CN110208541A - SAE2 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit - Google Patents

SAE2 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit Download PDF

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CN110208541A
CN110208541A CN201910305504.7A CN201910305504A CN110208541A CN 110208541 A CN110208541 A CN 110208541A CN 201910305504 A CN201910305504 A CN 201910305504A CN 110208541 A CN110208541 A CN 110208541A
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cell carcinoma
clear cell
sae2
kidney
leu
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林尧
王清水
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Fujian Normal University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

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Abstract

The present invention provides SAE2 to prepare the new opplication in clear cell carcinoma of kidney Postoperative determination assessment kit.The present inventor after extensive and in-depth study, has found for the first time, using relative expression quantity of the ImmunohistochemistryMethods Methods detection SAE2 in renal clear cell carcinoma, can judge that the risk of clear cell carcinoma of kidney relapse and metastasis occurs in clear cell carcinoma of kidney patient.The beneficial effects are mainly reflected as follows: the present invention provides SAE2 to prepare the application in clear cell carcinoma of kidney Postoperative determination assessment kit, prompt the albumen that can be used to prepare the protein molecular marker for judging clear cell carcinoma of kidney patient's prognosis, monitoring postoperative for clear cell carcinoma of kidney patient and sequential therapy also have important directive significance.

Description

SAE2 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit
Technical field
The present invention relates to SAE2 to prepare the application in clear cell carcinoma of kidney Postoperative determination assessment kit.
Background technique
Carcinoma of renal parenchyma is derived from the gland cancer of renal cells, and 85% is clear cell carcinoma, some is Granulocyte carcinoma and cell mixing cancer.Often there are bleeding, necrosis, capsule change and calcification in cancer.It is born in kidney essence, infiltrates, presses after growing up Compel, destroy renal plevis kidney calices, develop to outside kidney peplos, form hemangioma bolt or is transferred to lymph node and other organs.
The occurrence and development and prognosis of clear cell carcinoma of kidney and the activation of oncogene and tumor suppressor gene functionally inactive close association, It is multifactor induction, the complicated pathologic process that polygenes participates in.In recent years, the molecule machine of clear cell carcinoma of kidney early stage relapse and metastasis System is the heat subject in the field, deeply illustrates the molecular mechanism, and import magnetic target therapy measure in its specific link, It is expected to increase substantially very much the postoperative overall treatment effect of clear cell carcinoma of kidney.Therefore, effective clear cell carcinoma of kidney is sought Kidney hyaline cell cuts off early postoperation and recurs related early warning molecular marker, illustrates itself and clear cell carcinoma of kidney early stage relapse and metastasis Relationship, this to improve clear cell carcinoma of kidney aftertreatment effect, assessment clear cell carcinoma of kidney early stage risk of recurrence, judging prognosis and Individualized treatment has vital meaning.
SUMOization modification is the process of a dynamic reversible, and SUMO molecule in E1 activating enzymes, E2 desmoenzyme and E3 by connecting It connects and is covalently bound on substrate protein lysine residue under the participation of enzyme, regulate and control the structure and function of substrate protein, and SENPs is then By specifically to the modification of substrate target protein Go to SUMOization, adjusting the SUMOization state of substrate protein jointly with SUMO molecule, And then regulating cell function.SAE2 is one of E1 activating enzymes of SUMO, has important influence to SUMO signal path.
The research of the invention finds that the SAE2 expression in clear cell carcinoma of kidney with patient's Postoperative determination there are significant relationship, Imply that SAE2 can be used as effective early warning albumen of Postoperative determination of clear cell carcinoma of kidney.
Clear cell carcinoma of kidney threatens one of maximum tumour as to human health, its molecular mechanism occurred is not still so far It is clear, the molecular target of specificity is also lacked to its treatment, and SAE2 there is no at present as highly important tumour oncogene Document report SAE2 is related to clear cell carcinoma of kidney prognosis or clear cell carcinoma of kidney transfer is judged.
Summary of the invention
It is an object of the present invention to provide SAE2 to prepare the new opplication in clear cell carcinoma of kidney Postoperative determination assessment kit.
The technical solution adopted by the present invention is that:
SAE2 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit.
The present inventor after extensive and in-depth study, has found for the first time, saturating in kidney using ImmunohistochemistryMethods Methods detection SAE2 Relative expression quantity in clear cell carcinoma tissue can judge that clear cell carcinoma of kidney relapse and metastasis occurs in clear cell carcinoma of kidney patient Risk.Correlation based on SAE2 expression quantity with clear cell carcinoma of kidney relapse and metastasis, using the albumen as prognostic markers object to it Expression quantity, which carries out detection, can be used for instructing the Index for diagnosis of clear cell carcinoma of kidney, therefore can be using SAE2 as molecular labeling, benefit SAE2 is detected in renal clear cell carcinoma in conjunction with immunohistochemical experiment reagent with SAE2 monoclonal antibody or polyclonal antibody In relative expression quantity.
The kit specifically includes that source of people SAE2 monoclonal antibody or polyclonal antibody, immunohistochemical experiment reagent.Institute Stating immunohistochemical experiment reagent is the common agents in the immunohistochemical experiment of this field.
Inventor is having found that SAE2 expresses transparent higher than non-relapse and metastasis kidney in relapse and metastasis renal clear cell carcinoma Carcinoma tissues can speculate that SAE2 plays a significant role in the transfer of clear cell carcinoma of kidney postoperative recurrence.Domestic and foreign literature is consulted, The generation of SAE2 and clear cell carcinoma of kidney and the correlative study of relapse and metastasis are few, and in this experiment, SAE2 is in kidney hyaline cell Expression up-regulation in cancerous tissue, and downward is then expressed by the cancer and in normal kidney hyaline cell tissue, and with non-relapse and metastasis group It compares, SAE2 is expressed in relapse and metastasis group and also raised, and shows that SAE2 may participate in clear cell carcinoma of kidney hair as promotive factor Raw development process.It is analyzed by Kaplan-Meier survivorship curve, SAE2 expresses the prognosis of degree and clear cell carcinoma of kidney patient It is related, the highly expressed patient's prognosis mala (P < 0.05) of SAE2.
In conclusion SAE2 high expression in renal clear cell carcinoma, high expression and the clear cell carcinoma of kidney of SAE2 are suffered from The transfer of person's postoperative recurrence is related.SAE2 can be used as an important candidate molecular marker object of clear cell carcinoma of kidney prognosis.
Preferably, the source of people SAE2 polyclonal antibody is that SAE2 immune rabbit shown in SEQ ID NO.1 obtains as sequence , it can voluntarily prepare, commercially available commodity can also be used.
Specifically, the immunohistochemical experiment reagent includes: dimethylbenzene, ethyl alcohol, 3%H2O2(aqueous solution), 3%BSA closing Liquid (being prepared with PBS), DAB colour reagent, haematoxylin, horseradish peroxidase (for marking secondary antibody), PBS (pH7.4), 0.01M EDTA repairs liquid.
The application method of kit of the present invention is as follows:
(a) pathologic sampling of the Pathologic specimen in clear cell carcinoma of kidney patient biopsy or art, postoperative.
(b) ImmunohistochemistryMethods Methods utilize SP decoration method, the specific steps are as follows:
(c) renal clear cell carcinoma paraffin section is prepared, 60 DEG C of ovens are stayed overnight.
(d) it is sliced dewaxing.It successively impregnates: dimethylbenzene I:10min；Dimethylbenzene II:10min；Dimethylbenzene III:10min.
(e) it is sliced aquation.It successively impregnates: dehydrated alcohol: 3min；90% (v/v) ethyl alcohol: 3min；80% ethyl alcohol: 3min； 75% ethyl alcohol: 3min.
(f) PBS is cleaned 3 times, each 5min.
(h) EDTA antigen Pressure method: slice is put into 0.01M EDTA and repairs liquid immersion, and boiling water bath 5min is cooled to room Temperature.PBS is cleaned 3 times, each 5min.
(I) 3% (w/w) aqueous hydrogen peroxide solution of 300 μ L, 37 DEG C of 10min are added.PBS is cleaned 3 times, each 5min.
(J) 3% (w/w) the BSA confining liquid (PBS preparation) of 300 μ L, 37 DEG C of 1h are added.PBS is cleaned 3 times, each 5min.
(K) primary antibody: SAE2 antibody concentration: 1:500 is added, 4 DEG C of refrigerators take out after placing 16h, room temperature rewarming 15min, so PBS is washed 4 times afterwards, each 5min.
(L) secondary antibody is added dropwise, the secondary antibody is that horseradish peroxidase-labeled goat anti-rabbit igg (steps novel agent purchased from Foochow Company, instant, without dilution), 37 DEG C of 45min.PBS is washed 4 times, each 5min.
(M) PBS is washed 3 times, each 5min.Develop the color DAB (DAB colour reagent box, purchased from the raw work in Shanghai) 2-10min, under mirror Observation；Distilled water, which is washed, only to develop the color, and haematoxylin redyes 10s, is rinsed and is impregnated with tap water.
(N) it is dehydrated.It successively impregnates: 75% ethyl alcohol: 2min；80% ethyl alcohol: 2min；90% ethyl alcohol: 2min；Dehydrated alcohol: 2min。
(O) neutral gum, coverslip covering is added in electricity consumption blowing drying.
(P) 3 visuals field of renal clear cell carcinoma and cancer beside organism are randomly selected using microscope and imaging device to shoot, The photograph of tissue samples is scanned using Aperio Image Scope software, using the software after scanning Algorithms (Positive Pixel Count V9) program carries out positive strength calculating to each sample, calculates data such as Under:
(Q) the immunohistochemistry scoring of each tissue samples is calculated as Positivity × Log10 [255/Iavg], Middle Positivity=NPositive/NTotal, i.e. positive rate, calculation method are positive pixels quantity/colour developing total quantity； Iavg=(Iwp+Ip+Isp)/(Nwp+Np+Nsp), i.e., positive mean intensity, calculation method are positive mean intensity=(weak sun Property pixel overall strength+positive pixels overall strength+strong positive pixel overall strength)/(weakly positive pixel number+positive pixels quantity+strong Positive pixels quantity), the as immunohistochemistry scoring of the tissue is used for subsequent analysis.
(L) for statistical analysis using SPSS18.0, the enumeration data between test rating and clinical data uses Pearson Chi-square Test, measurement data are examined using t.The analysis of Testing index and clinical prognosis is raw using KaPlan-Meier Analysis is deposited, logarithm rank sum test (log-ranktest) compares the difference of survivorship curve.It is transparent thin that the invention shows SAE2 and kidney The prognosis of born of the same parents' cancer has significant correlation, to predict that relapse and metastasis and the postoperative survival rate of clear cell carcinoma of kidney provide one Completely new approach plays an important role to the prognosis of clear cell carcinoma of kidney patient.When the scoring of cancerous tissue SAE2 immunohistochemistry is higher than When 0.479, easily there is relapse and metastasis, the postoperative easy death of clear cell carcinoma of kidney patient in clear cell carcinoma of kidney.
The beneficial effects are mainly reflected as follows: the present invention provides SAE2 to prepare clear cell carcinoma of kidney Postoperative determination The application in kit is assessed, prompts the albumen that can be used to prepare the protein molecule mark for judging clear cell carcinoma of kidney patient's prognosis Note, monitoring postoperative for clear cell carcinoma of kidney patient and sequential therapy also have important directive significance.
Detailed description of the invention
Fig. 1 is SAE2 expression in the tissue samples of the postoperative no relapse and metastasis of clear cell carcinoma of kidney patient；
Fig. 2 is SAE2 expression in the tissue samples that clear cell carcinoma of kidney patient's postoperative recurrence shifts；
Fig. 3 is SAE2 low expression group and high expression group survivorship curve in renal clear cell carcinoma；
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:
Embodiment 1:
(a) pathologic sampling of the Pathologic specimen in clear cell carcinoma of kidney patient biopsy or art, postoperative.
(b) ImmunohistochemistryMethods Methods utilize SP decoration method, the specific steps are as follows:
(c) renal clear cell carcinoma paraffin section is prepared, 60 DEG C of ovens are stayed overnight.
(d) it is sliced dewaxing.It successively impregnates: dimethylbenzene I:10min；Dimethylbenzene II:10min；Dimethylbenzene III:10min.
(e) it is sliced aquation.It successively impregnates: dehydrated alcohol: 3min；90% (v/v) ethyl alcohol: 3min；80% ethyl alcohol: 3min； 75% ethyl alcohol: 3min.
(f) PBS is cleaned 3 times, each 5min.
(h) EDTA antigen Pressure method: slice is put into 0.01M EDTA and repairs liquid immersion, and boiling water bath 5min is cooled to room Temperature.PBS is cleaned 3 times, each 5min.
(I) 3% (w/w) aqueous hydrogen peroxide solution of 300 μ L, 37 DEG C of 10min are added.PBS is cleaned 3 times, each 5min.
(J) 3% (w/w) the BSA confining liquid (PBS preparation) of 300 μ L, 37 DEG C of 1h are added.PBS is cleaned 3 times, each 5min.
(K) primary antibody: SAE2 antibody concentration: 1:500 is added, 4 DEG C of refrigerators take out after placing 16h, room temperature rewarming 15min.PBS It washes 4 times, each 5min.
(L) secondary antibody is added dropwise, the secondary antibody is that horseradish peroxidase-labeled goat anti-rabbit igg (steps novel agent purchased from Foochow Company, instant, without dilution), 37 DEG C of 45min.PBS is washed 4 times, each 5min.
(M) PBS is washed 3 times, each 5min.Develop the color DAB (DAB colour reagent box, purchased from the raw work in Shanghai) 2-10min, under mirror Observation；Distilled water, which is washed, only to develop the color, and haematoxylin redyes 10s, is rinsed and is impregnated with tap water.
(N) it is dehydrated.It successively impregnates: 75% ethyl alcohol: 2min；80% ethyl alcohol: 2min；90% ethyl alcohol: 2min；Dehydrated alcohol: 2min。
(O) neutral gum, coverslip covering is added in electricity consumption blowing drying.
(P) 3 visuals field of renal clear cell carcinoma and cancer beside organism are randomly selected using microscope and imaging device to shoot, The photo of tissue samples is scanned using Aperio Image Scope software, using the software after scanning Algorithms (Positive Pixel Count V9) program carries out positive strength calculating to each sample, calculates data such as Under:
(Q) the immunohistochemistry scoring of each tissue samples is calculated as Positivity × Log10 [255/Iavg], Middle Positivity=NPositive/NTotal, i.e. positive rate, calculation method are positive pixels quantity/colour developing total quantity； Iavg=(Iwp+Ip+Isp)/(Nwp+Np+Nsp), i.e., positive mean intensity, calculation method are positive mean intensity=(weak sun Property pixel overall strength+positive pixels overall strength+strong positive pixel overall strength)/(weakly positive pixel number+positive pixels quantity+strong Positive pixels quantity), the as immunohistochemistry scoring of the tissue is used for subsequent analysis.SAE2 high low expression standard is with 30 kidneys The median (0.479) of SAE2 expression scoring is boundary in hyaline cell cancerous tissue.
(L) for statistical analysis using SPSS18.0, the enumeration data between test rating and clinical data uses Pearson Chi-square Test, measurement data are examined using t.The analysis of Testing index and clinical prognosis is raw using KaPlan-Meier Analysis is deposited, logarithm rank sum test (log-ranktest) compares the difference of survivorship curve.
According to the method described above, present invention testing result such as Fig. 1-2 in the tumor tissues of 30 clear cell carcinoma of kidney patients Shown: SAE2 is lower than relapse and metastasis group (Fig. 2) without the expression (Fig. 1) in relapse and metastasis group.
The relationship of SAE2 and clear cell carcinoma of kidney patient prognosis:
It is analyzed by Kaplan-Meier survivorship curve, the expression degree and the prognosis phase of clear cell carcinoma of kidney patient of SAE2 It closes (Fig. 3).
Embodiment 2:
It takes the postoperative tumor sample of certain clear cell carcinoma of kidney to carry out specimens paraffin embedding slices, and utilizes above-described immuning tissue Chemical method is detected, and is computed, and the SAE2 immunohistochemistry tissue scoring of cancerous tissue is 0.697.It is sent out by Follow-up After It is existing, 38th month after surgery generation clear cell carcinoma of kidney relapse and metastasis of the patient, postoperative death in 48 months.
Embodiment 3:
It takes the postoperative tumor sample of certain clear cell carcinoma of kidney to carry out specimens paraffin embedding slices, and utilizes above-described immuning tissue Chemical method is detected, and is computed, and the SAE2 immunohistochemistry tissue scoring of cancerous tissue is 0.396.It is sent out by Follow-up After Existing, which 5 does not find transfer and relapse every year after surgery, is still living and in good health.
By the above test result it is found that the method detection SAE2 molecule relative expression quantity by using immunohistochemistry can be predicted Clear cell carcinoma of kidney DISTANT METASTASES IN risk and the postoperative existence or death of patient.When the immunohistochemistry scoring of cancerous tissue SAE2 is high When 0.479, easily there is relapse and metastasis, the postoperative easy death of clear cell carcinoma of kidney patient in clear cell carcinoma of kidney.Obvious SAE2 and kidney Clear cell carcinoma has correlation, and therefore, kidney can be predicted by being detected using SAE2 as protein molecular marker to its expression quantity The events such as clear cell carcinoma recurrence after operation transfer, and judging prognosis.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent defines.
Sequence table
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Claims (2)

1.SAE2 preparing clear cell carcinoma of kidney Postoperative determination assessment kit in application, it is characterised in that: using SAE2 as Molecular labeling, using source of people SAE2 monoclonal antibody or source of people SAE2 polyclonal antibody, in conjunction with immunohistochemical experiment reagent, detection Relative expression quantity of the SAE2 in renal clear cell carcinoma.
2. SAE2 according to claim 1 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit, Be characterized in that: the source of people SAE2 polyclonal antibody is that SAE2 immune rabbit shown in SEQ ID NO.1 obtains as sequence.
CN201910305504.7A 2019-04-16 2019-04-16 SAE2 is preparing the application in clear cell carcinoma of kidney Postoperative determination assessment kit Withdrawn CN110208541A (en)

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