CN112481362B - Detection method for RGDFK inhibition PDL1 and application of detection method in preparation of anti-PDL 1 medicine - Google Patents
Detection method for RGDFK inhibition PDL1 and application of detection method in preparation of anti-PDL 1 medicine Download PDFInfo
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Abstract
The invention provides a method for detecting PDL1 inhibited by RGDFK, which comprises the following steps: (1) the expression regulation mechanism of PDL1 molecule on the surface of tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene and protein levels, respectively; (2) detecting the level of phosphorylation of ERK in the tumor cell; (3) the inhibition effect of RGDfK on tumor cell PDL1 is verified: after treatment of tumor cells with the RGDfK inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique. The invention provides that the inhibitor RGDfK of integrin alpha v beta 3 can enhance the immunotherapy effect of T cells in breast cancer, and is expected to provide feasible and theoretical basis for clinically treating breast cancer.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a detection method for RGDfK inhibition PDL1 and application of the detection method in preparation of a PDL1 resistant medicine.
Background
Breast cancer is a malignant tumor with a high incidence in the female population. In recent years, significant clinical effects have been achieved in the treatment of breast cancer, mainly by surgery, radiotherapy and chemotherapy, and then the survival rate is generally low in 5 years, especially in patients in middle and advanced stages. With the development of tumor biology and immunology, cancer immunotherapy has become a new research hotspot in the field of tumor therapy in recent years, is taken as a fourth tumor therapy mode, and mainly refers to a therapy method which applies immunological principles and methods, specifically removes tumor tiny residual focuses, inhibits tumor growth and breaks immune tolerance by activating immune cells in vivo and enhancing anti-tumor immune response of organisms. Tumor immunotherapy is intended to overcome the mechanism of tumor immune escape, thereby re-awakening immune cells to eliminate cancer cells. Generally, the immune response of tumor patients is suppressed, on one hand, due to the deficiency of cytotoxic T cell-associated antigens and helper T cells in vivo, and on the other hand, due to immunosuppressive molecules produced by tumor cells, resulting in immune tolerance. Research shows that immune checkpoint programmed death receptor 1 (PD 1) and ligand 1 (PDL 1) participate in forming a tumor microenvironment, can obviously inhibit T cell activity, and promote tumor cells to escape from monitoring and killing of an immune system of a body. PD1 is a cell surface receptor belonging to the immunoglobulin superfamily that is expressed only on activated T lymphocytes. Structurally, the mature PD1 protein comprises an extracellular recognition domain, a transmembrane region, and an intracellular tail. Functionally, PD1 plays an important role in inhibiting T cell activity, has two phosphorylation site tails, an immunoreceptor tyrosine-based inhibition motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), both of which negatively regulate T cell receptor signaling.
Breast cancer is the most common malignant tumor that endangers the health of women, and metastasis is the main reason for the failure of clinical treatment of breast cancer. Epithelial-mesenchymal transition (EMT) is a key step in tumor metastasis, and is involved in multiple malignant processes such as occurrence and infiltration of breast cancer. Fibronectin (FN) is a functional glycoprotein in the extracellular matrix. Clinical data studies have shown that FN is poorly expressed in normal adult breast tissue, while expression levels are significantly elevated in tumor stroma. Integrin is a transmembrane glycoprotein receptor located in cells and shown to mediate intracellular and extracellular signaling, and FN can regulate various physiological functions of cells by binding to the integrin receptor through an RGD site.
With the application of molecular biological methods such as signal pathway, apoptosis and the like in tumor research, breast cancer molecular targets and targeted therapy gradually become trends and hot spots for anti-breast cancer research. In view of the limitations of traditional therapeutic approaches, immunotherapy, with its high specificity and memory, has the potential to improve the survival and prognosis of tumor patients. Immune checkpoints are the main reason for hindering the body's immune cells from resisting tumors and are the chief culprit in the development of immune escape. At present, aiming at immune check points, a method of adopting a monoclonal antibody inhibitor is mainly adopted to improve the anti-tumor effect. PDL1 and PDL2 are core targets for current tumor immunotherapy, in particular immune checkpoint blockade therapy. The inhibition of PDL1 and PDL2 functions can activate the anti-tumor immunity of T cells and kill tumor cells. The invention aims to research the application of RGDfK in preparing anti-PD-L1 medicaments and provide more theoretical basis for clinical practice of Cyclo-RGDfK.
Disclosure of Invention
The invention aims to provide a detection method for inhibiting PDL1 by RGDfK and application thereof in preparing anti-PDL 1 medicines, and provides more theoretical basis for clinical practice of Cyclo-RGDfK.
In order to solve the above technical problem, an embodiment of the present invention provides a method for detecting PDL1 suppressed by RGDfK, including the following steps:
(1) the expression regulation mechanism of PDL1 molecule on the surface of tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene and protein levels, respectively;
(2) detecting the level of phosphorylation of ERK in the tumor cell;
(3) the inhibition effect of RGDfK on tumor cell PDL1 is verified: after treatment of tumor cells with the RGDfK inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.
Wherein, in the step (1), the small molecule inhibitor comprises an inhibitor RGDFK of integrin alpha v beta 3.
Wherein, the tumor cells comprise breast cancer cell lines SUM159 and MDA-MB-231.
Wherein the treatment concentrations of the integrin α v β 3 inhibitor RGDFK are 2 and 5 μ M.
Wherein, the Western Blot method and the real-time quantitative PCR method are adopted in the step (1) to detect the protein level and the gene level expression of PDL 1.
Wherein, the step of treating the tumor cells by using the small molecule inhibitor in the step (1) comprises the following steps:
(1-1) resuscitating the cells: taking out the freezing tube from-80 deg.C, directly soaking in warm water of 37 deg.C, and shaking to melt it as soon as possible;
(1-2) taking out the cryopreservation tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing;
(1-3) centrifuging at 1000rpm for 3min, and discarding the supernatant;
(1-4) adding 10% FBS-containing DMEM medium to resuspend the cells, and uniformly inoculating the cells in a 60mm culture dish with 5% CO2Standing and culturing in an incubator at 37 ℃; replacing the culture solution once the next day, and continuing culturing;
(1-5) when the cell density reaches 90%, digesting the cells by pancreatin, and determining the number of the paved holes according to subsequent experiments; continuing culturing;
(1-6) RGDFK inhibitor treatment: the concentration of the selected treatment is 2, 5 mu M in sequence, and the treatment time is 24 h.
Wherein, the Western blotting step in step (1) is:
(1-7) preparation of protein sample: preparing a lysis buffer solution and placing the lysis buffer solution in an ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells with a lysate containing PMSF, and lysing for 30min on ice bath; centrifuging at 4 deg.C for 15min, and completely sucking supernatant; after mixing the protein with 5 × Loading buffer, the ratio of 4: 1, boiling in 95 deg.C metal bath for 5min, and storing at-20 deg.C;
(1-8) electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; cutting PVDF membrane into size same as cut protein gel, and balancing in membrane buffer for 3 min; the stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing the PVDF membrane for 3min by TBST, adding milk or primary antibody diluted by primary antibody diluent, and incubating overnight at 4 ℃; rinsing with TBST for 5min × 3 the next day, adding appropriate amount of secondary antibody, and incubating at room temperature for 2 h; finally, TBST was rinsed for 10min × 3 and developed using ECL reagent.
Wherein, the immunofluorescence detection in the step (3) comprises the following steps:
(3-1) quantitative RT-PCR
(3-1-1) detecting through gene transcription level, designing RT-PCR primers of PDL1 and PDL2 respectively, and verifying in SUM159 cells and MDA-MB-231 cells respectively; the primers are respectively as follows:
PDL1-RT-F：GTAGCACTGACATTCATCTTC
PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC
PDL2-RT-F：CATAGCCACAGTGATAGCCCT
PDL2-RT-R：GGCTCCCAAGACCACAGGTTC；
(3-1-2) extraction of total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; adding 1mL of RNA isolator, and standing at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, turning upside down, mixing, and standing at-20 deg.C for 2 hr; centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; the supernatant was discarded and 1mL of pre-cooled 75% ethanol was added. 7500r, centrifuging at 4 deg.C for 5min, and discarding supernatant as much as possible; drying and precipitating in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 deg.C water bath for 5min, measuring concentration, and storing at-80 deg.C;
(3-2) cDNA Synthesis and qPCR detection:
and (3) reversing the RNA extracted in the step (3-1-2) according to the instruction of the qPCR kit to synthesize cDNA, wherein the method comprises the following steps:
(3-2-1) removing a genome, and preparing the following mixed solution in an RNase-free centrifugal tube, wherein the template RNA is 1 mug; 4 XgDNA wiper Mix, 4 μ L; adding RNase-free ddH2O to 16 μ L, gently blowing and beating with a pipette, and mixing well at 42 deg.C for 2 min;
(3-2-2) preparing a reverse transcription reaction system, and directly adding 4 mu L of 5 xHiScript II qRT SuperMix II into the reaction tube in the step (3-2-1);
(3-2-3) carrying out reverse transcription reaction at 50 ℃ for 15min and 85 ℃ for 5s, adding water to dilute the product by 8 times, and storing at-20 ℃; gene expression levels of PDL1 and PDL2 were subsequently detected using a quantitative PCR detection kit;
(3-3) immunofluorescence assay
(3-3-1) allowing the cells to grow on a circular cover glass put in a 24-hole cell culture plate, wherein the inoculation density reaches 60%, and the cells are treated by adding medicine for 24 hours;
(3-3-2) washing the cells 3 times with 500. mu.L of PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature;
(3-3-3) removing paraformaldehyde, washing the cells 3 times with 500. mu.L PBS per well for 5min each time;
(3-3-4) adding 500. mu.L of 1% BSA to each well, and blocking at room temperature for 2 h;
(3-3-5) removing the blocking solution, adding 200 mu L of primary antibody into each hole, and standing overnight at 4 ℃;
(3-3-6) removing the antibody, washing the cells 3 times per well with 500. mu.L PBS for 5min each time;
(3-3-7) adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature;
(3-3-8) removing the secondary antibody, washing the cells 3 times with 500 PBS per well for 5min each time;
(3-3-9) adding Hochest into each hole, washing the cells for 3 times (5 min each time) at room temperature for 10min by 500 mu LPBS;
(3-3-10) dropping a small drop of the fading resistant agent on the slide glass, and taking the cover glass out of the well and covering the slide glass on the fading resistant agent with the surface facing down so as to be in contact with the slide glass;
(3-3-11) the slide is left to dry in the dark for 5min, at which time observation can be carried out under a fluorescence microscope.
The invention also provides application of RGDFK in preparation of anti-PDL 1 drugs.
The technical scheme of the invention has the following beneficial effects: the invention adopts the RGDfK inhibitor of integrin alphavbeta 3 to inhibit the expression of an immune check point PDL1 of tumor cells, effectively weakens the function of a PD1/PDL1 for inhibiting a signal path, and increases the anti-tumor activity of T cells. The small molecule inhibitor is utilized to improve the cytotoxicity of the T cell, and has more advantages compared with a monoclonal antibody, such as easy large-scale production and low cost; the composition is easy to infiltrate into the tumor tissue, and the anti-tumor activity is effectively exerted; the small molecule inhibitor also has other anti-tumor mechanisms and can play a combined anti-tumor effect. The RGDFK adopted by the invention is a potential anti-tumor drug with good development prospect, the expression of PDL1 is obviously reduced at the gene level, and feasibility and theoretical basis are hopefully provided for clinically treating breast cancer.
Drawings
FIG. 1 is a graph showing the expression of WB-detected PDL1 after RGDfK treatment of SUM159 and MDA-MB-231 cells in accordance with the present invention;
FIG. 2 is a diagram showing the expression of PDL1 detected by RT-PCR after treating SUM159 and MDA-MB-231 cells with RGDfK according to the present invention;
FIG. 3 is a graph showing the expression of p-ERK detected by WB after RGDfK treatment of SUM159 and MDA-MB-231 cells in accordance with the present invention;
FIG. 4 is a graph showing the expression of PD-L1 in immunofluorescence assay after treating SUM159 cells with RGDfK at various concentrations in the present invention;
FIG. 5 is a graph showing the expression of PD-L1 detected by immunofluorescence after MDA-MB-231 cells were treated with RGDFK at various concentrations in the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.
The invention provides a method for detecting PDL1 inhibited by RGDFK, which comprises the following steps:
(1) the expression regulation mechanism of PDL1 molecule on the surface of tumor cells is explored: treating tumor cells by using a small molecular inhibitor, and detecting the protein level and the gene level expression of PDL1 by respectively adopting a Western Blot method and a real-time quantitative PCR (RTPCR) method;
the small molecule inhibitor in this step is preferably the inhibitor RGDfK of integrin α v β 3 at treatment concentrations of 0, 2 and 5 μ M.
(2) Detecting the level of phosphorylation of ERK in the tumor cell;
(3) the inhibition effect of RGDfK on tumor cell PDL1 is verified: after treatment of tumor cells with the RGDfK inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.
The tumor cells include breast cancer cell lines SUM159 and MDA-MB-231.
The invention also provides application of RGDFK in preparation of anti-PDL 1 drugs.
The technical solution of the present invention is described in detail below with reference to specific examples.
To explore the expression regulation mechanism of PDL1 molecules on the surface of tumor cells, tumor cells were treated with different small molecule inhibitors, and the expression of PDL1 was characterized by gene and protein levels, respectively. It was found that the expression of RGDfK, an inhibitor of integrin α v β 3, treated breast cancer cell lines SUM159 and MDA-MB-231, PDL1 and PDL2 was significantly reduced. In the experiment, treatment concentrations of the integrin α v β 3 inhibitor RGDfK were selected to be 0, 2 and 5 μ M, respectively, and the protein level and gene level expression of PDL1 were detected by Western Blot method and real-time quantitative PCR (RTPCR) method.
1. Cell processing
1.1, reviving cells: taking out the frozen tube from-80 deg.C, directly soaking in warm water of 37 deg.C, and shaking to melt as soon as possible (instant slow freezing).
1.2, taking out the freezing tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing.
Centrifuging at 1.3 rpm and 1000rpm for 3min, and discarding the supernatant.
1.4 addition of DMEM containing 10% FBS for cultureCells were suspended in nutrient media, inoculated evenly in 60mm petri dishes, 5% CO2Standing and culturing in an incubator at 37 ℃; the culture solution was changed the next day and the culture was continued.
1.5, when the cell density reaches 90%, the cells are digested by pancreatin, the number of the spread holes (6-hole plate) is determined according to the subsequent experiment, and the culture is continued.
1.6 RGDFK inhibitor treatment: the concentration of the selected treatment is 0, 2 and 5 mu M in sequence, and the treatment time is 24 h.
2. Western Blot (WB)
2.1, preparation of protein sample: preparing Lysis buffer (containing 10 μ L PMSF and 990 μ L lysine buffer) and placing in ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells in a lysate containing PMSF, and lysing for 30min on ice bath (shaker); the supernatant (protein) was completely aspirated after high speed centrifugation at 4 ℃ for 15 min. After mixing the protein with 5 × Loading buffer, the ratio of 4: sucking at a ratio of 1, boiling in metal bath at 95 deg.C for 5min, and storing at-20 deg.C.
2.2, electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; cutting PVDF membrane into size same as cut protein gel, and balancing in membrane buffer for 3 min; the stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing PVDF membrane with TBST for 3min, adding milk or primary antibody (1: 1000) diluted by primary antibody diluent, and incubating overnight at 4 deg.C; rinsing with TBST for 5min × 3 the next day, adding appropriate amount of secondary antibody (1: 2000), and incubating at room temperature for 2 h; finally, TBST was rinsed for 10min × 3 and developed using ECL reagent. The protein level detection results are shown in fig. 1, and the result analysis shows that RGDFK can significantly inhibit the expression of PDL1 protein in SUM159 and MDA-MB-231 cells. Previous results indicated that expression of PDL1 and PDL2 in SUM159 and MDA-MB-231 cells was dependent on activation of ERK in tumor cells. In addition, the phosphorylation levels of ERK were measured before and after RGDfK treatment in both cells, and the results are shown in fig. 3, and the analysis of the results shows that RGDfK can significantly inhibit the phosphorylation levels of ERK in SUM159 and MDA-MB-231 cells.
3. Quantitative RT-PCR
The invention carries out detection through gene transcription level, RT-PCR primers of PDL1 and PDL2 are respectively designed for the detection, and are respectively verified in SUM159 cells and MDA-MB-231 cells. The primers are respectively as follows:
PDL1-RT-F：GTAGCACTGACATTCATCTTC
PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC
PDL2-RT-F：CATAGCCACAGTGATAGCCCT
PDL2-RT-R：GGCTCCCAAGACCACAGGTTC
extracting total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; 1mL of RNA isolater (Novozam R401-01) was added and left at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, mixing by turning upside down, and standing at-20 deg.C for 2h (increasing precipitation); centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; the supernatant was discarded and 1mL of pre-cooled 75% ethanol (in DEPC water) was added. 7500r, centrifuging at 4 deg.C for 5min, and discarding supernatant as much as possible; drying the precipitate in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 water bath for 5min, measuring concentration, and storing at-80 deg.C.
Synthesis of cDNA and qPCR detection:
the RNA after the above measurements was inverted to synthesize cDNA according to the instructions of HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Norzan R223-01). The method comprises the following specific steps: removing a genome, and preparing a mixed solution with a template RNA of 1 mu g in an RNase-free centrifugal tube; 4 XgDNA wiper Mix, 4 μ L; RNase-free ddH2O was made up to 16. mu.L and gently pipetted and mixed well at 42 ℃ for 2 min. Preparing a reverse transcription reaction system, and directly adding 4 mu L of 5 XHiScript II qRT SuperMix II into the reaction tube in the step I. ③ carrying out reverse transcription reaction at 50 ℃ for 15min and 85 ℃ for 5 s. The product is diluted 8 times by adding water and stored at-20 ℃. The AceQ qPCR SYBR Green Master Mix (Novozam Q111-02) kit was then used to detect the gene expression levels of PDL1 and PDL 2.
The mRNA level detection results are shown in FIG. 2, and the result analysis shows that RGDfK can significantly inhibit the transcription of PDL1 gene in SUM159 and MDA-MB-231 cells.
To further verify the inhibitory effect of RGDfK on tumor cell PDL1, the protein expression level of PDL1 in the cells was examined using immunofluorescence techniques. Next, the expression level of PDL1 was detected in SUM159 and MDA-MB-231 cells using immunofluorescence techniques, with RGDFK treatment concentrations selected to be 0, 2, and 5. mu.M, respectively, in the experiment.
4. Immunofluorescence
Cells were grown on round coverslips placed in 24-well cell culture plates, the seeding density reached 60%, and cells were treated with drug for 24 h. Cells were washed 3 times with 500 μ L PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature; removing paraformaldehyde, washing cells with 500 μ L PBS for 5min each time for 3 times; adding 500 mu L of 1% BSA into each hole, and blocking for 2h at room temperature; removing blocking solution, adding 200. mu.L primary antibody (BSA dilution) to each well, and standing overnight at 4 ℃; removing antibody, washing cells with 500 μ LPBS for 5min 3 times per well; adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature; remove the secondary antibody, wash the cells 3 times with 500 PBS per well for 5min each time; add Hochest (1: 200PBS dilution) to each well, wash cells 3 times at room temperature for 10min, 5min each time at 500. mu. LPBS; dropping a small drop of anti-fade agent on the slide, removing the cover slip from the well, covering it face down onto the anti-fade agent, and gently touching it (carefully bubbling) onto the slide; the slide was left to air dry in the dark for 5min, at which time it was observed under a fluorescent microscope.
The detection results of SUM159 cells are shown in fig. 4, and the results analyze that the immunofluorescence intensity is obviously weakened after the SUM159 cells are treated by RGDfK, which indicates that the protein level of PDL1 is obviously reduced. Similar experimental results were also confirmed in MDA-MB-231 cells, and the results are shown in FIG. 5.
In conclusion, the RGDFK inhibitor of integrin α v β 3 can significantly inhibit the expression of PDL1 gene in breast cancer cells, and probably reduces the transcription of PDL1 gene by reducing the phosphorylation level of ERK, and finally inhibits the expression of PDL1 in tumor cells. The tumor immune escape of the breast cancer is avoided, so that the inhibitor RGDfK of the integrin alpha v beta 3 can enhance the immune treatment effect of T cells in the breast cancer, and is expected to provide feasible and theoretical basis for clinically treating the breast cancer.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of southeast Tong
<120> RGDfK PDL1 inhibition detection method and application thereof in preparation of anti-PDL 1 drugs
<141> 2020-12-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> Artificial Synthesis (Artificial Synthesis)
<400> 1
Gly Thr Ala Gly Cys Ala Cys Thr Gly Ala Cys Ala Thr Thr Cys Ala
1 5 10 15
Thr Cys Thr Thr Cys
20
Claims (7)
1. A method for detecting PDL1 inhibited by RGDFK is characterized by comprising the following steps:
(1) the expression regulation mechanism of PDL1 molecule on the surface of tumor cells is explored: treating tumor cells with a small molecule inhibitor, characterizing the expression of PDL1 by gene and protein levels, respectively;
wherein the small-molecule inhibitor is selected from an inhibitor RGDfK of integrin alpha v beta 3, and the treatment concentration of the inhibitor RGDfK of the integrin alpha v beta 3 is 2 and 5 mu M;
(2) detecting the level of phosphorylation of ERK in the tumor cell;
(3) the inhibition effect of RGDfK on tumor cell PDL1 is verified: after treatment of tumor cells with the RGDfK inhibitor, the protein expression level of PDL1 in the cells was detected by immunofluorescence technique.
2. The method for detecting PDL1 inhibited by RGDfK according to claim 1, wherein the tumor cells comprise breast cancer cell lines SUM159 and MDA-MB-231.
3. The method for detecting PDLK 1 inhibited by RGDfK according to claim 1, wherein the expression of PDL1 protein level and gene level is detected in step (1) by Western Blot method and real-time quantitative PCR method.
4. The method for detecting PDL1 inhibited by RGDFK according to claim 1, wherein the step of treating the tumor cells with the small molecule inhibitor in step (1) is:
(1-1) resuscitating the cells: taking out the freezing tube from-80 deg.C, directly soaking in warm water of 37 deg.C, and shaking to melt it as soon as possible;
(1-2) taking out the cryopreservation tube from the water bath at 37 ℃, adding 1mL of complete culture medium, and uniformly mixing;
(1-3) centrifuging at 1000rpm for 3min, and discarding the supernatant;
(1-4) adding 10% FBS-containing DMEM medium to resuspend the cells, and uniformly inoculating the cells in a 60mm culture dish with 5% CO2Standing and culturing in an incubator at 37 ℃; replacing the culture solution once the next day, and continuing culturing;
(1-5) when the cell density reaches 90%, digesting the cells by using pancreatin, determining the number of the paved holes according to a subsequent experiment, and continuously culturing;
(1-6) RGDFK inhibitor treatment: the concentration of the selected treatment is 2, 5 mu M in sequence, and the treatment time is 24 h.
5. The method for detecting PDL1 inhibited by RGDFK according to claim 1, wherein the Western blotting step in step (1) is:
(1-7) preparation of protein sample: preparing a lysis buffer solution and placing the lysis buffer solution in an ice bath for later use; washing cells by precooling PBS, transferring the cells to a sterilized centrifuge tube, centrifuging and removing a supernatant; resuspending the cells with a lysate containing PMSF, and lysing for 30min on ice bath; centrifuging at 4 deg.C for 15min, and completely sucking supernatant; after mixing the protein with 5 × Loading buffer, the ratio of 4: 1, boiling in 95 deg.C metal bath for 5min, and storing at-20 deg.C;
(1-8) electrophoresis glue running and film transferring color development: boiling the protein sample again for 5min at 105 ℃; preparing 10% separation gel according to the formula of the protein gel, preparing 5% concentrated gel on the upper layer, and adding 10 μ L protein sample into each hole; adjusting the voltage of the electrophoresis apparatus to 80V, and adjusting the voltage of the electrophoresis apparatus to 120V again when the bromophenol blue indicator reaches the lower layer separation gel; according to a standard protein marker, a sample area to be inspected is cut off; cutting PVDF membrane into size same as cut protein gel, and balancing in membrane buffer for 3 min; the stacking sequence is as follows: performing wet rotation for 1h at 300mA for a power supply anode, filter paper, a PVDF (polyvinylidene fluoride) membrane, gel, filter paper and a power supply cathode; putting the PVDF membrane in 5% skimmed milk powder, and sealing for 2 h; rinsing the PVDF membrane for 3min by TBST, adding milk or primary antibody diluted by primary antibody diluent, and incubating overnight at 4 ℃; rinsing with TBST for 5min × 3 the next day, adding appropriate amount of secondary antibody, and incubating at room temperature for 2 h; finally, TBST was rinsed for 10min × 3 and developed using ECL reagent.
6. The method for detecting PDL1 inhibited by RGDFK according to claim 1, wherein the step of immunofluorescence detection in step (3) is:
(3-1) quantitative RT-PCR
(3-1-1) detecting through gene transcription level, designing RT-PCR primers of PDL1 and PDL2 respectively, and verifying in SUM159 cells and MDA-MB-231 cells respectively; the primers are respectively as follows:
PDL1-RT-F：GTAGCACTGACATTCATCTTC
PDL1-RT-R：TTCCTTCCTCTTGTCACGCTC
PDL2-RT-F：CATAGCCACAGTGATAGCCCT
PDL2-RT-R：GGCTCCCAAGACCACAGGTTC；
(3-1-2) extraction of total RNA of cells: total RNA was extracted from the above treated cells by Trizol method as follows: discarding culture solution of cells, washing once by using PBS, adding pancreatin to digest cells and collecting in an EP tube without RNA enzyme; adding 1mL of RNA isolator, and standing at room temperature for 5 min; adding 200 μ L chloroform, shaking vigorously for 15s, and standing at room temperature for 3 min; centrifuging at 12000r at 4 deg.C for 15min, separating into three layers, carefully sucking the upper layer into a new EP tube without RNase, adding 0.5ml isopropanol, turning upside down, mixing, and standing at-20 deg.C for 2 hr; centrifuging at 12000r at 4 deg.C for 10min to obtain white precipitate at the bottom of the tube; discarding the supernatant, adding 1mL of precooled 75% ethanol, 7500r, centrifuging at 4 ℃ for 5min, and discarding the supernatant as much as possible; drying and precipitating in a fume hood for 10-30min, adding 30 μ L DEPC water to dissolve RNA, heating in 65 deg.C water bath for 5min, measuring concentration, and storing at-80 deg.C;
(3-2) cDNA Synthesis and qPCR detection:
and (3) reversing the RNA extracted in the step (3-1-2) according to the instruction of the qPCR kit to synthesize cDNA, wherein the method comprises the following steps:
(3-2-1) removing a genome, and preparing the following mixed solution in an RNase-free centrifugal tube, wherein the template RNA is 1 mug; 4 XgDNA wiper Mix, 4 μ L; adding RNase-free ddH2O to 16 μ L, gently blowing and beating with a pipette, and mixing well at 42 deg.C for 2 min;
(3-2-2) preparing a reverse transcription reaction system, and directly adding 4 mu L of 5 xHiScript II qRT SuperMix II into the reaction tube in the step (3-2-1);
(3-2-3) carrying out reverse transcription reaction at 50 ℃ for 15min and 85 ℃ for 5s, adding water to dilute the product by 8 times, and storing at-20 ℃; gene expression levels of PDL1 and PDL2 were subsequently detected using a quantitative PCR detection kit;
(3-3) immunofluorescence assay
(3-3-1) allowing the cells to grow on a circular cover glass put in a 24-hole cell culture plate, wherein the inoculation density reaches 60%, and the cells are treated by adding medicine for 24 hours;
(3-3-2) washing the cells 3 times with 500. mu.L of PBS per well for 5min each time; then adding 200ul of 4% paraformaldehyde into each hole, and fixing for 15min at room temperature;
(3-3-3) removing paraformaldehyde, washing the cells 3 times with 500. mu.L PBS per well for 5min each time;
(3-3-4) adding 500. mu.L of 1% BSA to each well, and blocking at room temperature for 2 h;
(3-3-5) removing the blocking solution, adding 200 mu L of primary antibody into each hole, and standing overnight at 4 ℃;
(3-3-6) removing the antibody, washing the cells 3 times per well with 500. mu.L PBS for 5min each time;
(3-3-7) adding 200 mu L of fluorescent secondary antibody into each hole, and incubating for 2h at room temperature;
(3-3-8) removing the secondary antibody, washing the cells 3 times with 500 PBS per well for 5min each time;
(3-3-9) adding Hochest into each hole, washing the cells for 3 times (5 min each time) at room temperature for 10min by 500 mu LPBS;
(3-3-10) dropping a small drop of the fading resistant agent on the slide glass, and taking the cover glass out of the well and covering the slide glass on the fading resistant agent with the surface facing down so as to be in contact with the slide glass;
(3-3-11) the slide is left to dry in the dark for 5min, at which time observation can be carried out under a fluorescence microscope.
7. Application of RGDFK in preparing anti-PDL 1 medicine is provided.
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