CN1692946A - Method for cnotrolling cell fector expression by heating, and its application for treating tumor - Google Patents

Abstract

A heating method for regulating the expression of cell factor to treat tumor is disclosed. The local heating is used to stimulate the genetic expression of cell factor for making the target expression in tumor and its peripheral tissue. Its method includes such steps as configuring artificial gene expression box, putting the promotor of thermal induced gene expression at up-stream of cell factor coding gene, transferring the box to tumor and its peripheral region, and local heating to induce cell factor gene expression, so eliminating local tumor and inducing whole antineoplastic immunity.

Description

A kind of with the method for heating regulating cell factor expression and the application in oncotherapy thereof

Technical field

The invention belongs to biomedical sector, relate to a kind of method, the method that especially adopts local heat control immunostimulatory cell factor gene to express, and the application in oncotherapy with heating regulating cell factor expression.

Background technology

Malignant tumor remains the topmost disease that threatens human health and life at present.Existing various Therapeutic Method (comprising operation, radiation and chemotherapy) is only effective to the tumor focus of limitation, and not good for the tumor efficiency of sending out or recurring.Therefore, press for and set up new tumor therapeuticing method, this also is one of ultimate challenge that mankind nowadays faced.

Gene therapy provides many good opportunities for the treatment problem that solves malignant tumor, and wherein main is because the tumor treatment window can be significantly widened in gene therapy.Along with the enforcement of the Human Genome Project and post genome project, the selection of therapeutic gene is more extensive, and the method that the regulation and control therapeutic gene is expressed also can be more.Therefore, might accomplish the selective killing tumor cell and less to normal cell and tissue injury.Not only therapeutic effect can be improved, toxicity, side effect can also be alleviated.

In order to reach the purpose that killing tumor cell did not to greatest extent simultaneously damage or seldom damaged normal tissue cell again, when design therapy of tumor strategy and method, must consider following 3 points.

1. potent antineoplaston gene;

2. efficiently therapeutic gene is transported in the tumor;

3. regulating and control therapeutic gene effectively expresses in tumor;

In the mammalian cell gene of having found at present, the strongest modulated gene is heat shock protein (heatshock protein) gene family.These genes have function widely, are to escort the escort of new synthetic protein to their subcellular fraction position; And when environment weight (stress) such as the stress reaction albumen (stress response protein) that is activated when infecting of thermal pressure, chemical pressure or inoculating microbe.The most outstanding feature of heat shock protein is that its expression can be raised thousands of times when temperature surpasses normal physiological body temperature 5-6 ℃, and the albumen synthesizer more than 90% all transfers to produce heat shock protein in the cell at this moment.Have and studies confirm that the regulation and control of heat shock protein mainly occur in transcriptional level, the promoter that is positioned at heat shock protein gene 5 ' end plays pivotal role.The heat shock protein promoter is one of promoter the strongest in the mammalian cell, and expressing for the regulation and control therapeutic gene provides best selection.Thermotherapy (hyperthermia) and heating ablation (thermalablation) technology have been applied to the treatment of various solid tumors at present, but the above-mentioned factor combination of no-trump still so far is used for the report of medical practice.

Summary of the invention

The purpose of this invention is to provide a kind of method, the method that especially adopts local heat control immunostimulatory cell factor gene to express with heating regulating cell factor expression.Further aim of the present invention provides the application of described method in oncotherapy.

The technical scheme of the inventive method comprises uses heat shock protein gene promoter and immunostimulatory cell factor gene to make up artificial gene, described artificial gene has the potentiality that the immuno-stimulatory genes targeting is expressed in tumor, adopt microwave or modes such as excusing from death or radio frequency to heat tumor then, make the tumor by local temperature increase the 3-5 degree, make the immunoregulation gene at the tumor by local selectivity, efficiently express, reach the purpose of removing local and general tumour.The immunostimulatory cell factor of the present invention comprises that interleukin II, 12,18 and 21 is as IL2, IL12,18,21; Interferon-ALPHA, β, γ (Interferons α, β, γ); Tumor necrosis factor (TNF-α), grain, giant cell colony stimulating factor (GM-CSF), and other tumor-killing gene such as TRAIL.

This method places therapeutic gene under the heat shock protein gene promoter and makes up artificial gene, expresses and then realize the effect of treatment tumor at tumor by local by tumor by local being heated the regulation and control therapeutic gene.Described method has bigger toxic therapeutic gene particularly favourable and important to normal cell and tissue again for those existing intensive antitumor actions.This method can be used for removing local tumor and excites the reaction of whole body antitumor.Can use separately, also can combine and be used for tumor treatment with radiotherapy, chemotherapy or thermotherapy (comprising heating ablation).

It is therapeutic gene that the present invention preferentially selects immuno-stimulatory genes for use, and it has the following advantages:

1. potent;

2. have a bystander effect;

3. when topical application, also can have and excite the reaction of whole body antitumor.

The key of thermoinducible cytokine gene treatment and immunization therapy is design and makes up artificial gene, being about to the cytokine encoding gene places under the thermoinducible promoter regulation, further artificial gene is inserted into then in virus or the non-viral gene treatment carrier, again virus or non-virus carrier are applied directly to tumor mass or tumor normal structure on every side, adopt the local heat treatment tumor.The method of described local heat comprises microwave, radio frequency, high strength supersonic etc., and tumor and temperature of tissue surrounding thereof are risen more than 3 ℃, and the promoter height of heat shock protein gene activation under this temperature conditions activates high-caliber therapeutic gene and expresses.

The heat shock protein gene promoter that the present invention adopts comprises following gene, but be not limited to following gene, be Hsp70, Hsp70B, spendable cytokine gene is including, but not limited to the independent of following gene or unite use, be IL2, IL4, IL12, IL18, Interferons, TNF-α, TRAIL, FasL, GM-CSF etc.

The combination of other gene expressions of the therapeutic gene expression of local heat, heat regulation and control and heat regulation and control will be played potent antitumous effect to local and general tumour in technique scheme.

The inventive method comprises the steps:

1. adopt the initiator sequence of PCR or other known method separation and human cloning heat shock protein gene;

2. clone immunostimulation/tumor-killing cell factor gene cDNA;

3. immunostimulation/tumor-killing cell factor gene cDNA is placed under the heat shock protein gene promoter, make up artificial gene;

4. the artificial gene that will have the polyA tail is inserted in virus or the non-virus carrier, and described viral vector comprises adenovirus, slow virus etc., and non-virus carrier comprises naked plasmid dna and liposome-plasmid dna complex compound etc.;

5. be injected into above-mentioned carrier in the tumor or tumor around tissue in;

6. tumor by local and surrounding tissue thereof are heated, heating can be adopted hot pad, microwave, radio frequency or local mode such as ultrasonic.

Said method also can combine with existing Therapeutic Method such as chemotherapy, radiotherapy and surgical intervention etc., and its most outstanding advantage of method of cytokine-expressing gene that the present invention heats regulation and control is as follows:

1. the therapeutic gene targeting is expressed in tumor and tumor surrounding tissue, has obviously reduced the toxicity, side effect to other position normal structures of health---targeting;

2. can start or close expression---the Modulatory character of cytokine gene as required;

3. although therapeutic gene is expressed at tumor by local, the immunostimulatory cell factor not only has the effect that kills and wounds local tumor, can also induce the whole body anti tumor immune response---systemic effect and potent.

Description of drawings

Fig. 1 is that the mIL12 expression vector establishment sketch map and the thermal induction cytokine gene expression of heating regulation and control implemented sketch map.

Fig. 2 is by the expression through thermal initiation of reporter gene green fluorescent protein in tumor cell and tumor tissues in adenovirus Jie road.

Wherein, A is that the mouse mastopathy cell 4T1 of In vitro culture infects through 20MOI Ad-HSP-GFP, heats 30 minutes in 42 ℃ of water-baths after 24 hours, cultivates after 24 hours for 37 ℃, and fluorescence microscope GFP expresses.Infecting and heating back cellular expression GFP obviously increases.

B is. with 1 * 10 ⁶Mouse mastopathy cell 4T1 is expelled to Balb/c mouse back tumor form model, when treating tumor growth to diameter 1mm size, with the 1ml syringe 50ul is contained 1 * 10 ⁸Pfu Ad-HSP-GFP is expelled in the form.To heat 45 minutes in 42 ℃ of water-baths of form immersion after 24 hours.Heat anesthetized mice after back 24 hours, observe under fluorescence microscope, heating back GFP expression obviously increases.

Fig. 3 is that the tumor cell or the interior growing tumors tissue of body of In vitro culture expressed the situation of mIL12 after Ad-HSP-mIL12 infects and heats.

Wherein A is. the mouse mastopathy cell 4T1 of In vitro culture infects through 20MOI Ad-HSP-mIL12, and collecting cell culture fluid after 24 hours heated 30 minutes in 42 ℃ of water-baths then, and 37 ℃ were continued to cultivate after 24 hours, once more the collecting cell culture fluid.Adopt R﹠amp; The mIL12 Elisa detection kit of D company detects before and after the heating mIL12 content in the cell culture fluid, and visible heating back cellular expression mIL12 obviously increases, mIL12 even can increase by 10 ⁴Doubly.

B is. the mIL12 of heating induction expresses in tumor tissues,

With 1 * 10 ⁶It is subcutaneous that mouse mastopathy cell 4T1 is expelled to Balb/c mice back leg, when treating tumor growth to diameter 7～8mm size, with the 1ml syringe 100ul contained 1 * 10 ⁸Pfu Ad-HSP-mIL12 is expelled in the tumor.To heat 45 minutes in 42 ℃ of water-baths of Subcutaneous tumor immersion after 24 hours.Heat back 48 hours anesthetized mices, take out tumor, Elisa detects mIL12 content after the homogenate, and compares with not injection group or not heating group.Injecting virus and heating group mIL12 expression obviously increase, and wherein the mIL12 expression can increase by 80 times.

Fig. 4 is that Ad-HSP-mIL12 suppresses mouse interior tumor growth figure.

Wherein, with 5 * 10 ⁶It is subcutaneous that mouse mastopathy cell 4T1 is inoculated into the mice back leg, when treating tumor growth to diameter 5～7mm, with 2 * 10 ⁸Ad-HSP-mIL12 or Ad-hsp-GFP are expelled in the tumor, will heat 45 minutes in 42 ℃ of water-baths of tumor immersion after 24 hours.Use vernier caliper measurement tumor size every other day once.Draw tumor growth curve, visible treatment group tumor growth obviously is suppressed.Among the figure, solid triangle does not heat group for injection Ad-hsp-GFP virus.Solid circles does not heat group for injection Ad-hsp-mIL12.Hollow square frame is injection Ad-hsp-GFP heating group.Hollow triangle is injection Ad-hsp-mIL12 heating group.As can be seen, have only the speed of growth of Ad-hsp-mIL12 heating group to be subjected to significant limitation.

Fig. 5 is thermoinducible gene therapy of use in conjunction and radiotherapy mouse interior tumor growth curve.

The specific embodiment

What enumerate below is the instantiation that how the present invention is put into practice.The promoter of HSP70B gene is used to reporter gene GFP in the example below, and the expression of therapeutic gene mIL12.The constructed adenovirus that carries GFP and mIL12 studies confirm that through experiment in vivo and vitro can be heated regulating and expressing, and has antitumous effect.

Embodiment 1:

1.PCR amplification HSP70B gene promoter:

As template, adopt following primer with the HEK-293 cell DNA, pcr amplification HSP70B gene 5 ' end regulation and control order.The pcr amplification condition be 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1 minute, 25 cycles.The long 420bp of PCR product, sequencing result show to have the sequence of sequence 1.

Forward primer 5 '-GGTCGAGGCGCGTCCTCAGAGCCA-3 '

Downstream primer 5 '-AGGTCGACTAGAAGCTTCTTGTCG-3 '

2. heat the structure of the GFP expression vector of regulation and control:

The PCR product is inserted earlier in the TA cloning carrier pCR-Blunt, and after restricted enzyme EcoRI digestion, (the EcoRI site in multiple clone site CA) makes up the pHSP-EGFP expression vector for Clontech, Carlbad to be connected to carrier pEGFP-1 again.

3. heat the GFP of regulation and control and the structure of mIL12 expression vector:

With restricted enzyme Sal I and Not I digestion mIL12 expression plasmid pNGVL-mIL12, separate mIL-12 encoding gene fragment behind the agarose gel electrophoresis, replace pHSP-EGFP expression vector GFP encoding gene, make up the pHSP-mIL-12 expression vector.The cDNA of described mIL12 gene has the sequence of sequence 2,3.

4. make up the shuttle vector of band genes of interest:

Adopt restriction enzyme A flII digestion pHSP-GFP and pHSP-mIL12 expression vector, polishing then earlier.Reuse EcoRI digestion separates behind the agarose gel electrophoresis and contains HSP70 promoter and reporter gene GFP or therapeutic gene mIL12, and the expressed intact box of polyA tail.Insertion is in the shuttle vector pENTR1A (Invitrogen company product) of SalI and EcoRI digestion.Structure contains the shuttle vector of GFP and mIL12, pENTR1A-HSP-GFP and pENTR1A-HSP-mIL12.

5. structure adenovirus vector:

With shuttle plasmid pENTR1A-HSP-GFP and pENTR1A-HSP-mIL12 and adenovirus skeleton carrier pAd-PL/DEST reaction back transfection recipient bacterium Top10, select resistance clone containing on the flat board of ampicillin.Enzyme action and electrophoresis are identified after the extracting plasmid DNA, determine to contain target gene fragment HSP-GFP or HSP-mIL12 after, rotaring redyeing 293 cell behind a large amount of extractings and the purification.

6. the packing of adenovirus:

With the adenovirus DNA pAd-HSP-GFP or the pAd-HSP-mIL12 transfection adenovirus packaging cell HEK293 (ATCC product) of purification, treat that collecting cell after the pathological changes, multigelation 3 times appear in cell.Infect 293 cells once more.Through 3 take turns plaque select after, select the duplicating efficiency height, do not have the adenopathy seed culture of viruses Ad-HSP-GFP and the Ad-HSP-mIL12 of wild poison.

7. the amplification of adenovirus and purification:

Further adopt HEK293 cell increase in a large number adenovirus pAd-HSP-GFP and pAd-HSP-mIL12.The cesium chloride density gradient centrifugation purification of adenoviral, or adopt the adenovirus purification kit purification of BD company, virus quantity can reach 3～20 * 10 ¹¹Pfu/ml.

8.Ad-HSP-GFP and the check and analysis expressed in the cultured cell in vivo and in vitro of Ad-HSP-mIL12:

The virus of purification meets 20MOI (multiplicity of infection) infecting mouse breast carcinoma cell strain 4T1, infects back 24 hours fluorescence microscopes (Ad-HSP-GFP infection) or collecting cell culture fluid (Ad-HSP-mIL12).The culture plate that will contain infected cell then with the Parafilm sealing after, put into 42 ℃ of water-baths heating 30 minutes, put into 37 ℃ CO again ₂Continue in the incubator to cultivate.Collecting cell culture fluid once more after 24 hours, fluorescence microscope, the intensity of cellular expression GFP before and after the heating relatively, or adopt R﹠amp; The mIL12 Elisa detection kit of D company detects before and after the heating mIL12 content in the cell culture fluid, and the result shows that the heating back expresses GFP or mIL12 obviously increases, mIL12 even can increase by 10 ⁴Doubly.

9.Ad-HSP-GFP and Ad-HSP-mIL12 is in the in-house expression of mouse interior tumor:

Select mouse breast cancer as model.With 1 * 10 ⁶It is subcutaneous that mouse mastopathy cell 4T1 is expelled to Balb/c mice back leg, when treating tumor growth to diameter 7～8mm size, with the 1ml syringe 100ul contained 1 * 10 ⁸Pfu Ad-HSP-GFP or Ad-HSP-mIL12 are expelled in the tumor.To heat 45 minutes in 42 ℃ of water-baths of Subcutaneous tumor immersion after 24 hours.Heat back 48 hours anesthetized mices, take out tumor, vibration section back fluorescence microscope, or Elisa detects mIL12 content after the homogenate, and compare with not injection group or not heating group.The result shows that injecting virus and heating back GFP and mIL12 expression obviously increase, and wherein the mIL12 expression can increase by 80 times.

10.Ad-HSP-mIL12 inhibitory action to the mouse interior tumor growth:

Select the mice carcinoma of prostate as model.With 5 * 10 ⁶It is subcutaneous that mice prostate gland cancer cell Tramp-C is inoculated into the mice back leg, when treating tumor growth to diameter 5～7mm, with 2 * 10 ⁸Ad-HSP-mIL12 is expelled in the tumor, will heat 45 minutes in 42 ℃ of water-baths of tumor immersion after 24 hours.Use vernier caliper measurement tumor size every other day once.Draw tumor growth curve.The result shows that treatment group tumor growth obviously is suppressed.

11. thermoinducible gene therapy of use in conjunction and radiotherapy are handled mice carcinoma of prostate animal model:

Earlier with 2 * 10 ⁶Carcinoma of prostate tumor cell TrampC is expelled under the right back shedding of C57/BL6 mice, and tumor growth is to diameter 5-7mm size after 3 weeks.Mice is divided into 6 groups:

First group is that the gene therapy matched group claims the AdhspGFP processed group again, only carries the adenovirus of thermal induction gene Hsp70 promoter and GFP encoding gene at locally injected into tumor.The adenovirus consumption is every mice 2 * 10 ⁸Pfu, volume 50 μ l.Injecting method is a multi-point injection in the tumor.

Second group is that the gene therapy experimental group claims the AdhspmIL12 processed group again, only carries the adenovirus of thermal induction gene Hsp70 promoter and mIL12 encoding gene at locally injected into tumor.

The 3rd group is that gene therapy+thermotherapy matched group claims the AdhspGFP+HT processed group again, and locally injected into tumor carries the adenovirus of thermal induction gene Hsp70 promoter and GFP encoding gene, and the adenovirus consumption is the same for reaching injecting method.Inject and gave heat treated in back 24 hours, heating means are as follows: be ready to water temperature earlier and be 42 ℃ water bath, will be fixing behind the mouse anesthesia, and the right rear leg that will carry tumor then is immersed in 42 ℃ the water, heats 30 minutes.Treating to send back to after mice revives Animal House raises again.

The 4th group is that gene therapy+thermotherapy experimental group claims Adhsp mIL12+HT processed group again, and locally injected into tumor carries the adenovirus of thermal induction gene Hsp70 promoter and mIL12 encoding gene, and the adenovirus consumption is the same for reaching injecting method.Inject and gave heat treated in back 24 hours, heating means are the same.

The 5th group is that gene therapy+thermotherapy+radiotherapy matched group claims Adhsp GFP+HT+RT processed group again.Make radiotherapy before injecting virus earlier, method is as follows: radiotherapy 3 times, and the cycle is 5 days, promptly begins the back the 1st, 3,5 day in radiotherapy respectively, tumor by local gives 6Gy radiation gamma, has a rest, and does not shine in the 2nd, 4 day.After the 3rd radiotherapy, locally injected into tumor carries the adenovirus of thermal induction gene Hsp70 promoter and GFP encoding gene, and the adenovirus consumption is the same for reaching injecting method.Inject and gave heat treated in back 24 hours, heating means are the same.

The 6th group is that gene therapy+thermotherapy+radiotherapy experimental group claims Adhsp mIL12+HT+RT processed group again.Make radiotherapy before injecting virus earlier, method is the same.After the 3rd radiotherapy, locally injected into tumor carries the adenovirus of thermal induction gene Hsp70 promoter and mIL12 encoding gene, and the adenovirus consumption is the same for reaching injecting method.Inject and gave heat treated in back 24 hours, heating means are the same.

After above-mentioned processing, use vernier caliper measurement tumor maximum diameter, path and height secondary weekly, calculate tumor volume, draw tumor growth curve.The result as seen, thermoinducible gene therapy of use in conjunction and radiotherapy, the mouse interior tumor volume obviously dwindles, then slowly the growth.With other each groups notable difference is arranged relatively, show that thermoinducible gene therapy of use in conjunction and radiotherapy have synergism to tumor.

The cDNA order of human interleukin-12 gene of the present invention is as sequence 4, the cDNA order of human interleukin 12 gene is IL12p35 such as sequence 5 wherein, IL12 p40 such as sequence 6, the cDNA order of human interferon-alpha gene is as sequence 7, the cDNA order of human interferon beta gene is as sequence 8, the cDNA order of human interferon gamma gene is as sequence 9, the cDNA order of human TNF alpha gene is as sequence 10, the cDNA order of human GM-CSF gene is as sequence 11, and the cDNA of people's cell death factor TRAIL order is as sequence 12.

Li Chuan source 1.txt

SEQUENCE?LISTING

＜110〉Lee, Chuan Yuan

Huang, pretty

＜120〉a kind of with the method for heating regulating cell factor expression and the application in oncotherapy thereof

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Li Chuan source 1.txt

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Li Chuan source 1.txt

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<400>7

atggcctcgc?cctttgcttt?actgatggtc?ctggtggtgc?tcagctgcaa?gtcaagctgc 60

tctctgggct?gtgatctccc?tgagacccac?agcctggata?acaggaggac?cttgatgctc 120

ctggcacaaa?tgagcagaat?ctctccttcc?tcctgtctga?tggacagaca?tgactttgga 180

tttccccagg?aggagtttga?tggcaaccag?ttccagaagg?ctccagccat?ctctgtcctc 240

catgagctga?tccagcagat?cttcaacctc?tttaccacaa?aagattcatc?tgctgcttgg 300

gatgaggacc?tcctagacaa?attctgcacc?gaactctacc?agcagctgaa?tgacttggaa 360

gcctgtgtga?tgcaggagga?gagggtggga?gaaactcccc?tgatgaatgc?ggactccatc 420

ttggctgtga?agaaatactt?ccgaagaatc?actctctatc?tgacagagaa?gaaatacagc 480

ccttgtgcct?gggaggttgt?cagagcagaa?atcatgagat?ccctctcttt?atcaacaaac 540

ttgcaagaaa?gattaaggag?gaaggaataa 570

Li Chuan source 1.txt

<210>8

<211>564

<212>DNA

<213>human

<400>8

atgaccaaca?agtgtctcct?ccaaattgct?ctcctgttgt?gcttctccac?tacagctctt 60

tccatgagct?acaacttgct?tggattccta?caaagaagca?gcaattttca?gtgtcagaag 120

ctcctgtggc?aattgaatgg?gaggcttgaa?tactgcctca?aggacaggat?gaactttgac 180

atccctgagg?agattaagca?gctgcagcag?ttccagaagg?aggacgccgc?attgaccatc 240

tatgagatgc?tccagaacat?ctttgctatt?ttcagacaag?attcatctag?cactggctgg 300

aatgagacta?ttgttgagaa?cctcctggct?aatgtctatc?atcagataaa?ccatctgaag 360

acagtcctgg?aagaaaaact?ggagaaagaa?gatttcacca?ggggaaaact?catgagcagt 420

ctgcacctga?aaagatatta?tgggaggatt?ctgcattacc?tgaaggccaa?ggagtacagt 480

cactgtgcct?ggaccatagt?cagagtggaa?atcctaagga?acttttactt?cattaacaga 540

cttacaggtt?acctccgaaa?ctga 564

<210>9

<211>438

<212>DNA

<213>human

<400>9

atgcaggacc?catatgtaaa?agaagcagaa?aaccttaaga?aatattttaa?tgcaggtcat 60

tcagatgtag?cggataatgg?aactcttttc?ttaggcattt?tgaagaattg?gaaagaggag 120

agtgacagaa?aaataatgca?gagccaaatt?gtctcctttt?acttcaaact?ttttaaaaac 180

tttaaagatg?accagagcat?ccaaaagagt?gtggagacca?tcaaggaaga?catgaatgtc 240

aagtttttca?atagcaacaa?aaagaaacga?gatgacttcg?aaaagctgac?taattattcg 300

gtaactgact?tgaatgtcca?acgcaaagca?atacatgaac?tcatccaagt?gatggctgaa 360

ctgtcgccag?cagctaaaac?agggaagcga?aaaaggagtc?agatgctgtt?tcgaggtcga 420

agagcatccc?agtaatga 438

<210>10

<211>702

<212>DNA

<213>human

<400>10

atgagcactg?aaagcatgat?ccgggacgtg?gagctggccg?aggaggcgct?ccccaagaag 60

acaggggggc?cccagggctc?caggcggtgc?ttgttcctca?gcctcttctc?cttcctgatc 120

gtggcaggcg?ccaccacgct?cttctgcctg?ctgcactttg?gagtgatcgg?cccccagagg 180

gaagagttcc?ccagggacct?ctctctaatc?agccctctgg?cccaggcagt?cagatcatct 240

tctcgaaccc?cgagtgacaa?gcctgtagcc?catgttgtag?caaaccctca?agctgagggg 300

cagctccagt?ggctgaaccg?ccgggccaat?gccctcctgg?ccaatggcgt?ggagctgaga 360

gataaccagc?tggtggtgcc?atcagagggc?ctgtacctca?tctactccca?ggtcctcttc 420

aagggccaag?gctgcccctc?cacccatgtg?ctcctcaccc?acaccatcag?ccgcatcgcc 480

gtctcctacc?agaccaaggt?caacctcctc?tctgccatca?agagcccctg?ccagagggag 540

Li Chuan source 1.txt

accccagagg?gggctgaggc?caagccctgg?tatgagccca?tctatctggg?aggggtcttc 600

cagctggaga?agggtgaccg?actcagcgct?gagatcaatc?ggcccgacta?tctcgacttt 660

gccgagtctg?ggcaggtcta?ctttgggatc?attgccctgt?ga 702

<210>11

<211>435

<212>DNA

<213>human

<400>11

atgtggctgc?agagcctgct?gctcttgggc?actgtggcct?gcagcatctc?tgcacccgcc 60

cgctcgccca?gccccagcac?gcagccctgg?gagcatgtga?atgccatcca?ggaggcccgg 120

cgtctcctga?acctgagtag?agacactgct?gctgagatga?atgaaacagt?agaagtcatc 180

tcagaaatgt?ttgacctcca?ggagccgacc?tgcctacaga?cccgcctgga?gctgtacaag 240

cagggcctgc?ggggcagcct?caccaagctc?aagggcccct?tgaccatgat?ggccagccac 300

tacaagcagc?actgccctcc?aaccccggaa?acttcctgtg?caacccagac?tatcaccttt 360

gaaagtttca?aagagaacct?gaaggacttt?ctgcttgtca?tcccctttga?ctgctgggag 420

ccagtccagg?agtga 435

<210>12

<211>846

<212>DNA

<213>human

<400>12

atggctatga?tggaggtcca?ggggggaccc?agcctgggac?agacctgcgt?gctgatcgtg 60

atcttcacag?tgctcctgca?gtctctctgt?gtggctgtaa?cttacgtgta?ctttaccaac 120

gagctgaagc?agatgcagga?caagtactcc?aaaagtggca?ttgcttgttt?cttaaaagaa 180

gatgacagtt?attgggaccc?caatgacgaa?gagagtatga?acagcccctg?ctggcaagtc 240

aagtggcaac?tccgtcagct?cgttagaaag?atgattttga?gaacctctga?ggaaaccatt 300

tctacagttc?aagaaaagca?acaaaatatt?tctcccctag?tgagagaaag?aggtcctcag 360

agagtagcag?ctcacataac?tgggaccaga?ggaagaagca?acacattgtc?ttctccaaac 420

tccaagaatg?aaaaggctct?gggccgcaaa?ataaactcct?gggaatcatc?aaggagtggg 480

cattcattcc?tgagcaactt?gcacttgagg?aatggtgaac?tggtcatcca?tgaaaaaggg 540

ttttactaca?tctattccca?aacatacttt?cgatttcagg?aggaaataaa?agaaaacaca 600

aagaacgaca?aacaaatggt?ccaatatatt?tacaaataca?caagttatcc?tgaccctata 660

ttgttgatga?aaagtgctag?aaatagttgt?tggtctaaag?atgcagaata?tggactctat 720

tccatctatc?aagggggaat?atttgagctt?aaggaaaatg?acagaatttt?tgtttctgta 780

acaaatgagc?acttgataga?catggaccat?gaagccagtt?ttttcggggc?ctttttagtt 840

ggctaa 846

Claims (14)

1, a kind of method with heating regulating cell factor expression is characterized in that adopting the promoter regulation immuno-stimulatory genes of thermal induction expressing gene to express, and comprises the steps:

(1) initiator sequence of separation and human cloning heat shock protein gene;

(2) clone immunostimulation/tumor-killing cell factor gene cDNA;

(3) above-mentioned immunostimulation/tumor-killing cell factor gene cDNA is placed under the heat shock protein gene promoter, make up artificial gene;

(4) above-mentioned artificial gene is inserted in virus or the non-virus carrier;

(5) be injected into above-mentioned carrier in the tumor or tumor around tissue in;

(6) tumor by local and surrounding tissue thereof are heated.

2, by the described method of claim 1, wherein said promoter is any promoter that just is activated after increasing more than 3 ℃ or 3 ℃ than normal physiological body temperature.

3, by the method for claim 1 or 2, wherein said promoter can be derived by the promoter of Hsp70B gene.

4, by the method for claim 3, wherein said promoter has the sequence of sequence 1.

5, by the process of claim 1 wherein that the described immunostimulatory cell factor comprises that interleukin II, 12,18 and 21 is as IL2, IL12,18,21; Interferon-ALPHA, β, γ (Interferons α, β, γ); Tumor necrosis factor (TNF-α), grain, giant cell colony stimulating factor (GM-CSF), and other tumor-killing gene such as TRAIL.

6, by the process of claim 1 wherein that described viral vector comprises adenovirus and slow virus, non-virus carrier comprises naked plasmid dna and liposome-plasmid dna complex compound.

7, by the process of claim 1 wherein that its mode of described heating adopts hot pad, microwave, radio frequency or local ultrasonic mode of heating.

8, by the method for claim 1 or 2 or 3, wherein said promoter adopts the local heat mode to activate.

9, by the method for claim 1 or 2 or 3, wherein said promoter adopts the focus supersonic heater to activate.

10, by the method for claim 1 or 2 or 3, wherein said promoter adopts microwave heating equipment to activate.

11, by the method for claim 1 or 2 or 3, wherein said promoter adopts radio frequency heating apparatus to activate.

12, the described purposes of method in preparation treatment solid tumor drugs of claim 1 with heating regulating cell factor expression.

13, the described purposes of method in preparation and radiotherapy combined treatment solid tumor drugs of claim 1 with heating regulating cell factor expression.

14, the described purposes of method in preparation and chemotherapy combined treatment solid tumor drugs of claim 1 with heating regulating cell factor expression.

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