WO2012045286A1

WO2012045286A1 - Recombinant vector and fibroin membrane modified by transgenic bone stromal cell, and application thereof

Info

Publication number: WO2012045286A1
Application number: PCT/CN2011/080570
Authority: WO
Inventors: 杨吉成; 缪竞诚; 盛伟华; 韩亚丽; 刘铁连; 张晓锋
Original assignee: 苏州大学
Priority date: 2010-10-09
Filing date: 2011-10-09
Publication date: 2012-04-12
Also published as: CN102242148A

Abstract

Provided is a recombinant plasmid pAdTrack-CMV-VEGF165-PolyA-promoter-Ang-1 and pAdTrack-CMV-VEGF165-IRES-Ang-1, collection codes CCTCC M 2010221 and CCTCC M 2010220 respectively. Also provided is a recombinant adenovirus produced when said plasmid is homologously recombined with an adenovirus, and then packaged in a QBI-293A cell. Also provided is a fibroin membrane modified by a transgenic bone stromal cell. After being injected into the corneal limbus of a rabbit, the recombinant adenovirus successfully induces the growth of new corneal blood vessels; after the fibroin membrane is implanted into the corneal layer of a rabbit, new blood vessels grow into the area of the cornea implanted with fibroin membrane, and the blood vessel density significantly increases, hence VEGF165, Ang-1, and CD34 have successful expression in the corneal layer of a rabbit. A rat wound repair experiment further proves that the fibroin membrane can also promote the repair and the growth of blood vessels in wound tissues. The recombinant adenovirus and the fibroin membrane modified by transgenic bone stromal cell in the present invention have value in medical applications.

Description

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Recombinant vector and transgenic bone marrow stromal cell modified silk fibroin film and application thereof

This application claims priority to Chinese Patent Application No. 201010512520.2, entitled "Recombinant Vector and Transgenic Bone Marrow Membrane Modified Silk Fibroin Film and Its Application", filed on October 09, 2010 The content is incorporated herein by reference. Technical field

The present invention relates to the field of biology, and in particular to recombinant vectors and transgenic bone marrow matrix-modified silk fibroin membranes and uses thereof. Background technique

The mechanism of body damage repair must first generate microvessels, and then form new granulation tissue, and then input the various cells and nutrients needed to promote the repair of damaged tissue. Angiogenesis is a complex, step-by-step process that requires not only the stimulation of stimuli, the involvement of many cells, but also the involvement of various cytokines, growth factors, and extracellular matrices. Ang-1, VEGF, FGF and PDGF play an important role in wound granulation tissue formation, vascular proliferation and collagen fiber synthesis.

VEGF is a dimeric glycoprotein which is cross-linked by two disulfide bonds of two identical subunits. Its molecular weight is 40-45KD. Its gene is located at chromosome 6p21.3, 14kb in length, and consists of 8 exons and 7 introns. Sub-composition, due to different splicing methods of mRNA, forms multiple forms of existence. Among them, VEGF121 and VEGF165 are secreted soluble proteins, which can promote vascular endothelial cell proliferation, migration and increase vascular communication by binding to adjacent vascular endothelial receptors. The role of permeability. In angiogenesis, VEGF and its receptors are considered to be the most potent and specific key regulators. VEGF is a specific mitogen of vascular endothelial cells, which promotes the growth of endothelial cells in vitro and induces angiogenesis in vivo.

In recent years, the important regulatory effects of Ang-1 and Tie2 on the formation, remodeling and maturation of new blood vessels during angiogenesis are gaining attention. Ang-1 and VEGF play a synergistic role in the regeneration of blood vessels. VEGF acts on the early stage of vascular formation and promotes the formation of primitive vascular network, while Ang acts on subsequent vascular remodeling, shaping, and promoting shape. It is a mature and spatially structured vascular network, so gene therapy for co-expression of VEGF and Ang-1 genes may be an innovative exploration to promote angiogenesis.

At present, the construction strategies of the two-gene co-expression vector mainly include: 1. The genes are linked by an IRES (internal ribosome entry site) sequence; 2. The double promoter expression vector; 3. The shear vector 4. The genes are ligated with the cleavage target sequence of Furin; 5. The genes are linked by 2A sequence; 6. The fusion gene is expressed.

The genes are linked by an IRES (internal ribosome entry site) sequence, and the IRES sequence and the gene linked thereto can be simultaneously transcribed, and the translation of the distal mRNA is initiated in a cap-independent manner. Different proteins were translated on the same transcript; a polyA-promotor (CMV) dual promoter expression cassette was inserted between the genes to construct a double gene co-expression vector with two expressions of independent promoters of polyA-promotor (CMV) The cassette is constructed on a vector in which the two genes independently transcribe two different mRNAs and independently translate different proteins.

It is well known to those skilled in the art: Adenoviral vectors are a promising gene therapy viral vector, and adenovirus is a double-stranded DNA virus. Adenoviral vectors have many advantages over other vectors: 1. Wide range of hosts , low pathogenicity to humans; 2. High infection rate, infection of both dividing and non-dividing cells; 3. Ability to carry 4 long therapeutic genes and simultaneous expression of multiple genes; 4. Not integrated into chromosomes, No insertion mutagenicity, good safety; 5. It can effectively proliferate, high titer, easy to process and prepare. The use of pAdEasy adenovirus expression system has obvious advantages: 1. The homologous recombination of adenoviral plasmid can be efficiently carried out in Escherichia coli BJ5183, and the bacteria multiply rapidly and the homologous recombination ability is strong, thus fundamentally overcoming the intracellular homologous recombination rate. Low defects; 2. Adenovirus can insert llkb gene fragment or insert multiple gene fragments at the same time; 3. Due to the GFP reporter gene contained in the adenovirus backbone, it is very convenient to monitor the success of virus packaging and detect virus titer. And infection efficiency. All of these avoid the plaque purification process of the virus and greatly reduce the virus preparation time.

Construction of a two-gene co-expression recombinant transfer vector is one of the effective ways to treat gene therapy for tumors. However, so far, there is no report on the construction of VEGF and Ang-1 double gene vectors by IRES strategy at home and abroad. In addition, the use of constructing polyA +promoter dual promoter strategy to construct a double gene recombinant transfer vector, although the theory has related research, but in practice There is no related method, and the method has the following difficulties: In the process of constructing the double gene recombinant transfer vector by constructing the polyA+promoter double promoter strategy, it is found that there is a pad cleavage site in polyA, and it is impossible to adopt the cleavage linearity of the pad. Adenovirus packaging was performed in 293A cells after transformation, affecting the normal expression of the double genes.

As a new type of medical biomaterial, silk fibroin film has many excellent properties such as non-toxic, non-irritating, moisture permeability, etc. It can be used as a wound dressing, and can also be used as a scaffold material for tissue repair, and can even be developed into artificial skin. . However, how to make the scaffold material adequately vascularized, and further development and preparation of small-caliber artificial blood vessels combined with vascular-induced silk fibroin-based scaffold materials and MSC have not progressed. Summary of the invention

An object of the present invention is to provide a recombinant vector of VEGF165 and Ang-1 which have high gene expression efficiency and high blood vessel inducibility.

The VEGF165 and Ang-1 double transgenic recombinant plasmid pAdTrack-CMV-VEGF165-PolyA-promoter-Ang-1 provided by the present invention has the accession number: CCTCC M 2010221.

The plasmid-transferred gene VEGF165 is available in Genebank under accession number AF016050.1 and the gene Ang-1 is disclosed in NCBI Reference Sequence: NM 001146.3.

The present invention also provides that the recombinant plasmid M 2010221 is homologously recombined with adenovirus pAdEasy-1, and the recombinant adenovirus Ad-VEGF165-PolyA-promoter-Ang-1 produced in QBI-293A cells is packaged.

The VEGF165 and Ang-1 double transgenic recombinant plasmid pAdTrack-CMV-VEGF 165-IRES-Ang-1 provided by the present invention has the accession number; CCTCC

The present invention also provides that the recombinant plasmid Μ 2010220 is homologously recombined with the adenovirus pAdEasy-1, and the recombinant adenovirus Ad-VEGFl 65- IRES-Ang- 1 is packaged in QBI-293A cells.

Subcloning through Ad-polyA+promoter and Ad-IRES empty vectors VEGF165 and Ang-1 construct an adenovirus expression vector for VEGF 165 and Ang-1 double gene. PCR, restriction enzyme digestion and gene sequencing showed that the polyA-promoter/IRES-mediated co-expression vector of VEGF165 and Ang-1 was successfully constructed. The results of RT-PCR showed that the above VEGF165 and Ang-1 genes co-expressed adenoviral vectors. It can mediate the transcription of exogenous VEGF 165 and Ang-1 genes, and successfully obtain recombinant adenovirus co-expressing VEGF165 and Ang-1 genes.

The results of ELISA confirmed that not only the polyA-promoter was more efficient than the IRES-mediated double gene expression, but also further demonstrated that the expression levels of the Ang-1 and VEGF165 genes downstream of the IRES or PolyA-promoter were expressed in the adenovirus expression vector. Significantly lower than the amount of upstream gene expression.

The invention also provides the use of the recombinant adenovirus Ad-VEGF165-PolyA-promoter-Ang-1, Ad-VEGF165-IRES-Ang-1 in the preparation of a pro-angiogenic drug.

Rabbit limbal injection The recombinant adenovirus Ad-VEGF 165-PolyA-promoter-Ang-1 and Ad--VEGF165-IRES-Ang-1 of the present invention can successfully induce corneal neovascularization, and in adenovirus expression vector, VEGF165 The gene is located upstream of the IRES or polyA-promoter to induce corneal neovascularization better than it is located downstream. This result is also consistent with the expression levels of VEGF 165 and Ang-1 cell factors detected by ELISA.

Rabbit corneal limb injection of recombinant adenovirus Ad-VEGF 165-PolyA-promoter-Ang-1 and Ad-VEGF 165-IRES-Ang-1 can successfully induce corneal neovascularization, and the former is more effective than the latter, regardless of Whether it is an IRES vector or a polyA-promoter vector, the VEGF165 gene is not able to induce corneal neovascularization well when it is located downstream of the vector. This may be related to the level of expression of the VEGF165 gene when it is downstream of the vector. Because angiogenesis is the direct or indirect effect of multiple positive and negative regulators, VEGF/VEGFR and Ang/Tie 2 can only be finely regulated in space, time and quantity to form functional blood vessels. VEGF165 acts on blood vessel formation. In the early stage, the formation of the original vascular network is promoted, while Angl acts on subsequent vascular remodeling and shaping to promote the formation of a mature, spatially structured vascular network. VEGF plays a major role in this process, and Angl plays a synergistic role. When VEGF165 is located downstream of the vector, the expression level is low, only the upstream gene list. The amount of 30%-40% was reached. Therefore, after injection of Ad-Ang-1-polyA-promoter-VEGF165 and Ad-Ang-1-IRES-VEGF165 at the edge of rabbit cornea, almost no corneal neovascularization was observed. Good induction of corneal neovascularization.

Another object of the present invention is to provide a method of preparing a transgenic cell-modified silk fibroin film comprising the steps of:

The bone marrow stromal cells are seeded on the silk fibroin membrane, and the recombinant adenovirus is added

Ad-VEGF 165-PolyA-promoter-Ang-1 or Ad-VEGF 165- IRES-Ang-1, the regenerated silk fibroin membrane has a bone marrow stromal cell concentration of 0.5χ 10 ⁵ ·-1 χ 10 ⁵ per square centimeter. The recombinant adenovirus was inoculated at 20-30 ΜΟΙ and cultured at 37 ° C under 5% CO ₂ .

Marrow stromal cells (MSCs) have typical stem cell characteristics, multi-directional differentiation potential and strong proliferative capacity. Bone marrow stromal cells are easy to obtain, and can be isolated and purified by cell adhesion during cell culture. And the hematopoietic cells are gradually removed during the fluid exchange, which is convenient for autologous transplantation.

Preferably, the bone marrow stromal cells are in a logarithmic growth phase.

Preferably, the bone marrow stromal cells are primary cultured bone marrow stromal cells, and the cell concentration is lx lO ⁸ ! ¹ .

Preferably, the incubation time is 48-72 hours.

The invention also provides a transgenic cell modified silk fibroin membrane prepared by the method.

The invention also provides the use of the transgenic cell modified silk fibroin film for the preparation of a vascular inducible medical biomaterial.

Silk fibroin is a new biomedical biomaterial with good biocompatibility synthesized by silk fibroin. It is not only non-cytotoxic, has no effect on seed cytogenetic properties, and has no adverse effects on cell cycle and apoptosis. The effect does not cause inflammatory reaction to the body, does not cause hemolysis, and exhibits excellent biocompatibility. The cells adhere well, expand and proliferate on silk fibroin materials, and facilitate endothelialization to form blood vessel-like tissues. In vivo tests can repair bone defects and heal skin wounds.

In an embodiment of the present invention, the silk fibroin film is a physically cross-linked regenerated silk fibroin film, and the silk fibroin is a natural protein composed of 18 kinds of amino acids such as acetyl acid, a hydrogenic acid, and serine, and Na _{2 is} used for silk silk. C0 ₃ solution degumming, CaCl ₂ 'CH ₃ CH ₂ OH'H ₂ 0 (molar ratio 1: 2: 8) The ternary solvent is dissolved, and after dialysis and filtration, it is dried by a casting method at 60 ° C to form a film. After being sterilized by Co-60 irradiation, it is sealed and stored for use, which is a physical cross-linked regenerated silk fibroin film. The following tube is called a regenerated silk. Membrane.

The rabbit bone marrow stromal cells in the logarithmic growth phase were digested to a cell concentration of 1 10 ⁵ /ml, and inoculated into a 24-well cell culture plate with regenerated silk fibroin membrane at 1 ml/well. The experimental group was the same as before, and the virus was infected for 48 hours. After that, the supernatants of each group were taken, and the contents of Angl and VEGF were determined according to the ELISA test kit. The results showed that rabbit bone marrow stromal cells without transgenic modification could only secrete autologous VEGF165 and Ang-1 cytokines at low levels, but the expression of VEGF165 and Ang-1 was significantly increased by inoculation of the recombinant adenovirus group of the present invention.

HE staining of SF+MSC+Ad-VEGF165-Ang-l group in the corneal stroma after implantation of Ad- VEGF 165-PolyA-promoter- Angl gene-modified regenerated silk fibroin membrane showed abundant capillaries, CD34 immunohistochemistry The analysis showed a strong positive, microvessel density was (17 ± 3.5) / high power field, while SF + MSC + Ad-GFP group and SF + MSC group corneal stromal capillaries were rare, no obvious CD34 positive expression, the difference is statistically Academic significance (* Ρ<0·05).

VEGF 165 and Ang-1 were positively expressed in the corneal stroma of SF+MSC+Ad-VEGF165-Ang-1 group after VS165 and Ang-1 gene-modified regenerated silk fibroin membranes were implanted into rabbit cornea. There was no significant expression of VEGF and Ang-1 in the corneal stroma of SF+MSC+Ad-GFP group and SF+MSC group, the difference was statistically significant (P<0.05).

The expression of HIF-a and bFGF in SF+MSC+Ad-VEGF 165-Ang-1 group after VEGF165 and Ang-1 gene-modified regenerated silk fibroin membrane was brownish black, which was significantly higher than SF. +MSC+Ad-GFP group and SF+MSC control group (PO.05); EGF, PDGF expression and SF+MSC+Ad-GFP group and SF+MSC control group were brown, no statistical significance between the groups. (P>0.05), indicating that the regenerated silk fibroin membrane modified by VEGF165 and Ang-1 gene cells can not only effectively express VEGF165 and Ang-1 gene in the corneal layer, but also promote the autocrine HIF-a of bone marrow stromal cells. The level of bFGF is increased, and the normal and stable expression of autocrine EGF and PDGF in bone marrow stromal cells is maintained.

The PolyA-promoter and IRES structures provided by the present invention are compared with the prior art. VEGF165 and Ang-1 double gene recombinant transfer plasmid pAdTrack-CMV-VEGF 165-PolyA-promoter-Ang-1 (Accession number is CCTCC M 2010221) and pAdTrack-CMV-VEGF165-IRES-Ang-l (Accession number is CCTCC M 2010220 ) , and Ad-VEGF 165-PolyA-promoter-Ang-1 and Ad-VEGF 165-IRES-Ang-1 recombinant adenoviral vectors produced by QBI-293A cell packaging make VEGF 165 and Ang-1 genes ideal. The successful combination is successfully expressed on the same vector, and the combination of the two gene expression products promotes angiogenesis and wound repair.

The rat wound repair experiment further proves that the transgenic bone marrow stromal cell modified silk fibroin material of the present invention can also promote angiogenesis of wound tissue, and creates conditions for further development of a blood vessel-inducing medical biomaterial such as a small-caliber artificial blood vessel. Biomaterial preservation information description:

1. pAdTrack-CMV-Ang- 1 -IRES- VEGF 165 plasmid, deposited at the China Center for Type Culture Collection on September 15, 2010, at Wuhan University, China, with the accession number CCTCC M 2010220, named as the large intestine Bacillus

DH5a/pAdTrack-CMV-hVEGF 165-IRES-hAng-1.

2. pAdTrack-CMV-VEGFl 65-PolyA-promoter-Ang-1 plasmid, deposited at the China Center for Type Culture Collection on September 15, 2010, at Wuhan University, China, with the accession number CCTCC M 2010221, named It is Escherichia coli DH5a/pAdTrack-CMV-hVEGF 165-polyA-Promter-hAng-1.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 shows the AdTrack-CMV-PolyA-promoter plasmid map of the present invention.

Figure 2 shows a legend for the calculation of corneal neovascularization area in rabbits.

Figure 3 shows the ability of HE and immunohistochemistry to detect angiogenesis after injection of the limbus. Figure 4 shows the RT-PCR electrophoresis pattern of VEGF165 and Ang-1 genes in rabbit bone marrow stromal cells on regenerated silk fibroin membrane.

1. SF+MSC group; 2. SF+MSC+Ad-GFP group; 3. SF+MSC+Ad-VA group. Figure 5 shows the effect of transgenic cell modified regenerated silk fibroin membrane on corneal neovascularization after implantation into rabbit cornea; SF: regenerated silk fibroin membrane; MSC: rabbit bone marrow stromal cells.

Figure 6 shows VEGF165 in the rabbit corneal layer of the regenerated silk fibroin membrane modified with transgenic cells.

Immunohistochemical analysis of Ang-1, HIF-a, bFGF, EGF, and PDGF.

Figure 7 shows the expression of VEGF in the wound healing tissues of rats by immunohistochemistry: SFMSCVA group compared with PBS group, SF group, SFMSC group, △ P < 0.05; SFMSC group compared with PBS group, SF group, * P < 0.05.

Figure 8 shows the expression of Ang-1 in the wound healing tissues of rats by immunohistochemistry: SFMSCVA group compared with PBS group, SF group, SFMSC group, △ P < 0.05; SFMSC group compared with PBS group, SF group, * P < 0.05.

Figure 9 shows the expression of CD34 in the wound healing tissues of rats by immunohistochemistry: SFMSCVA group compared with PBS group, SF group, SFMSC group, △ P < 0.05; SFMSC group compared with PBS group, SF group, * P < 0.05. detailed description

In order to further understand the invention, the preferred embodiments of the present invention are described in the accompanying claims.

The effects of the present invention are described below by way of specific examples, but the scope of the present invention is not limited by the following examples.

The invention discloses a double gene co-expression vector and a regenerated silk fibroin film modified by transgenic bone marrow stromal cells, and those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.

According to the present invention, the injection of the recombinant adenovirus of the present invention can successfully induce corneal neovascularization; the rabbit bone marrow stromal cells cultured on the silk fibroin membrane are described in the present invention. After recombinant adenovirus-infected cells, VEGF165 and Ang-1 genes can be successfully expressed; after the transgenic cell-modified silk fibroin membrane is implanted into the rabbit cornea, the neovascularization grows into the cornea of the silk fibroin implantation area for more than 1 month. HE staining and immunohistochemistry showed a marked increase in vascular density, and VEGF165, Ang-1, and CD34 were successfully expressed in the rabbit cornea. The rat wound repair experiment further proves that the transgenic bone marrow stromal cell modified silk fibroin membrane of the invention can also promote angiogenesis of wound tissue, indicating that the recombinant adenovirus of the invention and the transgenic bone marrow stromal cell modified silk fibroin membrane have potential Value.

The effects of the present invention are described below by way of specific examples, but the scope of the present invention is not limited by the following examples. Example 1 : pAdTrack-CMV-Ang-1 -PolyA-promoter-VEGF 165 double gene recombinant transfer plasmid map

The polyana 296-298-promoter (PolyA-promoter) and IRES sequences are disclosed in the Chinese Patent Application Specification No. 200810244301.3. The double gene recombinant transfer plasmid of the present invention is based on the pAdTrack-CMV-PolyA-promoter transfer plasmid, and the Ang-1 fragment is inserted between the Bgl II and Sal I cleavage sites, and the Not I and Xho I cleavage sites are inserted. The VEGF165 fragment was inserted between the spots, and the plasmid map is shown in Fig. 1. Example 2: Cloning of the gene of interest

Comparing the conserved sequences of Ang-1, primers PI and P2 were designed (Table 1), and Bgl II and Sai l restriction sites were introduced at both ends. The pAdTrack-CMV-containing Ang-1 fragment was constructed in the department. The Ang-1 -IRES-VEGF 165 plasmid was used as a template, and the PCR product of the Ang-1 target fragment obtained by PCR amplification was identified by agarose gel electrophoresis.

Comparing the conserved sequences of VEGF165, primers P3 and P4 were designed (Table 1;), and the Not I and Xho I restriction sites were introduced at both ends, respectively. The pAdTrack-CMV-Ang-1 containing the VEGF165 fragment was constructed in the department. -IRES-VEGF165 plasmid as a template, PCR amplification The PCR product of the VEGF165 target fragment obtained was identified by agarose gel electrophoresis.

Table 1 PCR primers

Sense Antisense

Ang- 1 Ρ1 :5' -tagagatctatgacagtt Ρ2:5' -Accgtcgacctcaaaaatct

( Bgl II, Sal I ) ttcctttcctttgc -3 ' aaaggtcgaatcatc-3 '

VEGF165 P3 : 5 ' -gcagcggccgcatga P4 : 5 ' -Taactcgagtcaccgcctcg

( Not I , Xho I ) actttctgctgtcttgggtg-3 ' gcttgtcacatc-3 '

VEGF165 P5 : 5 ' -gcaagatctatgaacttt P6 : 5 ' -Taagtcgacctcaccgcctc

(Bgl II, Sal I ) ctgctgtcttgggtg -3, ggcttgtcacatc-3,

Ang-1 P7 : 5 ' -taggcggccgcatgac P 8 : 5 ' -accctcgagtcaaaaatctaa

( Not I , Xho I ) agttttcctttcctttgc-3 ' aggtcgaatcatc-3 '

The PCR conditions were: pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 50 s, annealing at 58 °C for 50 s, extension at 72 °C for 50 s, 30 cycles in total, and extension for 10 min at 72 °C.

The PCR system is as follows:

lOxPCR buffer (containing Mg2+) 5μ1

dNTPmix( 1 OmM each) 1 μΐ

Primer PI ( 25μΜ ) Ιμΐ

Primer Ρ 2 ( 25μΜ ) Ιμΐ

pAdTrack-CMV-Ang- 1 -IRES-VEGF 165

Plasmid template

ddH ₂ 0 40μ1

Taq DNA polymerase ( 5υ/μ1 ) 1 μΐ

Final volume 50μ1 Example 3: Construction of pAdTrack-CMV-Ang-1 -PolyA-promoter single gene recombinant transfer plasmid

The PCR product of the Ang-1 gene purified by the DNA cleaning kit and the transfer plasmid pAdTrack-CMV-PolyA-promoter extracted by the small amount of plasmid extraction kit were double-digested with Bgl II and Sal I at 37 ° C for 5 h, respectively. Recycling the target fragment, recycling according to the kit Methods and Procedures, and then ligated overnight at 4 °C with T4 DNA ligase, and then the ligated product was transformed into E. coli DH5a, and positive monoclonals were selected in Kana-containing (5 (^g/ml) resistant plates). After identification by PCR, double enzyme digestion and DNA sequencing, the positive clones were kept at -20 °C in the refrigerator.

The double digestion system is as follows: pAdTrack-CMV-PolyA-promoter plasmid double digestion system Ang-1 PCR product double digestion system lOxBuffer H 2μ1 lOxBuffer H 3μ1 pAdTrack-CMV-PolyA-promoter plasmid 15μ1 VEGF165 PCR product 23μ1

Bgl II 1.5μ1 Bgl II

Sal T fil SaH M Final volume 20μ1 Final volume The connection system is as follows:

Purified double digestion Ang-1 PCR product 8μ1

Purified double digestion pAdTrack-CMV-PolyA-promoter large fragment 8 1

lOxBuffer 2μ1

Τ4 DNA ligase 2 1

Final volume 20μ1

The preparation of DH5a E. coli competent cells by CaCl ₂ method and the transformation technique are as follows:

One day before the transformation, DH5a was inoculated with 1 ml of 5 ml LB medium at 37 ° C overnight, and cultured in 1% of 5 ml LB medium at 37 ° C until the OD 600 nm was about 0.2 (about 1.3 h for culture), and the bacterial solution was poured. In 5 ml centrifuge tube, 5000r/min, centrifuge for 5min to collect the bacteria, add 5ml pre-cooled lOOmM CaC12, suspend the cells at 0 °C, 20 ~ 30min, 5000 r / min, centrifuge for 5min, collect the cells with about 600μ1 lOOmM Pre-cooled CaC12 suspension cells, and transformed according to the following table, and cultured at 37 °C for 16h in a water-isolated incubator to identify positive clones, thus obtaining pAdTrack-CMV-Ang-1-PolyA-promoter single gene recombinant transfer plasmid The methods of plasmid extraction, PCR and double enzyme digestion are the same as above. Transformation group Receptor DNA culture plate Positive group 60μ1 Ιμΐ Plasmid Kana negative group 60μ1 One Kana experimental group 60μ1 20μ1 ligation solution Kana Example 4: Construction of pAdTrack-CMV- Ang-1 -PolyA-promoter- VEGF 165 double gene recombinant transfer plasmid

Based on the VEGF165 gene clone and the pAdTrack-CMV-Ang-1 -PolyA-promoter single gene recombinant transfer plasmid constructed in Example 2, the VEGF165 gene fragment and p which were double-digested with Not l and Xho I and recovered by gel were used. The AdTrack-CMV-Ang-1 -PolyA-promoter plasmid fragment was ligated with T4 DNA ligase to construct a pAdTrack-CMV-Ang-1 -PolyA-promoter- VEGF 165 double gene recombinant transfer plasmid. Example 5: Construction of pAdTrack-CMV-VEGF 165 -PolyA-promoter- Ang-1 double gene recombinant transfer plasmid

Referring to Examples 1-4, primers P5, P6 and P7, P8 (see Table 1) were designed for the conserved sequences of VEGF165 and Ang-1, and Bgl ll and Sal I restriction sites were introduced at both ends of VEGF165 primers; -1 primers were introduced into the Not I and Xho I restriction sites, and the VEGF165 and Ang were amplified by PCR using the pAdTrack-CMV-Ang-l-IRES-VEGF165 plasmid containing VEGF165 and Ang-1 fragments as a template. The PCR product of the -1 fragment was identified by agarose gel electrophoresis.

Double-cleavage of the target gene PCR product and pAdTrack-CMV-PolyA-promoter empty vector plasmid, the gel was recovered and ligated with T4 DNA ligase overnight at 4 °C, and then the ligated product was transformed into E. coli DH5a and containing Kana (50 g/ml). The LB agar plate was screened to select a monoclonal antibody, thereby inserting a VEGF165 fragment between the Bgl II and Sal I cleavage sites in the multiple cloning site (MCS), and inserting Ang- between the Not l and Xho I cleavage sites. 1 fragment. PCR, double enzyme digestion and DNA sequencing were performed, and the positive clones were kept for survival. Construction of pAdTrack-CMV-VEGF 165-IRES-Ang-1 double-gene recombinant transfer plasmid: Based on the construction of pAdTrack-CMV-VEGF 165-PolyA-promoter-Ang-1 double gene recombinant transfer plasmid, Bgl ll, The VEGF 165 gene fragment of pAdTrack-CMV-VEGF165-PolyA-promoter-Ang-1 and the large fragment of pAdTrack-CMV-IRES plasmid were digested with Sal I and ligated with T4 DNA ligase to construct pAdTrack-CMV- VEGF 165-IRES single gene recombinant transfer plasmid. The construction method is the same as that of the embodiment 3.

An Ang-1 gene fragment of pAdTrack-CMV-VEGF1 65- PolyA-promoter-Ang-1 and a large fragment of pAdTrack-CMV-VEGF165-IRES single gene recombinant transfer plasmid, which were digested with Not I and Xho I and recovered by gel, The cells were ligated overnight at 4 °C with T4 DNA ligase, and the ligated product was transformed into E. coli DH5a and LB agar plates containing Kana (5 (^g/ml) to select monoclonal clones, and identified by PCR, double enzyme digestion, positive clones. The method was the same as in Example 4. The double-gene recombinant transfer plasmid pAdTrack-CMV-VEGF 165-PolyA-promoter-Ang-1 was deposited with the China Center for Type Culture Collection on September 15, 2010. The deposit number is CCTCC M 2010221.

The pAdTrack-CMV-VEGF1 65-IRES-Ang-1, pAdTrack-CMV-Ang-1 -IRES-VEGF 165 plasmid was constructed by the above method, wherein the double gene recombinant transfer plasmid pAdTrack-CMV- VEGF 165-IRES-Ang- 1 Deposited with the China Type Culture Collection on September 15, 2010, with the accession number CCTCC M 2010220. Example 6: Identification of double gene recombinant transfer plasmid

The recombinant vector pAdTrack-CMV-Ang-l-PolyA-promoter-VEGF165 plasmid was used as a template, and Pl and P2 were used as primers to amplify the target gene fragment of Ang-1, and its size was the theoretical value of the expected Ang-1 amplification. Consistent with 1497 bp, P1 and P4 were used as primers to amplify the VEGF165 target gene fragment, and its size was consistent with the expected size of VEGF165 (576 bp). The 1 ng bp Ang-1 fragment was cleavage by Bgl ll and Sal I, and the 576 bp VEGF 165 was released by Not I and Xho I digestion. In the paragraph, it was shown that the Ang-1 and VEGF165 target gene fragments were successfully subcloned into the pAdTrack-CMV-PolyA-promoter engineered empty transfer plasmid. DNA sequencing further confirmed the successful construction of the pAdTrack-CMV-Ang-1 -PolyA-promoter-VEGF 165 double gene recombinant transfer plasmid.

According to the above method, other double gene recombinant transfer plasmids pAdTrack-CMV-VEGF 165-PolyA-promoter-Ang-1, pAdTrack-CMV-VEGF165-IRES-Ang-1, etc. were identified and DNA sequence was determined, which confirmed the successful construction. . Example 7: Construction and identification of homologous recombinant adenoviral plasmids

The pAdTrack-CMV-Ang-1 -PolyA-promoter-VEGF 165, pAdTrack -CMV-VEGF 165-PolyA-promoter-Ang-1 and pAdTrack-CMV-VEGF165-IRES-Ang-1 recombinant transfer plasmids were ligated to the backbone plasmid pAdEasy, respectively. -1 picked up the cloned plasmid after homologous recombination in BJ5183 bacteria, and initially screened according to molecular weight

pAdEasy-1 -pAd-Ang- 1 -PolyA-promoter-VEGF 165, pAdEasy-1 - pAd- VEGF 165-PolyA-promoter-Ang-1

pAdEasy-1 -pAd-VEGF 165-IRES-Ang-1 homologous recombination positive clone, agarose electrophoresis results showed that 5 of the pAd-Ang-1 -PolyA-promoter-VEGF 165 and pAdEasy-1 co-transformation groups were selected. 4 clones were positive clones; pAd-

Five of the 6 clones selected by VEGF 165-PolyA-promoter-Ang-1 and pAdEasy-1 co-transformation groups were positive clones; pAd-VEGF165-

Two of the four clones selected by the IRES-Ang-1 and pAdEasy-1 co-transformation groups were positive clones; the identification of the genomic DNA fragment of about 30 kb and the unique 4.5 kb or 3.0 kb size were obtained by pad digestion. Ari and kanamycin resistance coding gene fragments. Example 8: Packaging, amplification and titer detection of recombinant adenovirus

Pac I linearized pAdEasy- 1-pAd-Ang-l -PolyA-promoter-VEGF 165, pAdEasy-1 -pAd-VEGF 165-PolyA-promoter-Ang-1 and pAdEasy-1 -pAd-VEGF 165-IRES- Ang-1 homologous recombinant adenovirus plasmid LipofectamineTM2000 Operational Instructions After transfection of 293A cells with Lipofectamin liposomes, GFP expression was observed under fluorescence microscopy, and the fluorescence intensity increased with the prolongation of culture time. Ad-Ang-1 -PolyA-promoter-VEGF 165 , Ad-VEGF 165-PolyA-promoter-Ang-1 , Ad-VEGF 165-IRES-Ang-1 and Ad-Ang-1 -IRES were collected after 10 days of transfection. - VEGF 165 first generation adenovirus, secondary infection of 293 A cells, fluorescence observed under inverted fluorescence microscope, and CPE appeared, the results showed that the four viruses were successfully packaged in 293A cells. After multiple rounds of infection and amplification, the virus with a titer of up to 5 l0 ⁹ pfu/ml is finally stored at -80 °C for storage. Example 9: RT-PCR Identification of Recombinant Adenovirus

QBI-293A cells infected with Ad-Ang-1 -PolyA-promoter-VEGF 165 , Ad-VEGF 165-PolyA-promoter-Ang-1, Ad-VEGF 165-IRES-Ang- ¹ were collected and ¹ J was collected. The virus-infected QBI-293A cells were extracted from total RNA for RT-PCR, and the prepared three groups of cDNAs were used as templates, Pl and P2; P3 and P4 were primers to identify VEGF 165, Ang-1, and internal reference β-actin genes, respectively. Transcription in QBI-293A cells. It can be seen that the 576 bp VEGF 165 and 1497 bp Ang-1 products of the expected size can be produced in each infected group; the QBI-293A uninfected cell control group did not produce the above bands at the corresponding positions. The results of RT-PCR showed that the recombinant adenovirus expression vector co-expressed by IRES-mediated and polyA+promoter-mediated three sets of Ang-1 and VEGF165 genes was successfully constructed. Example 10: Comparison of VEGF165 and Ang-1 cytokine secretion levels in WI-38 cells by four recombinant adenoviruses by ELISA

WI-38 cells in logarithmic growth phase were digested to a cell concentration of lxlO ⁸ L-1, seeded on a 24-well cell culture plate at 1 ml/well, and cultured for 24 h with 50 MOI of Ad-Ang-1 -PolyA- promoter-VEGF 165, Ad-VEGF 165-PolyA-promoter- Ang-1, Ad-Ang-1 -IRES- VEGF 165, Ad-VEGF 165-IRES-Ang-1 and empty vector Ad-GFP adenovirus infection WI- For 38 cells, 3 replicate wells were set in each group. After 48 hours of infection, the cell growth morphology and fluorescence intensity after virus infection were observed. After 48 hours of virus infection, the supernatants of each group were cultured, and the contents of VEGF165 and Ang-1 in the supernatant were determined by ELISA. The results showed that: WI-38 cells in each group of PBS group and Ad-GFP group. Secretion of lesser levels of VEGF165 and Ang-1 cytokines, but IRES/PolyA-promoter-mediated VEGF165 and Ang-1 double genomes allow expression of VEGF- and Ang-1 genes in WI-38 cells The levels were significantly increased (P<0.05); the IRES-mediated Ang-1 and VEGF165 genes, both upstream and downstream of the IRES, were less expressed than the Ang-1 mediated by the polyA-promoter. The expression of VEGF165 gene (P < 0.05), and the expression of downstream gene of Ang-1/VEGF165 in different positions on the same vector and downstream were significantly lower than that of upstream gene expression (Ρ < 0.05).

The results showed that adenovirus-mediated VEGF165 and Ang-1 double gene vectors could be efficiently expressed in WI38 cells; polyA-promoter was more efficient than IRES-mediated double gene expression, and the expression levels of both downstream genes were significantly lower than those of the two genes. The amount of upstream gene expression. Example 11: Comparison of corneal limbal injection IRES/PolyA-promoter-mediated VEGF 165 and Ang-1 double-gene viruses inducing corneal neovascularization

Ten New Zealand white rabbits were randomly divided into five groups and injected with Ad-Ang-1 -PolyA-promoter- VEGF 165, Ad- VEGF 165-PolyA-promoter- -Ang-1 and Ad-Ang-1 -IRES- VEGF 165 , Ad- VEGF 165-IRES-Ang-1 and Ad-GFP virus. Intramuscular injection of ketamine 5mg / kg general anesthesia, rabbits wiped the Anil iodine disinfection, each eye drops 1 ~ 2 drops of Nobel hi surface anesthesia, open the device, rinse the binocular conjunctiva with gentamicin injection. The above five viruses were injected into the corneal stroma at the 12-point angle of the sclera at the top of each rabbit eye. The colitis was injected and the conjunctival sputum was washed again, and the chlortetracycline eye ointment was applied. Corneal stromal injection of virus on the third day, the first week, the first month, respectively, the corneal neovascularization was recorded. The corneal neovascular growth area was calculated according to the Robert computer mathematical model formula: C/12x3.1416[r2-(r-l)2] (Fig. 2). Where C is the number of corneal circumferential clock points where neovascularization occurs, r is the corneal radius, and 1 is the length of the blood vessel.

The results showed: injection of Ad- VEGF 165-polyA-promoter- Ang-1, Ad- VEGF 165-IRES-Ang-1 rabbit corneal margin, lw posterior limbal injection area near the conjunctiva Moderate hyperemia of the blood vessels, congestion is confined to the area of virus injection; conjunctival hyperemia near the limbus is slightly relieved 2 weeks after injection, and the brush-like neovascularization grows into the cornea; the conjunctival hyperemia at the 4th after injection is significantly relieved, and corneal neovascularization has been seen. Extending to the edge of the pupil, the tip of the bifurcation, partially interlaced in a network; and the former is more significant than the latter (p<0.05); and injection of Ad-Ang-1-polyA-promoter- VEGF 165, Ad-Ang-1 -IRES- VEGF 165 and Ad-GFP rabbit corneal margin, lw only caused mild conjunctival congestion near the injection area of the limbus, conjunctival hyperemia was significantly reduced 2 weeks after injection, only a small number of blood vessels into the cornea, and limited to the cornea At the edge, the conjunctival hyperemia disappeared completely at the 4th week after injection, and only a small amount of new blood vessels existed in the cornea. There was no statistically significant difference between the three groups (p>0.05).

It indicated that the injection of Ad-VEGF165-PolyA-promoter-Ang-l and Ad- VEGF 165-IRES-Ang-1 into the limbus of rabbits can successfully induce corneal neovascularization, and the former is more effective than the latter, regardless of the IRES vector. Or the polyA-promoter vector, the VEGF165 gene can successfully induce corneal neovascularization only when it is located upstream of the vector, and it can not induce corneal neovascularization when it is located downstream of the vector. Example 12: Immunohistochemistry was used to detect the expression of VEGF165 and Ang-1 in rabbit cornea.

After the rabbit cornea was injected with the virus for one month according to the method described in Example 10, the rabbits were sacrificed by injection, and the eyeballs were removed. The whole cornea was immediately fixed with 10% formalin, embedded in paraffin, and serially sliced perpendicular to the corneal surface. About 5μηι. The expression of VEGF165 and Ang-1 factors were detected by immunohistochemical SP method in the embedded corneal tissue. The results were as follows: cytoplasm yellow (+); brown (++); brownish black (+++).

The results showed that VEGF 165 and Ang-1 were positively expressed in rabbit corneal layer containing VEGF165 and Ang-1 virus, and VEGF165 and Ang-1 were positive in the upstream of polyA-promoter/IRES than in the downstream. The expression was more pronounced, brownish black (+++), and no positive expression of VEGF165 and Ang-1 was observed in the corneal layer injected with Ad-GFP virus. Example 13: HE staining and CD34 immunohistochemistry were used to detect neovascular density in rabbit cornea. The rabbit limb injection was injected with the double gene virus according to the method described in Example 10, respectively. After 1 month of poisoning, the experimental rabbits were sacrificed by air injection, and the eyeballs were removed. The whole corneal membrane was immediately fixed with 10% formalin, embedded in paraffin, and serially sliced perpendicular to the corneal surface, about 5 μηι thick. HE staining and CD34 immunohistochemistry were used to detect microvessel density in rabbit cornea. The number of positive blood vessels was determined by red blood vessel-containing red blood vessel lumen (HE staining) or brown-yellow blood vessel lumen (CD34 staining). Immunohistochemical examination showed that each slice was observed under a light microscope of 100 times, and a total of 10 fields of view were counted for analysis and statistics. All data were expressed as ±S. All experimental data were statistically processed using SAS software, and analysis of variance was used, P < 0.05.

The results are shown in Figure 3. Injection of Ad-VEGF165-polyA-promoter-Ang-l, Ad-VEGF 165-IRES-Ang-1 rabbit cornea and injection of Ad-Ang-1 -polyA-promoter- VEGF 165 , Ad- Compared with the cornea of Ang-1 -IRES-VEGF 165 and Ad-GFP, HE staining showed abundant capillaries. CD34 immunohistochemical analysis showed strong positive, microvessel density was (11.9 ± 2.4) / high power field, the difference was Statistical significance (PO.05), and the former is more significant than the latter (PO.05); whereas Ad-Ang-1 -polyA-promoter- VEGF 165, Ad-Ang-1 -IRES- VEGF 165 and Ad The capillaries in the margins of the three groups of GFP were rare, and no significant expression of CD34 was observed. The difference was not statistically significant (P>0.05). Example 14: Isolation and culture of rabbit bone marrow stromal cells

Take a New Zealand white rabbit (Suzhou University Experimental Animal Center), obtain the rabbit bone marrow primary cells by bone marrow aspiration, and add it to the synthetic cell culture medium (EBM-2-MV) containing 5% fetal bovine serum at 37 °C. Incubate under 5 % C0 ₂ . After 3 days, change the solution to remove unattached blood cells. 7 ~ 14d cells can be covered with cell culture bottoms. Rabbit bone marrow mesenchymal stem cells (BMSCs) grow in the shell wall and form field packs. The cells are cleared and the stromal cells are purified and continue to culture.

0.25% trypsin-digested primary cultured bone marrow stromal cells [MSC], adjusted to a cell concentration of lxl0 ⁸ Ll, and inoculated into a 24-well culture plate with regenerated silk fibroin membrane [SF] at 1 ml/well, divided into three groups. : Experimental group (SF+MSC+Ad-VEGF 165-polyA-promoter-Ang-1 group with 50MOI Ad-VEGF 165-polyA-promoter-Ang-1 added, cartridge: SF+MSC+Ad-VA group), negative control group (SF+MSC+Ad-GFP group with 50MOI Ad-GFP), cell control group (SF+MSC group), three replicate wells per group, at 37 The cells were observed under the condition of °C and 5%C0 ₂ for 72 h, and the morphological changes and fluorescence infection were observed under inverted fluorescence microscope.

The rabbit bone marrow stromal cells grown on the regenerated silk fibroin membrane adhered to the wall, and the state was good, the cells were transparent, the morphology was intact, and the spindle shape was stretched. Infected adenosis Ad-VEGF165-PolyA-promoter-Ang-1, Ad-GFP rabbit bone marrow stromal cells showed strong green fluorescence under fluorescence microscopy, while cells not infected with adenovirus showed no fluorescence. This shows that the regenerated silk fibroin membrane has no adverse effects on the growth of transgenic cells. Example 15: Detection of cytokine transcription levels in Ad- VEGF165-PolyA-promoter-Angl transgenic rabbit bone marrow stromal cells on regenerated silk fibroin membrane by RT-PCR

The rabbit bone marrow stromal cells cultured on the regenerated silk fibroin membrane were infected with 50 MOI of Ad-VEGF165-PolyA-promoter-Angl and Ad-GFP virus for 48h, then the cells were collected, washed 2-3 times with PBS, and the cells were extracted according to the kit instructions. Total RNA, using VEGF165 primer P5: 5 '-gcaagatctatgaactttctgctgtcttgggtg-3 ' ,

P6: 5 '-taagtcgacctcaccgcctcggcttgtcacatc-3 ' ,

Angl Primer P7: 5 ' -taggcggccgcatgacagttttcctttcctttgc-3 ' ,

P8: 5'-accctcgagtcaaaaatctaaaggtcgaatcatc-3' was identified by RT-PCR. The PCR conditions were 94 °C for 4 min, 94 °C for 50 sec, 58 °C for 50 sec, 72 °C for lmin, 35 cycles, 72 °C for 10 min, and finally The ΙΟμΙ product was electrophoresed with DNA Marker row agarose gel.

The results are shown in Figure 4: VEGF165, Ang-1 gene and β-actin in the SF+MSC+Ad-VA group showed positive bands; while SF+MSC group and SF+MSC+Ad-GFP group only had β-actin A positive band appears. Example 16: ELISA assay for cytokine secretion of Ad-VEGF165-PolyA-promoter-Angl transgenic rabbit bone marrow stromal cells on regenerated silk fibroin membrane. Rabbit bone marrow stromal cells (MSC) in logarithmic growth phase were digested to cell concentration. Lxl0 ⁵ /ml, inoculated at 1ml/well on a 24-well cell culture plate with regenerated silk fibroin film (SF), the experiment was grouped as before, feeling After 48 hours of virus infection, the supernatants of each group were taken, and the contents of VEGF and Angl were determined according to the ELISA test kit. The results showed that: rabbit bone marrow stromal cells in SF+MSC group and SF+MSC+Ad-GFP group only secrete autologous VEGF165 and Ang-1 cytokines at low levels, but

The expression level of SF+MSC+Ad-VA group was significantly improved (*PO.05). Example 17: Observation and measurement of neovascularization after implantation of rabbit corneal layer by Ad-VEGF165-PolyA-promoter-Angl transgenic rabbit bone marrow stromal cells

Intramuscular injection of ketamine 5mg / kg general anesthesia, rabbits wipe the Anil iodine disinfection, each eye drops 1 ~ 2 drops of Nobel hi surface anesthesia, open the device, rinse the double eye conjunctiva with gentamicin injection. The corneal stroma was cut in the "L" shape (5 4 mm) centered at the 12 o'clock direction of the cornea, and the depth was 1/2 corneal thickness. The corneal stroma was separated horizontally with a diamond knife, and a 5x4 mm pouch was formed in the corneal stroma, and 4 x 3 mm transgenic cells were formed. The modified regenerated silk fibroin film was buried between the corneal layers, and the corneal incision was sutured for 3 needles. After the implantation, the gentamicin injection was again washed with the conjunctival sputum and the chlortetracycline ointment was applied.

Corneal neovascularization was recorded photographicly in the first week, the second week, and the first month after implantation of the transgenic cells modified by the cornea. The corneal neovascular growth area was calculated according to the Robert computer mathematical model formula: C / 12x3.1416 [r ² -(rl) ² ] (Fig. 2). Where C is the number of corneal circumferential clock points where neovascularization occurs, r is the corneal radius, and 1 is the length of the blood vessel. The statistical results are shown in Fig. 5.

After the rabbit corneal layer was implanted with the regenerated silk fibroin membrane modified by transgenic cells, moderate conjunctival hyperemia occurred in the limbus of the five groups of rabbits, and congestion was confined around the surgical incision; corneal mild edema in the silk fibroin material (SF) implantation area The capillary can be seen in the small capillary capillaries; after the implantation of the regenerated silk fibroin material, the conjunctival hyperemia near the limbus was reduced in each group, and the corneal edema in the silk fibroin implant area was slightly reduced; SF+MSC+Ad-GFP group And the small blood vessels around the limbus of the SF+MSC group gradually subsided, while SF+MSC+Ad-A (Ad- PolyA-promoter-Ang-1), SF+MSC+Ad-V (Ad- VEGF- PolyA- Promoter), SF+MSC+Ad-VA group brush-like neovascularization thickened, extending from the limbus into the silk fibroin implantation area, and SF+MSC+Ad-V, SF+MSC+Ad-VA group than SF +MSC+Ad-A group was more obvious; conjunctival hyperemia disappeared in each group after 4th implantation; capillaries at the limbus were completely Regression; the cornea of the SF implantation area is gradually transparent, and the neovascularization in the SF+MSC+Ad-GFP group and the SF+MSC group regenerated silk fibroin implantation area is basically ablated, while SF+MSC+Ad-A, SF+MSC +Ad-V, SF+MSC+Ad-VA group had more vascular formation, and SF+MSC+Ad-VA group had more obvious blood vessel formation. The thick neovascularization had completely covered the regenerated silk fibroin membrane transplantation area and reached the center of the cornea. Area (* P<0.05). Example 18: HE staining and CD34 immunohistochemical method for detecting neovascular density in rabbit cornea after implantation of transgenic cell modified regenerated silk fibroin membrane

The transgenic modified regenerated silk fibroin membrane was implanted into the cornea for 1 month, and the rabbits were sacrificed by air injection to remove the eyeballs. The full-angle membrane was immediately fixed with 10% formalin, embedded in conventional paraffin, and serially sliced perpendicular to the surface of the cornea, about 5 μηι thick. HE staining and CD34 immunohistochemistry were used to detect microvessel density in rabbit cornea. Immunohistochemical staining (Wuhan Dr. Company provides primary antibody, Shanghai Gene Company provides secondary antibody) The specific method is as follows: Paraffin section, 0.3% hydrogen peroxide incubation for 30 min, soaking in PBS, adding 10% normal goat serum (diluted with PBS) lOmin Add VEGF (1: 30)-antibody, wash with PBS, add a broad-spectrum biotin-labeled secondary antibody at 37 °C, develop ABC color reagent, counterstain, and seal. The number of positive blood vessels was determined by red blood cell-containing red blood vessel lumen (HE staining) or brown-yellow blood vessel lumen (CD34 staining). Immunohistochemical examination showed that each slice was observed under a light microscope of 100 times, and a total of 10 fields of view were counted for analysis and statistics.

The results showed that HE staining of SF+MSC+Ad-VEGF165-Ang-l group in the corneal stroma after regeneration of Ad-VEGF165-Angl gene-modified regenerated silk fibroin membrane showed abundant capillaries, and immunohistochemical analysis of CD34 was performed. Significantly strong positive, microvessel density was (17 ± 3.5) / high power field, while SF + MSC + Ad-GFP group and SF + MSC group corneal stromal capillaries were rare, no significant CD34 positive expression, the difference was statistically significant (* P<0.05). Example 19: Immunohistochemical method was used to observe the expression of VEGF165, Ang-1, HIF-a, bFGF, EGF and PDGF in the cornea after transfection of modified transgenic silk fibroin membranes.

Rabbit cornea was implanted with transgenic modified regenerated silk fibroin film. After one month, the experimental rabbits were sacrificed and the eyeballs were removed. The whole corneal membrane was immediately fixed with 10% formalin, embedded in paraffin, and sag. Continuously sliced directly to the surface of the cornea, about 5 μηι thick. The expression of VEGF165, Ang-1, HIF-a, bFGF and other factors were detected by immunohistochemical SP method. The staining results were: cytoplasm yellow (+); brown (++); brown black (+ ++). Immunohistochemistry was performed under 400-fold light microscopy. A total of 10 fields were recorded. Image-Pro Plus 6.0 image analysis software was used to measure VEGF165, Ang-1, HIF-a, bFGF, EGF in the collected corneal tissue. , Ratio of positive area of PDGF, calculate the average value. All data were expressed as ±S, and all experimental data were statistically processed using SAS software, using analysis of variance, P < 0.05.

The results are shown in Fig. 6. It is shown that: VEGF165 and Ang-1 were positive in the corneal stroma of SF+MSC+Ad-VEGF165-Ang-l group after implantation of VEGF165 and Ang-1 gene-modified regenerated silk fibroin membrane. The expression was brown-black (+++), but there was no significant expression of VEGF and Ang-1 in the corneal stroma of SF+MSC+Ad-GFP group and SF+MSC group, the difference was statistically significant (P<0.05).

The expression of HIF-a and bFGF in SF+MSC+Ad-VEGF165-Ang-l group after VEGF165 and Ang-1 gene-modified regenerated silk fibroin membrane was brownish-black (+++). Significantly higher than SF+MSC+Ad-GFP group and SF+MSC control group (PO.05); EGF, PDGF expression and SF+MSC+Ad-GFP group and SF+MSC control group were brown (++) There was no statistically significant difference between the two groups (P>0.05), indicating that the regenerated silk fibroin membrane modified by VEGF165 and Ang-1 gene cells can not only effectively express VEGF165 and Ang-1 genes in the corneal layer, but also promote bone marrow. The levels of autoimmune HIF-a and bFGF in stromal cells are increased, and the normal and stable expression of autocrine EGF and PDGF in bone marrow stromal cells is maintained. Example 20: Isolation and culture of rat bone marrow stromal cells

Male Sprague-Dawley rats (Experimental Animal Center of Suzhou University) were taken. After the neck was sacrificed, the bilateral tibia and femur were taken under aseptic conditions. The metaphysis was cut off and the medullary cavity was rinsed with EBM-2-MV complete medium. Added SingleQuots containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF2), R3 insulin growth factor (R3-IGF-1), endothelial growth factor (EGF), ascorbic acid and 5% fetal calf Serum and other related components, a synthetic cell culture medium (EBM-2-MV) containing 5% fetal bovine serum, and collecting the rinsing liquid in the culture medium. Gently blow and incubate at 37 ° C, 5 % C0 ₂ . After 3 d, change the solution to remove unattached blood cells. 7 ~ 14d cells can be used to cover the bottom of the cell culture bottle. Rat bone marrow stromal cells (rMSCs) grow adherently and form cell colonies. After 2-3 changes, non-adherent Most of the cells were cleared, and the bone marrow stromal cells were initially purified and cultured.

The bone marrow stromal cells isolated and cultured were digested with 0.25% trypsin, adjusted to a cell concentration of lxlO ⁸ L" ¹ , and inoculated into a 24-well culture plate with regenerated silk fibroin membrane at 1 ml/well. The cells were cultured for 24 h with 50 MOI. Ad-VEGF165-Angl recombinant adenovirus (Ad-AV group) and empty vector Ad-PolyA-promoter and Ad-IRES (empty vector control group), and a virus-infected cell control group. Example 21: Rat wound Repair experiment

1, experimental methods

Forty-eight healthy male Sprague-Dawley rats, weighing from 180 to 220 g, were anesthetized with intraperitoneal injection of 3.5% hydrated chlorine at a dose of 10 ml/kg body weight, and a square incision of about 2 cm x 2 cm on both sides of the lumbar spine. , deep into the skin is the full layer of the skin. Rats were divided into 4 groups: regenerated silk fibroin (SF) group, regenerated silk fibroin + MSC cells (SFMSC) group, regenerated silk fibroin membrane + MSC cells + Ad- VEGF-Angl (SFMSCVA) group and blank control. (PBS) group, 10 in each group. A total of two time points were selected at 7 and 14 days after injury, and each group had 10 wounds per phase (two wounds per rat). 0.5 cm from the wound, the silk fibroin materials prepared in Example 20 were sutured to the wound with the surgical line at the upper, lower, left and right sides, and the blank control group was left untreated; the wound was coated on the wound surface after surgery. Erythromycin ointment to prevent infection, the animals are fed in a single cage, free to eat, water.

The wound healing of the animals was observed every day after operation. The blood vessel distribution was observed after intraperitoneal injection of chlorinated chlorine for 7 and 14 days. The tissue was placed in 10% neutral furfural solution for fixation and paraffin embedding. The sections were subjected to HE staining, and Ang-1, VEGF and CD34 antibodies were used to detect the expression of the target gene and the blood vessel density in the tissues.

The paraffin sections were tested for the expression of VEGF, Ang-1 and CD34 by immunohistochemical staining procedure according to the SP immunohistochemical kit instructions, and the mechanism of wound healing was preliminarily studied. Each The sections were observed under a 400-fold light microscope, and a total of 10 fields of view were recorded. Image-Pro Plus 6.0 image analysis software was used to measure the percentage of positive area of VEGF, Ang-1 and CD34 in the wounds of the whole skin defect collected (Ratio of positive area). , calculate the average value. All data were expressed as ±S. All experimental data were statistically processed using SAS software, and analysis of variance was used, P < 0.05.

2 results

2.1 General observation of wounds

After wound Id, SFMSC group of regenerated silk fibroin + MSC, SFMSCVA group of regenerated silk fibroin + MSC + Ad-VA began to shrink and showed a little redness, indicating that granulation tissue was growing, while other groups were wet; In the last 3 days, the granulation tissue of the SFMSC group and the SFMSCVA group was significantly higher than that of the blank control PBS group and the regenerated silk fibroin SF group, and the skin contraction was obvious. On the 7th day after injury, the former had dry wounds, no infection, and the wound bottom and the wound edge were different. Newborn capillaries, while the latter are moist and oozing. On the 14th day after injury, the former epidermal hyperplasia of the rat margin covered the part of the wound, the granulation tissue became mature, and the granulation tissue in the latter was loose, and the SFMSCVA group was higher than the SFMSC group.

2.2 HE staining observation

The results showed that at the 7th day after operation, the SFMSC group and SFMSCVA group had denser bottom and wound granulation tissue, and capillary and fibroblasts were abundant. However, there were fewer new capillaries in the PBS group and SF group, and deep granulation. The tissue was loose. At 14 days after operation, the epidermal hyperplasia of the SFMSC group and the SFMSCVA group was covered with partial wounds. The granulation tissue became mature, the fibroblasts were abundant, the capillaries and the wounds grew vertically, and the SFMSCVA group was higher than the SFMSCVA group. SFMSC group.

2.3 immunohistochemical observation

The tissue sections of wound healing were detected by VEGF antibody immunohistochemical SP method. The results are shown in Figure 7-9. It is shown that VEGF is mainly distributed in the cytoplasm of fibroblasts and vascular endothelial cells. 7 days after operation, VEGF group and SF group VEGF Positive, while SFMSC group, SFMSCVA group was strongly positive, the difference was statistically significant (P < 0.05). At 14 days after operation, the expression of VEGF was decreased in all groups, but the SFMSC group and SFMSCVA group were still higher than the PBS group and SF group (P < 0· 05), and the SFMSCVA group was higher than the SFMSC group, but there was no significant difference between the PBS group and the SF group.

The tissue sections of wound healing were detected by Ang-1 antibody immunohistochemical SP method. The results showed that Ang-1 was positive in PBS group and SF group at 7 days after operation, while strong positive in SFMSC group and SFMSCVA group. Statistical significance (Ρ < 0· 05 ). After Md, the expression of Ang-1 was decreased in all groups, but the SFMSC group and SFMSCAV group were still higher than PBS group and SF group, and SFMSCVA group was higher than SFMSC group (P < 0.05), while PBS group and SF group. There were no significant differences in the groups.

The tissue sections of wound healing were detected by CD34 antibody immunohistochemical staining. The results showed that the cytoplasmic CD34 of dermal endothelial cells was positive in the PBS group and SF group, and the SFMSC group was positive. The SFMSCVA group was strongly positive, and the difference was statistically significant (P < 0.05). At 14 days after surgery, the number of CD34-positive capillaries decreased in each group, but the SFMSC group and SFMSCVA group had full-thickness skin defect wounds. The number of capillaries was still higher than that of PBS group and SF group, and SFMSCVA group was higher than SFMSC group (P < 0.05), but there was no significant difference between PBS group and SF group. Example 22: Detection of the effects of three other double gene recombinant adenoviruses

According to the methods described in Examples 14-21, three additional double gene recombinant adenoviruses were used.

Ad-Ang-1 -IRES-VEGF 165, Ad-VEGF 165-IRES-Ang-1, Ad-Ang-1 - PolyA-promoter- VEGF 165 Detection of transgenic cell modified regenerated silk fibroin membrane after implantation of rabbit cornea Medium and new vascular density, expression of VEGF165, Ang-1, HIF-a, bFGF, EGF, PDGF in the corneal layer, cytokine transcription level of rabbit bone marrow stromal cells and rat wound repair experiments, the results showed that only double gene recombination Adenovirus Ad- VEGF 165-IRES-Ang-1 has a similar effect on promoting angiogenesis as Ad-VEGF 165-PolyA-promoter-Angl recombinant adenovirus, and its transdermal bone marrow stromal cells modified silk fibroin material can promote wound tissue Angiogenesis, the experimental process will not be repeated here.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims

Rights request

1. A recombinant plasmid pAdTrack-CMV-VEGF 165-PolyA-promoter-Ang-1 having the accession number: CCTCC Μ 2010221.

2. The recombinant adenovirus Ad-VEGF 165-PolyA-promoter-Ang-1 produced by packaging the recombinant plasmid of claim 1 and the recombinant adenovirus pAdEasy-1 in QBI-293A cells.

3. A recombinant plasmid pAdTrack-CMV-VEGF 165-IRES-Ang-1 with the accession number; CCTCC Μ 2010220 ο

4. The recombinant plasmid of claim 3 is homologously recombined with adenovirus pAdEasy-1, and the recombinant adenovirus Ad-VEGF 165- IRES-Ang-1 is packaged in QBI-293A cells.

The use of the recombinant adenovirus according to claim 2 or 4 for the preparation of a pro-angiogenic drug.

6. A method of preparing a transgenic cell modified silk fibroin film, comprising the steps of:

The bone marrow stromal cells seeded in fibroin film 2 or the addition of adenovirus to claim 4, the regenerated silk fibroin membrane per square centimeter of bone marrow stromal cells at a concentration of ^{^{0.5χ 10 5 · -1 χ 10 5}} , a recombinant The inoculation amount of adenovirus was 20-30 ΜΟΙ, and the culture was carried out at 37 ° C under 5% CO ₂ .

7. A method according to claim 5 wherein the bone marrow stromal cells are in a logarithmic growth phase.

The method according to claim 5, wherein the bone marrow stromal cells are primary cultured bone marrow stromal cells, and the cell concentration is lx lO ⁸ ! ¹ .

9. A transgenic cell modified silk fibroin membrane prepared by the method of any of claims 6-8.

10. Use of a transgenic cell modified silk fibroin membrane according to claim 9 for the preparation of a vascular inducing and wound repairing medical biomaterial.