CN115372617A - Application of UBD protein in preparation of brain glioblastoma multiforme prognosis evaluation marker - Google Patents
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Abstract
The invention belongs to the technical field of medical biological detection, and discloses application of UBD protein in preparation of a brain glioblastoma prognosis evaluation marker, wherein the prognosis evaluation marker comprises a prognosis evaluation reagent or a prognosis evaluation kit; the invention also discloses a kit and a detection method for carrying out the prognosis evaluation of the glioblastoma multiforme by using the quantitative detection of the UBD protein, wherein the prognosis evaluation kit comprises a UBD protein antibody system and a reagent system matched with the quantitative detection of the UBD protein.
Description
Technical Field
The invention relates to the technical field of medical biological detection, in particular to a new application of UBD protein, and specifically relates to an application of UBD protein in preparation of a glioblastoma multiforme prognosis evaluation marker.
Background
Brain gliomas are the most common primary tumors of the central nervous system and can be classified into grade I to IV according to their pathological and clinical features, wherein Glioblastoma (GBM) is the highest grade malignant subtype of glioblastoma. Due to the aggressive growth of glioblastoma, despite recent advances in standardized therapy-based multi-treatments, the overall prognosis for glioblastoma patients has not improved significantly, with a median survival of only 14-16 months.
The prognosis of glioblastoma patients depends on certain clinical factors, such as age of onset, KPS score, extent of surgical resection, type of pathology, etc. At present, the diagnosis of glioblastoma is based on morphology only, and is difficult to objectively and comprehensively reflect the biological behavior characteristics of tumors, so that the prognosis of patients cannot be accurately judged and postoperative clinical treatment cannot be guided. Therefore, there is a need for a gene/protein that can effectively differentiate glioblastoma molecule subtypes and accurately determine prognosis, and screen high-level patients to guide clinical treatment.
UBD, also known as FAT10, is a member of the ubiquitin-like protein (UBL) family, comprising 165 amino acids, consisting of two tandem ubiquitin-like domains, with a relative molecular mass of 18kDa. The C-terminus of UBD proteins contains a Gly-Gly motif that is critical for covalent binding to substrates and to themselves, and thus UBD is involved in a variety of important cellular developmental processes, including immune-mediated inflammation, apoptosis, cell cycle progression and proliferation. Studies have shown that FAT10 has the ability to bind to and disrupt the function of the spindle assembly checkpoint protein Mad2 during mitosis, leading to a shortened mitotic phase and chromosomal aneuploidy replication, and thus UBD promotes tumor development and proliferation. UBD expression is elevated in liver cancer, stomach cancer, neuro-glioblastoma and other cancer tissues, particularly in poor prognosis of glioblastoma. Higher UBD levels are indispensible in connection with a short post-operative progression-free survival of glioblastoma patients. However, there is no relevant research description of the application of UBD to glioblastoma multiforme.
Disclosure of Invention
The invention aims to provide application of UBD protein in preparing a brain glioblastoma prognosis evaluation marker, and the relative expression quantity of the UBD protein in a brain glioblastoma tissue is detected by an immunohistochemical method, so that the prognosis of a patient with the brain glioblastoma can be judged; based on the correlation between the relative expression level of the UBD protein and the glioblastoma multiforme, the expression level of the UBD protein is detected by taking the UBD protein as a molecular marker, so that the UBD protein can be used for guiding the prognosis judgment of a patient with the glioblastoma multiforme.
In order to achieve the above purpose, the invention provides the following technical scheme:
the application of the UBD protein in preparing a brain glioblastoma prognosis evaluation marker, wherein the prognosis evaluation marker comprises a prognosis evaluation reagent or a prognosis evaluation kit.
Further, the prognostic evaluation marker is a prognostic evaluation reagent, and the prognostic evaluation reagent is a reagent for detecting the relative expression amount of UBD protein in a biological sample.
Further, the prognostic evaluation marker is a prognostic evaluation kit, and the prognostic evaluation kit comprises a reagent for detecting the relative expression amount of UBD protein in a biological sample.
Further, the reagent for detecting the relative expression quantity of the UBD protein in the biological sample comprises an antibody of the UBD protein and a reagent matched with the quantitative detection of the UBD protein.
Further, the UBD protein antibody is a monoclonal antibody or a polyclonal antibody.
Further, the quantitative detection of the UBD protein is immunohistochemical detection, and reagents required by the immunohistochemical detection comprise dimethylbenzene and ethanol、H 2 O 2 Volume fraction of 3% of H 2 O 2 Methanol solution, 1% BSA blocking solution, DAB chromogenic reagent, hematoxylin and horseradish peroxidase.
Further, the biological sample is a tumor specimen section obtained by surgical resection of a patient with glioblastoma.
The prognosis evaluation kit of the UBD protein in preparing the brain glioblastoma prognosis evaluation marker comprises a UBD protein antibody system and a reagent system matched with the quantitative detection of the UBD protein.
A method for carrying out prognosis evaluation on glioblastoma utilizing a prognosis evaluation kit of UBD protein in preparation of a glioblastoma multiforme prognosis evaluation marker comprises the following steps:
(a) Labeling rabbit anti-human IgG by using an immunohistochemical experimental reagent in a prognosis evaluation kit, and performing immunohistochemical staining on a glioblastoma multiforme tissue section;
(b) Observing the tissue staining condition of the glioblastoma multiforme under a light microscope, and judging and scoring the positive rate of UBD protein antibody staining cells and the UBD protein antibody staining intensity in nuclei of tumor cells;
(c) Judging high expression or low expression of UBD protein in the glioblastoma multiforme according to the comprehensive score.
Further, the scoring method in step (b) is:
observing positive staining under a microscope, selecting five high-power microscope visual fields for each specimen, counting 100 cells in each visual field, and calculating the percentage of positive cells; the brown/yellow staining in the nucleus is taken as positive, and the staining is divided into four grades according to the depth of the staining: no staining is scored as 0 point, light yellow is scored as 1 point, tan is scored as 2 points, and tan is scored as 3 points; taking cells with brown/yellow staining in the nucleus as positive cells, calculating the number of the positive cells in 100 cells, and scoring the standard as: marking no positive cells as 0 point, marking the positive cells with the proportion less than 5% as 1 point, marking the positive cells with the proportion between 5% and 10% as 2 points, and marking the positive cells with the proportion more than or equal to 10 as 3 points; multiplying the staining depth integral by the positive rate integral to obtain the final integral of the UBD protein expression quantity in the corresponding tumor tissue in the sample point;
the criteria for judging high expression or low expression in step (c) are:
the score for staining of UBD protein in the nucleus was less than or equal to 3 and high expression was recorded when the score was greater than 3.
The technical scheme has the beneficial effects that:
the invention takes UBD protein as a molecular marker, and analyzes the relative expression quantity of the UBD protein in the glioblastoma multiforme tissue by using UBD monoclonal or polyclonal antibodies and the quantitative detection of the UBD protein. The discovery of the correlation between the UBD protein and the glioblastoma multiforme provides a brand-new way for predicting the recurrence risk of the glioblastoma multiforme, has an important function for judging the prognosis of a patient with the glioblastoma multiforme, and also has important guiding significance for the postoperative monitoring and the sequential treatment of the patient with the glioblastoma multiforme: when immunohistochemistry scores were higher than 3 points, brain glioblastoma were prone to recurrence.
In terms of technology, the detection of the expression level of UBD protein is essentially the immunohistochemical quantitative detection of tumor tissue sections, has the characteristics of simple operation, sensitive detection, good specificity, high repeatability and the like, and is increasingly applied to clinical examination technology nowadays.
The invention utilizes immunohistochemical technology and system scoring to measure the expression level of UBD protein in the glioblastoma multiforme tissue, and combines postoperative follow-up information to determine the correlation between the expression level of the UBD protein and the prognosis of a patient with post-operation glioblastoma multiforme, and the UBD protein can be used for preparing protein molecular markers for judging the prognosis of the patient with the glioblastoma multiforme, and has important guiding significance for molecular typing of high-grade glioblastoma multiforme, postoperative monitoring and sequential treatment of the patient.
Drawings
FIG. 1 is a graph showing immunohistochemical staining results of UBD in glioblastoma tissue and normal glial cell tissue; in the figure, the UBD in the glioblastoma tissue is seen to be strongly positively colored, and the normal hard brain glial cell tissue is not colored;
FIG. 2 is a graph showing the results of UBD expression in glioblastoma tissue and normal glial cell tissue; in the figure, the expression integral values of UBD in the glioblastoma tissue and the normal glial cell tissue are respectively 2.15 +/-1.23 and 0, and the difference between the two tissues has a significant statistical significance (p is less than 0.001);
FIG. 3 is a graph of the results of immunohistochemical staining of UBD in glioblastoma tissue; in the figure, UBD shows wide and strong positive coloration in tumor cell nucleus (the integral is more than 3 points, and UBD is highly expressed in tumor tissues);
FIG. 4 is a graph of immunohistochemical staining of UBD in glioblastoma tissue; in the figure, UBD in the nucleus of tumor cells is scattered in weaker positive staining (the integral is less than or equal to 3 points, UBD is low expressed in tumor tissues);
FIG. 5 is a K-M plot of high and low expression of UBD protein versus progression-free survival PFS between two groups of patients.
Detailed Description
The following embodiments are implemented on the premise of the technical scheme of the present invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the present invention is not limited to the following embodiments.
Description of the drawings: the reagents and starting materials used in the invention are commercially available or can be prepared according to literature methods; the UBD protein antibody can be prepared by a conventional preparation method of the antibody, and the UBD monoclonal antibody is a commercial antibody, and the preparation method is shown in the literature: hipp MS, kalveram B, raasi S, groettrup M, schmidke G.FAT10, a ubiquitin-independent signal for procedural degradation [ J ]. Mol Cell Biol,2005,25 (9): 3483-3491. Experimental procedures without specific conditions noted in the following examples, generally according to conventional conditions or according to conditions recommended by the manufacturer;
in the following examples, tumor tissue samples from patients with glioblastoma were obtained from Shanghai Hospital and were clearly diagnosed as glioblastoma by 2 pathologists;
the inventors collected surgical specimens of 30 patients who underwent glioblastoma multiforme resection during the period from 2016 to 2020 and 1 month and 15 patients who underwent craniocerebral trauma decompression therapy from 2018 to 2020 and 1 month (the specimens were obtained by examination and approval by the ethical committee of the first subsidiary hospital of the university of naval military medical science). All patients were obtained from the Shanghai Changhai Hospital neurosurgery and were confirmed by 2 pathologists, as described in further detail below with reference to the accompanying drawings.
The first embodiment is as follows:
paraffin sections of 30 cases of tumor tissues after operation of patients with glioblastoma and 15 cases of brain tissue parts needing to be cut off after craniocerebral trauma are randomly selected.
In this embodiment, an immunohistochemical method is used to detect the expression of UBD protein in glioblastoma tissue and normal glial cell tissue and score them, specifically comprising the following steps:
(1) Preparing a paraffin section of a high-grade brain glioblastoma tissue, and standing overnight in an oven at 60 ℃;
(2) Dewaxing and hydrating the slices (xylene (1): 10min → xylene (2): 10min → xylene (3): 10min → 100% ethanol 5min → 95% ethanol 5min → 85% ethanol 5min → 75% ethanol 5min → double distilled water 5 min);
(3)3％H 2 O 2 standing the methanol solution at room temperature for 20min;
(4) Double-steaming and washing with water for 5min × 3;
(5) Antigen retrieval: boiling the slices in 0.01M citrate buffer solution (pH6.0) for 5min, heating for 10min, and boiling for 5min;
(6) Naturally cooling to room temperature, and washing with double-distilled water for 5min × 3;
(7) 1% BSA blocking solution incubated at 37 ℃ for 30min;
(8) Removing the serum blocking solution, and dropwise adding primary antibody (rabbit anti-human UBD monoclonal antibody) at 37 deg.C for 60min;
(9) Taking out at 4 ℃, rewarming at room temperature for 15min, and then washing with 0.01M PBS for 5min × 4;
(10) Dropwise adding a secondary antibody (Envision secondary antibody kit), and incubating according to the instruction of the secondary antibody kit;
(11) Washing with 0.01M PBS for 5min × 4, DAB developing for 2-10min, and observing under the mirror;
(12) Stopping color development by double distilled water, and counterstaining with hematoxylin for 10 seconds;
(13) After differentiation, the tap water turns blue, and is soaked in distilled water;
(14) Dehydrating and transparent, and covering with a cover glass;
(15) Positive staining was observed under microscope: each specimen selects 5 high power lens fields, each field counts 100 cells, and the percentage of positive cells is calculated; the brown/yellow staining in the nucleus is taken as positive, and the staining is divided into four grades according to the depth of the staining: no staining was scored as 0min, light yellow as 1 min, tan as 2 min, tan as 3 min; taking cells with brown/yellow staining in the nucleus as positive cells, calculating the number of the positive cells in 100 cells, and scoring the standard as: marking no positive cells as 0 point, marking the positive cells with the proportion less than 5% as 1 point, marking the positive cells with the proportion between 5% and 10% as 2 points, and marking the positive cells with the proportion more than or equal to 10 as 3 points; and multiplying the staining depth integral by the positive rate integral to obtain the final integral of the UBD protein expression quantity in the corresponding tumor tissue in the sample point.
As shown in fig. 1, UBD was seen to be broadly and strongly positively stained in the nuclei of glioblastoma cerebri (fig. 3), showing significantly high expression; the control normal brain glial tissue was weakly stained (fig. 4), and the expression integral value of UBD in the brain glioblastoma and normal brain glial tissue was 2.15 ± 1.23, 0, as shown in fig. 2, and the difference between each two groups had significant statistical significance (p < 0.001).
Example two:
randomly taking a tumor specimen of a patient with the glioblastoma multiforme after surgical resection, preparing a tissue section of the tumor of the patient with the glioblastoma multiforme, detecting the relative expression of UBD protein in the tissue of the glioblastoma multiforme by adopting the experimental steps of the immunohistochemical method in the first embodiment, and calculating an immunohistochemical score.
30 cases of brain glioblastoma tissues are detected in total, and the expression integral of UBD in 15 tumor tissues is found to be less than or equal to 3 points, and the expression integral of UBD in 15 tumor tissues is found to be more than 3 points. Patients with glioblastoma were grouped according to the level of UBD protein expression (high expression when immunohistochemistry final score was greater than 3 and low expression when the score was less than or equal to 3), and a progression-free survival graph was plotted (fig. 5). The results indicate that patients with high expression of UBD in tumors have significantly shorter post-operative progression-free survival times than low-expressors.
Example three:
sample preparation: the relative expression level of UBD protein in the glioblastoma multiforme tissue is detected by adopting the experimental steps of the immunohistochemical method on the tissue section of the tumor of a certain glioblastoma multiforme patient. A micrograph of glioblastoma is shown in fig. 3. The immunohistochemistry score for this tissue was calculated to be 6.
The patient can be found to have tumor recurrence after 6 months of operation by postoperative follow-up.
Example four:
sample preparation: the relative expression level of UBD protein in the glioblastoma multiforme tissue is detected by adopting the experimental steps of the immunohistochemical method on the tissue section of the tumor of a certain glioblastoma multiforme patient. A micrograph of glioblastoma is shown in fig. 4. The immunohistochemical score for this tissue was calculated to be 0.
The postoperative follow-up shows that the patient is still healthy after 63 months of operation and the tumor has no relapse.
From the above experimental results, it can be known that the progression-free survival time and the overall survival time of the brain glioblastoma patient after the operation can be predicted by detecting the relative expression amount of the UBD protein molecules by an immunohistochemical method. When the immunohistochemistry score is larger than 3 minutes, the glioblastoma is easy to relapse after operation, and the prognosis is poor. Obviously, the UBD protein has correlation with the prognosis of glioblastoma, so that the detection of the expression level of the UBD protein as a protein molecular marker can predict the relapse and other events after the operation of the glioblastoma, and judge the prognosis. Accordingly, antibodies specific for UBD proteins, including monoclonal and polyclonal antibodies, can be used to prepare kits for determining the prognosis of glioblastoma surgery.
In summary of 4 examples, the present invention uses UBD protein as a molecular marker, and uses a monoclonal or polyclonal antibody against UBD and quantitative detection of UBD protein to analyze the relative expression of UBD protein in glioblastoma tissue. The discovery of the correlation between the UBD protein and the glioblastoma multiforme provides a brand-new way for predicting the recurrence risk of the glioblastoma multiforme, has an important function for judging the prognosis of a patient with the glioblastoma multiforme, and also has important guiding significance for the postoperative monitoring and the sequential treatment of the patient with the glioblastoma multiforme: when the immunohistochemistry score is higher than 3 minutes, the brain glioblastoma is easy to relapse;
in terms of technology, the detection of the expression level of UBD protein is essentially the immunohistochemical quantitative detection of tumor tissue slices, has the characteristics of simple and convenient operation, sensitive detection, good specificity, high repeatability and the like, and is increasingly applied to clinical examination technology nowadays;
the invention utilizes immunohistochemical technology and system scoring to measure the expression level of UBD protein in the glioblastoma multiforme tissue, and combines postoperative follow-up information to determine the correlation between the expression level of the UBD protein and the prognosis of a patient with post-operation glioblastoma multiforme, and the UBD protein can be used for preparing protein molecular markers for judging the prognosis of the patient with the glioblastoma multiforme, and has important guiding significance for molecular typing of high-grade glioblastoma multiforme, postoperative monitoring and sequential treatment of the patient.
The above description is only an example of the present invention, and the common general knowledge of the technical solutions or characteristics known in the solutions is not described herein too much. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, and these should also be considered as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be defined by the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (10)
- The application of the UBD protein in preparing a brain glioblastoma prognosis evaluation marker is characterized in that: the prognostic evaluation marker comprises a prognostic evaluation reagent or a prognostic evaluation kit.
- 2. The use of the UBD protein according to claim 1 for preparing a marker for prognosis of glioblastoma multiforme, wherein: the prognostic evaluation marker is a prognostic evaluation reagent, and the prognostic evaluation reagent is a reagent for detecting the relative expression quantity of UBD protein in a biological sample.
- 3. The use of the UBD protein according to claim 1 for preparing a marker for prognosis of glioblastoma multiforme, wherein: the prognostic evaluation marker is a prognostic evaluation kit, and the prognostic evaluation kit comprises a reagent for detecting the relative expression amount of UBD protein in a biological sample.
- 4. Use of the UBD protein according to any one of claims 2 or 3 for the preparation of a marker for the prognosis of glioblastoma, characterized in that: the reagent for detecting the relative expression quantity of the UBD protein in the biological sample comprises a UBD protein antibody and a reagent matched with the quantitative detection of the UBD protein.
- 5. The use of the UBD protein according to claim 4, for preparing a marker for prognosis evaluation of glioblastoma multiforme, wherein: the UBD protein antibody is a monoclonal antibody or a polyclonal antibody.
- 6. The use of the UBD protein according to claim 4 for preparing a marker for prognosis of glioblastoma multiforme, wherein: the quantitative detection of the UBD protein is immunohistochemical detection, and reagents required by the immunohistochemical detection comprise dimethylbenzene, ethanol and H 2 O 2 Volume fraction of 3% of H 2 O 2 Methanol solution, 1% BSA blocking solution, DAB chromogenic reagent, hematoxylin and horseradish peroxidase.
- 7. The use of the UBD protein according to claim 2 for preparing a marker for prognosis of glioblastoma multiforme, wherein: the biological sample is a tumor specimen section which is excised by a brain glioblastoma patient operation.
- 8. The prognostic evaluation kit for use of the UBD protein according to claim 3 for preparing a marker for prognostic evaluation of glioblastoma multiforme, characterized in that: the prognosis evaluation kit comprises a UBD protein antibody system and a reagent system matched with the quantitative detection of the UBD protein.
- 9. A method for prognostically assessing glioblastoma using a prognostic assessment kit for use of the UBD protein according to claim 8 in the preparation of a marker for prognostically assessing glioblastoma, comprising the steps of:(a) Labeling rabbit anti-human IgG by using an immunohistochemical experimental reagent in a prognosis evaluation kit, and performing immunohistochemical staining on a glioblastoma multiforme tissue section;(b) Observing the tissue staining condition of the glioblastoma multiforme under a light microscope, and judging and scoring the positive rate of UBD protein antibody staining cells and the UBD protein antibody staining intensity in nuclei of tumor cells;(c) Judging high expression or low expression of UBD protein in the glioblastoma multiforme according to the comprehensive score.
- 10. A method for performing prognosis evaluation of glioblastoma using a prognosis evaluation kit for the use of the UBD protein according to claim 9 in preparing a marker for prognosis evaluation of glioblastoma, the method comprising: the scoring method in step (b) is:observing positive staining under a microscope, selecting five high-power microscope visual fields for each specimen, counting 100 cells in each visual field, and calculating the percentage of positive cells; the brown/yellow staining in the nucleus is taken as positive, and the staining is divided into four grades according to the depth of the staining: no staining was scored as 0min, light yellow as 1 min, tan as 2 min, tan as 3 min; taking cells with brown/yellow staining in the nucleus as positive cells, calculating the number of the positive cells in 100 cells, and scoring the standard as: marking no positive cells as 0 point, marking the positive cells with the proportion less than 5% as 1 point, marking the positive cells with the proportion between 5% and 10% as 2 points, and marking the positive cells with the proportion more than or equal to 10 as 3 points; multiplying the staining depth integral by the positive rate integral to obtain the final integral of the UBD protein expression quantity in the corresponding tumor tissue in the sample point;the criteria for judging high expression or low expression in step (c) are:the score for staining of UBD protein in the nucleus was less than or equal to 3 and high expression was recorded when the score was greater than 3.
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