CN110286218A - Death-associated protein kinase 1 is preparing the application in postoperative gastric cancer prognosis evaluation reagent kit - Google Patents
Death-associated protein kinase 1 is preparing the application in postoperative gastric cancer prognosis evaluation reagent kit Download PDFInfo
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Abstract
The present invention provides death-associated protein kinases 1 to prepare the new opplication in postoperative gastric cancer prognosis evaluation reagent 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 death-associated protein kinase 1 in stomach organization, can judge that the risk of recurrence of gastric cancer transfer occurs in patients with gastric cancer.The beneficial effects are mainly reflected as follows: the present invention provides death-associated protein kinases 1 to prepare the application in postoperative gastric cancer prognosis evaluation reagent kit, prompt the albumen that can be used to prepare the protein molecular marker for judging patients with gastric cancer prognosis, monitoring postoperative for Patients with Gastric Cancer and sequential therapy also have important directive significance.
Description
Technical field
The present invention relates to death-associated protein kinases 1 to prepare the application in postoperative gastric cancer prognosis evaluation reagent kit.
Background technique
Gastric cancer is initiated by the malignant tumour of gastric epithelial, and disease incidence ranks first in the various malignant tumours in China, stomach
Cancer morbidity has apparent region gender gap, is evident as with coastal region in east China incidence gastric cancer rate than southern area in the northwest in China
It is high.The hair age, the ratio between men and women's disease incidence was 2:1 at 50 years old or more well.Due to the change of dietary structure, operating pressure increase and
The reasons such as the infection of helicobacter pylori, so that rejuvenation tendency is presented in gastric cancer.Gastric cancer can betide any position of stomach, wherein half
Number is above to betide antrum portion, and greater curvature, lesser curvature and front and rear wall can be involved.Most gastric cancers belong to gland cancer, in early days without
Manifest symptom, or the nonspecific symptoms such as epigastric discomfort, belch occur, often with the stomaches chronic disease symptom phase such as gastritis, gastric ulcer
Seemingly, it is easily ignored, therefore, the early diagnostic rate of China's gastric cancer is still lower at present.The prognosis of gastric cancer and pathological staging, the portion of gastric cancer
Position, organization type, biological behaviour and remedy measures are related.
Death-associated protein kinase 1 is a kind of serine/threonine protein kitase that calcium ion/calmodulin is adjusted, dead
Related protein kinase 1 may participate in numerous intracellular signaling pathway, and proliferation, apoptosis, autophagy, attachment and the mistake nest for adjusting cell wither
It dies.So far from nineteen ninety-five discovery, effect of the death-associated protein kinase 1 in cancer, apoplexy and cardiovascular disease is just gradually
It is revealed.Research finds that death-associated protein kinase 1 can promote mammary gland in the breast cancer cell that p53 is mutated or is lacked
The proliferation of cancer cell, and death-associated protein kinase 1 promotes Apoptosis in p53 wild-type cell.In cancer early stage, extremely
Dying related protein kinase 1 can prevent cancer cell from spreading by inhibiting cell transformation.
The research of the invention finds that the expression of death-associated protein kinase 1 in gastric cancer exists significantly with patient's Postoperative determination
Relationship implies that death-associated protein kinase 1 can be used as effective early warning albumen of Postoperative determination of gastric cancer.
Gastric cancer threatens one of maximum tumour as to human health, and the molecular mechanism of its generation is still unclear so far, right
Its treatment also lacks the molecular target of specificity, and death-associated protein kinase 1 is as highly important tumour oncogene, mesh
It is preceding that there is no literature reported on death-associated protein kinases 1 is related to gastric cancer prognosis or Metastasis of Gastric Cancer is judged.
Summary of the invention
It is an object of the present invention to provide death-associated protein kinases 1 to prepare newly answering in postoperative gastric cancer prognosis evaluation reagent kit
With.
The technical solution adopted by the present invention is that:
Death-associated protein kinase 1 is preparing the application in postoperative gastric cancer prognosis evaluation reagent kit.
The present inventor after extensive and in-depth study, has found for the first time, detects dead related egg using ImmunohistochemistryMethods Methods
Relative expression quantity of the white kinases 1 in stomach organization can judge that the risk of recurrence of gastric cancer transfer occurs in patients with gastric cancer.Based on dead
The correlation that 1 expression quantity of related protein kinase is shifted with recurrence of gastric cancer is died, using the albumen as prognostic markers object to its expression quantity
Carrying out detection can be used for instructing the Index for diagnosis of gastric cancer, therefore can regard death-associated protein kinase 1 as molecular labeling, utilize
1 monoclonal antibody of death-associated protein kinase or polyclonal antibody detect death-associated protein in conjunction with immunohistochemical experiment reagent
Relative expression quantity of the kinases 1 in stomach organization.
The kit specifically includes that 1 monoclonal antibody of source of people death-associated protein kinase or polyclonal antibody, immune group
Change experiment reagent.The immunohistochemical experiment reagent is the common agents in the immunohistochemical experiment of this field.
Inventor is having found that death-associated protein kinase 1 is lower than non-relapse and metastasis in relapse and metastasis expression in gastric carcinoma
Stomach organization can speculate that death-associated protein kinase 1 plays a significant role in Postoperative Recurrence of Gastric Cancer transfer.Consult domestic foreign language
It offers, death-associated protein kinase 1 and the generation of gastric cancer and the correlative study of relapse and metastasis are few, in this experiment, dead related
Expression of the protein kinase 1 in stomach organization is lowered, and up-regulation is then expressed by cancer and in normal gastric mucosa, and is turned with not recurring
Shifting group is compared, and death-associated protein kinase 1 is expressed in relapse and metastasis group and also lowered, and shows that death-associated protein kinase 1 may
Gastric cancer occurrence and development process is participated in as promotive factor.It is analyzed by Kaplan-Meier survivorship curve, death-associated protein swashs
It is related with the prognosis of patients with gastric cancer that enzyme 1 expresses degree, patient's prognosis mala of 1 low expression of death-associated protein kinase (P <
0.05)。
In conclusion the low expression in stomach organization of death-associated protein kinase 1, the low table of death-associated protein kinase 1
Up to related with patients with gastric cancer postoperative recurrence transfer.Death-associated protein kinase 1 can be used as an important candidate of gastric cancer prognosis
Molecular marked compound.
Preferably, 1 polyclonal antibody of source of people death-associated protein kinase is dead shown in SEQ ID NO.1 as sequence
The acquisition of 1 immune rabbit of related protein kinase is died, 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 patients with gastric cancer biopsy or art, postoperative.
(b) ImmunohistochemistryMethods Methods utilize SP decoration method, the specific steps are as follows:
(c) stomach organization 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: 1 antibody concentration of death-associated protein kinase: 1:500 is added, 4 DEG C of refrigerators take out after placing 16h, room temperature
Rewarming 15min, then PBS is washed 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 stomach organization and cancer beside organism are randomly selected using microscope and imaging device to shoot, is utilized
Aperio Image Scope software is scanned the photograph of tissue samples, and the Algorithms of the software is used after scanning
(Positive Pixel Count V9) program carries out positive strength calculating to each sample, and it is as follows to calculate data:
(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.The invention shows death-associated proteins to swash
The prognosis of enzyme 1 and gastric cancer has significant correlation, to predict that relapse and metastasis and the postoperative survival rate of gastric cancer provide one entirely
New way plays an important role to the prognosis of patients with gastric cancer.When the scoring of 1 immunohistochemistry of cancerous tissue death-associated protein kinase is lower than
When 0.423, easily there is relapse and metastasis, the postoperative easy death of patients with gastric cancer in gastric cancer.
The beneficial effects are mainly reflected as follows: the present invention provides death-associated protein kinases 1 to prepare Gastric Carcinoma
Application in prognosis evaluation reagent kit afterwards prompts the albumen that can be used to prepare the protein molecule mark for judging patients with gastric cancer prognosis
Note, monitoring postoperative for Patients with Gastric Cancer and sequential therapy also have important directive significance.
Detailed description of the invention
Fig. 1 is the expression in the tissue samples of the postoperative no relapse and metastasis of patients with gastric cancer of death-associated protein kinase 1；
Fig. 2 is the expression in the tissue samples that patients with gastric cancer postoperative recurrence shifts of death-associated protein kinase 1；
Fig. 3 is 1 low expression group of death-associated protein kinase and high expression group survivorship curve in stomach organization；
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 patients with gastric cancer biopsy or art, postoperative.
(b) ImmunohistochemistryMethods Methods utilize SP decoration method, the specific steps are as follows:
(c) stomach organization 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: 1 antibody concentration of death-associated protein kinase: 1:500 is added, 4 DEG C of refrigerators take out after placing 16h, room temperature
Rewarming 15min.PBS is washed 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 stomach organization and cancer beside organism are randomly selected using microscope and imaging device to shoot, is utilized
Aperio Image Scope software is scanned the photo of tissue samples, and the Algorithms of the software is used after scanning
(Positive Pixel Count V9) program carries out positive strength calculating to each sample, and it is as follows to calculate data:
(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.The high low expression of death-associated protein kinase 1
Standard is with the median (0.423) of the expression of death-associated protein kinase 1 scoring in 30 stomach organizations for boundary.
(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 in the tumor tissues of 30 Patients with Gastric Cancer is as shown in Figs. 1-2: dead
Related protein kinase 1 is higher than relapse and metastasis group (Fig. 2) without the expression (Fig. 1) in relapse and metastasis group.
The relationship of death-associated protein kinase 1 and patients with gastric cancer prognosis:
It is analyzed by Kaplan-Meier survivorship curve, the expression degree of death-associated protein kinase 1 and patients with gastric cancer
Prognosis correlation (Fig. 3).
Embodiment 2:
It takes certain postoperative gastric cancer tumor sample to carry out specimens paraffin embedding slices, and utilizes above-described immunohistochemical method
It is detected, is computed, 1 immunohistochemistry tissue of the death-associated protein kinase scoring of cancerous tissue is 0.356.By it is postoperative with
Visit discovery, patient generation recurrence of gastric cancer transfer in 20th month after surgery, postoperative death in 32 months.
Embodiment 3:
It takes certain postoperative gastric cancer tumor sample to carry out specimens paraffin embedding slices, and utilizes above-described immunohistochemical method
It is detected, is computed, 1 immunohistochemistry tissue of the death-associated protein kinase scoring of cancerous tissue is 0.479.By it is postoperative with
Discovery is visited, which 3 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 by using immunohistochemistry detects 1 molecule phase of death-associated protein kinase
Gastric cancer DISTANT METASTASES IN risk and the postoperative existence or death of patient can be predicted to expression quantity.When cancerous tissue death-associated protein swashs
When the immunohistochemistry scoring of enzyme 1 is lower than 0.423, easily there is relapse and metastasis, the postoperative easy death of patients with gastric cancer in gastric cancer.Obviously death phase
Closing protein kinase 1 and gastric cancer has correlation, therefore, using death-associated protein kinase 1 as protein molecular marker to its table
The events such as relapse and metastasis after Surgery for Gastric Carcinoma, and judging prognosis can be predicted by carrying out detection up to amount.
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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1330 1335 1340
Thr Ser Asn Gly Ala Pro Lys Asp Phe Leu Pro Ser Pro Leu His Ala
1345 1350 1355 1360
Leu Leu Arg Glu Trp Thr Thr Tyr Pro Glu Ser Thr Val Gly Thr Leu
1365 1370 1375
Met Ser Lys Leu Arg Glu Leu Gly Arg Arg Asp Ala Ala Asp Phe Leu
1380 1385 1390
Leu Lys Ala Ser Ser Val Phe Lys Ile Asn Leu Asp Gly Asn Gly Gln
1395 1400 1405
Glu Ala Tyr Ala Ser Ser Cys Asn Ser Gly Thr Ser Tyr Asn Ser Ile
1410 1415 1420
Ser Ser Val Val Ser Arg
1425 1430
Claims (2)
1. death-associated protein kinase 1 is preparing the application in postoperative gastric cancer prognosis evaluation reagent kit, it is characterised in that: with death
Related protein kinase 1 is used as molecular labeling, utilizes the dead related egg of 1 monoclonal antibody of source of people death-associated protein kinase or source of people
White 1 polyclonal antibody of kinases detects phase of the death-associated protein kinase 1 in stomach organization in conjunction with immunohistochemical experiment reagent
To expression quantity.
2. death-associated protein kinase 1 according to claim 1 is preparing answering in postoperative gastric cancer prognosis evaluation reagent kit
With, it is characterised in that: 1 polyclonal antibody of source of people death-associated protein kinase is dead shown in SEQ ID NO.1 as sequence
Die the acquisition of 1 immune rabbit of related protein kinase.
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