CN102191209A - VEGF165 and Ang-1 double-gene co-expression vector and application thereof - Google Patents
VEGF165 and Ang-1 double-gene co-expression vector and application thereof Download PDFInfo
- Publication number
- CN102191209A CN102191209A CN 201010263189 CN201010263189A CN102191209A CN 102191209 A CN102191209 A CN 102191209A CN 201010263189 CN201010263189 CN 201010263189 CN 201010263189 A CN201010263189 A CN 201010263189A CN 102191209 A CN102191209 A CN 102191209A
- Authority
- CN
- China
- Prior art keywords
- ires
- ang
- vegf165
- gene
- epcs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a VEGF165 and Ang-1 double-gene co-expression vector. The double-gene co-expression vector contains a human gene VEGF165 and a human Ang-1 gene; the double-gene co-expression vector is pAdTrack-CMV-Ang-1-IRES-VEGF165; the double genes are recombined to transfer vectors; the double genes are in homologous recombination with the adenovirus framework plasmid pAdEasy-1 and then are packed and augmented in QB 1-293A cells so as to obtain an Ad-Ang-1-IRES-VEGF165 double-gene co-expression recombination adenovirus; and finally a double-gene recombination adenovirus carrying the VEGAF and the Ang-1 infects vessel endothelial progenitor cells so as to promote the angiogenesis of ischemic myocardium and improve functions of the heart.
Description
Technical field
The invention belongs to the recombinant vectors field, be specifically related to the application in VEGF165 and the Ang-1 double gene coexpression preparing carriers promotion angiogenesis medicament.
Background technology
As everyone knows, in angiogenesis, there are many cells and chemokines to participate in regulation and control.Think at present: VEGF (vascular endothelial growth factor) has specificity to be promoted endothelial cell proliferation, brings out the new vessel growth, forms biological actions such as side Zhi Xunhuan then, in the initial and maintenance stage of angiogenesis effect is arranged all; FGF (fibroblast growth factor) mainly participates in the initial stage process; And Ang-1 (angiogenin), IGF (rhIGF-1) mainly influence the maturation of blood vessel with stable with PDGF (Thr6 PDGF BB).Experimental study discloses: the exogenous VEGF gene therapy may obviously improve the perfusion and the function of ischemic myocardium, and obtained certain curative effect (referring to Losordo DW, Vale PR, Symes JF, et al.1998,98:2800-2804.).But some experiments prompting successively recently: when using VEGF separately, even what use is therapeutic dose, the obvious oedema that might cause local organization is (referring to Dor Y, DjionovV, Abramovitch R, et al.2002,21 (8): 1939-1947.), the necrosis of ischemic limb is (referring to Masaki L, Yonemitsu Y, Yamashita A, et al.2002,90 (9): 966-973.), vascular tumor forms and heart function deterioration etc. (referring to Lee RJ, Springer ML, Blanco-Bose WE, et al.2000,102:898-901.).Therefore, when effectively bringing into play the therapeutic action of VEGF angiogenesis, inquiring into scheme how to avoid and reduce its untoward reaction, is the clinical problem that presses for solution, and the combination therapy of gene may be one of terms of settlement.
In recent years, gene combination therapy myocardial ischemia becomes one of focus of research, has research to use VEGF and two kinds of genes of Ang-1 respectively at the ischemic local injection, and the area of myocardial infarction dwindles, point out this two kinds of gene therapy synergies, in view of similar research all is twice or carries out gene transfection respectively, be mostly to be applied in the part of ischemic tissue, as ischemic lower limb or cardiac muscle (referring to Jho D, Mehta D, Ahmmed G, etal.2006,96 (12): 1282-1290; Shyu KG, Chang H, Isner JM.Life Sciences, 2003,73 (20): 563-579.).Combined gene therapy previously all is that two carriers carry two genes respectively, successively or simultaneously gives, and needs twice gene transfection when therapeutic transgene, and two genes when giving respectively dosage be difficult to shortcomings such as Coordination calculation and gene transfering efficiency are low.Do not see as yet: carry two kinds of genes that promote vasculogenesis by same adenovirus carrier, and in endothelial progenitor cell (EPCs), express jointly, the associating gene promotes ischemic region angiogenesis and the report that improves heart function in conjunction with the cellular replacement therapy ischemic myocardium.
Summary of the invention
The object of the invention provides a kind of VEGF165 and Ang-1 double gene coexpression carrier.
For achieving the above object, the technical solution used in the present invention is: a kind of VEGF165 and Ang-1 double gene coexpression carrier, described double gene coexpression carrier contains people's gene VEGF165 and people Ang-1 gene, and described double gene coexpression plasmid is pAdTrack-CMV-Ang-1-IRES-VEGF165.
Further in the technical scheme, after dual-gene recombinant transfer plasmid of above-mentioned pAdTrack-CMV-Ang-1-IRES-VEGF165 and adenovirus skeleton plasmid pAdEasy-1 homologous recombination, packing and amplification obtain Ad-Ang-1-IRES-VEGF165 double gene coexpression recombinant adenovirus in the QBI-293A cell, so that infect endothelial progenitor cell (EPCs), promote the angiogenesis of ischemic myocardium and improve heart function with the dual-gene recombinant adenovirus that carries VEGF and Ang-1.
Therefore, the application of the dual-gene recombinant transfer vector of the claimed pAdTrack-CMV-Ang-1-IRES-VEGF165 of the present invention in the angiogenesis medicament of preparation promotion ischemic myocardium.
Simultaneously, the application of the claimed Ad-Ang-1-IRES-VEGF165 double gene coexpression of the present invention recombinant adenovirus in the angiogenesis medicament of preparation promotion ischemic myocardium.
Adopt routine techniques that the dual-gene recombinant transfer plasmid of above-mentioned pAdTrack-CMV-Ang-1-IRES-VEGF165 is imported in the host bacterium intestinal bacteria, carry out preservation then, this colibacillary preservation information is: depositary institution: Chinese typical culture collection center; Address: Chinese Wuhan University; Preservation date: on May 16th, 2010; Deposit number CCTCC NO:M 2010117; Classification name: bacillus coli DH 5 alpha/pAdTrack-CMV-hAng-1-IRES-hVEGF165; Escherichia coli DH5 α/pAdTrack-CMV-hAng-1-IRES-hVEGF165.
The preparation method of the dual-gene recombinant transfer vector of above-mentioned pAdTrack-CMV-Ang-1-IRES-VEGF165 may further comprise the steps:
(1) IRES (the internal ribosome insertion site) conserved sequence of reporting according to GenBank, design primer P1, P2, to contain the segmental pGEZ-Term plasmid of IRES is template pcr amplification IRES target gene fragment, subclone to pAdTrack-CMV transferring plasmid makes up the pAdTrack-CMV-IRES double gene coexpression and shifts empty plasmid, and identifies through PCR, double digestion and dna sequencing;
(2) the people VEGF165 gene order of reporting according to GenBank, design primer P3, P4, with the pSV-K3-VEGF165 plasmid that contains people VEGF165 gene fragment is template pcr amplification people VEGF165 target gene fragment, subclone makes up pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid to pAdTrack-CMV-IRES, and identifies through PCR, double digestion and dna sequencing;
(3) the people Ang-1 gene order of reporting according to GenBank, design primer P5, P6, with the total RNA reverse transcription of human bone marrow cell synthetic cDNA is template PCR (RT-PCR) amplification people Ang-1 target gene fragment, and subclone makes up pAdTrack-CMV-Ang-1-IRES single-gene recombinant transfer plasmid and the dual-gene recombinant transfer plasmid of pAdTrack-CMV-Ang-1-IRES-VEGF165 and identifies through PCR, double digestion and dna sequencing to pAdTrack-CMV-IRES and pAdTrack-CMV-IRES-VEGF165 respectively.
Further in the technical scheme, the pAdTrack-CMV-IRES empty plasmid that above-mentioned structure is correct, pAdTrack-CMV-IRES-VEGF165 and pAdTrack-CMV-Ang-1-IRES single-gene recombinant transfer plasmid and the dual-gene recombinant transfer plasmid of pAdTrack-CMV-Ang-1-IRES-VEGF165 after Pme I enzyme is cut respectively with pAdEasy-1 adenovirus skeleton plasmid homologous recombination in the BJ5183 intestinal bacteria, the recombinant adenovirus plasmid pAdEasy-1-pAdTrack-CMV-IRES (pAd-IRES) that obtains, pAdEasy-1-pAdTrack-CMV-IRES-VEGF165 (pAd-IRES-VEGF165), pAdEasy-1-pAdTrack-CMV-Ang-1-IRES (pAd-Ang-1-IRES) and pAdEasy-1-pAdTrack-CMV-Ang-1-IRES-VEGF165 (pAd-Ang-1-IRES-VEGF165) distinguish transfection QBI-293A cell with liposome again after Pac I enzyme is cut, after infecting and increase, many wheels obtain Ad-IRES empty carrier adenovirus, Ad-IRES-VEGF165 single-gene recombinant adenovirus, the dual-gene recombinant adenovirus of Ad-Ang-1-IRES single-gene recombinant adenovirus and Ad-Ang-1-IRES-VEGF165, and to utilize RT-PCR and Western blot to detect adenovirus be carrier mediated exogenous VEGF 165 and the transcript and expression of Ang-1 gene in the QBI-293A cell.
Found that: respectively with the pGEZ-Term plasmid, the total RNA reverse transcription of pSV-K3-VEGF165 plasmid and human bone marrow cell synthetic cDNA is the IRES that template PCR all can amplify 581bp, the people Ang-1 target gene fragment of the people VEGF165 of 576bp and 1497bp, the IRES of subclone, it is in full accord that VEGF165 and Ang-1 gene fragment its sequencing result and GenBank report, successfully made up the pAdTrack-CMV-IRES empty plasmid, pAdTrack-CMV-IRES-VEGF165 single-gene recombinant plasmid, the dual-gene recombinant plasmid of pAdTrack-CMV-Ang-1-IRES single-gene recombinant plasmid and pAdTrack-CMV-Ang-1-IRES-VEGF165, and successfully obtained Ad-IRES empty carrier adenovirus, Ad-IRES-VEGF165 single-gene recombinant adenovirus, the dual-gene recombinant adenovirus of Ad-Ang-1-IRES single-gene recombinant adenovirus and Ad-Ang-1-IRES-VEGF165; RT-PCR and Western blot confirm that the exogenous VEGF 165 of Ad-IRES-VEGF165, Ad-Ang-1-IRES and Ad-Ang-1-IRES-VEGF165 mediated by recombinant adenovirus and Ang-1 gene can successful transcript and expressions in the QBI-293A cell.
Because endothelial progenitor cell (endothelial proganitor cells, EPCs) be the endotheliocyte precursor cell that a kind of marrow and peripheral blood exist, the height proliferation potential is arranged, can be in the brephic vascularization mode of vasculogenesis-be, participate in the compensatory reconstructing blood vessel of human body ishemic part.When blood vessel injury and tissue ischemia, mobilize EPCs in the marrow in peripheral blood, to discharge and to the ischemic region chemotactic, in view of the promotion angiogenesis of EPCs itself and the function of chemotactic, therefore selecting EPCs for use is very reasonable and effective as the Vectors in Gene Therapy cell.
Therefore, further in the technical scheme, on the basis of endothelial progenitor cell (EPCs) separation and Culture and the dual-gene recombination adenovirus construction of Ad-Ang-1-IRES-VEGF165, with Ad-Ang-1-IRES-VEGF165 Infection in Vitro EPCs cell, be the carrier mediated VEGF165 and the EPCs of the dual-gene modification of Ang-1 angiogenic growth factor with the preparation adenovirus; Result of infection shows that VEGF165 and Ang-1 recombinant adenovirus can efficiently infect the EPCs cell, and its efficiency of infection can reach more than 90%; Indirect immunofluorescence and ELISA detected result further confirm adenovirus mediated exogenous VEGF 165 and Ang-1 gene can be in the EPCs cell coexpression.
Further discover, endothelial progenitor cell through VEGF165 and the dual-gene modification of Ang-1, through behind the catheter delivery, can significantly promote allogenic gene expression and new vessel in vivo to form, improve the left ventricle contractile function, alleviate ventricle and enlarge, effect is better than the blood vessel progenitor cell that single-gene is modified.
Because the technique scheme utilization, the present invention compared with prior art has following advantage:
1, the adenovirus carrier of VEGF165 that makes up with IRES of the present invention and Ang-1 double gene coexpression and can be used as the medicine of treatment myocardial ischemia with the endothelial progenitor cell (EPCs) that Ad-Ang-1-IRES-VEGF165 modifies.
2, the present invention treats the treatment plan of myocardial ischemia with the dual-gene modification endothelial progenitor cell of Ad-Ang-1-IRES-VEGF165, utilized the characteristic of EPCs to myocardial ischemia district chemotactic, carry out gene therapy, avoid causing the side effects such as angiogenesis of non-ischemic region effectively.
Description of drawings
Colibacillary preservation information is: depositary institution: Chinese typical culture collection center; Address: Chinese Wuhan University; Preservation date: on May 16th, 2010; Deposit number CCTCC NO:M 2010117; Classification name: bacillus coli DH 5 alpha/pAdTrack-CMV-hAng-1-IRES-hVEGF165; Escherichia coli DH5 α/pAdTrack-CMV-hAng-1-IRES-hVEGF165.
Fig. 1 is the VEGF165 and the dual-gene adenovirus coexpression of the Ang-1 mode chart of IRES mediation among the embodiment one;
Fig. 2 is that pAdTrack-CMV-IRES transforms structure and the evaluation figure that idle running moves plasmid among the embodiment one, and wherein, (A) PCR obtains the IRES fragment: the 1.pGEZ-Term plasmid is the IRES PCR product of template; M.DL2000marker; (B) pAdTrack-CMV-IRES plasmid PCR identifies: the 1.pAdTrack-CMV-IRES plasmid is the IRES PCR product of template; M.DL2000marker; (C) pAdTrack-CMV-IRES plasmid double digestion is identified: 1.pAdTrack-CMV-IRES plasmid SalI, Not I double digestion; M.DL2000marker;
Fig. 3 is the structure and the evaluation figure of pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid among the embodiment one, and wherein, (A) PCR obtains the VEGF165 fragment: the 1.pSV-K3-VEGF165 plasmid is the VEGF165 PCR product of template; M.DL2000 marker; (B) pAdTrack-CMV-IRES-VEGF165 plasmid PCR identifies: the 1.pAdTrack-CMV-IRES-VEGF165 plasmid is the VEGF165PCR product of template; M.DL2000marker; (C) pAdTrack-CMV-IRES-VEGF165 plasmid Xho I, EcoR V double digestion are identified: 1.pAdTrack-CMV-IRES-VEGF165 plasmid Xho I, EcoR V double digestion; M.DL2000marker; (D) pAdTrack-CMV-IRES-VEGF165 plasmid Sal I, EcoR V double digestion are identified: 1.pAdTrack-CMV-IRES-VEGF165 plasmid SalI, EcoR V double digestion; M.DL2000marker;
Fig. 4 is the structure and the evaluation figure of pAdTrack-CMV-Ang-1-IRES single-gene recombinant transfer plasmid among the embodiment one, and wherein, (A) RT-PCR obtains the Ang-1 fragment: 1. the total RNA of medullary cell is the Ang-1RT-PCR product of template; M.DL2000marker; (B) pAdTrack-CMV-Ang-1-IRES plasmid PCR identifies: the 1.pAdTrack-CMV-Ang-1-IRES plasmid is the Ang-1PCR product of template; M.DL2000marker; (C) pAdTrack-CMV-Ang-1-IRES plasmid double digestion is identified: 1.pAdTrack-CMV-Ang-1-IRES plasmid Bgl II, Sal I double digestion; M.DL2000marker; (D) pAdTrack-CMV-Ang-1-IRES plasmid double digestion is identified: 1.pAdTrack-CMV-Ang-1-IRES plasmid Bgl II, Not I double digestion; M.DL2000 marker;
Fig. 5 is the structure and the evaluation figure of the dual-gene recombinant transfer plasmid of pAdTrack-CMV-Ang-1-IRES-VEGF165 among the embodiment one, and wherein, (A) RT-PCR obtains the Ang-1 fragment: 1. the total RNA of medullary cell is the Ang-1RT-PCR product of template; M.DL2000 marker; (B) pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmid PCR identifies: the 1.pAdTrack-CMV-Ang-1-IRES plasmid is the Ang-1PCR product of template; M.DL2000marker; (C) pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmid double digestion is identified: 1.pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmid Bgl II, SalI double digestion; M.DL2000marker; (D) pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmid double digestion is identified: 1.pAdTrack-CMV-Ang-1-IRES-VEGF165 plasmid Bgl II, EcoR V double digestion; M.DL2000marker;
Fig. 6 is that homologous recombination cloned plasmids (BJ5183) agarose electrophoresis molecular weight size is identified figure among the embodiment one, and wherein, (A) pAd-IRES homologous recombination cloned plasmids (BJ5183) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-IRES; 3～8. is 6 clones that chosen, and wherein 3,7,8 are the homologous recombination clone; 4,5,6 is non-homogeneous recombinant clone; (B) pAd-IRES-VEGF165 homologous recombination cloned plasmids (BJ5183) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-IRES-VEGF165; 3～8. is 6 clones that chosen, and wherein 3,4,6,7 are the homologous recombination clone; 5,8 is non-homogeneous recombinant clone; (C) pAd-Ang-1-IRES homologous recombination cloned plasmids (BJ5183) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-Ang-1-IRES; 3～8. is 6 clones that chosen, and wherein 3,4,5,6,7,8 is the homologous recombination clone; (D) pAd-Ang-1-IRES-VEGF165 homologous recombination cloned plasmids (BJ5183) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-Ang-1-IRES-VEGF165; 3～8. is 6 clones that chosen, and wherein 3,4,5,6,7 are the homologous recombination clone; 8 is non-homogeneous recombinant clone;
Fig. 7 is that homologous recombination cloned plasmids among the embodiment one (DH5 α) agarose electrophoresis molecular weight size is identified figure, and wherein, (A) pAd-IRES homologous recombination cloned plasmids (DH5 α) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-IRES; 3～5. are respectively 3 homologous recombination clones among Fig. 7 (A); (B) pAd-IRES-VEGF165 homologous recombination cloned plasmids (DH5 α) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-IRES-VEGF165; 3～6. are respectively 4 homologous recombination clones among Fig. 7 (B); (C) pAd-Ang-1-IRES homologous recombination cloned plasmids (DH5 α) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-Ang-1-IRES; 3～8. are respectively 6 homologous recombination clones among Fig. 7 (C); (D) pAd-Ang-1-IRES-VEGF165 homologous recombination cloned plasmids (DH5 α) molecular weight size is identified: 1.pAdEasy-1; 2.pAdTrack-CMV-Ang-1-IRES-VEGF165; 3～7. are respectively 5 homologous recombination clones among Fig. 7 (D);
Fig. 8 is homologous recombination plasmid PCR evaluation figure among the embodiment one, and wherein, 1.pAd-IRES is a template, and P1, P2 are primer I RES PCR positive products; 2.pAd-IRES be template, P3, P4 are that primer VEGF165PCR product is negative; 3.pAd-IRES be template, P5, P6 are that primer Ang-1PCR product is negative; 4.pAd-IRES-VEGF165 be template, P1, P2 are primer I RES PCR positive products; 5.pAd-IRES-VEGF165 be template, P3, P4 are primer VEGF165PCR positive products; 6.pAd-IRES-VEGF165 be template, P5, P6 are that primer Ang-1PCR product is negative; 7.pAd-Ang-1-IRES be template, P1, P2 are primer I RES PCR positive products; 8.pAd-Ang-1-IRES be template, P3, P4 are that primer VEGF165PCR product is negative; 9.pAd-Ang-1-IRES be template, P5, P6 are primer Ang-1PCR positive products; 10.pAd-Ang-1-IRES-VEGF165 be template, P1, P2 are primer I RES PCR positive products; 11.pAd-Ang-1-IRES-VEGF165 be template, P3, P4 are primer VEGF165PCR positive products; 12.pAd-Ang-1-IRES-VEGF165 be template, P5, P6 are primer Ang-1PCR positive products; M.DL2000marker;
Fig. 9 is that homologous recombination plasmid PacI enzyme is cut evaluation figure among the embodiment one, and wherein, (A) pAd-IRES plasmid PacI enzyme is cut evaluation: 1.pAd-IRES PacI enzyme is cut; M. λ-Hind III digest DNAmarker; (B) pAd-IRES-VEGF165 plasmid PacI enzyme is cut evaluation: the 1.pAd-IRES-VEGF165PacI enzyme is cut; M. λ-Hind III digest DNA marker; (C) pAd-Ang-1-IRES plasmid PacI enzyme is cut evaluation: 1.pAd-Ang-1-IRES PacI enzyme is cut; M. λ-Hind III digest DNA marker; (D) pAd-Ang-1-IRES-VEGF165 plasmid PacI enzyme is cut evaluation: the 1.pAd-Ang-1-IRES-VEGF165PacI enzyme is cut; M. λ-Hind III digest DNA marker;
Figure 10 respectively organizes recombinant adenovirus to infect QBI-293A cell light microscopic and fluorescence photo among the embodiment one, wherein, and A.QBI-293A; B.QBI-293A+Ad-IRES; C.QBI-293A+Ad-IRES-VEGF165; D.QBI-293A+Ad-Ang-1-IRES; E.QBI-293A+Ad-Ang-1-IRES-VEGF165;
Figure 11 is that VEGF165 and Ang-1 gene are transcribed evaluation (RT-PCR) figure among the embodiment one in the QBI-293A cell, wherein, figure A 1.QBI-293A, P3, P4 are primer; 2.QBI-293A P1, P2 are that primer I RES RT-PCR product is negative; 3.QBI-293A P5, P6 are primer; M.DL2000marker; Figure B 1.QBI-293A+Ad-IRES, P1, P2 are primer; 2.QBI-293A+Ad-IRES P3, P4 are primer; 3.QBI-293A+Ad-IRES P5, P6 are primer; M.DL2000marker; Figure C 1.QBI-293A+Ad-IRES-VEGF165, P1, P2 are primer; 2.QBI-293A+Ad-IRES-VEGF165 P3, P4 are primer; 3.QBI-293A+Ad-IRES-VEGF165 P5, P6 are primer; M.DL2000marker; Scheme D.1.QBI-293A+Ad-Ang-1-IRES, P1, P2 are primer; 2.QBI-293A+Ad-Ang-1-IRES P3, P4 are primer; 3.QBI-293A+Ad-Ang-1-IRES P5, P6 are primer; M.DL2000marker; Scheme E.1.QBI-293A+Ad-Ang-1-IRES-VEGF165, P1, P2 are primer I RESRT-PCR product; 2.QBI-293A+Ad-Ang-1-IRES-VEGF165 P3, P4 are primer VEGF165RT-PCR product; 3.QBI-293A+Ad-Ang-1-IRES-VEGF165 P5, P6 are primer Ang-1RT-PCR product; M.DL2000marker;
Figure 12 is that the ELISA method detects Ad-Ang-1/VEGF165 VEGF165 and the proteic expression level figure of Ang-1 in the 293A cells and supernatant among the embodiment one;
Figure 13 is VEGF165 and the short CAM angiogenic activity detection figure of Ang-1 recombinant adenovirus among the embodiment two;
Figure 14 respectively organizes recombinant adenovirus to infect EPCs cell light microscopic and fluorescence photo among the embodiment two, wherein, and A.EPCs; B.EPCs+Ad-IRES; C.EPCs+Ad-IRES-VEGF165; D.EPCs+Ad-Ang-1-IRES; E.EPCs+Ad-Ang-1-IRES-VEGF165;
Figure 15 is a VEGF165ELISA typical curve among the embodiment two;
Figure 16 is an Ang-1ELISA typical curve among the embodiment two;
Figure 17 is among the embodiment three
125I-Tf (Fe)
2The EPCs of mark dynamic observes figure what the myocardial infarction rabbit in vivo distributed;
Figure 18 is the EPCs injection distribution plan in each internal organs after 24 hours among the embodiment three, wherein, and 1. blood; 2. brain; 3. lung; 4. the heart; 5. liver; 6. spleen; 7. kidney; 8. intestines; 9. muscle; 10. Tiroidina;
Figure 19 is that RT-PCR identifies exogenous VEGF 165 and Ang-1 gene transcribing in the rabbit ischemic myocardium among the embodiment three, wherein, 1. not treatment group, P1, P2 are that primer I RES RT-PCR product is negative; 2. not treatment group, P3, P4 are that primer VEGF165RT-PCR product is negative; 3. not treatment group, P5, P6 are that primer Ang-1RT-PCR product is negative; 4.EPCs the treatment group, P1, P2 are that primer I RES RT-PCR product is negative; 5.EPCs the treatment group, P3, P4 are that primer VEGF165RT-PCR product is negative; 6.EPCs the treatment group, P5, P6 are that primer Ang-1RT-PCR product is negative; 7.EPCs+Ad-IRES the treatment group, P1, P2 are primer I RES RT-PCR product; 8.EPCs+Ad-IRES the treatment group, P3, P4 are that primer VEGF165RT-PCR product is negative; 9.EPCs+Ad-IRES P5, P6 are that primer Ang-1RT-PCR product is negative; 10.EPCs+Ad-IRES-VEGF165 the treatment group, P1, P2 are primer I RES RT-PCR product; 11.EPCs+Ad-IRES-VEGF165 the treatment group, P3, P4 are primer VEGF165RT-PCR product; 12.EPCs+Ad-IRES-VEGF165 the treatment group, P5, P6 are that primer Ang-1RT-PCR product is negative; 13.EPCs+Ad-Ang-1-IRES the treatment group, P1, P2 are primer I RES RT-PCR product; 14.EPCs+Ad-Ang-1-IRES the treatment group, P3, P4 are that primer VEGF165RT-PCR product is negative; 15.EPCs+Ad-Ang-1-IRES the treatment group, P5, P6 are primer Ang-1RT-PCR product; 16.EPCs+Ad-Ang-1-IRES-VEGF165 the treatment group, P1, P2 are primer I RES RT-PCR product; 17.EPCs+Ad-Ang-1-IRES-VEGF165 the treatment group, P3, P4 are primer VEGF165RT-PCR product; 18.EPCs+Ad-Ang-1-IRES-VEGF165 the treatment group, P5, P6 are primer Ang-1RT-PCR product; M.DL2000marker;
Figure 20 is the immunohistochemical staining photo of the vWF factor among the embodiment three;
Figure 21 is the immunohistochemical staining photo of α-actin among the embodiment three.
Embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment one: the structure and the evaluation of VEGF165 and the dual-gene recombinant vectors of Ang-1, and the VEGF165 and the dual-gene adenovirus coexpression of the Ang-1 pattern that adopt IRES to mediate, synoptic diagram is as shown in Figure 1.
(1) material
Bgl II, Sal I, Not I, Xho I and EcoR V restriction enzyme, T4DNA ligase enzyme, DL2000marker, Taq archaeal dna polymerase, dNTPmix, Oligo d (T)
18Available from TaKaRa company; Reversed transcriptive enzyme MMLV, λ-Hind III digest DNA marker and low molecular weight protein (LMWP) marker are available from MBI company; Daily type plasmid extraction test kit, PCR product cleaning agents box, DNA glue in a small amount reclaims test kit all available from the clean biochemical technology of Hangzhou Wei Te company limited; PGEZ-Term plasmid, pSV-K3-VEGF165 plasmid, bacillus coli DH 5 alpha become professor to be so kind as to give by medical college of University Of Suzhou cell with the Yang Ji of molecular biology teaching and research room; The pAdTrack-CMV transferring plasmid of GFP mark, adenovirus skeleton plasmid pAdEasy-1, intestinal bacteria BJ5183, adenovirus packaging cell QBI-293A are so kind as to give by professor Zhong Jiang of microbiology teaching and research room of life science institute of Fudan University; RPMI1640 is available from GIBCO company; Calf serum is available from Hangzhou folium ilicis chinensis company; 24 holes and 6 porocytes are cultivated available from CORNING company; Anti-goat IgG-the AP two of people VEGF165 antibody [VEGF (A-20): sc-152-G], people Ang-1 antibody [Ang-1 (C-19): sc-6320] and rabbit resists available from Santa Cruz company; Biochemical reagents such as primer (as table 1), the total RNA extraction agent of UNIQ-10 pillar Trizol box, yeast extract, Tryptones, agar powder are given birth to worker Bioisystech Co., Ltd available from Shanghai.
Table 1 pcr amplification the primer and sequence thereof
(2) pAdTrack-CMV-IRES transforms the structure that idle running moves plasmid
PCR obtains IRES purpose fragment: the conserved sequence of comparison IRES, design primer P1, P2 (as table 1), to contain the segmental pGEZ-Term plasmid of IRES is template pcr amplification IRES purpose fragment, two ends are introduced Sal I, Not I restriction enzyme site respectively, and the IRES PCR product of acquisition is identified through agarose electrophoresis.The result shows: amplify IRES target gene fragment size and the IRES theoretical value size (581bp) consistent [Fig. 2 (A)] of expecting amplification.
The IRES fragment is subcloned on the pAdTrack-CMV transferring plasmid: after the transferring plasmid pAdTrack-CMV that extracts of plasmid extraction test kit uses 37 ℃ of double digestion 5h of Sal I, Not I respectively in a small amount with the IRES PCR product of DNA cleaning agents box purifying and daily type, the purpose fragment is reclaimed in rubber tapping, and its 4 ℃ of connections are spent the night with the T4DNA ligase enzyme, then connect product transformed into escherichia coli DH5 α and Kana (50 μ g/ml) screening and choose mono-clonal, and identify and determined dna sequence that with PCR, double digestion it is kind standby that the positive colony bacterium protects.The result shows: recombinant vectors pAdTrack-CMV-IRES can amplify about 581bp size through PCR band [Fig. 2 (B)] and Sal I, Not I double digestion can discharge the fragment [Fig. 2 (C)] of identical size equally, show that all the IRES target gene fragment successfully is subcloned in the pAdTrack-CMV transferring plasmid.The further confirmation of determined dna sequence has successfully made up pAdTrack-CMV-IRES transformation idle running and has moved plasmid.
(3) structure of pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid
With the pSV-K3-VEGF165 plasmid that contains the VEGF165 gene fragment is template pcr amplification VEGF165 purpose fragment, and two ends are introduced Xho I, EcoR V restriction enzyme site respectively, and the VEGF165PCR product of acquisition is identified through agarose electrophoresis.The result shows: amplify the VEGF165 target gene fragment, its size and the VEGF165 theoretical value size (576bp) consistent [Fig. 3 (A)] of expecting amplification.
After the extractive transformation transferring plasmid of plasmid extraction test kit pAdTrack-CMV-IRES uses 37 ℃ of double digestion 5h of Xho I, EcoRV respectively in a small amount with the VEGF165PCR product of purifying and daily type, the purpose fragment is reclaimed in rubber tapping, and its 4 ℃ of connections are spent the night with the T4DNA ligase enzyme, then connect product transformed into escherichia coli DH5 α and Kana screening and choose mono-clonal, and identify and determined dna sequence that with PCR, double digestion it is kind standby that the positive colony bacterium protects.The result shows: recombinant vectors pAdTrack-CMV-IRES-VEGF165 can amplify the band [Fig. 3 (B)] of about 576bp size through PCR; Can discharge the fragment [Fig. 3 (C)] of 576bp size equally through Xho I, EcoR V double digestion; Can discharge the IRES+VEGF165 fragment [Fig. 3 (D)] of 1157bp size through Sal I, EcoR V double digestion, show that all the VEGF165 target gene fragment successfully is subcloned on pAdTrack-CMV-IRES and transforms idle running and move in the plasmid.The further confirmation of determined dna sequence has successfully made up pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid.
(4) structure of pAdTrack-CMV-Ang-1-IRES single-gene recombinant transfer plasmid
The clone of Ang-1 gene: according to the GenBank (number of landing: NM-001146) Bao Dao people Ang-1cDNA sequences Design upstream and downstream primer P5, P6 (as table 1), with the total RNA reverse transcription of medullary cell synthetic cDNA is template pcr amplification Ang-1 target gene fragment, two ends are introduced Bgl II, Sal I restriction enzyme site respectively, and the Ang-1RT-PCR product of acquisition is identified through agarose electrophoresis.The result shows: amplify the Ang-1 target gene fragment, its size and the Ang-1 theoretical value size consistent [Fig. 4 (A)] of expecting amplification.
After the extractive transferring plasmid pAdTrack-CMV-IRES of plasmid extraction test kit uses 37 ℃ of double digestion 5h of Bgl II, Sal I respectively in a small amount with the Ang-1RT-PCR product of above-mentioned purifying and daily type, the purpose fragment is reclaimed in rubber tapping, and its 4 ℃ of connections are spent the night with the T4DNA ligase enzyme, then connect product transformed into escherichia coli DH5 α and Kana screening and choose mono-clonal, and identify and determined dna sequence that with PCR, double digestion it is kind standby that the positive colony bacterium protects.The result shows: recombinant vectors pAdTrack-CMV-Ang-1-IRES can amplify the band [Fig. 4 (B)] of about 1497bp size through PCR; Can discharge the fragment [Fig. 4 (C)] of 1497bp size equally through Bgl II, Sal I double digestion; Can discharge the Ang-1+IRES fragment [Fig. 4 (D)] of 2078bp size through Bgl II, Not I double digestion, show that all the Ang-1 target gene fragment successfully is subcloned on pAdTrack-CMV-IRES and transforms idle running and move in the plasmid.The further confirmation of determined dna sequence has successfully made up pAdTrack-CMV-Ang-1-IRES single-gene recombinant transfer plasmid.
(5) structure of the dual-gene recombinant transfer plasmid of pAdTrack-CMV-Ang-1-IRES-VEGF165
On the basis that pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid makes up, to be template with the cDNA of the total RNA of reverse transcription synthetic medullary cell, P5, P6 further be subcloned on the dual-gene recombinant transfer plasmid of structure pAdTrack-CMV-Ang-1-IRES-VEGF165 in the pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid for the Ang-1 target gene fragment [Fig. 5 (A)] of primer RT-PCR amplification.Recombinant vectors pAdTrack-CMV-Ang-1-IRES-VEGF165 can amplify the band [Fig. 5 (B)] of about 1497bp size through PCR; Can discharge the fragment [Fig. 5 (C)] of 1497bp size equally through Bgl II, SalI double digestion; Can discharge the Ang-1+IRES+VEGF165 fragment [Fig. 5 (D)] of 2654bp size through B gl II, EcoR V double digestion, show that all the Ang-1 target gene fragment successfully is subcloned in the pAdTrack-CMV-IRES-VEGF165 single-gene recombinant transfer plasmid.The further confirmation of determined dna sequence has successfully made up the dual-gene recombinant transfer plasmid of pAdTrack-CMV-Ang-1-IRES-VEGF165.
(6) structure of recombinant adenovirus plasmid
PAdTrack-CMV-IRES with above-mentioned structure, pAdTrack-CMV-IRES-VEGF165, after pAdTrack-CMV-Ang-1-IRES and pAdTrack-CMV-Ang-1-IRES-VEGF165 recombinant transfer plasmid are used 37 ℃ of single endonuclease digestion 2h of Pme I linearizing, respectively with pAdEasy-1 adenovirus skeleton plasmid Calcium Chloride Method cotransformation BJ5183 competence bacterium, mono-clonal extracting plasmid is chosen in the Kana screening, agarose electrophoresis is according to molecular weight size preliminary screening pAdEasy-1-pAdTrack-CMV-IRES (abbreviating pAd-IRES as), pAdEasy-1-pAdTrack-CMV-IRES-VEGF165 (abbreviating pAd-IRES-VEGF165 as), pAdEasy-1-pAdTrack-CMV-Ang-1-IRES (abbreviating pAd-Ang-1-IRES as) and pAdEasy-1-pAdTrack-CMV-Ang-1-IRES-VEGF165 (abbreviating pAd-Ang-1-IRES-VEGF165 as) homologous recombination clone, agarose electrophoresis (Fig. 6) is the result show, 6 clones that pAdTrack-CMV-IRES and pAdEasy-1 cotransformation group are chosen have 3 positive clones; 6 clones that pAdTrack-CMV-IRES-VEGF165 and pAdEasy-1 cotransformation group are chosen have 4 positive clones; 6 clones that pAdTrack-CMV-Ang-1-IRES and pAdEasy-1 cotransformation group are chosen are positive colony; 6 clones that pAdTrack-CMV-Ang-1-IRES-VEGF165 and pAdEasy-1 cotransformation group are chosen have 5 positive clones.The above-mentioned homologous recombination positive colony of respectively organizing is transformed extracting plasmid (Fig. 7) behind the DH5 α respectively, and select a certain preliminary homologous recombination positive colony of determining to carry out PCR and identify that the PCR product of (Fig. 8) all visible expection size and PacI enzyme cut ori and kalamycin resistance encoding gene fragment that evaluations (Fig. 9) all can obtain the 4.5kb size of the adenoviral gene group fragment of 30kb size and uniqueness.
(7) packing of recombinant adenovirus, the detection of increasing and tire
PAd-IRES, pAd-IRES-VEGF165, pAd-Ang-1-IRES and the pAd-Ang-1-IRES-VEGF165 homologous recombination adenoviral plasmid glue after Pac I linearizing that makes up is reclaimed big fragment, press Lipofectamine
TM2000 operation instructionss are the adherent QBI-293A cell of transfection 70% respectively.The linearizing pAd-IRES of Pac I, pAd-IRES-VEGF165, pAd-Ang-1-IRES and pAd-Ang-1-IRES-VEGF165 homologous recombination plasmid are behind Lipofectamin liposome difference transfection QBI-293A cell, the expression of visible GFP under the fluorescent microscope, and fluorescence intensity prolongs enhancing gradually with incubation time.Collect Ad-IRES, Ad-IRES-VEGF165, Ad-Ang-1-IRES and Ad-Ang-1-IRES-VEGF165 first-generation virus behind the transfection 10d respectively, superinfection QBI-293A cell again, under inverted fluorescence microscope, can be observed fluorescence, and CPE appears, the result shows that Ad-IRES, Ad-IRES-VEGF165, Ad-Ang-1-IRES and Ad-Ang-1-IRES-VEGF165 pack successfully in the QBI-293A cell.Behind the superinfection 4-6d, collecting cell, suspend with PBS, multigelation obtains Ad-IRES, Ad-IRES-VEGF165, Ad-Ang-1-IRES and the sub-crude extract of Ad-Ang-1-IRES-VEGF165 third generation recombinant virus, and behind many wheel infection, amplification and CsCl gradient centrifugation purifications, finally obtain 5 * 10 respectively
10The Ad-IRES virion, 2 * 10 of pfu/ml titre
10The Ad-IRES-VEGF165 virion, 3 * 10 of pfu/ml titre
10The Ad-Ang-1-IRES virion and 4 * 10 of pfu/ml titre
10The Ad-Ang-1-IRES-VEGF165 virion of pfu/ml titre is standby in-80 ℃ of preservations.Ad-IRES, Ad-IRES-VEGF165, Ad-Ang-1-IRES and Ad-Ang-1-IRES-VEGF165 infect QBI-293A cell light microscopic CPE and fluorescence photo such as Figure 10.
Wherein, the method that detects of tiring of recombinant adenovirus is: with the QBI-293A cell of logarithmic phase, after the trysinization, adjusting cell concn is 1 * 10
5Individual/ml, on 96 orifice plates,, behind the cultivation 24h, Ad-Ang-1-IRES-VEGF165, Ad-Ang-1-IRES, Ad-IRES-VEGF165 recombinant adenovirus and the empty carrier Ad-IRES adenovirus of results are done 10 by every hole 100 μ l inoculating cells
-4, 10
-5, 10
-6, 10
-7After the dilution, each extent of dilution is inoculated 3 holes by every hole 100 μ l, and 37 ℃, 5%CO
2After cultivating 18h in the cell culture incubator, under the fluorescent microscope, carry out the fluorescence counting, press virus titer (pfu/ml)=(every hole fluorescence mean number * 10)/extent of dilution formula and calculate virus titer.
(8) evaluation of recombinant adenovirus
RT-PCR identifies: after infecting QBI-293A cell 48h respectively with Ad-Ang-1-IRES-VEGF165, Ad-Ang-1-IRES, Ad-IRES-VEGF165 recombinant adenovirus and the empty carrier Ad-IRES adenovirus of 10MOI, the centrifugal 5min of 1000r/min collects the QBI-293A cell contrast of above-mentioned virus infected cell and uninfecting virus, PBS washing 2～3 times, respectively organize the QBI-293A cell total rna by the total RNA extraction agent of UNIQ-10 pillar Trizol box specification sheets operation extraction, with above-mentioned primer P1, P2; P3, P4; P5, P6 carry out RT-PCR, identify IRES, VEGF165 and Ang-1 gene transcribing in the QBI-293A cell respectively.Total RNA extraction and RT-PCR system and condition are the same.
The results are shown in Figure 581bpIRES, 576bp VEGF165 and 1497Ang-1PCR product that the 11:Ad-Ang-1-IRES-VEGF165 infected group can produce the expection size; The Ad-IRES-VEGF165 infected group all can produce the 581bp IRES and the 576bp VEGF165PCR product of expection size, and the corresponding position does not produce the 1497Ang-1PCR product; The Ad-Ang-1-IRES infected group can produce the 581bpIRES and the 1497Ang-1PCR product of expection size, and the corresponding position does not produce 576bp VEGF165PCR product; The Ad-IRES infected group can produce the 581bp IRES PCR product of expection size, and the corresponding position does not produce 576bp VEGF165 and 1497Ang-1PCR product; The cell control group of QBI-293A uninfecting virus does not all produce above-mentioned band in the corresponding position.The RT-PCR qualification result shows and has successfully made up Ad-Ang-1-IRES-VEGF165 (dual-gene), Ad-Ang-1-IRES (single-gene), Ad-IRES-VEGF165 (single-gene), Ad-IRES (empty carrier) adenovirus.
The ELISA method detects Ad-Ang-1/VEGF165 VEGF165 and the proteic expression of Ang-1 in the 293A cell: after infecting QBI-293A cell 48h respectively with Ad-Ang-1-IRES-VEGF165, Ad-Ang-1-IRES, Ad-IRES-VEGF165 recombinant adenovirus and the empty carrier Ad-IRES adenovirus of 10MOI, collect and respectively to organize cells and supernatant, measure the content of Ang-1 and VEGF in the supernatant according to the ELISA detection kit respectively.
The results are shown in Figure 12 and table 2, show that further the VEGF165 of IRES mediation and external source VEGF165 and Ang-1 gene that the dual-gene adenovirus expression carrier of Ang-1 imports all can successfully express.
Table 2 ELISA method detects Ad-Ang-1/VEGF165 VEGF165 and proteic expression level of Ang-1 (pg/ml) in the 293A cells and supernatant
| The experiment group | Ang-1(pg/ml) | VEGF165(pg/ml) |
| The contrast of 293A cell | 2490.86±550.95 | 6455.33±653.68 |
| The Ad-IRES blank | 2323.20±260.55 | 6074.51±629.58 |
| Ad-IRES-Ang1 | 47022.53±3685.57 | 6274.51±609.59 |
| Ad-IRES-VEGF165 | 38783.13±3449.95 | 10367.12±1401.22 |
| Ad-IRES-Ang1-VEGF | 72652.53±4286.02 | 9558.13±1221.21 |
Embodiment two: embodiment one gained VEGF165 and the dual-gene adenovirus carrier Infection in Vitro of Ang-1 peripheral blood endothelial progenitor cell
(1) material
8d chicken embryo is available from the hatcher, Suzhou City; Zhengguangmycin A5 (PYM) is available from First Affiliated Hospital of Soochow University,Suzhou; 24 holes and 6 porocytes are cultivated available from CORNING company; RPMI1640 is available from GIBCO company; Calf serum is available from Hangzhou folium ilicis chinensis company; People VEGF165 antibody [VEGF (A-20): sc-152-G], people Ang-1 antibody [Ang-1 (C-19): sc-6320] are available from Santa Cruz company; Immunofluorescence dyeing test kit-anti-goat Cy3 is available from green skies biotechnology research institute; People VEGF, Ang-1ELISA detection kit are available from crystalline substance U.S. company.
(2) activity of VEGF165 and Ang-1 recombinant adenovirus detects (CAM test)
The good chicken embryo of growth conditions of hatching 8d is divided into 6 groups at random: Ad-IRES empty carrier group, Ad-IRES-VEGF165 single-gene group, Ad-Ang-1-IRES single-gene group, dual-gene group of Ad-Ang-1-IRES-VEGF165, NS group and Zhengguangmycin A5 (PYM) group (as negative control), every group of 6 chicken embryos.Ad-IRES empty carrier group, Ad-IRES-VEGF165 single-gene group, Ad-Ang-1-IRES single-gene group and dual-gene group of 4 groups of adenovirons of Ad-Ang-1-IRES-VEGF165 all are adjusted into 1 * 10 with the PBS dilution
8The concentration of individual/ml; Zhengguangmycin A5 (PYM) disposes with PBS, and concentration is 10mg/L.With the air chamber end eggshell of tincture of iodine cotton balls wiping chicken embryo, and prick an aperture and tear inner shell membrane, expose CAM with aseptic nipper.With sterilized diameter is that the filter paper sequin of 5mm soaks the above-mentioned central authorities that 50 μ l sample liquid are placed on the content-addressable memory face that respectively organize, and dosing finishes and uses the adhesive tape sealing label.After hatching 3d, tear adhesive tape, the stationary liquid of adding formaldehyde and acetone (1: 1) is 10min fixedly, takes off CAM, and it is tiled on the slide glass, blood vessel is classified, major blood vessel is decided to be the one-level blood vessel, and the branch vessel of major blood vessel is decided to be the secondary blood vessel, and the following branch of secondary blood vessel is decided to be three grades of blood vessels, calculate filter paper interior blood vessel trunk and the branch at different levels of 5mm scope on every side, to detect the short CAM angiogenic activity of adenovirus mediated VEGF165 and Ang-1.
The result is referring to Figure 13 and table 3, as seen: Ad-IRES-VEGF165 group, Ad-Ang-1-IRES group and Ad-Ang-1-IRES-VEGF165 group are more vigorous than NS group CAM angiogenic growth, capillary vessel is abundanter, be and fill the air shape, radial vascularization, and Ad-IRES empty carrier group and NS compare indifference; Two groups of single-gene groups of Ad-IRES-VEGF165 and Ad-Ang-1-IRES compare, and its short CAM angiogenic activity Ad-Ang-1-IRES is better than Ad-IRES-VEGF165; Two groups of single-gene groups of dual-gene group of Ad-Ang-1-IRES-VEGF165 and Ad-IRES-VEGF165 and Ad-Ang-1-IRES compare, and its short CAM vascularization ability all is better than Ad-Ang-1-IRES and Ad-IRES-VEGF165 single-gene group.The result shows that constructed VEGF165 and Ang-1 recombinant adenovirus have significantly short CAM angiogenic activity, and is significant synergistic effect.
The angiopoietic comparison of short CAM of table 3 VEGF165 and Ang-1 (
N=6)
| Group | The one-level blood vessel # | The secondary blood vessel $ | Three grades of blood vessels & |
| NS | 3.50±0.60 a | 15.80±2.86 a | 62.00±9.65 a |
| Ad-IRES 1) | 3.55±0.58 a | 15.90±2.82 a | 61.50±9.57 a |
| Ad-IRES-VEGF 165 2) | 3.70±0.61 a | 22.30±3.12 b | 78.30±11.53 b |
| Ad-Ang-1-IRES 3) | 3.85±0.57 a | 27.80±4.10 c | 86.20±12.36 c |
| Ad-Ang-1-IRES-VEGF 165 4) | 4.38±0.62 b | 36.60±6.15 d | 98.60±13.82 d |
| PYM | 2.82±0.56 c | 10.70±2.13 e | 40.70±6.81 e |
#F=9.132, P＜0.001, the q check: a between group in the table, b, the c letter is identical, two group difference not statistically significants (P＞0.05), letter is different in the table, and two group differences have statistical significance (P＜0.05)
$F=38.357, P＜0.0001, the q check: a between group in the table, b, c, d, e have letter identical, and it is identical not have letter between two group difference not statistically significants (P＞0.05) group, and two group differences have statistical significance (P＜0.05)
﹠amp; F=27.246, P＜0.0001, the q check: a between group in the table, b, c, d, e have letter identical, two group difference not statistically significants (P＞0.05), it is identical not have letter between group, and two group differences have statistical significance (P＜0.05)
(3) carry out the separation and Culture and the evaluation of EPCs cell according to prior art, referring to document: Lv Haitao, Yang Xiangjun, Cheng Cheng, etc. the endothelial progenitor cell amplification in vitro research in rabbit peripheral blood source. University Of Suzhou's journal medicine, 2007. (3): 341-346).
(4) embodiment one gained VEGF165 and Ang-1 recombinant adenovirus Infection in Vitro EPCs cell
The EPCs of 0.25% trysinization separation and Culture of former generation, the centrifugal 5min harvested cell of 1000r/min, PBS clean the RPMI1640 perfect medium of using 10%FBS after 2 times and suspend, and it is 1 * 10 that cell concn is adjusted in the counting back
5/ ml, every hole 500 μ l are inoculated in 24 well culture plate overnight incubation.Carry out VEGF165 and Ang-1 recombinant adenovirus Infection in Vitro EPCs cell next day, be divided into 5 groups: be respectively dual-gene group of the empty virus control group of EPCs cell control group, EPCs+Ad-IRES, EPCs+Ad-IRES-VEGF165 single-gene group, EPCs+Ad-Ang-1-IRES single-gene group and EPCs+Ad-Ang-1-IRES-VEGF165,3 parallel holes are respectively established in experiment.VEGF165 and Ang-1 recombinant adenovirus Infection in Vitro EPCs cell method: discard substratum, the every porocyte of the soft washing of PBS 2 times, in above-mentioned 5 groups of cells, add PBS (100l) respectively, Ad-IRES virus liquid (100 μ l viral dilution liquid, the suitable 50MOI of dosage), Ad-IRES-VEGF165 virus liquid (100 μ l viral dilution liquid, the suitable 50MOI of dosage), Ad-Ang-1-IRES virus liquid (100 μ l viral dilution liquid, the suitable 50MOI of dosage) and Ad-Ang-1-IRES-VEGF165 virus liquid (100 μ l viral dilution liquid, the suitable 50MOI of dosage), shake up gently, viral liquid is fully contacted with cell.Put 5%CO
2, cultivate in 37 ℃ of incubators, the RPMI1640 perfect medium of adding 900 μ l 10%FBS behind the 2h continues to cultivate.Observe GFP green fluorescence expression under the EPCs cell growthhabit and the fluorescence visual field observing under the common light microscopic visual field respectively behind the 48h, to judge that 50MOI recombinant adenovirus dosage is to the influence of EPCs cell growth with to the efficiency of infection of EPCs cell.
The result shows: after infecting 48h, observe under the common light microscopic visual field under fluorescent microscope that respectively to organize the equal form of recombinant adenovirus infected group EPCs cell normal, well-grown and does not infect adenovirus EPCs cell control group indifference; Under the fluorescence visual field, each is organized the nearly all cell of recombinant adenovirus infected group EPCs cell and can be observed green fluorescence (Figure 14), the result shows the nontoxicity of 50MOI dosage virus infection EPCs cell pair cell own, and is very high efficiency of infection, and it can reach more than 90%.
(5) expression in the EPCs cell of exogenous VEGF 165 and Ang-1 gene is identified
Get common clean cover glass and in 70% ethanol, soak 5min or longer time, with sterilizing-drying behind the solution washings such as PBS or 0.9%NaCl 3 times.Cover glass is placed under aseptic condition in 6 orifice plates, plant the EPCs cell cultures and spend the night, make to be about 50%-80% completely.Carry out VEGF165 and Ang-1 recombinant adenovirus Infection in Vitro EPCs cell next day, experiment grouping and the same step of infection method (4).Infect to collect behind the 48h and respectively organize recombinant adenovirus and infect EPCs creep plate cell and culture supernatant thereof and carry out indirect immunofluorescence and ELISA respectively and detect adenovirus mediated exogenous VEGF 165 and the expression of Ang-1 gene in the EPCS cell.
(6) indirect immunofluorescence detects
Adenovirus mediated exogenous VEGF 165 and the expression of Ang-1 gene in the EPCs cell utilize immunofluorescence dyeing test kit-anti-goat Cy3 to carry out indirect immunofluorescence and detect, and method is undertaken by immunofluorescence dyeing test kit-anti-goat Cy3 specification sheets.
Ad-IRES empty carrier adenovirus, Ad-IRES-VEGF165 single-gene recombinant adenovirus, Ad-Ang-1-IRES single-gene recombinant adenovirus and the dual-gene recombinant adenovirus of Ad-Ang-1-IRES-VEGF165 are infected the EPCs cell of creep plate in 6 orifice plates respectively with 50MOI dosage.After infecting 48h, collect EPCs creep plate cell, utilize immunofluorescence dyeing test kit-anti-goat Cy3 to carry out the indirect immunofluorescence detection and detect adenovirus mediated exogenous VEGF 165 and the expression of Ang-1 gene in the EPCs cell respectively.
The result shows, red fluorescence appears in all EPCs (not infecting and infected group) when detecting VEGF165 and Ang-1 protein expression respectively, but when detecting VEGF165, EPCs cell control group, EPCs+Ad-IRES empty carrier adenovirus group and EPCs+Ad-Ang-1-IRES single-gene adenovirus group fluorescence intensity all are weaker than EPCs-Ad-IRES-VEGF165 single-gene adenovirus group and the dual-gene adenovirus group of EPCs-Ad-Ang-1-IRES-VEGF165; And when detecting Ang-1, EPCs cell control group, EPCs+Ad-IRES empty carrier adenovirus group and EPCs-Ad-IRES-VEGF165 single-gene adenovirus group also all are weaker than EPCs+Ad-Ang-1-IRES single-gene adenovirus group and the dual-gene adenovirus group of EPCs-Ad-Ang-1-IRES-VEGF165.The indirect immunofluorescence detected result shows that EPCs endothelial progenitor cell itself is also expressed certain VEGF165 and Ang-1 angiogenic growth factor, and adenovirus mediated exogenous VEGF 165 and Ang-1 gene can be in the EPCs cell successful expression.
(7) the ELISA protein quantification is identified
ELISA detects and respectively to organize the protein concentration that recombinant adenovirus infects VEGF165 and Ang-1 in the EPCs cells and supernatant.
According to VEGF165 and Ang-1 standard substance concentration and OD value (table 4) thereof drawing standard curve and carry out statistical test and get linear regression equation (Figure 15 and Figure 16) respectively.Calculate EPCs cells and supernatant VEGF165 and Ang-1 concentration respectively according to each group EPCs cells and supernatant VEGF165 and measured OD and the linear regression equation thereof of Ang-1 again, result's (table 5, table 6) shows that ELISA detects: the dual-gene infected group EPCs culture supernatant of Ad-IRES-VEGF165 single-gene infected group and Ad-Ang-1-IRES-VEGF165 VEGF165 concentration is respectively 1998.4 ± 21.7,1992.9 ± 14.8pg/mL; The dual-gene infected group EPCs culture supernatant of Ad-Ang-1-IRES single-gene infected group and Ad-Ang-1-IRES-VEGF165 Ang-1 concentration is respectively 1298.7 ± 62.3,1309.2 ± 32.9pg/mL.Each organizes EPCs cells and supernatant VEGF165 and Ang-1ELISA detection by quantitative result further shows, adenovirus mediated exogenous VEGF 165 and Ang-1 gene can successful expression and secretions in the EPCs cell.
Table 4 VEGF165 and Ang-1 standard substance concentration and OD value thereof
| VEGF165 standard substance OD |
0 | 0.09 | 0.15 | 0.21 | 0.36 | 0.59 | 1.06 | 1.43 |
| VEGF165 standard substance concentration (pg/ml) | 0 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 |
| Ang-1 standard substance OD |
0 | 0.12 | 0.18 | 0.25 | 0.39 | 0.62 | 1.15 | 1.68 |
| Ang-1 standard substance concentration (pg/ml) | 0 | 31.25 | 62.5 | 125 | 250 | 500 | 1000 | 2000 |
Table 5 respectively organize EPCs cells and supernatant VEGF165 protein ELISA detected result (
N=6) *
| Group | Culture supernatant VEGF165 concentration (pg/ml) |
| EPCs | 2519.92±272.97 |
| EPCs+Ad-IRES | 2362.31±272.97 |
| EPCs+Ad-IRES-VEGF 165 | 25908.03±1477.38 |
| EPCs+Ad-Ang-1-IRES | 2510.85±152.75 |
| EPCs+Ad-Ang-1-IRES-VEGF165 | 25055.07±1477.38 |
* one-way analysis of variance F=49627.1, P=0.00;
The q check: except EPCs, EPCs+Ad-IRES and EPCs+Ad-Ang-1-IRES group and EPCs+Ad-IRES-VEGF165 and EPCs+Ad-Ang-1-IRES-VEGF165 group VEGF165 concentration difference not statistically significant, difference has statistical significance (P＜0.05) between other group.
Table 6 respectively organize EPCs cells and supernatant Ang-1 protein ELISA detected result (
N=6) *
| Group | Culture supernatant Ang-1 concentration (pg/ml) |
| EPCs | 2630.67±180.83 |
| EPCs+Ad-IRES | 2526.27±180.83 |
| EPCs+Ad-IRES-VEGF 165 | 3899.48±1039.68 |
| EPCs+Ad-Ang-1-IRES | 4499.75±1039.68 |
| EPCs+Ad-Ang-1-IRES-VEGF 165 | 5226.01±125.01 |
* one-way analysis of variance F=3026.84, P=0.00;
The q check: except EPCs, EPCs+Ad-IRES and EPCs+Ad-IRES-VEGF165 group and EPCs+Ad-Ang-1-IRES and EPCs+Ad-Ang-1-IRES-VEGF165 group Ang-1 concentration difference not statistically significant, difference has statistical significance (P＜0.05) between other group.
Embodiment three: the peripheral blood endothelial progenitor cell of embodiment two gained VEGF165 and the dual-gene modification of Ang-1 promotes the vascularization of rabbit ischemic myocardium.
Promote the neovascularization effect of ischemic tissue in vivo for the EPCs that observes embodiment two gained VEGF165 and Ang-1 genetic modification, in the present embodiment, on the rabbit myocardial infarction and ischemia model, be transplanted to ischemic myocardium with in-vitro multiplication and through the EPCs of VEGF165 and Ang-1 genetic modification from the artery approach, observe to promote the curative effect of the new vessel of ischemic tissue, to estimate the biological function of the EPCs that VEGF165 and Ang-1 double gene coexpression carrier and single-gene modify.
(1) material
Selecting for use a cleaning level new zealand white rabbit (male, body weight 2.5-3.1kg) to provide [credit number: SCXK (Soviet Union) 2002-0008] by University Of Suzhou experimentation on animals center, is to raise under 18-25 ℃ the condition in temperature.Edible normal diet, the drink ortho-water; No. 5/0 terylene woven line of heart operation special use and 3/8 medical no wound sewing needle are available from Shanghai big vast medical apparatus and instruments factory of unit; Apotransferrin (apo-transferrin) and Iodogen are available from SIGMA company; Ferric citrate amine is available from last seamount Pu chemical industry company limited; Na
125I is available from center Qualcomm; PD-10 chromatography column (Sephadex G25) is available from U.S. GE company; The polyclonal antibody (Novus company) that rabbit vWF is relevant, anti-rabbit unstriated muscle actin antibody (Lab visionNeoMarkers company).
(2) set up rabbit ischemic myocardium model according to prior art, referring to: Zhao Jiangmin, Li Ruixiang, sheep Hui Jun etc., rabbit myocardial infarction model preparation method's improvement and the application in M RI thereof, West China medical university journal, 2002; 33 (4): 640～644.
1% vetanarcol (30mg/L) are anaesthetized through auricular vein, routine electrocardiogram and cardiac monitoring before the art, get mid-sternal incision, successively cut skin, muscle under from the 2-5 intercostal to xiphoid-process to breastbone, along and be close to left border of sternum and slowly cut the 3-4 rib, machine for chest-opening fully exposes the heart of beating, and attention is avoided damaging pleura and caused pneumothorax; Follow with two anodontia tweezers tweezer pericardiums, and cut off pericardium, clamp is pericardium fixedly, Smooth forceps is the fixing apex of the heart fast, exposes left anterior descending branch, with No. 5/0 terylene woven line of heart operation special use and 3/8 medical no wound sewing needle in the nearly apex of the heart end of left anterior descending branch 0.5cm place's ligation, as seen perfusion area is pale gradually, dyskinesis occurs, electrocardiogram(ECG relatively before and after the cardiac monitoring synchronously, ligation, confirm to form myocardial infarction, observe 2-3min, after situation is stable, successively close chest with No. 4 lines.3 days intramuscular injection penicillin 400,000/skies of postoperative.
Electrocardiogram(ECG in the operation or cardiac monitoring can be observed the limb leads II after the ligation of anterior descending coronary part, III, the last ST section of aVF damaging significantly " hunchbacked sample " is raised, and the prompting acute myocardial ischemia takes place, ligation is effective, and the Modelling success of myocardial ischemia is described.
(3) comparative study that distributes in the artery and vein approach infusion endothelial progenitor cell body
Present embodiment with the saturated Transferrins,iron complexes of radioiodinated iron [
125I-Tf (Fe)
2] as tracer agent (among the embodiment, the iodine labeling of Transferrins,iron complexes [
125I-Tf (Fe) 2] mark rate be 88.12 ± 1.77%, radiochemical purity is 97.01 ± 0.21%, the radioactive concentration of mark product is 0.91mCi/mL), as target spot, monitor the endothelial progenitor cell DYNAMIC DISTRIBUTION in vivo of transplanting with the TfR on the endothelial progenitor cell with the scintiscanning imaging.
The mark of endothelial progenitor cell:
1. the preparation of the saturated Transferrins,iron complexes of iron: be dissolved in PBS (0.01mol/L, apotransferrin pH=7.4) (10mg/mL) be dissolved in 0.01mol/L NaHCO
3Ferric citrate amine (0.1mg/mL) incubated at room 4h, excessive iron is spent the night for 4 ℃ by PBS dialysis and is removed.The final concentration of the saturated Transferrins,iron complexes of iron passes through OD
454nmMeasure, saturation ratio should be more than 90% usually.
2.
125I-Tf (Fe)
2Preparation (iodination reaction): with 20 μ g Iodogen be applied to the pipe end, add 100 μ L0.25mol/L PB, add 10 μ L (1mg/mL) Fe-transferrin, add 1.8mCi Na
125I, room temperature reaction 5min adds 0.01mol/L PBS to cumulative volume 500 μ L termination reactions.
3.
125I-Tf (Fe)
2Purifying: the PD-10 post is with 20mL 0.01mol/L PBS drip washing, and 5mL1%BSA is saturated, uses 10mL 0.01mol/L PBS drip washing again, and application of sample is with 0.01mol/L PBS wash-out.
4.
125I-Tf (Fe)
2Radiochemical purity measure: product carries out radiochemical purity and measures behind the purifying, and stationary phase is No. 1 chromatographic paper of Xinhua, and mobile phase is a propyl carbinol: dehydrated alcohol: 50% ammoniacal liquor=5: 1: 2 (v/v).Product Rf=0.0, free-iodine Rf=0.5～0.6.
5.
125I-Tf (Fe)
2The test that combines with endothelial progenitor cell: the endothelial progenitor cell number is 1 * 10
6/ 100 μ L, every pipe add the above-mentioned marked product of 700 μ L, and activity is 23.68MBq (640 μ Ci), hatches 1h for 37 ℃, chooses 6 of myocardial infarction model rabbits, is divided into two groups at random: intravenous injection and intra-arterial injection approach group, 3 every group.
The endothelial progenitor cell of SPECT video picture and infusion distributes in vivo and measures
30min, 2h, 24h carry out the SPECT video picture after injection, adopt static the collection, and the 250k counting can the peak be 37keV, and window width 60% is drawn region of interest and carried out semi-quantitative analysis.24h video picture posterior vein injection air is put to death rabbit, gets blood and each internal organs, measures with gamma counter after the weighing, calculates every gram and organizes the percentage injected dose.
Endothelial progenitor cell is the precursor cell of endotheliocyte, is rich in TfR, with ectogenic highly purified Transferrins,iron complexes [Tf (Fe)
2] avidity is very big, so, with the saturated Transferrins,iron complexes of radioiodinated iron [
125I-Tf (Fe)
2] as tracer agent, with the TfR on the endothelial progenitor cell as target spot,
125I-Tf (Fe)
2The DYNAMIC DISTRIBUTION of 0.5h, 2h and 24h in vivo after transplanting with scintiscanning imaging monitoring endothelial progenitor cell.Result (as Figure 17) shows: intra-arterial injection group tracer agent 0.5h mainly concentrates at the heart, liver and kidney, and 2h mainly concentrates at the heart, liver, kidney and bladder, and 24h mainly concentrates at the heart, liver, Tiroidina, kidney and bladder; And intravenous injection group 0.5h mainly concentrates at liver, the heart and kidney, and 2h mainly concentrates at liver, the heart, kidney and bladder, and 24h mainly concentrates at liver, the heart, Tiroidina, kidney and bladder.
In the semi-quantitative results of body prompting (as table 7): through three-factor analysis of variance, group (artery group and vein group) there are differences (p=0.039), and the stem cell of the spike that as seen the artery group is gathered at heart is more than the vein group; Between the artery and vein group different time view-point also variant (p=0.0081), there is significant difference between 3 time points such as 0.5h, 2h, 24h in each group of prompting, as if artery and vein all have a common trend for two groups: promptly the EPCs of mark fades behind the 24h, compare with the video picture of 2h, counter have enhancing trend.Prompting EPCs has chemotaxis to Ischemic Heart.
Table 7
125I-Tf (Fe)
2The semi-quantitative results of the SPECT video picture of the EPCs of mark
(average counter of each internal organs region of interest and liver ratio)
Annotate: three-factor analysis of variance: F
Group=6.432, p=0.039; F
Time=7.210, p=0.0081; F
Internal organs=7.238, p=0.009
As Figure 18, artery, intravenous injection group are collected at every gram of heart and organize the percentage injected dose to be respectively 1.207%, 1.067%.The EPCs of cue mark may go back to the nest to ischemic myocardium, and the intra-arterial injection approach is better than the intravenous injection approach.
(4) EPCs of dual-gene modification treatment rabbit myocardial ischemia transplantation experiments
Anaesthetize in the time of the 14th day once more rabbit model success back, and under the aseptic condition, heparin-saline soaks vasodilation sheath and conduit.The puncture right carotid artery is put 4F vasodilation sheath, after the conventional anti-freezing of heparin 250U/Kg, sends the soft conduit that shows the way of 4F, judges that according to pressure curve conduit enters left ventricle.Return and to remove conductor housing and place on the main moving arteries and veins Vital-fixture, the perfusion of warp-wise aorta ascendens is according to following experiment 1 * 10 after each the personal 50MOI adenovirus infection of dividing into groups
6Individual EPCs cell suspension 1mL, not treatment group infusion equivalent substratum then pushes physiological saline 5mL, the EPCs that may be detained in the flushing line.With 30 of the rabbits of modeling success and survival, be divided into 6 groups at random, 5 every group, experiment is grouped as follows: the 1. dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165: the EPCs that VEGF165 and the dual-gene adenovirus carrier of Ang-1 infect is carried in perfusion; 2. EPCs+Ad-Ang-1-IRES single-gene treatment group: perfusion contains the EPCs that Ang-1 single-gene adenovirus carrier infects; 3. EPCs+Ad-IRES-VEGF165 single-gene treatment group: perfusion contains the EPCs that VEGF165 single-gene adenovirus carrier infects; 4. the empty viral therapy group of EPCs+Ad-IRES: the EPCs of perfusion empty carrier adenovirus infection; 5. simple EPCs treatment group: perfusion EPCs (not infecting empty virus, single-gene and dual-gene virus); 6. not treatment group: perfusion equivalent substratum is as blank.
(5) heart function detects
Respectively at 4 weeks under narcosis, detecting heart function before the Transplanted cells and after transplanting with the CDFI cardiograph.The measurement index of chamber, left side contractile function comprises: super left ventricular ejection mark (LVEF), the left chamber Dd (LVDD) measured of left chamber long axis view or the horizontal M of left chamber minor axis papillary muscle.
Behind the rabbit myocardial ischemia, the M ultrasound observation arrives: not treatment group rabbit left ventricular wall motion is almost straight, and fluctuating range obviously lowers, and the left chamber motion of 5 transplantation group all recovers to some extent than before transplanting.Heart function between each experimental group changes following characteristics: simple EPCs treatment group, the empty viral therapy group treatment group of EPCs+Ad-IRES are compared with not treatment group: left ventricular ejection fractional rangeability is little, the degree that ventricle enlarges slightly is better than not treatment group, but no difference of science of statistics (P＞0.05); EPCs+Ad-Ang-1-IRES single-gene treatment group, EPCs+Ad-IRES-VEGF165 single-gene treatment group and the dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 cardiac systolic function, left chamber diastasis diameter all are better than untransfected genome (simple EPCs treatment group, the empty viral therapy group of EPCs+Ad-IRES and not treatment group), and difference has statistical significance (P＜0.05); And the left systolic heart of the dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 and left chamber diastasis diameter index obviously are better than single-gene treatment group (EPCs+Ad-IRES-VEGF165 single-gene treatment group, EPCs+Ad-Ang-1-IRES single-gene treatment group) (P＜0.05).Heart function before and after each group is transplanted is relatively as table 8.
Heart function before and after each group of table 8 is transplanted relatively (
N=5)
LVEF and LVDD were relatively before and after each group was transplanted: * P＜0.05 * * P＜0.01
6 groups of LVEF differences compare F=23.16, P＜0.0001, and the q check: a between group in the table, b, the c letter is identical, two group difference not statistically significants (P＞0.05), letter is different in the table, and two group differences have statistical significance (P＜0.05);
6 groups of LVDD differences compare F=8.59, P＜0.0001, and the q check: a between group in the table, b, c have letter identical, two group difference not statistically significants (P＞0.05), nothing is alphabetical identical between group, and two group differences have statistical significance (P＜0.05).
(6) RT-PCR and immunohistochemical methods detect
Leaving and taking of sample: in 4 weeks after the Transplanted cells, after the heart colorful ultrasonic finishes, inject 10% Repone K injection liquid (2mL.kg fast from auricular vein
-1), make asystole diastole.Take off heart immediately, behind the normal saline flushing, keep left chamber, get part rabbit ischemic myocardial tissue after the weighing and extract total RNA and carry out RT-PCR, detect exogenous VEGF 165 and Ang-1 gene transcribing in the rabbit ischemic myocardium; All the other are the fixing 24h of 10% formaldehyde solution at once, and dehydration, paraffin embedding carry out conventional H E dyeing and immunohistochemical methods detection perpendicular to the ventricle long axis direction every the 4um serial section.
RT-PCR detects exogenous VEGF 165 and Ang-1 gene transcribing in the rabbit ischemic myocardium:
Extract not treatment group, simple EPCs treatment group, the empty viral therapy group of EPCs+Ad-IRES, EPCs+Ad-IRES-VEGF165 single-gene treatment group, EPCs+Ad-Ang-1-IRES single-gene treatment group and the total RNA of each group cardiac muscle of the dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 by the operation of the total RNA extraction agent of UNIQ-10 pillar Trizol box specification sheets, with above-mentioned primer P1, P2; P3, P4; P5, P6 carry out RT-PCR, identify IRES, VEGF165 and Ang-1 gene transcribing in the rabbit ischemic myocardium respectively.
The result can produce 581bp IRES, 576bp VEGF165 and the 1497Ang-1PCR product of expection size referring to the dual-gene treatment group of Figure 19: EPCs+Ad-Ang-1-IRES-VEGF165; EPCs+Ad-IRES-VEGF165 single-gene treatment group all can produce the 581bp IRES and the 576bp VEGF165 PCR product of expection size, and the corresponding position does not produce 1497Ang-1 PCR product; EPCs+Ad-Ang-1-IRES single-gene treatment group can produce the 581bp IRES and the 1497Ang-1PCR product of expection size, and the corresponding position does not produce 576bp VEGF165 PCR product; The empty viral therapy group of EPCs+Ad-IRES can produce the 581bp IRES PCR product of expection size, and the corresponding position does not produce 576bp VEGF165 and 1497Ang-1PCR product; Simple EPCs treatment group and not treatment group all do not produce above-mentioned band in the corresponding position.The RT-PCR qualification result shows that the EPCs that VEGF165 and the dual-gene recombinant adenovirus of Ang-1 are modified can mediate transcribing of VEGF165 and Ang-1 equally in the rabbit ischemic myocardium.
Conventional H E dyeing and ordinary optical microscope inspection.
Immunohistochemical staining is checked the new vessel of ischemic myocardium:
With the immunohistochemical methods method dyeing of VIII factor related antigen, with the quantity of quantitative each experimental group new capillary vessel.
With anti-unstriated muscle α-action antibody mediated immunity group method dyeing arteriole, with the arteriolar quantity of quantitative new life.
Capillary density is observed: the capillary vessel of immunohistochemical staining is pale brown look, and method of counting is that 3 the highest zones of blood vessel are selected in every section, counts the blood vessel number that is averaged under the light microscopic in the single visual field.The judging criterion of blood vessel: in the section of anti-VIII factor immunohistochemical methods, do not occur determining whether to be blood vessel, also do not count capillary vessel whether tube chamber to occur with erythrocytic.Every pale brown look, several endotheliocytes or endotheliocyte dyed is bunch all as 1 capillary vessel counting, and all tube chambers are bigger, have the blood vessel of thicker flesh layer, all do not count to be capillary vessel.On anti-unstriated muscle α-action immunohistochemical methods sheet: all stained positive, tube chamber is bigger, or has the blood vessel of flesh layer, and all counting is arteriole.
Nascent blood vessel density is estimated (as table 9, Figure 20, Figure 21): EPCs+Ad-IRES-VEGF165 single-gene treatment group EPCs+Ad-Ang-1-IRES single-gene treatment group and the dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 all have tangible angiogenesis (P＜0.05) with respect to not treatment group, simple EPCs treatment group, the empty viral therapy group of EPCs+Ad-IRES, all there is distribution blood vessel infarct central area and marginarium, but more obvious with the marginarium.Wherein, the dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 angiogenesis quantity is again obviously more than single-gene treatment group group (P＜0.05).Though the empty viral therapy group of simple EPCs treatment group and EPCs+Ad-IRES also has angiogenesis, compares not statistically significant (P＞0.05) with the blank group; The dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 new vessel number and single-gene treatment group (EPCs+Ad-IRES-VEGF165 single-gene treatment group and EPCs+Ad-Ang-1-IRES single-gene treatment group) relatively have notable difference (P＜0.05).
Before and after table 9 is transplanted myocardium new capillary vessel count the comparing of arteriole number (
N=5)
| Group | New capillary vessel is counted difference before and after transplanting # | Arteriole is counted difference before and after transplanting $ |
| Not treatment group | 14.6±1.7 a | 1.4±0.6 a |
| Simple EPC treatment group | 18.4±2.1 a | 1.6±0.6 a |
| The empty viral therapy group of EPCs+Ad-IRES | 17.6±2.4 a | 2.2±1.3 a |
| EPCs+Ad-IRES-VEGF165 single-gene treatment group | 25.2±1.9 b | 4.4±1.1 b |
| EPCs+Ad-Ang-1-IRES single-gene treatment group | 24.8±2.9 b | 4.0±1.0 b |
| The dual-gene treatment group of EPCs+Ad-Ang-1-IRES-VEGF165 | 35.0±2.7 c | 6.6±1.5 c |
New capillary vessel was counted difference relatively before and after the #6 group was transplanted: F=50.52, P＜0.0001, q check: a between group in the table, b, the c letter is identical, two group difference not statistically significants (P＞0.05), letter is different in the table, and two group differences have statistical significance (P＜0.05)
Arteriole was counted difference relatively before and after the $6 group was transplanted: F=17.63, and P＜0.0001, the q check: a between group in the table, b, the c letter is identical, two group difference not statistically significants (P＞0.05), letter is different between group, and two group differences have statistical significance (P＜0.05)
Therefore, embodiment three shows endothelial progenitor cell transplantation treatment energy induction of vascular growth factor release dual-gene or the autologous peripheral blood source that single-gene is modified, promotes ischemic myocardium angiogenesis and heart function to improve.
Claims (5)
1. intestinal bacteria that contain people VEGF165 and people Ang-1 double gene coexpression carrier, this colibacillary preservation information is: depositary institution: Chinese typical culture collection center; Address: Chinese Wuhan University; Preservation date: on May 16th, 2010; Deposit number CCTCC NO:M 2010117; Classification name: bacillus coli DH 5 alpha/pAdTrack-CMV-hAng-1-IRES-hVEGF165; Escherichiacoli DH5 α/pAdTrack-CMV-hAng-1-IRES-hVEGF165; Described double gene coexpression carrier is pAdTrack-CMV-Ang-1-IRES-VEGF165.
2. one kind contains people VEGF165 and people Ang-1 double gene coexpression carrier, it is characterized in that described double gene coexpression carrier is pAdTrack-CMV-Ang-1-IRES-VEGF165.
3.VEGF165 with Ang-1 double gene coexpression recombinant adenovirus, described double gene coexpression recombinant adenovirus is Ad-Ang-1-IRES-VEGF165.
4.pAdTrack-CMV-Ang-1-IRES-VEGF165 the application of dual-gene recombinant transfer vector in the angiogenesis medicament of preparation promotion ischemic myocardium.
5.Ad-Ang-1-IRES-VEGF165 the application of double gene coexpression recombinant adenovirus in the angiogenesis medicament of preparation promotion ischemic myocardium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010263189 CN102191209A (en) | 2010-08-26 | 2010-08-26 | VEGF165 and Ang-1 double-gene co-expression vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010263189 CN102191209A (en) | 2010-08-26 | 2010-08-26 | VEGF165 and Ang-1 double-gene co-expression vector and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102191209A true CN102191209A (en) | 2011-09-21 |
Family
ID=44600084
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010263189 Pending CN102191209A (en) | 2010-08-26 | 2010-08-26 | VEGF165 and Ang-1 double-gene co-expression vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102191209A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012045286A1 (en) * | 2010-10-09 | 2012-04-12 | 苏州大学 | Recombinant vector and fibroin membrane modified by transgenic bone stromal cell, and application thereof |
| CN102533831A (en) * | 2012-02-16 | 2012-07-04 | 郑州大学 | Tissue-specific double gene silencing RNAi expression vector having self-splicing locus |
| CN103602625A (en) * | 2013-08-16 | 2014-02-26 | 苏州大学 | Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids |
| CN104428315A (en) * | 2012-07-13 | 2015-03-18 | 罗氏格黎卡特股份公司 | Bispecific anti-VEGF/anti-ANG-2 antibodies and their use in the treatment of ocular vascular diseases |
| CN110760542A (en) * | 2019-11-18 | 2020-02-07 | 天津大学 | Plasmid for coexpression of ZNF580 and VEGF165 double genes and application thereof |
-
2010
- 2010-08-26 CN CN 201010263189 patent/CN102191209A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 20041215 刘翔 《心肌内注射Ad-VEGF165和Ad-Ang1治疗大鼠急性心肌梗死的实验研究》 E062-100-7-28页 5 , 第4期 * |
| 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 20090915 刘铁连 《Ang-1或/和VEGF165转基因细胞修饰的再生丝素膜诱导血管形成效应及分子机制研究》 E080-47-3-12页 1-3 , 第9期 * |
| 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 20090915 刘铁连 《Ang-1或/和VEGF165转基因细胞修饰的再生丝素膜诱导血管形成效应及分子机制研究》 E080-47-3-12页 4-5 , 第9期 * |
| 《中国协和医科大学》 20010131 郑哲 《心肌注射VEGF121质粒治疗慢性缺血性心脏病的实验研究》 第27-30页 4 , * |
| 《国外医学情报》 20060331 诸葛素 《VEGF基因转染内皮前体细胞能促进血管新生》 第16页 5 第24卷, 第3期 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012045286A1 (en) * | 2010-10-09 | 2012-04-12 | 苏州大学 | Recombinant vector and fibroin membrane modified by transgenic bone stromal cell, and application thereof |
| CN102533831A (en) * | 2012-02-16 | 2012-07-04 | 郑州大学 | Tissue-specific double gene silencing RNAi expression vector having self-splicing locus |
| CN104428315A (en) * | 2012-07-13 | 2015-03-18 | 罗氏格黎卡特股份公司 | Bispecific anti-VEGF/anti-ANG-2 antibodies and their use in the treatment of ocular vascular diseases |
| CN104428315B (en) * | 2012-07-13 | 2017-09-29 | 罗氏格黎卡特股份公司 | Bispecific anti-vegf/antibody of anti-ANG 2 and its application in treatment Ocular Vessels disease |
| CN103602625A (en) * | 2013-08-16 | 2014-02-26 | 苏州大学 | Escherichia coli containing recombinant adenovirus plasmids and applications of recombinant adenovirus plasmids |
| CN103602625B (en) * | 2013-08-16 | 2015-09-30 | 苏州大学 | Intestinal bacteria containing recombinant adenovirus plasmid and the application of recombinant adenovirus |
| CN110760542A (en) * | 2019-11-18 | 2020-02-07 | 天津大学 | Plasmid for coexpression of ZNF580 and VEGF165 double genes and application thereof |
| CN110760542B (en) * | 2019-11-18 | 2022-07-26 | 天津大学 | Plasmid for coexpression of ZNF580 and VEGF165 double genes and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102191209A (en) | VEGF165 and Ang-1 double-gene co-expression vector and application thereof | |
| CN109517791B (en) | Preparation of conditioned medium for enhancing autophagy umbilical cord mesenchymal stem cells and application of conditioned medium in angiogenesis | |
| CN104622902B (en) | It is a kind of for treating the stem cell medicine of liver fibrosis | |
| CN112121063B (en) | Application of exosome in preparation of medicine for treating pulmonary fibrosis | |
| CN112384614B (en) | Oncolytic virus expressing interferon and uses thereof | |
| Liu et al. | Transplantation of parthenogenetic embryonic stem cells ameliorates cardiac dysfunction and remodelling after myocardial infarction | |
| CN107384858A (en) | A kind of preparation method and applications of hypoxic tolerance type mescenchymal stem cell | |
| CN113278591B (en) | Cardiac targeting genetic engineering exosome and preparation method and application thereof | |
| CN105647872A (en) | Liver injury targeted mesenchymal stem cell and preparation method and application thereof | |
| CN113559273B (en) | Pretreatment method of adult stem cells for intravenous injection, adult stem cell injection and application | |
| CN101591658B (en) | ING4 and IL-24 dual gene co-expression vector and application thereof | |
| CN107385033B (en) | PiRNA-5938 and application of antisense nucleic acid thereof in diagnosis and treatment of ischemic heart diseases | |
| Wang et al. | KSHV enhances mesenchymal stem cell homing and promotes KS-like pathogenesis | |
| CN104524599A (en) | Antisense nucleotide MiRNA-532 containing pharmaceutical composition and application thereof | |
| CN106222174A (en) | CircRNA CHIF nucleotide, containing the medical composition and its use of this nucleotide | |
| CN106065401B (en) | Treatment use of the lentivirus mediated CXCR7 high expression engineering endothelial progenitor cells in ischemic disease | |
| CN115369077A (en) | MEFLC cell strain, and construction method and application thereof | |
| CN101219222B (en) | Application of PDCD4 recombined expression vector in preparing medicament for treating gonad cancer | |
| CN102533982B (en) | The novelty teabag of people KLF8 gene in oncotherapy | |
| CN106854660B (en) | The recombined adhenovirus of carrier's miR486 gene, the carrier for preparing the virus and application | |
| CN107164331B (en) | Liver injury targeted mesenchymal stem cell based on miR-221 and preparation method and application thereof | |
| CN114099537B (en) | Application of preparation for knocking down VAMP5 gene expression in preparation of medicine for treating brain glioma | |
| CN102888403A (en) | Application of micro ribonucleic acid (miRNA)-539 and miRNA-539antisense nucleotide, and medicinal composition for miRNA-539 antisense nucleotide | |
| CN111840327B (en) | Mesenchymal stem cell preparation for treating diabetic foot and application thereof | |
| CN102559753A (en) | IL-24 and OSM double-gene co-expression recombinant plasmid, recombinant adenovirus and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20110921 |