CN101020081A - Cell transplating composition for repairing infarcted cardiac muscle and reconstructing blood vessel - Google Patents

Abstract

The object of the present invention is to provide a cell transplantation composition for infarcted myocardium repair and blood vessel reconstruction, the composition includes transplantation auxiliary cells and a genetic engineering carrier for preparing myocardial precursor cells in vitro. Transplant helper cells can promote the formation of blood vessels in the myocardial infarction area after being implanted into the scar tissue of the myocardial infarction area, and at the same time improve the survival rate of myocardial precursor cell transplantation; the genetic engineering carrier is used in vitro to obtain bone marrow stem cells from patients (or animals) Preparation of high-purity myocardial precursor cells for myocardial repair. The composition of the present invention also includes a drug corresponding to the suicide gene on the above-mentioned genetic engineering carrier. Neoplastic myocardial tissue precursor cells are removed from the host. Since cardiomyocyte precursor cells can be derived from the patient's bone marrow, no immunosuppressant is needed to maintain the survival of cardiomyocyte precursor cells.

Description

A kind of cell transplantation composition for infarcted myocardium repair and blood vessel reconstruction

technical field

The invention belongs to the field of biotechnology. The invention relates to a cell transplant composition for infarcted myocardium repair and blood vessel reconstruction.

Background technique

Significance and existing problems of cell transplantation in the prior art:

At present, there is a lack of effective therapeutic drugs and treatment methods for congestive heart failure (CHF) caused by myocardial injury. Since cardiomyocytes in adult mammals cannot regenerate, CHF caused by cardiomyocyte loss is an irreversible pathological process. So cell transplantation is the fundamental way to treat CHF in the future.

The key to cell transplantation is to solve the problem of cell source. Because stem cells have the potential to differentiate into various tissue cells, especially bone marrow stem cells (Born marrow stem cells, BMSCs), which are easy to obtain, can also achieve autologous transplantation, and do not require immunosuppressants to maintain graft survival. It is considered to be an ideal seed cell for tissue repair. According to current research, myocardial tissue precursor cells can be obtained by inducing differentiation culture of bone marrow mesenchymal stem cells (MSCs) in BMSCs in vitro, such as using specific chemical inducers 5-AZA and bFGF to induce MSCs Differentiate into cardiomyocyte-like cells in vitro (Pei Xuetao, Niu Lili, etc., Method for In vitro Expansion and Directional Induction of Adult Bone Marrow Mesenchymal Stem Cells into Cardiomyocyte-like Cells, Chinese Patent Publication No.: CN1536076A), etc.

However, this does not mean that we can obtain a large number of tissue precursor cells that can be used for transplantation and repair. Because BMSCs are induced to differentiate into various tissue precursor cells in vitro, not all BMSCs can differentiate into the target cells we need. Therefore, in a BMSC induction differentiation culture system, there are often undifferentiated BMSCs and a variety of tissue precursor cells, and the myocardial tissue precursor cells we need only account for a part of them. If the tissue precursor cells that have not been screened and purified are transplanted into the body, it may cause the implanted cells to undergo neoplastic changes in the body, thereby bringing potential danger to the patient. Therefore, how to separate and purify myocardial precursor cells from the mixed cell system produced by BMSC induced differentiation culture is the key to solving the problem of cell source. Regarding the separation and purification method of cells, the most commonly used method at present is flow cytometry based on specific protein markers on the cell surface. Unfortunately, there are still academic debates about what specific protein markers exist on the surface of myocardial precursor cells.

In order to obtain purified myocardial precursor cells from the culture system after stem cell differentiation, Klug and Field et al. used an expression vector containing the α-cardiac myosin heavy chain (MHC) promoter to drive the aminoglycoside phosphotransferase neo ^r , transferred to the induced differentiation culture system of embryonic stem cells, and then screened with G418 to successfully isolate and purify myocardial precursor cells (J. Clin. Invest. 98: 216, 1996). Perhaps the method of Klug and Field is an ideal method for isolating and purifying myocardial tissue precursor cells that meet the requirements of cell transplantation from the in vitro induced differentiation system of stem cells.

However, this method is only applicable to the isolation and purification of myocardial precursor cells from the embryonic stem cell differentiation culture system, but it is difficult to apply to the isolation and purification of cells that need to be repaired by the myocardium from the BMSC differentiation culture system. This is because embryonic stem cells have a strong ability to expand in vitro, while BMSCs have a very limited ability to expand in vitro. Generally, 10 to 20 passages are limited in vitro, which cannot meet the requirements of genetic engineering operations. Especially after the differentiation of BMSCs, the cells will soon be apoptotic, and it is impossible to carry out the selection and culture of positive cell clones after transgenesis.

Telomerase and Cell Expansion

Telomerase (Telomerase) plays an important role in maintaining telomere stability, genome integrity, long-term cell activity and potential continued proliferation. Most of the tissue cells in mammals lose their telomerase activity after birth, so tissue cells can only be cultured for limited passages in vitro, which restricts the application of genetic engineering technology in animal tissue cells. It is generally believed that the reactivation of telomerase activity can maintain the length of telomeres, and delaying the clonal aging of cells is a key step in maintaining cell immortality. Many studies have shown that reconstitution of telomerase activity in tissue cells that have lost telomerase activity can lead to increased cell expansion in vitro while maintaining the original cell function.

After the telomerase activity is reactivated, the cells will continue to divide and clone, but at the same time, the risk of cell neoplasia and cancer is relatively increased, because the uncontrolled division of cells is very aggressive to the individual and destructive. However, moderately and transiently increasing the telomerase activity in tissue cells can improve the ability of cells to expand in vitro, facilitate genetic engineering operations, and at the same time increase the ability of cells to integrate with body tissues after transplantation.

Cell transplantation initially nonfunctional

The biggest difference between cell transplantation and organ transplantation is that the implanted organ has a complete anatomical structure. After the operation is completed, the implanted organ can obtain the nutrients needed for survival through its own vascular system. After the implanted cells enter the body, they can only rely on the interstitial fluid in the implanted environment to provide nutrients and oxygen. Unfortunately, in most cases, the environment where cells need to be implanted is the scar tissue formed after some tissue damage, such as: scar tissue of infarcted myocardium, etc. In these tissues, blood vessels are often sparsely distributed. Except for tumor cells, Normal cells have difficulty surviving in these tissues during the initial stages of implantation. Therefore, the remodeling of vasculature in scar tissue after myocardial injury is the key to the survival of myocardial precursor cell transplantation.

As mentioned above, how to effectively solve the problem of how to screen and purify myocardial tissue precursor cells after stem cell differentiation, how to rebuild the cardiovascular system in necrotic tissue sites, and how to effectively control the tumorigenesis of cells after implantation in the body is the key to the treatment of congestive diseases by cell transplantation. The key to heart failure. Therefore, the present invention provides a cell transplant composition for infarcted myocardium repair and vascular reconstruction, which undoubtedly brings hope to patients with congestive heart failure.

Contents of the invention

The object of the present invention is to provide a cell transplantation composition for infarcted myocardium repair and blood vessel reconstruction, the composition includes transplantation auxiliary cells and a genetic engineering carrier for preparing myocardial precursor cells in vitro. Transplant helper cells can promote the formation of blood vessels in the myocardial infarction area after being implanted into the scar tissue of the myocardial infarction area, and at the same time improve the survival rate of myocardial precursor cell transplantation; the genetic engineering carrier is used in vitro to obtain bone marrow stem cells from patients (or animals) Preparation of high-purity myocardial precursor cells for myocardial repair.

The composition of the present invention also includes a drug corresponding to the suicide gene on the above-mentioned genetic engineering carrier. Neoplastic myocardial tissue precursor cells are removed from the host. Since cardiomyocyte precursor cells can be derived from the patient's bone marrow, no immunosuppressant is needed to maintain the survival of cardiomyocyte precursor cells.

The technical solution adopted to achieve the above-mentioned purpose is as follows: a kind of cell transplantation composition for repairing infarcted myocardium and vascular reconstruction is provided, the composition includes transplantation helper cells and genetic engineering of myocardial precursor cells prepared in vitro Vector; the transplant helper cells are human or animal hepatic sinusoidal endothelial cell lines or other tumor cell lines, which are obtained through screening after transfection with virus vectors expressing suicide genes, and can stably express suicide gene products; the genetic engineering The vectors include bicistronic adenoviral vectors that transduce cardiomyocyte-specific promoters and internal ribosome entry site sequences (IRES) to regulate the expression of neozyme resistance gene and telomerase catalytic subunit, and tumor cell-specific promoters A lentiviral vector that regulates the expression of suicide genes.

The drug corresponding to the suicide gene is also included in the composition.

In the above composition: the tumor cell-specific promoter is selected from the group consisting of telomerase catalytic subunit promoter, alpha-fetoprotein promoter, carcinoembryonic antigen promoter, prostate specific promoter, retinoic acid effect growth/differentiation factor promoter, kidney cytokine promoter, tyrosine kinase promoter, and one of the above-mentioned tumor cell-specific promoter mutation sequences artificially synthesized.

The cardiomyocyte characteristic promoter is selected from one of the myocardial myosin heavy chain (MHC) promoter and the myocardial myosin light chain 2 (MLC2v) promoter.

The suicide gene is selected from one of cytosine deaminase gene, herpes simplex virus thymidine kinase gene, varicella herpes virus thymidine kinase gene, cytochrome P450 2B1 gene, and nucleoside phosphorylase gene.

The drug corresponding to the suicide gene is selected from one of 5-fluorocytosine, guanosine, 6-methoxypurine arabinoside, cyclophosphamide, and 6-methylpurine deoxynucleoside.

The above composition can be prepared by currently mature genetic engineering and tissue culture techniques, wherein:

1. the preparation process of described transplant helper cell comprises: (1) in vitro culture of human hepatic sinusoidal endothelial cell line (or other tumor cells); (2) with the viral vector transfection described in step (1) of expressing suicide gene Cell; (3) carry out selection culture with the corresponding antibiotic culture medium of eukaryotic resistance gene on the virus carrier described in step (2); (4) carry out suicide gene phenotype identification to the cell that step (3) obtains; (4) ) expanding and culturing the cell clone capable of stably expressing the suicide gene product according to a conventional method; (5) washing the cells obtained by (4) with PBS before transplantation;

2. Construction of the bicistronic adenovirus shuttle plasmid vector:

(1) Design PCR primers based on the MHC or MLC2v promoter sequence found in NCBI, and amplify MHC or MLC2v promoter fragments of different lengths from human (or animal) tissue cell genomic DNA; MHC Or insert the MLC2v promoter fragment into the promoterless adenovirus shuttle plasmid vector; insert the reporter gene at the 3′ end of the MHC or MLC2v promoter; package the newly constructed adenovirus shuttle plasmid vector and the adenovirus genome plasmid into an adenovirus in vitro Virus particles; co-cultivate the packaged adenovirus particles with a culture system containing cardiomyocytes or myocardial precursor cells; after 24 to 48 hours of culture, detect the expression of reporter genes, and select those with strong cardiomyocyte-specific expression of reporter genes The shortest MHC or MLC2v promoter fragment was used for the next step. (2) Insert the MHC or MLC2v promoter fragment at the 5' end of the multi-cloning site on the common promoter-free adenovirus shuttle plasmid vector (such as: pdc312, etc. can be purchased from the market); (3) Insert the IRES fragment into the MHC Or the middle of the MLC2v promoter fragment and the PolyA fragment; (4) insert the hTERT coding sequence between the MHC or MLC2v promoter fragment and the IRES fragment; (5) insert the neo ^r coding sequence between the IRES and the PolyA fragment; (6) Finally, PCR was used to identify the plasmid vector obtained in the previous step.

3. The tumor-specific expression CD lentiviral vector construction: (1) According to the telomerase catalytic subunit promoter, alpha-fetoprotein promoter, carcinoembryonic antigen promoter, prostate specific promoter, One of the retinoic acid-responsive growth/differentiation factor promoters, kidney cytokine promoters, tyrosine kinase promoters, and artificially synthesized tumor cell-specific promoter sequences was designed to amplify from human or animal genomic DNA. Increase the promoter fragment, or use an artificially synthesized tumor cell-specific promoter fragment; (2) replace the promoter fragment of the lentiviral vector with this fragment; (3) replace the tumor cell-specific promoter of the vector obtained in the previous step 3' end, inserting the reporter gene; (4) Packaging the vector and the corresponding packaging plasmid obtained in the previous step for virus particle packaging in vitro, and infecting the primary cultured cells of tumor cells and normal tissues with the obtained virus particles (5) 24 to 48 hours after virus particles infect the cells, detect the reporter gene expression of each cultured cell, and select a tumor cell-specific expression reporter gene carrier for the next step of operation. (6) According to one of the cytosine deaminase gene, herpes simplex virus thymidine kinase gene, varicella herpes virus thymidine kinase gene, cytochrome P450 2B1 gene, and nucleoside phosphorylase coding sequence recorded in NCBI Primer design, and use PCR to amplify the corresponding suicide gene segment; (7) Insert the suicide gene segment into and replace the reporter gene segment in the vector obtained in step (3), thereby obtaining a lentiviral vector with a new sequence tumor-specific expression of the suicide gene;

4. The drug corresponding to the suicide gene: it can be obtained through any drug market.

The application process of the above composition in infarcted myocardium repair and vascular reconstruction:

(1) Use the bicistronic shuttle plasmid vector provided by the composition and the tumor cell-specific expression suicide gene lentiviral vector to be packaged into adenoviral particles and lentiviral particles respectively according to conventional methods, and separate and concentrate the adenoviral particles and lentiviral particles ;

(2) Isolation and culture of BMSC;

(3) use 5-Az to induce the differentiation of BMSC cultured in vitro;

(4) transfect differentiated BMSCs with adenovirus particles and lentivirus particles;

(5) After transfecting the virus, transfer the BMSCs to a medium containing G418 and antibiotics corresponding to eukaryotic resistance genes (such as: blasticidin, etc.) on the lentiviral vector for screening and culture, and the obtained myocardial precursor cells are washed with PBS After dropping eukaryotic antibiotics, it can be used for transplantation.

(6) Surgically implant the transplantation assisting cells in the composition of the invention into the patient's myocardial infarction area, and inject cyclosporine A 1 mg/kg body weight intramuscularly every day;

(7) Monitor the growth of the transplanted helper cells with an ultrasonic detector or CT, and when a tumor focus with a diameter of 3 to 5 mm is formed at the transplant site, implant the cells obtained in the operation step (5) into the tumor focus;

(8) 1 to 2 weeks after cardiomyocyte implantation, intravenous or intramuscular injection of appropriate amount of drugs corresponding to suicide genes, such as 5-fluorocytosine, etc., and continuous administration until the transplanted helper cells are completely eliminated.

The beneficial effects of the present invention are as follows:

The composition includes transplantation helper cells and a genetic engineering carrier for preparing myocardial precursor cells in vitro, wherein the transplantation helper cells can promote the formation of blood vessels in the myocardial infarction area after being implanted into the scar tissue of the myocardial infarction area, and at the same time improve the myocardial precursor cells. The role of transplant survival rate; genetic engineering carrier can prepare high-purity myocardial precursor cells for myocardial repair from bone marrow stem cells of patients (or animals). After the myocardial precursor cells obtained by the method of the present invention are transplanted and survived in the myocardial infarction area and form the vascular system of the transplanted helper cells and the myocardial precursor cells, specific drugs can be used to treat the transplanted helper cells and the myocardial tissue with neoplastic changes. Somatic cells are eliminated from the host body. Therefore, using the composition of the present invention to repair infarcted myocardium and rebuild blood vessels not only has a high survival rate of myocardial precursor cell transplantation, but also avoids the possibility of bone marrow stem cells undergoing tumorigenesis after implantation due to factors such as the end of differentiation. Potentially dangerous, so it has the characteristics of safe use.

Description of drawings

Figure 1: Flowchart of the implementation and application scheme of cell transplantation for infarcted myocardium repair and vascular reconstruction of the present invention.

Figure 2: Cell map after adding 5-azacytidine to induce differentiation in BMSC cultured in vitro for 72 hours (10×20)

As shown in the figure, the second-generation BMSC cells were isolated and cultured by the method described in the literature. After adding 5-Az to induce differentiation and culture for 48 hours, most of the cells became wider and began to show signs of aging, and it was difficult for the cells to continue to proliferate.

Figure 3: After adding 5-azacytidine and simultaneously transfecting the adenovirus Ad-MHCp-hTERT-IRES-neo ^r , the cells were cultured for 72 hours (10×20)

As shown in the figure, using the method provided by the present invention to isolate and culture BMSC second-generation cells, after adding 5-Az to induce differentiation and culture for 48 hours, most of the cells were in the shape of long spindles, and the number of cells increased significantly, showing a strong expansion capability.

Figure 4: TRAP chart of telomerase activity assay of myocardial precursor cells after transfection with adenovirus Ad-MHCp-hTERT-IRESneo. As shown in the figure, compared with the control group before transfection, adenoviral particles containing MHC promoters driving the expression of hTERT, 24 hours after transfection of myocardial precursor cells, the activity of cell telomerase was significantly improved. After 72 hours, the cell telomerase activity reached the highest.

Figure 5: Cell image (10×20) after 48 hours of G418 and blasticidin double antibody screening. As shown in the figure, the BMSCs transfected with recombinant adenovirus and lentivirus particles by adding 5-azacytidine at the same time, after adding G418 and blasticidin double antibody screening culture for 48 hours, most of the cells were killed and were suspended .

Figure 6: Cell image (10×20) after 72 hours of G418 and blasticidin double antibody screening. As shown in the figure, after 5-azacytidine was added to BMSCs transfected with recombinant adenovirus and lentiviral particles at the same time, after adding G418 and blasticidin double antibody screening culture for 72 hours, some cells aggregated together, and high-frequency creep.

Figure 7: Diagram of myocardial precursor cells after adding G418 and blasticidin for double antibody screening for 96 hours (10×20). As shown in the figure, after 5-azacytidine was added to BMSCs transfected with recombinant adenovirus and lentivirus particles at the same time, after adding G418 and blasticidin double antibody screening and culturing for 96 hours, the cells began to show obvious cardiomyocyte appearance characteristics. Between cells, there is even a structure similar to a disc.

Fig. 8: Confocal microscope image of cells infected with pdc315-MHCp-EGFP adenovirus particles and differentiated for 72 hours (10×63). As shown in the figure, BMSCs transfected with recombinant adenovirus and lentivirus particles by adding 5-azacytidine at the same time, after being infected by pdc315-MHCp adenovirus particles, some cells expressed EGFP, and the cells expressing EGFP had myocardial the shape of the cell.

Fig. 9: Confocal laser microscope image (10×63) for functional identification of recombinant hTERT promoter. As shown in Figure A, lentiviral particles loaded with hTERT promoter to drive the expression of EGFP can express EGFP visible to the naked eye after infecting telomerase-positive tumor cells; as shown in Figure B, the lentiviral particles loaded with hTERT promoter to drive EGFP-expressing lentiviral particles cannot express visible EGFP after infecting telomerase-negative normal tissue primary culture cells; indicating that the artificially synthesized hTERT promoter has strong specificity in telomerase-positive tumor cells expression activity.

Detailed ways

The foregoing of the invention can be further illustrated by the following non-limiting examples. Materials other than the composition required for the practice are commercially available.

Application example 1: Using the composition to repair infarcted myocardium and revascularization in rats

1. Establishment of rat model of myocardial infarction: SD rats with a weight of about 200 grams were taken, anesthetized with ether, fixed on the table, and connected to a special ventilator for rats. After the chest was clipped and sterilized with clippers, the chest cavity was surgically opened to expose the heart. After ligating the left anterior descending branch of the coronary artery with silk thread, squeeze the chest cavity by hand to expel the air, and then suture the chest cavity. After two months, it can be used for the next step.

2. Preparation of an adenoviral shuttle plasmid vector of adenoviral bicistronic specific expression of hTERT and neor in rat cardiomyocytes: (1) use rat genomic DNA as a template, and design primers according to the sequence X04267 contained in NCBI, wherein The upstream primer is: 5'-CGGTCTAGATGGGAGGAAGAGGC 3', the downstream primer is: 5'-TTGAATTCGTTGGCTGCAAAACA-3', and the rat cardiac myosin heavy chain (MHC) promoter sequence is amplified by PCR. See SEQ ID NO: 1 in the sequence listing. (2) The promoter fragment of step (1) is then taken as the mCMV promoter with the adenovirus shuttle plasmid pdc315, and the reporter gene EGFP fragment is inserted into the multiple cloning site, and the new vector obtained is named: pdc315-pMHC-EGFP . After it was packaged into virus particles, it was transfected into a cell culture system after induction of differentiation by BMSC, and the expression of EGFP was identified by confocal fluorescence microscopy. The results are shown in FIG. 8 . (3) with restriction endonuclease EcoRI, Not I digestion plasmid pGRN145 (can buy from the market) and plasmid pIRESneor3, electrophoresis and gel recovery hTERT fragment and linear pIRESneor3 fragment, use T4 ligase to hTERT fragment and linear pIRESneor3 fragment Connected, the adenovirus shuttle plasmid obtained is named: phTERT-IRES-neo ^r ; (4) digest the plasmid phTERT-IRES-neo ^r with restriction endonuclease EcoRI, XbaI, and digest the adenovirus with EcoR I and NheI Shuttle plasmid pdc315-pMHC, hTERT-IRES- ^neor fragment and linear adenovirus shuttle plasmid pdc315-pMHC fragment were recovered respectively, and the recovered two fragments were connected with T4 DNA ligase, and the recombinant plasmid obtained was named: pdc315-pMHC- hTERT-IRES- ^neor is an adenoviral bicistronic adenoviral shuttle plasmid vector specifically expressing hTERT and ^neor in cardiomyocytes according to the present invention, and its nucleotide sequence is shown in SEQ ID NO.2 in the sequence table.

3. Construction of lentiviral vectors for tumor-specific expression of CD and EGFP: (1) Design the promoter sequence based on the base sequence between 2930 and 3762 of the sequence AB016767 in NCBI, in order to improve the expression of the promoter in tumor cells Transcriptional activity, while reducing the expression activity in telomerase-negative cells, some cis-acting element sequences were inserted into the sequence (see the sequence table SEQ ID NO: 3.); for the next step, it can easily replace the lentiviral vector The promoter on the above, introduced the ClaI restriction endonuclease site recognition sequence at the 5' end of the sequence, introduced the BamHI restriction endonuclease site recognition sequence at the 3' end of the sequence, and finally synthesized by artificial chemical synthesis , and subcloned it into PUC57, and named it PUC57-phTERT; (2) Design primers according to the sequence S56903 registered by NCBI. In order to facilitate connection with the vector, we introduced BamHI restriction internal Dicer recognition site sequence, introduce the AgeI restriction endonuclease recognition site sequence at the 5 end of the downstream primer, thus, the upstream primer is: 5'-ATAGGATCCGACAGCAGCAATGAC-3', and the downstream primer is: 5'-TATACCGGTGCGTTGGAGGAAT-3' . Carry out PCR amplification then; (3) use PCR method to amplify EGFP fragment, its upstream primer is: 5'-CACCATGGTGAGCAAGGGC-3', downstream primer is: 5'-ACAACACTCAACCCTATCTCGGTCT-3', PCR product and kit pLenti6/ The linear lentiviral vector in the V5 Directional TOPO(R) Cloning Kit (can be purchased from Invitrogen) carries out TOP connection, and the vector obtained is called pLenti-EGFP; (4) digestion step (2) with restriction endonuclease BamH I, Age I ) PCR product, and the lentiviral vector plenti-EGFP, and finally the CD fragment and the linear vector fragment were recovered by agarose gel electrophoresis; The lentiviral vector plasmid is named after: pLenti-CMVp-CD; (5) with restriction endonuclease Cla I, BamH I digestion step (1) gained plasmid PUC57-phTERT, and lentiviral vector pLenti-CMVp-CD, use respectively The phTERT promoter fragment and the promoterless lentiviral vector pLenti-CD fragment were recovered by gel electrophoresis, and the recovered two fragments were connected with ligase, and the obtained plasmid vector was a tumor-specific lentiviral vector expressing CD, and Named: pLenti-phTERT-CD, its nucleotide composition is shown in the sequence table SEQ ID NO.4; (6) after replacing the CMV promoter fragment on the lentiviral vector pLenti-EGFP obtained in step (3) with the hTERT promoter , and the resulting plasmid was named: pLenti-phTERT-EGFP.

4. In vitro packaging and purification of lentiviral particles: (1) Transform the lentiviral plasmid vectors plenti-hTERT-CD and plenti-hTERT-EGFP obtained from the above operations into DH5α competent E. coli for amplification, and prepare a large amount of These two ultrapure plasmids were mixed with Invitrogen's lentiviral packaging plasmids plp1, plp2, plpVSV-G and cationic liposomes in a certain proportion, and then transferred to 293FT cells for packaging and isolation and purification of the virus Particles, (the specific operation is carried out according to the instruction manual of pLenti6/V5 Directional TOPO® Cloning Kit); (2) the lentiviral particles packaged by plasmids plenti-hTERT-EGFP and plenti-CMV-EGFP are respectively combined with telomerase-positive: hLSEC cells, colon cancer cells (SW480), human embryonic renal epithelial tumor cells (293FT), liver cancer cells (HepGII), gastric cancer cells (SGC7901), melanoma cells (B16), etc., and human osteosarcoma cells negative for telomerase expression (U20S), W38 cells, and normal tissue cells were co-cultured, and after 48 hours, the expression of EGFP was observed with a laser confocal microscope. The results are shown in Figure 9.

5. Packaging identification of adenovirus particles: (1) Co-transfect human embryonic kidney with the shuttle plasmid pdc315-MHCp-hTERT-IRES-neo ^r loaded with cardiomyocyte-specific expression of hTERT and neo ^r and the adenovirus packaging plasmid pBHGloxDeltaEl, 3Cre 293 cells (HEK293); (2) When 60% to 80% of the packaging cells appear rounded, and some cells begin to appear detachment cytopathic phenomenon (CPE), the cells are broken and centrifuged by ultrasonic waves, and the cells containing The supernatant of virus particles, the virus particles obtained by packaging are named: Ad-MHCp-hTERT-IRES-neor, after purification, virus particles are measured and double-primer PCR identification is carried out. The specific operation is carried out according to the method provided by Yuanyang Company. The virus titer obtained by this method can reach more than 5×10 ⁹ VP/ml (note: if the OD260 is less than 0.1, use cesium chloride gradient ultrahigh-speed centrifugation to concentrate the virus recovery solution before testing). The primers for PCR identification of MHC fragments are 5'-CTAGGTCCTAACAGAC-3 for upstream primers, 5'-TGTCTCTCCTAGATTC-3' for downstream primers (the product size is 247bp); the primers for identifying hTERT fragments are 5'-TCTGGGATGCGAACGG-3' for downstream primers It is 5'-ACGGATGGGTGGGAGTG-3' (product size is 309bp).

6. Preparation of myocardial precursor cells: From healthy 2-month-old SD rats with a body weight of 150-200 g, after ether inhalation anesthesia, remove the femur and tibia under aseptic conditions, rinse with PBS, remove the bone ends, and expose Bone cavity; rinse the bone cavity with 5 ml of DMEM/F12 (ratio: 1:1) containing 10% fetal year serum; repeatedly blow the rinse solution with a 5 ml pipette until no clot is seen, inoculate in 50 ml In the cell culture flask; after continuing to culture for 24 hours, change the medium to remove non-adherent cells; continue to culture in a 37°C carbon dioxide incubator until 80% of the cells are confluent; absorb the culture medium, and digest the cells with 0.25% trypsin at room temperature 3 ~4 minutes; add 15 ml of DMEM/F12 containing 10% fetal year serum to stop the digestion, use a 5 ml pipette gun to repeatedly pipette the cells to a suspension state, and inoculate in three 50 ml cell culture flasks; 37 ° C carbon dioxide incubator After culturing in medium for 20 minutes, gently transfer the non-adherent cell suspension to another culture flask; repeat this operation 2 times, and culture in a 37°C carbon dioxide incubator until the cells are 40% to 60% confluent; Aspirate off the medium, add 5-azacytidine (5-Az) with a final concentration of 10uM, 10 ⁶ ～10 ⁹ pfu/ml adenovirus Ad-MHCp-hTERT particles, 10 ⁵ ～10 ⁹ VP/ml slow The culture medium of the virus pLenti-hTERTp-CD particles was cultured in a carbon dioxide incubator at 37°C; after 72 hours, replace the 5-Az-free medium containing G418 with a final concentration of 50ug/ml and blasticidin at a final concentration of 50ug/ml for double antibody After that, every time the solution was changed, the concentration of G418 and blasticidin increased by 50ug/ml, and the concentration of blasticidin increased by 2ug/ml. Finally, the final concentration of G418 was maintained at 400ug/ml, and the final concentration of blasticidin was maintained at 10ug/ml; when the cells were passed to passage 10, the cells needed for the repair of infarcted myocardium could be collected, as shown in Figure 7. And wash away antibiotics with PBS solution before surgical transplantation.

7. Genetic engineering transformation and preparation of helper cells for transplantation: inoculate well-growing HLSECs on a culture medium plate, and when the cells grow to 40-60% confluent state, replace the culture medium, and add a final concentration of 10 ⁶ to 10 ⁹ VP/ml lentivirus Lenti-CMVp-CD particle culture medium, cultured in a 37°C carbon dioxide incubator for 72 hours, then replace the medium with a medium containing blasticidin with a final concentration of 10ug/ml for resistance screening culture . The cells obtained after screening were cloned and cultured, and after 5-10 generations of resistance selection and culture, the CD drug sensitivity test was carried out. Cell clones sensitive to CD were selected for expansion and culture, and washed with PBS solution before surgical transplantation.

8. Operations of cell transplantation to reconstruct blood vessels and repair infarcted myocardium: (1) surgically open the chest cavity of rat myocardial infarction model, expose the heart, and implant 104 to 106 CD-sensitive HLSEC cells (helper cells) in the area of myocardial infarction ), and then suture the chest cavity (the specific operation is the same as the establishment method of the rat myocardial infarction model); (2) intramuscularly inject 1 mg of cyclosporine A (GsA) every day to suppress the immune rejection of the transplanted helper cells in rats; (3) Use B-ultrasound or CT to detect the growth of the transplant helper cells. When the implantation point of the transplant helper cells forms a tumor focus with a size of 3 to 5 mm, use the method of step (3) to implant 10 ⁵ to 10 ⁸ of the method of the present invention. Prepared cardiomyocyte precursor cells derived from BMSCs.

9. Drugs to remove helper cells and neoplastic myocardial precursor cells in the recipient: 2 to 5 weeks after cell transplantation, start intravenous injection of 5-Fc three times a day, each dose of 50 to 200 mg/kg body weight, Continuous medication for 3 to 5 days can achieve the purpose of eliminating HLSEC and cancerous myocardial precursor cells.

Application Example 2: Using the Composition of the Present Invention to Repair Human Infarcted Myocardium and Reconstruction of Blood Vessels

1. Preparation of adenoviral shuttle plasmid vector of adenoviral bicistronic specifically expressing hTERT and neo ^r in human cardiomyocytes: replace the promoter sequences in the sequence table SEQ ID NO.1 and SEQ ID NO.2 with human MHC or The MLC2v promoter sequence can be used to obtain the adenoviral bicistronic adenoviral shuttle plasmid vector specifically expressing hTERT and neo ^r in human cardiomyocytes.

2. Preparation of human myocardial precursor cells: After extracting 5-10 ml of bone marrow from a patient with myocardial infarction to be transplanted, the human myocardial precursor cells were prepared in the same way as in "Application Example 1".

3. Subsequent steps and methods are the same as "Application Implementation Case 1".

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tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120

atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180

tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240

aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300

gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360

actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420

gggtcaaagt tggcgtttta ttattatagt cagntctaga tgggaggaag aggccatgtc 480

aattaaaaag cagttcaaac tgcctctgcc attttctgct cagcatcctc tctttgagga 540

atgcctttac ttatttttat tatggtattt aacttgggat gttctgttct ccatacctgt 600

acttctcaga aaaggaagac aaagtgtcaa aagtggcatt tcaaaggctc cttcaccaga 660

ctgaaagaat caagagacac ttggattaaa cagattctaa gcccatggca tacagtagca 720

actcaacaaa cacgagtgga ttatgttttc ccagtgctga gccctcgtgt gaagctccac 780

tgggcatggg acaggccaga tctccttgac cttgtctgag aggctgccat ccacttcagg 840

gtgacaggtt gatactagga tgggcaggag gctgccggct gccggctgcc ggcccaggga 900

gcagctgggc tgactggcag tttgttttgc agccaactgt tttgcagcca acgaattccc 960

cccgcgatgc cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac 1020

cgcgaggtgc tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg 1080

gtgcagcgcg gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg 1140

ccctgggacg cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag 1200

gagctggtgg cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc 1260

ttcggcttcg cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc 1320

gtgcgcagct acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg 1380

ctgctgctgc gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc 1440

tttgtgctgg tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc 1500

ggcgctgcca ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga 1560

tgcgaacggg cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc 1620

ccgggtgcga ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc 1680

aggcgtggcg ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac 1740

ccgggcagga cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc 1800

gccgaagaag ccaccctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc 1860

gtgggccgcc agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac 1920

acgccttgtc ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag 1980

gagcagctgc ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg 2040

aggctcgtgg agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg 2100

ttgccccgcc tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg 2160

aaccacgcgc agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg 2220

gtcaccccag cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc 2280

gaggaggagg acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc 2340

tggcaggtgt acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg 2400

ggctccaggc acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg 2460

aagcatgcca agctctcgct gcaggagctg acgtggaaga tgagcgtgcg ggactgcgct 2520

tggctgcgca ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag 2580

gagatcctgg ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg 2640

tctttctttt atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag 2700

agtgtctgga gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg 2760

cgggagctgt cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg 2820

tccagactcc gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac 2880

gtcgtgggag ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg 2940

aaggcactgt tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc 3000

tctgtgctgg gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg 3060

gcccaggacc cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac 3120

accatccccc aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg 3180

tactgcgtgc gtcggtatgc cgtggtccag aaggccgccc atggcacgt ccgcaaggcc 3240

ttcaagagcc acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct 3300

cacctgcagg agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg 3360

aatgaggcca gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg 3420

cgcatcaggg gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc 3480

acgctgctct gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg 3540

cgggacgggc tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc 3600

cacgcgaaaa ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg 3660

aacttgcgga agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct 3720

tttgttcaga tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg 3780

accctggagg tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc 3840

accttcaacc gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg 3900

cggctgaagt gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc 3960

accaacatct acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag 4020

ctcccatttc atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac 4080

acggcctccc tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc 4140

aagggcgccg ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc 4200

ctgctcaagc tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca 4260

gcccagacgc agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca 4320

gccaacccgg cactgccctc agacttcaag accatcctgg actgatgtaa actaaagcgg 4380

ccgcatagat aactgatcca gtgtgctgga attaattcgc tgtctgcgag ggccagctgt 4440

tggggtgagt actccctctc aaaagcgggc atgacttctg cgctaagatt gtcagtttcc 4500

aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgcg 4560

tccatctggt cagaaaagac aatctttttg ttgtcaagct tgaggtgtgg caggcttgag 4620

atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc acaggtgtcc 4680

actcccaggt ccaactgcag gtcgagcatg catctagggc ggccaattcc gcccctctcc 4740

ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 4800

ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 4860

ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 4920

tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 4980

tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 5040

aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 5100

ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 5160

ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 5220

tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 5280

tttcctttga aaaacacgat gataagcttg ccacaacccg ggataattcc tgcagccaat 5340

atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 5400

aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 5460

cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 5520

aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 5580

gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 5640

ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 5700

gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 5760

aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 5820

ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc 5880

atgcccgacg gcgatgatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 5940

gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 6000

tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 6060

gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 6120

cgccttcttg acgagttctt ctgaggggat caattctcta gcaaggatcc agcttgtcga 6180

cttcgagcaa cttgtttat gcagcttata atggttacaa ataaagcaat agcatcacaa 6240

atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 6300

atgtatctta tcatgtctgg atcgtctagc atcgaagatc caataacttc gtatagcata 6360

catttatacga agttataagt agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 6420

attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 6480

ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 6540

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 6600

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 6660

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 6720

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 6780

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 6840

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 6900

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 6960

cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt 7020

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 7080

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 7140

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 7200

agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 7260

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 7320

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 7380

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 7440

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 7500

ttaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 7560

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 7620

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 7680

gtgctgcaat gataccgcga gacccacgct caccggctcc agattatca gcaataaacc 7740

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 7800

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 7860

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 7920

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 7980

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 8040

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 8100

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 8160

cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca 8220

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 8280

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 8340

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 8400

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 8460

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 8520

cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat 8580

taacctataa aaataggcgt atcactctag gcaaaatagc accctcccgc tccagaacaa 8640

catacagcgc ttcacagcgg cagcctaaca gtcagcctta ccagtaaaaa agaaaaccta 8700

ttaaaaaaac accactcgac acggcaccag ctcaatcagt cacagtgtaa aaaagggcca 8760

agtgcagagc gagtatatat aggactaaaa aatgacgtaa cggttaaagt ccacaaaaaa 8820

cacccagaaa accgcacgcg aacctacgcc cagaaacgaa agccaaaaaa cccacaactt 8880

cctcaaatcg tcacttccgt tttcccacgt tacgtaactt cccattttaa gaaaactaca 8940

attcccaaca catacaagtt actccgccct aaaacctacg tcacccgccc cgttcccacg 9000

ccccgcgcca cgtcacaaac tccaccccct catttatcata ttggcttcaa tccaaaataa 9060

ggtatagacc 9066

<210>3

<211>295

<212>DNA

<213> Synthetic

<220>

<221>promoter

<222>(1)...(295)

<400>3

gacagacgcc caggaccgcg cttcccacgt ggcggagggc atggggaccc gggcacccgt 60

cctgcccctt caccttccag ctccgcctcc tccgcgcgga ccccgccccg tcccgacccc 120

tcccgggtcc ccggcccagc cccctccggc cctccccagcc cctccccttc ctttccgcgg 180

ccccgccctc tcctcgcggc gcgagtttca ggcagcgctg cgtcctgctg cgcacgtggg 240

aagccctggc cccggccact cgaggacgca cgtgggcgca cgtgggcgca cgtgg 295

<210>4

<211>8048

<212>DNA

<213> Lentiviral vector

<220>

<221>promoter

<222>(1799)...(2095)

<400>4

aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg gtaacgatga gttagcaaca 60

tgccttacaa ggagagaaaa agcaccgtgc atgccgattg gtggaagtaa ggtggtacga 120

tcgtgcctta ttaggaaggc aacagacggg tctgacatgg attggacgaa ccactgaatt 180

gccgcattgc agagatattg tattaagtg cctagctcga tacataaacg ggtctctctg 240

gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 300

tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 360

taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 420

aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 480

gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 540

actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 600

attagatcgc gatgggaaaa aattcggtta aggccagggg gaaagaaaaa atataaatta 660

aaacatatag tatgggcaag cagggagcta gaacgattcg cagttaatcc tggcctgtta 720

gaaacatcag aaggctgtag acaaatactg ggacagctac aaccatccct tcagacagga 780

tcagaagaac ttagatcatt atataataca gtagcaaccc tctattgtgt gcatcaaagg 840

atagagataa aagacaccaa ggaagcttta gacaagatag aggaagagca aaacaaaagt 900

aagaccaccg cacagcaagc ggccgctgat cttcagacct ggaggaggag atatgaggga 960

caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat taggagtagc 1020

accccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg gaataggagc 1080

tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt caatgacgct 1140

gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca atttgctgag 1200

ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca agcagctcca 1260

ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg ggatttgggg 1320

ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt ggagtaataa 1380

atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgagggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcgatc 1800

acagacgccc aggaccgcgc ttcccacgtg gcggagggca tggggacccg ggcacccgtc 1860

ctgccccttc accttccagc tccgcctcct ccgcgcggac cccgccccgt cccgacccct 1920

cccgggtccc cggcccagcc ccctccggcc ctccccagccc ctccccttcc tttccgcggc 1980

cccgccctct cctcgcggcg cgagtttcag gcagcgctgc gtcctgctgc gcacgtggga 2040

agccctggcc ccggccactc gaggacgcac gtgggcgcac gtgggcgcac gtggggatcc 2100

gacagcagca atgacgcatg tggaggctaa cagtgtcgaa taacgcttta caaacaatta 2160

ttaacgcccg gttaccaggc gaagaggggc tgtggcagat tcatctgcag gacggaaaaa 2220

tcagcgccat tgatgcgcaa tccggcgtga tgcccataac tgaaaacagc ctggatgccg 2280

aacaaggttt agttataccg ccgtttgtgg agccacatat tcacctggac accacgcaaa 2340

ccgccggaca accgaactgg aatcagtccg gcacgctgtt tgaaggcatt gaacgctggg 2400

ccgagcgcaa agcgttatta acccatgacg atgtgaaaca acgcgcatgg caaacgctga 2460

aatggcagat tgccaacggc attcagcatg tgcgtaccca tgtcgatgtt tcggatgcaa 2520

cgctaactgc gctgaaagca atgctggaag tgaagcagga agtcgcgccg tggattgatc 2580

tgcaaatcgt cgccttccct caggaaggga ttttgtcgta tcccaacggt gaagcgttgc 2640

tggaagaggc gttacgctta ggggcagatg tagtgggggc gattccgcat tttgaattta 2700

cccgtgaata cggcgtggag tcgctgcata aaaccttcgc cctggcgcaa aaatacgacc 2760

gtctcatcga cgttcactgt gatgagatcg atgacgagca gtcgcgcttt gtcgaaaccg 2820

ttgctgccct ggcgcaccat gaaggcatgg gcgcgcgagt caccgccagc cacaccacgg 2880

caatgcactc ctataacggg gcgtatacct cacgcctgtt ccgcttgctg aaaatgtccg 2940

gtattaactt tgtcgccaac ccgctggtca atattcatct gcaaggacgt ttcgatacgt 3000

atccaaaacg tcgcggcatc acgcgcgtta aagagatgct ggagtccggc attaacgtct 3060

gctttggtca cgatgatgtc ttcgatccgt ggtatccgct gggaacggcg aatatgctgc 3120

aagtgctgca tatggggctg catgtttgcc agttgatggg ctacgggcag attaacgatg 3180

gcctgaattt aatcacccac cacagcgcaa ggacgttgaa tttgcaggat tacggcattg 3240

ccgccggaaa cagcgccaac ctgattatcc tgccggctga aaatgggttt gatgcgctgc 3300

gccgtcaggt tccggtacgt tattcggtac gtggcggcaa ggtgattgcc agcacacaac 3360

cggcacaaac caccgtatat ctggagcagc cagaagccat cgattacaaa cgttgaacga 3420

ctgggttaca gcgagcttag tttatgccgg atgcggcgtg aacgccttat ccggcctacg 3480

tagagcactg aactcgtagg cctgataagc gtagcgcatc aggcaattcc agccgctgat 3540

ctgtgtcagc ggctaccgtg attcattccc gccaacaacc gcgcattcct ccaacgcacc 3600

ggttagtaat gagtttggaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 3660

ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 3720

aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 3780

tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 3840

tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 3900

gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 3960

tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca 4020

attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca 4080

tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca 4140

acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc 4200

gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg 4260

tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa 4320

atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc 4380

atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc 4440

gtgaattgct gccctctggt tatgtgtggg agggctaagc acaattcgag ctcggtacct 4500

ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg 4560

ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg 4620

tctctctggt tagacccagat ctgagcctgg gagctctctg gctaactagg gaacccactg 4680

cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt 4740

gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt 4800

agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag 4860

agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 4920

aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 4980

aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc 5040

taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttattttg 5100

cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 5160

gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc 5220

cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 5280

agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 5340

ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc 5400

ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc 5460

tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct 5520

aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa 5580

acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc 5640

tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact 5700

caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg 5760

gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct 5820

tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 5880

taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 5940

tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 6000

gcggcattt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 6060

gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 6120

cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 6180

tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 6240

tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 6300

atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 6360

ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 6420

gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 6480

gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 6540

gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 6600

gcaggacac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 6660

gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 6720

cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 6780

atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 6840

tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 6900

ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 6960

gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 7020

tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 7080

ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt 7140

ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 7200

gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 7260

ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac gggggttcg 7320

tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 7380

ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 7440

agggtcggaa caggagagcg cacgaggggag cttccagggg gaaacgcctg gtatctttat 7500

agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 7560

gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 7620

tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 7680

accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 7740

gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 7800

attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 7860

gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 7920

gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 7980

catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa 8040

gcttgcga 8048

Claims (8)

1, a kind of cell transplantation compositions that is used for infarcted myocardium reparation and reconstructing blood vessel, said composition comprise the engineering carrier of the myocardium precursor of transplanting accessory cell and external preparation; Described transplanting accessory cell is to obtain through screening after the viral vector transfection of expressing suicide gene, human or animal's that can stably express suicide gene product sinusoidal endothelial cell strain or other tumor cell lines; Described engineering carrier comprises that myocardial cell specificity promoter and internal ribosome entry site sequence regulate and control the bicistronic mRNA adenovirus vector that the plain resistant gene of new enzyme and telomerase catalytic subunit are expressed simultaneously, and the tumor cell specific promoter regulation suicide gene slow virus carrier of expressing.

2. be used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel according to claim 1, it is characterized in that: also comprise in the compositions and the corresponding medicine of suicide gene.

3. be used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as claimed in claim 1 or 2, it is characterized in that: described tumor cell specific promoter is selected from telomerase catalytic subunit promoter, afp promoter, carcinoembryonic antigen promoter, prostate specific promoter, tretinoin effect growth/differentiation factor promoter, nephrocyte factor promoter, network propylhomoserin kinase promoter, synthetic tumor cell specific and expresses a kind of in the promoter.

4. be used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as claimed in claim 1 or 2, it is characterized in that: described myocardial cell characteristic promoter is selected from a kind of in MHC promoter, the MLC2v promoter.

5. be used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as claimed in claim 1 or 2, it is characterized in that: described suicide gene is selected from a kind of in the thymidine kinase gene, herpes virus varicellae thymidine kinase gene, Cytochrome P450 2B1 gene, nucleoside phosphorylase gene of cytosine desaminase gene, herpes simplex virus.

6. as being used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as described in the claim 3, it is characterized in that: it is the sequence of artificial synthesizing ribonucleotide that the tumor cell specific of described synthetic is expressed promoter, and its nucleotide consists of SEQ ID No.3.

7. be used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as claimed in claim 1 or 2, it is characterized in that: the nucleotide of the slow virus carrier that described tumor cell specific promoter regulation suicide gene is expressed consists of SEQ ID No.4.

8. as being used for the cell transplantation compositions of infarcted myocardium reparation and reconstructing blood vessel as described in the claim 2, it is characterized in that: described and the corresponding medicine of suicide gene are selected from a kind of in 5-flurocytosine, gancyclovir, acycloguanosine, 6-methoxy purine galactoside, cyclophosphamide, the 6-methyl purine deoxynucleoside.

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