CN105617359A - Application of Wnt signal path protein Frzb in preparation of anti-hepatoma medicine - Google Patents

Abstract

The invention relates to an application of a Wnt signal path protein Frzb in preparation of an anti-hepatoma medicine and belongs to the technical field of biological medicines. In the invention, it is determined that the expression of Frzb in a hepatoma patient liver tissue sample library and a hepatoma cell line is relatively low by means of a protein immunoblotting and immunohistochemistry technology, and it is discovered that Frzb presents an expression characteristic of gradual reduction along with the rising of a pathological grade in hepatoma patient liver tissues; and by means of a nude mouse subcutaneous xenograft and caudal vein pulmonary metastasis model, a research shows that Frzb remarkably inhibits growth of a nude mouse subcutaneously transplanted tumor and formation of pulmonary metastasis, and the inhibition ratios are 85.4 percent and 68.6 percent (P is smaller than 0.01).

Description

Wnt signaling pathway protein Frzb application in preparing anti-liver cancer drug

Technical field

The present invention relates to the application of a kind of Wnt signaling pathway protein, be specifically related to a kind of Wnt signaling pathway protein Frzb application in preparing medicines resistant to liver cancer, belong to biomedicine technical field.

Background technology

Hepatocarcinoma serious threat human health, still lacks effective medicine at present. Hepatocarcinoma (HepatocellularCarcinoma, HCC) is China's malignant tumor occurred frequently, there are about 130,000 people every year and dies from hepatocarcinoma, accounts for the 40% of whole world PLC mortality number. Although the Therapeutic Method of hepatocarcinoma includes the kinds of schemes such as operation, but therapeutic effect is still extremely limited so far, not yet significantly extend the life cycle of patient, and for the current first clinical treatment medicine having become advanced liver cancer patient of inhibitors of kinases Sorafenib (Sorafenib) of the Mutiple Targets such as VEGFR, RAF, again show that the targeted molecular for tumor cell nonspecific signal transduction network regulation has significant clinical efficacy and researching value.

Wnt signal path plays a significant role in the pathological processes such as fetal development, stem cell, immune system and tumor generation. The generation development of the multiple human tumors such as Wnt signal path and colon cancer, hepatocarcinoma, pulmonary carcinoma, carcinoma of prostate, renal carcinoma, the melanoma as one of direction, biomedical sector forward position is closely related. Research confirms, Wnt signal path plays an important role in the physiological process such as liver development, liver normal growth, liver cell regeneration, liver metabolism, and this path aberrant continuation activates the progress significant correlation with multiple chronic hepatopathy and liver tumor. Transgenic mice liver cancer model and human hepatoma cell Histological research it turned out in hepatocarcinoma Carcinogenesis Wnt path the gene mutation such as ��-catenin, APC, Axin1 and Axin2, and the imbalance of Wnt and Frizzled receptor element is likely to also be one of early hepatocarcinoma phenomenon. In addition, B-mode, hepatitis C virus and Wnt path close association, hepatitis B viruses (HBV) X protein can activate Wnt path and participate in the formation of hepatocarcinoma, at least part of and Wnt-1 the intervention of hepatitis C virus D8L energy cell cultured supernatant growth course is closely related, and in hepatitis C virus oncogenic process, ��-catenin sudden change chance is higher with frequency ratio hepatitis B. Therefore, it is suppressed that or block the protein antagonist of Wnt signal path or chemical small molecule and can provide new molecular targeted strategy for the preventing and treating of hepatocarcinoma.

Frizzleds associated protein (the secretedfrizzled-relatedprotein of secreted, sFrp) it is the natural agonist of Wnt signal path, Frzb (is also referred to as sFrp3) and is positioned human chromosomal 2q31-33 region as first member of secreted frizzled related protein sFRP family in Wnt signal path in separated clone in 1996, but its function of preventing and treating in hepatocarcinoma was also never in the news.

Summary of the invention

Activating, for Wnt signal path aberrant continuation, the dependency that the development with multiple chronic hepatopathy and liver tumor exists, the present invention proposes a kind of Wnt signaling pathway protein Frzb application in preparing anti-liver cancer drug.

Wnt signaling pathway protein Frzb of the present invention can directly as drug use, and all the other adjuvants are acceptable on materia medica, humans and animals is nontoxic and inertia pharmaceutical carrier.

The present invention first passes through the Western Immuno marking and immunohistochemistry technique specify that in hepatocarcinoma patient liver organization Sample Storehouse and hepatoma cell strain, the expression of Frzb is relatively low, it was found that Frzb presents, in hepatocarcinoma patient liver organization, the expression characteristic being gradually lowered with the rising of pathological grading; Secondly, build slow virus recombinant expression plasmid pLVX-Puro-Frzb, it is thus achieved that Frzb recombinant slow virus suspension, screen the hepatoma cell strain successfully obtaining stably express Frzb through puromycin; Again, MTT experiment shows that Frzb not only reduces the multiplication rate of hepatoma carcinoma cell in time dependence mode, and Frzb significantly inhibits the formation of HepG2 cell migration and pseudopodium; Finally, by nude mice by subcutaneous xenotransplantation and tail vein Lung metastases model, research shows that Frzb significantly inhibits the formation of Xenografts in nude mice growth and Lung metastases, and its suppression ratio is 85.4% and 68.6% (P < 0.01) respectively.

Accompanying drawing explanation

Fig. 1 is Frzb low expression figure in hepatocarcinoma; A illustrates the expression of Frzb protein level in hepatoma cell line; In the contiguous non-tumor hepatic tissue of B show, the expression of Frzb is apparently higher than the expression in its liver cancer tissue.

Fig. 2 is the vegetative map that can substantially suppress hepatoma carcinoma cell after process LAN Frzb in vitro; A illustrates and has illustrated the expression of Frzb albumen in HepG2/Frzb and HepG2/pLVX in slow-virus transfection Frzb HepG2/Frzb and HepG2/pLVX, the A obtained after process LAN in hepatoma cell line HepG2; B illustrates that MTT experiment draws the growth curve of hepatoma carcinoma cell.

Fig. 3 is the process LAN Frzb invasion and attack motor capacity figure that can substantially suppress hepatoma carcinoma cell; The number of HepG2/Frzb and the HepG2/pLVX cell traverse that A observes after illustrating 24 hours under 100 power microscopes; B illustrates cut healing state after 48 hours in cell cut Healing Experiments, and C illustrates that the actin adopting rhodamine phalloidin labelling liver cancer cell is with observation of cell matrix morphology.

Fig. 4 is process LAN Frzb propagation and the transfer figure that can substantially suppress hepatoma carcinoma cell in vivo; A illustrates that HepG2/Frzb and HepG2/pLVX cell infusion is to nude mice by subcutaneous, is put to death by nude mice after one month, and gained subcutaneous tumors is taken pictures volume; B illustrates the tumor volume vs figure that in A, HepG2/Frzb and HepG2/pLVX cell is obtained; C illustrates the obtained lung tissue Nodules formational situation of nude mice after injection HepG2/Frzb and HepG2/pLVX cell.

Detailed description of the invention

Below in conjunction with accompanying drawing, embodiments of the present invention are described in detail.

Embodiment one

1, cell is cultivated

1.1 cell recoveries

Hepatoma cell strain used is from American Type Culture Collecti (ATCC). Cell cryopreservation tube takes out rapidly from liquid nitrogen, after 75% alcohol disinfecting, is immediately placed in 37 DEG C of constant water bath box, is shaken gently for accelerating it up and down and melts. Aseptically carefully open cryopreservation tube, cell suspension is proceeded in the centrifuge tube of the complete DMEM culture fluid being previously added 5mL, 1000rpm after mixing, centrifugal 5min, abandon supernatant, add blow and beat up and down containing 10% calf serum DMEM culture fluid be seeded to after cell for several times p100 culture dish put into be placed in 37 DEG C, containing 5%CO₂, cultivate in saturated humidity incubator, change a culture fluid next day.

1.2 passages

When cell density reaches 80%-90%, suck culture fluid, add 3mL containing EDTA-0.25% pancreatin after 37 DEG C of digested cell 2min of incubator, take out, pat Tissue Culture Dish edge gently until cell completely falls off, add the 3mL DMEM culture fluid containing 10% newborn calf serum and terminate digestion, suck culture fluid with pipet and repeatedly blow and beat attached cell, make it disperse, it is transferred to centrifuge tube, 1000rpm, supernatant is abandoned after centrifugal 5min, add to blow and beat up and down containing 10% calf serum DMEM culture fluid and be seeded to p100 culture dish for several times after cell and put into and be placed in 37 DEG C, containing 5%CO₂, cultivate in saturated humidity incubator.

1.3 cells are cultivated

With the DMEM height sugar culture fluid that with the addition of 10% (v/v) calf/hyclone, it is incubated in 37 DEG C of steril cell incubators, maintains with 5%CO₂, saturated humidity air.

1.4 cell countings

By cell from CO₂Incubator takes out, after adding the 3mL 0.25% trypsinization containing EDTA, makes single cell suspension, draw a small amount of cell suspension with 20 �� L liquid-transfering guns, add cell counting count board, under inverted microscope, observe the cell number in the grid of counting chamber corner 16 with 40 �� object lens, calculate summation. Substitute into below equation and obtain cell density: cell number (individual/mL)=(summation/4) �� 10⁴��

1.5 cell cryopreservations

When after cell length to 80% degree of converging, sucking culture medium, rinse with 1mLPBS and suck, add 3mL trypsinization liquid, 37 DEG C of digestion start to shrink to cell and become round, and blot pancreatin, add 2mL cells frozen storing liquid, suspension cell, make cell density be not less than 1 �� 10⁶, 1mL/ pipe is sub-packed in 1.8mL cryopreservation tube; In 4 DEG C, cryopreservation tube being placed 30min, goes to-20 DEG C and place 60min, being immediately placed on-80 DEG C of refrigerator middle or short terms deposits, and can proceed to the medium-term and long-term preservation of liquid nitrogen after overnight.

2, the structure of stable expression cell strain

The structure of 2.1 expression vectors

The cDNA nucleotide sequence of the cattle source Frzb gene adopted is such as shown in SEQNO.1, and correlation molecule biological experiment technology carries out according to a conventional method, and constructed recombiant plasmid confirms and called after pLVX-puro-Frzb through order-checking.

Forward primer: 5 '-CTAGTCTAGACCGCGGCCCCAGAAGTCTTA-3 '

Reverse primer: 5 '-CTAGGTTTAAACTTACTTGTCATCGTCGTCCT-3 '

The preparation of 2.2 recombinant slow virus

Recombinant slow virus expression vector pLVX-puro-CRD-WD is at liposome-mediated lower and packaging plasmid (pSPAX2), envelope plasmid (pMD2G) cotransfection 293T cell, cultivate 48-72 h before harvest supernatant culture fluid, the concentrated slow virus obtaining restructuring also measures its titre, and this recombinant slow virus is for the infection of host cell subsequently.

The structure of 2.3 stable expression cell strains

The recombinant slow virus of above-mentioned preparation is infected hepatoma Hep G 2 cells, the puromycin screening that concentration is 2 �� g/mL is added after 48h, through the lasting screening of two weeks, obtain positive colony cell, extract total protein of cell, expressing so that it is determined that the cell strain of Frzb stably express by Westernblot detection fusion albumen.

3, protein immunoblotting

3.1 total protein of cell extract

Take the logarithm trophophase cell, after the trypsinization of 0.25% goes down to posterity, be placed in culture dish and cultivate, culture dish is placed in 37 DEG C, 5%CO₂Cell culture incubator is cultivated 24h. With pre-cooling 1 �� PBS washed cell culture dish secondary. Each culture dish adds the RIPA cell pyrolysis liquid 300 �� L containing Roche cocktail protease inhibitor, cell pyrolysis liquid and cell is made to be fully contacted so that cracking completely, after placing 10min on ice, with clean cell scraper, cell is scraped, then blowing and beating cell up and down with pipettor makes for tens times it crack fully, ice bath 10min. Transferring the sample in the aseptic EP centrifuge tube of 1.5mL with 1mL liquid-transfering gun, 4 DEG C, 14000rpm is centrifuged 30min. Use pipettor Aspirate supernatant, carefully transfer in clean aseptic EP centrifuge tube, carry out labelling good after standby in-80 DEG C of Refrigerator stores.

3.2Bio-RadDCProteinAssay detects protein concentration

The dilution of BSA standard protein: take 2mg/mLBSA and add deionized water dilution respectively so that it is final concentration is followed successively by 0.0625,0.125,0.25,0.5,1.0,2mg/ml; The dilution of protein sample to be detected: albumen distilled water to be detected is diluted 10 times; The ratio mixing of the reagent A by DCProteinAssay: S=50:1; Quantity per sample, after being sequentially added into 25 �� LA:S mixed liquors, adds the diluted protein solution of 5 �� LBSA protein standard liquid or concentration to be detected in each hole, and distilled water is used as blank, is eventually adding 200 �� L reagent B, lucifuge incubated at room 15min; Measuring each hole OD value with BioRADiMarkTMmicroplatereader in 750nm place, drawing standard curve also calculates each sample protein matter concentration.

3.3Westernblot

Glass glue plate, 10 holes or 15 hole comb and electrophoresis tanks are cleaned before experiment, standby after air-dry. Glue to be separated is poured between two blocks of glass glue plates rapidly after preparing mixing, irrigates along a side glass plate wall during encapsulating, it is to avoid produces bubble in colloid, and reserves the perfusion concentration glue requisite space of about 2-3cm. After having filled glue, then draw a small amount of aseptic double-distilled water and be slowly covered on separation gel, to completely cut off air and to compress separation gel upper limb and make its level; After glue to be separated is polymerized completely, remove upper strata distilled water. Concentration glue irrigates rapidly after having configured and on the separation gel being polymerized, slowly inserts the comb of wash clean after filling immediately, about after half an hour, standby after gel is polymerized completely. After the offset plate configured is put into electrophoresis tank, add 1 �� RunningBuffer to submergence offset plate, extract comb gently, use pipettor application of sample, be sequentially added into albumen Marker5 �� L or the every hole of protein sample 80-100 �� g/. Concentration glue institute making alive is constant voltage 75V, when protein sample pressing is a line, changes voltage into 100V. Separation gel required voltage is 100V constant voltage, until bromophenol blue arrives after bottom separation gel, electrophoresis terminates. Carefully gel is taken out from glass plate, carry out labelling according to the position in albumen Marker. Filter paper, 7.5 �� 8cmNC film, sponge are soaked in 5min in 1 �� TransferBuffer buffer; Opening clamping plate, black negative pole down, is put sponge, filter paper 3, gel, NC film successively, then is sequentially placed filter paper 3, sponge, notes not formed between filter paper, gel and film bubble, and the clamping plate that close are installed in electrophoresis tank after forming sandwich structure; Black faces toward negative pole, red switches on power facing to positive pole, adjusts voltage to 100V constant voltage transferring film 60-90min (transferring film time opsin's matter molecular size range and determine). After transferring film completes, take off NC film, swing gently with 1 �� TBST liquid and wash once, film is put into 5% skim milk (1 �� TBST preparation) is closed 1h. The primary antibodie liquid liquid-transfering gun diluted after illustratively ratio 5%BSA is diluted to 2mL, is proceeded in plastic foil, is then carefully placed into by NC film by primary antibodie, make primary antibodie diluent liquid submergence NC film, with finger by after removal of bubbles, then by sealing machine high-temperature compression molding sealer, 4 DEG C of overnight incubation. After primary antibodie hatches end, take out NC film, with appropriate TBST shake washing 3 times, each 10min. Anti-with 1:5000 dilution proportion, incubated at room 1h by the two of the horseradish peroxidase-labeled of suitable source with 5% skim milk. Wash film, two anti-hatch end after, take out NC film, with appropriate TBST shake washing 3 times, each 10min. NC film is suitably drained on filter paper, is combined with the one of albumen and faces up tiling. Size according to NC film, by appropriate ECL reagent A liquid, the mixing of B liquid equal-volume, dropping, on NC film, makes liquid cover whole film. After reaction 5min, being mentioned by whole film, EDGE CONTACT filter paper, drained by ECL reactant liquor, be clipped in the preservative film that two-layer is clean and put into magazine, blend compounds band is fixed. Darkroom carries out X-ray exposure and grasps time of exposure flexibly according to fluorescence signal intensity on film. Fixing and development it is sequentially carried out after X-ray exposure, cleaner with tap water, target stripe is carried out labelling, scanner scanning band, Photoshop processes the view data obtained.

4, mtt assay measures cell growth rate

Trophophase cell of taking the logarithm is inoculated in 24 well culture plates, every hole 2 �� 10⁴Individual cell, 3 multiple holes, after 24h, every hole adds 1mg/mLMTT working solution 500 �� L, hatches 3h for 37 DEG C, abandons supernatant, and 500 �� L, the 200rpm shaking table concussion 5min that every hole adds 0.4% acidifying isopropanol are dissolving crystallized; Draw 200 �� L to 96 hole ELISA Plate and detect each hole absorbance (OD value) at 570nm wavelength place in full-automatic microplate reader.

Computing formula: proliferation inhibition rate (%)=(1-experimental port A value/control group A value) �� 100%

Growth vigor (%)=experimental port A value/control group A value �� 100%.

5, Wound-Healing analyzes

6 empty orifice plates are inverted laterally disposed, along one vertical line of middle line drawing of culture hole, then laterally draw the straight line of 4-6 bar level again, intersect vertically with middle vertical line. It is centrifuged after cell dissociation, resuspended by culture medium, according to every hole 1 �� 10⁶The density of individual cell is inoculated in 6 orifice plates, carefully rocks and makes cell be uniformly distributed. Second day, with the wound parallel with vertical line of standardized bar on the rifle head of 20 �� L cellular layer in hole so that it is edge is straight clearly. Line rifle head must selected shape rule, the product of neat in edge. Different holes, uses identical dynamics as far as possible, makes the wound width marked roughly the same. Remove culture medium, rinse cell 3 times with aseptic PBS, to remove the cell and cell debris come off. The propagation of cell also can promote speed of wound healing, in order to reduce cell proliferation rate, uses the DMEM culture medium culturing cell containing 2%FBS. Select 0,12,24, tri-time points of 48h carry out Taking Pictures recording. Different photo opporunity point, it should the coordinate according to being drawn in bottom culture dish positions, and is taken pictures in same region.

6, Transwell cell migration assay

Use the culture medium re-suspended cell of serum-free, take 2 �� 10⁴Individual cell, adds to 400 �� L by culture medium by volume. 24 orifice plates add 500 �� L complete mediums, transwell cell is kept flat in hole. Cell mixing is joined transwell chamber, and weak vibrations makes it be evenly distributed, and puts into cultivation 24h in incubator. A new hole adds 500 �� L methanol, transwell cell is taken out from foramen primum, be put into equipped with, in the hole of methanol, then adding the methanol of 200 more than �� L in the little indoor of transwell after sucking supernatant, fixing 20-30min. The hole that another is new adds 500 �� L violet staining liquid, transwell cell is taken out from original hole, being put in the hole of violet staining liquid after sucking methanol, then add the violet staining liquid of 200 more than �� L in the little indoor of transwell, dye 30-60min. The hole that another is new adds 500 �� LPBS, transwell cell is taken out from original hole, puts into after sucking dyeing liquor in hole, then add 400 �� LPBS in the little indoor of transwell, rocking gently with tweezers and wash, this step is in triplicate. PBS in transwell cell is removed, gently cellular layer is scraped totally in inside with medical cotton stick. The hole that another is new adds 500 �� LPBS, the transwell cell after cleaning is put into hole, adds 200 �� LPBS at transwell chamber. With inverted microscope, transwell cell being taken pictures, each transwell cell randomly chooses three visuals field and each takes a picture, and the cell quantity in photo is counted the tally function in the photo photoshop software taken. Carry out 2-3 independent experiment, then experimental result is carried out statistical analysis.

7, immunofluorescence dyeing

Being outwelled by DMEM complete medium, 1 �� PBS washes three times. Adding 5%BSA, 4% paraformaldehyde, pH7.2-7.3 in grown cultures liquid, cell adds the fixing 10min of this fixative at 4 DEG C. Cell adds the 1 �� PBS of 0.1%TritonX-100 and washes five times, each 5min. With the dilution proportion FITC-phalloidin according to 1:100 of the PBS containing 5%BSA and 0.1%TritonX-100. The dyer droplets configured is added on cell, and room temperature lucifuge hatches 60min. 1 �� PBS washes five times, each 5min. 1 �� PBS washes three times, each 5min, drips DAPI after adding anti-cancellation solution, and after adding coverslip, nial polish mounting preserves. Confocal laser scanning microscope result.

8, nude mice experiment in vivo

8.1 prepare cell

Seed cells in p100 culture dish. When in culture dish, degree of converging reaches 80%-90% to cell, 0.25% trypsin digestion cell, add the DMEM culture medium containing 10%FBS and terminate digestion. Part cell detects protein content for Westernblot, collects residue most cells, and 1500rpm is centrifuged 5min, abandons supernatant. With the DMEM culture medium re-suspended cell without FBS, 1500rpm is centrifuged 5min, abandons supernatant. Clean cell in the same fashion three times with sterilized 1 �� PBS, wash serum and the antibiotic of residual off. Collect cell, prepare cell suspension with 1 �� PBS. Blood counting chamber counts. Cell concentration is adjusted to 5 �� 10⁷cells/mL��

8.2 subcutaneous xenograft model

Choose female BAl BIc/C nude mice, treat that it grows into 6 weeks and carry out tumor generation experiment. Experiment is divided into two groups, often group ten. Take 1mL syringe (brown syringe needle, 0.45 �� 16RWLB) and aseptically draw the cell suspension (5 �� 10 prepared⁷Cells/mL), every nude mice right neck injection 100 �� L cell suspension (5 �� 10⁶Cells/mouse). Labelling every nude mice, aseptically raises. Measure the length of nude mouse tumor major axis and short axle every three days, according to formula V=0.5236 �� L₁��L₂ ²(L₁For tumor major axis, L₂For the short axle of tumor) calculate gross tumor volume. Euthanasia nude mice after 30 days, peels off tumor, weighs tumor weight. Tumor is after PBS, and 4% formaldehyde is fixing overnight, paraffin embedding, the microscope slide that section and preparation can be observed for microscope. Tissue slice carries out haematoxylin & eosin (H&E) dyeing and SABC.

8.3 tumor pulmonary metastasis models

Tumor pulmonary metastasis model is set up by tail vein injection. Choose female BAl BIc/C nude mice, be placed in SPF level Animal House and raise 6 week old. Take 1mL syringe (0.33mm �� 12.7mm) and aseptically draw the cell suspension (5 �� 10 prepared⁷Cells/mL), every nude mice tail vein injection 100 �� L cell suspension (5 �� 10⁶Cells/mouse). Labelling every nude mice, aseptically raises. Observe a nude mice every three days, by its euthanasia after 30 days. Taking nude mice liver, spleen and lung, lung meridian Bouin ' ssolution is fixing overnight, checks that transitivity tuberosity is taken pictures and counts. After lung meridian PBS, 4% formaldehyde is fixing overnight, paraffin embedding, the microscope slide that section and preparation can be observed for microscope. Carry out haematoxylin & eosin (H&E) dyeing.

9, the expression of His-Frzb recombiant protein, purification and antitumor application

First, by above-mentioned molecular biology method, Frzb it is subcloned on prokaryotic expression carrier pET28a and converts to e. coli bl21 (DE3), when transformant grow to OD600 value be 0.5 time, the IPTG adding final concentration of 0.4mM induces the expression of destination protein Frzb under 16 DEG C of conditions, 4 DEG C, the centrifugal 15min of 2000 �� g collects cell, adopt ice bath lysis buffer (1 �� PBS, 300mmol/LNaCl, pH7.4) re-suspended cell, in 4 DEG C after ultrasonic degradation, the centrifugal 30min of 10000 �� g collects supernatant soluble fraction. Secondly, immobilized metal affinity chromatography (IMAC) method purification Frzb recombiant protein is adopted. Take 1mL nickel-NTA agarose gel FF prepacked column, use 10mL equilibration buffer, broken supernatant 10mL sample is with 0.5mL/min loading, collection is in charge of by 2mL/ pipe, with the buffer solution elution destination protein containing 500mM imidazoles, destination protein removes imidazoles by SephadexG15 gel chromatography subsequently, and buffer system is replaced by 20mMTris-HCl, pH8.0. Finally, the recombiant protein of acquisition is applied to mice antitumor action. Build Transplanted tumor model by the Female nude mice of human liver cancer cell HepG2 subcutaneous injection 4-6 week old, be specially every nude mice by subcutaneous inoculation 5 �� 10⁶Individual HepG2 cell, after 7 days, when tumor length to diameter 6-7mm, nude mice is randomly divided into three groups, respectively matched group (PBS negative control), small dose group (5mg/kg), heavy dose of group (20mg/kg), then the fusion protein after purification is processed the nude mice after packet by hypodermic mode, between be administered every two days, it is administered 10 times, monitors the growing state of tumor every day, treat that the gross tumor volume of matched group reaches 2000mm³Time, all mouse are put to death, dissects, measure gross tumor volume and weight. Final experimental result shows, compared with matched group, the growth of high low dose group transplanted tumor is all subject to obvious suppression, its suppression ratio respectively 83% and 62%, during experimental therapy, the weight of animals increases to some extent, general status is good, it was shown that animal can tolerate given dosage. Therefore, recombiant protein Frzb can substantially suppress and killing hepatoma cell.

Embodiment two

In embodiment one specific as follows about the result of Frzb albumen:

1, Frzb albumen chooses multiple hepatoma cell strain respectively in significant low expression characteristic in hepatoma carcinoma cell and liver cancer tissue, extract albumen, Frzb expression in these cell strains is detected by protein immunoblotting, shown in Figure 1A, it has been found that in hepatoma cell strain, Frzb is significant low expression characteristic. Then, in order to further determine that the relation between Frzb and hepatocarcinoma patient pathological characters, fresh hepatocarcinoma patient tissue and contiguous non-liver cancer tissue is chosen from hepatocarcinoma patient tissue bank sample, utilize Immunohistochemical Method detection Frzb difference between liver cancer tissue and contiguous non-liver cancer tissue, result is as shown in Figure 1B, compared with contiguous non-liver cancer tissue, Frzb substantially lowers in liver cancer tissue, presents the expression characteristic being gradually lowered with the rising of pathological grading. The above results shows, the expression of Frzb is closely related with liver cancer genesis and development.

2, Frzb significantly reduces growth and the motion transfer ability of hepatoma carcinoma cell in vitro

For in vivo and in vitro Frzb active anticancer in hepatoma carcinoma cell. First molecular cloning build pLVX-Frzb-puro slow virus and compare virus accordingly. Then the recombinant slow virus obtained is infected HepG2 cell, and screen through puromycin puromycin, thus obtain can in hepatoma carcinoma cell the cell strain of stably express Frzb, as shown in Figure 2 A, the overexpression (Flag label) of Frzb is substantially detected at HepG2/Frzb cell strain. Carry out series of experiments subsequently to confirm Frzb inhibitory action in hepatoma carcinoma cell. Growth of Cells MTT experiment shows, the growth rate (Fig. 2 B) that the cell growth rate of HepG2/Frzb compares significantly lower than HepG2/pLVX. Show that Frzb can significantly inhibit the multiplication capacity of hepatoma carcinoma cell. Meanwhile, Transwell attacks cell and experiments show that the cell invasion ability of HepG2/Frzb is significantly lower than HepG2/pLVX compared with control cells, result is as shown in Figure 3A, after 24 hours, HepG2/Frzb cell is considerably less than HepG2/pLVX compared with control cells through the number of cell, and in Fig. 3 B, cell cut Healing Experiments then shows that the healing ability of HepG2/Frzb cell substantially reduces; Additionally, experiments show that by the actin of rhodamine phalloidin labeled cell, under 400 times of fluorescence microscopies, see that Frzb significantly inhibits the formation (Fig. 3 C) of cell pseudopodium. Many results show that Frzb significantly reduces liver cancer cell growth and motion invasive ability above.

3, Frzb significantly inhibits formation and the Lung metastases of tumor in mouse species body

In order to study Frzb active anticancer in vivo further, build nude mouse tumor subcutaneous xenograft model and tail vein injection tumor pulmonary metastasis model. In animal body, experimental result is as shown in Figure 4 A, the gross tumor volume that HepG2/pLVX compared with control cells is formed is significantly higher than the subcutaneous tumors volume that HepG2/Frzb cell is formed, and tumor growth in vivo curve clearly shows that Frzb can significantly inhibit the Tumor formation (Fig. 4 B) of hepatoma carcinoma cell in vivo. Meanwhile, the lung tissue transitivity tuberosity number of HepG2/pLVX compared with control cells tumor-bearing mice is significantly more than HepG2/Frzb cell metastatic nodules, it was shown that Frzb can suppress the invasive ability of hepatoma carcinoma cell in vivo. Statistical data shows, Frzb significantly inhibits the formation of Xenografts in nude mice growth and Lung metastases, and its suppression ratio is 85.4% and 68.6% (P < 0.01) respectively.

Claims (1)

1. the Wnt signaling pathway protein Frzb application in preparing anti-liver cancer drug.