CN103852585B - Rna plymerase ii the 5th subunit Function protein is preparing the application carried out in the reagent of prognosis of HCC or complementary TACE prognosis - Google Patents
Rna plymerase ii the 5th subunit Function protein is preparing the application carried out in the reagent of prognosis of HCC or complementary TACE prognosis Download PDFInfo
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Abstract
The present invention relates to rna plymerase ii the 5th subunit Function protein (RMP) and prepare the application carried out in the reagent of prognosis of HCC or complementary TACE prognosis.To disclose in hepatocellular carcinoma prognosis after RMP high expressed Hepatocellular Carcinoma Be Indicated resection operation first more undesirable; Meanwhile, the postoperative complementary TACE of its high expressed indication has validity.Therefore, the present invention establishes a kind of Postoperative determination method of the hepatocellular carcinoma based on RMP.
Description
Technical field
The invention belongs to bioengineering technical field; Specifically, the present invention relates to the tag application of rna plymerase ii the 5th subunit Function protein (RMP) and complementary TACE curative effect bad for assessment of primary hepatoma Postoperative determination.
Background technology
Primary carcinoma of liver is one of common clinically malignant tumour, and China is the hotspot of primary carcinoma of liver, about has the new cases of global half to occur in China every year.Hepatocellular carcinoma (HepatoCellular Carcinoma, HCC) is histological type common in primary carcinoma of liver, and proportion is about 70-85%.Current excision is the first-selected therapeutic modality of primary liver cancer.But the high relapse rate after Surgical Resection of Hepatic Carcinoma remains the principal element causing liver cancer patient prognosis not good.Complementary after Liver Cancer Operation is one of method of the postoperative recurrence of the excision that prevents liver cancer commonly used clinically through transcatheter arterial TAE (Adjuvant transcatheter arterial chemoembolization, Adjuvant TACE).But because it may affect the function of postoperative residue liver, so its curative effect remains dispute.Think that the patient's (such as, diameter of tumor is greater than 5 centimetres, has TTPV formation etc.) that there is liver cancer recurrence high risk factor should accept complementary TACE clinically at present, and can benefit.But this sorting technique exists certain drawback, there are some patients may there are the hazards of recurrence of PHC, but not yet reach the standard applying complementary TACE, just may cause omission.Therefore more careful molecule parting is needed; set up the system of many indexes associated prediction; make up deficiency (the Wang Z of single index; Li Z, Ji Y.Postoperative transcatheter arterial chemoembolization should be recommended in the hepatocellular carcinoma treatment guidelines of the American Association for the Study of Liver Diseases.Hepatology2011; 54:1489-149).
RMP (RPB5-mediating protein) is the Function protein of rna plymerase ii (RNA polymerase II, RNAP II) the 5th subunit, has another name called C19orf2 or URI.Far-Western immunoblot method is adopted the earliest by Cheong etc., with
32recombined human RPB5 probe examination from hepatoma cell strain cDNA library of P mark obtains.There are some researches show, RMP is an important transcription regulaton factor, participate in the transcriptional regulatory of several genes, and at oophoroma (Theurillat JP, Metzler SC, et al.URI is an oncogene amplified in ovarian cancer cells and is required for their survival.Cancer Cell2011; 19:317-332) etc. present obvious high expressed in cancerous tissue.
But, the correlativity of this area complementary TACE after not knowing RMP and Liver Cancer Operation at present.
Summary of the invention
The object of the present invention is to provide the application of rna plymerase ii the 5th subunit Function protein in the reagent preparing prognosis of HCC assessment or complementary TACE prognosis evaluation.
In a first aspect of the present invention, provide the purposes of rna plymerase ii the 5th subunit Function protein, for the preparation of carrying out the assessment of hepatocellular carcinoma Postoperative determination or carrying out the postoperative complementary diagnostic reagent assessed through transcatheter arterial TAE curative effect (prognosis).
In another aspect of this invention, there is provided a kind of for carrying out the assessment of hepatocellular carcinoma Postoperative determination or carrying out the postoperative complementary kit through transcatheter arterial TAE curative effect evaluation, described kit comprises: the reagent of specific detection rna plymerase ii the 5th subunit Function protein expression.
In a preference, in described kit, the reagent of described specific detection rna plymerase ii the 5th subunit Function protein expression is the antibody of anti-rna plymerase ii the 5th subunit Function protein.
In another preference, in described kit, described antibody is polyclonal antibody.
In another preference, also comprise in described kit: the detection antibody (two resist) carrying detectable signal molecule.
In another preference, described detectable signal molecule is selected from (but being not limited to): biotin, horseradish peroxidase, alkaline phosphatase, glucose oxidase, β – D-galactosidase, urase, hydrogen peroxidase or glucoamylase.
In another preference, also comprise in described kit: SABC reagent.
In another preference, described SABC reagent comprises: chromogenic reagent, dimethylbenzene, ethanol, H
2o
2methanol solution, antigen retrieval buffers, confining liquid, PBS, neutral resins.
In another aspect of this invention, there is provided the purposes of the reagent of specific detection rna plymerase ii the 5th subunit Function protein expression, for the preparation of carrying out the assessment of hepatocellular carcinoma Postoperative determination or carrying out the postoperative complementary kit through transcatheter arterial TAE curative effect evaluation.
In a preference, the reagent of described specific detection rna plymerase ii the 5th subunit Function protein expression is the antibody of anti-rna plymerase ii the 5th subunit Function protein.
In another aspect of this invention, provide a kind of method of carrying out the assessment of hepatocellular carcinoma Postoperative determination, described method comprises:
(1) kit described in application detects in experimenter's isolated liver cancerous tissue and surveys rna plymerase ii the 5th subunit Function protein expression;
(2) analyzing proteins expression;
If survey rna plymerase ii the 5th subunit Function protein expression high expressed, then this experimenter's prognosis is undesirable.
In another aspect of this invention, provide a kind of and carry out the postoperative complementary method through transcatheter arterial TAE curative effect evaluation, described method comprises:
(1) kit described in application detects in experimenter's isolated liver cancerous tissue and surveys rna plymerase ii the 5th subunit Function protein expression;
(2) analyzing proteins expression;
If survey rna plymerase ii the 5th subunit Function protein expression high expressed, then this experimenter is applicable to carrying out postoperative complementary through transcatheter arterial TAE.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
RMP ImmunohistochemistryResults Results comparison diagram in Fig. 1, liver cancer clinical sample.
Figure 1A utilizes ImmunohistochemistryMethods Methods, detects cancer beside organism in liver cancer clinical sample (278 example), the expression of cancerous tissue and TTPV (20 example) middle RMP.
Figure 1B is SABC scoring situation in each group of sample.
The relation of Fig. 2, RMP and liver cancer shorter survival.Figure is according to SABC appraisal result, in liver cancer tissue height express RMP patient excision after survivorship curve.
The curative effect of Fig. 3, RMP complementary TACE after can judging Liver Cancer Operation.
Wherein Fig. 3 A curative effect of complementary TACE after Liver Cancer Operation in the patient of RMP high expressed; Fig. 3 B is the curative effect of complementary TACE after Liver Cancer Operation in the patient of the low expression of RMP.
Embodiment
The present inventor is through extensive and deep research, and in Late Cambrian hepatocellular carcinoma, after rna plymerase ii the 5th subunit Function protein (RMP) high expressed Hepatocellular Carcinoma Be Indicated resection operation, prognosis is more undesirable; Meanwhile, the postoperative complementary TACE of its high expressed indication has validity.On this basis, the present invention establishes a kind of Postoperative determination method of the hepatocellular carcinoma based on RMP.
Primary and foremost purpose of the present invention is to provide a kind of RMP to may be used for assessing the mark of the bad and complementary TACE curative effect of primary hepatoma Postoperative determination, to solve the problem at present biological diagnosis of hepatocellular carcinoma and postoperative complementary TACE curative effect being lacked to suitable biological marker.
As optimal way of the present invention, set up a kind of detection kit, the reagent containing specific detection RMP expression in described kit.
As optimal way of the present invention, the reagent of described specific detection RMP expression is the antibody of anti-RMP.
The technology of Dispersal risk is well known in the art.Antibody of the present invention can be have specific monoclonal antibody to RMP.Monoclonal antibody can utilize hybridoma technology to prepare (see people such as Kohler, Nature256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Described monoclonal antibody can utilize RMP albumen or protein fragments or functional areas, is obtained by immunological technique.In addition, recombination method can also be utilized to prepare or utilize Peptide synthesizer to synthesize.
Antibody of the present invention also can be have specific polyclonal antibody to RMP albumen.Described polyclonal antibody is prepared by the method for routine, such as, obtains by being imported in animal by described RMP albumen, such as, and immune animal after RMP albumen is mixed according to proper proportion (as 1:1) with Freund's adjuvant.Immunization method can use Animal Skin hemostasis.Described animal can be selected from rabbit, sheep, ox etc.Also can be commercial antibody, such as described polyclonal antibody be the product of Proteintech.
After the reagent obtaining specific detection RMP protein expression level, the detection kit for specific detection RMP albumen can be prepared easily.Except containing except described antibody in described kit, can also comprise: the detection antibody (for being combined with anti-RMP antibody) carrying detectable signal molecule, and epidemic disease group reagent.Described SABC reagent includes but not limited to: chromogenic reagent, dimethylbenzene, ethanol, H
2o
2methanol solution, antigen retrieval buffers, confining liquid, PBS, neutral resins etc.
As used herein, described " detectable signal molecule " refers to that whether and the mark of the amount existed existence for determining RMP albumen in detected sample.Determining specific antibody that kit of the present invention adopts and after detecting antibody, this area can adopted conventional for being combined with detection antibody the various detectable signal molecules carrying out detecting.Such as, detectable signal molecule can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β – D-galactosidase, urase, hydrogen peroxidase or glucoamylase.
When employing some enzymes as implied above divide the period of the day from 11 p.m. to 1 a.m as detectable signal, also need the substrate adopting some to be combined with corresponding enzyme, thus report detectable signal molecule by modes such as colour developings there is situation or amount.Described substrate is such as: for o-phenylenediamine (OPD), tetramethyl benzidine (TMB), the ABTS of horseradish peroxidase; For the p-nitrophenyl phosphate (p-nitrophenyl phosphate, p-NPP) of alkaline phosphatase; Etc..Those skilled in the art according to the kind of adopted detectable signal molecule and characteristic, can select suitable substrate.
Based on new discovery of the present invention, panimmunity correlation technique can be utilized to detect the expression of RMP albumen in sample, and these methods are all in the present invention involved.In one embodiment, described method comprises step: detected RMP expression in the liver cancer of the patients with hepatocellular carcinoma postoperative paraffin embedding of excision and the other normal liver tissue of cancer by SABC and mark to coloration result, according to its SABC appraisal result, judge the curative effect of this patient's prognosis and the complementary TACE of Hepatectomy, instruct the individualized treatment that liver cancer patient is postoperative.
To sum up, according to the expression of RMP, liver cancer patient prognosis is assessed.The present invention proposes utilize the expression of RMP to carry out detecting and predict the events such as hepatocellular carcinoma recurrence after operation transfer first, and judging prognosis, for the postoperative monitoring of hepatocellular carcinoma patient and sequential therapy, also there is important directive significance.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.
Embodiment 1, the RMP expression in liver cancer tissue sample
Hospital of liver and gall surgical department chooses 278 routine hepatocellular carcinoma excision samples and 20 routine TTPV samples in the Orient.The characteristic parameter of 278 routine samples is as shown in table 1.
The characteristic parameter of table 1, test group patient
Use multi-clone rabbit source RMP antibody (1:75, Proteintech), utilize immunohistochemical method to detect RMP protein expression level in tissue.Immunohistochemical method comprises step detects RMP in the postoperative paraffin-embedded hepatocellular carcinoma of hepatocellular carcinoma patients excision and the other normal liver tissue of cancer expression by immunohistochemical method.Antigen retrieval 2 minutes, primary antibodie uses multi-clone rabbit source RMP antibody 1:75, proteintech, and 4 DEG C of overnight incubation, using normal rabbit source IgG antibody 1:75 as negative control; Biotin labeled two resist 37 DEG C hatches 1 hour, and DAB develops the color.
The result obtained carries out SABC scoring according to IRS points-scoring system.
Result shows, cancer beside organism, and the representational SABC figure of expression of cancerous tissue and TTPV (20 example) middle RMP is as Figure 1A.In clinical sample, in cancer beside organism, RMP majority presents low expression, and in liver cancer tissue sample, RMP expression is higher, most in high expressed state in TTPV.
Therefore, the case SABC that RMP antibody may be used for hepatocellular carcinoma detects, and can distinguish the grade malignancy of hepatoma carcinoma cell, has good specificity and susceptibility.
According to foregoing immune group method obtain tissue in RMP protein expression level, carry out IRS scoring, method is as follows: all samples often open section Stochastic choice five visuals field, each visual field counts 100 cells; According to the number percent of counting cells, the expression of RMP is divided 4 grades: 1 is divided into 1-25%, and 2 are divided into 26-50%, and 3 are divided into 51-75%, and 4 are divided into more than 76%; Be divided into 4 grades according to the expression of the intensity RMP of cell color again: 0 is divided into non-coloring, 1 is divided into light yellow particle, and 2 are divided into tawny particle, and 3 are divided into dark brown speckles.Often open the corresponding fractions of section and painted mark; both are added final score (organization chip only carries out intensity ratings) (the Wang Q being this section; Tan YX; Ren YB, et al.Zinc finger protein ZBTB20expression is increased in hepatocellular carcinoma and associated with poor prognosis.BMC Cancer2011; 11:271.).
Result as shown in Figure 1B.
Embodiment 2, Kaplan-Meier Measures compare RMP height expresses the post-operative survival rates time
According to IRS scoring, (according to the scoring of SABC tinctorial strength, 0,1 point is defined as low expression sample to be divided into RMP high expressed group and low expression group; 2,3 points are defined as high expressed), as table 2.
The correlativity (n=278) of RMP protein expression and Clinicopathologic Features in table 2, test group
In table 2, P value represents, the expression of RMP is obviously relevant to sex, and expression and the vein of RMP are invaded obviously relevant, and the expression of RMP is obviously relevant to tumor size, the expression of RMP is by stages obviously relevant to BCLC, and the expression of RMP is by stages obviously relevant to TNM.Be exactly that RMP expression in male patient is higher specifically; RMP expression is higher, and patient's vein infringement probability is higher; RMP expression is higher, and diameter of tumor is larger; RMP expresses higher, and BCLC is by stages higher; RMP expresses higher, and TNM is by stages higher.
Application SPSS18.0 analysis software, draw survivorship curve, result is as Fig. 2.
Result show, in liver cancer tissue high expressed RMP operation in patients after life span be significantly shorter than low expression RMP patient, as Fig. 2.
As seen from Figure 2, in liver cancer tissue, the expression of RMP is obviously relevant to primary carcinoma of liver shorter survival.According to the display of SABC marking result, high expressed RMP group patient prognosis is obviously poor, and low expression RMP group patient prognosis is obviously better.
The above results is pointed out further, and the high expressed of RMP is relevant to the biological behaviour of tumour.
Embodiment 3, different RMP expressions can judge the curative effect of postoperative complementary TACE
In the patient of embodiment 2, collect the situation of the complementary TACE of postoperative enforcement, carry out layering according to the scoring of RMP SABC, application SPSS18.0 analysis software draws prognosis survivorship curve, and result is as Fig. 3 A-B.
Result shows, and in this part patient of high expressed RMP, postoperative complementary TACE can significantly extend the survival of patients time, and curative effect is significantly better than the curative effect of control group patient; And in the patient of low expression RMP, postoperative complementary TACE can not improve patient's prognosis.
The clinical practice of embodiment 4, detection kit
Obtain the postoperative liver cancer tissue of 5 clinical patients with hepatocellular carcinomas, the method as embodiment 1 prepares SABC section, carries out SABC detection, and marks to testing result.The scoring of the tinctorial strength of section is added with painted scale scores, is less than 4 points and thinks the low expression of RMP; Be more than or equal to 4 points and think RMP high expressed.
In conjunction with RMP expression, go the prognosis predicting patient.Result is as follows:
2 low expression of patient RMP, think that hepatocellular carcinoma Postoperative determination is better; But need regular return visit inspection, treat if desired.
3 patient RMP high expresseds, think that hepatocellular carcinoma Postoperative determination is undesirable, advise follow-uply actively checking and treating; Meanwhile, think applicable and accept complementary TACE.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after having read above-mentioned instruction content of the present invention.
Claims (4)
- The purposes of 1.RNA polymerase II the 5th subunit Function protein, for the preparation of carrying out the assessment of hepatocellular carcinoma Postoperative determination or carrying out the postoperative complementary diagnostic reagent through transcatheter arterial TAE curative effect evaluation.
- 2. the purposes of the reagent of specific detection rna plymerase ii the 5th subunit Function protein expression, for the preparation of carrying out the assessment of hepatocellular carcinoma Postoperative determination or carrying out the postoperative complementary kit through transcatheter arterial TAE curative effect evaluation.
- 3. purposes as claimed in claim 2, it is characterized in that, the reagent of described specific detection rna plymerase ii the 5th subunit Function protein expression is the antibody of anti-rna plymerase ii the 5th subunit Function protein.
- 4. purposes as claimed in claim 3, it is characterized in that, described antibody is polyclonal antibody.
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| WO2017139276A1 (en) * | 2016-02-08 | 2017-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Gene signature predictive of hepatocellular carcinoma response to transcatheter arterial chemoembolization (tace) |
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Non-Patent Citations (2)
| Title |
|---|
| RMP Plays Distinct Roles in the Proliferation of Hepa-tocellular Carcinoma Cells and Normal Hepatic Cells;Sijun Yang et al;《International Journal of Biological Sciences》;20130705;第9卷(第6期);第637－648页 * |
| RMP基因沉默对肝癌SMMC2-7721细胞增殖和迁移能力的影响;连晓宁等;《肿瘤》;20100131;第30卷(第1期);第15－20页 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017139276A1 (en) * | 2016-02-08 | 2017-08-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Gene signature predictive of hepatocellular carcinoma response to transcatheter arterial chemoembolization (tace) |
| CN108884495A (en) * | 2016-02-08 | 2018-11-23 | 美国政府(由卫生和人类服务部的部长所代表) | Genetic marker predicts hepatocellular carcinoma to the response through transcatheter arterial chemoembolization (TACE) |
| CN108884495B (en) * | 2016-02-08 | 2023-08-22 | 美国政府(由卫生和人类服务部的部长所代表) | Gene markers predict the response of hepatocellular carcinoma to Transcatheter Arterial Chemoembolization (TACE) |
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