CN103543266B - Applications of reagent for detecting PGRMC1 to preparation of kidney cancer diagnosis reagent and kit - Google Patents
Applications of reagent for detecting PGRMC1 to preparation of kidney cancer diagnosis reagent and kit Download PDFInfo
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- 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
- G01N33/53—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
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
Abstract
The invention belongs to the field of oncomolecularbiology, and particularly relates to a kidney cancer diagnosis reagent, a kit and medicaments for prevention and treatment. The kidney cancer diagnosis reagent contains a reagent capable of detecting protein expression level and/or phosphorylation level of a progesterone receptor membrane component 1. The kidney cancer diagnosis reagent provided by the invention can effectively diagnose kidney cancer diseases at the early stage, thus improving the survival rate of patients greatly. The kidney cancer diagnosis reagent provided by the invention can diagnose effectively only through less kidney cancer tissue so as to prevents hurt to the patients, and is beneficial to pertinent treatment on the patients through selecting appropriate medicaments and methods selected by doctors.
Description
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to a kind of Diagnosis of Renal Cell Carcinoma reagent, kit and protective agents.
Background technology
Kidney is one of common malignant tumour of urinary system, and account for the 2-3% of all malignant tumours, account for 90% of tumor of kidney, its incidence of disease accounts for the second of urological cancer.Kidney originates from renal cells, and about 75% is clear cell carcinoma of kidney.The M & M of kidney all has the trend increased in recent decades, the incidence of disease with annual 2% speed increment.Owing to lacking effective early diagnosis marker, hinder the timely diagnosis to kidney, 50% patients with renal cell carcinoma has been late period when making a definite diagnosis, and the patients with renal cell carcinoma of about 1/4th has shifted when finding, prognosis is poor.
The treatment of current kidney still based on radical surgery, but has chemicotherapy repellence because of kidney, and postoperative about have the patient of 20-40% to occur recurrence.Therefore, the early screening of kidney and early diagnosis extremely important for the control of kidney, and to develop the relapse and metastasis reagent of effective monitoring kidney, kit and research and develop new kidney target therapeutic agent be the great demand being badly in need of important topic and the national health solved.
PGRMC1, namely PgR membrane component 1 (PGRMC1) is a 28kD, albumen (the SEQ ID No.1:MAAEDVVATG ADPSDLESGG LLHEIFTSPL NLLLLGLCIF LLYKIVRGDQ PAASGDSDDD EPPPLPRLKR RDFTPAELRR FDGVQDPRIL MAINGKVFDV TKGRKFYGPE GPYGVFAGRD ASRGLATFCL DKEALKDEYD DLSDLTAAQQ ETLSDWESQF TFKYHHVGKL LKEGEEPTVY SDEEEPKDES ARKND be made up of 195 amino acid, http://www.uniprot.org/uniprot/O00264), belong to film associated progesterone receptors protein family, recent research display PGRMC1 albumen plays a significant role [1 in cancer, 2].PGRMC1 albumen usually at mammary gland, colon and thyroid neoplasm tissue and colon, thyroid gland, ovary, process LAN [3,4] in the cell of lung and cervical carcinoma origin.Microarray analysis has detected the expression of PGRMC1 albumen in colon, lung, ovary and tumor of breast [5-7].The analysis of proteomics shows that in breast cancer, PGRMC1 albumen increases, and PGRMC1 albumen is the new molecular marker [8,9] of estrogen receptor.In addition, PGRMC1 albumen also regulates cancer cell to the susceptibility [10] of chemotherapy.Up to the present, the report of PGRMC1 and kidney relation is had no.Have no and realize the diagnostic reagent of kidney and the relevant report of kit by detecting PGRMC1 expression and phosphorylation level, also have no by reducing its expression or suppressing its phosphorylation level to reach the report of the object for the treatment of kidney.
This area needs to provide the diagnosis especially early diagnosis of new kidney, the testing product of curative effect of medication monitoring and protective agents at present.
Summary of the invention
First technical matters to be solved by this invention is to provide a kind of Diagnosis of Renal Cell Carcinoma reagent.This diagnostic reagent contains the reagent that can detect PgR membrane component 1 protein expression level and/or phosphorylation level.
Wherein, above-mentioned detection PgR membrane component 1 protein expression level and/or the reagent of phosphorylation level comprise polyclonal antibody or the monoclonal antibody of PgR membrane component 1.
Wherein, above-mentioned detection PgR membrane component 1 protein expression level and/or the reagent of phosphorylation level comprise the PCR primer of energy specific amplified PgR membrane component 1 gene mRNA.
The Second Problem that the present invention solves there is provided a kind of kidney detection kit or biochip.This kidney detection kit or biochip contain above-mentioned Diagnosis of Renal Cell Carcinoma reagent.
The purposes in Diagnosis of Renal Cell Carcinoma reagent prepared by the reagent simultaneously also providing above-mentioned detection PgR membrane component 1 protein expression level and/or phosphorylation level.Also provide the reagent of above-mentioned detection PgR membrane component 1 protein expression level and/or phosphorylation level further at preparation kidney detection kit or biochip
The 3rd problem that the present invention solves there is provided a kind of medicine for the treatment of kidney.The medicine of this treatment kidney is the medicine that can reduce the mRNA content that the protein expression level and/or phosphorylation level that change PgR membrane component 1 or its encoding gene are transcribed.
Wherein, above-mentioned medicine is the expression vector that the shRNA molecule of PgR membrane component 1 maybe can express the shRNA molecule that can detect PgR membrane component 1.
Wherein, above-mentioned medicine is the antagonist of PgR membrane component 1 or polyclonal antibody or monoclonal antibody.
Present invention also offers the method for the above-mentioned medicine of screening.The method comprises the following steps:
A, set up the cell model of kidney or the model group of animal model, and set up Normal group;
B, with medicine to be screened according to dosage gradient effect in model group, then detect and wherein can detect the protein expression level of PgR membrane component 1 albumen or the mRNA content of its gene;
C, testing result and Normal group to be compared, the medicine to be screened that can detect the protein expression level of PgR membrane component 1 albumen or the mRNA content remarkable convergence Normal group of its gene in model group can be made namely by sieve.
Innovation of the present invention and beneficial effect are: Diagnosis of Renal Cell Carcinoma reagent of the present invention can efficient diagnosis kidney disease in early days, substantially increases the survival rate of patient; Diagnosis of Renal Cell Carcinoma reagent of the present invention only needs a small amount of renal carcinoma tissue to carry out efficient diagnosis, avoids the injury caused to patient; Be conducive to medicine that doctor selects to be applicable to and method carries out immunotherapy targeted autoantibody to patient.The present invention prevents and treats the medicine of kidney disease, for the medication of clinical kidney disease provides a kind of selection newly.The present invention's screening prevents and treats the method for the medicine of kidney disease, providing a kind of new approach, having broad application prospects for finding the medicine preventing and treating kidney disease.
Accompanying drawing explanation
Fig. 1 Mass Spectrometric Identification result.Identified by the quanti-fication proteomics based on Leu-d3, in renal carcinoma tissue, the other normal kidney tissue of the expression ratio cancer of PGRMC1 raises 3.91 times.Wherein: Figure 1A is one section of peptide section finger-print of PGRMC1 in the other normal kidney tissue of cancer and HEK293 mixing with cells sample, comprise the representative isotope labeling peptide section (m/z1019.99/1018.49 that pair of sequences is " GDQPAASGDSDDDEPPPLPR; SEQ ID No.2 ", 2+), the relative expression quantity of PGRMC1 in the other normal kidney tissue of quantitative cancer and HEK293 cell is carried out with it.Figure 1B is one section of peptide section finger-print of PGRMC1 in renal carcinoma tissue and HEK293 mixing with cells sample.The relative expression quantity of PGRMC1 in the other normal kidney tissue of quantitative cancer and HEK293 cell is carried out with representative isotope labeling peptide section (m/z1019.99/1018.49,2+) that pair of sequences is " GDQPAASGDSDDDEPPPLPR, ".Fig. 1 C is peptide section GDQPAASGDSDDDEPPPLPR(m/z1018.49) tandem mass spectrometry figure.
The expression of Fig. 2 PGRMC1 normal kidney tissue by renal carcinoma tissue and cancer.Result shows that the other normal kidney tissue of the expression ratio cancer of PGRMC1 in renal carcinoma tissue is much higher.
Fig. 3 PGRMC1 is in renal carcinoma tissue (A-E) and the expression in the other normal kidney tissue (F-J) of cancer, and in renal carcinoma tissue, the expression of PGRMC1 is higher than the other normal kidney tissue of cancer.(A)-(D) represents the negative expression of PGRMC1 in renal carcinoma tissue respectively, weak positive expression, moderate positive are expressed and the dye levels of strong positive expression.(F)-(I) represents the negative expression of PGRMC1 in the other normal kidney tissue of cancer respectively, weak positive expression, moderate positive are expressed and the dye levels of strong positive expression.(E) and (J) represent the phosphorylation level of the PGRMC1 detected in the other kidney normal structure of renal carcinoma tissue and cancer respectively.In figure, horizontal line represents 100 μm (original amplifications 400 times).
Fig. 4 identifies the phosphorylation site Thr74 of PGRMC1 albumen in three pairs of renal carcinoma tissues and the other normal kidney tissue of cancer by Western blot.In renal carcinoma tissue, the phosphorylation level of PGRMC1 is higher than the other normal kidney tissue of cancer.
The Thr74 position phosphorylation level change of PGRMC1 albumen after Fig. 5 variable concentrations BAY 43-9006 process kidney OS-RC-2 cell 24h.
Embodiment
Foundation of the present invention is the research based on there is development associated protein to kidney, and by the research to 45 routine patients, Analysis and Identification goes out kidney associated protein PgR membrane component 1.
The present invention is the expression of application immunohistochemistry technology research PgR membrane component 1 albumen in renal carcinoma tissue further also, and functional study prompting and kidney closely-related PgR membrane component 1 albumen can be applied in clinical diagnosis, Index for diagnosis and drug development.
On the basis of above-mentioned a large amount of pioneering research, those skilled in the art just easily can using the mRNA of above-mentioned PgR membrane component 1 albumen or its gene as the Diagnosis of Renal Cell Carcinoma reagent and the kit that detect target; Simultaneously can the mRNA of above-mentioned PgR membrane component 1 albumen or its gene as the method for the screening treatment kidney medicine of target; Also invented the medicine preventing and treating kidney, such as: the molecule of energy antagonism PgR membrane component 1 albumen, such as its monoclonal antibody or polyclonal antibody, as PGRMC1 polyclonal antibody (ab48012); And obtain the good shRNA molecule of prevention effect and the plasmid of shRNA molecule of PgR membrane component 1 can be expressed.
Embodiment one, use the present invention detect kidney
1. biological cells and tissues samples sources
Human embryonic kidney cell line HEK293 (HEK293): be derived from ATCC, this laboratory is preserved; Human renal carcinoma cell strain OS-RC-2, is derived from ATCC, is purchased from Chinese Academy of Sciences's cell bank;
45 routine renal carcinoma tissues and the other normal kidney tissue of 45 routine kidneys, provide by Huaxi Hospital Attached to Sichuan Univ Urology Surgery, and sample supplier Post operation signature Informed Consent Form, every example is organized and all passed through pathological biopsy.
2. reagent source
Cell culture fluid DMEM: purchased from Gibco BRL; Leu-d
3: purchased from Britain Camb isotopic laboratory; Dialysis serum is purchased from Gibco; Containing Leu-d
3the DMEM of mark: autogamy; Hyclone (FBS), digestive juice pancreatin (Trypsin) are purchased from Gibco BRL.
Mass spectrum level trypsase Trypsin Gold: purchased from Promega; Acetonitrile (ACN), trifluoroacetic acid (TFA): be chromatographically pure level, purchased from Fisher Scientific; NH4HCO3: chromatographically pure, purchased from Merck; Alpha-cyano-4-hydroxycinnamic acid (CHCA): purchased from Sigma; Protease inhibitors-Cocktail: purchased from Sigma.
PGRMC1 polyclonal antibody (ab48012): purchased from Abcam; Phospho-Threonine-Proline antibody (#9391): purchased from Cell signalingtechnology; Mouse anti human β-actin antibody: purchased from doctor's moral biotechnology company; Horseradish peroxidase (HRP) marks goat anti-mouse igg, the anti-sheep IgG of rabbit, goat anti-rabbit igg all purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.
The super quick S-P(mouse/rabbit of instant SABC) kit (KIT-9710) wins Bioexperiment Materials Research Laboratories purchased from Beijing Lay.DAB chromogenic reagent box (AR1022) is purchased from Wuhan Boster Biological Technology Co., Ltd..
3. method
Stable isotope (the Leu-d of 3.1 cells
3) mark
HEK293 cell is containing Leu-d
3cultivate with in the DMEM nutrient solution of 10% dialysis hyclone (D-FBS), changed a nutrient solution every 3 days, treat that cell grows to about 90% and goes down to posterity.Often for cell stay 1/3 continuation cultivate, collect all the other cells and with ice-cold PBS wash put for 3 times-80 DEG C for subsequent use.After cultivating 6-8 generation, extract each generation total protein of cell, be separated through SDS-PAGE, coomassie brilliant blue staining, cuts after decolouring often for the β-actin band of cell protein.Enzymolysis, carry peptide after, MALDI-TOF MS detect often for isotope integration, until completely mark till.Conservation after cell marks completely.
The preparation of 3.2 protein samples, separation and enzymolysis
By the nephridial tissue sample of the cell that gathers or grind into fine powder, the RIPA lysate cracking of 500 μ L of general one bottle (area 80cm culture flask) cell, tissue sample adds RIPA lysate by the concentration of 5mg/mL (i.e. 5mg tissue 1mL RIPA lysate cracking), add protease inhibitors cocktail (RIPA/cocktail=50/1 (V/V) simultaneously, namely 10 μ L cocktail are added in 500 μ L RIPA by volume), piping and druming mixing.After placing 30-40min on ice, utilize ultrasonic treatment: use probe type ultrasonication to carry out the very brief impact of appropriate frequency on ice, 5-8 time altogether, each 5-10sec, midfeather 10-20sec.Cleavage mixture in 4 DEG C, 15000r/min centrifugal (the centrifugal 30min of lysis potpourri, the centrifugal 60min of Tissue Lysis potpourri).Draw supernatant to manage in new Ep, measure protein concentration with Protein Assay Kit.-80 DEG C save backup or test for downstream immediately.
3.2SDS-PAGE electrophoresis and dyeing
Protein sample prepares: according to mensuration concentration (measure protein concentration be generally 6-12mg/mL), by the cell protein of mark respectively with the histone mixed in equal amounts of two states,
Loading electrophoresis: add equal-volume 2 × SDS sample-loading buffer in the sample to which, the centrifugal 5min of boiling water boiling 5-10min, 12000g, retains supernatant.Starting potential 80V, until bromophenol blue indicator enters separation gel, adjustment voltage is 120V, arrives bottom margin to indicator, stops electrophoresis, takes out gel corner cut and marks.Coomassie brilliant G-250 jolting stained over night, destainer decolouring is removed completely to background.To gel image scanning imaging, store.
3.4 enzymolysis, carry peptide, Mass Spectrometric Identification and analysis
Glue is washed 2-3 time, to remove residual acetic acid and ethanol with Milli-Q before cutting glue.Cut film with plastics and glue is cut into 1mm
3the micelle of size, is contained in 0.6mL EP pipe.Add the band of 100ul(for little glue) 50% acetonitrile (Acetonitrile, ACN)/50%50mM NH
4hCO
3solution, DL 10-30min on whirlpool mixed instrument, removes supernatant, repeats 1-2 time, until decolour completely.Add 100 μ L ACN and mix micelle, ambient temperatare puts 5-10min, and visible micelle bleaches, and sucks ACN; Repeat 1-2 time, place 10-20min at 37 DEG C, allow residual ACN volatilize to the greatest extent.Often pipe adds 12.5ng/ μ L pancreatin and was not had by micelle.37 DEG C, enzymolysis spends the night.Transferred to by enzymolysis liquid in new 0.6mL EP pipe, add 50-80 μ L50%ACN/5%TFA, ultrasonic 10-15 minute in micelle, centrifugal, supernatant merges; Add 20-50 μ L50%ACN/5%TFA again, repeat 1-2 time, supernatant merges.Seal up parafilm film, prick hole ,-80 DEG C of freezing supernatants, freeze-drying, carries out Mass Spectrometric Identification.
3.5 SABC detect expression and the phosphorylation level of PGRMC1 albumen
3.5.1 organized processing and section
(1) get people's kidney and cancer beside organism, be cut into small pieces, thickness is no more than 5mm, carries out mark, and fix more than 24h by 10% neutral formalin, tap water spends the night;
(2) graded ethanol is crossed: 75% alcohol, 1 time, 1h → 85% alcohol, 1 time, 1h → 95% alcohol, 1 time, 1h → 100% alcohol, 2 times, each 30min;
(3) dimethylbenzene, 2 times, each 30min;
(4) paraffin soaks, 3 times, each 30min;
(5) with paraffin-embedded tissue;
(6) cut into slices: thickness is 3 ~ 5 μm.
3.5.2 immunohistochemical staining
(1) paraffin section de-waxing is to water: paraffin section is placed in 60 DEG C and heats 2h, be placed in rapidly dimethylbenzene I dewaxing 10min, be placed in dimethylbenzene II dewaxing 10min again, use each 2min of 100%, 95%, 80%, 70% ethanol postincubation respectively, distilled water washs 2 times (being placed in shaking table), each 5min.
(2) hydrogen peroxide (Maixin-Bio, KIT-9710a) closes endogenous peroxydase: 3%H2O2, room temperature lucifuge process 10min, then with distilling washing 2 times, each 5min.
(3) antigen retrieval: section be placed in antigen retrieval buffers (10mM sodium citrate buffer solution, pH6.0), boil rear hyperbaric heating 5min, naturally cool to room temperature, PBS develops a film 2 times, each 5min.
(4) normal serum is closed: take out section, moisture (keeping tissue in moisture state) around section back side moisture and section face weave is sucked with filter paper, drip normal serum (with second antibody isogenic animal serum) (Maixin-Bio, KIT-9710b), hatch 15min for 37 DEG C, PBS washes 2 times, each 5min.
(5) primary antibodie is hatched: inhale unnecessary serum with filter paper, and directly drip goat-anti people's PGRMC1 polyclonal antibody or Phospho-Threonine-Proline antibody (1:200), 4 DEG C of night incubation, PBS washes 2 times, each 5min.
(6) two anti-hatch: drip biotin labeled second antibody (Maixin-Bio, KIT-9710c), hatch 40min for 37 DEG C, PBS washes 2 times, each 5min.
(7) three anti-hatch: drip enzyme mark streptavidin compound (Maixin-Bio, KIT-9710d) 37 DEG C and hatch 40min, PBS washes 2 times, each 5min.
(8) DAB colour developing: filter paper sucks section surrounding liquid, drip Fresh DAB working fluid (dilution 20 times after developer A: developer B: developer C1:1:1 mixing) colour developing, Microscopic observation, uses tap water color development stopping in good time.
(9) haematoxylin is redyed: room temperature 30sec, and tap water returns blue 15min.
(10) gradient alcohol dehydration: 80% alcohol, 2min → 95% alcohol, 2min → 100% alcohol, 2 times, each 5min.
(11) neutral gum mounting is used, light Microscopic observation
4. result and analysis
Mass Spectrometric Identification result is as Fig. 1.By based on Leu-d
3quanti-fication proteomics qualification, in renal carcinoma tissue, the other normal kidney tissue of the expression ratio cancer of PGRMC1 raises 3.91 times.
For verifying with Leu-d further
3for interior scalar quantity histone group method is applicable to the quantitative comparison newly identifying differential protein, we have done Western Blot and immunohistochemical analysis to PGRMC1.Western Blot(Fig. 2) and the display of SABC (Fig. 3) result, with conservative protein β-actin for contrast, compared with the other normal kidney tissue of cancer, PGRMC1 expresses much higher in renal carcinoma tissue.
The phosphorylation site of existing bibliographical information PGRMC1 albumen has Ser-57, Ser-181, Tyr-139, Tyr-180, Thr-74[11,12].We apply LC-ESI-MS/MS mass-spectrometric technique, have identified phosphorylation site Thr-74(Fig. 4 of PGRMC1 albumen first in kidney).In three pairs of renal carcinoma tissues and the other normal kidney tissue of cancer, the phosphorylation site Thr-74 of PGRMC1 albumen is identified by Western blot.In renal carcinoma tissue, the phosphorylation level of PGRMC1 is higher than the other normal kidney tissue of cancer.
In above-mentioned quantitative proteomics Analysis and Identification renal carcinoma tissue PGRMC1 albumen basis on, systematically analyze the relative expression levels of PGRMC1 albumen in kidney and the relation of kidney clinical scale first, the signal path that PGRMC1 protein phosphorylation participates in and biological function, these results of study so far there are no domestic and international report.We find that the expression of PGRMC1 albumen and kidney develop and have clinical correlation (see table 1), and PGRMC1 albumen obviously raises (p<0.01) in most of kidney.In 45 routine clear-cell carcinomas, in 1 example (2.2%) sample, PGRMC1 feminine gender is expressed, 44 examples (97.8%) positive expression, and wherein 20 examples (44.4%) are strong positive expression; And in the other nephridial tissue of cancer, 3 examples (6.7%) are negative expression, 42 examples (93.3%) are positive expression, and wherein 7 examples (15.6%) strong positive is expressed.
In table 1. kidney and cancer beside organism, PGRMC1 expresses
1.Student’s t test,p<0.001。
2.a: negative; B: the weak positive; C: moderate positive; D: strong positive.
Meanwhile, the mean age of 45 routine patients is 58 years old, and wherein 30 examples are the male sex, and other 15 examples are women.In 45 routine patients, we do not find the expression of PGRMC1 and the correlativity (p>0.05) of patient gender.But we find expression and patient's clear-cell carcinoma (renal cell carcinoma, RCC) Fuhrman rank correlation (p<0.05) (see table 2) of PGRMC1.Fuhrman Nuclear grading is widely used organizational hierarchy system in RCC tissue, Fuhrman Nuclear grading is the Nuclear grading system being widely used in clear-cell carcinoma at present most, whether this hierarchy system is mainly obviously divided into 4 grades according to nucleus size, shape and kernel, has well predict prognostic value to classic RCC.
The correlativity (n=45) of table 2.PGRMC1 expression and patient RCC Fuhrman grade
| Fuhrman grade * | Number of cases (%) | Score | Expression |
| G2 | 20(44.44%) | 6.40±3.87 | ++ |
| G3 | 23(51.11%) | 8.77±3.28 | ++/+++ |
| G4 | 2(4.44%) | 12 | +++ |
1.One-way ANOVA analyzes, p<0.05; LSD-t test, p<0.05(G2 compare G3; G2 is than G4).
2.+: weak expression; ++: moderate is expressed; +++: strongly expressed.
Result shows, patient RCC Fuhrman higher grade, and the expression of PGRMC1 is higher.Illustrate that the expression of PGRMC1 becomes positive correlation with patient's RCC tumor grade, show the malignancy that can be characterized RCC by the expression of PGRMC1.
On this basis, we, with 15 μMs of BAY 43-9006 process kidney OS-RC-2 cell 24h, find that the expression change of PGRMC1 albumen is little, but the Thr74 position phosphorylation level change of PGRMC1 albumen obviously (Fig. 5), and can affect human renal carcinoma cell growth.
Result of study of the present invention illustrates PGRMC1 albumen, in kidney, developing effect occurs, for optimizing current available therapy and the new target molecules of development kidney diagnosis and treatment is significant, especially there is important using value instructing exploitation PGRMC1 targeting proteins to be used for the related reagent of kidney early diagnosis or kit and kidney medicine direction.
List of references
【1】Cahill MA:Progesterone receptor membrane component1.an integrative review.J Steroid Biochem Mol Biol2007,105:16-36.
【2】Losel RM,Besong D,Peluso JJ,Wehling M:Progesterone receptor membrane component1--many tasks for a versatile protein.Steroids2008,73:929-934.
【3】Crudden G,Loesel R,Craven RJ:Overexpression of the cytochrome p450activator hpr6(heme-1domain protein/human progesterone receptor)in tumors.Tumour Biol2005,26:142-146.
【4】Peluso JJ,Liu X,Saunders MM,Claffey KP,Phoenix K:Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1.J Clin Endocrinol Metab2008,93:1592-1599.
【5】Difilippantonio S,Chen Y,Pietas A,Schluns K,Pacyna-Gengelbach M,Deutschmann N,et al:Gene expression profiles in human non-small and small-cell lung cancers.Eur J Cancer2003,39:1936-1947.
【6】Dressman HK,Hans C,Bild A,Olson JA,Rosen E,Marcom PK,et al:Gene expression profiles of multiple breast cancer phenotypes and response to neoadjuvant chemotherapy.Clin Cancer Res2006,12:819-826.
【7】Irby RB,Malek RL,Bloom G,Tsai J,Letwin N,Frank BC,et al:Iterative microarray and RNA interference-based interrogation of the SRC-induced invasive phenotype.Cancer Res2005,65:1814-1821.
【8】Neubauer H,Clare SE,Wozny W,Schwall GP,Poznanovic S,Stegmann W,et al:Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1.Breast Cancer Res2008,10:15.
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Claims (3)
1. the purposes in Diagnosis of Renal Cell Carcinoma reagent prepared by the reagent detecting PgR membrane component 1 protein expression level and/or phosphorylation level.
2. purposes according to claim 1, is characterized in that: described detection PgR membrane component 1 protein expression level and/or the reagent of phosphorylation level comprise polyclonal antibody or the monoclonal antibody of PgR membrane component 1.
3. purposes according to claim 1, is characterized in that: described detection PgR membrane component 1 protein expression level and/or the reagent of phosphorylation level comprise the PCR primer of energy specific amplified PgR membrane component 1 gene mRNA.
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| CN201310460771.4A CN103543266B (en) | 2013-09-30 | 2013-09-30 | Applications of reagent for detecting PGRMC1 to preparation of kidney cancer diagnosis reagent and kit |
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| CN201310460771.4A CN103543266B (en) | 2013-09-30 | 2013-09-30 | Applications of reagent for detecting PGRMC1 to preparation of kidney cancer diagnosis reagent and kit |
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