CN106053837A - Method and kit for prognosis judgment of colon cancer - Google Patents
Method and kit for prognosis judgment of colon cancer Download PDFInfo
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Abstract
The invention provides a method for prognosis judgment of colon cancer. The method comprises the step of detecting the amino acid sequence of a polypeptide, wherein the amino acid sequence is the sequence shown in SEQ NO.1 or a sequence with 99% of display content identical with the display content of the sequence shown in SEQ NO.1. A novel marker, namely HOXB-AS3 polypeptide, for prognosis evaluation and judgment of colon cancer is also provided. Clinical study proves that low expression or expression failure of the HOXB-AS3 polypeptide in colon cancer tissue is positively related to poor prognosis of colon cancer patients, and patients with low HOXB-AS3 polypeptide level in colon cancer tissue have higher death rate and shorter lifetime. Therefore, by using the HOXB-AS3 polypeptide as the biomarker, the positive coincidence rate is effectively increased when the kit is clinically applied to prognosis judgment of colon cancer. Furthermore, by detecting the level of the HOXB-AS3 polypeptide in a biological sample tissue slice, generation, malignancy grade and metastatic potential of colon cancer can be judged easily.
Description
Technical field
The present invention relates to diagnosis and the treatment field of tumor, in particular to a kind of for colon cancer Index for diagnosis
Method and test kit.
Background technology
Colon cancer is a kind of common alimentary system malignant tumour, and its sickness rate arranges the 3rd in all kinds of tumors.
The annual new cases in the whole world are about 1,200,000 examples, and annual death toll is up to 600,000.In recent years, carrying along with people's living standard
Height, life style, the change of dietary structure, the aggravation of aged tendency of population, colon cancer sickness rate rises year by year in China, and in
Revealing the trend of rejuvenation, its M & M occupies the 3rd and 5 of China's tumor incidence and mortality rate respectively.
5 years survival rates of limitation colorectal cancer patients are up to 90% in early days, but only have the colorectal cancer patients less than 40%
Confirmed in early days.When most of colorectal cancer patients are gone to a doctor, its middle and advanced stage and general 40～50% there occurs and turn at a distance
Moving, Advanced Colon Cancer survival then drops to about 11%.Along with the development of colon cancer Clinics, colon cancer pre-
After had the biggest improvement, but the prognosis of Advanced Colon Cancer patient is the most as one wishes.The main reason of clinical treatment failure
It is that colon cancer there occurs transfer or recurrence.Therefore, identify and can be used for the new label pointing out colon cancer transfer and relapse, contribute to
Assess the prognosis of colorectal cancer patients clinically, it is judged that the need of further assisting treatment (such as chemotherapy) after excision.
At present, TNM is the most important Clinical criteria judging colorectal cancer patients prognosis by stages.But, many colon cancer
Even if patient TNM belongs in early days by stages, still there is transfer and recurrence in tumor, the current clinic diagnosis of declaratives technically finds
There is not the colon cancer of transfer, however it remains the potential of transfer and relapse.Therefore, it is sought after researching and developing preferably for commenting
Estimate the novel marker of colorectal cancer patients prognosis.
Summary of the invention
In view of this, the invention provides a kind of method for colon cancer Index for diagnosis, including detecting a peptide species
Aminoacid sequence；Described aminoacid sequence is selected from sequence shown in SEQ NO.1 or selected from showing at least with sequence shown in SEQ NO.1
The sequence of 99% homogeneity.
In some embodiments, described polypeptide is HOXB-AS3 polypeptide.
In some embodiments, the epitope peptide sequence of described detection HOXB-AS3 polypeptide is selected from SEQ NO.2 or choosing
From the sequence showing at least 99% homogeneity with sequence shown in SEQ NO.2.
In some embodiments, the purity of described epitope peptide > 85%.
In some embodiments, described epitope peptide coupling has keyhole limpet hemocyanin as carrier protein, strengthens
The immunogenicity of epitope peptide.
In some embodiments, before the step of the aminoacid sequence of described detection one peptide species, following step is also included
Rapid:
The biological sample to be tested separated is provided；Described biological sample is carried out paraffin embedding and makes tissue and cut
Sheet.
In some embodiments, after the step of the aminoacid sequence of described detection one peptide species, following step is also included
Rapid:
According to the diaminobenzidine of the anti-upper coupling of described goat-anti rabbit two, colon cancer cell in described tissue slice is dyeed
Intensity and area mark；The intensity ratings of described cell dyeing and the stained area scoring of described cell are added, it is thus achieved that
The final scoring of HOXB-AS3 polypeptide expression level；Judge that HOXB-AS3 polypeptide is in colon cancer tissue according to described final scoring
Expression, thus realize the diagnosis to colon cancer or Index for diagnosis.
In some embodiments, the step that the section of colon cancer tissue immunohistochemical staining is carried out imaging is also included.
Present invention also offers a kind of test kit for colon cancer Index for diagnosis, described test kit application colon cancer prognosis
The method judged carries out colon cancer Index for diagnosis.
A kind of being for the method for colon cancer Index for diagnosis and the advantage of test kit of present invention offer:
(1) a kind of colon cancer prognosis evaluation is provided to judge novel marker: HOXB-AS3 polypeptide.Clinical research confirmation, its
In colon cancer tissue, low expression or do not express is proportionate with colorectal cancer patients poor prognosis, HOXB-AS3 polypeptide in colon cancer tissue
Low-level patient has higher mortality rate and shorter life cycle.It follows that use HOXB-AS3 polypeptide as biology
Mark effectively raises and uses test kit to carry out the positive coincidence rate of colon cancer Index for diagnosis in clinic.
(2) the polypeptide HOXB-AS3 of the present invention can be as the biomarker of colon cancer Index for diagnosis.And by using
The specific antibody of the polypeptide of the present invention detects the polypeptide of present invention level in the tissue slice of biological sample, can have
Help judge the generation of colon cancer and grade malignancy thereof and transfer.
(3) before making the present invention, the most do not go out to now refer to what protein polypeptide of the present invention diagnosed for colon cancer prognosis evaluation
Open report, does not more occur confirming HOXB-AS3 long-chain non-coding RNA (long non-coding RNA, lncRNA) coding
, the most there is not HOXB-AS3 polypeptide for public affairs that colon cancer prognosis evaluation diagnoses in the open report of a kind of active polypeptide
Open report.
In sum, the present invention has above-mentioned many advantages and practical value, and there are no similar in like product
Method publish or use and really belong to innovation.
Accompanying drawing explanation
It should be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore it is not construed as model
The restriction enclosed, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to according to these
Accompanying drawing obtains the accompanying drawing that other are relevant.
Fig. 1 is that in the embodiment of the present invention 2, anti-HOXB-AS3 antibody can specific recognition and the knot of detection HOXB-AS3 polypeptide
Fruit figure；
Fig. 2 is that the HOXB-AS3 polypeptide of the embodiment of the present invention 3 is at the different Invasion and Metastasis potential colon cancer cell in same source
The result of middle expression；
Fig. 3 is that the HOXB-AS3 polypeptide of the embodiment of the present invention 4 is corresponding at the colon cancer tissue that the 3 pairs of Fresh Frozens are paired
Result in Ai Bang colon.
In Fig. 4 embodiment of the present invention 5, HOXB-AS3 polypeptide is expressed in colon cancer and corresponding Ai Bang normal colonic tissue
Schematic diagram；Fig. 4 a represents corresponding Ai Bang normal colonic tissue；Fig. 4 b represents colon cancer tissue；
Fig. 5 is that the paired corresponding cancer of colon cancer tissue is close to knot 90 by the HOXB-AS3 polypeptide of the embodiment of the present invention 5
Expression of results in intestinal paraffin organization；
Fig. 6 is HOXB-AS3 polypeptide expression level height and colorectal cancer patients mortality rate relation in the embodiment of the present invention 5
Result；
Fig. 7 be in the embodiment of the present invention 5 HOXB-AS3 polypeptide difference expression to colorectal cancer patients Index for diagnosis
Kaplan-Meier analysis graph.
Detailed description of the invention
The claim of the present invention is described in further detail by the mode below in conjunction with specific embodiment, following
Description elaborates a lot of detail so that fully understanding the present invention.
But the present invention can implement to be much different from alternate manner described here, and those skilled in the art are permissible
Doing similar improvement in the case of intension of the present invention, therefore the present invention is not limited by following public being embodied as.
The invention provides a kind of method for colon cancer Index for diagnosis, including the aminoacid sequence detecting a peptide species
Row；Described aminoacid sequence is selected from sequence shown in SEQ NO.1 or selected from showing that at least 99% is same with sequence shown in SEQ NO.1
The sequence of property.
Further, described polypeptide is HOXB-AS3 polypeptide.
Above-mentioned, it is to be understood that to provide a kind of colon cancer prognosis evaluation to judge novel marker: HOXB-AS3 polypeptide.Face
Bed research confirms, its low expression or do not express in colon cancer tissue is proportionate with colorectal cancer patients poor prognosis, colon cancer group
Knit the middle low-level patient of HOXB-AS3 polypeptide and there is higher mortality rate and shorter life cycle.It follows that use HOXB-
AS polypeptide effectively raises as biomarker and uses test kit to carry out the sun of diagnosis of colon cancer or Index for diagnosis in clinic
Property coincidence rate.
It is understood that the polypeptide HOXB-AS3 of the present invention can be as the biomarker of colon cancer Index for diagnosis.And
And the specific antibody of the polypeptide of the application of the invention detects the polypeptide of the present invention in the tissue slice of biological sample
Level, can help to judge the generation of colon cancer and grade malignancy thereof and transfer.
Before making the present invention, protein polypeptide of the present invention is not the most gone out to now refer to for public affairs that colon cancer prognosis evaluation diagnoses
Open report, more do not occur confirming HOXB-AS long-chain non-coding RNA (long non-coding RNA, lncRNA) coding one
The open report of active polypeptide, does not the most occur that the disclosure that HOXB-AS3 polypeptide diagnoses for colon cancer prognosis evaluation is reported
Road.
Further, the epitope peptide sequence of described detection HOXB-AS3 polypeptide selected from SEQ NO.2 or is selected from and SEQ
Sequence shown in NO.2 shows the sequence of at least 99% homogeneity.
Further, the purity of described epitope peptide > 85%.
Further, described epitope peptide coupling has keyhole limpet hemocyanin KLH as carrier protein, enhancement antigen table
The immunogenicity of position peptide.
Further, before the step of the aminoacid sequence of described detection one peptide species, also comprise the following steps:
The biological sample to be tested separated is provided；Described biological sample is carried out paraffin embedding and makes tissue and cut
Sheet.
Further, after the step of the aminoacid sequence of described detection one peptide species, also comprise the following steps:
Resist the diaminobenzidine (DAB) of upper coupling to colon cancer cell in described tissue slice according to described goat-anti rabbit two
Intensity and the area of dyeing are marked；The intensity ratings of described cell dyeing and the stained area scoring of described cell are added,
Obtain the final scoring of HOXB-AS3 polypeptide expression level；Judge that HOXB-AS3 polypeptide is in colon cancer group according to described final scoring
Knit middle expression, thus realize the diagnosis to colon cancer or Index for diagnosis.
Further, the step that the section of colon cancer tissue immunohistochemical staining is carried out imaging is also included.
Present invention also offers a kind of test kit for colon cancer Index for diagnosis, this test kit is applied to colon cancer prognosis
The method judged carries out colon cancer Index for diagnosis.
For the ease of understanding the present invention, further illustrate technical scheme below in conjunction with embodiment.Applicant
Statement, the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment, but the present invention not office
It is limited to above-mentioned detailed process equipment and technological process, does not i.e. mean that the present invention has to rely on above-mentioned detailed process equipment and technique
Flow process could be implemented.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, each to product of the present invention
The equivalence of raw material is replaced and the interpolation of auxiliary element, concrete way choice etc., all falls within protection scope of the present invention and disclosure
Within the scope of.
Embodiment 1
Anti-HOXB-AS3 antibody, and the titer of the anti-HOXB-AS3 antibody of detection preparation, concrete operations are prepared for explanation
Journey is as follows:
(1) (gill biochemical (Shanghai) has the epitope peptide for HOXB-AS3 polypeptide of design to carry out chemosynthesis
Limit company), the epitope peptide coupling keyhole limpet hemocyanin then synthesized is as immunizing antigen, and this immunizing antigen uses high pressure
Liquid chromatograph (HPLC) is purified.
Take 2 body weight and be respectively designated as rabbit 1 and rabbit 2 not less than 2kg new zealand rabbit.The above-mentioned rabbit ear arterial blood 2 of each collection
～3ml, separating and obtain serum, this serum is negative control sera.
(2) above-mentioned immunizing antigen after purification carries out immunizing antigen multi-point injection.Detailed process is as follows:
First, by the epitope peptide of keyhole limpet hemocyanin coupling, dissolve with PBS, then according to quality 1:1 geometric ratio with
Freund's complete adjuvant (Freund ' s complete adjuvant, FCA), or incomplete Freund's adjuvant (Freund ' s
Incomplete adjuvant, FIA) mixing and emulsifying.
Secondly, the 500ug immunizing antigen of FCA emulsifying carries out multi-point injection at new zealand rabbit back.After 2 weeks, then rabbit
The 500ug immunizing antigen of back multi-point injection FCA emulsifying.Use the 250ug immunizing antigen of FIA emulsifying at new zealand rabbit after 2 weeks again
Back carries out multi-point injection.Afterwards every 1 week, carry out multiple spot note with the 250ug immunizing antigen of FIA emulsifying at new zealand rabbit back
Penetrate, carry out altogether 4 times.
(3) immunizing antigen multi-point injection process terminates first 1 week, collects rabbit blood 2～3ml by arteria auricularis, isolated and purified
Go out serum, carry out enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) and analyze, sentence
The antibody titer of disconnected preparation.
Meanwhile, the above-mentioned isolated and purified rabbit ear serum obtained and above-mentioned immunizing antigen carry out affinity purification, thus obtain
Anti-HOXB-AS3 antibody.
(4) with PBS, this immunizing antigen is diluted to 5 μ g/ml, corresponding every hole of 96 orifice plates adds 100ul dilution
Good immunizing antigen, 4 DEG C overnight.Clap by liquid in hole the most afterwards, wash above-mentioned 96 orifice plate 3 times with distillation.Then, every hole adds
200ul confining liquid (PBS, 1%BSA, 0.05%TWEEN-20), 37 DEG C of calorstats hatch 2 hours.Afterwards, clap 96 orifice bores
Middle liquid, washes 96 orifice plate 5 times with distillation.Every hole add 100ul sample diluting liquid (dilution ratio is respectively 1:3125,1:6250,
1:12500,1:25000,1:50000, sample diluting liquid is the BSA solution of 2%).Above-mentioned negative control sera dilution ratio 1:
3000 every holes add 100ul, and 37 DEG C of calorstats are hatched 1 hour, wash 96 orifice plate 5 times with distillation.Every hole adds 100ul Radix Cochleariae officinalis peroxidating
Goat anti-rabbit igg (the HRP-labeled goat anti-rabbit IgG) diluent of thing enzyme (HRP) labelling, dilution ratio is 1:
5000,37 DEG C of calorstats are hatched 45 minutes, wash 96 orifice plate 5 times with distillation.Every hole adds 100ul substrate tetramethyl benzidine
(TMB) nitrite ion, ambient temperatare is put 5～8 minutes, adds 2M sulphuric acid and terminates reaction.Plate is read the most again in microplate reader.
As seen from the results in Table 1, the antibody titer of the anti-HOXB-AS3 polypeptide prepared based on rabbit 1 and rabbit 2 is all higher than 1:
50000, the measurement result of titer is all preferable, and antibody titer is the highest, thus obtains the anti-HOXB-AS3 polypeptide of high-titer
Antibody.
Table 1 anti-HOXB-AS3 antibody titer measurement result
Embodiment 2
The anti-HOXB-AS3 antibody specific detection to HOXB-AS3 polypeptide, specifically comprises the following steps that
Build HOXB-AS3-GFP integrative gene expression vector.Wherein, wild type GFP (GFPwt) gene translation starts
Sub-ATGGTG is sported ATTGTT (GFPmut), thus destroys the independent translation of GFP gene, becomes and utilizes HOXB-AS3 base
Because upper translation promoter ATG is translated, produce HOXB-AS3-GFP fusion protein.Utilize liposome Lipo2000 (Life
Technology) transfection HeLa cell (ATCC, Number:CCL-2) is after 24 hours, is utilized respectively anti-GFP antibody and makes above
The antibody of standby anti-HOXB-AS3 polypeptide, the method using immunoblotting, the expression of detection HOXB-AS3-GFP albumen.Result is such as
Shown in Fig. 1 a, anti-GFP antibody and anti-HOXB-AS3 antibody respectively can be with specific detection to this HOXB-AS3-GFP fusion protein
Expression.But use negative control sera (pre-serum) to carry out corresponding immune-blotting method, then albumen can not be detected
Band.And anti-HOXB-AS3 antibody test to HOXB-AS3-GFP fusion protein pillar location arrive with anti-GFP antibody test
HOXB-AS3-GFP fusion protein pillar location completely the same, illustrate that anti-HOXB-AS3 antibody specific detection has arrived HOXB-AS3
Polypeptide.Using detection β-actin as internal reference.
Further, use for HOXB-AS3 2 siRNA#1 and siRNA#2 silence Colon Carcinoma (ATCC,
Number:CCL-228) expression of HOXB-AS3 gene in, the method using immunoblotting, the expression of detection HOXB-AS3 polypeptide
Level.By using reverse transcription-gene amplification (RT-PCR) to find in siRNA silence Colon Carcinoma cell
After the expression of HOXB-AS3RNA, result sees Fig. 1 b, with intracellular before and after anti-HOXB-AS3 antibody test siRNA silence
The change of HOXB-AS3 peptide level, finds that HOXB-AS3 peptide level significantly reduces.Also illustrate that the anti-HOXB-AS3 of preparation resists
Body can specific recognition and detection HOXB-AS3 polypeptide, result is shown in Fig. 1 c.Using detection β-actin as internal reference.
Embodiment 3
The method using immunoblotting, utilizes anti-HOXB-AS3 antibody test HOXB-AS3 polypeptide prepared above same
Expression in the different Invasion and Metastasis potential colon cancer cell in source, specifically comprises the following steps that
Use this anti-HOXB-AS3 antibody, utilize western blotting method to have detected 2 Invasion and Metastasis different to same source and dive
Can Colon Carcinoma and SW620 (ATCC, Number: be respectively CCL-228 and CCL-228), HTC-116 (ATCC,
And HTC-116 Number:CCL-247)highHOXB-AS3 peptide level between (this cell is set up by the screening of this laboratory early stage)
Difference, find HOXB-AS3 peptide level height attack metastatic potential colon cancer cell SW620 and HTC-116highMiddle downward,
Result is shown in Fig. 2, to detect β-actin as internal reference.
Embodiment 4
Collect 2015 and be diagnosed as colon cancer patients undergoing hepatectomy 3 in attached 3rd hospital of Guangzhou medical university, take
Its tumor tissues and corresponding cancer are close to normal structure.All colorectal cancer patients are preoperative does not all accept any antineoplaston, and not
Suffer from other tumor disease, all male, 62.3 ± 5.6 years old age.
Use liquid nitrogen to grind colon cancer and Ai Bang normal colonic tissue, the tissue after cracking grinding respectively, extract tissue thin
Born of the same parents' albumen.Utilizing anti-HOXB-AS3 polypeptide antibody prepared above, the method using immunoblotting, detection HOXB-AS3 polypeptide exists
Level difference in these 3 pairs of colon cancer tissue corresponding Ai Bang normal colonic tissues.
Result is as it is shown on figure 3, relative to Ai Bang normal colonic tissue, under HOXB-AS3 peptide level is in colon cancer tissue
Adjust.Using detection β-actin as internal reference.
Embodiment 5
For proving HOXB-AS3 peptide level and colon cancer pathological characteristics and colon in larger scale clinical samples further
Relation between the cancer survival of patients phase, detailed process is as follows:
Gather be diagnosed as colorectal cancer patients (male 48 examples, female 42 example, 69.7 ± 11.1 years old age) colon cancer tissue and
Its corresponding Ai Bang normal colonic tissue, and it is fabricated to organization chip (Shanghai Xin Chao company, HCol-Ade180Sur-06).
Use Use immunohistochemistrySP SP.Specifically comprise the following steps that the Ai Bang normal colonic tissue of colon cancer tissue and pairing
Organization chip routine dewaxing aquation, inserts in 1mM EDTA (pH 8.0) buffer, carries out antigen with high steam repairing method and repair
Multiple.It is then cooled to room temperature, utilize the H of 3%2O2Block endogenous peroxydase, hatch 15min.PBS (2%)
Rinse 3 times, each 3min.Then, lowlenthal serum confining liquid, incubated at room temperature 25min are dripped.Drip 50ul more prepared above
Anti-HOXB-AS3 antibody (1:300), 4 DEG C of overnight incubation, PBS (2%) rinses 5 times, each 5min.Drip 50ul again
The goat-anti rabbit Ig G (two resist) (1:2000) of HRP labelling, incubated at room 45min, PBS (2%) flushing 5 times, every time
5min, diaminobenzidine (Diaminobenzidine, DAB) develops the color 1～3min, and haematoxylin is redyed, and conventional dehydration is transparent, in
Property natural gum mounting.
The sxemiquantitative ImmunohistochemistryResults Results determination methods set up according to this laboratory early stage, analyzes immunohistochemical staining figure
Sheet.
Particularly as follows: according to staining power, be 0 point without brown color；Light brown yellow is 1 point；Brown color is 2 points；Sepia is 3
Point.Identical thing Microscopic observation positive cell number, no positive cell number is 0 point；Positive cell number≤10% is 1 point；Positive cell
Several 11%～50% is 2 points；Positive cell number > 50% it is 3 points.
The score of staining power score and positive cell number percentage ratio being added, the HOXB-AS3 being this sample expresses water
Flat score.The colon cancer tissue sample score of HOXB-AS3 compared with corresponding Ai Bang normal colonic tissue score, the i.e. table less than 1
Showing HOXB-AS3 low expression (low) in colon cancer tissue, other is then high expressed (high).As shown in Figure 4, Fig. 4 is result
The HOXB-AS3 polypeptide of the embodiment of the present invention 5 expression knot in colon cancer tissue and its corresponding Ai Bang normal colonic tissue
Really.Wherein, Fig. 4 a represents corresponding Ai Bang normal colonic tissue (Adjacent tissues), and Fig. 4 b represents colon cancer tissue
(Cancer tissues)。
Using SPSS12.0 statistical package to analyze, GraphPad Prism 5 software is drawn, the knot of HOXB-AS3 polypeptide
Intestinal cancer tissue obtains demultiplexing Mann-Whitney U test method and carries out statistical analysis with corresponding Ai Bang normal colonic tissue.Result
As it is shown in figure 5, compared with corresponding Ai Bang normal colonic tissue (N), HOXB-AS3 peptide level is notable in colon cancer tissue (T)
Lower (p < 0.0001).
On this basis, according to HOXB-AS3 polypeptide expression level, with the colon cancer tissue sample score of HOXB-AS3 with
Corresponding Ai Bang normal colonic tissue score is compared, and i.e. represents HOXB-AS3 low expression (low) in colon cancer tissue less than 1, its
It is then high expressed (high), thus is divided into HOXB-AS3 height and expresses 2 groups.Have evaluated further HOXB-AS3 peptide level with
The dependency of colon cancer pathological hallmarks, including the sex of patient, age, organizational hierarchy (Histological Grade), T divides
Phase (pT status), N (pN status) by stages, clinical stages (Clinical stage).Result is as shown in table 2, Wo Menfa
Existing HOXB-AS3 polypeptide low expression group has worse clinical stages (p=0.037).
Dependency between table 2 HOXB-AS3 peptide level and colon cancer pathological characteristics
*Mean age
#American Joint Committee on Cancer classification(Version 7)(AJCC)
Further, according to HOXB-AS3 expression height packet above, it has been found that HOXB-AS3 high expressed
(high) colorectal cancer patients only has the mortality rate of 37.48%, and HOXB-AS3 low expression (low) colorectal cancer patients is up to
The mortality rate of 75.47%, the mortality rate of HOXB-AS3 low expression colorectal cancer patients group is that HOXB-AS3 high expressed colon cancer group is dead
(result is shown in Fig. 6) more than 2 times of rate of dying.And then use Kaplan-Meier method and log-rank inspection, analyze HOXB-AS3 table
Reach the dependency of height and colorectal cancer patients life cycle.Result is as it is shown in fig. 7, HOXB-AS3 low expression colorectal cancer patients group is survived
Rate is lower, prognosis is worse (p < 0.0001)；HOXB-AS3 low expression colorectal cancer patients mean survival time is 46 months, and
HOXB-AS3 high expressed colorectal cancer patients mean survival time is 75 months.
Claims (9)
1. the method for colon cancer Index for diagnosis, it is characterised in that: include the aminoacid sequence detecting a peptide species；Institute
State aminoacid sequence selected from sequence shown in SEQ NO.1 or selected from showing at least 99% homogeneity with sequence shown in SEQ NO.1
Sequence.
2. the method for colon cancer Index for diagnosis as claimed in claim 1, it is characterised in that: described polypeptide is HOXB-
AS3 polypeptide.
3. the method for colon cancer Index for diagnosis as claimed in claim 1, it is characterised in that: described detection HOXB-AS3 is many
The epitope peptide sequence of peptide is selected from SEQ NO.2 or selected from the sequence showing at least 99% homogeneity with sequence shown in SEQ NO.2
Row.
4. the method for colon cancer Index for diagnosis as claimed in claim 3, it is characterised in that: described epitope peptide pure
Degree > 85%.
5. the method for colon cancer Index for diagnosis as claimed in claim 3, it is characterised in that: described epitope peptide coupling
There is keyhole limpet hemocyanin as carrier protein, the immunogenicity of enhancement antigen epitope peptide.
6. the method for colon cancer Index for diagnosis as claimed in claim 5, it is characterised in that: described detection one peptide species
Before the step of aminoacid sequence, also comprise the following steps:
The biological sample to be tested separated is provided；Described biological sample is carried out paraffin embedding and makes tissue slice.
7. the method for colon cancer Index for diagnosis as claimed in claim 6, it is characterised in that: described detection one peptide species
After the step of aminoacid sequence, also comprise the following steps:
According to the diaminobenzidine of the anti-upper coupling of described goat-anti rabbit two to colon cancer cell dyeing strong in described tissue slice
Degree and area are marked；The intensity ratings of described cell dyeing and the stained area scoring of described cell are added, it is thus achieved that HOXB-
The final scoring of AS3 polypeptide expression level；Judge that HOXB-AS3 polypeptide is expressed in colon cancer tissue according to described final scoring
Level, thus realize the Index for diagnosis to colon cancer.
8. the method for colon cancer Index for diagnosis as described in any one in claim 1～7, it is characterised in that: also wrap
Include the step that the section of colon cancer tissue immunohistochemical staining is carried out imaging.
9. the test kit for colon cancer Index for diagnosis, it is characterised in that: in described test kit application claim 1～7
The method for colon cancer Index for diagnosis described in any one carries out colon cancer Index for diagnosis.
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| CN106957354A (en) * | 2017-04-14 | 2017-07-18 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | HOXB AS3 polypeptides and the ORFs for encoding it |
| CN110777206A (en) * | 2019-12-04 | 2020-02-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of LOC90024RNA and product for diagnosis, prognosis evaluation and treatment of tumors |
| CN110865184A (en) * | 2019-12-04 | 2020-03-06 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of SRSP protein and SRSP epitope peptide and product for diagnosing and treating tumors |
| CN111848747A (en) * | 2020-08-07 | 2020-10-30 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Polypeptide for preventing and/or treating malignant tumor, application and medicament |
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| CN106957354A (en) * | 2017-04-14 | 2017-07-18 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | HOXB AS3 polypeptides and the ORFs for encoding it |
| CN106957354B (en) * | 2017-04-14 | 2020-06-30 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | HOXB-AS3 polypeptide and open reading frame for coding same |
| CN110777206A (en) * | 2019-12-04 | 2020-02-11 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of LOC90024RNA and product for diagnosis, prognosis evaluation and treatment of tumors |
| CN110865184A (en) * | 2019-12-04 | 2020-03-06 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of SRSP protein and SRSP epitope peptide and product for diagnosing and treating tumors |
| CN110865184B (en) * | 2019-12-04 | 2023-04-07 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Application of SRSP protein and SRSP epitope peptide and product for diagnosing and treating tumors |
| CN111848747A (en) * | 2020-08-07 | 2020-10-30 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Polypeptide for preventing and/or treating malignant tumor, application and medicament |
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| CN106053837B (en) | 2017-12-15 |
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