CN103045634A - Cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as preparation method and anti-tumor effect thereof - Google Patents

Abstract

The invention belongs to the field of gene treatment, provides a new gene therapy product, and specifically relates to a cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid as well as a preparation method and an anti-tumor effect thereof. The gene expression carrier contains genes encoding human LKB1 protein and can express the human LKB1 protein in eukaryotic cells. Based on experiments, the cationic liposome compound of cancer suppressor gene LKB1 eukaryotic expression plasmid, disclosed by the invention, has excellent functions of resisting tumor growth and invasion and metastasis so as to provide a new selection for the tumor treatment.

Description

Cationic liposome complex of cancer suppressor gene LKB1 eukaryon expression plasmid and preparation method thereof and antitumor action

Technical field

The invention belongs to the genetically engineered field, be specifically related to cationic liposome complex and preparation method thereof and the antitumor action of cancer suppressor gene LKB1 eukaryon expression plasmid.

Background technology

Lung cancer is the common malignant tumour of world, also is that whole world M ﹠ M growth is the fastest, and human health and life are threatened maximum malignant tumour.According to the WHO recent statistics, the annual whole world estimates at and surpasses 2,000,000 new patients with lung cancer, dead about 1,800,000 people [1].The incidence and mortality of China's lung cancer is zooming trend, and lung cancer has become first of China's urban population Death Cause for Malignant Tumors.National governments have all dropped into a large amount of man power and materials, in the hope of haveing breakthrough at lung cancer therapy and research field.At present, the primary treatment mode of lung cancer is surgical operation therapy, radiotherapy, chemotherapy etc.But the result for the treatment of of lung cancer is unsatisfactory, even carry out combined chemotherapy, it is efficient also to only have 30-40%, and survival rate only had 4-15% in 5 years.The pitiful survival rate of lung cancer requires to have the appearance of new methods for the treatment of under the routine treatment.Gene Therapy for Lung Cancer becomes methods for the treatment of extremely promising after radiotherapy chemotherapy and operative treatment.The in recent years gene therapy of lung cancer has obtained significant progress, and the research that has has entered clinical experimental stage [2,3].

Studies show that all there is the inactivation of cancer suppressor gene in almost half human tumor, cancer suppressor gene inactivation and tumor growth have close relationship.The inactivation that a plurality of cancer suppressor genes of research report are arranged all has collaborative promoter action [4,5] to generation and the development of tumour.Therefore, normal cancer suppressor gene being imported tumour cell, remove the cancer suppressor gene therapeutic strategy that compensates and replace suddenling change or lack, may be a kind of important treatment pattern in the therapy of tumor.

People's the LKB1 assignment of genes gene mapping is in 19p13.3, and whole gene span is 23kb, and its a kind of protein serine/threonine of encoding comprises 433 amino acid, and relative molecular mass is 50000.The LKB1 wide participation many cellular metabolism processes such as signal transduction, energy stress, cell polarity etc. [6,7].The dysfunction that studies show that LKB1 in recent years is not only the syndromic paathogenic factor of PJSPeutz-Jeghers, simultaneously LKB1 or a newfound important cancer suppressor gene [8].LKB1 is relevant with the generation of the kinds of tumors such as lung cancer, colorectal carcinoma, carcinoma of small intestine, mammary cancer and ovarian cancer, brings into play important regulative [9,10,11] in the multiple biological process of tumour and signal transduction pathway.Particularly nearest research finds that LKB1 is one of the most normal five genes of undergoing mutation in the adenocarcinoma of lung, in the adenocarcinoma of lung of 17-54%, there is LKB1 inactivation sudden change [12], in other nonsmall-cell lung cancers beyond the adenocarcinoma of lung, also have the mass mutation of LKB1, LKB1 plays very important effect as a new cancer suppressor gene in the generation of lung cancer, development, biological processes and the signal transduction pathway [13 a large amount of with cell proliferation, cell cycle, apoptosis, invasion and attack migration and the vasculogenesis etc. of Tumor-assaciated have been regulated and control in participation, 14,15].As a multi-functional kinases and tumor-inhibiting factor, LKB1 has not only participated in the carcinogenesis of human of lung cancer, and the target spot of the prospect that is rich in also is provided for the treatment of lung cancer.Also do not utilize at present both at home and abroad LKB1 to be used for Gene Therapy for Lung Cancer and LKB1 unites the experiment report that other genes are used for the lung cancer combined gene therapy.The antineoplastic molecular mechanism of relevant LKB1 also waits further investigation.

In addition, the primary technical problem of That Gene Therapy Facing is the import system of gene.The target gene import system that adopts in the at present gene therapy is mainly virus vector and non-virus carrier.In virus vector, adenovirus because of specificity with lung, can infect division and Unseparated Cell, higher, the unconformability of transformation efficiency becomes the most frequently used a kind of carrier of Gene Therapy for Lung Cancer to the medium characteristics of karyomit(e) in lung.Although the treatment of the local tumor by adenovirus mediated gene has obtained some achievements, owing to the adenovirus proteins encoded can excitating organism immune response, make the very fast removing of virus vector; Simultaneously, because the adenovirus carrier systemic administration may produce fatal anaphylaxis, can only be used for the local injection treatment of tumour, obviously there is certain limitation in the treatment that this carrier is used for the tumor disease of the general diffusions such as lung cancer, mammary cancer, colorectal carcinoma.Therefore, need to develop effectively for the treatment of the tumor disease of the generals such as lung cancer diffusions but can not the excitating organism immune response, and the target gene carrier of the gene therapy of safety that can the whole body administration.Tool is typically cationic-liposome (cationic liposome) carrier in the non-virus carrier, but it has natural degradation, non-immunogenicity, be fit to produce in enormous quantities, to its goal gene unbounded size system that shifts, can repeat the advantage such as transfection, the approval by NIH (NIH) and Recombinant DNA Advisory Committe (RAC) enters cancer therapy II clinical trial phase as gene therapy vector, is the most promising gene therapy vector [16] that developed recently gets up.The transgenosis of the suitable especially lung tumor of research report cationic-liposome is arranged, when vein gives liposome complex, the lung tumors tissue is the highest organ of destination gene expression level, and systemic injection uses cationic-liposome not find obvious correlation among organs toxicity [17,18].Cationic-liposome has caused more concerns in the lung tumors gene therapy in recent years, and exploring high-efficiency low-toxicity cationic-liposome-mediated polygene coexpression combination therapy lung cancer may be a new way of Gene Therapy for Lung Cancer.This area especially needs to have new selection at present aspect oncotherapy.

Summary of the invention

First technical problem to be solved by this invention provides a kind of expression vector that can express people LKB1 albumen.This carrier is recombinant plasmid vector, is mounted with the gene of encoding human LKB1 albumen, can be at eukaryotic expression people LKB1 albumen.

Wherein, the gene of above-mentioned encoding human LKB1 albumen is under the control of Pcmv promotor and expresses.Wherein, the formation of the gene place expression cassette of above-mentioned encoding human LKB1 albumen is: from 5 ' to 3 ' direction is followed successively by Pcmv promotor, the gene of encoding human LKB1 albumen, BGHpA tailing signal.

Further, the nucleotides sequence of the gene place expression cassette of above-mentioned encoding human LKB1 albumen is classified as shown in the SEQ ID No.1.

Further, above-mentioned recombinant plasmid vector has the nucleotide sequence shown in the SEQ ID No.2.

Second technical problem to be solved by this invention provides the liposome complex of above-mentioned recombinant plasmid vector.The weight ratio of liposome and recombinant plasmid vector is 6～8:1 in this liposome complex.Further, the weight ratio of liposome and recombinant plasmid vector is 6.5～7.5:1 in the described liposome complex.Preferably, the weight ratio 7:1 of liposome and recombinant plasmid vector in the described liposome complex.

Wherein, above-mentioned liposome is that DOTAP and CHOL make, and consumption proportion is mol ratio 1:0.9～1.1.Be preferably the mol ratio 1:1 of DOTAP and CHOL.

The 3rd technical problem to be solved by this invention provides above-mentioned recombinant plasmid vector or the purposes of liposome complex in the preparation antitumor drug.

The 4th technical problem to be solved by this invention provides a kind of antitumor drug.This antitumor drug is that above-mentioned recombinant plasmid vector or liposome complex add pharmaceutically acceptable complementary composition as main active ingredient and be prepared from.

According to a first aspect of the invention, provide a kind of Eukaryotic expression recombinant vector that contains above-mentioned LKB1 gene.These can carry the recombinate various carriers of LKB1 gene of the present invention is known in the art, as: adenovirus, plasmid and other protokaryon and the eukaryotic vector that after processing, can contain LKB1 gene order of the present invention.

Because the adenovirus carrier unconformability is to karyomit(e), thereby lose with the cell fission meeting, can not obtain stable and express enduringly; The adenovirus proteins encoded can excitating organism immune response, virus vector is eliminated very soon, simultaneously because the intravenous systemic medication may produce fatal anaphylaxis, can only be used for the oncotherapy of local injection, affect stable result for the treatment of, the shortage specificity.Although the local tumor treatment by adenovirus mediated gene has obtained some achievements, but because the toxicity of virus vector and can not effectively goal gene be sent to target organ, obviously there is certain limitation in the tumor disease that uses this therapeutic modality to be used for the treatment of such as these whole bodies diffusions such as lung cancer, mammary cancer, colorectal carcinoma.Therefore, need to select effectively but can not the excitating organism immune response drug-loading system of the gene therapy of safety that can the whole body administration.

Preferably, described carrier is the DNA plasmid vector.Owing to will realize the gene transfection eukaryotic cell, therefore, further preferred plasmid vector is pVAX.Made up the pVITRO-LKB1-FUS1 plasmid vector with the RT-PCR method from this laboratory and amplified the LKB1 gene fragment, then with LKB1 gene fragment clone provided by the invention to the pVAX carrier, obtain the pVAX-LKB1 expression vector.

Another aspect of the present invention provides a kind of antitumor drug.This antitumor drug is to add pharmaceutically by above-mentioned recombinant plasmid vector or its liposome complex that the complementary composition of acceptable is prepared from.Preferably, described Antioncogene medicine is LKB1 gene liposome complex (Lipo-PVAX-LKB1 is hereinafter to be referred as P-L).It is scheme known in the art that the composition of liposome and preparation method can adopt.Wherein, described tumour is preferably lung tumors.

In addition, the present invention also provides a kind of preferred liposome scheme, is DOTAP and cholesterol (Chol) composition liposome, and preferred proportioning is mol/mol=1:0.9～1.1.The ratio of preferred liposome and dna vector is 6.5～7.5:1.Adding LKB1 gene cationic-liposome drug injection that pharmaceutically acceptable complementary composition is prepared into can be by promoting apoptosis of tumor cells, suppressing tumor vascular growth and produce anti-lung cancer, the effect of especially anti-lung cancer metastasis.

The present invention also provides the method for preparing above-mentioned liposome complex.The method may further comprise the steps:

Accurately take by weighing DOTAP and cholesterol by mole 1:1, every 20ml chloroform dissolving 58mg DOTAP and 32mg cholesterol are steamed in the bottle in revolving, rotary evaporation is removed chloroform on rotatory evaporator afterwards, then in placing Vacuumdrier to take out after dry 12 hours, add an amount of 5% glucose solution 60 degrees centigrade of lower supersound process 8 minutes, gained liquid is continued namely to get cationic-liposome 60 degrees centigrade of lower hatchings 1.5 hours;

In the cationic liposome complex of every preparation 200 μ l, use recombinant plasmid vector plasmid 20 μ g; Get two EP pipes and be labeled as A and B, add 80 μ l5% glucose among the A, add 81.5 μ l5% glucose among the B, the recombinant plasmid vector plasmid is mixed with the solution 20 μ l of 1 μ g/ μ l, add mixing among the A; With 5% glucose solution will prepare DOTAP:CHOL concentration be adjusted to 4mM, get 18.5 μ l and add the B mixing; At last the whole sucking-offs of plasmid solution among the A are added mixing among the B, then place 4 degree to preserve, namely get liposome complex.

Need to prove that especially the concrete technological method of more than producing and operate recombinant vectors disclosed by the invention and antineoplastic complex injection is well known by persons skilled in the art, and can finish according to the technology of having described.

The present invention adopts LKB1 to make up gene eucaryon expression plasmid and implements gene therapy, by intravenous injection LKB1 gene cationic liposome complex is imported in the body, confirm obviously to reduce in vivo the number of lung tumors metastasis and the growth of inhibition tumour, obviously prolonged the lifetime of tumor animal.This mainly is because quiding gene expression in vivo and in vitro can effectively promote apoptosis of tumor cells, suppresses the Invasion and Metastasis of tumour, produces obvious antitumor action, for Gene Therapy for Lung Cancer provides new approach.In addition, cationic-liposome of the present invention have suit especially some the tissue such as the application in the transgenosis of lung tumor.When vein gave liposome complex, the lung tumors tissue was the highest organ of destination gene expression level, and this is main relevant to the affinity height of cation lipid with the respiratory tract cell.And the HE of animal tissues dyeing finds that systemic injection uses this gene cationic liposome complex not find obvious correlation among organs toxicity.Improved the transfection efficiency of genomic medicine, simultaneously can continuous repeated multiple times use, the toxicity of virus vector and shortcoming that can not prolonged and repeated use have been avoided, made things convenient for further clinical application, improved antineoplastic action, especially the treatment for lung cancer provides extremely promising gene therapy medicament.

Description of drawings

Fig. 1. the PCR product electrophoretogram (1%Agarose electrophoresis) of people's gene LKB1 full length fragment.M:marker; 1: total length LKB1PCR product, the about product clip size of full length fragment is about 1300bp, conforms to LKB1 gene size.

The structure restriction enzyme digestion and electrophoresis figure of Fig. 2 .pVAX-LKB1 gene eucaryon co-expression carrier.M:Marker; 1:pVAX-LKB1 plasmid .2.pVAX-LKB double digestion fragment, the 3.pVAX-LKB1EcoRI single endonuclease digestion; 4.pVAX-LKB1XbaI single endonuclease digestion.

Fig. 3. the LKB1 gene is in the expression (24h) of protein level in the checking gene eucaryon expression plasmid.1: control group; 2:pVAX; 3:pVAX-LKB.Transfected Recombinant Plasmid A549 cell, with the A549 cell of untransfected and the only negative contrast of cell of transfection empty carrier pVAX, Western blot detected result shows in the A549 cell of LKB1 behind transfection pVAX-LKB1 behind the 24h all expression, and relative molecular mass is about 16kDa; All conform to the expection size.

Fig. 4. inducing apoptosis of tumour cell (100X) behind the external LKB1 gene eucaryon expression plasmid transfection A549 cell.A: control group (GLu) B:pVAX; C:pVAX-LKB1.Hochest fluorescent staining microscopically is observed transfection A549 cellular change after 48 hours, and the visible A549 nucleus of pVAX-LKB1 group (C) is concentrated, fragment, smaller volume and a large amount of apoptotic body form, and empty carrier group (B) has a small amount of apoptotic body to occur; Control group (A) cell does not significantly change.Show that pVAX-LKB1 has good expression efficiency external, thereby the LKB1 encoding sequence that connects can be translated the function of exercising cell death inducing in the A549 cell.

Fig. 5 .LKB1 gene eucaryon expression plasmid DNA liposome complex particle size determination result.The cation lipid body and function Malvern ParticleSizer for preparing detects, and shows its granulometric curve.

Fig. 6. tumor migration inhibition ability (200X) behind external LKB1 gene eucaryon expression plasmid transfection A549 and the H460 cell.Conntrol: control group p:pVAX; P-L:pVAX-LKB1.Because lung carcinoma cell migration plays an important role in the evolution of lung cancer, therefore, our method by Transwell has detected the ability whether LKB1 can suppress the NSCLC cell migration.Untreated NSCLC cell migration rate is made as 100%, compares by the cell quantity with the cell quantity after the otherwise processed and untreated fish group and obtain relative mobility.As shown in Figure 6.PVAX-LKB1 individual curing cell can be obtained the ability of comparatively desirable inhibition tumor cell migration, can reach about 60%～70% two kinds of its inhibiting rates of NSCLC.

Fig. 7. suppress tumor invasion ability (X200) behind external LKB1 gene eucaryon expression plasmid transfection A549 and the H460 cell.Control: control group p:pVAX; P-L:pVAX-LKB1.Similar to the lung carcinoma cell transfer ability, the lung carcinoma cell invasion ability plays an important role in the evolution of lung cancer too, and its grade of malignancy of NSCLC that invasive ability is higher is also higher.Therefore, we have detected the ability whether LKB1 can suppress the NSCLC cell invasion by Boyden cell method.The vitro invasion experimental result is compared with control group as shown in Figure 7, can suppress 75% left and right sides cell invasion ability after single-gene is processed in two kinds of lung carcinoma cells.

Anti-lung trans effect (A549 nude mice model) in Fig. 8 .LKB1 gene eucaryon expression plasmid body.A: control group B:pVAX; C:pVAX-LKB1.The result shows that the pVAX-LKB1 group is compared with glucose group with empty carrier pVAX group, and nude mice lung metastasis obviously reduces, and shows obvious tumor growth and suppresses, and inhibiting rate reaches 80%.

Anti-lung cancer prolongs lifetime (A549 nude mice model) in Fig. 9 .LKB1 gene eucaryon expression plasmid body.Conntrol: control group p:pVAX; P-L:pVAX-LKB1.Compare with glucose group (A) with empty carrier pVAX group (B), nude mice obviously prolongs lifetime, until behind all glucose treatment groups and the pVAX treatment group dead mouse, pVAX-LKB1 treatment group mouse all none example is dead.We further analyze and draw, and glucose treatment group mouse median survival interval only 70 days, result show that the pVAX-LKB1 drug treatment all can significant prolongation tumor-bearing mice survival time.

The concise and to the point structure schema of Figure 10 .pVAX-LKB1.

Embodiment

The invention will be further described below in conjunction with the description of accompanying drawing by embodiment, but this is not limitation of the present invention, those skilled in the art are according to basic thought of the present invention, can make various modification or improvement, only otherwise break away from basic thought of the present invention, all within the scope of the present invention.

Embodiment one makes up people LKB1 gene eukaryotic expression vector

With the sequence NM000455 design primer of LKB1 in GenBank, after total RNA reverse transcription of people's lung fibroblast MRC-5 cell, react the LKB1 gene fragment of this gene of amplification by nest-type PRC.Then with the LKB1 gene fragment clone to the pVAX carrier, obtain the pVAX-LKB1 expression vector, make up flow process and see Figure 10.

Upstream primer (SEQ ID No.3): 5 '-

CCAGCATGGAGGTGTGGAC-3 '.

Downstream primer (SEQ ID No.4): 5 '-

TCACTGCTGCTTGCAGG'CCGA-3 '.

Wherein mark section of boldface type institute is respectively 5' end EcoRI, 3' end XbaI restriction enzyme site, and italics mark part is the protection base.The EcoRI restriction enzyme site is added in LKB1 gene fragment upstream, and the XbaI site is added in the downstream.The sequence of the people LKB1 gene that amplification obtains is SEQ ID No.1.

2): the said gene product is built into the gene eucaryon expression recombinant vectors;

The LKB1 eukaryon expression plasmid called after pVAX-LKB1 that the present invention makes up, result such as Fig. 1 are shown in 2.Its sequence is shown in SEQ ID No.5.

Identify with restriction enzyme EcoRI and the reaction of XbaI double digestion, obtained fragment and the treaty 3kb left and right sides fragment of a treaty 1.4kb, consistent with expected results, deliver order-checking, Sequence Identification is correct, and LKB1 gene eucaryon expression plasmid pVAX-LKB1 successfully constructs.

The preparation of the liposome complex of embodiment two people LKB1 gene eukaryotic expression vectors

Utilize QIAGEN company to remove the extraction test kit extracting PVAX-LKB1 (P-L) of endotoxic a large amount of plasmids, PVAX (P) vector plasmid is also quantitative.

The present invention is through a large amount of trial test in early stage; determine DOTAP([1-(2; the 3-dioleoyl)]-and N, N, N-Trimethylamine 99 the third Methylsulfate; 1; 2-Dioleoyl-3-Trimethyllammonium-Propane, C43H83NO4, molecular weight; 698.55) and the liposome jointly made of cholesterol Chol be more suitable in the mixture of preparation with PVAX-LKB1, both proportionings are mol/mol=1:0.9～1.1.Be preferably the mol ratio 1:1 of DOTAP and CHOL.

Obtain by the following method dual-gene cationic liposome complex.

1, the preparation of cationic-liposome:

Accurately take by weighing DOTAP 58mg, cholesterol Chol 32mg(mol/mol=1:1), revolve in 100ml and to steam in the bottle, add the dissolving of 20ml chloroform, afterwards on rotatory evaporator rotary evaporation 45 minutes to remove chloroform, place Vacuumdrier after dry 12 hours, take out, add a certain amount of 5% glucose solution, 60 degrees centigrade of lower Probe Ultrasonic Searchings (power is 400W) 8 minutes, gained liquid is continued namely to get cationic-liposome 60 degrees centigrade of lower hatchings 1.5 hours.Be placed in after packing under the 4-8 degrees celsius and preserve.

2, the preparation of DOTAP:CHOL and dual-gene plasmid composite and particle size determination

Preparation process is as follows:

1.4mM DOTAP:CHOL solution preparation: the EP pipe of getting two 1.5ml respectively adds 800 μ l, 5% glucose, and the liposome solutions that take out a pipe 20mM from 4 degree refrigerators are drawn respectively 200 μ l and added in the aforementioned EP pipe and namely be diluted to 4mM, and mixing is for subsequent use.

2.1 the preparation of μ g/ μ l plasmid solution: get plasmid and be diluted to 1 μ g/ μ l from-70 ℃ of refrigerators, mixing is for subsequent use.

3. the preparation of plasmid and DOTAP:CHOL mixture: as: preparation plasmid P-F is that 20 μ g end-bodies are the mixture of 200 μ l.Get two EP pipes and be labeled as A and B, add 80 μ l5% glucose among the A, add 81.5 μ l5% glucose among the B, the P-L plasmid solution 20 μ l of the 1 μ g/ μ l for preparing are added among the A gently mixing three times, the DOTAP:CHOL 18.5 μ l of the 4mM for preparing are added gently mixing three times of B, at last the whole sucking-offs of plasmid solution among the A are added among the B, add fashionable should the adding gently at the liquid level place and mixing three times, action must softly avoid precipitation to occur during mixing.Whole process for preparation rapidly, places 4 to spend night as far as possible at last.

4. the mensuration of gel retardation assay, particle diameter and current potential and treatment of animals: morning takes out institute from refrigerator joins after the mixture room temperature leaves standstill 1 hour, is used for particle diameter and the potential measurement for the treatment of of animals experiment and mixture.

In the preparation of DNA liposome complex, the present invention passes through DNA liposome complex gel retardation assay, the ratio of comprehensive evaluation liposome and DNA is come in several each and every one aspects of the mensuration of particle diameter and current potential and transfection efficiency, the advantage ratio that draws is that the weight ratio of liposome and plasmid is 6-7:1, and optimum proportion is 7:1.The liposome complex that the present invention has all taked this ratio to prepare in the experiment in vivo.Detect the liposome complex particle diameter with Malvern ParticleSizer, measurement result shows for the continuous liposome complex homogeneous grain diameter for the treatment of repeatedly, particle size range about 240, meet liposome in vivo the particle diameter in the gene therapy require (see figure 5).

Experimental example three. people LKB1 gene eucaryon expression plasmid is at external expression and anti-tumor activity

1, LKB1 protein expression in the transfection recombinant plasmid cell

Transfected Recombinant Plasmid A549 cell, with the A549 cell of untransfected and the only negative contrast of cell of transfection empty carrier pVAX, Western blot detected result shows that LKB1 has expression in the A549 of transfection PVAX-LKB1 cell behind the 24h, relative molecular mass is about 54kDa, and (Fig. 3) all conforms to the expection size.

2.LKB1 gene expression plasmid detects at anti tumor activity in vitro

Process cell with the pVAX-LKB1 plasmid, with the A549 cell of untransfected and the only negative contrast of cell of transfection empty carrier pVTRO2, observing Growth of Cells behind the transfection 24h is suppressed, in order further to inquire into the type of necrocytosis, we carry out Hoechst33258 fluorescent staining microscopically observation of cell and change (Fig. 4), visible cell through the pVAX-LKB1 transfection under the mirror, send bright blue-fluorescence during ultraviolet excitation, nuclear pyknosis occurs and is cracked into fragment, produce obvious apoptotic body, and transfection the cell growth state of empty carrier or untransfected good, this result shows that the LKB1 expression vector has an effect of obviously inducing the lung cell A549 apoptosis external, and apoptosis-promoting effect is because the result of LKB1 genetic expression.

1.LKB1 the vitro inhibition tumor invasion of gene expression plasmid migration:

2.1:pVAX-LKB1 suppress NSCLC cell migration capability study

Transfected Recombinant Plasmid A549 or H460 cell are with A549 cell or H460 cell and the only negative contrast of cell of transfection empty carrier pVAX of untransfected.A549 or H460 cell spread 6 orifice plates, and transfection is also changed liquid, change and continue cellar culture spend the night (about 12h) behind the liquid.Millicell is placed in the 24 orifice plate apertures, and the chamber adds 600 μ l and contains the 1%FBS1640 fresh culture under the cell, and trysinization is also collected the cell of different treatment group, and is resuspended and to adjust cell concn be 1 * 10 with containing the 1%FBS1640 fresh culture ⁵/ ml, in the upper chamber of cell adding Millicell with different treatment, 400 μ l/well, 3 parallel holes of every group of Treatment Design.Continue the backward taking-up cell of cellar culture 24h and in cell plate, add methyl alcohol fixed cell 15min, use thereafter 0.1% violet staining 15min; Dyeing is used ddH after finishing ₂O washes 2 times; The variation of the invasion and attack transfer ability of several groups of cells of Microscopic observation finds that the cell number of the genomic cell-penetrating basilar membrane arrival of transfection LKB1 microporous membrane lower floor is compared obvious minimizing than control group.Result (Fig. 6) shows that the A549 cellular migration inhibition rate of pVAX-LKB1 treatment group is 60-70%; Adopt the SPSS11.0 statistical package, carry out T detection analysis demonstration pVAX-LKB1 group and compare with control group, the P value is all less than 0.05; The inhibition that proof LKB1 expresses the transporting action of NSCLC cell has significance.

2.2 pVAX-LKB1 suppresses the capability study of NSCLC cell invasion

Transfected Recombinant Plasmid A549 or H460 cell are with A549 cell or H46 cell and the only negative contrast of cell of transfection empty carrier pVAX of untransfected.A549 or H460 cell spread 6 orifice plates, and transfection is also changed liquid, change continue behind the liquid cellar culture spend the night (～12h).The cell of different treatment is added in the upper chamber of Boyden 3 parallel holes of every group of Treatment Design of 400 μ l/well().Cell shifts to an earlier date 1h with matrigel and two coated in 37 ℃ of the ratios of 1:6 without 1640 substratum, and with the serum-free RPMI 1640 fresh culture aquations that contain 1%BSA.The cell that adds cell continues to take out cell with methyl alcohol fixed cell 15min, thereafter with 0.1%crystal violet dyeing 15min behind the cellar culture 24h; Dyeing is used ddH after finishing ₂O washes 2 times; Carefully wipe confluent monolayer cells on the cell with cotton balls.The variation of the invasion and attack transfer ability of several groups of cells of Microscopic observation finds that the cell number of the genomic cell-penetrating basilar membrane arrival of transfection LKB1 microporous membrane lower floor is compared obvious minimizing than control group.Result (Fig. 7) shows that the A549 cell invasion inhibiting rate of pVAX-LKB1 treatment group is 75%; Adopt the SPSS11.0 statistical package, carry out T detection analysis demonstration pVAX-LKB1 group and compare with control group, the P value is all less than 0.05; The inhibition that proof LKB1 expresses the invasion and attack effect of NSCLC cell has significance.

Experimental example four LKB1 plasmid DNA liposome complexes in vivo anti-tumor activity detect

1, experimental technique

1.1 anti-lung cancer metastasis animal pattern test in the body

Model human lung cancer cell A549 nude mice Pulmonary metastases model of the present invention, adopt pVAX, the cationic liposome complex of pVAX-LKB1 is 4 tail intravenously administrable treatment tumours every other day, dosage is 20ug/mice, second week is dissected mouse after the treatment, the number of counting lung tumors metastasis calculates the metastasis inhibition rate.Experimental result has confirmed LKB1 gene eucaryon expression plasmid DNA liposome complex anti-tumor activity in vivo.

1.2 lung shifts the nude mice prolongation test of lifetime

The present invention has then carried out the LKB1 gene eucaryon expression plasmid and has prolonged the experiment of lifetime of lung transfer nude mice.On the A549 nude mice interior tumor experiment lung metastasis model of setting up, the 8th day begin treatment behind the inoculation A549 cell, liposome complex particle mean size is about 242nm, giving glucose, liposome empty carrier mixture (Lip-pVITRO2), liposome pVITRO2-LKB1 gene composite etc. by the tail vein respectively treats, treat every other day 1 time, dosage is 20ug/mice, totally 4 times.After treatment finished, weighing nude mice body weight was observed the nude mice survival condition weekly.When obvious hogback appears in nude mice, listless, during the dying phenomenon such as appetite stimulator, observe every day, record nude mice date of death, until nude mice of control group is all dead, then do the figure statistics.

2, experimental result

A, each group of experimental session are not all observed obvious toxic side effects, and no significant difference between treatment group and each tissue of control group is found in HE dyeing.

After B, treatment finished for 2 weeks, etherization was put to death nude mice, by the oral cavity through the Intratracheal instillation Indian ink, dissect and take out lung tissue, be immersed in the AAF damping fluid, statistics lung surface metastasis number, liposome pVAX-LKB1 treatment group metastasis number obviously is less than control group.Carrying out mean through variance analysis relatively shows, compare with glucose group, liposome pVAX-LKB1 group neoplasm metastasis minimum number, tumour inhibiting rate is 80%, utilize SPSS software to do the T check, result's demonstration is compared with glucose group, and liposome pVAX-LKB1 group has significant difference (P＜0.05) (Fig. 8).

C. after treatment finished, weighing nude mice body weight was observed the nude mice survival condition weekly.When obvious hogback appears in nude mice, listless, during the dying phenomenon such as appetite stimulator, put to death nude mice, record date of death, and do statistical study (Fig. 9).The result shows, dextrose treatment group nude mice mean survival time (MST) is 70 days, 105 days mean survival time (MST)s of liposome empty carrier mixture treatment group (Lip-pVAX) nude mice, and the experiment that arrives ends the whole nude mices survivals of liposome pVAX-LKB1 mixture treatment group nude mice when finishing, other group is all dead.Adopt SPSS statistics software to analyze carrying out Log Rank (Mantel-Cox) its lifetime, liposome pVAX-LKB1 mixture significance has prolonged nude mice lifetime (p＜0.05).

To sum up, the restructuring LKB1 gene eucaryon expression cationic liposome complex that is made up by the present invention can suppress tumor invasion and shift by promoting apoptosis of tumor cells, effectively suppresses in vivo growth and metastasis of tumours.

Simultaneously by this area general knowledge as can be known, if different requirements is arranged, the consumption of restructuring pVAX-LKB1 gene can change in a big way at one in the antitumor injection of the present invention.Those skilled in the art can be according to some known factors, such as the kind of disease, and coincident with severity degree of condition, patient body weight, formulation, selected routes of administration etc. are determined at an easy rate.

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Claims (14)

1. be mounted with the gene of encoding human LKB1 albumen, can be at the recombinant plasmid vector of eukaryotic expression people LKB1 albumen.

2. recombinant plasmid vector according to claim 1, the gene that it is characterized in that described encoding human LKB1 albumen are under the control of Pcmv promotor expresses.

3. recombinant plasmid vector according to claim 2, the aminoacid sequence that it is characterized in that described people LKB1 albumen is shown in the Seq ID No.2.

4. recombinant plasmid vector according to claim 2, the encoding gene that it is characterized in that described people LKB1 albumen is shown in the Seq ID No.1.

5. recombinant plasmid vector according to claim 2, the formation that it is characterized in that stating in the carrier gene place expression cassette of coding LKB1 albumen is: from 5 ' to 3 ' direction is followed successively by gene, the BGHpA tailing signal of Pcmv promotor, coding LKB1 albumen.

6. recombinant plasmid vector according to claim 1 is characterized in that having the nucleotide sequence shown in the SEQ ID No.5.

7. the liposome complex of each described recombinant plasmid vector of claim 1～6.

8. liposome complex according to claim 7, it is characterized in that: the weight ratio of described liposome and plasmid is 6～8:1.

9. liposome complex according to claim 8, it is characterized in that: the weight ratio of described liposome and plasmid is 6.5～7.5:1.

10. liposome complex according to claim 9, it is characterized in that: the weight ratio of described liposome and plasmid is 7:1.

11. liposome complex according to claim 7 is characterised in that: described liposome is that DOTAP and CHOL form, and the consumption proportion scope of DOTAP and CHOL is 1:0.9～1.1 in molar ratio.

12. each described recombinant plasmid vector of claim 1～6 or each described liposome complex of claim 7～11 purposes in the preparation antitumor drug.

13. antitumor drug adds pharmaceutical excipient by each described recombinant plasmid vector of claim 1～6 or each described liposome complex of claim 7～11 as main active ingredient and is prepared from.

14. prepare the method for each described liposome complex of claim 7～11.

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