CN110568191A - oral cavity phosphorus cell cancer marker and application thereof - Google Patents
oral cavity phosphorus cell cancer marker and application thereof Download PDFInfo
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Abstract
The application provides an oral cavity phosphorus cell cancer marker and application thereof, and more particularly provides a kit for detecting a marker reagent in a saliva exosome, application of the reagent for detecting the oral cavity exosome marker in preparation of the kit and a detection analysis system. The kit is for use in at least one of: detecting squamous cell carcinoma; determining the stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for squamous cell carcinoma, and wherein the kit comprises: a reagent for detecting a marker in a saliva exosome, the marker comprising POSTN. The POSTN protein can be used as a biomarker of phosphorus cell cancer.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to an oral cavity phosphorus cell cancer marker and application thereof, and more particularly relates to a kit for detecting a marker reagent in saliva exosomes, application of the reagent for detecting the oral cavity exosome marker in preparation of the kit and a detection and analysis system.
background
Oral Squamous Cell Carcinoma (OSCC) is a widespread malignancy of the oral and maxillary tissues. Oral squamous cell carcinoma is prone to lymph node metastasis.
exosomes are vesicles in the human body fluid. The diameter of the exosome is 30-120 nm, and the exosome has the function of transporting various substances in a long distance.
However, how to rapidly screen patients with oral squamous cell carcinoma and how to screen patients with oral squamous cell carcinoma metastasis easily are key problems in the active prevention and treatment of oral squamous cell carcinoma and the confirmation of a suitable treatment scheme.
Disclosure of Invention
the present application is based on the discovery by the inventors of the following problems and facts:
The inventors screened saliva exosomes of 20 oral cavity phoma patients for proteins, 10 of the 20 oral cavity phoma patients were metastatic oral cavity phoma cancer, 10 were non-metastatic oral cavity phoma cancer, and 10 healthy patients without oral cavity phoma cancer were enrolled as controls, and the 30 subjects were similar in age and sex. The inventors surprisingly found that POSTN is highly expressed in saliva exosomes of oral squamous cell carcinoma patients, while POSTN is not substantially expressed in saliva exosomes of healthy people; in addition, the inventors found that the expression level of POSTN in patients with metastatic squamous cell carcinoma was significantly higher than that in patients with non-metastatic squamous cell carcinoma. Meanwhile, the inventors performed protein expression profiling in salivary exosomes and found that the other two proteins, TNC and TGFBI, had similar expression differences in patients and healthy persons as POSTN.
based on the above findings, in a first aspect of the present invention, the present invention provides a kit. According to an embodiment of the invention, the kit is for at least one of: detecting squamous cell carcinoma; determining the stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for squamous cell carcinoma, and wherein the kit comprises: a reagent for detecting a marker in a saliva exosome, the marker comprising POSTN. As mentioned above, the POSTN protein can be used as a biomarker of phosphorus-like cell carcinoma. As described above, the inventors found that the expression level of POSTN in saliva exosomes is significantly different between healthy subjects and patients with squamous cell carcinoma, and the expression level of POSTN in saliva exosomes in patients with non-metastatic squamous cell carcinoma and patients with metastatic squamous cell carcinoma is also significantly different, so that the detection of the expression level of POSTN in saliva exosomes by using the kit according to the embodiment of the present invention can effectively and accurately detect squamous cell carcinoma and determine the stage of squamous cell carcinoma in a subject; if a squamous cell carcinoma patient is to receive a certain treatment, then with a kit according to an embodiment of the present invention, the treatment effect of the squamous cell carcinoma patient as well as the optimal treatment regimen for the squamous cell carcinoma can also be determined.
According to an embodiment of the present invention, the kit may further comprise at least one of the following additional technical features:
According to an embodiment of the invention, the squamous cell carcinoma is an oral squamous cell carcinoma and the kit further comprises an agent for isolating salivary exosomes. According to a specific embodiment of the invention, the reagent is a 10% polyethylene glycol solution. By using the kit provided by the embodiment of the invention, the saliva exosomes can be efficiently separated, the obtained exosomes have high purity without damaging the active ingredients in the exosomes, and further the expression quantity of POSTN in the saliva exosomes is detected, the quantitative accuracy of POSTN is further improved, and the detection accuracy of oral squamous cell carcinoma is further improved.
According to an embodiment of the invention, the marker further comprises at least one selected from TNC and TGFBI. As described above, the inventors found that the expression levels of TNC, TGFBI and POSTN in exosomes change consistently in normal humans and cancer patients, and therefore, further detecting at least one of TNC and TGFBI using the kit of the embodiment of the present invention can further improve the sensitivity and accuracy of detecting squamous cell carcinoma.
according to an embodiment of the invention, the reagent for detecting the marker is adapted to detect the marker by at least one of western blotting, enzyme linked immunosorbent assay, and time-of-flight mass spectrometry. The expression amount or the relative expression amount of the marker can be effectively quantified or semi-quantified by using the method according to the embodiment of the invention, and the accuracy and the sensitivity of detecting the phosphorus cell carcinoma by using the kit according to the embodiment of the invention are further improved.
In a second aspect of the invention, the invention proposes the use of an agent for detecting an oral exosome marker in the preparation of a kit. According to an embodiment of the invention, the oral exosome marker comprises POSTN, the kit being for use in at least one of: detecting squamous cell carcinoma; determining the diseased stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for the squamous cell carcinoma. It should be noted that the detection of squamous cell carcinoma as described herein can refer to the detection of actual ongoing squamous cell carcinoma, the detection of the risk of oral squamous cell carcinoma in healthy persons, or the risk of recurrence of squamous cell carcinoma after other treatment regimens have been performed. According to the embodiment of the invention, the expression level of the POSTN is detected by using a reagent for detecting the oral exosome marker POSTN, and then whether the patient has the phosphorus-like cell carcinoma or not, whether the patient has the risk of having the phosphorus-like cell carcinoma or not, whether the patient has the risk of relapsing the phosphorus-like cell carcinoma or not, determining the diseased stage of the phosphorus-like cell carcinoma, determining the treatment effect of the phosphorus-like cell carcinoma and determining the treatment scheme of the phosphorus-like cell carcinoma are determined according to whether the marker is expressed or the expression level is high or low.
In a third aspect of the invention, a detection analysis system is presented. According to an embodiment of the invention, the system is adapted for at least one selected from the group consisting of: detecting squamous cell carcinoma; determining the stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for squamous cell carcinoma, and the detection analysis system comprises: a detection device for determining the amount of a marker comprising at least one selected from POSTN, TNC and TGFBI in saliva exosomes of a subject, optionally the subject has undergone treatment or treatment with a candidate treatment regimen; an analysis device connected to the detection device and adapted to determine, based on the content of the marker: whether the subject is suffering from squamous cell carcinoma; a diseased stage of squamous cell carcinoma in the subject; the therapeutic effect of the squamous cell carcinoma patient; a treatment regimen for said squamous cell carcinoma. With the detection analysis system according to the embodiment of the present invention, it is possible to effectively detect squamous cell carcinoma, including whether a subject has squamous cell carcinoma, is in a metastatic stage of the few, if treated, how effective the treatment is, and whether the treatment regimen is effective. The detection analysis system for detecting squamous cell carcinoma has high detection accuracy and high sensitivity.
According to an embodiment of the present invention, the detection analysis system may further include at least one of the following additional technical features:
According to an embodiment of the invention, the detection and analysis system further comprises a saliva exosome-isolating means for isolating exosomes from saliva of the subject. And then the detection device utilizes the exosomes separated by the saliva exosome separation device to determine the content of the marker in the saliva exosomes.
According to an embodiment of the invention, the detection device further comprises at least one of the following units: a western blot unit; an enzyme-linked immunoassay unit; and a time-of-flight mass spectrometry unit. At least one of the units is utilized to realize the quantification or semi-quantification of the content of the marker, so that the quantification of the marker is more accurate and the reliability is higher.
According to an embodiment of the invention, the analysis device comprises: a comparison unit for comparing the content of the marker of the subject with at least one of: a first predetermined threshold determined based on marker levels in salivary exosomes of individuals known not to suffer from squamous cell carcinoma; a second predetermined threshold determined based on the marker content in salivary exosomes of individuals known to be in a metastatic stage of squamous cell carcinoma; (ii) marker levels in saliva exosomes of the patient prior to the treatment; (ii) marker levels in saliva exosomes of controls not subjected to the treatment regimen; a determination unit coupled to the comparison unit for determining at least one of the following based on the comparison unit: whether the subject suffers from squamous cell carcinoma, wherein a level of the marker in the subject above the first threshold is indicative of the subject suffering from squamous cell carcinoma; a diseased stage of squamous cell carcinoma in the subject, wherein a level of the marker in the subject above the second threshold is indicative of a metastatic stage of squamous cell carcinoma; a therapeutic effect of said squamous cell carcinoma patient, wherein an amount of said marker in said subject that is not higher than an amount of a marker in saliva exosomes of said patient prior to said treatment is indicative of said treatment being effective; a treatment regimen for said squamous cell carcinoma, wherein a candidate treatment regimen having the ability to alter the level of said marker in said subject is selected as a final regimen. With the above-described analysis device according to the embodiment of the present invention, it is possible to effectively determine whether a subject suffers from squamous cell carcinoma, a diseased stage of squamous cell carcinoma of the subject, a therapeutic effect of a squamous cell carcinoma patient, or a treatment regimen of squamous cell carcinoma.
Drawings
FIG. 1 is a schematic diagram of a detection and analysis system according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a detection and analysis system according to an embodiment of the present invention;
FIG. 3 is a schematic structural view of an analysis apparatus according to an embodiment of the present invention;
FIG. 4 is a picture of salivary exosomes from metastatic oral squamous cell carcinoma patients, non-metastatic oral squamous cell carcinoma patients and healthy control groups and experimental validation results for exosomes extracted according to an embodiment of the present invention;
FIG. 5 is a graph of the results of comparing salivary exosome proteins in metastatic oral squamous cell carcinoma patients to non-metastatic oral squamous cell carcinoma patients according to an embodiment of the present invention; and
FIG. 6 is a graph showing the results of detecting the expression levels of POSTN, TNC and TGFBI in healthy control group, non-metastatic oral squamous cell carcinoma patients and metastatic oral squamous cell carcinoma patients by Western blotting, according to an embodiment of the present invention.
Detailed Description
the following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
reagent kit
in a first aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit is for at least one of: detecting squamous cell carcinoma; determining the stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for squamous cell carcinoma, and wherein the kit comprises: a reagent for detecting a marker in a saliva exosome, the marker comprising POSTN. The inventor unexpectedly found in experiments that the POSTN protein is expressed at a high level in saliva exosomes of phosphorus cell carcinoma patients, but is not expressed in saliva exosomes of healthy people, and the expression of the POSTN protein in saliva exosomes of metastatic phosphorus cell carcinoma patients is higher than that of non-metastatic phosphorus cell carcinoma patients. Thus, the POSTN protein can be used as a biomarker for phosphoid cell carcinoma and an indicator for determining whether or not phosphoid cell carcinoma metastasizes. By using the kit provided by the embodiment of the invention to detect the expression amount of POSTN in saliva exosomes, squamous cell carcinoma can be effectively and accurately detected, the stage of the squamous cell carcinoma of a subject can be determined, the treatment effect of a squamous cell carcinoma patient can be determined, and the treatment scheme of the squamous cell carcinoma can be determined.
according to a particular embodiment of the invention, the squamous cell carcinoma is oral squamous cell carcinoma and the kit further comprises an agent for isolating salivary exosomes, in particular, the agent is a 10% polyethylene glycol solution. By using the kit provided by the embodiment of the invention, the saliva exosomes can be efficiently separated, and the expression quantity of POSTN in the saliva exosomes can be further detected. The kit provided by the embodiment of the invention is used for detecting the oral cavity phosphorus cell carcinoma, so that the accuracy is further improved.
according to still another specific embodiment of the present invention, the marker further comprises at least one selected from TNC and TGFBI. The inventor finds that the expression quantity change trends of TNC and TGFBI in exosomes in normal human bodies and cancer patients are consistent with that of POSTN through protein mass spectrometry, and the sensitivity and the accuracy of detecting the phosphorus cell carcinoma can be further improved by further detecting at least one of TNC and TGFBI by using the kit provided by the invention.
The detection means suitable for the reagent for detecting a marker is not particularly limited as long as the expression amount or relative expression amount of the marker can be quantified or semiquantified. According to a specific embodiment of the present invention, the reagent for detecting a marker is suitable for detecting the marker by at least one of western blotting, enzyme linked immunosorbent assay, and time-of-flight mass spectrometry. The expression amount or the relative expression amount of the marker can be effectively quantified or semi-quantified by using the method according to the embodiment of the invention, and the accuracy and the sensitivity of detecting the phosphorus cell carcinoma by using the kit according to the embodiment of the invention are further improved.
Use of
in a second aspect of the invention, the invention proposes the use of an agent for detecting an oral exosome marker in the preparation of a kit. According to an embodiment of the invention, the oral exosome marker comprises POSTN, the kit being for use in at least one of: detecting squamous cell carcinoma; determining the diseased stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for the squamous cell carcinoma. The detection of squamous cell carcinoma as described herein can refer to either the detection of actual ongoing squamous cell carcinoma, the detection of a healthy person at risk of oral squamous cell carcinoma, or the detection of the presence or absence of recurrent squamous cell carcinoma following the implementation of other treatment regimens. According to the embodiment of the invention, the reagent for detecting the oral exosome marker POSTN is utilized to detect whether a patient has the phosphorus-like cell carcinoma, whether the patient has the risk of relapse of the phosphorus-like cell carcinoma, determine the diseased stage of the phosphorus-like cell carcinoma, determine the treatment effect of the phosphorus-like cell carcinoma and determine the treatment scheme of the phosphorus-like cell carcinoma according to the expression level of the marker.
unless otherwise stated, the reagent for detecting oral exosome markers described in the present application includes a reagent capable of specifically recognizing oral exosome markers, such as an antibody, a probe, and the like, and the reagent is suitable for western blotting, enzyme linked immunosorbent assay, time-of-flight mass spectrometry, and the like, so that the expression amount or relative expression amount of the markers can be effectively detected.
detection system
In a third aspect of the invention, a detection analysis system is presented. According to an embodiment of the invention, the system is adapted for at least one selected from the group consisting of: detecting squamous cell carcinoma; determining the stage of squamous cell carcinoma in the subject; determining the efficacy of treatment of a patient with squamous cell carcinoma; and determining a treatment regimen for squamous cell carcinoma, and the detection analysis system comprises, with reference to fig. 1: a test device 100, the test device 100 for determining the level of a marker comprising at least one selected from POSTN, TNC and TGFBI in saliva exosomes of a subject, optionally the subject has undergone treatment or treatment with a candidate treatment regimen; an analysis device 200, said analysis device 200 being connected to said detection device 100 and being adapted to determine, based on the content of said marker: whether the subject is suffering from squamous cell carcinoma; a diseased stage of squamous cell carcinoma in the subject; the therapeutic effect of the squamous cell carcinoma patient; a treatment regimen for said squamous cell carcinoma. By utilizing the detection analysis system provided by the embodiment of the invention, the phosphorus cell carcinoma can be effectively detected, and the detection accuracy and the sensitivity are high.
According to yet another embodiment of the present invention, referring to fig. 2, the detection and analysis system further comprises a saliva exosome-isolating device 300 for isolating exosomes from saliva of the subject. Further, the detection apparatus 100 determines the content of the marker in the saliva exosomes by using the exosomes separated by the saliva exosome separation apparatus 300.
According to yet another embodiment of the present invention, the detecting device 100 further comprises at least one of the following units: a protein co-immunoprecipitation unit; a western blot unit; an enzyme-linked immunoassay unit; a time-of-flight mass spectrometry unit; and a protein sequencing unit. At least one of the units is utilized to realize the quantification or semi-quantification of the content of the marker, so that the quantification of the marker is more accurate and the reliability is higher.
According to still another embodiment of the present invention, referring to fig. 3, the analysis apparatus 200 includes: a comparison unit 210, the comparison unit 210 for comparing the content of the marker of the subject with at least one of: a first predetermined threshold determined based on marker levels in salivary exosomes of individuals known not to suffer from squamous cell carcinoma; a second predetermined threshold determined based on the marker content in salivary exosomes of individuals known to be in a metastatic stage of squamous cell carcinoma; (ii) marker levels in saliva exosomes of the patient prior to the treatment; (ii) marker levels in saliva exosomes of controls not subjected to the treatment regimen; a determining unit 220, the determining unit 220 being connected to the comparing unit 210 and configured to determine at least one of the following based on the comparing unit 210: whether the subject suffers from squamous cell carcinoma, wherein a level of the marker in the subject above the first threshold is indicative of the subject suffering from squamous cell carcinoma; a diseased stage of squamous cell carcinoma in the subject, wherein a level of the marker in the subject above the second threshold is indicative of a metastatic stage of squamous cell carcinoma; a therapeutic effect of said squamous cell carcinoma patient, wherein an amount of said marker in said subject that is not higher than an amount of a marker in saliva exosomes of said patient prior to said treatment is indicative of said treatment being effective; a treatment regimen for said squamous cell carcinoma, wherein a candidate treatment regimen having the ability to alter the level of said marker in said subject is selected as a final regimen. With the above-described analysis device according to the embodiment of the present invention, it is possible to effectively determine whether a subject suffers from squamous cell carcinoma, a diseased stage of squamous cell carcinoma of the subject, a therapeutic effect of a squamous cell carcinoma patient, or a treatment regimen of squamous cell carcinoma.
method for detecting squamous cell carcinoma
in a fourth aspect of the invention, a method of detecting squamous cell carcinoma is presented. According to an embodiment of the invention, the method comprises: (1) determining the amount of a marker comprising POSTN in saliva exosomes of a subject; (2) determining the risk of the subject suffering from squamous cell carcinoma based on the content of the marker in step (1). With the above method, the risk of the subject suffering from squamous cell carcinoma can be effectively determined.
it should be noted that the term "content" as used herein is to be understood in a broad sense, and can refer to both quantitative and semiquantitative results, both absolute and relative. In addition, the term "determining the risk of the subject for squamous cell carcinoma" as used herein may include having cancer as well as having the potential to have cancer.
according to a particular embodiment of the invention, the squamous cell carcinoma is an oral squamous cell carcinoma.
According to yet another embodiment of the present invention, the above method may further comprise isolating saliva exosomes of the subject by: (1) placing the frozen saliva sample in a room-temperature water bath until the saliva sample is completely dissolved, and storing the sample at 4 ℃ or on an ice bath after the saliva sample is dissolved; (2) centrifuging the sample at 2000g for 10 minutes, and taking the supernatant to 2 new tubes; (3) taking 1mL of supernatant, and adding 0.5mL of separation solution A; (4) mixing with vortex oscillator to obtain homogeneous term; (5) incubating for 1 hour at 4 ℃ in a refrigerator; (6) centrifuging at 4 ℃ for 1 hour at 10000 g; (7) sucking off the supernatant and retaining the bottom precipitate; (8) centrifuging at 4 ℃ for 5 minutes at 10000 g; (9) sucking off the supernatant and retaining the bottom precipitate; (10) adding 100 μ L of separation solution B for resuspension and precipitation, and mixing with vortex oscillator or pipettor; (11) samples were stored in aliquots and stored at 4 ℃ for one week or frozen for longer periods. The inventor finds that the separation liquid A10% polyethylene glycol solution is a key reagent for separating saliva exosomes, can efficiently separate the saliva exosomes without damaging active ingredients in the exosomes, and B is PBS. By utilizing the method provided by the embodiment of the invention, the saliva exosomes can be efficiently separated, the active ingredients of the obtained saliva exosomes are not lost, and the content of the marker in the saliva exosomes is more accurately and reliably detected.
According to a specific embodiment of the present invention, the marker further comprises at least one selected from TNC and TGFBI. The inventor finds that TNC and TGFBI are highly expressed in saliva exosomes of oral cavity phosphorus cell carcinoma patients in experiments, and the expression in the saliva exosomes of metastatic phosphorus cell carcinoma patients is higher than that of non-metastatic phosphorus cell carcinoma. By further detecting the change of the content of TNC and TGFBI by using the detection method of the embodiment of the invention, the accuracy of determining the risk of the subject suffering from squamous cell carcinoma based on the content of POSTN, TNC and TGFBI can be further improved.
According to an embodiment of the invention, in step (2), the marker is detected by at least one of western blotting, enzyme linked immunosorbent assay, time-of-flight mass spectrometry.
According to a specific embodiment of the present invention, the western blotting method can realize the detection of the saliva exosome marker by the following method: extracting total protein of saliva exosome, separating the total protein by polyacrylamide gel electrophoresis, and further transferring the total protein from the polyacrylamide gel to a PVDF membrane. The PVDF membrane to which the protein has been transferred is incubated with an antibody specific to the marker (hereinafter referred to as "primary antibody"). The greater the amount of marker, the more primary antibody binds to it. The primary antibody was washed away, and the membrane was further incubated with a secondary antibody (hereinafter referred to as "secondary antibody") having a detectable label to bind the primary antibody to the secondary antibody. The amount of the marker can be qualitatively determined by washing off the free secondary antibody and detecting the amount of the secondary antibody bound to the primary antibody, i.e., the greater the amount of the marker, the stronger the signal of the detected secondary antibody.
according to the specific embodiment of the invention, the enzyme-linked immunosorbent assay can realize the detection of the saliva exosome marker by the following modes: total saliva exosome proteins are extracted and the marker proteins are bound to a solid support by incubation (e.g.40-well polystyrene well plates). Further incubation with primary antibody was used to bind the marker protein to the primary antibody. The free primary antibody is washed away. An enzyme-labeled secondary antibody is added and the primary antibody is combined with the enzyme-labeled secondary antibody. And washing off free enzyme-labeled secondary antibody. Adding enzyme reaction substrate and stopping reaction timely. By detecting the amount of the enzyme reaction product, the amount of the marker can be qualitatively and quantitatively judged. I.e., the greater the amount of marker, the greater the amount of enzyme reaction product detected.
according to a specific embodiment of the present invention, the time-of-flight mass spectrometry can achieve the detection of salivary exosome markers by: and extracting total protein of the saliva exosome to prepare a sample. The sample is analyzed by a time-of-flight mass spectrometer, and a spectrogram can be obtained. By analyzing the spectrogram, the content of the marker protein can be qualitatively and quantitatively analyzed.
According to an embodiment of the present invention, in the step (2), further comprising: comparing the level of the marker in saliva exosomes of the subject to a predetermined threshold, wherein a level of the marker above a first predetermined threshold is indicative of the subject suffering from squamous cell carcinoma. According to a particular embodiment of the invention, said first predetermined threshold is determined based on the amount of markers in saliva exosomes of individuals known not to suffer from squamous cell carcinoma. According to a further embodiment of the present invention, the first predetermined threshold is determined based on a statistical analysis of the levels of markers in saliva exosomes of a plurality of individuals known not to suffer from squamous cell carcinoma.
wherein the first predetermined threshold is determined based on the method used to determine the amount of marker. According to an embodiment of the present invention, when the amount of marker POSTN in saliva exosomes of the subject is determined by western blot method, since the amount of marker POSTN in saliva exosomes of individuals not suffering from squamous cell carcinoma is zero, it is an indication that the subject suffers from squamous cell carcinoma as long as the expression of marker POSTN in saliva exosomes of the subject is detected.
Method for determining the diseased stage of squamous cell carcinoma in a subject
In a fifth aspect of the invention, a method of determining the diseased stage of squamous cell carcinoma in a subject is presented. According to an embodiment of the invention, the method comprises: (1) determining the amount of a marker comprising POSTN in saliva exosomes of a subject; (2) determining the disease stage of the subject based on the amount of the marker in step (1).
in particular, in the method for determining the diseased stage of squamous cell carcinoma in a subject set forth above, the squamous cell carcinoma is oral squamous cell carcinoma. Optionally, the marker further comprises at least one selected from TNC and TGFBI. Optionally, in step (2), the marker is detected by at least one of western blotting, enzyme linked immunosorbent assay, time of flight mass spectrometry.
According to an embodiment of the present invention, in the step (2), further comprising: comparing the level of the marker in saliva exosomes of the subject to a predetermined threshold, wherein a level of the marker above the second predetermined threshold is indicative of the subject's squamous cell carcinoma being in a metastatic stage. According to a particular embodiment of the invention, said predetermined threshold is determined on the basis of the content of markers in saliva exosomes of individuals known to suffer from squamous cell carcinoma in metastatic stage or on the basis of a statistical analysis of the content of markers in saliva exosomes of a plurality of individuals known to suffer from squamous cell carcinoma in metastatic stage.
Method for determining the efficacy of a treatment for a patient with squamous cell carcinoma
in a sixth aspect of the invention, a method of determining the effectiveness of a treatment for a patient with squamous cell carcinoma is presented. According to an embodiment of the invention, the method comprises: subjecting saliva exosomes from a patient undergoing treatment to levels of markers comprising at least one selected from POSTN, TNC and TGFBI, so as to obtain first marker levels; comparing the first marker level to a second marker level obtained from a sample from the pre-treatment patient; determining an effect of the treatment based on the comparison, wherein the first marker level is not higher than the second marker level is an indication that the treatment is effective. By using the method provided by the embodiment of the invention, the treatment effect of the squamous cell carcinoma patient can be effectively determined, and necessary evaluation basis is provided for selecting whether to continue the previous treatment mode or improve or change the previous treatment mode.
Method for determining treatment regimen for squamous cell carcinoma
In a seventh aspect of the invention, a method of determining a treatment regimen for squamous cell carcinoma is presented. According to an embodiment of the invention, the method comprises: (a) treating a squamous cell carcinoma patient with a candidate treatment regimen, and detecting the salivary exosome marker content of the squamous cell carcinoma patient undergoing the candidate treatment regimen, the marker comprising at least one selected from the group consisting of POSTN, TNC and TGFBI; (b) determining whether the candidate treatment has the ability to alter the level of the marker based on the test results obtained in step (a); (c) selecting a final treatment regimen based on the results of step (b). By utilizing the method for determining the treatment scheme of the squamous cell carcinoma, which is disclosed by the embodiment of the invention, necessary technical basis is provided for determining the optimal treatment scheme of the squamous cell carcinoma.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
the experimental reagents, materials and experimental methods used in the present application are as follows:
Saliva sample collection and preparation
20 patients were recruited from the oral medical college of Beijing university in 2013-2014, of which 10 patients with oral squamous cell carcinoma had cancer cell metastasis and 10 patients did not. The inventors recruited 10 healthy persons without oral squamous cell carcinoma as controls. The patients selected were untreated patients who had just been diagnosed with oral squamous cell carcinoma, and were similar in age, gender, and systemic disease to the healthy controls. Specimens were taken at 8:00 to 10:00 in the morning. All volunteers had a10 minute rest before specimen collection. All volunteers then gargle, sit upright, and place the tip of the tongue against the sublingual gland. Thus, saliva is secreted for about 5 minutes, and the inventors collected about 2mL of naturally secreted saliva. During this process, the volunteer was unable to speak. After collection, all saliva samples were placed on ice and centrifuged (10,000rpm,10min,4 ℃) to remove impurities and cellular debris. After collection the samples were stored at-80 ℃.
the study was approved by the ethical committee of biomedical sciences of the university of beijing. All participants provided written approval. The volunteer information is shown in table 1.
Table 1:
Exosome separation and transmission electron microscope
exosome extractions were performed according to the Invitrogen's instruction manual (Total Exosome Isolation, catalog number: 4484453). The exosome suspension was stored at-20 ℃ for later study. The inventors observed the shape and size of the extract using an electron microscope to confirm that the extract was exosome. After extraction, the exosomes were suspended in 1 × PBS. Next, a 5. mu.L portion of the sample was placed in a carbon-coated grid (pre-treated with a plasma cleaner; Ted Pella Inc., Calif., USA). After 30 seconds, the excess sample was blotted off with filter paper. Staining with 2% uranium acetate for 1 min. The grid was observed with a FEI T12 electron microscope at 120kV voltage. The micrographs were imaged by Gatanultrascan 4K X4K.
Western blotting method for detecting exosome membrane specific protein and specific protein in exosome of metastatic oral squamous cell carcinoma patient
The Exosome was cleaved with Invitrogen reagent (Total Exosome RNA and Proteins Isolation Kit) according to the manual instructions to obtain Exosome Proteins. The extracted proteins were stored at-80 ℃ for further analysis. The exosome proteins were electrophoresed through 10% SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were blocked and then diluted with primary antibodies (anti-beta actin, cat. No. ab6275; anti-CD63, cat. No. ab8219; all of the above-mentioned primary antibodies were purchased from Abcam Inc.) and a diluent (diluted 1:1000 with TBST solution in which skim milk powder was dissolved). Primary antibody was incubated with nitrocellulose membrane overnight. The nitrocellulose membrane was then washed 3 times with TBST. Next, the nitrocellulose membrane was incubated with anti-IgG fluorescent antibody (mixed well with TBST/skim milk powder solution at 1:15,000) for 1 hour, after which the membrane was washed. And finally, performing imaging analysis on the nitrocellulose membrane to detect a target protein band.
Shotgun mass spectrometry
Exosome proteins from 10 patients with metastatic oral squamous cell carcinoma, 10 patients with non-metastatic oral squamous cell carcinoma and 10 healthy people were used for shotgun mass spectrometry. The exosome protein is subjected to 10% SDS-PAGE electrophoresis, and a sample gel strip is divided into 5-6 parts. The gel was dissolved in methanol and spotted on an LTQ OrbitrapVelos device. Protein ID and primary abundance values were obtained from LC-MS/MS mass spectra.
Data analysis
The inventors queried the raw data from LC-MS/MS analysis on the Uniprot website. Meanwhile, GO analysis was performed at the DAVID website. The build-in decoy option is used to look up on the SwissProt database for human entries.
the applicant carried out the above-mentioned related experiments, and the experimental conclusions obtained are as follows:
Electron microscope picture and membrane protein identification
Figures 4A, B and C show pictures of salivary exosomes from patients with metastatic oral squamous cell carcinoma (OCM), non-metastatic oral squamous cell carcinoma (OUCM) and healthy control group (HC). The scale bar is 200 nm. The diameter of the exosome is 30-120 nm, and most of the exosome is nearly spherical in shape. The bilayer lipid membrane was visible. There was no significant difference in exosome size and shape. From this it can be determined that exosomes are present in the saliva sample. The exosome-specific protein CD63 and the housekeeping gene β -actin were used to detect vesicle membrane-specific proteins using western blotting (fig. 4D). Despite the expression differences between the three groups, CD63 and β -actin were detected, demonstrating that exosomes have been extracted.
Saliva exosome protein of healthy control group and oral squamous cell carcinoma patients
The protein expression of salivary exosomes differed between patients with metastatic and non-metastatic oral squamous cell carcinoma, and the protein expression of salivary exosomes differed between oral squamous cell carcinoma patients and healthy humans. 1151 proteins were detected in healthy control groups and 1580 proteins were detected in oral squamous cell carcinoma patients (including both metastatic and non-metastatic). Of these, 819 proteins were expressed in both oral squamous cell carcinoma patient groups and healthy control groups. The raw data peptide matching profiles were automatically generated from the system database, which is a useful comparative data between two groups of proteins, showing mainly the abundance of some proteins. Protein screening criteria were as follows: PSM ratio greater than 2 or less than 0.5; 2. unique peptides (Unique peptides) are greater than 2 and values greater than 10. Through the above screening, the inventors confirmed 233 proteins and their encoding genes that differed between oral squamous cell carcinoma patients and healthy controls (as shown in table 2).
Table 2:
These 233 genes were analyzed at the biochemical, molecular function and cellular component level using the DAVID database with the GO analysis method. Most of the highly abundant GO numbers are associated with exosome structures, immune responses and inflammation.
Comparison of salivary exosomes proteins in metastatic oral squamous cell carcinoma patients with non-metastatic oral squamous cell carcinoma patients
The salivary exosome proteins of the metastatic oral squamous cell carcinoma patients and the non-metastatic oral squamous cell carcinoma patients are analyzed by a shotgun mass spectrum. The untransferred group showed 1170 proteins, and the transferred group showed 1211 proteins (FIG. 5A). As with the protein screening method for oral squamous cell carcinoma patient groups and healthy control groups, there were 143 proteins with significant differences in expression between metastatic and non-metastatic oral squamous cell carcinomas (as shown in table 2), and they were also subjected to GO analysis (fig. 5B). Some of the high abundance components, such as those associated with cell attachment and bio-attachment, were associated with exosome formation and migration by GO analysis. According to the inventors' prediction, it is reasonable to enrich for genes associated with these functions. However, the high abundance of acute inflammatory response components prompted the inventors that this is a tumor sample. These abundantly expressed genes are not only expressed poorly in non-metastatic oral squamous cell carcinoma patients, but also in healthy controls with little to no expression. Thus, the inventors selected periodins (POSTN, NCBI GENE ID:10631), tenascin C (TNC, NCBI GENE ID:3371), and transforminggrowth factor-. beta. -induced proteins (TGFBI, NCBI GENE ID:7045) associated with cell attachment as target proteins (FIG. 5C). POSTN and TNC are expressed about 10-fold higher in patients with metastatic oral squamous cell carcinoma than in patients with non-metastatic oral squamous cell carcinoma (see table 2). POSTN, TNC and TGFBI are associated with cancer metastasis. In fact, TNC and POSTN may be specifically associated with oral cancer metastasis. Therefore, the inventors focused on these 3 proteins.
detection of selected specific proteins by Western blotting
POSTN, TNC and TGFBI were detected in individual samples by Western blotting. The results are shown in FIG. 6 by Western blotting. The results show that the levels of POSTN and TGFBI are significantly lower in healthy controls and in non-metastatic oral squamous cell carcinoma patients, and highest in metastatic oral squamous cell carcinoma patients. Due to the small amount of vesicular protein and the large molecular weight of TNC (241kDa), Western blot detection of TNC is difficult. Therefore, the inventors combined the samples to increase the concentration of TNC.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (9)
1. A kit, wherein the kit is for at least one of:
detecting squamous cell carcinoma;
Determining the stage of squamous cell carcinoma in the subject;
Determining the efficacy of treatment of a patient with squamous cell carcinoma; and
Determining a treatment regimen for the squamous cell carcinoma,
And, the kit comprises:
A reagent for detecting a marker in a saliva exosome, the marker comprising POSTN.
2. the kit of claim 1, wherein the squamous cell carcinoma is oral squamous cell carcinoma and the kit further comprises reagents for isolating salivary exosomes;
Preferably, the agent for isolating salivary exosomes is a 10% polyethylene glycol solution.
3. the kit of claim 1, wherein the marker further comprises at least one selected from the group consisting of TNC and TGFBI.
4. The kit of claim 1, wherein the reagent for detecting the marker is suitable for detecting the marker by at least one of western blotting, enzyme linked immunosorbent assay, and time-of-flight mass spectrometry.
5. use of a reagent for detecting an oral exosome marker comprising POSTN in the preparation of a kit for at least one of:
Detecting squamous cell carcinoma;
Determining the diseased stage of squamous cell carcinoma in the subject;
Determining the efficacy of treatment of a patient with squamous cell carcinoma; and
Determining a treatment regimen for squamous cell carcinoma.
6. A detection analysis system for at least one selected from the group consisting of:
detecting squamous cell carcinoma;
Determining the stage of squamous cell carcinoma in the subject;
Determining the efficacy of treatment of a patient with squamous cell carcinoma; and
determining a treatment regimen for the squamous cell carcinoma,
And, the detection analysis system includes:
a detection device for determining the amount of a marker comprising at least one selected from POSTN, TNC and TGFBI in saliva exosomes of a subject, optionally the subject has undergone treatment or treatment with a candidate treatment regimen;
an analysis device connected to the detection device and adapted to determine, based on the content of the marker:
Whether the subject is suffering from squamous cell carcinoma;
A diseased stage of squamous cell carcinoma in the subject;
Therapeutic effect of the squamous cell carcinoma patient
A treatment regimen for said squamous cell carcinoma.
7. the assay analysis system according to claim 6, further comprising a saliva exosome-isolating device for isolating exosomes from saliva of the subject.
8. The detection analysis system of claim 6, wherein the detection device further comprises at least one of:
A western blot unit;
An enzyme-linked immunoassay unit; and
a time-of-flight mass spectrometry unit.
9. The detection analysis system of claim 6, wherein the analysis device comprises:
a comparison unit for comparing the content of the marker of the subject with at least one of:
A first predetermined threshold determined based on marker levels in salivary exosomes of individuals known not to suffer from squamous cell carcinoma;
A second predetermined threshold determined based on the marker content in salivary exosomes of individuals known to be in a metastatic stage of squamous cell carcinoma;
(ii) marker levels in saliva exosomes of the patient prior to the treatment;
(ii) marker levels in saliva exosomes of controls not subjected to the treatment regimen;
A determination unit, connected to the comparison unit, for determining at least one of the following based on the comparison unit:
Whether the subject suffers from squamous cell carcinoma, wherein a level of the marker in the subject above the first threshold is indicative of the subject suffering from squamous cell carcinoma;
A diseased stage of squamous cell carcinoma in the subject, wherein a level of the marker in the subject above the second threshold is indicative of a metastatic stage of squamous cell carcinoma;
a therapeutic effect of said squamous cell carcinoma patient, wherein an amount of said marker in said subject that is not higher than an amount of a marker in saliva exosomes of said patient prior to said treatment is indicative of said treatment being effective;
a treatment regimen for said squamous cell carcinoma, wherein a candidate treatment regimen having the ability to alter the level of said marker in said subject is selected as a final regimen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112201356A (en) * | 2020-10-09 | 2021-01-08 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
| CN113834927A (en) * | 2020-06-08 | 2021-12-24 | 南京市口腔医院 | Salivary metabolism marker for predicting canceration of oral mucosa precancerous lesion patient and application thereof |
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2018
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113834927A (en) * | 2020-06-08 | 2021-12-24 | 南京市口腔医院 | Salivary metabolism marker for predicting canceration of oral mucosa precancerous lesion patient and application thereof |
| CN112201356A (en) * | 2020-10-09 | 2021-01-08 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
| CN112201356B (en) * | 2020-10-09 | 2022-02-01 | 郑州大学第一附属医院 | Construction method of oral squamous cell carcinoma diagnosis model, marker and application thereof |
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