CN110669145B - Method for modifying mesenchymal stem cells by tripolymer TRAIL fusion protein gene and application thereof - Google Patents
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Abstract
The invention relates to a method for modifying mesenchymal stem cells by using trimer TRAIL fusion protein genes and application thereof, wherein the nucleic acid sequence of the trimer TRAIL fusion protein is shown as SEQ ID NO. 1. The trimeric TRAIL fusion protein can form a triple-helix parallel coil structural domain by covalent combination of disulfide bonds on the molecular structure, thereby helping the protein fold into a trimer. Because the trimer structural domain is combined by covalent bond, the addition of the structural domain at the N terminal of the TRAIL protein can obviously enhance the stability and the antitumor activity of the TRAIL trimer.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a tripolymer TRAIL fusion protein, a method for modifying mesenchymal stem cells by using the tripolymer TRAIL fusion protein and application of the tripolymer TRAIL fusion protein.
Background
As a disease seriously harming human health, the incidence rate and the fatality rate of malignant tumor are increased year by year, however, the tumor can not be effectively treated by clinical means such as operation, radiotherapy, chemotherapy and the like, and other treatment methods are extremely needed to be searched.
The stem cell has been widely researched as a gene carrier for transferring to the tumor, researches show that the mesenchymal stem cell has the characteristic of specially tracking and migrating to the tumor part in vivo under the action of inflammatory factors and chemotactic factors, the mesenchymal stem cell is used as a targeting carrier of an anti-cancer medicament and carries an exogenous cancer suppressor to act on the tumor, and the mesenchymal stem cell is an effective treatment means at present.
The TRAIL can selectively target tumor cells and induce the apoptosis of cancer cells by activating the apoptosis-promoting Death Receptors (DRs)4 and 5 on the surface of the tumor cells, has no toxicity or low toxicity to most normal cells, has strong tumor cell killing selectivity, and is a novel antitumor drug with great potential. Research proves that the umbilical cord mesenchymal stem cells modified by the soluble TRAIL gene can migrate to the periphery of tumor tissues and can stably and continuously release TRAIL, thereby inhibiting the growth of tumor cells, promoting the apoptosis of tumors, prolonging the service life of model animal models and not influencing the survival time of negative control animals.
The TRAIL protein monomer molecule has high oligomerization tendency, and can form dimer with molecular weight of 48KDa and trimer with molecular weight of 66KDa, wherein the trimer can be further aggregated into hexamer. The configuration stability and aggregation form of the protein have close relationship with the tumor killing activity, receptor affinity, stability and the like of the protein, and when TRAIL forms a tripolymer structure in space, the anti-tumor activity of the TRAIL is optimal and the stability is highest.
Chinese patent publication No.: CN107287224A discloses a mesenchymal stem cell secreting TRAIL and its use in treating brain tumor, the TRAIL recombinant protein is combined with TRAIL coding gene by leucine zipper or isoleucine zipper recombinant gene, thereby improving the stability of trimeric TRAIL. However, the leucine zipper structure is a non-covalent weak interaction that employs hydrophobic interaction forces, and the presence of Zn atoms is critical. When the pH value and the ion concentration of the solution are changed or the couplant is increased to lose zinc atoms, the protein can be depolymerized, so that the TRAIL protein can be depolymerized into a dimer or aggregated into a hexamer, the antitumor activity is reduced, and the solubility is reduced.
It was found that lung surfactant protein-D (SPD) is covalently bound to the molecular structure via disulfide bonds to help the protein fold into dodecamers. The invention has the following patents: CN104160027B discloses a carrier capable of expressing dodekamer TRAIL and HSV-TK suicide gene and its anti-tumor action, the dodekamer TRAIL adopts the method of combining SPD with TRAIL to make TRAIL form dodekamer. However, in practical application, it is found that although dodecameric TRAIL has good tumor killing effect, the toxic and side effects are significantly higher than those of trimeric TRAIL or other oligomers TRAIL (animal safety evaluation, not disclosed). Our studies also found that retention of the SPD-C terminal domain (SPDcc), through covalent bonding, can form a triple-helical parallel-coiled-coil domain (trimeric coiled-coil domain), thereby helping the protein to fold into a trimer, significantly enhancing the stability of TRAIL trimer, and maintaining its anti-tumor activity as well as reducing liver toxicity.
In order to solve the problem of the insufficient stability of TRAIL protein trimer, we use genetic engineering means to construct SPDcc-TRAIL mutant genes in vitro, and transfect mesenchymal stem cells through virus vectors, so that the mesenchymal stem cells highly express soluble trimer TRAIL, thereby preparing anti-tumor genetic engineering stem cells.
Disclosure of Invention
In view of the above, it is necessary to provide a trimeric TRAIL fusion protein with good stability against the technical problem of poor stability of the trimeric TRAIL protein.
A trimeric TRAIL fusion protein has the nucleic acid sequence shown as SEQ ID NO. 1.
In some embodiments, the fusion protein comprises:
a secretory signal protein selected from tPA, IL-2 and SEC, preferably tPA, having a nucleic acid sequence as shown in SEQ ID NO. 2;
the coding region of the lung surfactant protein is amino acid of 224-255, and the nucleic acid sequence is shown as SEQID NO. 3;
the coding region of the humanized tumor necrosis factor related apoptosis-inducing ligand is 114-281 amino acid residues, and the nucleic acid sequence is shown as SEQ ID NO. 4.
The invention also provides a method for modifying mesenchymal stem cells by the trimeric TRAIL fusion protein, which comprises the following steps:
s1, obtaining the trimeric TRAIL fusion protein;
s2, mixing the fusion protein in the step S1 with auxiliary plasmid for culture, and transfecting cells;
s3, transfecting the transfected cells into mesenchymal stem cells to obtain the mesenchymal stem cells capable of expressing the SPDcc-TRAIL protein.
In some embodiments, the transfection is selected from the following: viral transfection, lipofection, electrotransfection, gene editing transfection and mRNA transfection.
In some embodiments, the transfection is a viral transfection.
In some embodiments, the helper plasmid comprises: pMDLg, pRSV/REV and pMDNA2. G.
In some embodiments, in step S2, the step of culturing the fusion protein in admixture with a helper plasmid comprises:
(1) mixing the fusion protein plasmid with the auxiliary plasmids pMDLg, pRSV/REV and pMDNA2.G, dissolving the mixed plasmid in an Opti-MEM culture medium, and standing for 5 min;
(2) adding PLUSTMStanding the reagent for 5 min;
(3) will be provided with
Mixing LTX reagent with Opti-MEM solutionStanding for 5min, mixing LTX reagent with PLUS containing plasmid vectorTMMixing the reagents uniformly, standing for 30min to obtain
LTX-plasmid mixed medium.
In some embodiments, the mesenchymal stem cells are from: bone marrow tissue, adipose tissue, umbilical cord tissue, or placental tissue.
In some embodiments, the umbilical cord tissue-derived mesenchymal stem cells are genetically engineered.
The invention also provides the application of the SPDcc-TRAIL protein in preparing tumor drugs.
The trimeric TRAIL fusion protein can form a triple-helix parallel coil structural domain by covalent combination of disulfide bonds on the molecular structure, thereby helping the protein fold into a trimer. Because the trimer structure domain is combined by covalent bond, the addition of the structure domain at the N terminal of the TRAIL protein can obviously enhance the stability of the TRAIL trimer and keep the antitumor effect.
The invention utilizes the characteristics of the specific chemotaxis of the mesenchymal stem cells to the tumor tissues and the selective targeting of the mesenchymal stem cells to the tumor cells, and uses a gene modification method to modify the tripolymer TRAIL fusion protein with good stability prepared in the embodiment to the mesenchymal stem cells to specifically express the tripolymer TRAIL protein, so that the mesenchymal stem cells can specifically kill the tumor cells and repair the damaged tissues and organs, thereby achieving the purpose of treating malignant solid tumors.
Through comparison tests, compared with a control group, the SPDcc-TRAIL gene modified MSC constructed by the method has higher stability of the expressed TRAIL, and is not easy to degrade at 37 ℃ (P is less than 0.01); furthermore, through tumor killing experimental analysis, the SPDcc-TRAIL gene modified MSC can obviously inhibit the proliferation of the lung cancer cell line A549 cells and promote the apoptosis of the tumor cells, and has better tumor treatment effect (P <0.01) compared with a control group.
Drawings
FIG. 1 is a schematic flow chart of a method for modifying mesenchymal stem cells with a trimeric TRAIL fusion protein in one embodiment disclosed herein;
FIG. 2 is a diagram of the structure of LV-tPA-SPDcc-TRAIL and LV-tPA-TRAIL plasmids;
FIG. 3 is a diagram of ELISA detection of TRAIL expression by genetically modified mesenchymal stem cells;
figure 4 is a stability assay graph of a TRAIL protein secreted by genetically modified mesenchymal stem cells;
FIG. 5 is a graph of the inhibition of non-small cell lung cancer A549 cells by genetically modified mesenchymal stem cells;
FIG. 6 is a diagram of apoptosis of non-small cell lung cancer A549 cells promoted by genetically modified mesenchymal stem cells.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "MSC" as used herein refers to mesenchymal stem cells.
The term "Opti-MEM" as used herein is an antiserum medium.
The term "PLUS" as used hereinTM"is a transfection reagent for pre-complexing DNA that enhances cationic lipid-mediated transfection of DNA into a number of cultured eukaryotic cells.
Terms used herein "
LTX' is a new advanced transfection reagent, suitable for common and difficult placesGene expression of rational cell types.
The term "DMEM" as used herein is a medium containing various amino acids and glucose.
The terms "pMDLg, pRSV/REV and pMDNA2.G" third generation lentiviral packaging plasmid combination as used herein.
The culture medium, reagent, kit and the like in the following examples are all conventional biological products and are commercially available.
Example 1
A trimeric TRAIL fusion protein has the nucleic acid sequence shown as SEQ ID NO. 1. The fusion protein comprises:
a secretory signal protein selected from tPA, IL-2 and SEC, preferably tPA, having a nucleic acid sequence as shown in SEQ ID NO. 2;
a lung Surfactant Protein (SPD), the coding region of which is 224-255 amino acid, and the nucleic acid sequence is shown as SEQ ID NO. 3;
a humanized tumor necrosis factor related apoptosis inducing ligand (homo sapiens tumor necrosis factor-related apoptosis inducing ligand, hTRAIL) has a coding region of amino acid residues 114-281 and a nucleic acid sequence shown in SEQ ID NO. 4.
The preparation method of the trimeric TRAIL fusion protein comprises the following steps:
(1) designing and synthesizing gene sequences of tPA, SPDcc, TRAIL, etc.
(2) The sequences of tPA, SPDcc, TRAIL, etc. are connected successively into tPA-SPDcc-TRAIL by means of molecular biological method.
(3) The sequences are loaded into a fourth generation lentiviral vector system by utilizing a molecular cloning technology to construct an LV-tPA-SPDcc-TRAIL fusion protein lentiviral expression plasmid.
Comparative example 1
LV-tPA-TRAIL and LV-tPA-SPDc-TRAIL fusion proteins were prepared in the same manner as in example 1, using LV-tPA-TRAIL as a control virus for the LV-tPA-SPDc-TRAIL fusion protein, and FIG. 2 shows plasmid structures of LV-tPA-TRAIL and LV-tPA-SPDc-TRAIL (upper graph is LV-tPA-TRAIL, and lower graph is LV-tPA-SPDc-TRAIL).
Example 2
A method for modifying mesenchymal stem cells by trimeric TRAIA fusion protein comprises the following steps:
s1, obtaining trimer TRAIL fusion protein;
among them, the preparation method of trimeric TRAIL fusion protein was the same as that of example 1.
S2, mixed culture of the fusion protein and the helper plasmid, and cell transfection;
among the modes of transfection, viral transfection, lipofection, electrotransfection, gene editing transfection and mRNA transfection are included.
The mixed culture of the trimeric TRAIL fusion protein and the helper plasmid comprises the following steps:
(1) mixing the fusion protein plasmid with the auxiliary plasmids pMDLg, pRSV/REV and pMDNA2.G, dissolving the mixed plasmid in an Opti-MEM culture medium, and standing for 5 min;
(2) adding PLUSTMStanding the reagent for 5 min;
(3) will be provided with
Mixing LTX reagent with Opti-MEM solution, standing for 5min, and mixing LTX reagent with PLUS containing plasmid vectorTMMixing the reagents uniformly, standing for 30min to obtain
LTX-plasmid mixed medium.
Further, the mode of transfection is preferably viral transfection.
Specifically, the step of transfecting 293T cells comprises:
recovering 293T cells, culturing on a cell culture dish, and performing the following operations when the state is good: mixing the above
Adding the LTX-plasmid mixed culture medium to a cell culture dish, incubating for 6h, removing the culture medium, adding a DMEM fresh culture medium, and continuing to culture for 72 h;
the cell culture supernatant containing the virus was aspirated into an EP tube, centrifuged, transferred to a new EP tube, filtered and stored at-80 ℃ to obtain lentiviral particles.
S3, transfecting the transfected cells into mesenchymal stem cells to obtain the mesenchymal stem cells capable of expressing the SPDcc-TRAIL protein.
Specifically, the obtained lentiviral particles are transfected into mesenchymal stem cells, puromycin is added after 24 hours to screen the virus-transfected mesenchymal stem cells, and the mesenchymal stem cells can express the SPDcc-TRAIL protein.
Wherein the source of the mesenchymal stem cells comprises bone marrow tissue, adipose tissue, umbilical cord tissue or placenta tissue. Further, the source of the mesenchymal stem cells is preferably umbilical cord tissue, the umbilical cord tissue obtains the isolated mesenchymal stem cells by a climbing sheet culture method, and the specific method is as follows: the human umbilical cord is from a healthy pregnant woman supplier in full-term pregnancy and cesarean section, is provided by qualified national hospitals above two, and is signed by a pregnant woman or family members thereof to enter an informed consent form and an umbilical cord collection registration form. The extraction, culture, cell bank establishment and other work of the umbilical cord mesenchymal stem cells are all finished in a GMP production workshop of the company. Carrying out separation culture and passage amplification on the cells until the cells reach the 2 nd generation, and then carrying out detection on exogenous microorganisms, viruses, endotoxins and the like; and simultaneously detecting the immunophenotype, differentiation capacity, cell biological efficacy and the like of the cells. And (3) taking the qualified cells as seed bank cells, and storing the seed bank cells in a liquid nitrogen tank at the temperature of-196 ℃.
Comparative example 2
The plasmids of LV-tPA-TRAIL and LV-tPA-SPDcc-TRAIL obtained in comparative example 1 were used to modify mesenchymal stem cells in the same manner as in example 2, and the virus-uninfected mesenchymal stem cells were used as negative controls.
ELISA detection of TRAIL expressed by two genetically modified MSCs in comparative example 1
The cell supernatant obtained in example 2 was assayed for TRAIL content using TRAIL ELISA assay kit. The expression condition of TRAIL in the fusion protein is detected by an ELISA detection kit, and the results show that: both genetically modified MSCs can highly express TRAIL protein with no significant difference in expression (P >0.05) as shown in fig. 3.
Example 3. stability test of TRAIL protein secreted by genetically modified mesenchymal stem cells culture supernatants of two genetically modified mesenchymal stem cells obtained in example 2 were simultaneously placed in a 37 ℃ warm bath, and a small amount of supernatants were aspirated at different time intervals (0, 24h, 48h, 72h, and 96h) and rapidly stored in a refrigerator at-70 ℃. After sampling was completed at all time points, the TRAIL protein content of each sample was determined as described in example 3.
Calculating the TRAIL protein residual rate of each treatment group at each time point according to the following formula by taking the protein content of each treatment group as percent:
residual rate ═ 100% (TRAIL concentration/0 hTRAIL concentration) at each time point
As a result, it was found that SPDcc-TRAIL gene modified MSC constructed by the method of the present invention expresses TRAIL with higher stability (P <0.05) compared to the control group (TRAIL gene modified MSC) (fig. 4).
Example 4: proliferation inhibition of non-small cell lung cancer A459 by genetically modified MSC
Non-small cell lung cancer A549 cells were inoculated into a 96-well plate at 5000/well, cultured in A5% CO2 incubator at 37 ℃ for 12 hours, and then the culture solution was carefully discarded. Adding the culture solution and diluted conditioned medium supernatant into A549 cells in groups, wherein the culture solution and the conditioned medium supernatant are divided into the following groups: 200ul of complete culture solution, 200ul of MSC supernatant, 200ul of TRAIL-MSC supernatant and 200ul of SPDcc-TRAIL-MSC supernatant, wherein the groups are respectively cultured for 24h, 48h, 72h, 96h and 120h, after the culture, the proliferation condition of each group of tumor cells is respectively detected by adopting a CCK-8 kit, the detection of each group of cells is repeated for 6 times, and the detection result is shown in figure 5.
The result shows that the mesenchymal stem cells after gene modification can obviously inhibit the proliferation level of A549 cells, and compared with the comparative example 2, the SPDcc-TRAIL gene modified MSC constructed by the method has more obvious inhibition effect on A549 cells.
Example 5: apoptosis effect of gene modified MSC on non-small cell lung cancer A459
Collecting the mesenchymal stem cell culture supernatant obtained in the comparative example 2 for later use; a549 cells are divided into 4 groups, the groups are respectively inoculated into 6-well plates according to the ratio of 2x10^ 5/well, the plates are placed in A5% CO2 incubator at 37 ℃ for 12h, and then culture solution is carefully discarded.
In the culture process, 2ml of complete culture solution, 2ml of MSC supernatant, 2ml of TRAIL-MSC supernatant and 2ml of SPDc-TRAIL-MSC supernatant are respectively added into 4 groups of cells, the cells are cultured for 48h and 72h respectively, EDTA-free pancreatic enzyme digestion and cell collection are adopted, the apoptosis condition of tumor cells is detected by adopting an Annexin V-FITC/PI flow cytometry method, and the cells of each group are repeated for 3 times.
The results show that the mesenchymal stem cells after gene modification can remarkably promote apoptosis of A549 cells, and compared with comparative example 2 (TRAIL gene modified MSC), the SPDc-TRAIL gene modified MSC constructed by the method has more remarkable apoptosis effect on A549 cells, and the method is shown in figure 6.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
1. A preparation method of trimeric TRAIL gene modified mesenchymal stem cells is characterized in that the gene modified mesenchymal stem cells can highly express trimeric TRAIL fusion protein, and the nucleic acid sequence of the fusion protein is shown as SEQID NO. 1;
the fusion protein comprises:
a secretion signal protein selected from tPA, the nucleic acid sequence of which is shown as SEQ ID NO. 2;
the lung surfactant protein has a nucleic acid sequence shown as SEQ ID NO. 3;
the nucleic acid sequence of the humanized tumor necrosis factor related apoptosis inducing ligand is shown as SEQID NO. 4;
the method comprises the following steps:
s1, obtaining a plasmid containing the trimeric TRAIL fusion protein;
s2, culturing the plasmid containing the fusion protein in the step S1 and an auxiliary plasmid in a mixed way, and transfecting cells;
s3, transfecting the transfected cells into mesenchymal stem cells to obtain mesenchymal stem cells capable of expressing the SPDcc-TRAIL protein;
the helper plasmid comprises: pMDLg, pRSV/REV and pMDNA2. G;
in step S2, the step of culturing the plasmid containing the fusion protein in admixture with a helper plasmid comprises:
(1) mixing the fusion protein plasmid with the auxiliary plasmids pMDLg, pRSV/REV and pMDNA2.G, dissolving the mixed plasmid in an Opti-MEM culture medium, and standing for 5 min;
(2) adding PLUS antibody, and standing for 5 min;
(3) mixing the Lipofectamine LTX reagent with the Opti-MEM solution, standing for 5min, uniformly mixing the LTX reagent with the PLUS cassette reagent containing the plasmid vector, and standing for 30min to obtain the Lipofectamine LTX-plasmid mixed culture medium.
2. The method of claim 1, wherein the transfection is a lentiviral transfection.
3. The method of claim 1 or 2, wherein the mesenchymal stem cells are from: bone marrow tissue, adipose tissue, umbilical cord tissue, or placental tissue.
4. Use of a trimeric TRAIL fusion protein as claimed in claim 1 for the preparation of an antitumor medicament.
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