CN101642577A

CN101642577A - Application of human ING4 gene serving as radiation sensitizer

Info

Publication number: CN101642577A
Application number: CN200910029793A
Authority: CN
Inventors: 杨吉成; 苏成海; 田玥; 翟红艳; 井莹莹
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2009-04-09
Filing date: 2009-04-09
Publication date: 2010-02-10

Abstract

The invention discloses an application of a human ING4 gene serving as a tumor radiation sensitizer and also provides a method for enhancing the sensitivity of in-vivo/in-vitro tumor cells to radiation. The method comprises the following steps: introducing a human ING4 gene into the in-vivo/in-vitro tumor cells before radiation so as to enable the human ING4 gene to be expressed in the tumor cells, and then radiating the human tumor cells. According to the application of the human ING4 gene serving as the radiation sensitizer and the method for enhancing the sensitivity of in-vivo/in-vitro tumor cells to radiation, the ING4 gene can be applied to a tumor radiation process, thereby obviously promoting the sensitivity of different tumor cells to radiation, effectively restraining the growthof the tumor cells and inducing the tumor cells to die.

Description

Human ING4 gene is as the application of radiotherapeutic sensitizer

Technical field

The present invention relates to genomics, molecular biology, cytobiology, genetic engineering and clinical medicine, the present invention is specifically related to the application of human ING4 gene as the chemotherapeutics sensitizer.

Background technology

Radiotherapy is one of conventional means of oncotherapy, and 70%～80% malignant tumor patient need be accepted radiotherapy in therapeutic process.Tumor is insensitive or radiation produced negation to radiation, is the one of the main reasons that radiotherapy is difficult to effect a radical cure malignant tumor.In recent years, along with to the going deep into of tumor invasion molecular mechanism and the research of cell adjusting and controlling growth mechanism, therapy of tumor has been obtained some stem-winding achievements, but usually can not reach desirable therapeutic at the therapeutic transgene of single link.Weichselbaum equals proposition oncogene-radiocurable new approaches in 1994, its principle is with the regulating and controlling sequence of radiation-induced type gene and tumor-killing gene coupling connection mutually, transfection tumor cell, tumor is implemented local irradiation inducible gene expression simultaneously, by the dual function killing tumor cells of ray and gene, the purpose of reduce exposure dose to reach, alleviating normal tissue injury.Therefore, radiotherapy is combined with gene therapy, get twice the result with half the effort, become one of important directions of oncotherapy research, oncogene associating at present radiocurable relevant mechanism mainly contain following several (referring to field Yue, Su Chenghai. international radiological medicine Journal of Nuclear Medicine .200832 (1)): 1. immunogene treatment and radiocurable associating; 2. antioncogene and radiocurable associating; 3. suicide gene therapy and radiocurable associating; 4. angiogenesis inhibitor gene therapy and radiocurable associating; 5. specificity promoter gene therapy and radiocurable associating; 6. anti-radiation protection gene therapy and radiocurable associating.

Publication number is that the Chinese invention patent prospectus of CN 1714871A discloses the purposes of human ING4 gene as the sensitizer of tumor chemotherapeutic drug, a kind of method that strengthens tumor cell to chemotherapy drug susceptibility is also disclosed, this method imports tumor cell with the ING4 gene before being included in chemotherapy, makes it to express; The ING4 gene can be used and the chemotherapy of tumors process, thereby obviously be promoted the drug susceptibility of different tumor cells, thereby can reduce the dosage of chemotherapeutics, and alleviate the toxic and side effects of chemotherapeutics the human normal cell to chemotherapeutics.

But the application as tumor radiotherapy sensitive-increasing agent does not but appear in the newspapers about human ING4 gene, and as well known to those skilled in the art: the chemotherapy sensitizing medicine may not can be used as radiotherapy hypersitization medicine and use; Radiosensitizing effect mainly proposes at that part of anoxic cell that radiation in the tumor is resisted, refer to some chemicals mass-energy and strengthen ray to the killing action of anoxic cell in the tumor and less the normal tissue injury of aerobic, these chemical substances are called radiotherapeutic sensitizer, and radiotherapeutic sensitizer commonly used mainly contains misonidazole (MISO), CMNa (CMNa), flavone acetic acid and oxygen; Some medicine not only can be used as radiotherapeutic sensitizer but also can be used as chemotherapeutic sensitizer, such as misonidazole (MISO), CMNa (CMNa), CMNa, cyclophosphamide, fluorouracil, VP-16, bleomycin, ametycin, (hydroxyl) camptothecine, paclitaxel, amycin, suitable (card) platinum etc.; But some medicine can only can not can overcome Drug resistance such as para-aminobenzenesulfonic acid, decitabine etc. as radiotherapy hypersitization medicine as the chemotherapy sensitizing medicine, and cancer is better treated.

And do not see that relevant human ING4 expression vector and gene coded protein thereof have the bibliographical information of radiotherapy sensitization effect at present.

Summary of the invention

The object of the invention provides the purposes of human ING4 gene as tumor radiotherapy sensitive-increasing agent;

Another object of the present invention provides a kind of method that strengthens tumor cell to radiation sensitivity;

For achieving the above object, the concrete technical scheme of the present invention is that human ING4 gene is as the purposes of tumor radiotherapy sensitive-increasing agent;

The present invention also provides a kind of method that strengthens the inside and outside tumor cell to radiation sensitivity, and this method imports the inside and outside tumor cell with people ING4 gene before being included in radiotherapy, and people ING4 gene is expressed in tumor cell;

Preferably, the described method that people ING4 gene is imported tumor cell is selected from: plasmid transfection or adenovirus mediated in a kind of.

The present invention also provides a kind of tumor radiotherapy sensitive-increasing agent pharmaceutical composition, comprises people ING4 gene or people ING4 gene coded protein and/or pharmaceutical carrier or excipient.

People ING4 gene of the present invention forms " human ING4 gene " by rat ING 4 gene after the humanization genetic modification, although gene order both had been different from the people, be different from again Mus ING4 sequence (referring to: well is sparkling, Yang Ji becomes etc., Chinese Journal of Immunology, 2009,25 (3)); ING4 gene coded protein of the present invention and publication number are that the disclosed people ING4 of the Chinese invention patent prospectus gene coded protein of CN 1714871A is identical.

Be example with adenovirus mediated method below, the technical solution used in the present invention be described, comprise following concrete steps:

(1), makes up the sub-Ad-hING4 of recombinant virus of adenovirus mediated people ING4 gene according to conventional method;

(2) with inside and outside human tumor cells or the transplanted tumor in nude mice cell of cultivating of Ad-hING4 infectosome, make people ING4 gene in people's tumor cell, obtain expressing;

(3) tumor cell to the described people of step (2) carries out radiotherapy, and the method for described radiotherapy is preferably the 125I particle and continues low dose rate radiation gamma method.

Because the technique scheme utilization, the present invention compared with prior art has following advantage:

People ING4 gene disclosed according to the present invention is as the purposes of tumor radiotherapy sensitive-increasing agent and strengthen the method for inside and outside tumor cell to radiation sensitivity, the ING4 gene can be applied to the tumor radiotherapy process, and obviously promote the sensitivity of different tumor cells, suppress the growth and the inducing apoptosis of tumour cell of inside and outside tumor cell effectively radiotherapy.

Description of drawings

5 groups of cancer of pancreas transplanted tumor treatment back change in volume curves among accompanying drawing 1, the embodiment three;

5 groups of comparisons that cancer of pancreas transplanted tumor treatment posterior tuberosity heavily changes among accompanying drawing 2, the embodiment three;

The comparison of 3 heavy tumour inhibiting rates of treatment group tumor among accompanying drawing 3, the embodiment three;

The comparison of AI, MVD, Survivin protein expression, Caspase-3 protein expression among accompanying drawing 4, the embodiment three.

The specific embodiment

Below in conjunction with reaching embodiment the present invention is further described:

Embodiment one, reorganization Ad-hING4 adenovirus infection Panc-1 human pancreatic cancer cell

1.1 experiment material, instrument

1.1.1 material and reagent

The Panc-1 pancreas cancer cell strain, QBI-293A incasing cells and empty carrier Ad are that University Of Suzhou's medical board cell and molecular biology teaching and research room preserve, Ad-hING4 is made up by University Of Suzhou's medical board cell and molecular biology teaching and research room, the hING4 gene primer is synthetic by Shanghai Sangon, MTT is available from Sigma company, RNA extraction agent box is given birth to worker's biotechnology company limited available from Shanghai, the MMLV reverse transcriptase is available from MBI company, cell culture medium is 3%, 8%, 10% calf serum DMEM is available from GIBCO company, newborn calf serum is matched happy biotechnology company available from Hangzhou, the Taq enzyme is available from dodging brilliant company, and anti-hING4 antibody is available from U.S. R﹠amp; D company, immunofluorescence dyeing test kit-anti-mice Cy3 and apoptosis-Hoechst staining kit are available from green skies biotechnology research institute.

1.1.2 instrument

Culture plate, culture bottle (U.S. COSTAR company), CO2 gas incubator (German Heraeus company), cell counting count board (Liuyi Instruments Plant, Beijing), operating theater instruments (the attached hospital operating room of University Of Suzhou) ESOEC-BAN-311 type electric heating constant temperature water bath (Japan), FA 1604S electronic balance (Shanghai balance equipment factory), superclean bench (Suzhou Decontamination Equipment Plant), XSZ-D2 inverted biological microscope (optical instrument factory, Chongqing), fluorescence microscope (Japanese Olympus company), MIKRO 12-24 type table model high speed centrifuge (German Hettich company), TGL-16G supercentrifuge (Shanghai medical analysis instrument plant), cryogenic refrigerator (U.S. Legaci company), full-automatic enzyme-linked immunologic detector (GIBCO company), EPICSXL flow cytometer (U.S. Coulter company).

1.2 method

1.2.1 the amplification of recombinant adenovirus (Ad-hING4) and empty carrier virus (Ad-GFP), tire detection and evaluation

1.2.1.1 the amplification of recombinant adenovirus

The propagation defective recombinant adenovirus Ad-hING4 that becomes seminar to make up the Yang Ji of University Of Suzhou infects the QBI-293A incasing cells of 70% adherent growth, next day is observation of cell pathological changes (cell pathologic effect under inverted fluorescence microscope, CPE), can observe green fluorescence; Centrifugal collecting cell behind the 48h (the centrifugal 5min of 1000r/min), get cell precipitation through aseptic PBS washing 2-3 time, multigelation 3 times (from-20 ℃ to 37 ℃) behind the reuse PBS suspension cell, the centrifugal 5min of 1500r/min, collect supernatant, said method can obtain high titre recombinant virus (Ad-hING4) in-80 ℃ of preservations after many wheel amplifications.Prepare Ad-GFP (as experiment contrast) with quadrat method.

The result shows: Ad-GFP, Ad-hING4 express in the QBI-293A cell well, can effectively increase.

The detection 1.2.1.2 recombinant adenovirus is tired

Recombinant virus (Ad-hING4) and Ad-GFP detect its virus titer with the fluorescence counting method: with incubation growth QBI-293A cell in order, and after the trypsinization, the cell counter counting, diluting cells concentration is 10 ⁵Behind individual/ml, on 96 orifice plates,, behind the cultivation 24h, Ad-hING4 recombinant virus and the empty carrier Adv adenoviron of results are pressed 10 by every hole 100 μ l inoculating cells ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9After doing dilution, each dilution factor is inoculated three holes by every hole 10 μ l, and 37 ℃, 5%CO ₂Add the DMEM complete culture solution 90 μ l that contain 5% calf serum after cultivating 2h in the cell culture incubator, behind the 18h, under the fluorescence microscope, carry out the fluorescence counting, press virus titer (pfu/ml)=(every hole fluorescence average * 10)/corresponding dilution factor formula and calculate virus titer.

The result shows: recombinant virus is after many wheel infection, amplification, and Ad-GFP, Ad-hING4 detect its virus titer titre through the fluorescence counting method and all reach 10 ¹⁰Pfu/ml.

1.2.1.3RT-PCR method is identified recombinant virus

Extract the total RNA of Panc-1 pancreatic cancer cell that handles through 0.1M PBS, 50MOIAd-GFP, 50MOI Ad-hING4 respectively with the total RNA extraction agent of UNIQ-10 pillar Trizol box, and use determined by ultraviolet spectrophotometry content, RT-PCR method amplification Ad-hING4 gene, agarose gel electrophoresis method is identified the PCR product.The upstream and downstream primer of ING4 is respectively:

P1：5’-GCGTCGACATGGATGATGGGATGTATTTGGAAC-3’，

P2：5’-GCAAGCTTCTAGTGGTGGTGGTGGTGGTGTTTCTTCTTCTTCC?GTTCTTGGGAG-3’。

The result shows: the total RNA of panc-1 pancreatic cancer cell that extracts the Ad-hING4 infection carries out RT-PCR, show behind its product agarose gel electrophoresis, can detect a specific band in the 500bp-750bp position, and PBS, occur the expection band in the Ad-GFP matched group, the result shows that adenovirus mediated Ad-hING4 gene can successfully transcribe in the panc-1 pancreatic cancer cell.

1.2.2Ad-hING4 the cell inner expression of in-vitro transfection Panc-1 and detection Ad-hING4

1.2.2.1Ad-hING4 in-vitro transfection Panc-1

1) Panc-1 of 0.25% trypsinization adhere-wall culture, 1000r/min, centrifugal 5min sedimentation cell, PBS wash the DMEM culture medium of using 10%NBS after 2 times and suspend, and adjusting cell density is 1 * 10 ⁵/ ml adds 100 μ l, 37 ℃, 5%CO2 overnight incubation in the every hole of 96 well culture plates.

2) abandon supernatant next day, adding serum-free RPMI DMEM culture fluid 10 μ l in 96 well culture plates respectively is blank cell contrast, and adding 10 μ l empty carrier Adv adenoviruss is empty virus control, add 10 μ l Ad-hING4 recombinant viruses and be experimental group, establish 3 multiple holes, put 37 ℃, 5%CO for every group ₂After hatching 2h in the incubator, the RPMI DMEM complete medium continuation cultivation that 90 μ l contain 10% calf serum is added in every hole.

3) in 24h, 48h behind the 72h, observes luciferase expression situation and Ad-hING4 to Panc-1 cell inhibiting growth effect under fluorescence microscope.

Observed result is: infect the Panc-1 cell through 50MOI Ad-hING4 dosage, 90% cell is all expressed GFP, presents very high efficiency of infection; And the Panc-1 cell that Ad-hING4 handles occur obviously becoming justify, come off, pathological changes such as shrinkage, and pathological changes such as change is justified, come off, shrinkage all appear through the Panc-1 cell that PBS, 50MOI Ad-GFP handle.The result shows that Ad-hING4 has the effect of obvious suppression cell growth to the Panc-1 pancreatic cancer cell.

1.2.2.2 indirect immunofluorescence detects the expression of exogenous ING4 gene in the Panc-1 cell

In being covered with 24 orifice plates of coverslip by 2 * 10 ⁴Individual/hole inoculation Panc-1 cell, add 0.1mol/L PBS behind the 24h respectively, 50MOI Ad-GFP, 50MOI Ad-hING4, cultivate the operating procedure of 72h by green skies biotechnology research institute immunofluorescence dyeing test kit-anti-mice Cy3 description again:

1) the fixing 4-6h of 4% paraformaldehyde.

2) 0.01mol/L TBST (containing 0.1%TritonX-100pH 7.4) rinsing 5min * 3/ time.

3) add and contain 0.1%TritonX-100pH 7.4 and 2%BSA and soaked 30 minutes.

4) add anti-spending the night.

5) 0.01mol/L TBST rinsing 5min * 3/ time.

6) dripping fluorescently-labeled two of suitably dilution resists.

7) 0.01mol/L TBST rinsing 5min * 3/ time.

8) buffering glycerol mounting is observed under inverted fluorescence microscope.

Observed result shows: the Panc-1 cell specimen that Ad-hING4 handles all can be observed red fluorescence, and the Panc-1 cell specimen that PBS, Ad-GFP matched group handle is not observed red fluorescence, and the result shows in the Panc-1 cell that Ad-hING4 handles the proteic expression of exogenous ING4.

Embodiment two, unite to continue the human pancreas cancer cell strain panc-1 that the low dose rate radiation gamma imports the hING4 gene

1.1 key instrument

Autoclaving pot, super-clean bench, all kinds of centrifuge, constant water bath box, ultra cold storage freezer, PCR instrument, nucleic acid and protein electrophoresis instrument, nucleic acid electrophoresis groove, gel imaging and analyser, protein nucleic acid analyser, electronic balance, protein electrophoresis groove, albumen transfer groove, gas bath, shaking bath.Multi-tumor marker protein chip detection system corollary equipment (providing) by Huzhou Health-Digit Co., Ltd..

1.2 main agents

The total RNA extraction agent of UNIQ-10 pillar Trizol box is given birth to worker's technology company limited available from Shanghai, and MMLV reverse transcriptase and Taq enzyme are available from Fermentas company.Primer is given birth to worker technology company by Shanghai and is synthesized.Antibody is the Neomarkers product available from crystalline substance U.S. company.Anti-and the Ecl of HRP two strengthens the luminescence reagent box available from green skies technical research institute.Cancer of pancreas Panc-1 cell strain, QBI-293A incasing cells and empty carrier Ad are provided by University Of Suzhou's medical board institute cell and molecular biology teaching and research room, and Ad-hING4 is made up and preserved by this teaching and research room.The 125I particle is produced (6711 type) by the former rich biomedical engineering company limited in Beijing.The hING4 gene primer is synthetic by Shanghai Sangon, MTT is available from Sigma company, RNA extraction agent box is given birth to worker's biotechnology company limited available from Shanghai, the MMLV reverse transcriptase is available from MBI company, newborn calf serum is matched happy biotechnology company available from Hangzhou, the total RNA extraction agent of UNIQ-10 pillar Trizol box is given birth to worker's technology company limited available from Shanghai, and MMLV reverse transcriptase and Taq enzyme are available from Fermentas company.Primer is given birth to worker technology company by Shanghai and is synthesized.β-actin is the Neomarkers product available from crystalline substance U.S. company.Anti-and the Ecl of HRP two strengthens the luminescence reagent box available from green skies technical research institute.Activated caspase-3 of mouse-anti people and two anti-goat-anti rat HRP-IgG (H+L), BeyoECL Plus are all available from green skies biotechnology research institute.

1.3 method

1.3.1 inverted microscope and fluorescence inverted microscope are observed the morphological change of panc-1

Get cell exponential phase of growth, after trypsinization, make the cell single-cell suspension in culture dish.Behind sample microscopically blood cell counting plate counting,, connect 2 * 10 with the dilution of gradient dilution method ⁵Individual cell in the 35mm diameter culture dish that is added with capacity DMEM culture fluid, treat that 15 independent culture dishs have been inoculated cell after, place CO ₂In the incubator one day, treat that iuntercellular reaches 60% processing that begins to experimentize when merging.The 1st day, experiment was divided into 5 groups (every groups repeat 3 wares): 1. normal control group (0.1mol/L PBS); 2. Ad negative control group (50MOI Ad-GFP); 3. Ad-hING4 processed group (50MOI Ad-hING4); 4. simple ¹²⁵I particle processed group placed device in the 2nd day, continued low dose rate gamma-rays 2Gy irradiation (45h 36min); 5. Ad-hING4 reaches ¹²⁵I particle Combined Treatment group added Ad-hING4 earlier and handles, and placed device to carry out the 2Gy irradiation in the 2nd day.All culture dishs are placed 37 ℃, 5%CO ₂Incubator is cultivated.To be illuminated unite group exposure dose reach 2Gy after, 5 groups of cells all take out, and observe GFP and express and the cell growthform under the fluorescence inverted microscope.

¹²⁵I particle predose rate: 4.44cGy/h;

During dosage 2Gy, irradiation time should be 1.9 days (45h 36min).

Observed result shows: infect the Panc-1 cell of handling through Ad-hING4,90% cell is all expressed GFP, presents very high efficiency of infection; And Ad-hING4 processed group, simple ¹²⁵The Panc-1 cell of I particle processed group, Combined Treatment group all occur obviously becoming justify, come off, pathological changes (CPE) such as shrinkage, and pathological changes such as change is justified, come off, shrinkage all appear through the Panc-1 cell that PBS, Ad-GFP handle.The result shows Ad-hING4 and continue the low dose rate gamma-rays all the Panc-1 pancreatic cancer cell is had tangible cytotoxicity, and is the most remarkable to unite the group effect especially.

1.3.2. flow cytometer detects the panc-1 apoptosis

Gather in the crops respectively and respectively organize cell and supernatant after the above-mentioned processing.After collecting cell was washed 2-3 time with the PBS phosphate buffer, 4 ℃ of 70% cold ethanol fixedly more than the 24h, were used flow cytometer and are detected apoptosis rate.The same first of concrete grammar.

The results are shown in Table 2,, compare no significant difference (P＞0.05) with the PBS group although the Ad-GFP group has lower cytotoxicity.Ad-hING4 processed group, simple ¹²⁵I particle processed group, Combined Treatment group are compared the apoptosis rate that all can significantly increase the Panc-1 cell with PBS group and Ad-GFP group, be respectively (17.80 ± 3.03) %, (23.43 ± 0.86) %, (51.23 ± 5.05) %, and the Combined Treatment group are than simple ¹²⁵I particle processed group apoptosis rate obviously increases, up to (51.23 ± 5.05) %.Ad-hING4 is described and continues the low dose rate gamma-rays and all can induce the Panc-1 apoptosis, and The combined processing back apoptosis rate obviously increases, and synergy is arranged.

Table 2 apoptosis rate (%) relatively

Grouping apoptosis rate (%)

PBS group 1.96 ± 0.40

Ad-GFP group 2.77 ± 0.67

Ad-hING4 processed group 17.80 ± 3.03

¹²⁵I particle processed group 23.43 ± 0.86

Combined Treatment group 51.23 ± 5.05

The PBS group is compared p＞0.05 with the Ad-GFP group; PBS group and Ad-hING4 processed group, simple ¹²⁵I particle processed group, Combined Treatment group compare p＜0.001; Each group compares p＜0.001 with the Combined Treatment group.

1.3.3. colony-forming test detects cell proliferation

Collecting cell, 500/ware are inoculated in the 35mm diameter culture dish, add culture medium and also rock gently, make it into unicellular distribution.After treating cell attachment, the experiment grouping is the same with processing.All culture dishs are placed 37 ℃, 5%CO ₂Incubator is cultivated 10d, cleans 2 times with PBS is careful, and air drying, formaldehyde fixed 15min, the Giemsa 15min that dye, the slow flush away dye liquor of flowing water, air drying again, microscopically is counted the clone greater than 50 cells.Be calculated as follows then: cloning efficiency (%)=(clone's number/inoculating cell number) * 100%;

Suppression ratio (%)=[1-(irradiation group cloning efficiency/matched group cloning efficiency)] * 100%.

Testing result sees Table 3 and shows, the cloning efficiency of each experimental group: PBS group for (32.93 ± 0.61) %, Ad-GFP group for (32.13 ± 0.41) %, Ad-ING4 group for (21.87 ± 0.81) %, ¹²⁵I particle group is that (12.93 ± 0.12) %, Combined Treatment group are (7.73 ± 0.76) %.The Combined Treatment group is compared with the Ad-GFP group with the PBS group, and very evident difference (P＜0.001) is all arranged.Ad-hING4 processed group and simple ¹²⁵Clone's suppression ratio of I particle processed group is respectively (33.56 ± 3.67) % and (60.83 ± 0.88) %, and the suppression ratio of Combined Treatment group is up to (76.51 ± 2.37) %, and the latter is apparently higher than the above two (all P＜0.001).The result shows, continues low dose rate gamma ray radiation therapeutic and ING4 gene therapy, can produce coordinating effect, suppresses the propagation of panc-1 pancreatic cancer cell.

Table 3 cloning efficiency and suppression ratio (%) are relatively

1.3.4.RT-PCR method detects the expression of pancreatic cancer cell IAP survivin gene mRNA

Handle the cell method and detect step with the streaming cell instrument, collecting cell, Trizol reagent extracting cell total rna is got total RNA2 μ g, operates by the explanation of reverse transcription test kit, with GAPDH internal reference, get RT product 2.0 μ l and carry out the PCR reaction.Get PCR product electrophoresis, dyeing, gel imaging system is taken pictures automatically.The RT-PCR step:

I. the extracting of total RNA

1) sample preparations

2) add 0.5ml Trizol, thermal agitation to cell precipitation dissolves disappearance soon;

3) use the 1ml syringe, twice of 26-G number (6#) syringe needle suction homogenate to be to shear genomic DNA, then directly from syringe with sample transfer in the 1.5ml centrifuge tube of aseptic Rnase-free;

4) in above-mentioned 1.5ml centrifuge tube, add chloroform/isoamyl alcohol (24: 1) 100 μ l, thermal agitation mixing 30s;

5) use desk centrifuge, 1,2000r/min, the centrifugal 5min of room temperature;

6) supernatant (about 450 μ l) carefully is transferred in the 1.5ml centrifuge tube of aseptic Rnase-free, adds dehydrated alcohol 150 μ l, mixing;

7) carefully complete soln is transferred to cover and be put in UNIQ-10 post central authorities in the collecting pipe, room temperature places behind the 2min 8, the centrifugal 1min of 000r/min room temperature;

8) carefully take out post, discard the waste liquid in the collecting pipe, post is put back in the collecting pipe, add the RPE Solution of 450 μ l, 10, the centrifugal 30s of 000r/min room temperature;

9) repeating step 8) once;

10) carefully take out post, discard the waste liquid in the collecting pipe, post is put back in the collecting pipe, 10, the centrifugal 30s of 000r/min room temperature;

11) carefully take out post, be put in the 1.5ml centrifuge tube of aseptic Rnase-free,, place 2min for 65 ℃ at the careful water that adds 50 μ l Rnase-free of post inner membrance central authorities;

12) 10, the centrifugal 1min of 000r/min room temperature.Solution in the centrifuge tube is the RNA sample.Can use immediately or-70 ℃ of preservations.

The II.RT-PCR reaction

Synthesize cDNA first chain with 20 μ l RT reaction systems, method is as follows:

1) gets one of the volumetrical little centrifuge tube of 200 μ l of Rnase-free, add total RNA 10 μ l and oligo (dT) ₁₈2 μ l place behind 65 ℃ of water-bath 5min cooled on ice 2min rapidly, make the RNA uncoiling, are beneficial to reverse transcription;

2) add 5 * Buffer (RT enzyme dedicated buffering liquid), 4 μ l, 10mmol/L dNTPs 2 μ l, RNase Inhibitor 1 μ l and M-MLV reverse transcriptase 1 μ l respectively, place 42 ℃ of effect 1h, place 70 ℃ of reactions promptly to get the cDNA template behind the 10min again;

As template, carry out pcr amplification with resulting cDNA with conventional primer P1 of Survivin gene and P2.

??ddH ₂O	??36μl
??ddH ₂O	??36μl	10 * Reaction Buffer (contains Mg ²⁺)	??5μl
??dNTPs(10mmol/L)	??1μl	10 * Reaction Buffer (contains Mg ²⁺)	??5μl
??dNTPs(10mmol/L)	??1μl	??P1(50μmol/L)	??1μl

??P2(50μmol/L)	??1μl
??P2(50μmol/L)	??1μl	The cDNA template	??5μl
The Taq archaeal dna polymerase	??1μl	The cDNA template	??5μl
The Taq archaeal dna polymerase	??1μl	Altogether	??50μl

Get rid of evenly through centrifuge, put and carry out amplified reaction in the PCR instrument:

Get the PCR product of 5 μ l and the DNA Marker of 5 μ l and carry out 1.5% agarose gel electrophoresis, with gel imaging analysis systematic observation and analytical electrophoresis result.

The result shows: RT-PCR detects the expression of apoptosis-related genes survivin and finds, after the radiotherapy, and pancreatic cancer cell survivin down-regulated expression, but in the Combined Treatment group, reduce more obvious.

1.3.5. immunoblotting (Western trace) detects the activated caspase-3 of cell death related protein

Behind the same processing cell, collecting cell extracts total protein, behind the SDS-PAGE electrophoresis, carries out gel successively and changes film, sealing, anti-and two anti-hybridization, at last with specific substrate benzidine colour developing, the exposure of X-ray sheet.

Concrete steps:

1) specimen adds people's cell pyrolysis liquid (containing PMSF), hatches on ice 30 fens.

2) it is about 20 to move into 4 ℃ of centrifuge tubes, 000g (about 15,000 change) 20 minutes.

3) draw supernatant ,-80 ℃ of preservations.

4) carry out Bradford colorimetric method for determining protein concentration.

5) get the cell pyrolysis liquid (volume * protein concentration) of equal in quality, and add 5 * electrophoresis sample loading buffer.

6) in the boiling water bath 5 minutes, stand-by.

7) glue: in the acrylamide gel preparation vessel, add freshly prepared 12%SDS-PAGE separation gel,, add after the horizontal positioned that distilled water binds so that separation gel and air insulated are beneficial to it solidifies to from about the about 2cm of preparation vessel upper limb.Distilled water is removed in gelling to be separated back admittedly, changes with freshly prepared 5%SDS-PAGE and concentrates sol solution till full, and insert the Teflon stripping fork rapidly, notes avoiding sneaking into bubble; Carefully pull out stripping fork after waiting to concentrate gelling admittedly.

8) go up the careful about 15 μ l of the above-mentioned sample for preparing that add of sample syringe with trace and in corresponding comb hole, prepare electrophoresis.

9) polyacrylamide gel electrophoresis keeps DC voltage 90V when sample is in the concentrated glue, treat to adjust voltage to 120V after sample enters separation gel, and swimming to bromjophenol blue stops electrophoresis at the bottom of glue.

10) change film: carefully take off the polyacrylamide gel behind the electrophoresis, extract the part of estimating to contain sample, good its size of amount prepare identical shaped and big or small Whatman filter paper number and (attention: the direct contact membranes of hands during the clip nitrocellulose filter) of nitrocellulose filter.This filter paper and nitrocellulose filter are put into the commentaries on classics film buffer for preparing, soak 10min to remove the bubble that may exist; The commentaries on classics film presss from both sides the filter paper after the negative electrode anode is placed cotton pad, immersion, the polyacrylamide gel that contains testing sample, the nitrocellulose filter after the immersion, filter paper and cotton pad successively.To change film and insert and put into electrophoresis tank, and fill it up with in the groove and change the film buffer, the 200mA unidirectional current changes film 2h under 4 ℃ of conditions.

11) sealing: the nitrocellulose filter after the electricity commentaries on classics is inserted in the plate of a modest size, add confining liquid.This plate lucifuge is put into shaking table and shaken 1-2h slowly in 37 ℃, with other all protein binding sites on the sealing nitrocellulose filter.

12) wash film: after sealing is finished, the nitrocellulose filter after the sealing is inserted another plate and added an amount of TBST, shake 5-10min slowly, absorption TBST in 37 ℃.So repeating 2-3 time gets final product.

13) anti-hatching: after washing film and finishing, film is inserted plate, pave, pour fresh preparation one anti-solution (by being diluted in the above-mentioned confining liquid for preparing at 1: 1000) into, generally get final product just to cover film.37 ℃ are shaken 1-2h slowly so that antibody combines with antigen is complete.

14) wash film again: wash film three times with step 12), anti-to remove unconjugated one.

15) two anti-hatching: after washing film again and finishing, film is inserted plate, pave, pour fresh preparation two anti-solution (by being diluted in the above-mentioned confining liquid for preparing at 1: 1500) into, generally get final product just to cover film.37 ℃ are shaken 1-2h slowly so that two anti-resist complete the combination with one.

16) wash film for the third time: wash film three times with step 12, anti-to remove unconjugated two.

17) strengthen luminescence reagent box A liquid, each 1ml of B liquid evenly with after mixing with Ecl, covering nitrocellulose filter 1-2 minute enters dark room operation.

18) exposure, development, photographic fixing.

The result shows that the ING4 gene transfection reaches and continues the low dose rate radiation gamma, all can raise the activated caspase-3 expression of gene of panc-1 pancreatic cancer cell, but the Combined Treatment group is more obvious.

1.3.5 the multi-tumor marker protein chip detects CA199, CA242, CA153

The supernatant 100 μ l that results are tested above directly carry out the multi-tumor marker protein chip and detect, and observe its combined effect.Operating procedure:

I. prepare before the experiment

A: standard substance dilution:

1) standard substance 4 usefulness 240 μ l redissolution water is redissolved;

2) standard substance 0 usefulness 600 μ l redissolution water is redissolved;

3) standard substance 4 after absorption 120 μ l redissolve add standard substance 0 dilution after 120 μ l redissolve, and obtain standard substance 3;

4) standard substance 3 after absorption 120 μ l redissolve add standard substance 0 dilution after 120 μ l redissolve, and obtain standard substance 2;

5) standard substance 2 after absorption 120 μ l redissolve add standard substance 0 dilution after 120 μ l redissolve, and obtain standard substance 1;

6) quality-control product is redissolved with 120 μ l redissolution water.

B: 15 times of distilled water dilutings of concentrated cleaning solution.

II. add sample to be tested, standard substance redissolution liquid, quality-control product redissolution liquid

Each draws 100 μ l, adds different chip surfaces.

III. incubation vibrates

37 ℃, 100rpm incubation vibration 30 minutes.Discard the liquid of chip surface then.

IV. washing

Chip put into wash box and add cleaning mixture, 37 ℃, the vibration of 250rpm incubation 8 minutes discard cleaning mixture.Wash altogether four times.

V. add reactant liquor

Each chip surface respectively adds 100 μ l reactant liquors.

VI. incubation vibrates

With step 3.

VII. washing

Peel off the top of protein chip integrated package, the back is with step 4.

VIII. detect

Add 20 μ l on the film surface of each chip and mixed 15 minutes detection liquid A and B mixed liquor, left standstill 1.5 minutes.

IX. read, analyze

The protein chip integrated package is added HD-2001 series of biologic chip detector, the automatic reading images of software (time 60s), make standard curve, analyze the test result and the printing data report form of each tested specimen.

CA153 critical reference value 35U/ml, CA199 critical reference value 35U/ml.

Tumor markers CA199, CA242 and the CA153 stronger at the cancer of pancreas specificity detect.The results are shown in Table 4, each organizes supernatant tumor markers CA199, CA242 and CA153, and the Ad-GFP group is compared no significant difference (P＞0.05) with the PBS group.Compare Ad-hING4 processed group, simple with PBS group and Ad-GFP group ¹²⁵Tumor markers CA199, CA242 and CA153 in I particle processed group, the Combined Treatment group supernatant all reduce, and each index Combined Treatment group descends the most remarkable.

Table 4 respectively organizes supernatant tumor markers CA199, CA242 and CA153 compares

1.3.6. statistical procedures: analyze with the SPSS10.0 statistical software, all (P＜0.05 is for there being statistical significance for X ± S) expression, group difference employing ANOVA variance analysis with mean ± standard deviation for each variable.

Embodiment one and embodiment two have illustrated Ad-hING4 gene therapy associating radioactivity ¹²⁵The I particle continues the experiment in vitro process of low dose rate gamma-ray therapy Panc-1 pancreatic cancer cell, by inverted microscope and the observation of fluorescence inverted microscope, flow cytometer detection, clone's formation experiment, RT-PCR method, Western-blotting and protein chip, inquire into the joint effect of gene-radiation therapeutic alliance malignant tumor, for its clinical practice provides experiment and theoretical basis.

The result shows: flow cytometer detects explanation Ad-hING4 and continues the low dose rate gamma-rays all can induce the Panc-1 apoptosis, and apoptosis rate is respectively (17.80 ± 3.03) %, (23.43 ± 0.86) %; And The combined processing back apoptosis rate obviously increases, and up to (51.23 ± 5.05) %, has synergy.

The clone forms experimental result demonstration Combined Treatment group and compares with the Ad-GFP group with the normal control group, and very evident difference (P＜0.001) is all arranged; Ad-hING4 processed group and simple ¹²⁵Clone's suppression ratio of I particle processed group is respectively (33.56 ± 3.67) % and (60.83 ± 0.88) %, and the suppression ratio of Combined Treatment group is up to (76.51 ± 2.37) %, and the latter is apparently higher than the above two (all P＜0.001).Presentation of results continues low dose rate gamma ray radiation therapeutic and the therapeutic scheme of uniting the ING4 gene, can produce coordinating effect, and significantly suppress the propagation of panc-1 pancreatic cancer cell.

Every experiment shows that Ad-hING4 and independent irradiation group all have tangible cytotoxicity to the Panc-1 pancreatic cancer cell separately, can promote its apoptosis, suppress its propagation.Though the Ad-GFP group has lower cytotoxicity, compare no significant difference (P＞0.05) with the PBS group.Yet it is the most remarkable to unite the effect of organizing cytotoxicity, the promotion apoptosis that presents and suppressing to breed, and the gamma-rays therapeutic alliance cancer of pancreas of ING4 and lasting low dose rate is described, has the obvious synergistic effect.

The mechanism of action that the gamma-rays associating of ING4 and lasting low dose rate can be treated cancer of pancreas is still clearly incomplete at present.Further result of study shows, the associating group can make survivin gene expression significantly reduce, strengthen the proteic expression of activated caspase-3, prompting is at cancer of pancreas panc-1 cell, the ING4 gene is united lasting low dose rate gamma ray radiation therapeutic may and activate activated caspase-3 protein expression by downward modulation survivin gene expression, and then promotion pancreatic cancer cell apoptosis.

In sum, continue low dose rate gamma ray radiation therapeutic associating ING4 gene therapy, can produce coordinating effect, suppress the propagation of panc-1 pancreatic cancer cell, promote its apoptosis, its mechanism may be relevant with the active caspase-3 protein expression of rise tool with downward modulation survivin gene expression.

Embodiment three, the potentiation of Ad-hING4 associating 125I particle anti-tumor in vivo

1.1 material

Cancer of pancreas Panc-1 cell strain, QBI-293A incasing cells and the adenovirus empty carrier Ad and the Ad-hING4 that carry the GFP green fluorescent protein are that cell and molecular biology teaching and research room provide by University Of Suzhou's preclinical medicine. ¹²⁵The I particle is produced (6711 type) by the former rich biomedical engineering company limited in Beijing, and activity is 14.8MBq.Age in 3-4 week, the only male BALB/c nu/nu of body weight 16-18g/ nude mice are available from Chinese Academy of Sciences's Shanghai Experimental Animal Center (credit number: SCXX (Shanghai) 2007-0005).The CD34 antibody assay kit is available from Shenzhen brilliant U.S. biotech firm; Survivin antibody, Caspase3 antibody assay kit are available from Shanghai brilliant U.S. company; RPMI-1640, newborn calf serum match happy biotechnology company available from Hangzhou.

1.2. method

1.2.1.Ad-hING4 the amplification of recombinant adenovirus

With example one 1.2.1.1

1.2.2. the mensuration of virus titer

With example one 1.2.1.2

1.2.3. the foundation of cancer of pancreas animal model

The Panc-1 pancreatic cancer cell is incubated in the RPMI-1640 culture medium that contains 10% calf serum, 37 ℃, 5%CO ₂Cultivate in the cell culture incubator.The Panc-1 cell of getting exponential phase of growth is after trypsinization disperses, and PBS washs 2 times, and the centrifugal 5min of 2000r/min is resuspended among the PBS, and adjusting cell concentration is 2 * 10 ⁷/ ml gets 100 μ l (2 * 10 ⁶Individual cell/only) be inoculated in respectively under the right ribbed hides of 25 nude mices, the SPF environment is raised down, treats experiment when treating the about 8-10mm of diameter of tumor.

1.2.4. experiment grouping and antineoplaston

Above-mentioned 25 tumor bearing nude mice randomizations are divided into 5 groups, 5 every group.Injection Ad-hING4 virus (1 * 10 in Ad-hING4 gene therapy group (Ad-hING4 group) the tumor body ⁷Pfu/50 μ l), inject the PBS and the empty carrier Ad-GFP of equivalent in PBS matched group (PBS group), Ad-GFP empty carrier matched group (Ad-GFP group) the tumor body respectively. ¹²⁵I corpuscular radiation treatment group ( ¹²⁵I particle group) earlier with radioactivity 14.8Mbq ¹²⁵The I particle places 2% glutaraldehyde soaking disinfection 30min, and is stand-by with physiological saline solution flushing back.Wear out skin with the 18G puncture needle in distance borderline tumor 5mm place then, subcutaneous moving under water enters tumor center, withdraws from nook closing member, with 1 piece ¹²⁵The I particle puncture needle of packing into pushes tumor center with nook closing member with particle, withdraws from puncture needle, and aseptic cotton carrier is put back to mouse cage with nude mice after gently pressing site of puncture 1min, and single cage is raised. ¹²⁵Implant in every tumor of I particle and Ad-hING4 therapeutic alliance group (therapeutic alliance group) 1 piece identical ¹²⁵The I particle is also injected Ad-hING4 virus (1 * 10 in the tumor body ⁷Pfu/50 μ l), the next day once, totally 6 times.

The result shows: present embodiment has successfully been set up people Panc-1 pancreatic cancer cell transplanted tumor in nude mice model, and tumor formation rate is 100%.

1.2.5 detect index and detection method

1.2.5.1. gross tumor volume, tumor weigh and tumour inhibiting rate

Treatment begins back per 5 days with measuring 1 gross tumor volume.Treat and put to death nude mice after 20 days, extract the tumor body, measure the long and short footpath of tumor and weigh gross tumor volume V=ab ²* 0,52 (a is a major diameter, and b is a minor axis).Calculate each treatment group tumour inhibiting rate, tumour inhibiting rate=(matched group tumor W-treatment group tumor W)/matched group tumor W * 100%.The cancer potentiation result that presses down of therapeutic alliance group judges with the equal method of Nintaus ^[8](calculating the q value), q value=E (A+B)/(EA+EB-EAEB), EA, EB are respectively the effect that A, B singly use in the formula, and molecule E (A+B) representative actual measurement merges effect, and denominator is that expectation merges effect.When q value=1 ± 0.15, be considered to summation action between two medicines, q value＞1.15 o'clock have been considered to synergism between two medicines, when q＜0.85, between two medicines antagonism are arranged.Tumor tissues specimen 10% neutral formalin fixedly spends the night, and the routine paraffin wax embedding is used to organize HE dyeing and immunohistochemistry to detect.

After the treatment, different treatment group gross tumor volumes, weight, tumour inhibiting rate the results are shown in Figure 1,2,3.The result shows: ¹²⁵The tumor body weight of I particle group, Ad-hING4 group, therapeutic alliance group is compared with the PBS matched group, and average tumour inhibiting rate reaches 34.19%, 31.50%, 67.15% respectively, and significant difference (p＜0.01) is arranged, and therapeutic alliance group tumour inhibiting rate apparently higher than ¹²⁵I particle combination radiotherapy group, Ad-hING4 gene therapy group (p＜0.01), q value＞1.15 (q=1.22) shows the Ad-hING4 associating ¹²⁵The I particle therapy can be worked in coordination with the growth that suppresses cancer of pancreas transplanted tumor, has the synergic synergism of tumor of pressing down, and shows that also Ad-hING4 is right simultaneously ¹²⁵The I particle has the radiotherapy sensitization effect, is a kind of ideal radiotherapeutic sensitizer.And the PBS matched group is compared there was no significant difference (p＞0.05) with empty carrier Ad-GFP group matched group.During raising treatment, do not find the toxic reaction of nude mice death and other body.

1.2.5.2. learning, routine pathology checks

Behind the conventional preparation paraffin section, HE dyeing, microscopically is observed cell growthform, degree of tissue damage and the scope in the tumor specimen respectively organized.

The result shows: the tissue slice of cancer of pancreas transplanted tumor dyes through conventional HE, microscopic examination, ¹²⁵I particle group, therapeutic alliance group are at the visible oncocyte degeneration necrosis in nearly particle place, the karyon dissolving, cellularity disappears, downright bad tumor tissues is homogenizing, redly dyes performance, but away from the particle place tumor cell of as seen surviving, the also visible more tumor cell degeneration necrosis of Ad-hING4 group, but its morphological change and distance relation are little, and PBS organizes, Ad-GFP group tumor cell degeneration necrosis is not obvious.

1.2.5.3. the detection of apoptotic index (apoptotic index AI)

Conventional H E dyeing, under low power lens (100 times), select earlier 5 visuals field (the treatment group should be chosen the visual field in the zone away from the visible survival tumor cell of particle) at random, again high power lens (400 times) down in each visual field of counting the apoptosis cancerous cell account for the percentage rate (%) of whole cancerous cell, percentile average represents that its criterion of apoptotic index (AI) of this tumor tissues inner cell is under 5 visuals field: tumor cell is as presenting smaller volume, Cytoplasm concentrates, nucleus diminishes, chromatin concentrates and the in bulk that condenses, it is poly-along the inboard nuclear limit of arranging of nuclear membrane chromatin to occur, even show as nucleus cracking formation apoptotic body (apoptoticbodies), then be judged as apoptosis of tumor cells.

The result shows: the tissue slice of cancer of pancreas transplanted tumor dyes through conventional HE, microscopic examination, ¹²⁵I particle group, Ad-hING4 group, all visible more apoptotic cell of therapeutic alliance group show as that cytoplasm concentrates, chromatin concentrates around nuclear membrane, are also shown in typical apoptotic body and form, and PBS group, Ad-GFP group apoptosis are then not obvious. ¹²⁵The apoptotic index (AI) of I particle group, Ad-hING4 group, therapeutic alliance group is respectively (36.28 ± 4.45) %, (32.83 ± 4.65) %, (58.30 ± 6.71) %, compares with the PBS group, and significant difference (P＜0.05) is arranged, and the therapeutic alliance group with ¹²⁵I particle group, Ad-hING4 group are compared, and difference also has significance meaning (P＜0.01), and there was no significant difference (P＞0.05) between PBS group (2.75 ± 0.60) %, Ad (4.81 ± 0.92) the % group shows ¹²⁵The I particle is implanted, the equal obvious apoptosis of inducing tumor cell of Ad-hING4 transfection, and the effect with the therapeutic alliance group is more remarkable especially.

1.2.5.4. the detection of tumor microvessel density (microvessel density MVD)

Adopt the dyeing of CD34 antibody mediated immunity group SP method to detect.Every section elder generation complete observation under low power lens (* 100), determine 5 vessel densities the highest " focus " district, under high power lens (* 400), carry out blood capillary counting (MVD) again, its criterion is: any pale brown color or the painted endotheliocyte of sepia or endotheliocyte bunch are designated as an independently blood vessel, but the flesh layer is thicker or the tube chamber area is not counted greater than the blood vessel of 8 red blood cell diameters.The microvessel count in 5 focus visuals field of record is got the MVD value of its average as this specimen.

The result shows: the tissue slice of cancer of pancreas transplanted tumor detects through the dyeing of CD34 antibody mediated immunity group SP method, and result (as Fig. 4) shows: ¹²⁵The microvessel density of I particle group, Ad-hING4 group, therapeutic alliance group is respectively 24.76 ± 5.17,28.44 ± 4.43,14.28 ± 3.08, is starkly lower than PBS group (P＜0.05), and the therapeutic alliance group with ¹²⁵I particle group, Ad-hING4 group is compared also has significant difference (P＜0.01), and there was no significant difference (P＞0.05) between the PBS group, Ad-GFP group shows ¹²⁵The I particle is implanted, the Ad-hING4 transfection can obviously suppress tumor vascular formation, suppresses the best results that tumor vessel forms with the therapeutic alliance group especially.

1.2.5.5. the detection that Apoptosis Inhibitor Protein Survivin is expressed

After adopting the dyeing of Survivin antibody mediated immunity group SP method, be the Survivin positive cell to occur being dispersed in or filling the air the brown yellow granule that shape distributes in cytoplasm or the nucleus.5 400 times of visuals field are counted in every example section at random, estimate the proteic expression of Survivin with the number of average each contained positive cell in the visual field.Getting on the same group all section averages adds up.

The result shows: the tissue slice of cancer of pancreas transplanted tumor is through the dyeing of Survivin antibody mediated immunity group SP method, and result (as Fig. 4) shows: ¹²⁵The Survivin positive cell number of I particle group, Ad-hING4 group, therapeutic alliance group is respectively 43.96 ± 3.29,46.40 ± 4.36,27.32 ± 3.34, is starkly lower than PBS group (P＜0.05), and the therapeutic alliance group with ¹²⁵I particle group, Ad-hING4 group is compared also has significant difference (P＜0.01), and there was no significant difference (P＞0.05) between the PBS group, Ad-GFP group shows ¹²⁵The I particle is implanted, the proteic expression of Survivin can be significantly reduced in the Ad-hING4 transfection, reduces the strongest of Survivin protein expression with the therapeutic alliance group especially.

1.2.5.6. the detection that the activated Caspase-3 of apoptosis-related protein expresses

After adopting the dyeing of activatory Caspase-3 antibody mediated immunity group SP method, be the Caspase-3 positive cell to occur being dispersed in or filling the air the brown yellow granule that shape distributes in the cytoplasm.5 400 times of visuals field are counted in every example section at random, estimate the proteic expression of Caspase-3 with the number of average each contained positive cell in the visual field.Getting on the same group all section averages adds up.

The result shows: the Caspase-3 antibody mediated immunity group SP method dyeing that the tissue slice of cancer of pancreas transplanted tumor is activated, and result (as Fig. 4) shows: ¹²⁵The Caspase-3 positive cell number of I particle group, Ad-hING4 group, therapeutic alliance group is respectively 32.04 ± 3.29,29.24 ± 2.81,48.32 ± 4.04, apparently higher than PBS group (P＜0.05), and the therapeutic alliance group with ¹²⁵I particle group, Ad-hING4 group is than also there being significant difference (P＜0.01), and there was no significant difference (P＞0.05) between the PBS group, Ad-GFP group shows ¹²⁵The I particle is implanted, the proteic expression of Caspase-3 can be obviously raised in the Ad-hING4 transfection, and the ability with therapeutic alliance group activation apoptosis-related protein Caspase-3 is the most remarkable especially.

Embodiment three has illustrated: ¹²⁵I particle, Ad-hING4 can significantly suppress the growth of nude mice PANC-1 cancer of pancreas transplanted tumor, and The combined is used has the synergic synergism of cancer of pressing down, and Ad-hING4 is expected to as a kind of new radiotherapeutic sensitizer; It presses down tumor mechanism may be to come inducing apoptosis of tumour cell, the formation of inhibition tumor vessel to wait and realize by downward modulation Survivin with the expression of raising Caspase3.

Claims

1. people ING4 gene is as the purposes of tumor radiotherapy sensitive-increasing agent.

2. the application of people ING4 gene in the preparation tumor radiotherapy sensitive-increasing agent.

3. the sub-Ad-hING4 of recombinant virus of adenovirus mediated people ING4 gene is as the purposes of tumor radiotherapy sensitive-increasing agent.

4. the application of the sub-Ad-hING4 of recombinant virus of adenovirus mediated people ING4 gene in the preparation tumor radiotherapy sensitive-increasing agent.

5. a method that strengthens the inside and outside tumor cell to radiation sensitivity is characterized in that: before the inside and outside tumor cell is carried out radiotherapy, people ING4 gene is imported the inside and outside tumor cell, people ING4 gene is expressed in tumor cell.

6. enhancing according to claim 5 inside and outside tumor cell is characterized in that the method for radiation sensitivity: the described method that people ING4 gene is imported the inside and outside tumor cell is selected from: plasmid transfection or adenovirus mediated in a kind of.

7. a tumor radiotherapy sensitive-increasing agent pharmaceutical composition is characterized in that: comprise people ING4 gene or people ING4 gene coded protein and/or pharmaceutical carrier or excipient.