CN103525941A - Application of CTHRC1 genes in preparation of drugs for detecting/treating cervical cancer - Google Patents
Application of CTHRC1 genes in preparation of drugs for detecting/treating cervical cancer Download PDFInfo
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Abstract
The invention provides an application of CTHRC1 (the Chinese name of CTHRC1 is triple helix repeat collagen I) genes in preparation of drugs for detecting and treating cervical cancer. According to the application, a Real-time PCR method is used for detecting expression of mRNA of CTHRC1 in cervical cancer tissue and expression of mRNA of CTHRC1 in normal control tissue, a tissue microarray technology is used for building a tissue chip containing 102 cases of cervical sqamous cell carcinoma, 30 cases of cervical adenocarcinoma and 31 cases of normal cervical tissue samples, an immunohistochemical technique is used for detecting expression of CTHRC1 in the tissue chip, the fact that the positive expression rate of CTHRC1 in normal cervical tissue is much lower than the positive expression rate of CTHRC1 in the cervical cancer is found, and the expression strength of CTHRC1 is positively associated with cervical cancer myometrial invasion depth and cervical cancer grading. Therefore, the CTHRC1 genes can be used for preparing the drugs for detecting or treating the cervical cancer. CTHRC1 can obviously distinguish tumor, non-tumor, high-risk tumor and low-risk tumor no matter from the cellular level or the protein level, repeatability of results is good, and the application of the CTHRC1 genes in preparation of the drugs for detecting/treating the cervical cancer has good clinical application prospects and high application value and social benefits.
Description
Technical field
The present invention relates to medicine, be specifically related to biological medicament, relate in particular to the application in detect in preparation/treatment of CTHRC1 gene cervical cancer medicine.
Background technology
Cervical cancer has been acknowledged as an infectious cancer, is common gynecological cancer in world wide, is also gynecologic malignant tumor main causes of death, accounts for global women because of the 4th of the lethal cause of disease of tumour.According to international cancer institute (IARC) updated statistics, show, within 2008, there are 52.98 ten thousand cervical cancer new cases in the whole world, 27.51 ten thousand deaths, within average 2 minutes, just there is 1 women to die from cervical cancer [Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D:Global cancer statistics.CA Cancer J Clin2011,61 (2): 69-90.].Within 2010, U.S.'s cervical cancer prediction neopathy number of cases is 12,170, estimates death 4,220 examples [Jemal A, Siegel R, Xu J, Ward E:Cancer statistics, 2010.CA Cancer J Clin2010,60 (5): 277-300.].The generation of cervical cancer and the infection of HPV are closely related, lasting HPV infects and has promoted the development [Hutchinson DJ, Klein KC (2008) .Humanpapillomavirus disease and vaccines.American journal of health-system pharmacy:AJHP:official journal of the American Society of Health-System Pharmacists65:2105 – 2112.] of precancerous lesion to cervical cancer.CTHRCl (collagen triple helix containin1, triple helical repeats collagen protein I) gene is a new gene of finding in 2005, is to find in to the differential expression sequence screening of normal rat artery and spherical damage artery at first.Its transient expression is in injured blood vessel position, by suppressing the deposition of collagen stroma and promoting cell migration to participate in the injury repairing process of blood vessel.（Pyagay,P.et?al.Collagen?triple?helix?repeat?containing1,a?novel?secreted?protein?in?injured?and?diseased?arteries,inhibits?collagen?expression?and?promotes?cell?migration.Circ.Res.,2005.96,261–268.）。The CTHRC1 expressing in vascular cell, by the response of regulation and control transforming growth factor-beta (TGF-β), thereby suppress the target gene of TGF-β and the expression (LeClair of collagen protein, R.et al.The role of collagen triple helix repeat containing1in injured arteries, collagen expression, and transforming growth factor beta signaling.Trends Cardiovasc.Med., 2007.17.202 – 205; LeClair RJ; Durmus T, Wang Q, Pyagay P; Terzic A, Lindner V.Cthrc1is a novel inhibitor of transforming growth factor-_signaling and neointimal lesion formation.Circ Res2007; 100:826-33).CTHRC1 has strong expression in brephic many organs; particularly in the tabular cartilage increasing and ground substance of bone; it is by regulating Smad2/3 and TGF signal control collagen deposition (LeClair RJ there; Durmus T; Wang Q; Pyagay P, Terzic A, Lindner V.Cthrc1is a novel inhibitor of transforming growth factor-_signaling and neointimal lesion formation.Circ Res2007; 100:826-33; Durmus T; LeClair RJ, Park KS, Terzic A; Yoon JK, Lindner V.Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1) .Gene Expr Patterns2006; 6:935 – 40).In normal adult tissue, CTHRC1 represents very low; but under pathological state, there will be the artery or skin wound (the Yamamoto S that increase sharply as damage; Nishimura O; Misaki K; Nishita M; Minami Y, Yonemura S, et al.Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex.Dev Cell2008; 15:23 – 36).CTHRC1 albumen is by promoting the formation of Wnt part and Frizzled receptor complex actively to regulate PCP-Wnt signal path (Li YW, Dudley AT.Noncanonical frizzled signaling regulates cell polarity of growth plate chondrocytes.Development2009; 136:1083 – 92).By PCP-Wnt signal pathway, cell movement during CTHRC1 regulation of embryonic development.In the development of bone tissue, PCP-Wnt signal path can regulate chondrocyte's maturation and cartilage to form.The change of the gene in path is along with there will be Chondrogenesis; the imbalance of collagen deposition; (Li YW, Dudley AT.Noncanonical frizzled signaling regulates cell polarity of growth plate chondrocytes.Development2009 with cartilage metamorphosis; 136:1083 – 92; Ahrens MJ, Li Y, Jiang H, Dudley AT.Convergent extension movements in growth plate chondrocytes require gpi-anchored cell surface proteins.Development2009; 136:3463 – 74).CTHRC1 transgenic mice shows osteoblastic bone forming to be increased, calcification (Kimura, H.et al.Cthrc1is a positive regulator of osteoblastic bone formation.PLoS ONE, 2008 of differentiation aggravation and osteogenic cell, 3, e3174).By interacting with Wnt5a, CTHRC1 has also participated in activation plane cell polarity path, and this hint CTHRC1 has the effect of regulation and control form in cell generating process.Grownup, CTHRC1 regulate tumor cell migration and invasion.The DNA array analysis of a human tumor complementation shows, CTHRC1 gene has expression at most of mankind's solid carcinoma.CTHRC1 has expression in infiltrating primary and metastasis melanin tumor, but nothing is expressed in benign nevus or Noninvasive sample.Suppress CTHRC1 and express external migration (the Tang L that can reduce melanoma cell; Dai DL; Su M; Martinka M; Li G, Zhou Y.Aberrant expression of collagen triple helix repeat containing1in human solid cancers.Clin Cancer Res2006; 12:3716 – 22).In most of dermatofibrosarcoma protuberans (tumour of the invasive frequent transfer in a kind of part), have CTHRC1 to express, but in the optimum fibrous histiocytoma of majority without expression
14.Wang,L.et?al.Collagen?triple?helix?repeat?containing-1in?the?differential?diagnosis?of?dermatofibrosarcoma?protuberans?and?dermatofibroma.Br.J.Dermatol.,2011,164,135–140）。In mammary cancer, the expression of CTHRC1 is significantly higher than healthy tissues or precancerous lesion tissue, and the relevant (Kharaishvili of risk shifting with bone, G.et al.Collagen triple helix repeat containing1protein, periostin and versican in primary and metastatic breast cancer:an immunohistochemical study.J.Clin.Pathol.2011,64,977 – 982.).CTHRC1 has high expression level in carcinoma of the pancreas; thereby by regulating the vigor of pancreatic cancer cell and adhesivity (the Park EH that plays a role at tumour progression with in shifting; Kim S; Jo JY, et al.Collagen triple helix repeat containing-1promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells.Carcinogenesis.2013Mar; 34 (3): 694-702.).In addition, in liver cancer CTHRC1 cross expression often and late tumor large with huge tumor first close.Multiplicity shows that CTHRC1 is an independent prognostic factor of liver cancer.Thereby CTHRC1 is by improving invasion and attack and transfer (the Chen YL that promotes tumour with transfer ability that stick of tumour cell in liver cancer; Wang TH; Hsu HC, et al.Overexpression of CTHRC1 in Hepatocellular Carcinoma Promotes Tumor Invasion and Predicts Poor Prognosis.PLoS One.2013Jul29; 8 (7): e70324.).Yet, the report that so far there are no CTHRC1 is relevant to other cancers for the treatment of.
Summary of the invention
Technical problem to be solved by this invention is the new indication of research and design CTHRC1, for the preparation of diagnoses and treatment cervical cancer medicine.
The invention provides the application in detect in preparation/treatment of CTHRC1 cervical cancer medicine.
Gene title: CTHRC1
Chinese name: triple helical repeats collagen protein I
English name: Collagen Triple Helix Repeat Containing 1
Sequence: see sequence table 1(SEQ ID NO.1).
Particularly, the invention provides the application in detect in preparation/treatment of CTHRC1 gene cervical cancer medicine.
The CTHRC1 gene of indication of the present invention comprises the DNA encoding sequence that it is complete, its RNA sequence and mutant thereof, with and function on active fragment.Need to understand, when the identical amino acid of coding, the replacement of the Nucleotide in codon is acceptable.It is also to be understood that, by Nucleotide replace and produce conservative aminoacid replacement time, the conversion of Nucleotide is also can be received.In the situation that obtained the nucleic acid fragment of CTHRC1, can design specific probe according to nucleotide sequence.
Nucleotide full length sequence or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.Once obtain relevant sequence, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, then by ordinary method separation from the host cell propagation, obtains relevant sequence.
In addition, also can synthesize relevant sequence by the method for synthetic, especially fragment length more in short-term.Conventionally, by first synthetic a plurality of small segments, and then connect and can obtain the fragment that sequence is very long.
At present, can be completely by chemosynthesis, obtain the DNA sequence dna of code book invention albumen (or its fragment, derivative).Then this DNA sequence dna can be introduced in various existing DNA moleculars as known in the art (or as carrier) and cell.
In the present invention, CTHRC1 polynucleotide sequence can be inserted in recombinant expression vector.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is conventionally to contain replication orgin, promotor, marker gene and translation controlling elements.
Method well-known to those having ordinary skill in the art can be for building containing the DNA sequence dna of CTHRC1 and suitable transcribing/the translate expression vector of control signal.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described DNA sequence dna can be effectively connected in the suitable promotor in expression vector, to instruct mRNA synthetic.Conversion carrier also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for the phenotypic character of the host cell of selection conversion, as eukaryotic cell is cultivated Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of use, or for colibacillary tsiklomitsin or amicillin resistance.
Comprise above-mentioned suitable DNA sequence dna and the suitable carrier of promotor or control sequence, can be for transforming suitable host cell, with can marking protein.
Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, the bacterial cell of streptomyces; Fungal cell is as yeast; Vegetable cell; Insect cell; Zooblast etc.
After having obtained nucleotide sequence, can design specific dna probe according to nucleotide sequence.The people such as the method for designing probe is this area routine, visible Sambrook, described in molecular cloning laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989).In detection of biological sample, whether exist the exemplary method of CTHRC1 albumen or nucleic acid to comprise the biological sample that obtains test subject, make this biological sample contact the nucleic acid probe of the mark that can hybridize with CTHRC1mRNA or genomic dna.Other probe for diagnostic test of the present invention is as described herein.
Nucleic acid probe contacts with the flag sequence of amplification.This probe is preferably connected to a kind of chromophoric group, but can be by radio-labeled.In another embodiment, probe is connected on a kind of binding partners, as antibody or vitamin H, or another kind of carry can the binding partners in detection architecture territory on.
In traditional method, detection can be carried out by Southern trace and with the probe hybridization of mark.The related technology of Southern trace is (referring to Sambrook etc., 1989) well-known to those skilled in the art.Conventional detection also has biochip, fluorography technology, cell streaming counting etc.
On the other hand, the present invention also comprises CTHRC1DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refers to that antibody capable is incorporated into CTHRC1 gene product or fragment." preferably " refer to that those can be combined with CTHRC1 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
The antibody of anti-CTHRC1 albumen can be used in immunohistochemistry technology, detects the CTHRC1 albumen in biopsy specimen, can also be as the specific treatment agent for preventing cervical cancer, ovarian cancer to shift and attack.
The direct mensuration of CTHRC1 in blood sample or urine can be used as the auxiliary diagnosis of tumour and the observation index of prognosis, also can be used as the foundation of early diagnosis of tumor.
Antibody can pass through ELISA, Western engram analysis, or with detection moiety coupling, by methods such as chemoluminescence, isotopic tracings, detect.
The present invention also comprises test kit, to carry out any method described herein.In a unrestriced example, described test kit comprises one or more in these reagent by the vessel form with suitable.Described test kit also can comprise for the reagent of the purifying of RNA separation, amplifying cells RNA, mark etc.
Detection of the present invention or treatment cervical cancer medicine comprise medicine or the test kit by RT-PCR, PCR, immunodetection, in situ hybridization, genechip detection or treatment cervical cancer.
The described medicine by RT-PCR detection cervical cancer at least comprises the primer of a pair of specific amplification CTHRC1 gene.
The described medicine by PCR detection cervical cancer at least comprises the primer of a pair of specific amplification CTHRC1 gene.
The described medicine by immunodetection detection cervical cancer comprises antibody, polyclonal antibody and the monoclonal antibody of being combined with CTHRC1 protein-specific.
The described medicine by situ hybridization detection cervical cancer comprises the probe with CTHRC1 nucleic acid array hybridizing.
The described medicine by genechip detection cervical cancer comprises the probe with CTHRC1 nucleic acid array hybridizing.
The described test kit for detection of cervical cancer comprises for the reagent of the purifying of RNA separation, amplifying cells RNA, mark etc.
The component of test kit can or be packed with the form of freeze-drying with the form of water medium.Container suitable in test kit at least comprises a kind of bottle, test tube, flask, PET bottle, syringe or other container conventionally, wherein can place a kind of component, and preferably, can carry out suitably decile.While existing more than a kind of component, in test kit, conventionally also by comprising second, third or other additional container, wherein place discretely additional component in test kit.Yet the component of various combination can be comprised in a bottle.Test kit of the present invention is conventionally also a kind of for holding the container of reactant by comprising, sealing is for commercial distribution.This container can comprise the plastic containers of injection molding or blowing mould, wherein can retain required bottle.
Another object of the present invention has been to provide the application in detect in preparation/treatment of CTHRC1 gene cervical cancer medicine.
Medicine of the present invention comprises: by RNA, disturb the protein that suppresses the double stranded RNA of CTHRC1 genetic expression, tumor vaccine based on CTHRC1 antigen protein or suppress CTHRC1 protein-active.
Detection/treatment cervical cancer medicine of the present invention can be by oral, skin or the administration of parenteral mode.
Detection/treatment cervical cancer medicine of the present invention can be made the formulations such as oral, suction, injection or suppository by this area routine techniques.
The inventor is by following test:
A. choose during 8 days-May 30 March in 2011 in informed consent the medical crowd in Women's Hospital, Senior Residents in Fengxian District of Shanghai that accepts voluntarily cervical HPV detection of nucleic acids and carry out cervical exfoliated cell sampling examination and epidemiology survey.To carrying out voluntarily further examiner, do again ThinPrep liquid-based cytology test (TCT) and vaginoscopy, the conventional section of cervical biopsy organization department dispensing pathology, it is frozen that part is positioned over-80 ℃ of refrigerators, extracts-80 ℃ of refrigerators of its serum placement frozen simultaneously.In above-mentioned crowd, choose cervical tissue (cervicitis, CIN and cervical cancer) 30 examples that HPV16 and 18 infects, patient's 30 examples of the HPV feminine gender of coupling age indifference, we with RT-PCR and method detect its CTHRC1 expression, its differential expression of comparison; Further by ELASA method, detect the expression of CTHRC1 in its serum simultaneously.
B. the 102 routine SCC that utilized micro-array tissue technique construction, 30 routine adenocarcinoma of the uterine cervix, the organization chip of 31 routine normal cervical tissues samples, and utilize immunohistochemistry technology to detect the expression of CTHRC1 in these samples, in uterus positive expression rate is far below positive expression rate in cervical cancer (16.68%vs73.25%) in film healthy tissues to find CTHRC1, and CTHRC1 expression intensity attacks the degree of depth (p < 0.0001) with cervical cancer flesh layer and Cervical Tumor classification (p=0.0167) is proportionate.
According to the above-mentioned experimental result especially analysis of clinical of large sample amount, there is positive connection in biological characteristics and the cervical cancer patient prognosis of the inventor is verified CTHRC1 gene and cervical cancer.The encoding histone product C THRC1 albumen of this gene, as a kind of secretor type glycoprotein, can be detected in serum, for the early diagnosis of the diseases such as cervical cancer and prognosis judgement provide a kind of new means.Therefore, CTHRC1 gene can be for the preparation of detect/treatment cervical cancer medicine.
The present invention's detection of CTHRC1 gene and product thereof in cervical cancer research and clinical cited field propose to utilize based on serum/tissue first reaches early diagnosis cervical cancer or the cervical cancer patient being treated surgically is carried out to prognosis judgement.The large sample clinical sample that experiment of the present invention is based upon on the checking of real-time quantitative PCR on gene level and protein level is verified, reliable results, and according to experimental result, no matter CTHRC1 is on cell levels or protein level, significantly to distinguish tumour and non-tumour, high-risk tumour and low danger tumour, result reproducible, have and become the potential quality of good clinical exercisable biomarker and good potential applicability in clinical practice, have larger using value and social benefit.
Accompanying drawing explanation
Fig. 1 HPV (-) cervical tissue and HPV(+) the mrna expression situation of CTHRC1 in cervical tissue
The mrna expression difference condition of CTHRC1 in Fig. 2 normal cervical tissues and cervical cancer tissues
Fig. 3 cervical cancer tissues immunohistochemical staining result (tumor tissues, strong positive)
Fig. 4 normal cervical tissues immunohistochemical staining result (contrast, feminine gender)
Fig. 5 is that the mrna expression water gaging of CTHRC1 in 5 strain endometrial carcinomas cells is flat, and ordinate zou represents the mrna expression level of CTHRC1 in clone, and X-coordinate from left to right represents cervical cancer tumer line HeLa, SiHa, HeLa229, MS751, Caski, C-33A successively
Fig. 6 is the cell invasion experimental result of cervical cancer cell MS751 cell after interference CTHRC1
Fig. 7 is the cell migration experimental result of cervical cancer cell MS751 cell after interference CTHRC1
Fig. 8 was cervical cancer cell HeLa cell invasion experimental result after expression CTHRC1
Fig. 9 was cervical cancer cell HeLa cell migration experimental result after expression CTHRC1
Embodiment
Embodiment 1, the CTHRC1 expression in Patients with Cervical Cancer tissue
Main agents
CTHRC1 monoclonal antibody is produced by Huaan, Hangzhou antibody company, and horseradish peroxidase-labeled two is anti-(purchased from Abmart company; HRP-bis-is anti-), diaminobenzidine (Diaminobenzidine, DAB) chromogenic substrate carries out the structure of immunostaining cervical cancer tissues chip array.
1.1 immunohistochemical staining
Before 1.4 dyeing, paraffin section is placed in to 80 ℃ of baking ovens and toasts 20 minutes, then press dimethylbenzene 10min, 1/2 dimethylbenzene 10min, 100% alcohol 10min, 95% alcohol 10min, 85% alcohol 10min, 75% alcohol 10min flow process dewaxing.The section that dewaxing is complete is dyeed by following flow process: tap water rinses 5-10min, antigen retrieval 10min after naturally cooling in 37 ℃ of 0.3%H2O2 3min with 1 * PBS, clean after the sealing of 10%BSA room temperature within 1 hour, hatch again 4 ℃ of primary antibodies and spend the night, second day is washed the 3 times anti-incubated at room 1-2h1 of HRP bis-* PBS with 1 * PBS and is cleaned then flowing water flushing 15min brazilwood extract dyeing 30-45s of rear DAB color development 10s, and tap water rinses 5min.Press afterwards 75% alcohol 5min, 85% alcohol 5min, 95% alcohol 5min, 100% alcohol 5min1/2 dimethylbenzene 5min, dimethylbenzene 5min flow process is dewatered, and uses resinene mounting, Microscopic observation after dehydration.
1.4 organization chip result judgements
In micro-Microscopic observation cervical cancer tissues sample, CTHRC1 expresses dyeing situation, and cervical cancer tissues sample expression amount is significantly higher than negative control group.(see Fig. 3,4 in)
1.5 statistical analysis
Further statistical analysis discovery cervical cancer tissues is compared with negative control group and is had significant significant difference.
Embodiment 2
Utilize slow virus interference carrier disturb in cervical cancer tumer line CTHRC1 gene and carry out cell function experimental study
CTHRC1 gene adopts the complete coding region (open reading frame) of people CTHRC1cDNA as goal gene, complete coding region sequence is with the ORF sequence in summary of the invention (NCBI Reference Sequence:NM_001256099.1) 1214bp, and the albumen of coding is asked for an interview sequence table 1(SEQID NO.1).
2.1 main agents
Cervical cancer cell strain HeLa, SiHa, MS751, Caski, C-33A is all purchased from Chinese Academy of Sciences's cell bank, and cell culture medium and foetal calf serum are all purchased from Gibco company, and CTHRC1 gene RNA disturbs Lentiviral purchased from Shanghai JiMa pharmacy Technology Co., Ltd.RNA extraction agent RNAiso Plus is purchased from precious biotechnology company limited, reverse transcription test kit High Capacity cDNA Reverse Transcription Kits is purchased from Invitrogen company, and PCR kit for fluorescence quantitative Power SYBR Green PCR Master Mix is purchased from Invitrogen company.CTHRC1 gene and house-keeping gene 18S primer are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
2.2 application real time fluorescence quantifying PCR methods detect the expression amount of CTHRC1 in five strain cervical cancer tumer lines
2.2.1. cell cultures: cervical cancer cell strain is cultivated based on 37 ℃ with the MEM of the foetal calf serum containing 10%, cultivates in the incubator of 5%CO2, treats that cell covers with extraction cell total rna.
2.2.2. cell total rna extracting:
Homogenate is transferred in centrifuge tube to standing 5 minutes of room temperature.12,000g4 ℃ centrifugal 5 minutes.The careful supernatant liquor of drawing, moves in other centrifuge tube.The chloroform that adds 1/5 volume of RNAiso Plus, thermal agitation 15 seconds, after solution is fully emulsified, more standing 5 minutes of room temperature (20-25 °), 12,000g4 ℃ is centrifugal 15 minutes.Drawing supernatant liquor is transferred in another new centrifuge tube.In supernatant, add isopyknic Virahol, after the centrifuge tube that turns upside down fully mixes, at 15～30 ℃ standing 10 minutes.12,000g4 ℃ centrifugal 10 minutes.Supernatant discarded, adds 75% ethanol 1ml along tube wall, the washing centrifuge tube tube wall that turns upside down, and 12,000g4 ℃ discards ethanol after centrifugal 5 minutes.Drying precipitated 2～5 minutes of room temperature (20-25 °), adds the RNase-free water dissolution of (40-80ul) in right amount with Nanodrop2000, to measure RNA concentration and purity after precipitating.
2.2.3. reverse transcription synthesizes cDNA
2.2.4Real-time?PCR
By following component preparation PCR reaction solution (reaction solution is formulated on ice and carries out).
Two-step approach is carried out PCR reaction, with 7300Real-Time PCR System,
PCR detects the primer:
F：AGTTACCCCAACGCGAAG
R：CTCTTGGTCACAGGATGACAC
Derived data, Realtime PCR result is analyzed by 2-△ △ CT method.Utilize Graphad Prism Software on Drawing to go out the chart (experimental result as shown in Figure 5) of CTHRC1 expression level in 5 strain cervical cancer cells.
2.3CTHRC1 gene RNA disturbs the cervical cancer cell strain MS751 of lentiviral vectors transfection CTHRC1 high expression level, with the cervical cancer cell strain HeLa cell strain of the low expression of CTHRC1 over-express vector transfection CTHRC1.
Utilize CTHRC1 gene RNA Gan Rao ∕ to cross expression Lentiviral and three packaging plasmid (GAG, REV, VSV) while cotransfection 293T cell, in cell, carry out viral packing, packaged pseudovirion is secreted in extracellular substratum, and centrifuging obtains the infection that can be used for host cell after supernatant liquor, after goal gene enters into host cell, through reverse transcription, be incorporated into genome, thereby play the function of related gene expression.Utilize tetracycline to screen metainfective cell, cell extraction RNA after screening, reverse transcription is that cDNA utilizes fluorescence quantitative PCR detection CTHRC1 Gan Rao ∕ to cross expression efficiency, found that the jamming effectiveness of two fragments wherein, more than 70%, crosses and express more than 100 times.
The 2.4 dry ∕ of disturbing carry out cell migration and cell invasion experiment after crossing expression CTHRC1
Cell migration experiment: by the hungry 24h of the cervical cancer cell in 6cm ware; With trysinization, after counting, be diluted to 20 * 104/ml, i.e. 4 * 104/200ul; Blow the cell suspension that even rear upper chamber vertically adds 200ul, lower chamber adds 600-800ul substratum, and for the purifying protein with 2%FBS dilution, (concentration gradient is respectively 0,5,50nmol/ml).After 17-20h, take out cell, suck the training liquid of chamber, PBS embathes 2-3 time, each 2min; With cotton swab or tweezers folder cotton-wool, clean gently upper chamber and do not attack cell; With 1% glutaraldehyde fixed cell 30min, PBS washes once, dries rear use 1% violet staining cell 30min, and PBS embathes 3 times; Cell faces up, and is placed on slide glass, is just putting microscopic examination, and 6 visual field film making countings are chosen in every hole at random.
Cell invasion experiment: matrigel4 ℃ of colloidal sol 0.5-1h, be melted into liquid state, prepare 24 orifice plates, transwell cell; Matrigel and serum free medium, with 1:4 dilution proportion, are placed on ice; Get 100ul dilution glue and evenly coat transwell cell filter membrane surface; In 37 ℃ of 5%CO2 temperature of saturation incubators, hatch 2-4h.The cell of taking the logarithm vegetative period, with serum-free or 2% serum free culture system cell, hungry 24h; Get the cell of hungry 24h, 0.25% trysinization, stops counting after digestion; With the nutrient solution diluting cells suspension of serum-free, making cell density is 1 * 106/ml; Every group of cell suspension got 100ul and added chamber, and making every ventricular cell number is 1 * 105/ml, and every group of cell established 2-3 multiple hole; In lower chamber, add 600-700ul to contain the nutrient solution of 5%FBS, during as motion chemoattractant ，Zhong Ban, between lower floor's substratum and cell, do not have bubble.After planting plate, continue to cultivate 48h; Take out cell, suck the training liquid of chamber, PBS embathes 2-3 time, each 2min; With cotton swab or tweezers folder cotton-wool, cleaning gently upper chamber does not attack cell and cleans artificial substratum glue; With 1% glutaraldehyde fixed cell 30min, PBS washes once, dries rear preparation dyeing; 1% violet staining cell 30min, PBS embathes inferior; With cell, face up, be placed on slide glass, micro-Microscopic observation is also taken pictures.
(1) cell invasion experimental result (as shown in Figure 6) and cell migration experimental result (as shown in Figure 7) after the rear cervical cancer cell MS751 of CTHRC1 interference
By upper figure experimental result, can find out that cervical cancer cell MS751 disturbs cell invasion and transfer ability after CTHRC1 gene all to decline.
(2) cross and to express the Matrigel result (as shown in Figure 8) of cervical cancer cell HeLa after CTHRC1 and to move experimental result (as shown in Figure 9).
By upper figure experimental result, can be seen to appear and express cervical cancer cell HeLa cell invasion and transfer ability after CTHRC1 gene and all obviously strengthen.
Claims (10)
- Application in detect in preparation/treatment of 1.CTHRC1 gene cervical cancer medicine, is characterized in that, described CTHRC1 gene order is SEQ ID NO.1.
- 2.CTHRC1 gene detects the application in cervical cancer medicine in preparation, it is characterized in that, described CTHRC1 gene order is SEQ ID NO.1.
- 3. application according to claim 2, is characterized in that, described detection cervical cancer medicine is medicine or test kit by RT-PCR, PCR, immunodetection, in situ hybridization or genechip detection cervical cancer.
- 4. application according to claim 3, is characterized in that, described detection at least comprises the primer of a pair of specific amplification CTHRC1 gene with the medicine of RT-PCR detection cervical cancer.
- 5. application according to claim 3, is characterized in that, described detection at least comprises the primer of a pair of specific amplification CTHRC1 gene with the medicine of PCR detection cervical cancer.
- 6. application according to claim 3, is characterized in that, described detection comprises antibody, polyclonal antibody or the monoclonal antibody of being combined with CTHRC1 protein-specific with the medicine of immunodetection cervical cancer.
- 7. application according to claim 3, is characterized in that, described detection comprises the probe with CTHRC1 nucleic acid array hybridizing with the medicine that in situ hybridization detects cervical cancer.
- 8. application according to claim 3, is characterized in that, described detection comprises the reagent of purifying or the CTHRC1 gene of mark separated for RNA, amplifying cells RNA for detection of the test kit of cervical cancer.
- The application of 9.CTHRC1 gene in preparation treatment cervical cancer medicine, is characterized in that, described CTHRC1 gene order is SEQ ID NO.1.
- 10. application according to claim 9, it is characterized in that, described treatment cervical cancer medicine comprises by RNA and disturbs double stranded RNA or the tumor vaccine based on CTHRC1 antigen protein that suppresses CTHRC1 genetic expression or the protein that suppresses CTHRC1 protein-active.
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