CN1793372A - Non-viruse gene transferring vector and its preparation and application thereof - Google Patents

Abstract

The invention relates to non-virus gene transfer vector. Its structure is as follows: NH2-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-COOH. The invention has good target direction property for non virus gene transfer vector, high transfection efficiency, and can linkage with bone matrix material to increase cell specificity adherence and form gene carrying material to realize cell directional transfection. The invention also discloses the non-virus gene transfer vector manufacturing method and its application in medicine experimental study and clinic gene therapy.

Description

A kind of non-viruse gene transferring vector and preparation thereof and application

Technical field

The invention belongs to life science, more particularly it relates to a kind of non-viral gene transfection carrier and preparation and the application in medical experiment and clinical gene therapy.

Background technology

Gene transfer vector comprises virus vector and non-virus carrier two big classes.Compare with virus vector, non-viral vector have preparation easily, carry that the foreign gene capacity is big, non-immunogenicity, safety, economic dispatch advantage, thereby become an important research direction of gene transfer technique research field in molecular biology and the organizational engineering.

The research focus that improves non-viruse gene transferring vector at present has two.One of them research focus is gene activation matrix (GAM), its method is that goal gene is coated on the organizational project substrate material with three-dimensional porous structure, this year gene substrate material, the Growth and Differentiation that both can be cell provides sufficient nutrition and three-dimensional the support; Have huge surface-area again, can fully contact, obtain the gene transfection of greater efficiency with the seed cell of being planted.But the gene of GAM research at present just is coated in membranaceous polymkeric substance (as collagem membrane) surface simply, do not combine with part at the specific seed cell, so transfection efficiency is still undesirable; Can not realize at bone marrow stroma stem cell (bone marrow stromal cells, BMSCs) specific gene transfection and cell adhesion [Richardson T, Murphy W, Mooney D.Polymericdelivery of proteins and plasmid DNA for tissue engineering and genetherapy[J] .Crit Rev Eukaryot Gene Expr, 2001,11 (1-3): 47-58.]

Part-cationoid polymerisation objects system is another focus of non-viruse gene transferring vector research.Its principle is that cationic polymers and foreign gene form fine and close polymer by electrostatic interaction, and part combines with the corresponding receptor-specific of cell surface and pass through receptoe mediated endocytosis mechanism with goal gene transfered cell inside.Part can promote the efficient adhesion of carrier and seed cell, and the fine and close compound physical efficiency of DNA/ polycation prevents to be degraded by lysosomal enzyme after DNA from entering cell, and this rotaring redyeing system has significantly improved the transfection efficiency of foreign gene.Compare with the virus vector of routine, it is a kind of safe, special and can realize having great application prospect the method for direct directed metastatic gene.The common optional majority polylysine of polymkeric substance, poly ethyleneimine, cationic-liposome, histone, nonhistones and cyclodextrin polymkeric substance etc.; Part is then decided alcohol and monoclonal antibody etc. according to corresponding acceptor selection asialoglycoprotein, Transferrins,iron complexes, RGD peptide, Urogastron, lactose, seminose, low-density lipoprotein, Regular Insulin, fibroblast growth factor, nerve growth factor, blood vessel intestines polypeptide (VIP), neurotensin, no saliva base Pp63 glycophosphoproteins, selection element, folic acid, c-kit, the fluorine piperazine of cell.People have been developed multiple part-polymkeric substance gene transfer system in recent years, a series of RGD peptide [the Hart SL that comprise Hart SL design, Harbottle RP, Cooper R, et al, Gene delivery and expressionmediated by an integrin-binding peptide.Gene Ther 1995; 2:552; HarbottleRP, Cooper RG, Hart SL, et al.An RGD oligolysine peptide:a prototypeconstruct for integrin-mediated gene delivery.Hum Gene Ther1998; 9:1037], but the targeting specific of these carriers when all reckoning without bone tissue engineer seed cell-bone marrow stroma stem cell mediate foreign gene transfection also reckons without the prospect that is applied to bone tissue engineer research.

Summary of the invention

The objective of the invention is to overcome above-mentioned the deficiencies in the prior art part, and a kind of non-viral gene transfection carrier is provided.It is a kind of new non-viruse gene transferring vector, can efficient mediate foreign gene transfection various kinds of cell.

Another object of the present invention provides a kind of method for preparing above-mentioned non-viral gene transfection carrier.

A further object of the present invention is that this novel vector is applied to bone tissue engineer, makes up the bionical substrate material of energy mediate foreign gene transfection.

The objective of the invention is to reach by following measure: a kind of non-viruse gene transferring vector, its structure sequence is: NH2-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-COOH (Gly wherein, G-glycine, Arg, R-arginine, Asp, the D-aspartic acid, Ser, S-Serine, Pro, the P-proline(Pro), Cys, the C-halfcystine), be abbreviated as K16GRGDSPC.

The method for preparing above-mentioned a kind of non-viruse gene transferring vector; it comprises the steps: to adopt FMOC/tBu solid-phase polypeptide synthesis method that first amino acid-halfcystine is linked on the resin; slough the Fmoc blocking group of first amino acid-halfcystine; and then connection proline(Pro); the above step that circulates is until SDGRG and 16 Methionins are all connected the generation aminoterminal; after small peptide is synthetic; with trifluoroacetic acid solution peptide chain is cut down from resin; peptide is dissolved in TFA and filters with gel-filtration column, then uses anti-phase C ₁₈Analytical column filters, and is lyophilized into powdery then.

The application of above-mentioned non-viruse gene transferring vector in medical experiment research and clinical gene therapy is characterized in that the carrier of this peptide as transgenosis.

Compare with other non-virus carrier, K16-GRGDSPC carrier of the present invention has following advantage: [1] target is good, can be specifically at bone tissue engineer seed cell bone marrow stroma stem cell; Other non-virus carrier lacks this targeting specific (Fig. 3, table 1 and table 2); [2] transfection efficiency is higher, can be suitable with business-like liposome under the optimal conditions; Preparation is simple, can realize producing in enormous quantities by Peptide synthesizer; Unit price is far beyond liposome cheap (Fig. 4); [3] can stablize commissure with extracellular matrix material, strengthen the specific adhesion of bone tissue engineer seed cell, and make up the directed transfection of carrying genetic material realization pair cell; And other non-virus carrier all can not be stablized compoundly with extracellular matrix material, also just can't realize the stable genetic modification of extracellular matrix material (Fig. 9); [4] molecular weight is little, and the shortcoming of easy activating complement has the intravital good prospect that is applied in the time of can avoiding other non-virus carrier to be applied in the body; Seldom produce immune response, can not produce transgenation causes tumour to take place, security is wanted high [Collins L more than other non-virus carrier, Gustafsson K, Fabre J.Tissue-binding properties of a synthetic peptideDNA vector targeted to cell membrane integrins:a possible universalnonviral vector for organ and tissue transplantation [J] .Transplantation, 2000,69 (6): 1041-1050].In addition, the base of dredging is arranged on its terminal C (halfcystine) of this carrier, be convenient to combine, help compound mediated gene transfection then of RGD peptide and materials chemistry and material compound bone marrow stroma stem cell, carry out the research of bone tissue engineer genetic modification with linking agent.Simultaneously K16 is placed the aminoterminal of peptide chain, avoided the first-generation to contain the RGD peptide and placed carboxyl terminal to cause the defective that is difficult to synthetic RGD three peptide domains so that yields poorly K16.

Description of drawings

Fig. 1 is the mass spectrum of K16GRGDSPC;

Fig. 2 is the high pressure liquid chromatography figure (HPLC) of K16GRGDSPC;

Fig. 3 be different substrates to BMSCs and fibronectin (fibronectin, Fn) and vitronectin (vitronectin, Vn) competitive inhibition of handling material adhesion figure as a result;

Fig. 4 is K16GRGDSPC and liposome-mediated green fluorescence protein gene transfection figure (comprising Fig. 4 .1, Fig. 4 .2);

Fig. 5 is the optimization series of drawing (comprising Fig. 5 .1, Fig. 5 .2, Fig. 5 .3, Fig. 5 .4, Fig. 5 .5, Fig. 5 .6, Fig. 5 .7) of K16GRGDSPC mediated gene transfection condition;

Fig. 6 identifies the transfection of K16GRGDSPC mediation TGF-β 1 gene and expresses figure for WesternBlot;

Fig. 7 is that XPS detects K16GRGDSPC and PLGA-[ASP-PEG] the commissure figure (comprising Fig. 7 .1, Fig. 7 .2, Fig. 7 .3, Fig. 7 .4, Fig. 7 .5, Fig. 7 .6) of timbering material;

Fig. 8 detects BMSCs at PLGA-[ASP-PEG for electronic scanning Electronic Speculum (SEM)]-adhesion and the propagation graph of a relation on g-K16GRGDSPC surface;

Fig. 9 is that RT-PCR detects PLGA-[ASP-PEG]-the expression figure of g-K16GRGDSPC mediation TGF-β 1 gene transfection;

Embodiment

Describe performance of the present invention in detail below in conjunction with accompanying drawing:

The method for preparing a kind of non-viruse gene transferring vector of the present invention comprises the steps; Adopt FMOC/tBu solid-phase polypeptide synthesis method that first amino acid-halfcystine is linked on the resin; slough the Fmoc blocking group of first amino acid-halfcystine; and then connection proline(Pro); the above step that circulates is until SDGRG and 16 Methionins are all connected the generation aminoterminal; after small peptide is synthetic; with trifluoroacetic acid solution peptide chain is cut down from resin, peptide is dissolved in TFA and filters with gel-filtration column, then uses anti-phase C ₁₈Analytical column filters, and is lyophilized into powdery then.

The structure sequence of a kind of non-viruse gene transferring vector of the present invention is: NH2-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-COOH (Gly wherein, G-glycine, Arg, the R-arginine, Asp, D-aspartic acid, Ser, the S-Serine, Pro, P-proline(Pro), Cys, the C-halfcystine), be abbreviated as K16GRGDSPC.

Contain RGD peptide sequence K16RGD, K16GRGD, K16GRGDS and K16GRGDSP with the Peptide synthesizer synthetic, mensuration by cell adhesion rate and adhesive power, find adhesion property the best of RGD peptide K16GRGDSP and bone marrow stroma stem cell, thereby we preferentially select K16GRGDSP as part (table 1);

Table 1. bone marrow stroma stem cell and different adhesion rate and the adhesive powers that contain between the RGD peptide

	K16RGD	K16GRGD	K16GRGDS	K16GRGDSP
					8 hours adhesion rate (%) adhesive powers (* 10 -10N)	56.3±5.4 488.4±22.6	64.8±6.1 522.8±24.3	75.2±6.8 596.7±27.5	85.3±7.4 657.4±29.2

The result shows, between bone marrow stroma stem cell and the RGD peptide sequence K16GRGDSP the strongest adhesion property is arranged, thereby the present invention selects the K16GRGDSP sequence as part, simultaneously according to the needs of engineering material of bone tissue commissure, add that at its end C (Cys) is beneficial to and the material commissure.With detecting and identify (Fig. 1 and Fig. 2) with mass spectrograph and high pressure liquid chromatography behind the synthetic peptide K16GRGDSPC of Peptide synthesizer.

Mass spectrum (Mass spectrometry) show peptide molecular-weight average is 2746.26m/z, and consistent with the synthetic peptide molecular-weight average of Theoretical Calculation design, other has two foreign material, and molecular-weight average is respectively 2662m/z and 3373.24m/z (see figure 1).

High pressure liquid chromatograph (HPLC) shows that the purity of synthetic peptide is 94%-95%, satisfies experiment demand (see figure 2).

Fig. 3 is that different substrates are to BMSCs and fibronectin (fibronectin, Fn) and vitronectin (vitronectin, Vn) the competitive inhibition result of processing material adhesion shows: exogenous RGD peptide GRGDSPC and K16GRGDSPC can significance suppress the adhesion (P＜0.05) that BMSCs and Fn and Vn handle storeroom, but not RGD peptide GRGESPC, BSA, PBS and K16 all do not have this competitive inhibition.

Table 2. Luci plasmid expression detected result (* 10 ⁹Cpm)

n	K16GRGDSPC	K16GRGDSPC+chloroquine	K16GRGDSPC +PEI	lipofectamine	K16
							5	*10.20±1.72	**25.36±3.85	***18.70±2.75	#26.54±3.48	##3.47±0.64

With ^*Relatively, ^*With ^{* *}The P value all less than 0.05, ^*With ^#Relatively the P value is greater than 0.05

Table 2 is the result show: contain RGD peptide K16GRGDSPC energy mediate foreign gene Luci plasmid effective expression, chloroquine and polymine (PEI) all can improve its transfection efficiency, especially the chloroquine effect is more obvious, can obtain the transfection efficiency (P＞0.05) close with liposome (lipofectamine).

Table 3. transfection competitive inhibition experimental result (n=5)

	Contrast	Add GRGDSPC	Add K16GRGDSPC	Add GRGESPC
					Luciferin enzymic activity (* 10 9cpm)	*10.20±1.72	**3.66±0.87	#3.52±0.80	##9.96±1.65

With ^*Relatively, ^*With ^##The P value all less than 0.05, and ^##The P value greater than 0.05

Table 2 is the result show: exogenous RGD peptide GRGDSPC and K16GRGDSPC can significantly suppress the transfection (P＜0.05) of K16GRGDSPC mediate foreign gene.

Promptly find by cell adhesion and the test of adhesion competitive inhibition: K16GRGDSPC can significantly improve the adhesion rate of bone marrow stroma stem cell, the adhesion and the transfection of K16GRGDSPC energy competitive inhibition DNA/ carrier complexes TGF β 1-K16GRGDSPC and bone marrow stroma stem cell.

Fig. 4 can obtain the transfection efficiency close with liposome for the transfection of K16GRGDSPC mediation green fluorescence protein gene from Fig. 4, and wherein Fig. 4 .1 is the transfection of K16GRGDSPC mediation green fluorescence protein gene, and 4.2 is liposome-mediated green fluorescence protein gene transfection; By K16GRGDSPC mediation green fluorescence protein gene transfection experiment, can observe intuitively K16GRGDSPC successfully mediate foreign gene change bone marrow stroma stem cell and effective expression over to.

Fig. 5 is the optimization series of drawing of K16GRGDSPC mediated gene transfection condition, and wherein Fig. 5 .1 shows that chloroquine plays indispensable effect in transfection process; Fig. 5 .2 shows the necessary sufficiently long (＞8 hours) of time that bone marrow stroma stem cell exposes in chloroquine solution; Fig. 5 .3 shows that bone marrow stroma stem cell exposure duration in carrier/plasmid composite too short (＜4 hours) or long (＞24 hours) are all unfavorable to transfection; Fig. 5 .4 shows that serum has obvious suppression to transfection; Fig. 5 .5 shows that the best plasmid concentration of transfection is 2～4 μ g/ holes (24 orifice plate); Fig. 5 .6 shows that plasmid/carrier mass ratio is in 1:(2～4) time can obtain best transfection effect; Fig. 5 .7 shows that Transferrins,iron complexes can strengthen the efficient that contains the transfection of RGD peptide, reaches best when 25 μ g.

Detect TGF-β 1 expression of gene by ELISA, optimized the condition of K16-GRGDSPC mediated gene transfection.

Fig. 6 is that WesternBlot identifies the transfection and the expression of K16GRGDSPC mediation TGF-β 1 gene, and wherein 1 and 2 is the transgenosis group, and 3 are transgenosis group not, and the result shows: K16GRGDSPC can mediate the transfection and the expression of TGF-β 1 gene.

Fig. 7 is that XPS detects K16GRGDSPC and PLGA-[ASP-PEG] commissure of timbering material.Wherein Fig. 7 .1 and Fig. 7 .2 are PLGA-[ASP-PEG] XPS of material figure, no element sulphur waveform; Fig. 7 .3 and 7.4 is PLGA-[ASP-PEG] with Sulfo-SPDP coupling agent commissure after XPS figure, show the element sulphur waveform, the bound energy of element sulphur is 162eV (sulphur and a phenyl ring key, S-Ben) with 164eV (disulfide linkage, S-S) two peaks, the element sulphur peak is too small, Fig. 7 .3 poor display.Reaction back C/S is 99.574: 0.4255.Fig. 7 .5 and 7.6 is for PLGA-[ASP-PEG] with the XPS figure that contains behind the RGD peptide K16GRGDSPC commissure, show the element sulphur waveform, the bound energy at element sulphur peak is at 164eV, C/S is 99.746: 0.1014, and polypeptide chain has been connected to PLGA-[ASP-PEG behind the proved response] with linking agent generation covalent attachment after disulfide bond (2-pyridyl disulfide) group of linking agent on.

Fig. 8 detects BMSCs at PLGA-[ASP-PEG for electronic scanning Electronic Speculum (SEM)]-adhesion and the propagation on g-K16GRGDSPC surface.

Table 4 shows K16GRGDSPC commissure PLGA-[ASP-PEG] after can significantly improve its adhesion property

	4h adhesion rate (%)	8h adhesion rate (%)	12h adhesion rate (%)
				The non-material modified film of material modified film (contrast)	55.6±6.4 31.7±5.2	78.4±8.6 48.8±5.9	85.7±9.1 54.2±6.3

Table 5 shows K16GRGDSPC commissure PLGA-[ASP-PEG] also there is promoter action the back to the growth of bone marrow stroma stem cell

The cell protein content measurement result

	4d	8d	12d
				The non-material modified film of material modified film (contrast)	68.8±3.5 40.2±2.8	108.4±6.6 83.7±5.4	116.3±7.1 110.8±6.9

Table 6 shows that ELISA detects PLGA-[ASP-PEG]-expression of g-K16GRGDSPC mediation TGF-β 1 gene transfection

	72h	The 1st week	The 2nd week	The 3rd week	The 4th week
						The transgenosis group is transgenosis group group not	48.8±2.8 3.5±0.5	19.8±1.7 3.7±0.6	25.6±1.9 3.6±0.4	49.2±2.6 3.9±0.5	52.4±2.8 4.0±0.6

Fig. 9 is that RT-PCR detects PLGA-[ASP-PEG]-expression of g-K16GRGDSPC mediation TGF-β 1 gene transfection.1 is transgenosis group not among Fig. 9, and 2 is the transgenosis group.

Above-mentioned accompanying drawing 6～Fig. 9 by XPS detect to find can be successfully with the K16-GRGDSPC commissure to macromolecular scaffold material PLGA-[ASP-PEG] on, that advances-go on foot studies show that the PLGA-[ASP-PEG that utilizes the K16-GRGDSPC modification] its biocompatibility of material significantly improves, and can realize transfection and the expression of mediate foreign gene to bone marrow stroma stem cell.

Concrete confirmatory experiment is as follows: non-viruse gene transferring vector of the present invention is done mass spectroscopy with final definite synthetic peptide molecular weight.Target peak is got sample after the freeze-drying, does stratographic analysis to determine its purity (seeing Fig. 1 and Fig. 2).The present invention designs synthetic peptide carrier K16GRGDSPC; the present invention places the aminoterminal of peptide chain with the K16 chain, because the fourth carboxyl that K16 protectiveness group produces has hydrophobicity, if K16 places carboxyl terminal; to suppress the combination of RGD peptide, then be difficult to synthetic RGD three peptide domains.Synthesize earlier and contain the RGD small peptide, add K16 at its aminoterminal again, then make it to become wetting ability.In addition, the base of dredging is arranged on the C (halfcystine), be convenient to combine, for following-step prepares with materials chemistry is compound with linking agent.Synthetic product is made into 1mg/ml solution with HEPES damping fluid (pH value 7.3) ,-20 ℃ of preservations after the room temperature shaken overnight.With synthetic RGD peptide sequence K16RGD, K16GRGD, K16GRGDS and the K16GRGDSP of containing of method.The above-mentioned RGD peptide 50 μ l of difference in 24 orifice plates, 37 ℃ are spent the night so that it fully is adsorbed on the diapire.The bone marrow stroma stem cell of taking the logarithm vegetative period (BMSCs) is adjusted cell concn to 1 * 10 with the DMEM substratum that contains 10%FBS ⁶/ ml, every hole 1.5ml, the conventional cultivation after 8 hours with the nutrient solution sucking-off, and with the 1.5mlPBS back sucking-off of vibrating gently, add the 1.5ml substratum again and continue to cultivate.Cell concentration (N) in the counting 3.0ml sucking-off liquid also calculates cell adhesion rate (adhesion rate=1-N/1.5 * 10 ⁶).Adhesive power between the flat board of employing microtubule sucking experimental system detection bone marrow stroma stem cell and coated RGD peptide.If the microtubule inside radius is R, the isolating critical negative pressure of cell and respective material film surface adhesion is Δ P, calculates adhesive power (F)=Δ P * π * R ²Every group is detected 30 cells (seeing Table 1).

Cell adhesion competitive inhibition experiment: with fibronectin (fibronectin, Fn) or vitronectin (vitronectin, Vn) (the two is the RGD peptide that contains the RGD structural domain) is made into 1mg/ml solution with PBS, and the every hole of 96 orifice plates adds 50 μ l, and 37 ℃ are spent the night so that it fully is adsorbed on the diapire.1h is handled with PBS37 ℃ that contains 3%BSA (bovine serum albumin) in every subsequently hole.The bone marrow stroma stem cell of taking the logarithm vegetative period (BMSCs) is adjusted cell concn to 1 * 10 with the DMEM substratum that contains 10%FBS ⁶/ ml adds peptide GRGDSPC, GRGESPC, K16, K16-GRGDSPC, BSA and PBS respectively in cell suspension, 1h gently vibrates under 4 ℃ of conditions.In 96 orifice plates of handling, add cell suspension 50 μ l/ holes (5 * 10 ⁴Individual), measure each hole absorbance by mtt assay behind 37 ℃ of conventional cultivation 4h.The result shows owing to containing the integrin receptor that RGD peptide GRGDSPC and K16GRGDSPC have sealed the bone marrow stroma stem cell surface, thereby antagonism part fibronectin (Fn) and vitronectin (Vn) combine with the specificity of the integrin receptor on bone marrow stroma stem cell surface, significantly reduced the adhesion of bone marrow stroma stem cell on the 96 orifice plate diapires of handling through Fn and Vn; In contrast, GRGESPC, K16, BSA and PBS pair cell adhere to not produce obviously influences (see figure 3).

With K16GRGDSPC is the detection of expression of carrier transfection Luci plasmid: get third generation bone marrow stroma stem cell, with 5 * 10 ⁴/ hole is inoculated in conventional cultivation the in 24 orifice plates, carries out transfection during 70% fusion.Preparation transfection composite: get Luci plasmid 2 μ g, mixing is in 100 μ lDMEM, other gets 6 μ g K16-GRGDSPC oligopeptides, also mixing is in 100 μ lDMEM, behind the 10min oligopeptide solution is joined in the plasmid solution, abundant incubated at room 30min behind the mixing joins with DMEM with the 0.8mlDMEM mixing and washs in three times 24 orifice plates.The conventional cultivation adds the DMEM substratum 1ml that contains 20% new-born calf serum in the incubator after 4 hours, removes plasmid-carrier complexes after 24 hours, changes the conventional cultivation of the DMEM substratum that contains 10% new-born calf serum.Is carrier transfection Luci plasmid with method with cationic-liposome (lipofectamine) and K16.Detect the expression of luciferase gene behind the transfection 48h.Press the explanation of Luci detection system, sop up the substratum in 24 orifice plates.Wash 3 times with PBS.Add 150 μ l histolysis damping fluids, cracking 15min scrapes cell with curet under the room temperature, changes in the EP pipe.12000r/min under the room temperature, centrifugal 10min changes supernatant liquor over to another EP pipe.Get 20 μ l supernatant liquors and add 100 μ l luciferin enzyme reaction substrates.On the BeckmanLS-5000 liquid scintillation counter, utilize the single photon counting program, measure the number of photons (cpm) that adds the 1min internal reaction system release behind the testing sample in the reactive system, come the activity of contained Luci in the assess sample with this.Observe the influence of lysosome inhibitor to oligopeptides carrier transfection efficiency simultaneously: (1) chloroquine adds chloroquine to influencing transfection composite solution and containing in the DMEM substratum of 20% new-born calf serum of oligopeptides carrier transfection efficiency, makes chloroquine concentration 100 μ M in the transfection solution.(2) PEI adds 5 μ lPEI to influencing of oligopeptides carrier transfection efficiency among the transfection composite DNA/K166RGDSPC, fully joins in the cell behind the mixing 10min.The result shows, K16GRGDSPC can successfully mediate the transfection and the expression of luciferase gene, but single is that the efficient of carrier mediated transfection is lower with K16GRGDSPC, and the use of lysosome inhibitor chloroquine and PEI can significantly improve the transfection efficiency of K16GRGDSPC carrier, and wherein the effect of chloroquine is more obvious.The efficient close with liposome lipofectamine transfection efficiency (seeing Table 2) of K166RGDSPC mediated gene transfection behind the adding chloroquine.

Transfection competitive inhibition experiment: before adding transfection composite K16GRGDSPC/DNA, add peptide GRGDSPC, GRGESPC and K16GRGDSPC earlier.After the result showed adding peptide GRGDSPC and K16GRGDSPC, the expression activity of Luci obviously descended, and GRGESPC does not have obvious influence (seeing Table 3) to transfection.This further specifies the K16GRGDSPC mediated gene transfection is to realize that with combining of its acceptor the destruction of RGB structural domain (as changing into RGE) will cause the forfeiture of the carrier mediated transgenosis function of peptide by the RGD structural domain.

With the K16GRGDSPC oligopeptides is carrier transfection pEGFP plasmid: get third generation bone marrow stroma stem cell, the conventional cultivation in 6 orifice plates carried out transfection during 70% fusion.Get plasmid 2 μ g, mix in 100 μ lDMEM, other gets 6 μ g K16GRGDSPC oligopeptides, also mix in 100 μ lDMEM, behind the 10min oligopeptides is joined in the plasmid solution, incubated at room 30min behind the mixing joins in 6 orifice plates with DMEM washing three times with the abundant mixing of 1.3mlDMEM again.The conventional cultivation adds the DMEM substratum 1.5ml that contains 20% new-born calf serum in the incubator after 4 hours, removes plasmid-carrier complexes after 24 hours, changes the conventional cultivation of the DMEM substratum that contains 10% new-born calf serum.More than all contain the chloroquine that final concentration is 100 μ M among the used DMEM.Continue to cultivate after 48 hours and under fluorescent microscope, observe.Be that carrier carries out transfection with method with liposome (lipofectamine) simultaneously.With the pcDNA3 transfection is control group.5 high power lens visuals field of picked at random under the fluorescent microscope, counting gene transfection efficient, and relatively the transfection efficiency of two kinds of carriers has indifference.Found that oligopeptides and liposome group be all visible bright green fluorescence under fluorescent microscope, control group does not have luciferase expression.Oligopeptides transfection group transfection positive rate is 18.6%, and liposome transfection group transfection positive rate is the no significance difference opposite sex (P＞a 0.05) (see figure 4) between 19.3%, two group.

WesternBlot identifies TGF β 1 expression of gene: transfection after 72 hours with the conditioned medium screening that contains G418 (400 μ g/ml) after positive colony occurs, choose clone and enlarged culturing, the final stably transfected cell line that obtains, called after BMSc-TGF β 1, identify TGP β 1 expression of gene by the method for WesternBlot, the DAB colour developing finds that positive band appears in experimental group, and control group does not then have (see figure 6).

Transfection pTGF β 1 emiocytosis TGF β 1 detects: the BMSc-TGF β 1 and the BMSc-pcDNA3 cell of the growth of taking the logarithm, serum-free culture was collected supernatant after 48 hours, behind the TGF β 1, be made into complex medium with the substratum that contains 0.2%BSA in 1: 1 ratio respectively in the activation supernatant.MV1 is pressed 5 * 10 ³Individual/hole is inoculated in 96 orifice plates, adds complex medium 100 μ l/ holes, with the substratum that contains 0.1%BSA in contrast.Adopt mtt assay to detect MV1 cell growth-inhibiting situation after 36 hours.MTT detected result BMSc-TGF β 1, BMSc-pcDNA3 and control group are respectively 0.762 ± 0.05l, 1.036 ± 0.042 and 0.984 ± 0.039.Compare with control group, growth has obvious inhibition (P＜0.05) to BMSc-TGF β 1 supernatant to MV1, and BMSc-pcDNA3 does not then show obvious suppression effect (P＞0.05).

In order to make the peptide carrier can bring into play maximum transfection usefulness, the present invention further optimizes its transfection conditions.Under the condition that keeps the other factors unanimity, screen certain optimum parameter by changing monofactor.We choose TGF β 1 as goal gene, detect its expression by the ELISA method.Get third generation marrow stromal cell, with 5 * 10 ⁴/ hole is inoculated in 24 orifice plates, carries out transfection during 70%～80% fusion.Preparation transfection composite: get plasmid 2 μ g, mixing is in 100 μ lDMEM, other gets 6 μ g K16GRGDSPC oligopeptides, also mixing is in 100 μ lDMEM, behind the 10min oligopeptide solution is joined in the plasmid solution, abundant incubated at room 30min behind the mixing adds the 0.8mlDMEM mixing again and joins in 24 orifice plates with serum-free DMEM washing 3 times.The conventional cultivation adds the DMEM substratum 1ml that contains 20% new-born calf serum in the incubator after 4 hours, removes plasmid-carrier complexes after 24 hours.Set the standard transfection step of above operation steps for this experiment.The chloroquine (chloroquine) that all contains final concentration 100 μ M among the above DMEM.Change the conventional cultivation of the DMEM substratum 2ml that contains 10% new-born calf serum after removing plasmid-carrier complexes.Expression with ELISA test kit detection TGF β 1 in 48 hours after the transfection.(1) effect of lysosome inhibitor chloroquine (chloroquine) in K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1 process we as can be seen, though do not use the chloroquine transfection efficiency to be height in the whole transfection process, be starkly lower than standard transfection group (P＜0.01) than the empty carrier group; And only preceding 4 hours without chloroquine, to transfection results influence influence and little (with standard group P＞0.05 relatively).This explanation chloroquine is indispensable reagent in transfection process, especially in middle and later periods of transfection.(2) BMSCs in chloroquine solution exposure duration to after the influencing BMSCs and in the carrier/plasmid composite that does not contain chloroquine, cultivate 4 hours of K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1, add the 20% new-born calf serum substratum 1ml that contains chloroquine 200 μ M, the chloroquine final concentration that makes transfection solution is 100 μ M.Continue cultivation 1,2,4,8,12,16 or change conventional culture medium culturing after 20 hours.The prolongation of the result shows BMSc in chloroquine solution exposure duration is favourable to the raising of transfection efficiency, and exposure duration should keep more than 8 hours at least.(3) BMSc in carrier/plasmid composite exposure duration to the influence of K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1.The removal transfection composite changes and contains the conventional cultivation of 10%NBS substratum (containing final concentration 100 μ M chloroquines in the nutrient solution all the time) BMSc exposes 0.5,1,2,4,8,12,16,20,24 and 30 hour respectively in carrier/plasmid composite after.Found that all there is disadvantageous effect BMSc exposure duration in carrier/plasmid composite too short (＜4 hours) or long (＞24 hours) to transfection.(4) serum adds NBS to the influence of K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1 in preceding 4 hours plasmid/carrier complexes of transfection, makes transfection composite contain the serum of different concns.The result shows that serum is to the obvious restraining effect of being combined with of carrier/plasmid composite and cell, and after especially serum-concentration reached 20%, the expression of TGF β 1 obviously was subjected to press down.(5) plasmid concentration remains at plasmid/carrier mass ratio the influence of K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1 under 1: 3 the situation, make every hole plasmid concentration be respectively 1 μ g, 2 μ g, 4 μ g, 8 μ g and 16 μ g, carry out transfection by standard step.Found that plasmid concentration transfection best results when 2～4 μ g, along with further increasing of plasmid concentration, transfection efficiency does not but increase thereupon.(6) plasmid/carrier mass ratio remains at plasmid concentration under the situation of every hole 2 μ g all the time to the influence of K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1, by changing the influence of plasmid/carrier mass ratio observation to the K16GRGDSPC transfection.The result shows, plasmid/carrier mass ratio is that higher transfection efficiency was arranged in 1: 2～4 o'clock; Higher mass ratio causes transfection efficiency to descend on the contrary.(7) Transferrins,iron complexes to K16GRGDSPC oligopeptides transfection pcDNA3-TGF β 1 influence 2 μ g pcDNA3-EGFP plasmids and 6 μ gK16-GRGDSPC oligopeptides add 5～45 μ g Transferrins,iron complexes transfection bone marrow stroma stem cells, transfection efficiency progressively improves with the increase of Transferrins,iron complexes concentration, transfection efficiency reaches maximum when 25 μ g, further increase Transferrins,iron complexes concentration, transfection efficiency does not increase.Externally add 25 μ g Transferrins,iron complexess with 6 μ gK16GRGDSPC oligopeptides and mediate 2 μ g pcDNA3-TGF β, 1 transfection bone marrow stroma stem cell, transfection records TGF β 1 after 72 hours expression amount is 64.4 ± 4.3ng/ml, expression amount 48.6 ± the 3.5ng/ml that does not add the control group TGF β 1 of Transferrins,iron complexes, (P＜0.01) (see figure 5) is significantly improved.

Studies show that more than K16GRGDSPC can strengthen sticking of cell, and,, under various optimal conditions, can obtain the transfection efficiency close with liposome successfully with the foreign gene transfered cell as the novel targeted property of a kind of ideal non-virus carrier.The polypeptide of the present invention design has the liposome can not be than the significant advantage of analogy, be exactly it can with the material commissure, increase the adhesion of cell on timbering material, improve the biocompatibility of timbering material, and the parcel several kinds of target gene is carried out body point gene expression decided at the higher level but not officially announced.The present invention selects for use TGF β 1 gene to describe as the research target gene.

PLGA-[ASP-PEG] with the combining and detection of small peptide: with PLGA-[ASP-PEG] be made as diameter 10mm, 3 thin slices that thickness 0.25mm is identical, be numbered thin slice A, B, C respectively, placed disinfection by ultraviolet light 4 hours, tri-distilled water flushing 5 times, use PBS liquid (phosphate buffered saline buffer PH=8) flushing 5 times at last, chips C places freeze-drying preservation under 4 ℃ of conditions.Thin slice A and B place reactor, add 1ml 1 * PBS liquid (PH=8) respectively, add each 10mg of Sulfo-LC-SPDP again, place oscillator reaction 48 hours under the normal temperature, finish the back and take out two PLGA-[ASP-PEG], wash repeatedly three times with PBS liquid, get thin slice B and preserve 4 ℃ of freeze-drying.Reacted thin slice A inserts and rinses well in the reactor, gets K16-GRGDSPC peptide solution 1ml (small peptide content is 1mg/ml) and adds in the reactor, and the concussion reaction is 48 hours under the room temperature, and reaction back thin slice A takes out, and 1 * PBS liquid (PH=8) washes three times repeatedly.XPS detects PLGA-[ASP-PEG] the grafting situation of material surface K16GRGDSPC peptide.

K16GRGDSPC modifies PLGA-[ASP-PEG] timbering material is to the influence of its cell in vitro biocompatibility: (1) precipitator method detect the adhesion rate of bone marrow stroma stem cell: material modified film is put in 24 orifice plates, with the compound cultivation of third generation bone marrow stroma stem cell, inoculating cell concentration is 1 * 10 ⁶/ ml, every hole 1.5ml.The conventional cultivation after 4 hours with the nutrient solution sucking-off, and with the 1.5mlPBS back sucking-off of vibrating gently, add the 1.5ml substratum again and continue to cultivate.Cell concentration (N) in the counting 3.0ml sucking-off liquid also calculates cell adhesion rate (adhesion rate=1-N/1.5 * 10 ⁶).With the non-material modified film of identical table area in contrast.Detect the adhesion rate of bone marrow stroma stem cell after 8 hours and 12 hours with method.(2) the microtubule sucking method adhesive power that detects bone marrow stroma stem cell adopts microtubule to suck experimental system and detects bone marrow stroma stem cell and modification PLGA-[ASP-PEG] adhesive power of film.Method is the same.(3) cell protein content detects: adopt the Xylene Brilliant Cyanine G assay method.Get 4,8,12 days the bone marrow stroma stem cell in inoculation back, put 100 ℃ of 30min, make the abundant cracking of cell with 0.4mol/LNaOH.Get cell pyrolysis liquid 100 μ l and add Xylene Brilliant Cyanine G 1.0ml mixing, measure solution absorbency value (A) at 595nm wavelength place with ultraviolet spectrophotometer behind the placement 10min.With the solvent is blank, with bSA (BSA) be the standard substance curve plotting as a reference.The result is presented at the cell adhesion rate of the material modified film of different time points and adhesive power all apparently higher than control group (P＜0.01); The speed of growth of material modified film compound cells is also obviously faster than control group (see Table 4 and table 5).These show that K16GRGDSPC can significantly improve the biocompatibility of timbering material.This is the advantage that other carrier can not have.

K16GRGDSPC commissure PLGA-[ASP-PEG] the material modified film that forms of back places 24 orifice plates, adds TGF β 1 cdna solution 1.0ml (mrna concentration is 0.05mg/ml), and normal temperature is hatched behind the 30min BMSCs with logarithmic phase with 1 * 10 ⁶The concentration of the every hole 1ml of/ml adds, and continues to cultivate, and detects the expression concentration of TGF β 1 after the transfection when 72h, 1 week, 2 weeks, 3 weeks, 4 weeks.The result shows, TGF β 1 gene continued for 4 weeks all apparently higher than control group, can think K16GRGDSPC-g-PLGA-[ASP-PEG] successfully mediate goal gene TGF β 1 and change BMSCs with the compound cultivation of timbering material over to, and TGF β 1 gene is more than at least 4 weeks of timbering material surface continuous expression (seeing Table 6).We identify K16GRGDSPC oligopeptides transfection pTGF β 1 plasmid expression by RT-PCR: transfection with tryptic digestion collecting cell BMSc, BMSc-TGF β 1 and BMSc-pcDNA3, was extracted total RNA and is carried out RT-polymerase chain reaction (RT-PCR) amplification TGF β 1cDNA after 72 hours.We have designed the TGF β 1cDNA of a pair of primer with amplification 582bp, introduce the KpnI enzyme at two ends and cut .P1:5 ' TCTGGTACCTGAACCCGTGTTGCT-3 '; P2: ' TTCGGTACCTTGCTGTACTGCGT-3 '.Reaction conditions is the first round 94 ℃ of sex change 4min, 94 ℃ of reaction 45s, and 64 ℃ are reacted 50 ℃, 72 ℃ of reaction 2min, 5 circulations.The 2nd takes turns 94 ℃ of reaction 45s, 65 ℃ of reaction 45s, 72 ℃ of reaction 1min, 35 circulations.Behind 72 ℃ of reaction 10min, electrophoresis observation finds that BMSc-TGF β 1 groups of cells has the specific PCR amplified production at about 580bp place, and BMSCs-pcDNA3 and BMSCs cell there is no positive band (see figure 9).Therefore, we can think the PLGA-[ASP-PEG that K16GRGDSPC modifies] material membrane K16GRGDSPC-g-PLGA-[ASP-PEG] can be the same with free K16GRGDSPC peptide, the mediate foreign gene transfection.But the K16GRGDSPC-g-PLGA-[ASP-PEG after modifying with the K16GRGDSPC peptide] have a special advantages, it can strengthen the adhesivity and the adhesive power of the seed cell of plantation, and make goal gene continuous expression product on material membrane of importing, performance biological effect.

Sequence table

＜110〉Wuhan Union Hospital

＜120〉a kind of non-viruse gene transferring vector and preparation thereof and application

＜130〉do not have

<160>1

<170>PatentIn version 3.1

<210>1

<211>23

<212>PRT

＜213〉artificial sequence

<400>1

COOH-Cys Pro Ser Asp Gly Arg Gly Lys Lys Lys Lys Lys Lys Lys Lys Lys

1 5 10 15

Lys Lys Lys Lys Lys Lys Lys-NH2

20

Claims (3)

1. a non-viruse gene transferring vector is characterized in that its structure is: NH2-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-lys-Gly-Arg-Gly-Asp-Ser-Pro-Cys-COOH.

2. the method for preparing the described a kind of non-viruse gene transferring vector of claim 1; it is characterized in that it comprises the steps: to adopt FMOC/tBu solid-phase polypeptide synthesis method that first amino acid-halfcystine is linked on the resin; slough the Fmoc blocking group of first amino acid-halfcystine; and then connection proline(Pro); the above step that circulates is until SDGRG and 16 Methionins are all connected the generation aminoterminal; after small peptide is synthetic; with trifluoroacetic acid solution peptide chain is cut down from resin; peptide is dissolved in TFA and filters with gel-filtration column, then uses anti-phase C ₁₈Analytical column filters, and is lyophilized into powdery then, promptly gets non-viruse gene transferring vector of the present invention.

3. the application of non-viruse gene transferring vector in medical experiment research and clinical gene therapy is characterized in that the carrier of this peptide as transgenosis.

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