CN105521482A - Combined application of HNF1[alpha] and HNF4[alpha] and FOXA3 for induced differentiation treatment for hepatocellular carcinoma - Google Patents

Abstract

The invention relates to combined application of HNF1[alpha], HNF4[alpha] and FOXA3 for induced differentiation treatment for hepatocellular carcinoma. Specifically, the invention relates to a method using hepatocyte nucleus factors HNF1[alpha], HNF4[alpha] and FOXA3 for inducing differentiation of human malignant hepatocellular carcinoma to be applied in treatment of malignant solid tumor and an application thereof. Researches indicate that through exogenous introduction of HNF1[alpha], HNF4[alpha] and FOXA3 genes to hepatocellular carcinoma cells, differentiation of hepatoma cells can be effectively induced, and thus a new tumor induced differentiation treatment method is provided.

Description

Use in conjunction HNF1 α, HNF4 α, FOXA3 induction-differential therapy hepatocarcinoma

Technical field

The present invention relates to molecular biology, cytobiology and medical domain.Relate to and utilize HNF HNF1 α, HNF4 α, the differentiation of FOXA3 human hepatocyte inducible cancer, thus be applied to method and the purposes of hepatocarcinoma treatment.

Background technology

Hepatocarcinoma is one of modal malignant tumor in the world, about has half patient to be distributed in China.Although the Clinics of hepatocarcinoma constantly progress in recent years, its early diagnosis difficulty, recurrence and metastatic rate are high, the insensitive present situation of chemicotherapy is not taken on a new look at all.Uniquely be approved for the target therapeutic agent Sorafenib of hepatocarcinoma, therapeutic effect is very undesirable.Therefore, the Therapy study of hepatocarcinoma needs adjust thinking badly, finds more effective treatment means.

Differentiation therapy is a kind of Therapeutic Method risen in recent years, by inducing PD tumor cell to ripe phenotypic differentiation, suppressing its propagation and transfer, promoting its apoptosis, thus reaching the object for the treatment of tumor.Differentiation therapy application is so far it is preferred that utilize Induction of Leukemic Cells Differentiation By Retinoic Acid, and a large amount of patients obtains good therapeutic effect.The success of neoplastic hematologic disorder differentiation therapy, has also driven the differentiation therapy of entity tumor to study.In succession occurred the research of applying all-trans-retinoic acid, dimethyl sulfoxide and arsenic agent treatment breast carcinoma, pulmonary carcinoma, colon cancer, but the research of the differentiation therapy of hepatocarcinoma at present also seldom.

HNF (Hepatocytenuclearfactor, HNF) be the transcription factor of class relative specificity high expressed in hepatocyte, play a significant role in the growth and hepatocyte function maintenance of liver, family member comprises HNF1, HNF3, HNF4, HNF6 and CCAAT/ enhancer binding protein (C/EBP) etc., they are transcription factor (SchremH of a series of predominant expression in liver, Liver-enrichedtranscriptionfactorsinliverfunctionanddeve lopment.PartI:thehepatocytenuclearfactornetworkandliver-specificgeneexpression, PharmacologicalReviews, 2002Mar, 54 (1): 129-58).Early stage, the present inventor place research team reported the application prospect (YinC of HNF4 α and HNF1 α in hepatocarcinoma differentiation therapy, Differentiationtherapyofhepatocellularcarcinomainmicewit hrecombinantadenoviruscarryinghepatocytenuclearfactor-4a lphagene, Hepatology, 2008Nov; 48 (5): 1528-39) (ZengX, Recombinantadenoviruscarryingthehepatocytenuclearfactor-1alphageneinhibitshepatocellularcarcinomaxenograftgrowth inmice, Hepatology, 2011Dec; 54 (6): 2036-47).But hepatocarcinoma heterogeneity is very strong, and in quite a few liver cancer tissue, the expression of HNF4 α or HNF1 α is not low, and this has also pointed out independent application HNF4 α Hepatoma therapy to have significant limitation.In addition, combined by multiple HNF theoretically and import that hepatoma carcinoma cell is collaborative to play a role, the effect of induction expression with human hepatocarcinoma cell differentiation may be better.Early stage, existing scholar was from the specific transcription factor of hepatocyte, and screening can induce human fibroblasts to the transcription factor of hepatocyte transdifferentiation, has finally been sieved to HNF1 α, HNF4 α and HNF3 γ (FOXA3).After these three kinds of factors are proceeded to human fibroblasts, people's induction type hepatocyte (hiHeps can be obtained, humaninducedhepatocytes), the similar normal liver cell of its form, and there is hepatocellular mark and biological function (HuangP, Directreprogrammingofhumanfibroblaststofunctionalandexpa ndablehepatocytes, CellStemCell, 2014Mar6; 14 (3): 370-84).The successful prompting of hiHeps: since fibroblast is induced hepatoblast by these three kinds of factors, hepatoma carcinoma cell induction is also probably " the normal function hepatocyte of band sudden change " (DNA mutation still exists, but hepatocyte function recovers) by it.

Therefore, the multiple HNF of exploitation use in conjunction is intended to induce expression with human hepatocarcinoma cell differentiation in this area, for liver cancer treatment provides a kind of new method.

Summary of the invention

The object of the present invention is to provide the multiple HNF of use in conjunction to induce expression with human hepatocarcinoma cell differentiation, for liver cancer treatment provides a kind of new method.Another object of the present invention is also to provide the new medical usage of three kinds of HNF HNF1 α, HNF4 α, FOXA3 gene and product albumen thereof.The third object of the present invention is to provide a kind of coupling medicine and this coupling medicine can as hepatocarcinoma differentiation therapy medicine.

A first aspect of the present invention, provides the purposes of three kinds of HNF HNF1 α, HNF4 α, FOXA3 gene and/or albumen, and they are used to the differentiation of inducing hepatocyte cancer.

The invention provides the application in preparation induction (pernicious) hepatocarcinoma differentiation agents or compositions of three kinds of HNF HNF1 α, HNF4 α, FOXA3 gene and/or albumen.

Described application refers to three kinds of HNF HNF1 α, HNF4 α, FOXA3 synergy (coupling).

Hepatocarcinoma differentiation therapy medicine of the present invention, refers to by inducing PD hepatoma carcinoma cell to ripe phenotypic differentiation, suppresses hepatoma cell proliferation and transfer, promotes its apoptosis, thus reach the object of Hepatoma therapy.

Reagent of the present invention is the reagent improving HNF1 α, HNF4 α, FOXA3 expression.

Compositions of the present invention is pharmaceutical composition.

Pharmaceutical composition of the present invention, the albumen containing three kinds of HNF HNF1 α, HNF4 α, FOXA3, coded sequence or containing the expression vector of described coded sequence and pharmaceutically acceptable carrier or excipient.

The dosage form of pharmaceutical composition of the present invention, is preferably injection.

HNF HNF1 α of the present invention, HNF4 α, FOXA3 are human hepatocytes nuclear factors.

The GENBANKID:NM_001306179.1 of HNF1 α;

The GENBANKID:NM_000457.4 of HNF4 α;

The GENBANKID:NM_004497.2 of FOXA3.

A second aspect of the present invention, provides a kind of coupling medicine, or claims drug combination use, and its active ingredient comprises following arbitrary:

(a) HNF1 α albumen, HNF4 α albumen and FOXA3 albumen;

The coded sequence of (b) HNF1 α albumen, the coded sequence of HNF4 α albumen and the coded sequence of FOXA3 albumen;

The expression vector of (c) coded sequence containing HNF1 α albumen, the coded sequence of HNF4 α albumen and the coded sequence of FOXA3 albumen.

Described expression vector simultaneously, also can express the coded sequence of these three kinds of factors respectively.

Described expression vector, includes but not limited to viral vector and the non-virus carriers such as adenovirus, slow virus, retrovirus and adeno-associated virus.

Described coupling medicine also comprises pharmaceutically acceptable carrier or excipient.

Present invention also offers described coupling medicine and preparing the application in cancer treatment drug, specifically for the preparation of hepatocarcinoma differentiation therapy medicine.

The invention provides HNF1 α, HNF4 α, the coupling of FOXA3 tri-factor, can be used for the formation suppressing liver cell tumor in body.

Coupling medicine of the present invention, can also comprise chemotherapeutics.

Coupling of the present invention, combination medicine, comprise three kinds of HNF HNF1 α, HNF4 α using effective dose simultaneously or sequentially, the albumen of FOXA3, coded sequence or the expression vector containing described coded sequence.

Pharmaceutically acceptable carrier of the present invention or excipient, refer to additive in addition to the active ingredient (s conventional in pharmaceutical field, such as diluent (starch based, saccharide, cellulose family and inorganic salts), excipient etc., filler is as starch sucrose, binding agent is as water, ethanol, cellulose derivative, gelatin and polyvinylpyrrolidone, disintegrating agent is as dried starch, carboxymethyl starch sodium, solubilizing agent is as poly yamanashi esters and polyoxyethylene fatty acid ester class etc., absorption enhancer, surfactant is as tween, span, absorption carrier, lubricant is as magnesium stearate, micropowder silica gel etc.In addition, other adjuvant can also be added in the composition as flavouring agent, sweeting agent etc.

Combination medicine of the present invention, can pharmaceutical composition form by oral, snuffing enters, the mode of rectum, parenteral or percutaneous dosing is applied to the patient needing this treatment.For time oral, conventional solid preparation can be made into as tablet, powder, granule, capsule, pill, slow-release micro-pill, solid dispersion, clathrate etc., the liquid preparation made is as suspensoid, Emulsion, melten gel agent, syrup, mixture, solution etc., during for parenteral, the solution of injection, water or Oil suspensions, Emulsion, liposome, microcapsule, microsphere, nanoparticle etc. can be made into, also can be made into various slow release, controlled release preparation.Preferential form is injection, the injection of preferential especially specific part Targeting delivery.

Pharmaceutical composition of the present invention, coupling medicine, also for suppressing the formation of solid tumor in body.

A third aspect of the present invention, provides a kind of induction or promotes the method that in mammal, hepatocarcinoma is broken up, and is also a kind of new liver cancer treatment method.

Described method comprises step: use in conjunction HNF HNF1 α, HNF4 α, FOXA3 albumen, their coded sequence or the expression vector of described coded sequence import in hepatoma carcinoma cell, suppress hepatoma cell proliferation and transfer, promote its apoptosis.

Hepatocarcinoma of the present invention, preferably people's primary hepatoma.

The present invention is that HNF provides new medical usage.

Present invention provides one kind of multiple HNFs to combine and import that hepatoma carcinoma cell is collaborative to play a role, induction expression with human hepatocarcinoma cell differentiation, the then new method of Hepatoma therapy.

Accompanying drawing explanation

Fig. 1 is AdHNF1 α, AdHNF4 α, AdFOXA3 tri-kinds of adenovirus infection hepatoma cell line LM3, and PLC, HepG2 cell is after 2 days, and Westernblot detects the protein expression situation of three factors.

Fig. 2 is that Realtime-PCR detects three factors various combination infection hepatoma carcinoma cell after 2 days, the quantitative analysis of hepatocyte correlation function molecule mrna expression.Wherein A is LM3 cell line, and B is PLC cell line, and C is HepG2 cell line, and D is SMMC-7721 cell line.

Fig. 3 is three factor adenovirus infections after hepatoma carcinoma cell LM315 days, and cellular morphology changes situation.Wherein A is that LM3 adds GFP adenovirus cellular morphology, and B is that LM3 adds three factor adenovirus cellular morphologies.

Fig. 4 is three factor adenovirus infections after hepatoma carcinoma cell LM315 days, Glycogen synthesis situation.Wherein A is that LM3 adds GFP adenovirus PAS (periodic acid Schiff stain) staining conditions, and B is that LM3 adds three factor adenovirus PAS staining conditions.

Fig. 5 is three factor adenovirus infections after hepatoma carcinoma cell LM315 days, lipogenesis situation.Wherein A is that LM3 adds GFP adenovirus oil red staining conditions, and B is that LM3 adds three factor adenovirus oil red staining conditions.

Fig. 6 is three factor adenovirus infections after hepatoma carcinoma cell LM315 days, urea secretion situation.

Fig. 7 is after three factor adenovirus infection hepatoma carcinoma cell LM3, suppresses situation to growth of tumour cell.

Fig. 8 is after three factor adenovirus infection hepatoma carcinoma cell LM3, suppresses situation to tumor cell migration.Wherein A is that LM3 adds GFP adenovirus and wears film migration situation, and B is that LM3 adds three factor adenoviruss and wears film migration situation, and C is that both wear the quantitative analysis of film migration situation.

Fig. 9 is after three factor adenovirus infection hepatoma carcinoma cell, the positive ratio of flow cytometer showed EpCAM.Wherein A is that LM3 adds GFP adenovirus EpCAM positive rate, and B is that LM3 adds three factor adenovirus EpCAM positive rates.

Figure 10 be intratumor injection three factor Adenoviral Therapy patient originate subcutaneous lotus tumor experiment.

Detailed description of the invention

The present invention is further illustrated below by drawings and Examples; embodiments of the invention are only used for the present invention is described; instead of limitation of the present invention, be all belong to the scope of protection of present invention to the simple modifications of the inventive method under concept thereof of the present invention.

Agents useful for same of the present invention and raw material all commercially maybe can be prepared by literature method.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as the people such as Sambrook " molecular cloning: lab guide " (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or conveniently condition, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise percentage ratio and number calculate by weight.

Embodiment 1: three kinds of adenoviruss of construction expression HNF1 α, HNF4 α, FOXA3, and detect three kinds of factor adenovirus infection hepatoma cell line LM3 with Western blotting (Westernblot), the expression of PLC, HepG2 cell.

Example is packaged as, in 35mm with FOXA3 ²cultivate the degree of converging of 293A cell to 80%-90% in dish, prepare skeleton plasmid pBHG11, vector plasmid pAdTrack-CMV-FOXA3 ().PBHG11 plasmid and pAdTrack-CMV-FOXA3 plasmid are mixed in the PBS of 100 μ l with the ratio of 2:1, are made into A liquid; 6 μ lPEI are mixed in the PBS of 100 μ l, are made into B liquid; After A liquid and B liquid fully being mixed, room temperature leaves standstill transfection 293A cell after 20min.After 12h, change the DMEM culture fluid containing 5% too Ox blood serum.After 7 to 10 days, after obvious plaque to be formed, draw the cell of plaque and add in 24 orifice plates of inoculated 293A, amplicon virus clone harvesting, in-80 DEG C and 37 DEG C of multigelations 3 times, obtain adenovirus supernatant.Get 5ul and carry out pcr amplification (PCR primer is provided by test kit) qualification.Observe the cell in 24 orifice plates, if all floating, absorption all cells and culture medium converge in order to infect in 10cm culture plate the 293A cell that rate is 80%.After 36h, collecting cell, multigelation 3 times, results employing virus cracking liquid, carries out purification with the Adenovirus Purification kit of BD company.Collect virion, by the titre of the adenovirus titer determination kit measurement adenovirus of BD company.HNF1 α, HNF4 α two kinds of adenovirus preparation methoies are the same.

LM3 is infected respectively by after AdHNF1 α, AdHNF4 α, AdFOXA3 mixing, HepG2, PLC, changed liquid after 4 hours, and 24 hours later cell lysates collect whole-cell protein, after protein standard is quantitative, respectively get 20ug in 10%SDS-PADE electrophoretic separation albumen, lower floor's filter paper, nitrocellulose filter (NC film), running gel, upper strata filter paper are superposed successively with transfering buffering liquid (TransferringBuffer) in balance after, be placed in and half-driedly turn electroporation, 15V, 70min.After 5% skim milk 20ml room temperature closing membrane 1h, after TBST washs 3 × 5min, with HNF1 α (purchased from SantaCruz company, sc-10791), HNF4 α is (purchased from Abcam company, ab92378), FOXA3 is (purchased from SantaCruz company, sc-74424) anti antibody (1:1000) incubated at room 1h, after TBST washs 3 × 5min, anti-(purchased from LI-COR company with rabbit two, ) (1:10000) incubated at room 1h, after TBST washs 3 × 5min, carry out gray scale scanning through Odyssey infrared laser imaging systems axiol-ogy fluorescence, hatch GAPDH (purchased from Abclonal company) antibody as internal reference simultaneously.

Result shows, and can significantly improve the expression (Fig. 1) of HNF1 α, HNF4 α, FOXA3 after three kinds of factor adenovirus infection Bel7402s.

Embodiment 2: three kinds of factor adenovirus infection hepatoma carcinoma cell, real-time quantitative PCR detects the expression of hepatocyte correlation function gene.

(1) by LM3, PLC, HepG2, SMMC-7721 kind in 6 orifice plates, to be cultured to after density 50%-60% by AdHNF1 α, AdHNF4 α, AdFOXA3 with MOI100 infection cell, 4 fresh cultures as a child changing 10% hyclone; TRIZOL1ml is added, the static 3min of room temperature after (2) 2 days; (3) 200 μ l chloroform thermal agitation 30sec are added, the static 3min of room temperature; (4) 4 DEG C, 12,000rpm, centrifugal 15min; (5) draw upper strata and contain the aqueous phase of RNA in new 1.5mlEp pipe, add 500 μ l isopropanol precipitating RNA, concuss 30sec, on ice static 10min; (6) 4 DEG C, 12,000rpm, centrifugal 15min; (7) abandon supernatant, add the washing with alcohol RNA precipitation of 0.5ml75%, 4 DEG C, 12,000rpm, centrifugal 5min; (8) remove ethanol, under hair-dryer, dry up RNA, with 30 μ lDEPC water dissolution RNA during transparence precipitation to appear; (9) content of RNA is measured with ultraviolet spectrophotometer ,-20 DEG C of preservations; (10) reverse transcription synthesis cDNA first chain:

(11) cDNA is obtained for fluorescence quantitative PCR detection.The relative expression quantity of genes of interest is through internal reference mark post analysis.

Real-TimePCR step

(12) mix, 95 DEG C of denaturation 10min, 95 DEG C of degeneration 30sec, 60 DEG C of annealing 30sec, 72 DEG C extend 40sec, 40 circulations, and 72 DEG C of whole ends extend 10min.By AppliedBiosystems7300/7900FastReal-TimePCRSystem software analysis result.

Table 1 people liver function primer sequence

Albumin (albumin, ALB); Cytochrome P450 family 1 α 2 (cytochromeP4501 α 2, CYP1 α 2); Phosphoenolpy ruvate carboxy kinase (phosphoenolpyruvatecarboxykinase, PEPCK); G-6-Pase (glucose-6-phosphatase, G-6-P); Glutamine synthetase (glutaminesynthetase, GS); Alpha antitrypsin (α 1-antitrypsin, AAT); Multidrug-associated protein 2 (multidrugresistance-associatedproteins2, MRP2).

Result shows, and the comprehensive function that three factor use in conjunction improve liver cancer function is significantly better than the effect (Fig. 2) of HNF1 α, HNF4 α, FOXA3 single-factor and combination of two (1+4,1+F, 4+F).

Embodiment 3: three kinds of factor adenovirus infections are after hepatoma carcinoma cell LM315 days, morphological observation

Infection three Summing Factor GFP adenovirus LM3/GFP, LM3/3F cell of 15 days is pressed respectively 2000 every hole kinds in 6 orifice plates, living cells form is observed in inverted phase contrast microscope (× 200 times) after 7 days, LM3/GFP cellular morphology can be observed little and justify by light microscopic, arrangement closely (as shown in Figure 3A), and LM3/3F cell is comparatively large, form flat roomy (as shown in Figure 3 B).

Embodiment 4: three kinds of factor adenovirus infections, after hepatoma carcinoma cell LM315 days, affect Glycogen synthesis.

LM3/GFP, LM3/3F cell is planted respectively in 12 orifice plates, after adherent one day, remove culture medium, PBS washes 2 times, adds 10% neutral formalin and fixes 10min, current rinse 1min, 1% periodic acid room temperature 5min, distillation washing 5min, then add Xi Fushi liquid chamber temperature 15min, 5min washed by distilled water 5, optical microphotograph Microscopic observation staining conditions.

Result shows, and LM3/3F cell is redder compared with LM3/GFP cell, Glycogen synthesis more (Fig. 4).

Embodiment 5: three kinds of factor adenovirus infections, after hepatoma carcinoma cell LM315 days, affect lipogenesis.

LM3/GFP, LM3/3F cell is planted respectively in 12 orifice plates, after adherent one day, remove culture medium, PBS washes 2 times, adds 10% neutral formalin and fixes 30min, 70% ethanol embathes 1min, the oil red stain dyeing 15min prepared by 70% ethanol, 70% ethanol embathes 1min, haematoxylin dyeing 90sec, the anti-blue 15sec of current, optical microphotograph Microscopic observation staining conditions.

Result shows, LM3/3F cell comparatively in LM3/GFP cell fat drip content more (Fig. 5).

Embodiment 6: three kinds of factor adenovirus infections, after hepatoma carcinoma cell LM315 days, affect urea secretion.

By LM3/GFP, LM3/3F cell by 10000 cell per well 96 holes finally, often kind of cell establishes 3 multiple holes, after adherent, every hole changes into does not cultivate containing the culture medium 100 μ L of serum, be positioned over 37 DEG C, draw supernatant after cultivating 24h in 5%CO2 incubator, carry out carbamide mensuration according to the operation instruction of QuantitChromTMUreaAssayKit (purchased from BioAssaySystems company); Remaining cell per well adds 100 μ LCCK8 working solutions, cultivate in incubator after 1 hour, put into microplate reader with 450nm wavelength detecting, read the OD value also calculating mean value in every hole, with LM3/GFP cell once proliferative conditions for standard, markization LM3/3F rises in value situation relatively, and surveyed urea content is obtained cell relative urea secretion numerical value divided by relative CCK8 numerical value.

Result shows, and LM3/3F cell can have more urea secretion (Fig. 6) relative to LM3/GFP cell.

After embodiment 7: three kinds of factor adenovirus infection hepatoma carcinoma cell LM3, situation is suppressed to growth of tumour cell.

To take the logarithm the LM3 cell of trophophase, plant in 26 orifice plates, infect three Summing Factor GFP adenoviruss respectively, liquid is changed after 4h, inoculate into 96 orifice plates by the density of 5000 cells in every hole after 24h and establish 3 multiple holes, every hole adds 200 μ L culture medium again, is positioned over 37 DEG C, 0d is cultivated in 5%CO2 incubator, 1d, 2d, 3d, 4d, 5d; Absorb culture medium in each time point, every hole adds 100 μ LCCK8 working solutions, cultivates after 1 hour in incubator, put into microplate reader with 450nm wavelength detecting, read the OD value in every hole and calculating mean value, take time as abscissa, OD value meansigma methods is vertical coordinate, be depicted as growth curve.

Survey two cell growth curves continuously to find through too much sky, three factor expressions have obvious inhibitory action to hepatoma carcinoma cell increment, and cellular control unit is bred obviously in time, and three factor set cell proliferation mild (Fig. 7).

After embodiment 8: three kinds of factor adenovirus infection hepatoma carcinoma cell LM3, situation is suppressed to tumor cell migration.

To take the logarithm the LM3 cell of trophophase, plant in 26 orifice plates, infect three Summing Factor GFP adenoviruss respectively, change liquid after 4h, inoculate into transwell upper strata cell after 24h by the density of 2 × 105 cells in every hole, every hole keeps 200 μ L culture medium, be placed in 24 orifice plates, in lower floor 24 orifice plate, inject the culture medium containing 20% too Ox blood serum, be positioned over 37 DEG C, cultivate in 5%CO2 incubator; Transwell is taken out after 20h, absorb upper strata cell culture medium, PBS washes 2 times, 10min is fixed with 10% neutral formalin, PBS washes 2 times, and 0.1% violet staining 15min, PBS wash 2 times, carefully wipe the little indoor staining cell in upper strata by swab stick, observe in inverted phase contrast microscope (× 100 times).

Result shows, and three factor set cells obviously can suppress the transfer ability (Fig. 8) of hepatoma carcinoma cell.

After embodiment 9: three kinds of factor adenovirus infection hepatoma carcinoma cell, EpCAM expression.

To take the logarithm the LM3 cell of trophophase, plant in 26 orifice plates, infect three Summing Factor GFP adenoviruss respectively, liquid is changed after 4h, 2nd day collecting cell, PBS washes 2 times, and EpCAM (purchased from BioLegend company) hatches 30min as primary antibodie 4 DEG C, every 5min clock is played and is once made abundant contact, through the ratio of flow cytomery EpCAM+ cell.

Result shows, and in the metainfective hepatoma carcinoma cell of three factors, the cell proportion of EpCAM+ obviously reduces (Fig. 9).EpCAM+ is the specific markers of tumor stem cell, and EpCAM+ cell proportion reduces, and shows that three factors can promote that tumor stem cell breaks up.

Embodiment 10 3 factor Adenoviral Therapy patient originates subcutaneous lotus tumor.

Patient to be originated liver cancer tissue, with about 0.5cm ³under volume is inoculated in nude mice armpit skin, treating after 1 month that tumor grows to size is 500cm ³after in the total pfu of intratumor injection (plaque forming unit) be 1 × 10 ¹⁰three factor adenoviruss, matched group is injected identical pfu (plaque forming unit) and is measured GFP adenovirus, injects every other day and measures tumorous size change.

Result shows, and after treating two weeks, experimental group nude mouse tumor comparatively matched group volume obviously reduces (Figure 10).

Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.

Claims (10)

1. three kinds of HNF HNF1 α, HNF4 α, FOXA3 gene and/or albumen coupling application in preparation induction hepatocarcinoma differentiation agents or compositions.

2. three kinds of HNF HNF1 α, HNF4 α according to claim 1, FOXA3 gene and/or the albumen coupling application in preparation induction hepatocarcinoma differentiation agents or compositions, described application refers to that the PD hepatoma carcinoma cell of induction is to ripe phenotypic differentiation, suppress hepatoma cell proliferation and transfer, promote its apoptosis.

3. three kinds of HNF HNF1 α, HNF4 α according to claim 1, FOXA3 gene and/or the albumen coupling application in preparation induction hepatocarcinoma differentiation agents or compositions, described reagent is the reagent improving HNF1 α, HNF4 α and FOXA3 expression.

4. three kinds of HNF HNF1 α, HNF4 α according to claim 1, FOXA3 gene and/or the albumen coupling application in preparation induction hepatocarcinoma differentiation agents or compositions, described compositions is pharmaceutical composition, the albumen containing three kinds of HNF HNF1 α, HNF4 α, FOXA3, coded sequence or containing the expression vector of described coded sequence and pharmaceutically acceptable carrier or excipient.

5. three kinds of HNF HNF1 α, HNF4 α according to claim 1, FOXA3 gene and/or the albumen coupling application in preparation induction hepatocarcinoma differentiation agents or compositions, described HNF HNF1 α, HNF4 α, FOXA3 are human hepatocytes nuclear factors.

6. a coupling medicine, its active ingredient comprises following arbitrary:

(a) HNF1 α albumen, HNF4 α albumen and FOXA3 albumen;

The coded sequence of (b) HNF1 α albumen, the coded sequence of HNF4 α albumen and the coded sequence of FOXA3 albumen;

The expression vector of (c) coded sequence containing HNF1 α albumen, the coded sequence of HNF4 α albumen and the coded sequence of FOXA3 albumen.

7. coupling medicine according to claim 6, is characterized in that, described expression vector comprises adenovirus, slow virus, retrovirus and adeno-associated virus and non-virus carrier.

8. coupling medicine according to claim 6, is characterized in that, described coupling medicine also comprises pharmaceutically acceptable carrier or excipient.

9. coupling medicine according to claim 6, is characterized in that, described coupling medicine also comprises chemotherapeutics.

10. the application of the coupling medicine as described in as arbitrary in claim 6 to 9 in preparation hepatocarcinoma differentiation therapy medicine.