of results
Green carries out green fluorescence and marks in advance, and dilution in proportion obtains the cell suspension of variable concentrations, and the essence under fluorescence microscope
Really count.
2. cell suspension mixes 3ml peripheral bloods after counting, and is obtained contains 6,9,15,30,90,300 NCI- respectively
The peripheral blood sample of H226 cells/3ml.
3., using circulating tumor cell disclosed in this invention feminine gender enrichment kit, peripheral blood is enriched with using said method
The green cells of CellTracker+ after being air-dried film-making, are counted by the circulating tumor cell in sample, swollen to circulation
The bioaccumulation efficiency of oncocyte feminine gender enrichment method, sensitivity are evaluated.
As can be seen from Figure 4, using circulating tumor cell according to the present invention feminine gender enrichment method to the NCI- without quantity
The peripheral blood sample of H226 cells is circulated the rate of recovery of tumor cell enrichment and reaches higher level, wherein, containing 9,
15th, the average recovery rate of the peripheral blood sample of 30 NCI-H226 cells/3ml is more than 85%, as can be seen from Figure 5, different numbers
The recycled in its entirety rate that amount circulating tumor cell is integrally enriched with is 77.66%, illustrates circulating tumor cell enrichment according to the present invention
Method has higher sensitivity.
In sum, the circulating tumor cell feminine gender enrichment method that the present invention is provided, using separation of lymphocytes pipe basis
On density gradient centrifugation pre-treatment is carried out to blood sample, simplify leucocyte separation application, shorten circulating tumor cell
Enrichment time, whole operation can complete in 2.5h；The quantity of leucocyte for staying is equivalent to blood sample leucoblast quantity
30-50%, to the leucocyte that stays is using the negative enrichment immunomagnetic beads CD45 mark leucocytes of self-control and removes, reduces and exempt from
The consumption of epidemic disease magnetic bead, greatlys save the cost of the negative enrichment of circulating tumor cell；The present invention adopts separation of lymphocytes pipe pair
Blood sample carries out pre-treatment, and circulating tumor cell feminine gender enrichment method is improved, and effectively increases enrichment procedure stability,
The rate of recovery of circulating tumor cell reaches more than 85%；The present invention also has highly sensitive advantage, can be few in quantity
Circulating tumor cell is stably detected in sample.
Embodiments of the invention are the foregoing is only, the scope of the claims of the present invention is not thereby limited, it is every using this
The equivalents that bright specification and accompanying drawing content are made, or the technical field of correlation is directly or indirectly used in, include in the same manner
In the scope of patent protection of the present invention.
Claims (9)
1. a kind of circulating tumor cell feminine gender enrichment method, it is characterised in that comprise the following steps：
S1：Density gradient separation liquid and tumor patient blood sample are mixed in separation of lymphocytes pipe, lavation buffer solution is added, is entered
Row is centrifuged for the first time；
S2：The baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred in the first centrifuge tube, is carried out
It is centrifuged for second, sucks supernatant, add erythrocyte cracked liquid, stands；
S3：The first centrifuge tube after standing is carried out into third time centrifugation, supernatant is sucked, magnetic bead incubation buffer is added, then
Negative enrichment immunomagnetic beads is added, is mixed, be incubated 20-40min, the negative enrichment immunomagnetic beads is combined with targeting leucocyte table
Face broad-spectrum marker CD45；
S4：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and is stood, supernatant is fully transferred to into second
In centrifuge tube, with lavation buffer solution the second centrifuge tube is rinsed；
S5：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, the second centrifuge tube supernatant is sucked, is added in the second centrifuge tube
It is applied to after cell fixer is resuspended on slide, dried overnight, obtains dry circulating tumor cell.
2. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that also including step：
S6：Using multiple-color immunofluorescence technology dry circulating tumor cell is carried out identifying and fluorescence is counted.
3. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that the S1 is specially：
Density gradient separation liquid is added in separation of lymphocytes pipe, brief centrifugation makes density gradient separation liquid, and all ttom of pipe is arrived in centrifugation；
The tumor patient blood sample of 3mL is added in separation of lymphocytes pipe equipped with density gradient separation liquid, and adds the washing more than 3mL
Buffer solution, carries out first time centrifugation, and the condition of the first time centrifugation is 25 DEG C, 800g/15min.
4. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that the S2 is specially：Will
The above-mentioned baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred in the first centrifuge tube, carries out second
Centrifugation, the condition of second centrifugation is 25 DEG C, 300g/10min, sucks supernatant, adds the erythrocyte cracked liquid of 5mL, quiet
Put 5min.
5. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that the S3 is specially：Will
The first centrifuge tube after standing carries out third time centrifugation, and the condition of the third time centrifugation is 25 DEG C, 300g/10min, is inhaled
Supernatant is removed, 5mL magnetic bead incubation buffers are added, the negative enrichment immunomagnetic beadses of 200uL are added, piping and druming is mixed, hung down in 4 DEG C of low speed
It is straight to mix, 30min is incubated, the negative enrichment immunomagnetic beads is combined with targeting leukocyte surface broad-spectrum marker CD45.
6. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that the S4 is specially：Will
Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and stands 5min, and supernatant is fully transferred to into the second centrifuge tube
In, rinse the second centrifuge tube 2 times with lavation buffer solution.
7. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that the S5 is specially：Will
Above-mentioned second centrifuge tube carries out the 4th centrifugation, and it is 100uL to suck supernatant to residual volume, is added in the second centrifuge tube
100uL cell fixers, it is resuspended after be applied on slide, 25 DEG C of dried overnights obtain dry circulating tumor cell, described
The condition of four centrifugations is 25 DEG C, 600g/5min.
8. circulating tumor cell according to claim 2 feminine gender enrichment method, it is characterised in that the multiple-color immunofluorescence
The dyestuff that technology is adopted is for CK-FITC, CD45-PE or DAPI.
9. circulating tumor cell according to claim 1 feminine gender enrichment method, it is characterised in that comprise the following steps：
S1：Density gradient separation liquid, brief centrifugation is added density gradient separation liquid is all centrifuged in separation of lymphocytes pipe
To ttom of pipe；The tumor patient blood sample of 3mL, and addition is added to be more than in the separation of lymphocytes pipe equipped with density gradient separation liquid
The lavation buffer solution of 3mL, carries out first time centrifugation, and the condition of the first time centrifugation is 25 DEG C, 800g/15min；
S2：The above-mentioned baffle plate upper solution in the separation of lymphocytes pipe of centrifugation for the first time is transferred in the first centrifuge tube,
Second centrifugation is carried out, the condition of second centrifugation is 25 DEG C, 300g/10min, sucks supernatant, adds the red blood cell of 5mL
Lysate, stands 5min；
S3：The first centrifuge tube after standing is carried out into third time centrifugation, the condition of the third time centrifugation is 25 DEG C,
300g/10min, sucks supernatant, adds 5mL magnetic bead incubation buffers, adds the negative enrichment immunomagnetic beadses of 200uL, and piping and druming is mixed
It is even, mix in 4 DEG C of slow speed verticals, 30min is incubated, the negative enrichment immunomagnetic beads is combined with targeting leukocyte surface wide spectrum mark
Note thing CD45；
S4：Above-mentioned the first centrifuge tube after incubation is placed on magnetic frame and stands 5min, supernatant is fully transferred to
In second centrifuge tube, with lavation buffer solution the second centrifuge tube 2 times is rinsed；
S5：Above-mentioned second centrifuge tube is carried out into the 4th centrifugation, it is 100uL to suck supernatant to residual volume, in the second centrifugation
It is applied on slide after adding 100uL cell fixers resuspended in pipe, 25 DEG C of dried overnights obtain dry circulating tumor thin
Born of the same parents, the condition of the 4th centrifugation is 25 DEG C, 600g/5min；
S6：Using multiple-color immunofluorescence technology dry circulating tumor cell is carried out identifying and fluorescence is counted, the polychrome is exempted from
The dyestuff that epidemic disease fluorescent technique is adopted is for CK-FITC, CD45-PE or DAPI.
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