CN105524973A - Method for screening antitumor drugs using autologous tumors and normally paired primary cells - Google Patents
Method for screening antitumor drugs using autologous tumors and normally paired primary cells Download PDFInfo
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Abstract
The invention discloses a method for screening antitumor drugs using autologous tumor and normally paired primary cells. The method comprises: S1, providing tumor cells of same tissue origin and normal primary cells normally paired, and culturing them separately; S2, inoculating the tumor cells and the normal primary cells, and using the tumor cells and the normal primary cells as an experimental group and a control group; S3, providing a plurality of alternative drugs for the tumor, and dosing the alternative drugs separately to the experimental group of the tumor cells and normal primary cells; S4, detecting and analyzing the inhibition rates of each alternative drug for the tumor cells and the normal primary cells by an ATP (adenosine triphosphate) biofluorescence efficacy detection method, and primarily screening to obtain candidate drugs; S5, subjecting the tumor cell set and the normal primary cells that the candidate drugs act on to flow apoptosis detection, screening from the candidate drugs a drug having highest inhibition rate for tumor cells and lowest inhibition rate for normal cells, and using the drug as a clinical recommended drug.
Description
Technical field
The present invention relates to biological technical field, more specifically, relate to and a kind ofly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine.
Background technology
In recent years, the incidence of tumour is in the trend increased year by year, the methods for the treatment of of current routine mainly contains operation, chemicotherapy, biotherapy etc., and wherein chemotherapy faced by colony account for major portion, chemotherapy be utilize chemicals stop cancer cells propagation, invade profit, shift until finally kill a kind of methods for the treatment of of cancer cells, it is a kind for the treatment of means of general.But because the selectivity of chemotherapeutics is not strong, while killing cancer cells, also inevitably can damages the cell of human normal, thus occur the untoward reaction of medicine; After how making patient accept chemotherapeutics, reach best antitumor action, the damage of chemotherapeutics to human body can be reduced to greatest extent again simultaneously, become the problem needing solution in current tumor therapeutic procedure badly.
Increasing evidence shows, the generation evolution of tumour is the result of the environmental interaction of tumour cell and its growth.Even if in tumorigenic very early time, the type survival out of control of tumour cell also needs the corresponding change of its environment to maintain to growth, because in-vivo tumour tissue has complicated structure more, comprise the cell of molecular marker for increased proliferation, resting cell and necrotic tissue etc., there is obvious heterogeneity, inner at same tumor tissues, different sites cell is different to the susceptibility of chemotherapeutics; Different patient, same tumor tissues is also different for the susceptibility of chemotherapeutics of the same race.So no matter be use tumour cell simulation human internal environment to carry out drug screening, or other some drug screening methods (as primary cell drug screening etc.), all really can not solve the select permeability of current oncotherapy Chinese traditional medicine.
Summary of the invention
The present invention is intended to one of solve the problems of the technologies described above at least to a certain extent.
For this reason, the present invention proposes a kind ofly to utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, and the method simple possible, can filter out the tumour medicine of good drug efficacy.
Utilizing autologous tumor and normally match primary cell and screen the method for tumour medicine according to the embodiment of the present invention, comprises the following steps: S1, the tumour cell providing tissue source identical and the Normal primary cell normally matched, and cultivates it respectively; S2, described tumour cell and described Normal primary cell to be inoculated respectively, and described tumour cell and described Normal primary cell are divided into experimental group and control group respectively; S3, provide multiple drug candidate for this tumour, and multiple described drug candidate is carried out agent-feeding treatment to the experimental group of described tumour cell and described Normal primary cell respectively; S4, being detected and analyze the inhibiting rate of each drug candidate to described tumour cell and described Normal primary cell by ATP bioluminescence Composition analyzed method, primary dcreening operation obtains drug candidate; S5, the described tumour cell group of drug candidate group effect and described Normal primary cell are carried out the detection of streaming apoptosis, to filter out in described drug candidate the highest and simultaneously to the medicine that described Normocellular inhibiting rate is minimum to the inhibiting rate of described tumour cell, as clinical recommendation medicine.
Utilizing autologous tumor and normally matching primary cell and screen the method for tumour medicine according to the embodiment of the present invention, different drug screening systems can be set up for different patient, judge the drug effect of different tumor candidate medicine, and the best tumour medicine of respective concentration is provided, ensure while effectively killing tumour cell, reduce the side effect of medicine for patient to greatest extent.
In addition, according to the above embodiment of the present inventionly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, the technical characteristic added as follows can also be had:
According to one embodiment of present invention, described step S1 comprises: S11, the tumour cell providing tissue source identical and the normal Normal primary cell matched, and described tumour cell is dispelled into individual cells respectively with described Normal primary cell; S12, described tumour cell and described Normal primary cell added in culture dish respectively and cultivates; S13, regularly described tumour cell and described Normal primary cell observed and taken pictures, carrying out passage simultaneously, after cultivating the scheduled time, collecting described tumour cell and described Normal primary cell.
According to one embodiment of present invention, in described step S12, the culture temperature of described tumour cell and described Normal primary cell is 36.5 DEG C-37.5 DEG C, and gas concentration lwevel is 4%-6%.
According to one embodiment of present invention, in described step S13, the described scheduled time is 5-7 days.
According to one embodiment of present invention, described step S1 also comprises: S14, by collect described tumour cell and described Normal primary cellular portions frozen.
According to one embodiment of present invention, described step S2 comprises: S21, described tumour cell carried out to precision detection and susceptibility test, filters out the described tumour cell meeting drug screening requirement, and determines the formal inoculation number of described tumour cell; S22, described tumour cell and described Normal primary cell to be seeded on 96 orifice plates according to formal inoculation number respectively, and described tumour cell and described Normal primary cell are divided into experimental group and control group respectively.
According to one embodiment of present invention, described step S21 comprises: S211, described tumour cell be divided into low number group, middle number group and high number group and inoculate respectively, wherein, the tumor cell number of described low number group is 400-600, the tumor cell number of described middle number group is 9000-11000, and the tumor cell number of described high number group is greater than 40000; S212, cultivate after the scheduled time, measure the RLU value of described low number group, middle number group and high number group respectively, the minimum tumor cell number of selection RLU mean value > 400000 is as formally inoculating number.
According to one embodiment of present invention, described step S3 comprises: S31, provide multiple drug candidate, and each described drug candidate is deployed into multiple concentration respectively; S32, the multiple described drug candidate of multiple concentration is carried out agent-feeding treatment to the experimental group of described tumour cell and described Normal primary cell respectively.
According to one embodiment of present invention, described step S4 comprises: S41, extract the ATP of each experimental group, utilizes bioluminescence principle to measure ATP content, result transmission computer; S42, by the different drug candidate of the software analysis different concns inhibiting rate to described tumour cell and described Normal primary cell, curve plotting; S43, pick out to the inhibiting rate of described tumour cell more than 60%, simultaneously to the inhibiting rate of described Normal primary cell lower than 50% drug candidate, primary dcreening operation obtains the concentration of drug candidate and drug candidate.
According to one embodiment of present invention, described step S5 comprises: S51, the described tumour cell group of collecting the effect of drug candidate group and described Normal primary cell, the cell count of each sample is 1 × 10
6; S52, each described sample is washed 1 time by incubation buffer respectively, the centrifugal 5min of 500-1000r/min; The label solution re-suspended cell of S53, use 100ul, under room temperature, lucifuge hatches 10-15min; The centrifugal 5min sedimentation cell of S54,500-1000r/min, and wash 1 time by incubation buffer; S55, cell is added fluorescent solutions, at 4 DEG C, hatch 20min, lucifuge also keeps vibration; S56, flow cytometer excitation wavelength 488nm, detect FITC fluorescence with the passband filter that a wavelength is 515nm, and the filter that another wavelength is greater than 560nm detects PI; S57, to filter out in described drug candidate the highest and simultaneously to the medicine that described Normocellular inhibiting rate is minimum to the inhibiting rate of described tumour cell, as recommendation medicine.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 is utilizing autologous tumor and normally matching primary cell and screen the schema of the method for tumour medicine according to the embodiment of the present invention;
Fig. 2 is the inhibiting rate graphic representation of drug candidate to stomach cancer cell;
Fig. 3 is the inhibiting rate graphic representation of drug candidate to normal primary cell;
Fig. 4 is the scatter diagram of bivariate flow cytometer.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Be exemplary below by the embodiment be described with reference to the drawings, be intended to for explaining the present invention, and can not limitation of the present invention be interpreted as.
Utilizing autologous tumor and normally matching primary cell and screen the method for tumour medicine according to the embodiment of the present invention is specifically described below in conjunction with Fig. 1.
As shown in Figure 1, utilizing autologous tumor and normally matching the method that primary cell screens tumour medicine and specifically comprise the following steps according to the embodiment of the present invention:
S1, the tumour cell providing tissue source identical and the normal Normal primary cell matched, and respectively it is cultivated.
S2, tumour cell and Normal primary cell to be inoculated respectively, and tumour cell and Normal primary cell are divided into experimental group and control group respectively.
S3, provide multiple drug candidate for this tumour, and multiple drug candidate is carried out agent-feeding treatment to the experimental group of tumour cell and Normal primary cell respectively.
S4, being detected and analyze the inhibiting rate of each drug candidate to tumour cell and Normal primary cell by ATP bioluminescence Composition analyzed method, primary dcreening operation obtains drug candidate.
S5, the tumour cell group of drug candidate group effect and Normal primary cell are carried out the detection of streaming apoptosis, to filter out in drug candidate the highest and simultaneously to the medicine that Normocellular inhibiting rate is minimum to the inhibiting rate of tumour cell, as recommendation medicine.
In other words, the autologous tumor that utilizes according to the embodiment of the present invention is the chemical drug filtering mode that one " customizes " based on tumour patient with normally match the method that primary cell screens tumour medicine, first this pattern turns out Tumor cell in specimens and normal cell composition pairing cell, again candidate's chemical drug is screened, select lethality maximum simultaneously to one group of medicine that normal cell damage is minimum.
Thus, utilizing autologous tumor and normally matching primary cell and screen the method for tumour medicine according to the embodiment of the present invention, different drug screening systems can be set up for different patient, judge the drug effect of different tumor candidate medicine, and the best tumour medicine of respective concentration is provided, ensure while effectively killing tumour cell, reduce the side effect of medicine for patient to greatest extent.
According to one embodiment of present invention, step S1 comprises: S11, the tumour cell providing tissue source identical and the normal Normal primary cell matched, and tumour cell is dispelled into individual cells respectively with Normal primary cell.
S12, tumour cell and Normal primary cell added in culture dish respectively and cultivates.
S13, regularly tumour cell and Normal primary cell observed and taken pictures, carrying out passage simultaneously, after cultivating the scheduled time, collecting tumour cell and Normal primary cell.
Wherein, in step s 12, the culture temperature of tumour cell and Normal primary cell is 36.5 DEG C-37.5 DEG C, and gas concentration lwevel is 4%-6%, and preferably, in step s 13, the scheduled time is 5-7 days, is preferably 6 days.
Thus, tumour cell and the Normal primary cell of needs can be collected by this step.
In embodiments more of the present invention, step S1 also comprises: S14, by collect tumour cell and Normal primary cellular portions frozen.
That is, in this application, except a part of tumour cell and Normal primary cell are used for carrying out except drug screening, also a part of tumour cell and Normal primary cell are carried out freezen protective, the cell of freezen protective can be recovered where necessary, when making the new departures such as clinician's consideration novel drugs, DC-CIK treatment, CAR-T cell therapy, gene therapy, Tumor cell can be used as most starting materials (as antigenic stimulation thing or look for mutant gene locus material) and most scientific be also to the most direct inspection tool of various treatment plan drug effect.
According to one embodiment of present invention, step S2 comprises: S21, tumour cell carried out to precision detection and susceptibility test, filters out the tumour cell meeting drug screening requirement, and determines the formal inoculation number of tumour cell.
S22, tumour cell and Normal primary cell to be seeded on 96 orifice plates according to formal inoculation number respectively, and tumour cell and Normal primary cell are divided into experimental group and control group respectively.
Particularly, step S21 comprises:
S211, tumour cell be divided into low number group, middle number group and high number group and inoculate respectively, wherein, the tumor cell number of low number group is 400-600, and the tumor cell number of middle number group is 9000-11000, and the tumor cell number of high number group is greater than 40000.
S212, cultivate after the scheduled time, measure the RLU value of low number group, middle number group and high number group respectively, the minimum tumor cell number of selection RLU mean value > 400000 is as formally inoculating number.
In other words, before drug screening is carried out to tumour cell group and Normal primary cell, also need to carry out precision detection and susceptibility test to tumour cell, thus filter out the tumour cell meeting drug screening requirement, and determine the inoculation number of cell, the requirement whether tumour cell of turning out meets drug screening can be weighed thus, for drug screening result afterwards provides stronger foundation.
In embodiments more of the present invention, step S3 comprises: S31, provide multiple drug candidate, and each drug candidate is deployed into multiple concentration respectively.
S32, multiple drug candidate of multiple concentration are carried out agent-feeding treatment to the experimental group of tumour cell and Normal primary cell respectively.
That is, when testing the experimental group of tumour cell and Normal primary cell, first filtering out may to the inhibited multiple drug candidate of tumour cell, then each drug candidate is deployed into multiple concentration respectively, and each concentration of each drug candidate is carried out agent-feeding treatment to the experimental group of tumour cell and Normal primary cell respectively.Thus, the accuracy of test can be improved further.
According to one embodiment of present invention, step S4 comprises: S41, extract the ATP of each experimental group, utilizes bioluminescence principle to measure ATP content, result transmission computer.
S42, by the different drug candidate of the software analysis different concns inhibiting rate to tumour cell and Normal primary cell, curve plotting.
S43, pick out to the inhibiting rate of tumour cell more than 60%, simultaneously to the inhibiting rate of normal primary cell lower than 50% drug candidate, primary dcreening operation obtains the concentration of drug candidate and drug candidate.
Thus, can filter out the inhibiting rate of tumour cell relatively high, and the concentration to the relatively low drug candidate of the inhibiting rate of normal primary cell and corresponding drug candidate.
Further, in embodiments more of the present invention, step S5 comprises:
S51, the tumour cell group of collecting the effect of drug candidate group and Normal primary cell, the cell count of each sample is 1 × 10
6.
S52, each sample is washed 1 time by incubation buffer respectively, the centrifugal 5min of 500-1000r/min.
The label solution re-suspended cell of S53, use 100ul, under room temperature, lucifuge hatches 10-15min.
The centrifugal 5min sedimentation cell of S54,500-1000r/min, and wash 1 time by incubation buffer.
S55, cell is added fluorescent solutions, at 4 DEG C, hatch 20min, lucifuge also keeps vibration.
S56, flow cytometer excitation wavelength 488nm, detect FITC fluorescence with the passband filter that a wavelength is 515nm, and the filter that another wavelength is greater than 560nm detects PI.
S57, to filter out in drug candidate the highest and simultaneously to the medicine that Normocellular inhibiting rate is minimum to the inhibiting rate of tumour cell, as recommendation medicine.
Thus, screening method according to the embodiment of the present invention can set up different drug screening systems for different patient, judge the drug effect of different tumor candidate medicine, and the best tumour medicine of respective concentration is provided, ensure while effectively killing tumour cell, reduce the side effect of medicine for patient to greatest extent.
Generally speaking, utilizing autologous tumor and normally match the cancer therapy drug that drug effect that method that primary cell screen tumour medicine utilizes the tumour of patient self and normal cultivation of matching primary cell to detect cancer therapy drug more can help patient selection useful to it intuitively according to the embodiment of the present invention, help tumour patient uses medicine targetedly.
The tumour that the patient that the present invention gets is autologous and corresponding primary cell are cultivated, and in cell cultivation process, analogue body inner cell growth conditions, after cell cultures completes, carries out cell drug screening and carry out assessment test to the selection result.
Utilizing autologous tumor and normally matching the method that primary cell screens tumour medicine and use patient's autologous tumor tissue according to the embodiment of the present invention, carries out cell cultures.Autologous tumor tissue has more cogency for the screening of medicine, and first, autologous tumour cell existence microenvironment agrees with the physical appearance of patient's reality completely; Next effectively prevent the algebra problem using tumor cell line cell; Use autologous tumor tissue simultaneously, drug screening can be carried out to the cell being in different tumour stage of growth simultaneously, make pharmacodynamic results more accurately reliable.
Utilizing autologous tumor and normally matching the primary cell that method that primary cell screens tumour medicine uses tumor tissues corresponding and carry out drug test simultaneously according to the embodiment of the present invention.There is certain otherness in the drug resistance of different patient, while selection tumour medicine, also need to consider the ability to bear of patient for this medicine, using primary cell to carry out drug test can help patient while selection effective antitumor medicine, reduces medicine to greatest extent to the damage of human body.
The autologous tumor that utilizes according to the embodiment of the present invention screens the method for tumour medicine in pharmacodynamic assessment with the normal primary cell that matches, and sets up ATP-TCA in conjunction with apoptotic evaluation system.ATP-TCA technology measures terminal using endogenous cellular ATP content as degree of activation of cells, and medicine all can reflect the effect of each cell cycle.This technology is responsive reliable, to sample can Assessment Rate more than 97%, be applicable to anticancer sensitivity and detect.Large quantity research shows, ATP-TCA technology is equipped with apoptosis detection method, and comparatively colony method (HTCA) and mtt assay have superior Sensitivity and Specificity in assessment cell viability.Meanwhile, this technical testing can be amplified to 1536 orifice plates from 96 orifice plates, 384 orifice plates, is applicable to miniaturization and high flux screening, has the good differentiation positive and the ability of negative control group sample.
Utilizing autologous tumor and normally matching the method that primary cell screens tumour medicine and utilize self pair tumour cell to screen to patient's medicine targetedly according to the embodiment of the present invention, for the accuracy that can improve medication patient, improve curative effect, reduce damage, also reduce medical expense simultaneously.
Utilizing autologous tumor and normally matching primary cell and screen the approach application autologous tumor of tumour medicine and corresponding primary cell carries out drug screening according to the embodiment of the present invention, the otherness that different patient reacts for tumour medicine can be solved well, medicine can be reduced for Normocellular infringement simultaneously.Drug screening is carried out relative to use tumor cell line, use autologous tumor cell effectively can avoid the heterogeneity of cell, namely autologous tumour cell suits the physical appearance of patient's reality more, the medicine screened also just meets the tumorigenic particular case of patient more, thus killing tumor cell more targetedly.
The autologous tumor that utilizes according to the embodiment of the present invention screens the method for tumour medicine while carrying out drug screening to autologous tumor cell with the normal primary cell that matches, normally match the drug screening of primary cell, some can killing tumor cells can to help patient selection, but the medicine little to physical impairment; Because different patients has certain difference for the tolerance of medicine, tumour medicine also can damage normal cell while killing tumour cell, so the drug screening carrying out pairing primary cell is also just necessary very much, is also one of advantage of the present invention.
Below in conjunction with specific embodiment utilizing autologous tumor and normally matching the method that primary cell screens tumour medicine and be described in detail the embodiment of the present invention.
Embodiments of the invention describe in detail for cancer of the stomach.
One: the primary cell culture of the normal gastric mucosa cell of gastric cancer tumor cell and pairing.
1, cancerous tissue and healthy tissues block (this step is provided by hospital doctor) is extracted.
2, after a sterilization being organized to clean in Bechtop, shred with scissors, add appropriate trysinization, digest 10 minutes.
3, after digestion, stop digestion with substratum, and with rifle head, postdigestive fragment of tissue is dispelled, dispel into individual cells.
4, the cell dispelled is proceeded to respectively be added with defined medium 10cm plate in, 37 degree, 5% carbonic acid gas cultivate.
5, regularly cell observed and take pictures, carrying out passage (trypsin digestion) simultaneously, cultivate after six days, collecting cell.
6, will collect cell, part is frozen, (this frozen storing liquid is specific cryoprotective agent, and the protected effect for cell is better) remaining inoculate into 96 orifice plates respectively, for effect experiment.
Two, stomach cancer cell precision and susceptibility test
Precision is tested: select the cell of tumour cell low number group, middle number group and high number group (500,10000,40000) to be seeded in 96 orifice plates, cultivate after 7 days, measuring four groups of RLU values respectively, selecting the minimum cell count of RLU mean value > 400000 as formally inoculating number.Test result, as table 1, requires: CV < 10%.
Susceptibility: 50 tumour cell > 440RLU, simultaneously background values < 200RLU.Weigh tumour cell and whether meet drug screening requirement.
Table 1, RLU value test result
Three, ATP bioluminescence Composition analyzed:
1, select different medicines to carry out agent-feeding treatment to the cell in orifice plate, and observation is taken pictures.Medicament categories has: 5-Fu (Ro 2-9757), DOC (Docetaxel), MMC (mitomycin), PTX (taxol), DDP (cis-platinum), EPI (pidorubicin).2, extract ATP, Fluorescence Scanner measures fluorescence intensity, result transmission computer.
3, by each control of the concentration rate of software analysis medicine, curve plotting, assessment drug effect.As shown in Figures 2 and 3, wherein, shown in Fig. 2 is the inhibiting rate of drug candidate to stomach cancer cell to the curve drawn, and shown in Fig. 3 is the inhibiting rate of drug candidate to normal primary cell.
Can find by carrying out analysis to Fig. 2 and Fig. 3, can the medicine more than 60% be the 5-FU of concentration more than 25%, the DOC of concentration more than 120%, the MMC of concentration more than 150% respectively to the inhibiting rate of this patient's stomach cancer cell in drug candidate, these three kinds of medicines to the inhibiting rate of pairing stomach Normal primary cell lower than 50% be 5-FU.
The drug candidate of primary dcreening operation to stomach cancer cell and Normocellular inhibiting rate as shown in table 2.
Table 2, drug candidate are to stomach cancer cell and Normocellular inhibiting rate
| Medicine | To the inhibiting rate of stomach cancer cell | To the Normocellular inhibiting rate of pairing |
| 25％5-FU | 63％ | 35％ |
| 50％5-FU | 82％ | 40％ |
| 100％5-FU | 88％ | 42％ |
| 200％5-FU | 92％ | 41％ |
According to table 2, selection 50%5-FU, 100%5-FU, 200%5-FU alternatively medicine carry out next step detection.
Four, flow cytometer detection apoptosis---AnnexinV/PI double-staining
1, cell harvesting: the cancer cells of 5-FU drug screening and normal stomach organization cell harvesting are got up, each sample cell count is 1 × 10
6.
2,1 time is washed by incubation buffer, the centrifugal 5min of 500-1000r/min.
3, with label solution (FITC-AnnexinV and the PI) re-suspended cell of 100ul, under room temperature, lucifuge hatches 10-15min.
4,500-1000r/min centrifugal 5min sedimentation cell incubation buffer washes 1 time.
5, hatch 20min at adding fluorescence (SA-FLOUS) solution 4 DEG C, lucifuge is also vibrated frequently.
6, flow cytometry analysis: flow cytometer excitation wavelength 488nm, detects FITC fluorescence with the passband filter that a wavelength is 515nm, and the filter that another wavelength is greater than 560nm detects PI.
7, result judge: apoptotic cell to all for cytoactive qualification dyestuff as PI has anti-metachromia, non-viable non-apoptotic cell then can not.Cytolemma has the DNA of the cell of damage can dye generation red fluorescence by PI, and the cell that cytolemma remains intact then does not have red fluorescence and produces.
Therefore, apoptotic early stage PI can not dye and there is no red fluorescent.Normal live cells similarly.On the scatter diagram of bivariate flow cytometer (as shown in Figure 4), left lower quadrant display viable cell, is (FITC-/PI-); Right upper quadrant is non-living cell, i.e. non-viable non-apoptotic cell, is (FITC+/PI+); And right lower quadrant is apoptotic cell, manifest (FITC+/PI-).
Can analyze from Fig. 4 and draw, the 5-Fu of 200% is better for this patient's stomach cancer cell fragmentation effect, simultaneously relatively little for the impact of patient's healthy tissues.
In sum: at present the efficient of chemotherapy of gastric cancer scheme mostly is about 50%, about half patient is to first chemotherapy regimen and insensitive (initial drug-resistant), and also some there will be secondary resistance.And use carrying out of the In Vitro Chemotherapy drug sensitivity test of cancer of the stomach and normal pairing primary cell, to selecting responsive chemotherapeutics, realize individualized treatment to tumour patient, reduce blindness and resistance there is extremely important clinical meaning.The present invention is compared with in vitro tests in the past, and this technology repeatability is strong, easy stdn, sample can assessment ratio high.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (10)
1. utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, comprise the following steps:
S1, the tumour cell providing tissue source identical and the normal Normal primary cell matched, and respectively it is cultivated;
S2, described tumour cell and described Normal primary cell to be inoculated respectively, and described tumour cell and described Normal primary cell are divided into experimental group and control group respectively;
S3, provide multiple drug candidate for this tumour, and multiple described drug candidate is carried out agent-feeding treatment to the experimental group of described tumour cell and described Normal primary cell respectively;
S4, being detected and analyze the inhibiting rate of each drug candidate to described tumour cell and described Normal primary cell by ATP bioluminescence Composition analyzed method, primary dcreening operation obtains drug candidate;
S5, the described tumour cell group of drug candidate group effect and described Normal primary cell are carried out the detection of streaming apoptosis, to filter out in described drug candidate the highest and simultaneously to the medicine that described Normocellular inhibiting rate is minimum to the inhibiting rate of described tumour cell, as clinical recommendation medicine.
2. according to claim 1ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S1 comprises:
S11, the tumour cell providing tissue source identical and the normal Normal primary cell matched, and described tumour cell is dispelled into individual cells respectively with described Normal primary cell;
S12, described tumour cell and described Normal primary cell added in culture dish respectively and cultivates;
S13, regularly described tumour cell and described Normal primary cell observed and taken pictures, carrying out passage simultaneously, after cultivating the scheduled time, collecting described tumour cell and described Normal primary cell.
3. according to claim 2ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, in described step S12, the culture temperature of described tumour cell and described Normal primary cell is 36.5 DEG C-37.5 DEG C, and gas concentration lwevel is 4%-6%.
4. according to claim 2ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, in described step S13, the described scheduled time is 5-7 days.
5. according to claim 2ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S1 also comprises:
S14, by collect described tumour cell and described Normal primary cellular portions frozen.
6. according to claim 1ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S2 comprises:
S21, described tumour cell carried out to precision detection and susceptibility test, filter out the described tumour cell meeting drug screening requirement, and determine the formal inoculation number of described tumour cell;
S22, described tumour cell and described Normal primary cell to be seeded on 96 orifice plates according to formal inoculation number respectively, and described tumour cell and described Normal primary cell are divided into experimental group and control group respectively.
7. according to claim 6ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S21 comprises:
S211, described tumour cell be divided into low number group, middle number group and high number group and inoculate respectively, wherein, the tumor cell number of described low number group is 400-600, and the tumor cell number of described middle number group is 9000-11000, and the tumor cell number of described high number group is greater than 40000;
S212, cultivate after the scheduled time, measure the RLU value of described low number group, middle number group and high number group respectively, the minimum tumor cell number of selection RLU mean value > 400000 is as formally inoculating number.
8. according to claim 1ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S3 comprises:
S31, provide multiple drug candidate, and each described drug candidate is deployed into multiple concentration respectively;
S32, the multiple described drug candidate of multiple concentration is carried out agent-feeding treatment to the experimental group of described tumour cell and described Normal primary cell respectively.
9. according to claim 1ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S4 comprises:
S41, extract the ATP of each experimental group, utilize bioluminescence principle to measure ATP content, result transmission computer;
S42, by the different drug candidate of the software analysis different concns inhibiting rate to described tumour cell and described Normal primary cell, curve plotting;
S43, pick out to the inhibiting rate of described tumour cell more than 60%, simultaneously to the inhibiting rate of described Normal primary cell lower than 50% drug candidate, primary dcreening operation obtains the concentration of drug candidate and drug candidate.
10. according to claim 1ly utilize autologous tumor and normally match primary cell and screen the method for tumour medicine, it is characterized in that, described step S5 comprises:
S51, the described tumour cell group of collecting the effect of drug candidate group and described Normal primary cell, the cell count of each sample is 1x10
6;
S52, each described sample is washed 1 time by incubation buffer respectively, the centrifugal 5min of 500-1000r/min;
The label solution re-suspended cell of S53, use 100ul, under room temperature, lucifuge hatches 10-15min;
The centrifugal 5min sedimentation cell of S54,500-1000r/min, and wash 1 time by incubation buffer;
S55, cell is added fluorescent solutions, at 4 DEG C, hatch 20min, lucifuge also keeps vibration;
S56, flow cytometer excitation wavelength 488nm, detect FITC fluorescence with the passband filter that a wavelength is 515nm, and the filter that another wavelength is greater than 560nm detects PI;
S57, to filter out in described drug candidate the highest and simultaneously to the medicine that described Normocellular inhibiting rate is minimum to the inhibiting rate of described tumour cell, as clinical recommendation medicine.
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