CN101380310B - Composition of bFGF modified liposome and shRNA expression vector targeting human VEGF gene and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of tumor gene therapy, and more particularly relates to a compound formed by RNA interference expression plasmid of targeted human VEGF genes and a cationic liposome modified by bFGF, and a preparation method and the use thereof. The first technical problem solved by the invention is that a gene therapy product with anti-tumor function is provided. The technical proposal for solving the problem is that a liposome compound is provided. The liposome compound takes liposome compounded by DOTAP, cholesterol and bFGF as an encapsulating layer with the following mixture ratio: the mol ratio of the DOTAP and the cholesterol is 2:1-2, and the weight of the tbFGF polypeptide is 20-80% of the total weight of the DOTAP and the cholesterol; and the encapsulating can express the compound formed by the expression vector of RNA interference sequence of human VEGF genes in human body. The compound has protective and slow release functions and good targeting effect on shRNA plasmid.

Description

The complex and the preparation thereof of the liposome that bFGF modifies and the shRNA expression vector of targeting human VEGF gene

Technical field

The invention belongs to field of tumor gene therapy, be specifically related to the rnai expression plasmid and the formed complex of modifying by bFGF of cationic-liposome of targeting human VEGF gene, its preparation method, and the purposes in preparation antitumor drug and used as adjuvant drug for antitumor.

Background technology

Tumor growth be unable to do without angiogenesis, and when diameter of tumor surpasses 2-3mm, growth of tumor will depend on the secreted various short angiogenesis factor of tumor cell and come induction of vascular to generate, thereby support tumor further growth and transfer.In promoting the tumor neogenetic blood vessels generative process, VEGF (vascular endothelial growth factor, VEGF, VEGF) be to act on one of the strongest stimulating factor, discover that in a large number VEGF gene normal tissue expression in the kinds of tumors tissue obviously increases, as pulmonary carcinoma, hepatocarcinoma, colon cancer, carcinoma of prostate, ovarian cancer, cancer of pancreas, cervical cancer, melanoma, vegf expression increases unusually in the malignant tumor such as incidence squamous cell carcinoma, detecting the VEGF gene expression dose in the tissue of patient that neoplasm metastasis and poor prognosis take place significantly raises, therefore, the VEGF gene is the important target spot of present tumor angiogenesis inhibitor treatment.

Treatment research at VEGF at present mainly comprises Antybody therapy (the antibody Avastin treatment of VEGF), micromolecular inhibitor treatment, VEGF growth inhibiting factor treatment (Endostatin endostatin treatment), immunization therapy etc.The RNA interference treatment is a kind of new disease treatment means of rising in recent years, RNA The Study of Interference at the VEGF gene also has report, siRNA can effectively suppress growth of tumor such as pulmonary carcinoma, colon cancer, carcinoma of prostate at the chemosynthesis of VEGF gene, shows RNA and disturbs the VEGF expression of gene that malignant tumor is had potential treatment prospect.But, at present reported in literature be mostly utilize the siRNA of chemosynthesis that tumor is treated, and there is following shortcoming in the siRNA of chemosynthesis: the siRNA consumption of chemosynthesis is big, the cost height; The siRNA operation of chemosynthesis requires high, and the time of effect is short in vivo.This area needs better scheme and solves the problems referred to above.

Summary of the invention

First technical problem to be solved by this invention provides a kind of gene therapy product with antitumor action.The technical scheme that addresses this problem has provided a kind of liposome complex.The liposome that this liposome complex is combined into DOTAP, cholesterol and bFGF is an encapsulated layer, its proportioning is: the mol ratio DOTAP of DOTAP and cholesterol: cholesterol=2: 1～2, the weight content of tbFGF polypeptide are by 20～80% of DOTAP and cholesterol gross weight; Seal the complex of the expression vector formation of the RNA interference sequence that can express human VEGF gene in vivo.

Further, the shRNAR interference sequence of above-mentioned human VEGF gene at be by target spot: 5 '-AAACCTCACCAAGGCCAGCAC-3 shown in the SEQ ID NO.1.

Further, express in the above-mentioned rnai expression carrier at being shown in the SEQ ID NO.2 by the positive-sense strand sequence of the expression framework of the RNA interference sequence of target spot shown in the SEQ ID NO.1 on the FAK gene, expressing framework antisense strand sequence is shown in the SEQ IDNO.3.Wherein, above-mentioned expression vector has the described nucleotide sequence of SEQ ID NO.4.

Wherein, the weight content of above-mentioned bFGF polypeptide is by 40～60% of DOTAP and cholesterol gross weight.

Wherein, above-mentioned bFGF polypeptide has the aminoacid sequence shown in the SEQ ID NO.5.That is:

n’-krlycknggfflrihpdgrvdgvreksdphiklqlqaeergvvsikgvcanrylamkedgrllaskcvtdecffferlesnnynty-C’。

Second technical problem to be solved by this invention provides the preparation method of above-mentioned liposome complex.This method may further comprise the steps:

A, take by weighing DOTAP, Chol places to revolve and steams bottle, adds chloroform and makes it to dissolve fully;

B, rotary evaporation in vacuo get liposome membrane, drying;

C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;

D, bFGF is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, obtain blank liposome, liposome after the hatching is pressed through 0.45 μ m polycarbonate membrane 4～8 times, the expression vector that can express the RNA interference sequence of human VEGF gene more in vivo mixes by 1: 2 ~ 3 weight ratio with liposome, at room temperature hatches 10～30 minutes, promptly.

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

The 4th technical problem to be solved by this invention provided a kind of antitumor drug or antitumour auxiliary drug.This antitumor drug or antitumour auxiliary drug are to add pharmaceutically by above-mentioned liposome complex as main active that the acceptable adjuvant is prepared from.

Further, above-mentioned antitumor drug or antitumour auxiliary drug also contain tumor chemotherapeutic drug as active component.

The present invention has at first made up the siRNA expression vector that can express at human VEGF gene, uses this expression vector to carry out gene therapy, and it is easier to operate, and can reduce production costs greatly, is easier to industrialization.

In addition, because basic fibroblast growth factor receptor (FGFR) high level expression in kinds of tumor cells, so the structure of basic fibroblast growth factor (bFGF) is transformed, the structure that has only kept bFGF and its receptors bind, the bFGF mutant of Chan Shenging has kept active with combining of FGFR like this, but can not stimulate cellular proliferation.The present invention is with this bFGF mutant and cationic-liposome coupling, be prepared into the cationic-liposome that the bFGF mutant is modified, it can carry out self assembly with plasmid DNA and form the liposome/plasmid dna complex compound with cancer target, therefore the cationic-liposome of bFGF mutant modification has the effect of target tumor and tumor vascular endothelial cell, can reduce the using dosage of therapeutic gene like this, produce better antitumor action and reduce toxic and side effects.

Compare with existing other technologies scheme, and the cationic-liposome parcel shRNA-VEGF plasmid that the present invention utilizes the bFGF mutant to modify forms the following advantage that has of complex: this complex has protection and slow releasing function to the shRNA plasmid, can stably this plasmid be imported in the body, RNA interferential action time can be more lasting like this, and can reduce drug dose and administration number of times; Have good targeting simultaneously, can targeting the surface have the FGF receptor various malignant tumor tissues, more can tell on.Owing to have targeting, the cationic-liposome parcel shRNA-VEGF plasmid that bFGF mutant of the present invention is modified forms the adoptable intravenous administration mode of complex, be better than in the tumor of existing reported in literature, administering mode such as intraperitoneal, use wider clinically, to nearly all tumor, the tumor that comprises transfer all is effective.In addition, the cation lipid nanocrystal composition that present patent application is modified shRNA plasmid/bFGF mutant is treated combined chemotherapy medicine cisplatin, can obviously strengthen the sensitivity of cisplatin chemotherapy, produces stronger antitumous effect.So the formed complex of the cationic-liposome that bFGF mutant of the present invention is modified parcel shRNA-VEGF plasmid has good application prospects.

Description of drawings

Fig. 1, pGensil-2 expression vector collection of illustrative plates.

The enzyme action of Fig. 2, rnai expression plasmid pGen-VEGF-328 is identified figure.

Fig. 3, A549 cell transfecting RNA interference plasmid are after 48 hours, and each organizes VEGF amount secreted in the supernatant.

Respectively organize the laboratory animal tumor weight after Fig. 4, the treatment.

Respectively organize the laboratory animal tumor growth curve after Fig. 5, the treatment.

Concrete embodiment

The structure of the shRNA expression plasmid of embodiment one targeting VEGF gene

The siRNA sequence that has the strongest inhibition VEGF gene expression according to reported in literature is VEGF-328:5 '-ACCTCACCAAGGCCAGCAC-3 ' (21nt) (people's VEGF gene coding region 328-348bp position): (humanVEGF:GenBank accession no:NM_001025366), and the shRNA plasmid expression vector makes up as follows:

1, synthetic following primer sequence:

The RNA of VEGF-328 disturbs target sequence (SEQ ID NO.1): 5 '-AAACCTCACCAAGGCCAGCAC-3 '

The primer structure:

Primer positive-sense strand (SEQ ID NO.2):

(design has the SacI restriction enzyme site)

Primer antisense strand (SEQ ID NO.3):

5’-AGCTTGAGCTCAAAAAAACCTCACCAAGGCCAGCACCGTCTTGAAGTGCTGGCCTTGGTGAGGTG-3’

2, synthetic sequence is connected on the pGensil-2 carrier

(1) annealing of strand target gene fragment connects:

Each dissolves above-mentioned Synthetic 2 OD strand target gene fragment with 50ul annealing buffer.

Respectively get 2ul (being 2ul VEGF-328-A+2ul VEGF-328-B)+16ul annealing buffer mixing.

94 ℃ of water-bath annealing naturally cool to room temperature.

Respectively get 1ul annealing product+99ul H ₂O does 100 times of dilutions

(2) the BamHI+HindIII double digestion of plasmid pGenesil-2,1% agarose gel reclaims big fragment.

(3) dilution annealing fragment respectively with being connected of linearisation pGenesil-2 plasmid expression vector.

The coupled reaction condition:

Dilution annealing fragment 1ul

Linearisation pGenesil-2 plasmid vector 1ul

10×Ligase?Buffer 1ul

T4?DNA?Ligase 1ul

H ₂O 6ul

22 ℃ of water-baths spend the night.

(4) respectively get 5ul and spend the night and connect product transformed competence colibacillus cell DH5, separate application is in containing Kana ^rOn the LB flat board of resistance (final concentration is 30ug/ml), 37 ℃ of calorstat overnight incubation.

(5) 3 monoclonal colony inoculations of each picking contain Kana in 3ml from each culture dish ^rIn the LB culture fluid of resistance (final concentration is 30ug/ml), 37 ℃ of constant temperature shaking tables (250rpm) overnight incubation.

(6) extract plasmid in a small amount with little extraction reagent kit, and do enzyme action with SacI respectively and identify:

Reaction condition is as follows:

Plasmid DNA 8ul

10×H?Buffer 1ul

SalI 1ul

37 ℃ of water-bath 3h.

The 1%Agase detected through gel electrophoresis, electrophoresis pattern is seen Fig. 2.Because plasmid expression vector pGenesil-2 the inside was the restriction enzyme site that does not have SacI originally; And recombiant plasmid can be by SacI institute enzyme action, the illustration purpose genetic fragment has been inserted in the plasmid vector pGenesil-2, identify that through enzyme action the siRNA of VEGF-328 expresses framework and has been connected in the pGenesil-2 plasmid expression vector called after pGen-VEGF-328

Constructed pGen-VEGF-328 plasmid expression vector total length 3936bp records its sequence shown in SEQ ID NO.4, wherein 655～720 expression frameworks for the siRNA shown in SEQ ID NO.3 of insertion.

The preparation of the cation lipid nanocrystal composition that embodiment two plasmid DNA-bFGF mutant is modified

1, the preparation of the cationic-liposome of bFGF mutant modification

Cationic-liposome DOTAP is mixed by 1: 1 mol ratio with neutral compound cholesterol (Chol), and the chloroform of mixture with the HPLC level dissolved, and place on the Rotary Evaporators 40 ℃ of backspins to steam film forming after 1 hour, vacuum drying spends the night.After taking out, the film that become is dissolved in 5% the glucose solution, under 55 ℃ of water bath condition, shook 1 hour then, mixture is forwarded in the test tube, and by 220nm polycarbonate membrane extruding 4 times, at last mixture is dissolved in an amount of 5% the glucose solution, obtains the DOTAP-Chol cationic-liposome suspension of 5mg/ml.According to cationic-liposome: bFGF mutant=2: 1 (mass ratio), in liposome solutions, slowly add the bFGF mutant protein, hatch down to spend the night for 4 degrees centigrade and form the cationic-liposome that the bFGF mutant is modified, the cationic-liposome that bFGF mutant after the hatching is modified pushes 4-8 time by 0.45 μ m polycarbonate membrane, obtains the cationic-liposome that the bFGF mutant is modified.

This bFGF mutant has aminoacid sequence as follows (SEQ ID NO.5):

n’-krlycknggfflrihpdgrvdgvreksdphiklqlqaeergvvsikgvcanrylamkedgrllaskcvtdecffferlesnnynty-C’。

2, preparation pGen-VEGF-328 plasmid DNA

Utilize the antibacterial high density fermentation jar of 50L to produce the pGen-VEGF-328 plasmid, utilize plasmid DNA purification instrument-AKTAPliot (General Electric Apparatus Co.(U.S.A.)), meet the plasmid DNA of national GMP standard with alkaline lysis preparation and purification, through detecting the requirement that purified plasmid meets experiment in vivo and vitro.

3, the preparation of the cationic-liposome of plasmid DNA-bFGF mutant modification

The pGen-VEGF-328 plasmid DNA is diluted, the cationic-liposome of modifying in the bFGF mutant: DNA=3 again: the cation lipid liquid solution that the ratio of 1 (W/W) is modified the bFGF mutant joins in the pGen-VEGF-328 plasmid DNA solution, eddy oscillating 2min at once afterwards, hatch 1h down at 4 degrees centigrade again, promptly get the cationic-liposome that expression plasmid DNA of the present invention and bFGF mutant are modified.

The experiment in vitro anti-tumor experiment of test example one pGen-VEGF-328 plasmid

Below the cationic-liposome modified of all bFGF mutant write a Chinese character in simplified form into bFGF-Liposome; PGen-VEGF-328 abbreviates pGen-VEGF as.

Experimental project is induced the apoptosis of human lung carcinoma cell for using the pGen-VEGF plasmid.

Human lung carcinoma cell line A549, the NCI-H446, the SPC-A1 that use below are the detailed experimental results of A549 cell strain, and other 2 strain cells also obtain the identical result of trend.

Experiment is carried out (6 orifice plate) by following grouping:

A) blank

B) irrelevant sequence (2 μ g pGen-HK)+bFGF-Liposome (5 μ g)

C)pGen-VEGF(2μg)+bFGF-Liposome(5μg)

Experimentation is as follows, preceding 24 hours of transfection with the A549 cell inoculation at 6 orifice plates (2 * 10 ⁵Cells/well), cell density is 40-50% during transfection, before transfection, the culture medium in 6 orifice plates was replaced by in 1-2 hour the RPMI1640 culture medium of serum-free, antibiotic-free, organizing added dosage by above grouping and each experimentizes, the operating process of plasmid DNA is seen in concrete operating process, and the complete medium that changed the culture medium in the hole into RPMI 1640+10% hyclone in 6 hours after the transfection is cultivated.

Experimental result is as follows: transfection is after 48 hours, 6 orifice plates are placed observation under the inverted microscope, find blank group and irrelevant sequence control group A 549 cell well-growns, pGen-HK plasmid transfection group has the 10-15% cell death, and the cell death of pGen-VEGF plasmid DNA transfection group 30-40%, show very strong inhibition growth of tumour cell and inducing apoptosis of tumour cell effect, and secreted VEGF reduced for 67% (as shown in Figure 3) than matched group in the experimental group cell conditioned medium.

The test example two pGen-VEGF plasmid/anti-people's pulmonary carcinoma of bFGF-Liposome complex animal experiment study

In order to study pGen-VEGF plasmid/bFGF-Liposome complex Graft Versus Tumor in vivo, in BALB/c nude mice (age in 6-8 week, female) subcutaneous people's pulmonary carcinoma transplanted tumor model of having set up, in brief, the human lung cancer cell A549 of In vitro culture is used trypsinization, and standardize solution is in 1640 culture medium of serum-free, antibiotic-free, and the cell concentration of adjustment is 5 * 10 ⁷Cells/ml is in the right side of every nude mice back of the body subcutaneous abdomen inoculation 5 * 10 ⁶Cells (0.1ml), cell inoculation can lay one's hand on after 7 days and to tumor, begin to treat (5 every group) by the following random packet of carrying out:

A) blank group: 5% glucose solution;

B) irrelevant sequence set: the pGen-HK/bFGF-Liposome complex places 5% glucose solution;

C) pGen-VEGF treatment group: the pGen-VEGF/bFGF-Liposome complex places 5% glucose.

Take the tail vein injection mode to treat, plasmid DNA/bFGF-Liposome complex proportioning is as follows: plasmid DNA (5 μ g): bFGF-Liposome (15 μ g)=1: 3 (mass ratio), and plasmid DNA-cation lipid nanocrystal composition is diluted in the glucose solution, and adjusts and make that the glucose final concentration is 5%.The per injection volume is 200 μ l, every other day treats, and treats altogether 12 times, measures 1 gross tumor volume in per 3 days, and gross tumor volume calculates by the following method: volume (mm ³)=0.52 * length (length) * wide ²(width ²).Tumor growth suppresses to use variance analysis, and P＜0.05 item thinks that statistical significance is arranged.When the execution animal is got tumor tissues each group tumor is weighed result's following (the results are shown in Figure 4).

Each group of experiment is not all observed obvious toxic and side effects, observes and mice is all put to death in 30 days.Find that from gross tumor volume curve (the results are shown in Figure 5), the tumor weight situation analysis of each above treated animal RNA interference treatment group tumor growth is slow, the pGen-VEGF/bFGF-Liposome complex shows strong antitumor action, compares inhibitory rate to 65% with matched group.

Above result shows that the pGen-VEGF/bFGF-Liposome complex can effectively suppress growth of tumor in people's lung cancer model, and what we used is a very low therapeutic dose.This points out us, in the clinical practice afterwards, can reduce the using dosage of pGen-VEGF/bFGF-Liposome complex greatly, improves therapeutic effect, reduces toxic and side effects simultaneously.

Test example three pGen-VEGF/bFGF-Liposome complex of the present invention and the test of cisplatin (cisplatin) coupling antitumor

In order to study the anti-pulmonary carcinoma effect that the pGen-VEGF/bFGF-Liposome complex adds chemotherapy, we are as previously mentioned in BALB/c nude mice (age in 6-8 week, female) subcutaneous people's pulmonary carcinoma transplanted tumor model of having set up, can lay one's hand on after 1 week and to tumor in cell inoculation, begin to carry out random packet treatment (5 every group) by following:

A) blank group: 5% glucose solution;

B) irrelevant sequence matched group: the pGen-HK/bFGF-Liposome complex places 5% glucose solution;

C) pGen-VEGF treatment group: pGen-VEGF/bFGF-Liposome places 5% glucose solution;

D) chemotherapy group: 0.1mg cisplatin (DDP);

E) irrelevant sequence adds chemotherapy group: the pGen-HK/bFGF-Liposome complex places 5% glucose solution, and the use of chemotherapeutics cisplatin DDP is as described below;

F) pGen-VEGF adds chemotherapy group: the pGen-VEGF/bFGF-Liposome complex places 5% glucose solution, and the use of chemotherapeutics cisplatin DDP is as described below.

Plasmid DNA/bFGF-Liposome complex takes the tail vein injection mode to carry out vivo medicine-feeding, plasmid DNA (5 μ g): bFGF-Liposome (15 μ g)=1: 3 (mass ratio), per injection 200 μ l volumes, every other day treat, treat 1 gross tumor volume of measurement in per 3 days altogether 12 times.

DDP takes the intraperitoneal administration mode to treat, and begins to carry out chemotherapy behind first time RNA interference treatment in 2 days, and weekly twice, in continuous 2 weeks, injected dose is the 5mg/kg body weight, the DDP of every mice per injection 0.1mg.Put to death animal at last and get tumor tissues, and each group tumor is weighed.Analysis result finds that the pGen-VEGF/bFGF-Liposome complex adds chemotherapeutics---cisplatin is used than these two kinds of medicine lists has stronger antitumous effect, total inhibitory rate to 80.7%, and simple pGen-VEGF treatment group (inhibitory rate to 61%) and plus cisplatin in treatment group (inhibitory rate to 47%), this shows that the RNA interference treatment of VEGF gene can strengthen the sensitivity of tumor to chemotherapeutics.Can be as the adjuvant drug of chemotherapy.

More than be result to people's lung cancer therapy, show this at the VEGF gene the RNA interference treatment and share the lung cancer therapy effect remarkable with chemotherapy.Vegf expression increases unusually in the malignant tumor such as pulmonary carcinoma, hepatocarcinoma, colon cancer, carcinoma of prostate, ovarian cancer, cancer of pancreas, cervical cancer, melanoma, incidence squamous cell carcinoma, the application by can this medicine of reasoning to the therapeutic outcome of pulmonary carcinoma and therapeutic modality above-mentioned other tumors are also had good therapeutic effect.

The above more excellent specific embodiment is that the present invention is further illustrated, but is not limitation of the scope of the invention, and those skilled in the art can make various modification or improvement according to basic thought of the present invention.Such as can be known by this area general knowledge, the length of Loop (catenation sequence) and form certain variation can be arranged in the expression cassette shelf structure among the present invention.And if different requirements arranged, the consumption of antineoplastic pharmaceutical compositions of the present invention and antitumour auxiliary drug compositions and occupation mode can change in a big way at one.Those skilled in the art can be according to some known factors, such as the kind of disease, and the degree that is in a bad way, patient body weight, dosage form, selected routes of administration etc. are determined at an easy rate.Only otherwise break away from basic thought of the present invention, these changes are all within the scope that spirit of the present invention and claims of being enclosed define.

SEQUENCE?LISTING

＜110〉Sichuan University

＜120〉complex and the preparation thereof of the shRNA expression vector of the liposome of bFGF modification and targeting human VEGF gene

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gcgcagatac?caaatactgt?ccttctagtg?tagccgtagt?taggccacca?cttcaagaac 3420

tctgtagcac?cgcctacata?cctcgctctg?ctaatcctgt?taccagtggc?tgctgccagt 3480

ggcgataagt?cgtgtcttac?cgggttggac?tcaagacgat?agttaccgga?taaggcgcag 3540

cggtcgggct?gaacgggggg?ttcgtgcaca?cagcccagct?tggagcgaac?gacctacacc 3600

gaactgagat?acctacagcg?tgagctatga?gaaagcgcca?cgcttcccga?agggagaaag 3660

gcggacaggt?atccggtaag?cggcagggtc?ggaacaggag?agcgcacgag?ggagcttcca 3720

gggggaaacg?cctggtatct?ttatagtcct?gtcgggtttc?gccacctctg?acttgagcgt 3780

cgatttttgt?gatgctcgtc?aggggggcgg?agcctatgga?aaaacgccag?caacgcggcc 3840

tttttacggt?tcctggcctt?ttgctggcct?tttgctcaca?tgttctttcc?tgcgttatcc 3900

cctgattctg?tggataaccg?tattaccgcc?atgcat 3936

<210>5

<211>86

<212>PRT

<213>artificial

<220>

<223>artificial

<400>5

Lys?Arg?Leu?Tyr?Cys?Lys?Asn?Gly?Gly?Phe?Phe?Leu?Arg?Ile?His?Pro

1 5 10 15

Asp?Gly?Arg?Val?Asp?Gly?Val?Arg?Glu?Lys?Ser?Asp?Pro?His?Ile?Lys

20 25 30

Leu?Gln?Leu?Gln?Ala?Glu?Glu?Arg?Gly?Val?Val?Ser?Ile?Lys?Gly?Val

35 40 45

Cys?Ala?Asn?Arg?Tyr?Leu?Ala?Met?Lys?Glu?Asp?Gly?Arg?Leu?Leu?Ala

50 55 60

Ser?Lys?Cys?Val?Thr?Asp?Glu?Cys?Phe?Phe?Phe?Glu?Arg?Leu?Glu?Ser

65 70 75 80

Asn?Asn?Tyr?Asn?Thr?Tyr

85

Claims (6)

1. liposome complex, it is characterized in that: the liposome that is combined into DOTAP, cholesterol and bFGF is an encapsulated layer, its proportioning is: the mol ratio DOTAP of DOTAP and cholesterol: cholesterol=2: 1～2, the weight content of bFGF polypeptide are by 20～80% of DOTAP and cholesterol gross weight; Seal the complex of the expression vector formation of the RNA interference sequence that can express human VEGF gene in vivo; Wherein, the aminoacid sequence of described bFGF is shown in SEQ ID NO.5, and the nucleotides sequence of the expression vector of the described RNA interference sequence that can express human VEGF gene is in vivo classified as shown in the SEQ ID NO.4.

2. liposome complex according to claim 1 is characterized in that: the weight content of described bFGF polypeptide is by 40～60% of DOTAP and cholesterol gross weight.

3. prepare the method for claim 1 or 2 described liposome complexes, it is characterized in that may further comprise the steps:

A, take by weighing DOTAP, cholesterol places to revolve and steams bottle, adds chloroform and makes it to dissolve fully;

B, rotary evaporation in vacuo get liposome membrane, drying;

C, in liposome membrane, add the ultrasonic aquation of 5% glucose solution and get blank liposome solutions;

D, bFGF is slowly joined in the blank liposome solution, slight concussion, hatching is spent the night, obtain blank liposome, liposome after the hatching is pressed through the polycarbonate membrane 4～8 times that the aperture is 0.45 μ m, the expression vector that can express the RNA interference sequence of human VEGF gene more in vivo mixes by 1: 2～3 weight ratio with liposome, at room temperature hatches 10～30 minutes, promptly.

4. claim 1 or 2 described liposome complexes are in the purposes of preparation in antitumor drug or antitumour auxiliary drug.

5. antitumor drug or antitumour auxiliary drug are to add pharmaceutically by claim 1 or 2 described liposome complexes as main active that the acceptable adjuvant is prepared from.

6. antitumor drug according to claim 5 or antitumour auxiliary drug is characterized in that: also contain tumor chemotherapeutic drug as active component.

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