CN103175817B - Method for detecting activity of antitumor drug on inhibiting adhesiveness between tumor cell and blood platelet - Google Patents
Method for detecting activity of antitumor drug on inhibiting adhesiveness between tumor cell and blood platelet Download PDFInfo
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Abstract
The invention relates to the field of biological chemistry, and discloses a method for detecting activity of an antitumor drug on inhibiting the adhesiveness between a tumor cell and a blood platelet. The method comprises the following steps of: labeling the blood platelets through fluorescently-labeled probes; respectively hatching the blood platelets with tumor cells without being treated by the antitumor drug and the tumor cells treated by the antitumor drug; on a flow cytometry, detecting the fluorescence rate of the tumor cells by utilizing an FL1 channel, so as to obtain the fluorescence rate serving as a control group and the fluorescence rate serving as a drug treated group; and analyzing the significant difference between the fluorescent rate in the control group and the fluorescent rate in the drug treated group, so as to obtain the detection result. According to the method, the tumor cells in a suspension state and the non-activated blood platelets are adopted for detecting, the process is in line with the adhesiveness process of the real tumor cells and the blood platelets; the cell density is adjusted by counting the cells before carrying out an adhesiveness test, so that the consistency of the density among the cells treated by the drug and that of cells in a contrast group can be ensured; and the quantity of the cells is detected under the setting of flow type analysis software, and therefore, the consistency of the quantity of the cells detected in different pre-treating groups can be ensured, the influence of different pre-treatments to the quantity of the tumor cells is avoided, and the accuracy of the result is improved.
Description
Technical field
The present invention relates to biochemical field, be specifically related to a kind of method detecting the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction.
Background technology
The Infiltration and metastasis of tumour is a complicated multi-step multifactorial process, comprises tumor cell secretion enzymolysis surrounding substrate, dissociates, enter blood circulation, survive and pass vascular wall in blood flow from primary tumor, then form a series of processes of metastasis.Tumour cell in blood circulation inevitably with the interaction between component in blood, and the immunocyte in blood can remove tumour cell, but blood platelet then can promote the transfer of tumour cell.Studies have found that, tumor patient often raises with blood platelet, and Antiplatelet therapy can Tumor suppression transfer.
Tumour cell and the effect of hematoblastic interaction in metastases process are mainly reflected in following several respects: 1, platelet adhesion reaction is in tumor cell surface, block the mechanical damage caused by wall shear stress, and help tumour cell to escape the attack of body immune system, thus promote that tumour cell is survived in blood flow; 2, adhere to the blood platelet of tumor cell surface, vascular endothelial cell can be adhered to simultaneously and cause vascular endothelial cell to shrink, thus promote that tumour cell stops in the blood vessel of remote part, and pass blood vessel arrival metastasis site; 3, blood platelet can discharge the cell factor such as platelet derived growth factor, vascular endothelial growth factor, can stimulate the growth of tumour cell, and the formation of tumour blood confession.
Therefore, detect antineoplastic to the influence degree of tumour cell and hematoblastic adhesive capacity for evaluate its whether have inhibition tumor cell shift and invasive ability most important.Method conventional at present mainly, by tumour cell kind in Tissue Culture Plate, treat that cell is completely adherent, and with antineoplastic process to be detected after merging completely, the blood platelet that fluorescence or isotope labeling are crossed is added in culture plate, after hatching certain hour, the blood platelet not adhering to tumour cell is removed in rinsing, after adding lysate cell lysis and blood platelet, detect fluorescence in culture plate or isotope level whether to decrease relative to without the fluorescence of antineoplastic process or isotope level, reflect whether medicine to be detected has the activity of inhibition tumor cell and platelet adhesion reaction, thus evaluate its effect in inhibition tumor cell Infiltration and metastasis.
But this detection technique of order has some limitations, be mainly reflected in following several respects: the real processes (1) cannot simulating tumour cell and platelet adhesion reaction, tumour cell is in adhered state, and in blood flow environment in vivo, tumour cell is in suspended state, and therefore the method can not react real tumour cell and platelet adhesion reaction process; (2) experimental implementation is complicated, need in advance by cell kind in culture plate, and make Growth of Cells to merging completely, experimental period is longer; (3) be difficult to quantitative detection, some antineoplastic can kill tumour cell or make tumour cell cannot be adherent, so cause the tumor cell number of drug treating group and control group inconsistent, truly cannot reflect whether be the effect reached due to inhibition tumor cell and platelet adhesion reaction; (4) adopt radioactive isotope to check, cause radiation injury and environmental pollution.Therefore a kind of accurate, easy, safety of exploitation is needed, and can the experimental technique of adhesion process in simulates real entity.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method detecting the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction, make described method safer, accurate, easy.
To achieve these goals, the invention provides following technical scheme:
Detect a method for the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction, it is characterized in that, comprise the following steps:
Step 1, fluorescence labeling probe and platelet rich plasma to be hatched, then centrifugal and draw the pellet platelets blood platelet cleansing solution washing of bottom, then use the blood platelet after the resuspended washing of platelet suspension, then in blood platelet re-suspension liquid, add CaCl
2, then adjusting PC is 10-100 × 10
6individual/mL and CaCl
2concentration is that 1mM is for subsequent use;
Step 2, the tumour cell without antineoplastic process to be detected is set to control group, tumour cell after antineoplastic process to be detected is drug treating group, two groups of cells wash with PBS respectively after trypsinization process, then obtain control group tumour cell re-suspension liquid and drug treating group tumour cell re-suspension liquid with tumor cell suspension is resuspended, adjusting two groups of TCDs is 5 × 10
6individual/mL is for subsequent use;
The blood platelet re-suspension liquid that step 3, two groups of tumour cell re-suspension liquid step 2 obtained obtain with step 1 is respectively hatched, and adopts FL1 Air conduct measurement tumour cell fluorescence rate to obtain drug treating group fluorescence rate and control group fluorescence rate after fixing with paraformaldehyde on flow cytometer;
Test group fluorescence rate and control group fluorescence rate are carried out statistical significant difference analysis and obtains testing result.
Wherein, described in step 1, PC is preferably 30 × 10
6individual/mL.
Of the present inventionly test group fluorescence rate and control group fluorescence rate are carried out statistical significant difference analysis obtain net result, refer to that both carry out significant difference analysis, if the fluorescence rate of drug treating group reduces and has significant difference relative to control group fluorescence rate, illustrate that antineoplastic to be measured has the activity of inhibition tumor cell and platelet adhesion reaction, can the Infiltration and metastasis of inhibition tumor cell.
Tumour cell of the present invention is the same with control group tumour cell before without antineoplastic process to be measured to be cultivated according to normal cultural method, and described process adds medicine in tumor cell culture liquid, and this belongs to processing mode known in this field.
Described in step 1 of the present invention, platelet rich plasma (PRP) can conventionally extract from blood, extracts as follows in embodiments of the present invention:
Venous blood samples 10mL, is placed in the test tube that mass percent is 3.8% citric acid trisodium anti-coagulants (volume ratio of anti-coagulants and blood sample is l:9).At room temperature centrifugal 1000rpm, 10min.Take out after hydro-extractor stops naturally, careful upper plasma of drawing is platelet rich plasma (PRP).
The defect compared with big error easily cannot be there is in real simulation tumour cell and platelet adhesion reaction process and testing process for existing detection technique, the present invention adopts the tumour cell of suspended state and non-activated blood platelet to carry out adhesion experiment, and eliminate test group because the lethal effect of tumour cell causes and the inconsistent problem of cellular control unit quantity by flow cytomery technology, meet real tumour cell and platelet adhesion reaction process.
Preferably, described blood platelet cleansing solution is prepared by following methods:
3.60g NaCl, 1.90g trisodium citrate, 3.00g glucose, is dissolved in 500ml distilled water, dissolves rear adjusted to ph to 6.5 and get final product.
Preferably, described platelet suspension is prepared by following methods:
Platelet suspension stoste 5ml, 1M HEPES125 μ L, 20% glucose 250 μ L, 1M MgCl
250 μ L, 0.05g BSA, is dissolved to 50ml with distilled water, dissolves rear adjusted to ph to 7.4 and get final product;
Described platelet suspension stoste is by 8.00g NaCl, 1.02g NaHCO
3, 0.195g KCl is dissolved in 100ml distilled water formulated.
Preferably, described fluorescence labeling probe is the BCECF-AM of Calcein-AM or 3 μM of DiI, 0.1-5 μM of 10 μMs CFDA-SE, 5-10 μM, is more preferably the CFDA-SE of 10 μMs.For convenience of preparation, CFDA-SE dissolves in dimethyl sulfoxide (DMSO) (DMSO) and is made into 10mM mother liquor, and carrying out dilution use more in actual applications, other fluorescence labeling probes all can be like this.
The present invention adopts CFDA-SE to mark blood platelet, relative fluorescence antibody or the hematoblastic technology of isotope labeling, the price of CFDA-SE more than antibody and isotope cheap, and CFDA-SE marking operation is convenient.If adopt antibody labeling blood platelet, after prepared by washing platelet, antibody labeling need be carried out again, need after antibody labeling again to wash to remove unnecessary antibody, avoid antibody directly in conjunction with tumour cell.And adopting CFDA-SE to mark blood platelet, unnecessary CFDA-SE, before washing platelet, removes by the step of mark while washing platelet, without the need to increasing washing times, simplifies experimental procedure, decreases the possibility that multioperation causes platelet activation.In addition, it is short that CFDA-SE marks the time, only needs 5-10min to complete fluorescence labeling, avoids the platelet activation that mark overlong time causes.CFDA-SE is nontoxic simultaneously, avoids the environmental pollution that isotope labeling causes.
As preferably, tumor cell suspension of the present invention is prepared by following methods:
1M CaCl
250 μ L, 1M MgCl
250 μ L, 0.05g BSA, be dissolved to 50ml with RPMI-1640 nutrient solution, dissolves rear adjusted to ph to 7.4 and get final product.
Preferably, the time of hatching described in step 1 and step 3 is 10-15min.
Preferably, described in step 2, PBS is prepared by following methods:
Take 8.00g NaCl, 0.20g KCl, 1.44g Na
2hPO
4, 0.24g KH
2pO
4be dissolved in 1000ml distilled water, adjusted to ph to 7.4 after dissolving, after autoclaving and get final product.
Preferably, pancreatin described in step 2 to be mass percent be 0.25% trypsin solution.
Preferably, described tumour cell is breast cancer cell.
Preferably, described in step 3, the volume ratio of tumour cell re-suspension liquid and blood platelet re-suspension liquid is 1:4.
In accuracy contrast test, the drug treating group fluorescent value conventionally detected and control group fluorescent value no significant difference, by having there is some spaces not having cell in the visible culture plate of the observation of fluorescent microscope, blood platelet can be seen in these gaps under light microscopic, show that error appears in the result that prior art detects, and adopt detection method to detect, cell and blood platelet hatch after in flow cytomery, the visible fluorescence rate adding antineoplastic processed group cell declines, and the decrease of platelet adhered on cell is described.
The present invention adopts the tumour cell of suspended state and non-activated blood platelet to carry out adhesion experiment, meet real tumour cell and platelet adhesion reaction process, adhesion experiment is carried out owing to adopting suspended state cell, cell density can be adjusted by cell count before adhesion experiment, cell and the cellular control unit consistent in density of agent-feeding treatment can be ensured, and detect cell number by flow cytometer showed software set, ensure that the cell number that different pretreatments group detects is consistent, avoid different pretreatments on the impact of tumor cell number object, result is more accurate.Meanwhile, the present invention is without the need to culture plate in testing process, and cell does not need to grow to merge completely yet, and sense cycle shortens greatly, and the fluorescence labeling probe relative radioactivity isotope adopted is safer.
Accompanying drawing explanation
Figure 1 shows that the flow cytomery collection of illustrative plates of embodiment 1;
Wherein, Fig. 1-A is control group flow cytomery collection of illustrative plates; Fig. 1-B is integrin alpha
2β
1blocking antibody processed group flow cytomery collection of illustrative plates; The collection of illustrative plates upper left corner is fluorescence rate score;
Figure 2 shows that the flow cytomery collection of illustrative plates of embodiment 2;
Wherein, Fig. 2-A is control group flow cytomery collection of illustrative plates, and Fig. 2-B is drug treating group (cantharidin processed group) flow cytomery collection of illustrative plates; Fig. 2-C is drug treating group (Norcantharidin processed group) flow cytomery collection of illustrative plates; The collection of illustrative plates upper left corner is fluorescence rate score;
Figure 3 shows that embodiment 3 prior art fluorescence microscopy figure;
Wherein, Fig. 3-A is control group fluorescence microscopy figure, Fig. 3-B is drug treating group (20 μMs of cantharidin processed group) optical microscope microscopy figure; Fig. 3-C is drug treating group (cantharidin processed group) optical microscope microscopy figure rectangle frame amplifier section; Fig. 3-D is drug treating group (200 μMs of Norcantharidin processed group) optical microscope microscopy figure; Fig. 3-E is drug treating group (Norcantharidin processed group) optical microscope microscopy figure rectangle frame amplifier section;
Fig. 4 is the flow cytomery collection of illustrative plates of embodiment 3 the method for the invention;
Wherein, Fig. 4-A is control group flow cytomery collection of illustrative plates, and Fig. 4-B is drug treating group (20 μMs of cantharidin processed group) flow cytomery collection of illustrative plates; Fig. 4-C is drug treating group (200 μMs of Norcantharidin processed group) flow cytomery collection of illustrative plates; The collection of illustrative plates upper left corner is fluorescence rate score;
Fig. 5 is the tumour cell fluorescence rate column diagram (obtaining based on Fig. 4 result) after the process of embodiment 3 detection method different pharmaceutical of the present invention;
Wherein cylindricality 1 is control group, and cylindricality 2 is 20 μMs of cantharidin processed group, and cylindricality 3 is 200 μMs of Norcantharidin processed group.
Embodiment
The invention discloses a kind of method detecting the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.The method of the invention and kit are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Just a kind of method detecting the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction provided by the present invention is described further below.
Embodiment 1: detect integrin alpha
2β
1blocking antibody suppresses the activity of breast cancer cell MCF-7 and platelet adhesion reaction
In order to verify that detection method to suppress the result accuracy of the activity of blood platelet and tumor cell adhesion to antineoplastic, the present embodiment selects integrin alpha
2β
1blocking antibody verifies (integrin alpha for the tumour cell process after resuspended
2β
1blocking antibody is different from the stage of antineoplastic process tumour cell, it adds process after tumour cell is resuspended, and antineoplastic adds process, this is the difference that reagent nature brings, and is still the central principle based on detection method).
1, related reagent preparation
Tumor cell suspension: 1M CaCl
250 μ L, 1M MgCl
250 μ L, 0.05g BSA, be dissolved to 50ml with RPMI-1640 nutrient solution, dissolves rear adjusted to ph to 7.4 and get final product
Blood platelet cleansing solution: 3.60g NaCl, 1.90g trisodium citrate, 3.00g glucose, is dissolved in 500ml distilled water, dissolves rear adjusted to ph to 6.5 and get final product;
Platelet suspension: platelet suspension stoste 5ml, 1M HEPES125 μ L, 20% glucose 250 μ L, 1M MgCl
250 μ L, 0.05g BSA, is dissolved to 50ml with distilled water, dissolves rear adjusted to ph to 7.4 and get final product;
Platelet suspension stoste: 8.00g NaCl, 1.02g NaHCO
3, 0.195g KCl is dissolved in 100ml distilled water formulated;
CFDA-SE mother liquor: CFDA-SE is dissolved in dimethyl sulfoxide (DMSO) (DMSO) and is made into 10mM mother liquor;
PBS: take 8.00g NaCl, 0.20g KCl, 1.44g Na
2hPO
4, 0.24g KH
2pO
4be dissolved in 1000ml distilled water, adjusted to ph to 7.4 after dissolving, after autoclaving and get final product.
2, detection method
Extract healthy volunteer's venous blood 10ml, all volunteers all do not take any anticoagulation and affect the medicine of platelet function in two weeks.Be placed in containing 3.8%(massfraction) test tube of citric acid trisodium anti-coagulants (volume ratio of anti-coagulants and blood sample is l:9), at room temperature centrifugal 1000rpm, 10min.Take out after hydro-extractor stops naturally, careful upper plasma of drawing is platelet rich plasma (PRP).
Adding 1000 × CFDA-SE mother liquor to final concentration in PRP is 10 μMs, incubated at room 10min.PRP after being marked by CFDA-SE is again centrifugal with 2500rpm, 10min, takes out after hydro-extractor stops naturally, and the careful pellet platelets drawing bottom, wash twice with blood platelet cleansing solution, avoid platelet activation in washing process, action is soft.Then by blood platelet settling flux in platelet suspension, then in blood platelet re-suspension liquid, add 1M CaCl
2, then adjusting PC is 30 × 10
6individual/mL and CaCl
2concentration is that 1mM is for subsequent use;
Take the logarithm breast cancer cell MCF-7 in growth period, PBS washing after the trypsin solution digestion of 0.25%, and tumor cell suspension is resuspended, counting cells, adjustment TCD to 5 × 10
6individual/mL is for subsequent use.
100 μ L5 × 10
6individual/mL concentration tumour cell and 30 × 10
6the washing platelet 400 μ L of individual/mL concentration hatches 15min as a control group, 100 μ L5 × 10
6individual/mL concentration tumour cell re-suspension liquid is through integrin alpha
2β
1after blocking antibody process, with 30 × 10
6the blood platelet re-suspension liquid 400 μ L of individual/mL concentration hatches 15min as drug treating group;
Respectively to adding the fixing 15min of 500 μ L4% paraformaldehydes (paraformaldehyde final concentration is 2%) in drug treating group and control group.Then respectively at flow cytometer adopting FL1 Air conduct measurement fluorescence rate, often group repeats 3-5 time, and statistical significant difference is analyzed two groups of data and whether had significant difference.
3, result
The results are shown in Figure other reproducible results figure of 1(slightly), as shown in Figure 1, after breast cancer cell MCF-7 and blood platelet re-suspension liquid are hatched, the cell of band fluorescence accounts for 41.9%(fluorescence rate).Integrin alpha
2β
1it is the key molecule of tumor cell surface mediated cell and platelet adhesion reaction, after the blocking antibody process of breast cancer cell MCF-7 integrin α2β1, hatch with blood platelet re-suspension liquid again, the cell of hatching rear band fluorescence accounts for 31.8%(fluorescence rate), after significant difference analysis, both have significant difference (P<0.05).Blocking antibody blocking-up tumour cell integrin alpha with integrin α2β1 is described
2β
1function after, the ability of tumour cell and platelet adhesion reaction declines, and testing result of the present invention is consistent with expected results, shows that detection method accuracy is high.
Embodiment 2: the activity detecting cantharidin and Norcantharidin suppression breast cancer cell MCF-7 and platelet adhesion reaction
1, related reagent preparation
Related reagent preparation is with reference to embodiment 1.
2, detection method
Extract healthy volunteer's venous blood 10ml, all volunteers all do not take any anticoagulation and affect the medicine of platelet function in two weeks.Be placed in containing 3.8%(massfraction) test tube of citric acid trisodium anti-coagulants (volume ratio of anti-coagulants and blood sample is l:9), at room temperature centrifugal 1000rpm, 10min.Take out after hydro-extractor stops naturally, careful upper plasma of drawing is platelet rich plasma (PRP).
Adding 1000 × CFDA-SE mother liquor to final concentration in PRP is 10 μMs, incubated at room 10min.PRP after being marked by CFDA-SE is again centrifugal with 2500rpm, 10min, takes out after hydro-extractor stops naturally, and the careful pellet platelets drawing bottom, wash twice with blood platelet cleansing solution, avoid platelet activation in washing process, action is soft.Then by blood platelet settling flux in platelet suspension, then in blood platelet re-suspension liquid, add 1M CaCl
2, then adjusting PC is 30 × 10
6individual/mL and CaCl
2concentration is that 1mM is for subsequent use;
The growth period breast cancer cell MCF-7 of taking the logarithm is set to control group, be drug treating group by the tumour cell after cantharidin and Norcantharidin process, three groups of cells wash with PBS respectively after trypsinization process, then obtain control group tumour cell re-suspension liquid and two drug treating group tumour cell re-suspension liquid with tumor cell suspension is resuspended, adjusting three groups of TCDs is 5 × 10
6individual/mL is for subsequent use
Three groups of tumour cell re-suspension liquid obtained above are respectively got 100 μ L respectively with 400 μ L blood platelet re-suspension liquid and hatch 15min, 500 μ L4% paraformaldehydes (paraformaldehyde final concentration is 2%) fix 15min, then on flow cytometer, adopt FL1 Air conduct measurement tumour cell fluorescence rate to obtain drug treating group fluorescence rate and control group fluorescence rate, often group repeats 3-5 time, and statistical significant difference is analyzed two groups of data and whether had significant difference.
3, result
The results are shown in Figure other reproducible results figure of 2(slightly), after breast cancer cell MCF-7 and blood platelet re-suspension liquid are hatched, the cell of band fluorescence accounts for 41.9%(fluorescence rate).Cantharidin and Norcantharidin be known can the antineoplastic of Tumor suppression Infiltration and metastasis, after breast cancer cell MCF-7 uses cantharidin and Norcantharidin process respectively, hatch with blood platelet re-suspension liquid again, the cell of hatching rear band fluorescence account for respectively 25.6% and 29.7%(fluorescence rate), after significant difference analysis and control group there is significant difference (P<0.05).Show that testing result of the present invention is consistent with expected results.
Embodiment 3: the contrast test of detection method and prior art
Prior art: by tumour cell kind in Tissue Culture Plate, treat that cell is completely adherent, and with antineoplastic process to be detected after merging completely, the blood platelet crossed by fluorescence labeling adds in culture plate, after hatching certain hour, the blood platelet not adhering to tumour cell is removed in rinsing, after adding lysate cell lysis and blood platelet, whether the fluorescence level detected in culture plate decreases relative to the fluorescence level without antineoplastic process, antineoplastic is cantharidin and Norcantharidin, tumour cell is breast cancer cell MCF-7, the results are shown in Figure 3;
The present invention: related reagent preparation and detection method are with reference to embodiment 2, and antineoplastic is cantharidin and Norcantharidin, and tumour cell is breast cancer cell MCF-7, the results are shown in Figure 4 and Fig. 5;
As shown in Figure 3, the tumour cell adopting prior art to carry out and the detection of platelet adhesion reaction, compared with control group, when after employing antineoplastic cantharidin (CAN) and Norcantharidin (NCTD) process, cell rounding, adhesion declines, occur in culture plate that some do not have the space of cell (referring to rectangle frame amplifier section microscopy figure), blood platelet can be seen in these gaps under light microscopic, visible green fluorescence under fluorescent microscope, illustrates that blood platelet has directly adhered to the gap on culture plate.Total fluorescence intensity after testing between each group is without significant difference, and cannot confirm drug on tumor cell and hematoblasticly be stained with inhibit activities, deviation appears in result.
From Fig. 4 and Fig. 5, tumour cell and blood platelet hatch after in flow cytomery, the visible fluorescence rate adding the cell of cantharidin and Norcantharidin processed group declines, the decrease of platelet adhered on cell is described, with control group result, there is significant difference (P<0.05), show that cantharidin and Norcantharidin have tumour cell and be hematoblasticly stained with inhibit activities, can the Infiltration and metastasis of inhibition tumor cell, be consistent with expected results, result is accurate.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. detect a method for the activity of antineoplastic inhibition tumor cell and platelet adhesion reaction, it is characterized in that, comprise the following steps:
Step 1, fluorescence labeling probe and platelet rich plasma to be hatched, then centrifugal and draw the pellet platelets blood platelet cleansing solution washing of bottom, then use the blood platelet after the resuspended washing of platelet suspension, then in blood platelet re-suspension liquid, add CaCl
2, then adjusting PC is 10-100 × 10
6individual/mL and CaCl
2concentration is that 1mM is for subsequent use;
Step 2, the tumour cell without antineoplastic process to be detected is set to control group, tumour cell after antineoplastic process to be detected is drug treating group, two groups of cells wash with PBS respectively after trypsinization process, then obtain control group tumour cell re-suspension liquid and drug treating group tumour cell re-suspension liquid with tumor cell suspension is resuspended, adjusting two groups of TCDs is 5 × 10
6individual/mL is for subsequent use;
The blood platelet re-suspension liquid that step 3, two groups of tumour cell re-suspension liquid step 2 obtained obtain with step 1 is respectively hatched, and adopts FL1 Air conduct measurement tumour cell fluorescence rate to obtain drug treating group fluorescence rate and control group fluorescence rate after fixing with paraformaldehyde on flow cytometer;
Drug treating group fluorescence rate and control group fluorescence rate are carried out statistical significant difference analysis and obtains testing result;
Described fluorescence labeling probe is the BCECF-AM of Calcein-AM or 3 μM of DiI, 0.1-5 μM of 10 μMs CFDA-SE, 5-10 μM.
2. method according to claim 1, it is characterized in that, described blood platelet cleansing solution is prepared by following methods:
3.60g NaCl, 1.90g trisodium citrate, 3.00g glucose, is dissolved in 500ml distilled water, dissolves rear adjusted to ph to 6.5 and get final product.
3. method according to claim 1, it is characterized in that, described platelet suspension is prepared by following methods:
Platelet suspension stoste 5ml, 1M HEPES 125 μ L, 20% glucose 250 μ L, 1M MgCl
250 μ L, 0.05g BSA, is dissolved to 50ml with distilled water, dissolves rear adjusted to ph to 7.4 and get final product;
Described platelet suspension stoste is by 8.00g NaCl, 1.02g NaHCO
3, 0.195g KCl is dissolved in 100ml distilled water formulated.
4. method according to claim 1, it is characterized in that, described tumor cell suspension is prepared by following methods:
1M CaCl
250 μ L, 1M MgCl
250 μ L, 0.05g BSA, be dissolved to 50ml with RPMI-1640 nutrient solution, dissolves rear adjusted to ph to 7.4 and get final product.
5. method according to claim 1, is characterized in that, the time of hatching described in step 1 and step 3 is 10-15min.
6. method according to claim 1, it is characterized in that, described in step 2, PBS is prepared by following methods:
Take 8.00g NaCl, 0.20g KCl, 1.44g Na
2hPO
4, 0.24g KH
2pO
4be dissolved in 1000ml distilled water, adjusted to ph to 7.4 after dissolving, after autoclaving and get final product.
7. method according to claim 1, is characterized in that, pancreatin described in step 2 to be mass percent be 0.25% trypsin solution.
8. method according to claim 1, it is characterized in that, described tumour cell is breast cancer cell.
9. method according to claim 1, it is characterized in that, described in step 3, the volume ratio of tumour cell re-suspension liquid and blood platelet re-suspension liquid is 1:4.
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| 宁忠华.血小板对NK细胞杀伤作用的影响.《中国优秀博硕士学位论文全文数据库(硕士) 医药卫生科技辑》.2005,(第1期), * |
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