CN102559753A

CN102559753A - IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application

Info

Publication number: CN102559753A
Application number: CN2011104331014A
Authority: CN
Inventors: 杨吉成; 缪竞诚; 刘济生; 盛伟华
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2011-12-21
Filing date: 2011-12-21
Publication date: 2012-07-11

Abstract

The invention relates to the field of biology, and discloses a double-gene co-expression recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM with the preservation number as China center for type culture connection number (CCTTCC NO.): M2011378, and a recombinant adenovirus Ad-IL-24-polyA-promoter-OSM prepared by the plasmid. An IL-24 gene and an OSM gene are subcloned between Ad-polyA-promoter empty carriers to construct the IL-24 and OSM double-gene co-expression recombinant plasmid, homologous recombination is performed between the recombinant plasmid and an adenovirus pAdEasy-1 to generate the recombinant adenovirus in packaging mode in a QBI-293A cell. The recombinant adenovirus has synergistic effect on inhibition of melanoma and cancer cells of nasopharyngeal darcinoma, and simultaneously has synergistic effect on radiosensitivity of the cancer cells of the nasopharyngeal darcinoma.

Description

IL-24 and OSM double gene coexpression recombinant plasmid and recombinant adenovirus and application

Technical field

The present invention relates to biological field, be specifically related to IL-24 and OSM double gene coexpression recombinant plasmid and recombinant adenovirus and application.

Background technology

In recent years, malignant melanoma of skin in the malignant tumour and nasopharyngeal carcinoma sickness rate are ascendant trend year by year.Wherein, Malignant melanoma of skin (cutaneous malignant melanoma; CMM) be the very high dermatoma of a kind of grade malignancy; Its generation often starts from those Pigmented positions, does not normally receive cell proliferation mechanism control deutero-by the living melanophore in the basal cell that is embedded in epidermis.Some nearest reports show that the sickness rate of China has the trend that increases gradually, owing to its seriousness insufficient recognition, often be late period when generally going to a doctor.And nasopharyngeal carcinoma (nasopharyngeal carcinoma NPC) is one of China's tumour occurred frequently, and Guangdong, Guangxi, Fujian, Hunan etc. are economized and to be domestic district occurred frequently.The incidence and development of nasopharyngeal carcinoma is the extremely complicated canceration process that a multifactor effect, polygene are participated in, the multistage pathology takes place, and its M & M all rises with age growth, is the significant threat of human health.

Tumour is one of principal disease of current social influence human health, the regulation and control of multiple reason in the incidence and development acceptor of tumour, and tumor suppressor gene is participated in the important molecule of these regulation and control just.The inactivation that some tumor suppressor gene is all arranged in a lot of tumours; In the cell of certain cancer suppressor gene of disappearance, import normal cancer suppressor gene; Then can reverse phenotype, inhibition tumor cell proliferation, the inducing death of neoplastic cells of tumour cell; Thereby reach therapeutic purpose, this method is referred to as gene therapy.Gene therapy has been the focus of medical circle extensive concern as the treatment means of a kind of high efficiency, specificity, target property, and appear as treatment malignant melanoma of skin and the nasopharyngeal carcinoma of gene therapy technology have indicated developing direction.

Present stage has had many pieces of articles to report the gene of relevant inhibition melanoma and nasopharyngeal carcinoma, for example interleukin II 4 (IL-24) gene and tumour inhibitor (OSM) gene.Interleukin II 4 (IL-24) is claimed melanoma differentiation correlation factor-7 (mda-7) again; Be the secretor type cytokine; Be a kind of tumor growth supressor of membrane receptor-mediated, it is that first had not only suppressed growth of tumour cell and vascularization and had induced its apoptosis simultaneously but also novel cancer suppressor gene that can immune stimulatory cell expressing cytokine.The effect that existing many up to now experiment proof IL-24 have significant inhibition tumour; Can optionally suppress the kinds of tumors growth; Inducing apoptosis of tumour cell comprises melanoma, neuroglia blastoma, osteosarcoma, mammary cancer, cervical cancer, colorectal carcinoma, lung cancer, cancer of the stomach, nasopharyngeal carcinoma, prostate cancer etc.This restraining effect does not rely on cancer suppressor genes such as p53, Rb and p16, and to not influence of normal cell.

Tumour inhibitor (OSM) is one of member of IL-6 cytokine family; It is a kind of multi-functional cytokine; Have the regulatory gene activity, regulate cell proliferation and differentiation, regulate immune, participate in inflammatory reaction, reinvent extracellular matrix, multiple functions such as hematopoiesis, neural system protection; In addition, OSM can also produce restraining effect to kinds of tumor cells.

Above-mentioned cancer suppressor gene is carried transfection or target cell infection through carrier, just can realize gene therapy malignant melanoma of skin and nasopharyngeal carcinoma, reach the effect that suppresses tumour cell.Application carrier can be divided into two types in the gene therapy: non-virus-type and virus-type.Virus-mediated method transfection efficiency is high in the two, and destination gene expression is stable, in existing gene therapy scheme, accounts for the overwhelming majority.The virus-type carrier commonly used that is used for gene therapy at present has retroviral vector, adenovirus carrier, adeno-associated virus (AAV) carrier, herpes simplex virus vector and chimeric vectors etc.And adenovirus carrier as well known to those skilled in the art and other virus vector are comparatively speaking, have many good qualities, as 1, host range is wide, and is pathogenic low to the people; 2, have the infection rate height, to the division and Unseparated Cell can both infect; 3, can carry very long therapeutic gene and express a plurality of genes simultaneously; 4, unconformability does not have the mutagenicity of insertion in karyomit(e), and security is good; 5, can effectively breed, titre is high, is prone to processing and preparing etc.And the pAdEasy gland virus expression system in the employing adenovirus carrier has more many special benefits: 1. the homologous recombination of adenoviral plasmid can efficiently be carried out in intestinal bacteria BJ5183; And bacterial reproduction is fast; The homologous recombination ability is strong, thereby fundamentally overcomes the low defective of homologous recombination rate in the cell; 2. can insert the gene fragment of 11kb in the adenovirus or insert a plurality of gene fragments simultaneously; 3. owing to the GFP reporter gene that in the adenovirus skeleton, contains, be convenient to very much monitor virus packets and whether dress up merit, detect virus titer and efficiency of infection etc.All these has avoided viral plaque clone purification process, has significantly reduced viral preparation time.

Though plurality of advantages is arranged through gene therapy malignant melanoma of skin and nasopharyngeal carcinoma; But be that the restraining effect to tumour cell is not high behind carrier homologous recombination IL-24 gene or the OSM gene with the adenovirus at present, limited the application of gene therapy technology in malignant melanoma of skin and nasopharyngeal carcinoma.

Summary of the invention

In view of this; The homologous recombination adenovirus that the purpose of this invention is to provide a kind of double gene coexpression recombinant plasmid and utilize this recombinant plasmid preparation; Make said homologous recombination adenovirus have synergism to the tumour cell that suppresses melanoma and nasopharyngeal carcinoma, the radio therapy sensitization to the nasopharyngeal carcinoma tumour cell has synergism simultaneously.

For realizing the foregoing invention purpose, the present invention provides following technical scheme:

A kind of IL-24 and OSM double gene coexpression recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM, its preserving number is CCTCC NO:M2011378.

The gene IL-24 of said double gene coexpression recombinant plasmid and gene OSM are open by Genebank, and the gene IL-24 nucleotide sequence that wherein the present invention introduced is shown in SEQ ID NO:1, and gene OSM nucleotide sequence is shown in SEQ ID NO:2.

The recombinant adenovirus plasmid pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM that the present invention also provides said double gene coexpression recombinant plasmid and adenovirus pAdEasy-1 homologous recombination to obtain.

The present invention also provides said double gene coexpression recombinant plasmid and adenovirus pAdEasy-1 homologous recombination, and the recombinant adenovirus Ad-IL-24-polyA-promoter-OSM that packing produces in packing cell (below be abbreviated as Ad-IL-24-OSM).Wherein, said packing cell is preferably QBI-293A.

The homologous recombination adenovirus problem not high to the single-gene recombinant expression acquisition that utilizes IL-24 or OSM to the tumour cell restraining effect of melanoma and nasopharyngeal carcinoma; The present invention is based on and between IL-24 and OSM are dual-gene, insert the conception that polyA-promotor (CMV) double-promoter expression cassette makes up the double gene coexpression carrier; On the basis of the pAdTrack-CMV-polyA-promoter transferring plasmid (plasmid map is seen Fig. 1) that the applicant successfully makes up; Between Bgl II, Sal I restriction enzyme site, insert the IL-24 fragment; Between Not I, Xho I restriction enzyme site, insert OSM fragment (see figure 2), construct double gene coexpression recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM according to the invention.

Double gene coexpression recombinant plasmid according to the invention shows through PCR and double digestion evaluation, by the IL-24 and the success of OSM double gene coexpression construction of recombinant plasmid of polyA-promoter mediation.In addition; RT-PCR and Western blot qualification result show; Recombinant adenovirus Ad-IL-24-OSM according to the invention can mediate exogenous IL-24 and OSM gene transcription and expression, shows to utilize double gene coexpression recombinant plasmid according to the invention successfully to obtain IL-24 and OSM double gene coexpression recombinant adenovirus.

Double gene coexpression recombinant plasmid according to the invention will have polyA-promotor (CMV) separately two expression cassettes of independent startup be implemented on the plasmid; IL-24 and OSM be dual-gene independently to transcribe out two kinds of different mRNA separately; In recombinant adenovirus, independently translate different albumen separately; These two kinds of albumen synergies increase the effect that suppresses melanoma and nasopharyngeal carcinoma tumour cell each other, and increase the expression amount of relevant short antiapoptotic factors, have significant synergies.

The present invention also provides said double gene coexpression recombinant plasmid and the application of recombinant adenovirus in preparation treatment malignant melanoma of skin and medicine for nasopharyngeal.

In each item test of vitro inhibition melanoma and nasopharyngeal carcinoma tumour cell; Recombinant adenovirus Ad-IL-24-OSM according to the invention makes an experiment to two kinds of tumour cells under the prerequisite of identical infection multiplicity with the homologous recombination adenovirus (hereinafter to be referred as Ad-IL-24 and Ad-OSM) that the single-gene recombinant expression of utilizing IL-24 or OSM obtains, and the result shows that Ad-IL-24-OSM suppresses the activity of the activity of growth of tumour cell apparently higher than the inhibition tumour cell of Ad-IL-24 and Ad-OSM.

Suppress in vivo in the animal model test of melanoma and nasopharyngeal carcinoma tumour cell; The present invention is through injecting the trial model that the tumour cell suspension is set up mice with tumor to nude mice; Guaranteeing under the identical infection multiplicity prerequisite mice with tumor model to be injected Ad-IL-24-OSM, Ad-IL-24 and Ad-OSM respectively then; Calculate tumour inhibiting rate through tumor weight, the result shows the tumour inhibiting rate of Ad-IL-24-OSM tumour inhibiting rate apparently higher than Ad-IL-24 and Ad-OSM, has the knurl synergism of pressing down.

Simultaneously; Nasopharyngeal carcinoma CNE-2Z cell and external melanoma A375 cell are detected the wherein relevant expression of urging apoptogene and suppressing apoptogene through the RT-PCR method; The result shows; The expression amount of the relevant short apoptogene in the tumour cell of injection Ad-IL-24-OSM is apparently higher than the expression amount of injection Ad-IL-24 and Ad-OSM; And the expression amount of anti-apoptotic genes expression obviously reduces; This and above-mentioned test-results external, the interior inhibition of body tumour cell match, and have shown that fully recombinant adenovirus Ad-IL-24-OSM according to the invention has synergism to the tumour cell that suppresses melanoma and nasopharyngeal carcinoma, can apply it in preparation treatment malignant melanoma of skin and the medicine for nasopharyngeal and go.

In addition, the present invention also provides said double gene coexpression recombinant plasmid and the application of recombinant adenovirus in preparation nasopharyngeal carcinoma radiotherapy sensitizer.Wherein, the ray of said radiotherapy is preferably cobalt 60 gamma-rays.

It is more hidden that nasopharyngeal carcinoma is grown in pharynx nasalis, its position at nasal cavity rear, and early stage normal non-evident sympton is out in the cold easily.And the only radiosensitive part tumour of independent radiotherapy has reasonable curative effect, but far the radiation resistance of the tumour cell of MET is higher for the primary lesion of nasopharyngeal carcinoma and nodus lymphoideus transferring rate kitchen range or hiding property.Therefore, in radiotherapy, need utilize radiotherapeutic sensitizer to reduce the radiation resistance of these tumours,, improve radiotherapeutic effect so that remove tumour cell comparatively completely.

Be in the not tumour cell of phase simultaneously of mitotic cycle, its radiosensitivity has very big difference, and is the most responsive with the cell in M phase and G2 later stage.And recombinant adenovirus Ad-IL-24-OSM according to the invention is in each item test of above-mentioned vitro inhibition melanoma and nasopharyngeal carcinoma tumour cell; Singly dye its influence of detection through PI to the nasopharyngeal carcinoma tumour cell cycle; The result shows; The nasopharyngeal carcinoma tumour cell cycle that adds recombinant adenovirus Ad-IL-24-OSM according to the invention obviously retardance occurs in the G2/M phase; And be in the tumor cell number of the tumor cell number in this period, show that it can be as the nasopharyngeal carcinoma radiotherapy sensitizer apparently higher than injection Ad-IL-24 and Ad-OSM

Simultaneously; Utilize nasopharyngeal carcinoma tumour cell nude mice model to inject interior, the in vitro tests of each item body of Ad-IL-24-OSM/Ad-IL-24/Ad-OSM+ radiotherapy; The result shows; Injected the test group of Ad-IL-24-OSM and radiotherapy; Its suppress tumour cell activity, relevant short apoptogene and proteic expression amount, radiation sensitivity etc. all be higher than Ad-IL-24+ radiotherapy test group, Ad-OSM+ radiotherapy test group, test-results shows that further recombinant adenovirus Ad-IL-24-OSM according to the invention can be applied to prepare in the nasopharyngeal carcinoma radiotherapy sensitizer.

Can know by above technical scheme; The present invention goes into IL-24 gene and gene constructed IL-24 of OSM and OSM double gene coexpression recombinant plasmid through subclone between the Ad-polyA-promoter empty carrier, and in the QBI-293A cell, packs generation Ad-IL-24-OSM recombinant adenovirus with adenovirus pAdEasy-1 homologous recombination.Said Ad-IL-24-OSM recombinant adenovirus has the knurl synergism of pressing down to the tumour cell of inhibition melanoma and nasopharyngeal carcinoma and the growth of transplanted tumor; In radiotherapy, have radio therapy sensitization and synergism simultaneously, can improve radiotherapeutic effect suppressing nasopharyngeal carcinoma tumour cell and growth of xenografted.

The explanation of biomaterial preservation information:

The pAdTrack-CMV-IL-24-polyA-promoter-OSM plasmid; Be preserved in Chinese typical culture collection center on November 7th, 2011; The address is Chinese Wuhan University; Deposit number is CCTCC NO:M 2011378, classification called after bacillus coli DH 5 alpha/pAdTrack-CMV-IL-24-polyA-promoter-OSM, Escherichia coli DH5 α/pAdTrack-CMV-IL-24-polyA-promoter-OSM.

Description of drawings:

Fig. 1 shows AdTrack-CMV-polyA-promoter plasmid map according to the invention;

Fig. 2 shows IL-24 and OSM double gene coexpression construction of recombinant plasmid synoptic diagram;

Fig. 3 shows the electrophoresis evaluation figure of IL-24 gene among the pAdTrack-CMV-IL-24-polyA-promoter-OSM;

A is the PCR evaluation figure of IL-24 gene, and M is 2000bp Marker, and 1 is IL-24 band swimming lane;

B is the double digestion evaluation figure of IL-24 gene, and M is 2000bp Marker, and 1 is IL-24 band swimming lane;

Fig. 4 shows the electrophoresis evaluation figure of OSM gene among the pAdTrack-CMV-IL-24-polyA-promoter-OSM;

A is the PCR evaluation figure of OSM gene, and M is 2000bp Marker, and 1 is OSM band swimming lane;

B is the double digestion evaluation figure of OSM gene, and M is 2000bp Marker, and 1 is OSM band swimming lane;

Fig. 5-A shows that IL-24 and the RT-PCR of OSM list/dual-gene recombinant adenovirus in melanoma A375 cell transcribe evaluation figure;

1 is the PBS group; 2 are the Ad-GFP group; 3 are the Ad-IL-24 group; 4 are the Ad-OSM group; 5 are the Ad-IL-24-OSM group; 6 is 2000bp Marker, is followed successively by 2000,1000,750,500 from top to bottom, 250bp;

Fig. 5-B shows IL-24 and the OSM list/dual-gene recombinant adenovirus Western blot evaluation figure in melanoma A375 cell;

A is the OSM protein band; B is the IL-24 protein band; C is confidential reference items β-actin;

1 is the Ad-IL-24 group; 2 are the Ad-IL-24-OSM group; 3 are the Ad-OSM group; 4 are the Ad-GFP group; 5 are the PBS group;

Fig. 6-A shows that IL-24 and the RT-PCR of OSM list/dual-gene recombinant adenovirus in nasopharyngeal carcinoma CNE-2Z cell transcribe evaluation figure;

Fig. 6-B shows IL-24 and the OSM list/dual-gene recombinant adenovirus Western blot evaluation figure in nasopharyngeal carcinoma CNE-2Z cell;

1 is the PBS group; 2 are the Ad-GFP group; 3 are the Ad-IL-24 group; 4 are the Ad-OSM group; 5 are the Ad-IL-24-OSM group;

Fig. 7 shows that mtt assay detects the column diagram of Ad-IL-24-OSM vitro inhibition melanoma A375 cell growth rate;

Ordinate zou is a percentage, and X-coordinate is a fate, and 4 groups of column diagrams are respectively Ad-GFP group, Ad-OSM group, Ad-IL-24 group, Ad-IL-24-OSM group from left to right;

Fig. 8 shows that mtt assay detects the broken line graph of Ad-IL-24-OSM vitro inhibition nasopharyngeal carcinoma CNE-2Z cell growth;

The a broken line is the PBS group; The b broken line is the Ad-GFP group; The c broken line is the Ad-OSM group; The d broken line is the Ad-IL-24 group; The e broken line is the Ad-IL-24-OSM group;

Ordinate zou is the OD value, and X-coordinate is fate (being 24h on the corresponding broken line, 48h, 72h, four time points of 96h), and Δ is represented P＜0.05, and * representes P＜0.01;

Fig. 9 shows that flow cytometer detects the column diagram that the melanoma A375 cell cycle changes;

Ordinate zou cell quantity per-cent, X-coordinate are the cell cycle, and 5 kinds of column diagrams are followed successively by PBS group, Ad-GFP group, Ad-IL-24 group, Ad-OSM group, Ad-IL-24-OSM group from left to right;

Figure 10 shows that PI singly dyes and detects flow cytometer that the nasopharyngeal carcinoma CNE-2Z cell cycle changes figure as a result;

Ordinate zou is a cell quantity, and X-coordinate is hour;

Figure 11 shows that flow cytometer detects the column diagram of melanoma A375 apoptosis rate;

Figure 12 shows that flow cytometer detects the column diagram of nasopharyngeal carcinoma CNE-2Z apoptosis rate, and Δ is represented P＜0.05, and * representes P＜0.01;

What Figure 13 showed that RT-PCR detects that Ad-IL-24-OSM infects genes involved behind the melanoma A375 cell transcribes the variation electrophorogram;

What Figure 14 showed that RT-PCR detects that Ad-IL-24-OSM infects genes involved behind the nasopharyngeal carcinoma CNE-2Z cell transcribes the variation electrophorogram;

Figure 15 shows that Ad-IL-24-OSM suppresses the knurl body weight column diagram of nude mice melanoma A375 Transplanted cells knurl;

Figure 16 shows the tumour inhibiting rate column diagram of Ad-IL-24-OSM to nude mice melanoma A375 Transplanted cells knurl;

Figure 17 shows that Ad-IL-24-OSM suppresses the knurl body weight column diagram of nude mice nasopharyngeal carcinoma CNE-2Z Transplanted cells knurl; Δ is represented P＜0.05, and * representes P＜0.01;

Figure 18 shows the tumour inhibiting rate column diagram of Ad-IL-24-OSM to nude mice nasopharyngeal carcinoma CNE-2Z Transplanted cells knurl; * represent P＜0.05;

Figure 19 shows that mtt assay detects the broken line graph of Ad-IL-24-OSM combined radiotherapy vitro inhibition nasopharyngeal carcinoma CNE-2Z cell growth;

The a broken line is the PBS group; The b broken line is the Ad-GFP group; The c broken line is a combination radiotherapy group; The d broken line is the Ad-IL-24+ combination radiotherapy group; The e broken line is the Ad-OSM+ combination radiotherapy group; The f broken line is the Ad-IL-24-OSM+ combination radiotherapy group;

4 time points are followed successively by 24h, 48h, 72h, 96h on the broken line, corresponding X-coordinate fate, and Δ is represented P＜0.05, * representes P＜0.01;

Figure 20-A and Figure 20-B show that PI singly dyes and detect flow cytometer that the nasopharyngeal carcinoma CNE-2Z cell cycle changes figure (combined radiotherapy) as a result;

Figure 21 shows that flow cytometer detects the column diagram of Ad-IL-24-OSM combined radiotherapy to nasopharyngeal carcinoma CNE-2Z apoptosis rate, and Δ is represented P＜0.05, and * representes P＜0.01;

What Figure 22 showed that RT-PCR detects that the Ad-IL-24-OSM combined radiotherapy infects genes involved behind the nasopharyngeal carcinoma CNE-2Z cell transcribes the variation electrophorogram;

1 is the PBS group; 2 are the Ad-GFP group; 3 is combination radiotherapy group; 4 is the Ad-IL-24+ combination radiotherapy group; 5 is the Ad-OSM+ combination radiotherapy group; 6 is the Ad-IL-24-OSM+ combination radiotherapy group;

The Ad-IL-24-OSM combined radiotherapy of showing Figure 23 suppresses the knurl body weight column diagram of nude mice nasopharyngeal carcinoma CNE-2Z Transplanted cells knurl; Δ is represented P＜0.05, and * representes P＜0.01;

Figure 24 shows the tumour inhibiting rate column diagram of Ad-IL-24-OSM combined radiotherapy to nude mice nasopharyngeal carcinoma CNE-2Z Transplanted cells knurl; * represent P＜0.05.

Embodiment:

The invention discloses a kind of IL-24 and OSM double gene coexpression recombinant plasmid and recombinant adenovirus and application, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Plasmid of the present invention, recombinant adenovirus and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.

Below in conjunction with embodiment, further set forth the present invention, related testing data adopts SPSS16.0 statistics software to carry out one-way analysis of variance among the embodiment, and P＜0.05, P＜0.01 are regarded as statistics there were significant differences.In addition; The recombinant adenovirus that all tests add such as undeclared is and adopts best infection multiplicity (promptly the best infection multiplicity to melanoma A375 cell is 100MOI; Best infection multiplicity to nasopharyngeal carcinoma CNE-2Z cell is 50MOI), each test group add-on is consistent simultaneously.

The collection of illustrative plates of embodiment 1:pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression recombinant plasmid

At application number is the open polyA of 200810244301.3 Chinese invention patent application specifications ^{Δ 296～298}-promoter (being called for short polyA-promoter) sequence.Double gene coexpression recombinant plasmid according to the invention is on the basis of pAdTrack-CMV-polyA-promoter transferring plasmid; Between Bgl II, Sal I restriction enzyme site, insert the IL-24 fragment; Between Not I, Xho I restriction enzyme site, insert the OSM fragment, said pAdTrack-CMV-polyA-promoter plasmid map is seen Fig. 1.

The clone of embodiment 2:IL-24 and OSM goal gene

The conserved sequence of comparison IL-24, design upstream primer IL-24-F, downstream primer IL-24-R (seeing table 1), two ends are introduced Bgl II, Sal I restriction enzyme site respectively; With the applicant made up to contain the segmental pAdTrack-CMV-IL-24-IRES plasmid of IL-24 be template, the IL-24 purpose fragment that pcr amplification obtains is identified with the expection size consistent through agarose electrophoresis.

The conserved sequence of comparison OSM; Design upstream primer OSM-F, downstream primer OSM-R (seeing table 1); Two ends are introduced Not I, Xho I restriction enzyme site respectively; With the applicant made up to contain the segmental pAdTrack-CMV-polyA-promoter-OSM plasmid of OSM be template, the OSM purpose fragment that pcr amplification obtains is identified with the expection size consistent through agarose electrophoresis.

Table 1 pcr amplification primer

Embodiment 3:pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression construction of recombinant plasmid

1, pAdTrack-CMV-IL-24-polyA-promoter single-gene construction of recombinant plasmid

After will using Bgl II, 37 ℃ of double digestion 5h of Sal I respectively by the transferring plasmid pAdTrack-CMV-polyA-promoter that the PCR product of the IL-24 gene of DNA cleaning agents box purifying and plasmid extraction test kit in a small amount extract; The purpose fragment is reclaimed in rubber tapping; Press test kit glue recovery method and procedure; With the T4DNA ligase enzyme it is spent the night 4 ℃ of connections then, will connect product transformed into escherichia coli DH5 α then again, and in containing Kana (50 μ g/ml) resistant panel, select positive monoclonal; After PCR, double digestion evaluation and determined dna sequence were identified, positive colony bacterium-20 a ℃ refrigerator guarantor planted subsequent use.

CaCl ₂Legal system is equipped with DH5 α competent escherichia coli cell and the transformation technology route is following:

Transform previous day; DH5 α is by 37 ℃ of overnight cultures of 1% inoculation 5ml LB substratum; Change kind of 37 ℃ of cultivations of 5ml LB substratum by 1%, be about 0.2 (cultivating 1.3h approximately) to OD600nm, pour bacterium liquid in 5ml centrifuge tube 5000r/min; Centrifugal 5min collects thalline, adds the 100mM CaCl of 5ml precooling ₂, behind the suspension thalline 0 ℃, behind the 20-30min, 5000r/min, centrifugal 5min collect thalline with about 600 μ l 100mM precooling CaCl ₂The suspension thalline; And according to the form below transforms; And put 37 ℃ of cultivations of water isolation type constant incubator 16h and choose the positive colony evaluation, promptly obtaining pAdTrack-CMV-IL-24-polyA-promoter single-gene recombinant plasmid, plasmid extraction, PCR and double digestion authentication method are the same.

The conversion group	Recipient bacterium	DNA	Culture plate
				Positive group	60μl		1 μ l plasmid	Kana
Negative group	60μl	--	Kana
				Experimental group	60μl	20 μ l connect liquid	Kana

2, pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression construction of recombinant plasmid

On the basis of the pAdTrack-CMV-IL-24-polyA-promoter single-gene recombinant plasmid that clone's OSM gene and embodiment 2 make up in embodiment 2; Will be through Not I, Xho I double digestion and the OSM gene fragment and the pAdTrack-CMV-IL-24-polyA-promoter plasmid fragment that reclaim through glue; Connect with the T4DNA ligase enzyme, make up pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression recombinant plasmid.Conversion, plasmid extraction, PCR and double digestion authentication method are the same.

The dual-gene recombinant transfer plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM of above-mentioned structure has been transformed on November 7th, 2011 and has been preserved in Chinese typical culture collection center in the bacillus coli DH 5 alpha, and deposit number is CCTCC NO:M 2011378.

Embodiment 4:pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression recombinant plasmid evaluation

Recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM with above-mentioned preservation is a template; IL-24-F, IL-24-R are that primer PCR can amplify the IL-24 target gene fragment; Its size and IL-24 theoretical value size (621bp) of expection amplification are consistent (sees Fig. 3-A); With OSM-F, OSM-R is that primer PCR can amplify the OSM target gene fragment (see Fig. 4-A), its size is consistent with OSM theoretical value size (750bp) of expection amplification.Cut the IL-24 fragment that discharges 621bp size through Bgl II, Sal I enzyme and (see Fig. 3-B); Not I, Xho I enzyme are cut the OSM fragment that discharges 750bp size and (are seen Fig. 4-B); The above results shows that all IL-24 and OSM target gene fragment successfully are subcloned on pAdTrack-CMV-polyA-promoter transformation idle running and move in the plasmid, has successfully made up pAdTrack-CMV-IL-24-polyA-promoter-OSM double gene coexpression recombinant plasmid.

Embodiment 5: packing, the amplification of the structure of homologous recombination adenoviral plasmid according to the invention and homologous recombination adenovirus

1, the structure of homologous recombination adenoviral plasmid pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM

With the pAdTrack-CMV-IL-24-ployA-promoter-OSM recombinant plasmid with Pme I single endonuclease digestion 2h linearizing after; Glue reclaims with the pAdEasy-1 adenovirus carrier and (is so kind as to give by professor Zhong Jiang of Fudan University; Be record in the 200810244301.3 China inventions at application number) with Calcium Chloride Method cotransformation BJ5183 competence, the LB card is received resistant panel and is selected mono-clonal, takes out plasmid, and enzyme is cut the evaluation positive colony; Obtain pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM homologous recombination adenoviral plasmid; Transform DH5 α competence bacterium, picking and a large amount of amplification positive colony, extracting plasmid.

2, the packing of homologous recombination adenovirus, amplification

To make up pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM homologous recombination adenoviral plasmid after Pac I enzyme linearizing enzyme is cut, big fragment is reclaimed in rubber tapping, presses Lipofecta-mine ^TM2000 operation instructionss are packed through Lipofecta-min liposome transfection QBI-293A cell, the expression of the down visible GFP of fluorescent microscope, and fluorescence intensity prolongs enhancing gradually with incubation time.Infect the QBI-293A cell again after the results first-generation adenovirus behind the transfection 10d, through too much wheel infection and amplification, the recombinant adenovirus Ad-IL-24-polyA-promoter-OSM of results (being called for short Ad-IL-24-OSM) is for use in-80 ℃ of preservations.

3, the homologous recombination adenovirus detection of tiring

With Ad-IL-24-OSM homologous recombination adenovirus infection QBI-293A cell, after 0.25% trysinization, PBS adjusts cell concn to 1 * 10 ⁵/ ml is inoculated in cell on 96 orifice plates by 100 μ l/ holes, in 37 ℃, 5%CO ₂After cultivating 24h in the cell culture incubator, with the homologous recombination adenovirus respectively with 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8Dilution, three multiple holes are respectively established in 100 μ l/ holes, continue to cultivate.Under fluorescent microscope, carry out the fluorescence counting behind the 18h, by formula: virus titer (pfu/m1)=(fluorescence number * 10)/extent of dilution calculates virus titer.After many wheels infected, increase, final acquisition can reach 5 * 10 ⁹The pfu/ml virion of tiring is subsequent use in-80 ℃ of preservations.

Embodiment 6: preparation IL-24 and two kinds of single-gene homologous recombination of OSM adenovirus

For in follow-up each item body, the contrast of in vitro tests; Method according to embodiment 3; The double digestion that adopts IL-24 and two kinds of gene pairss of OSM to answer; Being connected to pAdTrack-CMV-polyA-promoter goes up between Bgl II, Sal I restriction enzyme site and Not I, the XhoI restriction enzyme site; Construct single-gene recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter and pAdTrack-CMV-polyA-promoter-OSM, conversion, plasmid extraction, PCR and double digestion authentication method are with embodiment 3.

With the pAdTrack-CMV-IL-24-polyA-promoter of above-mentioned structure and pAdTrack-CMV-polyA-promoter-OSM method preparation single-gene homologous recombination adenovirus Ad-IL-24-polyA-promoter (being designated hereinafter simply as Ad-IL-24), Ad-polyA-promoter-OSM (being designated hereinafter simply as Ad-OSM) separately according to embodiment 5.

Embodiment 7: recombinant adenovirus is to the best infection multiplicity of melanoma A375 cell and nasopharyngeal carcinoma CNE-2Z cell

1, recombinant adenovirus is to the best infection multiplicity of melanoma A375 cell

It is 10 that the A375 cell preparation that will be in logarithmic phase becomes concentration ⁵The cell suspension of/ml is inoculated on 96 orifice plates by 100 μ l/ holes, continues to cultivate.Behind the 24h with Ad-GFP (empty plasmid adenovirus) with 1,10,25,50,100,150, the dosage of 200MOI infects the A375 cell.Establish 5 multiple holes, continue to cultivate for every group.The expression of green fluorescence (GFP) under the morphological change of observation A375 cell and the fluorescent microscope behind the 48h; The infective dose minimum with toxicity, that efficiency of infection is the highest is judged the best infection multiplicity MOI of recombinant adenovirus to the A375 cell; Through detecting, recombinant adenovirus is 100MOI to the best infection multiplicity of A375 cell.

2, recombinant adenovirus is to the best infection multiplicity of nasopharyngeal carcinoma CNE-2Z cell

It is 10 that the CNE-2Z cell preparation that will be in logarithmic phase becomes concentration ⁵The cell suspension of/ml is by 10 ⁴/ 100 μ l/ holes are inoculated on 96 orifice plates, continue to cultivate.Behind the 24h with Ad-GFP (empty plasmid adenovirus) with 1,10,25,50,100,150, the dosage of 200MOI infects the CNE-2Z cell.Establish 5 multiple holes, continue to cultivate for every group.The expression of green fluorescence (GFP) under the morphological change of observation CNE-2Z cell and the fluorescent microscope behind the 72h; The infective dose minimum with toxicity, that efficiency of infection is the highest is judged the best infection multiplicity MOI of recombinant adenovirus to the CNE-2Z cell; Through detecting, recombinant adenovirus is 50MOI to the best infection multiplicity of CNE-2Z cell.

Embodiment 8: the RT-PCR of each recombinant adenovirus and Western blot identify

1, IL-24 and OSM gene and proteic expression in the melanoma A375 cell

Press the best infection multiplicity of measuring among the embodiment 7 and infect the A375 cell; Collect the A375 cell that infects through Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP (empty plasmid adenovirus), PBS (negative control) respectively; The total RNA that extracts separately carries out RT-PCR; With IL-24-F, IL-24-R, OSM-F, OSM-R is that primer is identified IL-24, OSM gene transcribing in the A375 cell respectively, and the visible Ad-IL-24-OSM of result, Ad-IL-24, each group of Ad-OSM all can produce 621bp IL-245 fragment and the 750bp OSM fragment of expection size and (see Fig. 5-A); Two groups of Ad-GFP and PBS all do not produce above-mentioned band in the corresponding position.The RT-PCR qualification result tentatively shows IL-24 and OSM double gene coexpression recombinant adenovirus and each the single-gene recombinant adenovirus that has successfully made up by polyA-promoter mediation, and IL-24, OSM single-gene and dual-gene all can successfully transcribe in the A375 cell.

Best infection multiplicity infection to measure among the embodiment 7 is infected the A375 cell respectively with each recombinant adenovirus.Cell is respectively organized in collection after cultivating 72h, adds cell pyrolysis liquid (by 10 ⁷Cell: the ratio of 1ml cell pyrolysis liquid), get the total protein supernatant; The albumen supernatant is with 4: 1 ratio and 5 * SDS albumen sample-loading buffer mixing, carries out SDS-PAGE electrophoresis (separation gel, concentrate gum concentration be respectively 12%, 5%), changes film albumen is transferred on the pvdf membrane by gel; Pvdf membrane is with after the 5% skim-milk sealing, use mouse-anti human IL-2 4, mouse-anti people OSM and mouse-anti people β-actin antibody diluent effect respectively after, PBS washes film 3 times, adds corresponding two anti-diluents more respectively and makes its effect; At last pvdf membrane is fully contacted with luminous working fluid, incubated at room, compressing tablet exposure, development and photographic fixing in the darkroom (are seen Fig. 5-B).The result is visible, and Ad-IL-24, Ad-OSM group infect the A375 cell and can produce respectively and mouse-anti human IL-2 4 antibody and mouse-anti people OSM antibodies specific bonded band; Ad-IL-24-OSM infects the A375 cell and then can produce and mouse-anti human IL-2 4 antibody and mouse-anti people OSM antibodies specific bonded band simultaneously; Above-mentioned band does not all appear in empty infection group and viral infection group of Ad-GFP and PBS control group in the corresponding position.Western blot qualification result further shows IL-24 and OSM double gene coexpression recombinant adenovirus and each the single-gene recombinant adenovirus that has successfully made up by polyA-promoter mediation, IL-24, OSM single-gene and dual-gene all can be in the A375 cell successful expression albumen.

2, IL-24 and OSM gene and proteic expression in the nasopharyngeal carcinoma CNE-2Z cell

Detect according to the RT-PCR method in 1, the visible Ad-IL-24-OSM of result, Ad-IL-24, each group of Ad-OSM all can produce 621bp IL-245 fragment and the 750bp OSM fragment of expection size and (see Fig. 6-A); Two groups of Ad-GFP and PBS all do not produce above-mentioned band in the corresponding position.The RT-PCR qualification result tentatively shows IL-24 and OSM double gene coexpression recombinant adenovirus and each the single-gene recombinant adenovirus that has successfully made up by polyA-promoter mediation, and IL-24, OSM single-gene and dual-gene all can successfully transcribe in the CNE-2Z cell.

Western blot method according in 1 detects, and the result is visible (to see that Fig. 6-B), Ad-IL-24, Ad-OSM group infect the A375 cell and can produce respectively and mouse-anti human IL-2 4 antibody and mouse-anti people OSM antibodies specific bonded band; Ad-IL-24-OSM infects the A375 cell and then can produce and mouse-anti human IL-2 4 antibody and mouse-anti people OSM antibodies specific bonded band simultaneously; Above-mentioned band does not all appear in empty infection group and viral infection group of Ad-GFP and PBS control group in the corresponding position.Western blot qualification result further shows IL-24 and OSM double gene coexpression recombinant adenovirus and each the single-gene recombinant adenovirus that has successfully made up by polyA-promoter mediation, IL-24, OSM single-gene and dual-gene all can be in the CNE-2Z cell successful expression albumen.

Embodiment 9:MTT method detects the external antitumor activity of Ad-IL-24-OSM

1, mtt assay detects the vitro inhibition melanoma A375 cell activity of Ad-IL-24-OSM

To be in the A375 cell of logarithmic phase, by 5 * 10 ³Individual/hole is inoculated in 96 orifice plates, and 37 ℃, 5%CO ₂Test group adds 100MOIAd-IL-24, Ad-OSM, Ad-IL-24-OSM virus liquid respectively after cultivating 24h, and empty viral negative control group adds 100MOI Ad-GFP, and the PBS control group adds the RPMI-1640 substratum, establishes 5 multiple holes, 37 ℃, 5%CO for every group ₂Hatch under the condition.Add MTT (5mg/ml) 10 μ l/ holes respectively at 0d, 1d, 2d, 3d, 4d subsequently, add solvating agent [10%SDS+1%HCl (1mol/L)] 100 μ l/ holes after continuing to hatch 4-6h, after the crystallization of Dai Jia Za is dissolved fully to next day, on enzyme-linked immunosorbent assay instrument, survey OD ₅₇₀Value is drawn growth curve, and by inhibitory rate of cell growth=[(control group OD ₅₇₀Value-test group OD ₅₇₀Value)/control group OD ₅₇₀Value] * 100% calculating inhibitory rate of cell growth, the result sees Fig. 7.

Can know by the result; Ad-IL-24, Ad-OSM, each test group of Ad-IL-24-OSM and Ad-GFP group and PBS control group are relatively; The A375 cell there is the obvious suppression effect; Wherein Ad-IL-24-OSM obviously is superior to Ad-OSM (34.3%), Ad-IL-24 (31.6%) single-gene recombinant adenovirus (P＜0.05) to the growth inhibition ratio (57%) of A375 cell, has the cancer synergism of pressing down to suppressing the growth of A375 MC.

2, mtt assay detects the vitro inhibition nasopharyngeal carcinoma CNE-2Z cell activity of Ad-IL-24-OSM

It is 10 that the CNE-2Z cell preparation of logarithmic phase is become concentration ⁵The cell suspension of/ml is pressed cell 10 ⁴/ 100 μ l/ holes are inoculated on 96 orifice plates, and Ad-IL-24, Ad-OSM, each test group of Ad-IL-24-OSM and Ad-GFP group and PBS control group are established 3 multiple holes for every group.Abandon supernatant after cultivating 24h; Each group cell is handled according to the method described above, continued respectively subsequently to cultivate, add MTT (5mg/ml) 10 μ l/ holes in 24h, 48h, 72h, 96h; Add solvating agent [10%SDS+1%HCl (1mol/L)] 100 μ l/ holes after continuing to cultivate 5h, survey OD next day ₅₇₀Value, and by formula calculate inhibitory rate of cell growth, growth inhibition ratio=[(control group OD ₅₇₀Value-test group OD ₅₇₀Value)/control group OD ₅₇₀Value] * 100%, the result sees Fig. 8.

Measure the OD that respectively organizes cell respectively at 24h, 48h, 72h, 96h ₅₇₀Value, and draw growth curve (Fig. 8).The result shows; Ad-IL-24, Ad-OSM, Ad-IL-24-OSM all have growth-inhibiting effect in various degree to the CNE-2Z cell; And be time-dependent manner (the most obvious) in 96h; Inhibiting rate is respectively (23.96 ± 1.35) %, (23.01 ± 1.92) %, (32.57 ± 3.36) % through calculating; With PBS group and Ad-GFP group significant difference (P＜0.01) is arranged more all, wherein the Ad-IL-24-OSM group has significant difference (P＜0.01) than Ad-IL-24 group, Ad-OSM group, has the cancer synergism of pressing down.

Embodiment 10:PI singly dyes flow cytometer and detects the cell cycle influence of Ad-IL-24-OSM to melanoma A375 cell and nasopharyngeal carcinoma CNE-2Z cell

1, PI singly dyes the influence of flow cytometer detection Ad-IL-24-OSM to the melanoma A375 cell cycle

In the A375 cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively, 37 ℃, 5%CO ₂Collecting cell suspension behind the culturing cell 48h under the condition, PBS washing 2-3 time after 70% cold ethanol is fixing, adds the PI staining reagent, and flow cytometer detects the cell cycle variation, and the result sees Fig. 9.

Can know by the result; The A375 cell that infects through Ad-IL-24, Ad-OSM, Ad-IL-24-OSM tangible inferior G1 peak all occurs and produces G1 phase cell-cycle arrest in various degree; Dual-gene G1 phase (49.2%) of Ad-IL-24-OSM wherein, be significantly higher than Ad-IL-24 (31.1%), Ad-OSM (30.2%), Ad-GFP and organize (6%) and PBS control group (5.2%) (p＜0.05).

2, PI singly dyes the influence of flow cytometer detection Ad-IL-24-OSM to the nasopharyngeal carcinoma CNE-2Z cell cycle

In the CNE-2Z cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively, 37 ℃, 5%CO ₂Collecting cell suspension behind the culturing cell 72h under the condition, PBS washing 2-3 time after 70% cold ethanol is fixing, adds the PI staining reagent, and flow cytometer detects the cell cycle variation, and the result sees Figure 10.

Can know by the result; Ad-IL-24-OSM obviously retardance occurs in the G2/M phase; Can reach (42.52 ± 3.28) %; Not only be significantly higher than G2/M phase (13.52 ± 1.35) % of Ad-GFP group and G2/M phase (11.61 ± 1.26) % (P＜0.01) of PBS group, and be higher than the G2/M phase per-cent (P＜0.01) of Ad-IL-24 (30.22 ± 2.29) %, Ad-OSM (32.12 ± 2.29) %, compare there was no significant difference (P＞0.05) between the single-gene group.

The two staining streamings of embodiment 11:Annexin-V-PE/7-AAD detect Ad-IL-24-OSM to melanoma A375 cell and the apoptotic influence of nasopharyngeal carcinoma CNE-2Z

1, the two stainings of Annexin-V-PE/7-AAD detect Ad-IL-24-OSM to the apoptotic influence of melanoma A375

In the A375 cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively, 37 ℃, 5%CO ₂Collecting cell suspension behind the culturing cell 48h under the condition, 1-5 * 10 are collected in PBS washing 2-3 time ⁵Cell; Add 1 μ lAnnexin V-PE mixing after adding the Binding Buffer suspension cell of 100 μ l; 37 ℃ of water-bath 15min add 5 μ l 7-AAD dye liquor mixings then, and 37 ℃ of water-bath 15min add the Binding Buffer of 400 μ l then; Detect at 1h in-flow cell instrument, the result sees Figure 11.

The result is visible, and Ad-IL-24, Ad-OSM, Ad-IL-24-OSM all can induce the A375 apoptosis in various degree, and apoptosis rate is respectively 30.7%, 28.3%, 56.2% and organizes with Ad-GFP and PBS cell control group comparing difference has statistical significance (P＜0.05); Wherein dual-gene adenovirus Ad-IL-24-OSM obviously is better than single-gene adenovirus Ad-OSM and Ad-IL-24 (P＜0.05) to inducing the apoptotic effect of A375, and to inducing A375 MC apoptosis to have the knurl synergism of pressing down.

2, the two staining streamings of Annexin-V-PE/7-AAD detect Ad-IL-24-OSM to the apoptotic influence of nasopharyngeal carcinoma CNE-2Z

In the CNE-2Z cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively, 37 ℃, 5%CO ₂Collecting cell suspension behind the culturing cell 72h under the condition, PBS washing 2-3 time, buffer damping fluid re-suspended cell is regulated cell concn to 10 ⁶/ ml gets 10 ⁵In/100ul cell suspension to the streaming pipe, add Annexin-V-PE 10ul respectively, mixing on ice, lucifuge adds buffer damping fluid 380ul again after 15 minutes, add 7-AAD 10ul at last, and flow cytometer detects apoptosis rate, and the result sees Figure 12.

The result is visible; The cell death inducing rate that Ad-IL-24-OSM acts on CNE-2Z cell 72h is (30.66 ± 2.55) %; Organize (3.93 ± 1.17) % apparently higher than PBS group (1.94 ± 0.65) %, Ad-GFP; Significant difference (P＜0.01) is arranged, and Ad-IL-24-OSM organizes with Ad-IL-24 (23.64 ± 2.55) %, Ad-OSM (20.68 ± 2.66) % compares also has significant difference (P＜0.01), has the cancer synergism of pressing down.

The variation that genes involved is transcribed behind embodiment 12:RT-PCR detection Ad-IL-24-OSM infection melanoma A375 cell and the nasopharyngeal carcinoma CNE-2Z cell

1, the variation that genes involved is transcribed behind the RT-PCR detection Ad-IL-24-OSM infection melanoma A375 cell

In the A375 cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively; Virus infection 48h; Centrifugal collecting cell extracts total RNA; Use primer and carry out RT-PCR detection bcl-2, bax, p53 and the transcriptional expression level of p21 gene in the A375 cell respectively, the result sees Figure 13.

The result is visible; The short apoptogene of bax, p21 and p53 in the A375 cell of Ad-IL-24, Ad-OSM, Ad-IL-24-OSM effect is transcribed obvious rise; Bcl-2 suppresses then obviously downward modulation of apoptogene; With PBS group and Ad-GFP group significant difference (P＜0.05) is arranged relatively, and the effect of dual-gene Ad-IL-24-OSM group obviously is better than single-gene Ad-IL-24 and Ad-OSM group (p＜0.05).

2, RT-PCR detects the variation of transcribing that Ad-IL-24-OSM infects genes involved behind the nasopharyngeal carcinoma CNE-2Z cell

In the CNE-2Z cell that is logarithmic growth, add Ad-IL-24-OSM, Ad-IL-24, Ad-OSM, Ad-GFP, PBS respectively; Virus infection 72h; Centrifugal collecting cell extracts total RNA; Use primer and carry out RT-PCR detection bcl-2, survivin, p27 and the transcriptional expression level of p21 gene in the A375 cell respectively, the result sees Figure 14.

The result is visible; Organize relatively with PBS group, Ad-GFP; Ad-IL-24 group, Ad-OSM group, Ad-IL-24-OSM group all can obviously raise the expression of short antiapoptotic factors such as p21, p27, and the expression of obviously reducing survivins such as bcl-2, survivin, and the regulating effect that Ad-IL-24-OSM organizes the above-mentioned expression factor is superior to PBS group and Ad-GFP group (P＜0.01); And be superior to Ad-IL-24 group, Ad-OSM group, significant difference (P＜0.01) is arranged.Compare there was no significant difference (P＞0.05) between the single-gene group.

Embodiment 13:Ad-IL-24-OSM is to inhibition test in the body of melanoma and nasopharyngeal carcinoma animal model

1, the foundation of animal model

A375 cell/CNE-2Z cell uses the RPMI-1640 perfect medium that contains 10% calf serum in 5%CO ₂, 37 ℃ of conventional cultivations.A375 cell/CNE-2Z the cell that will be in logarithmic phase washs with PBS earlier; Through 0.25% trysinization; And end back with serum-free medium with the centrifugal 2-5min of the rotating speed of 1000-1500r/min; Harvested cell, PBS adjustment cell concn prepares cell suspension (A375 concentration of cell suspension 5.0 * 10 ⁵/ ml; CNE-2Z concentration of cell suspension 3.0 * 10 ⁷/ ml).Get 4 age in week 50 of SPF level BALB/c male nude mouses, An Er iodine behind the routine disinfection of the right front oxter of nude mice, every mouse bare subcutaneous injection cell suspension (A375 cell suspension: 1.0 * 10 ⁵/ 100 μ l; CNE-2Z cell suspension: 3.0 * 10 ⁶/ 100 μ l), treat that knurl body diameter acquires a certain degree, count the 0th day, be divided into 5 groups at random, 5 every group, begin test.

2, test is divided into groups and is handled

According to the therapeutic process that mouse model is carried out in the grouping and the processing of table 2, the situation of specifically dividing into groups and processing standard see the following form.

Table 2 test is divided into groups and is handled

3, knurl body weight and tumour inhibiting rate statistics

Behind the treatment 15d, nude mice is taken off neck put to death, tumor by local skin is with An Er iodine routine disinfection; Cut skin and win the knurl body; Electronic balance is claimed the tumour weight in wet base, according to knurl re-computation tumour inhibiting rate, and inhibiting rate (%)=(the average knurl of the average knurl weight/control group of 1-test group is heavy) * 100%.

4, interpretation of result

A375 cell mouse model result:

Can know by Figure 15; The average knurl body weight of Ad-IL-24 group, Ad-OSM group, Ad-IL-24-OSM group and PBS group, Ad-GFP group relatively have significant difference (P＜0.01); The knurl body weight of Ad-IL-24-OSM group is lower than Ad-IL-24 group, Ad-OSM group; Statistics has significant difference (P＜0.01), compares there was no significant difference (P＞0.05) between the single-gene group.

Can know that by Figure 16 the average tumour inhibiting rate of Ad-IL-24 group, Ad-OSM group, Ad-IL-24-OSM group reaches 49.3%, 45.3%, 73.9% respectively; With Ad-GFP group, PBS group significant difference is arranged relatively, and Ad-IL-24-OSM group tumour inhibiting rate organize (P＜0.01) apparently higher than Ad-IL-24 group, Ad-OSM, have but the knurl synergism suppressing the growth of A375 human melanoma cell transplanted tumor in nude mice.

CNE-2Z cell mouse model result:

Can know by Figure 17; The average knurl body weight

of Ad-IL-24 group, Ad-OSM group, Ad-IL24-OSM group is respectively 1.232 ± 0.052,1.263 ± 0.079,1.032 ± 0.062; With PBS group (1.981 ± 0.128), Ad-GFP group (1.927 ± 0.105) significant difference (P＜0.01) is arranged relatively; The knurl body weight of Ad-IL-24-OSM group is lower than Ad-IL-24 group, Ad-OSM group; Statistics has significant difference (P＜0.01), compares there was no significant difference (P＞0.05) between the single-gene group.

Can be known that by Figure 18 Ad-IL-24 group, Ad-OSM group, Ad-IL24-OSM group all have tangible tumor-inhibiting action to nude mice CNE-2Z transplanted tumor, tumour inhibiting rate is respectively (37.81 ± 3.42) %, (36.24 ± 3.88) %, (47.91 ± 5.68) %; Wherein Ad-IL24-OSM group tumour inhibiting rate has significant difference (P＜0.05) than Ad-IL-24 group, Ad-OSM group, has the knurl synergism of pressing down.

Embodiment 14:Ad-IL-24-OSM is to nasopharyngeal carcinoma CNE-2Z cell and transplanted tumor in nude mice radio therapy sensitization effect

1, different irradiation doses are to the influence of CNE-2Z apoptosis rate

To be exponential phase of growth, cell density adopts cobalt 60 gammairradiations for the 70%CNE-2Z cell is divided into 0,2,4,6, totally 5 groups of 8Gy under the room temperature, and absorbed dose rate is 1Gy/min, by University Of Suzhou's radiotherapy medical science radiotherapy center irradiation.37 ℃ of 5%CO ²Collecting cell behind the cultivation 72h under the same terms, PBS cleans 2 times, regulates cell to 1 * 10 with 1 * binding buffer ⁶Individual/ml, get cell 100 μ l in the streaming pipe, add 10 μ l Annexin-V-PE mixing on ice, lucifuge 15min.Add 1 * binding buffer, 380 μ l again, add 10 μ l 7-AAD again, the upflowing cell instrument detects, test triplicate, reference when being intended to filter out best irradiation dose for the biological function research of Ad-IL24-OSM combined radiotherapy.

Test-results is visible; Behind the CNE-2Z cell irradiation 72h;

Collection

0,2,4,6,8Gy respectively organize cell and carry out Flow cytometry simultaneously, and the apoptosis rate of each irradiation dose group is respectively (3.17 ± 0.60) %, (14.51 ± 1.98) %, (26.13 ± 1.98) %, (41.46 ± 2.26) % and (61.75 ± 2.83) %.For fear of the serious side effects of high-dose irradiation and be beneficial to and observe radio therapy sensitization and the synergism of Ad-IL24-OSM to low dose exposure; So irradiation dose of selecting the half irradiation dose (in vitro tests is 4Gy, and animal model test is 10Gy in the body) of conventional irradiation dose to use as the radio therapy sensitization test of follow-up inside and outside low dose exposure.

2, Ad-IL-24-OSM tests nasopharyngeal carcinoma CNE-2Z cells in vitro radio therapy sensitization

Test is divided into groups:

PBS group: 1 * 10 ⁵/ ml CNE-2Z+0.1mol/L PBS;

Ad-GFP group: 1 * 10 ⁵/ ml CNE-2Z+50MOI Ad-GFP;

Combination radiotherapy group: 1 * 10 ⁵/ ml CNE-2Z+ radiotherapy (4Gy);

Ad-IL-24+ combination radiotherapy group: 1 * 10 ⁵/ ml CNE-2Z+50MOI Ad-IL-24+ radiotherapy (4Gy)

Ad-OSM+ combination radiotherapy group: 1 * 105/ml CNE-2Z+50MOI Ad-OSM+ radiotherapy (4Gy)

Ad-IL-24-OSM+ combination radiotherapy group: 1 * 10 ⁵/ ml CNE-2Z+50MOIAd-IL24-OSM+ radiotherapy (4Gy)

Respectively nasopharyngeal carcinoma CNE-2Z cell being carried out external antitumor activity test, PI that mtt assay detects Ad-IL-24-OSM according to above-mentioned grouping singly dyes the two stainings of test, Annexin-V-PE/7-AAD that detect Ad-IL-24-OSM the nasopharyngeal carcinoma CNE-2Z cell cycle influence and detects Ad-IL-24-OSM detects genes involved behind the Ad-IL-24-OSM infection nasopharyngeal carcinoma CNE-2Z cell to test, the RT-PCR of the apoptosis influence of nasopharyngeal carcinoma CNE-2Z cell the test of transcribing variation; TP repeats no more at this with aforementioned each embodiment.

The result:

The MTT detected result:

Cell growth curve by Figure 19 can be known; Combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group, Ad-IL-24-OSM+ combination radiotherapy group all have growth-inhibiting effect in various degree to the CNE-2Z cell; And be time-dependent manner (the most obvious), calculate inhibiting rate and be respectively (35.4 ± 1.48) %, (46.16 ± 2.02) % in 96h; (45.09 ± 1.75) %; (56.93 ± 2.15) % more all has significant difference (P＜0.01) with PBS group and Ad-GFP group, and wherein the Ad-IL-24-OSM+ combination radiotherapy group has significant difference (P＜0.05) than combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group.

PI singly dyes detected result:

Can know by Figure 20-A and 20-B; The Ad-IL24-OSM+ combination radiotherapy group obviously retardance occurs in the G2/M phase; Can reach (62.64 ± 3.62) %; Not only be significantly higher than G2/M phase (13.52 ± 1.35) % of Ad-GFP group and G2/M phase (11.61 ± 1.26) % (P＜0.01) of PBS group, and be higher than the G2/M phase per-cent (P＜0.01) of combination radiotherapy group (39.24 ± 2.86) %, Ad-IL-24+ combination radiotherapy group (50.37 ± 3.17) %, Ad-OSM+ combination radiotherapy group (52.37 ± 3.01).

The two detected results of dying of Annexin-V-PE/7-AAD:

Can draw by Figure 21; The cell death inducing rate that the Ad-IL-24-OSM+ combination radiotherapy group acts on CNE-2Z cell 72h is (59.82 ± 3.91) %; Organize (3.93 ± 1.17) % apparently higher than PBS group (1.94 ± 0.65) %, Ad-GFP; Significant difference (P＜0.01) is arranged, and the same combination radiotherapy group of Ad-IL-24-OSM+ combination radiotherapy group (35.41 ± 3.22) %, Ad-IL-24+ combination radiotherapy group (48.82 ± 3.91) %, Ad-OSM+ combination radiotherapy group (45.83 ± 3.81) % compares significant difference is also arranged (P＜0.01).

The RT-PCR detected result:

Can know by Figure 22; Organize relatively with PBS group, Ad-GFP; Combination radiotherapy group, Ad-IL-24 group+combination radiotherapy group, Ad-OSM+ combination radiotherapy group Ad-IL-24-OSM+ combination radiotherapy group all can obviously raise the expression of short antiapoptotic factors such as p21, p27; And the expression of obviously reducing survivins such as bcl-2, survivin; And the Ad-IL-24-OSM+ combination radiotherapy group is superior to PBS group and Ad-GFP group (P＜0.01) to the regulating effect of the above-mentioned expression factor, and is superior to combination radiotherapy group, Ad-IL-24 group+combination radiotherapy group, Ad-OSM+ combination radiotherapy group, and significant difference (P＜0.05) is arranged.

3, Ad-IL-24-OSM is to radio therapy sensitization test in the body of nasopharyngeal carcinoma CNE-2Z cell

The foundation of mouse model is with embodiment 13, and subcutaneous vaccination 2 all left and right sides tumours are grown to 60-80mm ³During the left and right sides, count the 0th day, it is divided into 6 groups at random, 5 every group, test is divided into groups and is handled as follows:

PBS group: 50 μ l PBS/ only use in the knurl body injection to intervene medication, the next day once, inject altogether 6 times;

Ad-GFP group: 1 * 10 ⁸Pfu Ad-GFP/50 μ l/, treat-ment is organized with PBS;

Combination radiotherapy group: after anti-tumor experiment begins the 5th day, the local single fraction irradiation 10Gy/ of tumor bearing nude mice tumor site is only;

Ad-IL-24+ combination radiotherapy group: 1 * 10 ⁸Pfu Ad-IL-24/50 μ l/ only, treat-ment is organized with PBS, treat 2 times after in tumor by local single fraction irradiation 10Gy/;

Ad-OSM+ combination radiotherapy group: 1 * 10 ⁸Pfu Ad-OSM/50 μ l/ only, treat-ment is organized with PBS, treat 2 times after in tumor by local single fraction irradiation 10Gy/;

Ad-IL-24-OSM+ combination radiotherapy group: 1 * 10 ⁸Pfu Ad-IL-24-OSM/50 μ l/ only, treat-ment is organized with PBS, treat 2 times after in tumor by local single fraction irradiation 10Gy/;

Knurl body weight and tumour inhibiting rate statistics are with embodiment 13, and the result is following:

Can know by Figure 23; The average knurl body weight of combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group, Ad-IL-24-OSM+ combination radiotherapy group

is respectively 0.973 ± 0.042; 0.599 ± 0.033; 0.626 ± 0.038; 0.429 ± 0.038, with PBS group (1.981 ± 0.128), Ad-GFP group (1.927 ± 0.105) significant difference (g of unit, P＜0.01) is arranged relatively; The tumor-inhibiting action of Ad-IL-24-OSM+ radiotherapy combined group is superior to combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group, and statistics has significant difference (P＜0.05).

Can know by Figure 24; Combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group, Ad-IL-24-OSM+ combination radiotherapy group all have tangible tumor-inhibiting action to nude mice CNE-2Z transplanted tumor, are respectively (50.88 ± 4.51) %, (69.76 ± 5.46) %, (68.42 ± 5.78) %, (78.34 ± 6.12) %; Wherein Ad-IL-24-OSM+ radiotherapy combined group has significant difference (P＜0.05) than combination radiotherapy group, Ad-IL-24+ combination radiotherapy group, Ad-OSM+ combination radiotherapy group.

The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.

Claims

1. double gene coexpression recombinant plasmid pAdTrack-CMV-IL-24-polyA-promoter-OSM, its preserving number is CCTCC NO:M 2011378.

2. preserving number is the double gene coexpression recombinant plasmid of CCTCC NO:M 2011378 and the recombinant adenovirus plasmid pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM that adenovirus pAdEasy-1 homologous recombination obtains.

3. the preparation method of recombinant adenovirus Ad-IL-24-polyA-promoter-OSM; It is characterized in that; Preserving number is double gene coexpression recombinant plasmid and the adenovirus pAdEasy-1 homologous recombination of CCTCC NO:M 2011378, and the recombinant adenovirus plasmid pAdEasy-1-pAdTrack-CMV-IL-24-polyA-promoter-OSM of acquisition packs generation in packing cell.

4. according to the said preparation method of claim 3, it is characterized in that said packing cell is the QBI-293A cell.

5. the recombinant adenovirus Ad-IL-24-polyA-promoter-OSM of claim 3 or 4 said preparing methods preparation.

6. the application of the said double gene coexpression recombinant plasmid of claim 1 in preparation treatment malignant melanoma of skin and medicine for nasopharyngeal.

7. the application of the said double gene coexpression recombinant plasmid of claim 1 in preparation nasopharyngeal carcinoma radiotherapy sensitizer.

8. the application of the said recombinant adenovirus of claim 5 in preparation treatment malignant melanoma of skin and medicine for nasopharyngeal.

9. the application of the said recombinant adenovirus of claim 5 in preparation nasopharyngeal carcinoma radiotherapy sensitizer.

10. according to claim 7 or 9 said application, it is characterized in that the ray of said radiotherapy is cobalt 60 gamma-rays.