WO2017167209A1 - Trail-secreting mesenchymal stem cells and use thereof to treat brain tumors - Google Patents

Abstract

Provided in the present invention are a construct used to express soluble fragments of secreted TRAIL protein, and a lentivirus expression vector including said construct. Also provided are mesenchymal stem cells having said construct integrated into the genomes thereof and capable of expressing and secreting said TRAIL protein fragments. Also provided is the use of said construct, vector or mesenchymal stem cells to treat brain tumors.

Description

Mesenchymal stem cells secreting TRAIL and use thereof for treating brain tumors

The present application claims priority to Chinese Patent Application No. 201610194781.1, filed on March 30, 2016, entitled,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The priority of the Chinese Patent Application No. 201710184390.6, entitled "Hybridized Stem Cells Deriving TRAIL and Its Use for Treating Brain Tumors", is hereby incorporated by reference.

Technical field

The present invention relates to the field of genetic recombination and stem cell applications. In particular, the present invention provides a construct which expresses a soluble fragment of a secreted TRAIL protein, and a lentiviral expression vector comprising the construct, and a stem cell in which the construct is integrated on the genome, which is expressible And secreting the TRAIL protein fragment. The invention also provides the use of the construct or vector or stem cell for treating a brain tumor or preparing a brain tumor drug.

Background technique

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a member of the tumor necrosis factor superfamily, which is initiated by binding to cell death receptors (DR4 and DR5). An exogenous apoptotic pathway that induces apoptosis in tumor cells. Moreover, the apoptosis-inducing activity is mainly directed against tumor cells, and does not have a killing effect on normal cells, and thus has certain tumor specificity. The most economical way to get TRAIL protein It is a genetically engineered expression purification system, and the currently used expression system includes an E. coli prokaryotic expression system and a cellular eukaryotic expression system. Since TRAIL proteins are of mammalian origin, eukaryotic cell expression systems have better compatibility.

There are three problems in the process of eukaryotic expression of TRAIL protein. 1. Secretability of TRAIL: The general eukaryotic expression vector lacks an effective secretory signal peptide sequence, and cannot transfer the expressed target protein to the extracellular medium, but accumulates inside the cell, which affects on the one hand. The accumulation of the target protein, on the other hand, adds difficulties to the subsequent separation and purification process. 2. TRAIL protein solubility: The amino terminus of TRAIL protein molecule is a hydrophobic membrane transmembrane structure, which concentrates a large number of hydrophobic amino acid residues, resulting in poor water solubility of TRAIL protein, which tends to accumulate in solution and lose activity. 3. The genetic stability of the constructed genetically engineered cells, the common eukaryotic expression vector, can not be stably existed after the target gene fragment is introduced into the host cell genome, and the target gene will be gradually lost as the host cell undergoes the division process, so it is necessary Constant antibiotic selection and monoclonal cell screening.

There remains a need in the art to obtain constructs, vectors and host cells that are capable of stably and efficiently expressing and secreting active TRAIL proteins or fragments. There is a further need in the art for constructs, vectors and host cells that are capable of treating diseases and for the preparation of related drugs that are capable of stably and efficiently expressing and secreting active TRAIL proteins or fragments.

Summary of the invention

The present invention has been made in view of the above problems, and provides a mesenchymal stem cell in which an exogenous nucleic acid is integrated on its genome, which stably and efficiently expresses and secretes a TRAIL protein fragment. To this end, the present invention also constructs a lentiviral expression vector capable of expressing a soluble fragment of a secreted TRAIL protein, which stably transfects a mammalian host cell and secretes the soluble fragment of the expressed TRAIL protein into the host cell. The cell culture medium facilitates subsequent collection, isolation and purification. The present invention also provides the use of the mesenchymal stem cells for treating a TRAIL-related disease or preparing a drug, including cancer or tumor, Especially brain tumors.

In particular, the invention provides an isolated construct, wherein the construct comprises (1) a secretion signal peptide coding region; (2) a TRAIL protein trimer stable structural coding region; and (3) a TRAIL protein fragment Coding area,

Wherein the TRAIL protein fragment has an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

(a) 114 to 281 amino acid residues of TRAIL;

(b) having at least 90% identity with the amino acid sequence of (a), more preferably at least 95% identity, such as at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity Sexual and have TRAIL protein activity.

The secretory signal peptide of eukaryotes is present on the protein to be secreted or will become a transmembrane part, usually at the N-terminus of the protein. The signal peptide referred to in the present invention may be a heterologous signal peptide, i.e., a signal peptide that is not naturally operably linked to a protein or polypeptide. The use of a signal peptide allows the target protein or polypeptide to be secreted to the extracellular or increased secretion of the protein. Common signal peptides include fibrillin secretion signal peptide, growth factor signal peptide, hormone signal peptide, cytokine signal peptide and immune protein signal peptide. Examples of signal peptides include GDF signal peptides, IGF signal peptides, BMP signal peptides, neurotrophin signal peptides, PDGF signal peptides and EGF signal peptides, and hormone signal peptides (eg, production hormone, insulin, ADH, LH, FSH, ACTH, MSH). , TSH), or an interleukin signal peptide. Most of these signal peptides are derived from mammalian sources, such as humans, mice, rats, pigs, monkeys, etc. Preferably, the signal peptides used in the present invention are of mouse or human origin. The signal peptide can also be modified to enhance its ability to aid protein secretion.

Wherein the secretion signal peptide used in the construct of the present invention may be selected from the group consisting of a human fibrillin secretion signal peptide, a human growth hormone secretion signal peptide, a human immunoglobulin signal peptide, and a human interleukin 2 signal peptide. Wait.

The secretion signal peptide preferably used in the present invention is a secretion signal peptide derived from human fibrillin. Human fibrillin-1, or human fibrillin, by Sakai LY, Keene DR, Engvall E et al., 1986, J. Cell Biol. 103, "Fibrillin, a new 350-kD glycoprotein, is a component Of extracellular microfibrils" was reported. Human fibrillin is a secretable glycoprotein secreted by fibroblasts into the extracellular matrix. The human fibrillin secretion signal peptide has the following amino acid sequence: MRRGRLLEIALGFTVLLASYTSHGADA. Nucleic acid sequences encoding human fibrin secretion signal peptides can be used in the constructs of the invention. In the construct of the present invention, preferably the secretion signal peptide coding region has the sequence shown in SEQ ID NO: 2.

In the constructs of the invention, wherein the TRAIL protein trimer stabilizing structure is used to help form a stable TRAIL protein or a trimer form of a TRAIL fragment. The trimer form of the TRAIL protein or TRAIL fragment is more stable and more active than the monomer. Trimeric stable structural sequences useful in the present invention include isoleucine zipper structures, leucine zipper structures, and the like.

It is known in the art that a leucine zipper domain or an isoleucine zipper structure is found in a variety of natural proteins. The Leucine zippers structure refers to the presence of a leucine residue every 7 amino acid residues in a peptide chain of a trans-factor, and the helix formed by this peptide chain is composed of a bright ammonia. The hydrophobic surface formed by the acid residue and the other side is the hydrophilic surface composed of the hydrophilic amino acid residue. The hydrophobic surface composed of leucine residues is a leucine zipper strip, and two trans-factors having a leucine zipper strip can form a dimer or a multimer such as a trimer by hydrophobic interaction. The leucine zipper domain or the isoleucine zipper structure can interact to form a dimer or trimer. The leucine zipper domain or the isoleucine zipper structure contained in the construct of the present invention can promote oligomerization of the fusion protein and thus can increase stability. Suitable human leucine zipper domains are, for example, the leucine zipper domains contained in the human c-fos, c-jun, c-myc, max and mdx1 proteins. In the present invention, Optimizations and experiments have been performed to demonstrate that the expressed TRAIL protein fragment forms a trimer leucine zipper or isoleucine zipper structural sequence. The inventors obtained an optimized nucleic acid sequence encoding an isoleucine zipper structure sequence by design and testing. In the construct of the present invention, preferably the TRAIL protein trimer stable structural coding region has the sequence shown in SEQ ID NO: 3.

In the construct of the present invention, the TRAIL protein fragment is a fragment of amino acid sequence 114-281 amino acids of SEQ ID NO: 1.

Human TRAIL is known in the art to be a type II transmembrane protein consisting of 281 amino acids. Wiley, S. R. et al., is reported in Identification and Characterization of a New Member of the TNF Family that Induces Apoptosis. Immunity 3, 673-682 (1995). The amino acid sequence of the TRAIL protein is shown in SEQ ID NO: 5.

The protein encoded by the TRAIL coding region comprised by the construct of the present invention may be a fragment of the active TRAIL, i.e., a contiguous portion of the 281 amino acid amino acid sequence of TRAIL, which has activity of the TRAIL protein. Preferred TRAIL fragments of the invention include, for example, a fragment of 114-281 amino acid residues of TRAIL of SEQ ID NO: 1. It is known in the art that the intracellular N-terminus of TRAIL has no signal peptide and the active portion is located extracellularly from 114 to 281 amino acid residues. The 114-281 amino acid residues of human TRAIL can also form homotrimers with zinc binding sites near the top, which play an important role in maintaining TRAIL and stability.

The protein encoded by the TRAIL coding region comprised by the construct of the present invention may also have an amino acid sequence substantially identical to the 114-281 fragment of TRAIL. As used herein, "substantially identical amino acid sequence" refers to the substitution, deletion, addition or insertion of one to several (eg, 2, 3, 4 or 5) amino acids in an amino acid sequence which is identical to the amino acid sequence. Or similar or better activity.

The constructs of the invention can be prepared by using conventional DNA synthesis. The construct can be inserted into an expression vector by conventional genetic engineering methods; the host cell can be transformed with the obtained recombinant expression vector; Transformants; and collecting the polypeptide from the culture. This can be done, for example, by the method described in Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983).

The invention also provides a vector comprising the construct described above. The vector of the present invention may be a plasmid, a bacteriophage, a virus, or the like, and is capable of independently replicating and having a selection marker in a genetically engineered host capable of expressing a protein. The vector is amplified and expressed upon entry into the host cell. Suitable genetically engineered hosts are well known in the art and can be E. coli, yeast, insect cells, animal cells, and the like. Preferably, the vector of the present invention is a vector expressed in an animal cell, such as a non-viral vector, a baculovirus expression vector, an adenovirus vector, a retroviral vector, a lentiviral vector. The vector which can be used as the present invention is preferably a lentiviral vector such as a lentiviral vector pCDH, specifically pCDH having the aforementioned construct of the present invention. In one of the examples of the present invention, the present invention provides pCDH-seTRAIL prepared as in the examples.

The present invention also provides a cell, particularly a mammalian cell, such as a human, mouse, rat, porcine, or monkey cell, genomically integrating the above-described construct of the present invention, and expressing and secreting the TRAIL Protein fragment.

The mammalian cells of the present invention described above may be stem cells, lymphocytes, T cells, B cells, macrophages, fibroblasts, tumor cells, and the like. Preferably, the present invention provides a stem cell, such as a mesenchymal stem cell, which integrates the aforementioned construct of the present invention on its genome and which expresses and secretes a TRAIL protein fragment.

Mesenchymal stem cells (MSCs) are important members of the adult stem cell family and are derived from the mesoderm and ectoderm in early development. Mesenchymal stem cells have the characteristics of multi-directional differentiation, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. The study found that mesenchymal stem cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, heart muscle, endothelium, etc. under continuous induction conditions in vivo or in vitro, and successively subculture and freeze. Still after saving Have a multi-directional differentiation potential. According to the source, mesenchymal stem cells can be classified into umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose-derived mesenchymal stem cells.

In one aspect of the present invention, the present invention provides the above-described construct in which the construct of the present invention is integrated on a genome, and mammalian cells which can express and secrete the TRAIL protein fragment are prepared by the following method:

Constructing a construct of the invention as described above, the construct comprising (1) a secretion signal peptide coding region; (2) a TRAIL protein trimer stable structural coding region; and (3) a TRAIL protein fragment coding region ,

Wherein the TRAIL protein fragment has an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

(a) 114 to 281 amino acid residues of TRAIL;

(b) having at least 90% identity with the amino acid sequence of (a), more preferably at least 95% identity, such as at least 99% identity, and having TRAIL protein activity;

b. preparing a vector comprising the above construct, preferably, the vector is an animal cell expression vector, preferably a lentiviral vector, such as pCDH;

c. Infecting mammalian cells with the vector, preferably a stem cell, such as a mesenchymal stem cell.

The invention also provides a method for producing a TRAIL protein, the method comprising transforming a mammalian cell with the vector described above, expressing a TRAIL protein in the mammalian cell, the TRAIL protein being secreted into a culture outside the host cell In the liquid.

The present invention provides a pharmaceutical composition for treating a disease associated with TRAIL, which comprises any of the above-described constructs or vectors or cells of the present invention.

The present invention provides the use of any of the above constructs or vectors or cells of the present invention or a pharmaceutical composition thereof for the treatment of a disease associated with TRAIL. The present invention also provides any of the above uses of the present invention. Use of a construct or vector or cell or pharmaceutical composition thereof for the manufacture of a medicament for the treatment of a disease associated with TRAIL.

It is known in the art that TRAIL induces an apoptotic response through both DR4 and DR5 receptors. Thus, any of the constructs or vectors or cells of the invention or a pharmaceutical composition thereof can be used to treat a disease with a DR4 or DR5 receptor on a target cell.

Diseases treatable by the present invention include cancers and tumors such as brain cancer. The brain tumors treatable by the present invention include glioma and meningioma and the like. Glioma includes glioblastoma, anaplastic astrocytoma, gliosarcoma, anaplastic oligodendroglioma, and degenerative ganglion nerves. Glioma (anaplastic ganglioglioma), pineal cell tumor (pineoblasoma), medullobastoma (medullobastoma) and the like.

DRAWINGS

Fig. 1 is an agarose gel electrophoresis pattern of a secreted TRAIL protein soluble fragment-expressed gene recovered by double digestion. On the right is the DNA band of interest, and on the left is Invitrogen's 1Kb Plus DNA ladder molecular marker. The target nucleic acid fragment is approximately 711 bp.

Figure 2 is an electrophoresis map of the lentiviral expression vector pCDH plasmid DNA recovered by double digestion. The left side is the DNA band of interest, and the right side is the Invitrogen 1Kb Plus DNA ladder molecular marker used. The target nucleic acid fragment is approximately 8,000 bp.

Figure 3 is a fluorescence micrograph of HEK293 cells co-transfected with lentivirus-packaged four plasmids for 48 hours. GFP is a photo under fluorescence, Light is a white light photo in the same field of view, and Merge is an integrated photo of two fields of view.

Figure 4. Fluorescence and white light micrographs of MSC cells infected with lentiviral particles. GFP is a photo under fluorescence, Light is a white light photo in the same field of view, and Merge is an integrated photo of two fields of view.

Figure 5 shows an ELISA standard curve for detecting TRAIL protein. The abscissa is the absorbance at 450 nm and the ordinate is the TRAIL protein standard concentration (pg/ml).

Fig. 6 MTS assay detects the growth inhibitory effect of conditioned medium of MSC cells infected with lentivirus on U87 tumor cells.

Figure 7. Stability of TRAIL protein secreted by MSC cells infected with lentiviral vectors.

Figure 8. MSC cells infected with lentiviral vectors inhibit U87 solid tumors in mice.

detailed description

Example 1 Preparation of a construct encoding a soluble fragment of a secreted TRAIL protein

(1) A double-stranded DNA molecule having the nucleotide sequence of SEQ ID NO: 4 below was synthesized.

These include the XbaI site tctaga, the Kozak site gccatgggt, and

Secretion signal peptide coding region sequence:

Trimeric stable structure coding region sequence:

Nucleic acid sequence encoding 114-281 amino acids of TRAIL protein:

And BamHI site ggatcc.

(2) Double enzyme digestion and gel recovery of double-stranded DNA molecules:

The synthesized double-stranded DNA molecules were fully digested with restriction endonucleases Xba I and BamH I (NEB), and the digested products were analyzed by 1% agarose gel electrophoresis. The 711 bp nucleic acid fragment was purified by Qiagen's gel recovery kit. (figure 1).

Example 2 Construction of a secreted TRAIL protein soluble fragment lentiviral expression vector pCDH-seTRAIL

(1) Double digestion of pCDH vector: The pCDH vector plasmid (System Biosciences Catalog: CD513B-1) was fully digested with restriction endonucleases Xba I and BamH I (NEB). The product was analyzed by 0.8% agarose gel electrophoresis, and an approximately 8000 bp fragment was purified using Qiagen's gel recovery kit (Fig. 2).

(2) ligation and transformation of the digested product: the 711 bp fragment-encoding DNA recovered by the enzyme obtained in Example 1 and the 8,000 bp pCDH plasmid DNA recovered by the same digestion were mixed at a molar ratio of 5:1, and T4 DNA ligase (NEB) was ligated overnight at 16 ° C to construct the expression vector pCDH-seTRAIL.

The ligation product was transformed into E. coli stbls competent cells (stbls competent cells were purchased from Genecopoeia, transformed operations by F. Osbourne, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith K. Straer, "Guidelines for Editing Molecular Biology", John Wiley & Sons, New York, 1995, Third Edition, P39-40, screened for transformants with ampicillin resistance.

(3) Verification of the expression vector pCDH-seTRAIL: the selected positive transformants were expanded and plasmid DNA was extracted (plasmid DNA extraction method according to J. Sambrook, EF Ferric, T. Maniartis, Molecular cloning) The experimental guide, New York, Cold Spring Harbor Press, 2001, third edition, P27-30, and DNA sequencing confirmed that the connection was correct.

Example 3 Packaging and transfection of secreted TRAIL protein soluble fragment lentiviral particles

(1) Determination of concentration and purity of expression plasmid pCDH-seTRAIL and helper plasmid

Using the third generation lentiviral packaging system, in addition to the expression plasmid pCDH-seTRAIL, the packaging plasmid pMDLg/pRRE (purchased from Addgene, Catalog: 12251) was also used; the packaging plasmid pRSV-REV (purchased from Addgene, Catalog: 12253); The outer shell protein particle pMD2G (purchased from Addgene, Catalog: 12259) was used to complete the packaging of the virus particles.

The purity and concentration of each plasmid DNA were first analyzed by a NanoDrop spectrophotometer. The results are shown in Table 1.

Table 1 Results of analysis of concentration and purity of each plasmid DNA

(2) Plasmid co-transfection of HEK293 cell line

Co-transfection of plasmid formulations (per 15 cm dish):

*TE buffer (10 mM Tirs pH 8.0, 1 mM EDTA)

#HBS(280 mM NaCl, 50 mM HEPES, 1.5 mM Na ₂ HPO ₄ .H ₂ O, 10 mM KCl, 12 mM Dextrose)

Transfection:

A. The day before transfection, HEK293 cells (purchased from Clontech, Cat. No 632180) were inoculated into a 15 cm cell culture dish, and the cell density of the inoculation day was controlled to be about 80%;

B. Each plasmid component, CaCl ₂ solution, TE buffer is thoroughly mixed in a 50 ml centrifuge tube, and the above mixture is slowly added to another centrifuge tube in which the HBS solution has been placed;

C. Mix at medium speed and add to each cell culture dish and place in a cell culture incubator;

D. 16 hours after transfection, the medium was carefully aspirated and replaced with DMEM medium containing 10% FBS;

E. Incubation was continued for 48 hours, and the fluorescence signal of the cells was observed by a fluorescence microscope.

Figure 3 is a fluorescence micrograph of a HEK293 cell line co-transfected with four plasmids. The result is shown in Figure 3. GFP is a photo under fluorescence, Light is a white light photo in the same field of view, and Merge is a two-field view. Photo.

(3) Virus particle collection

A. collecting the above cell culture supernatant;

B. Centrifuge at 800 g for 10 min at 4 ° C, and collect the supernatant;

C. The supernatant is passed through a 0.45 μm microporous membrane and the filtrate is retained;

D. For each four volumes of filtrate, add one volume of pre-cooled PEG-it Virus Precipitation Solution (System Biosciences Catalog: LV810A-1), mix well, stand at 4 ° C overnight, centrifuge at 1,500 g 30 min, obtained virus particle precipitation;

E. Viral particles were resuspended as virus particle stocks in an appropriate amount of TBS solution (800 mg NaCl, 20 mg KCl, 300 mg Tris base in 100 ml deionized water, pH 8.0), dispensed and stored at -80 °C.

Example 4 Determination of secretable TRAIL lentivirus titer

1. HEK293 cells were seeded in 96-well plates, seeded at a density of 4,000 cells per well, and placed in a cell culture incubator overnight;

2. Prepared virus stock solution in DMEM medium (containing 10% FBS) containing 8 μg/ml of polybrene

3. Carefully aspirate the medium in the 96-well plate and add each well to the above medium containing the gradient-diluted virus particles, and repeat three wells in each concentration treatment group.

4. Incubation was continued for 48 hours. HEK293 cells with green fluorescence signal were observed under a fluorescence microscope, and the lowest dilution of the fluorescence signal was recorded (Table 2), and the titer of the virus particle stock solution was calculated by the following formula:

Titer (TU/μl)=1/10X10 ^(n-1)

n is the gradient dilution factor

Table 2 Titer determination of fluorescent signal detection results (√ indicates fluorescence, x indicates no fluorescence)

Calculated from the results of Table 2 and the above formula

pCDH-seTRAIL lentivirus titer is 1×10 ⁶ TU/μl

pCDH (empty vector control) lentivirus titer is 1 × 10 ⁶ TU / μl

Example 5 Expression of a secretible TRAIL lentivirus against human mesenchymal stem cells

1. Preparation of human umbilical cord derived human mesenchymal stem cells

Immediately after the delivery of the fetus, the umbilical cord is cut by the umbilical cord, and the umbilical cord is washed with physiological saline, and then disinfected with medical alcohol. The umbilical cord is placed in the umbilical cord preservation solution at a constant temperature of 2-8 ° C.

The obtained umbilical cord was washed with 0.9% sodium chloride injection and repeated 2 to 3 times to remove blood stains. 75% ethanol was immersed in the entire umbilical cord for sterilization. The sodium chloride injection was repeatedly washed to remove residual ethanol. The umbilical cord was cut into a number of about 2 to 5 cm with a sterile surgical scissors to remove congestion and clots in the small blood vessels of the umbilical cord. The white connective tissue between the amniotic membrane and the blood vessel is Huatong's glue, which is torn off with a gingival sputum, placed in a sterile dish, and an appropriate amount of 0.9% sodium chloride injection is added to wash the colloid. The weighed Huatong's colloid was cut into a tissue homogenate block of 1 to 4 mm ³ with a sterile tissue scissors, and 0.9% sodium chloride injection was added thereto, and the mixture was centrifuged at 800 to 900 g for 5 minutes. Collect the last wash solution for sterility. According to the weight of the colloid, add appropriate medium, and the concentration of the homogenized tissue homogenate is about 04-0.7g/ml. After pipetting the tissue homogenate block uniformly, the tissue homogenate block is inoculated into the T75 flask, and the medium is added. Mixed culture.

The culture flask is placed in a flat state to distribute the tissue homogenate block as evenly as possible over the entire bottom surface, and the culture flask is placed in a constant temperature and humidity incubator of carbon dioxide. Culture conditions: 37.0 ± 0.5 ° C, carbon dioxide volume fraction was 5.0 ± 0.2%.

The first change: the tissue was cultured until the 5th to 7th day for the full amount of liquid exchange. The unattached tissue blocks in the culture flask are combined with the old medium and transferred to a centrifuge tube, 800-900 g, centrifuged for 5 min, the supernatant is removed, and the residual tissue pieces after centrifugation are added to the appropriate amount of fresh medium. The liquid tube was blown evenly, and the same amount was divided into the original culture flask, and then the medium was added. The culture flask is placed flat so that the tissue block is evenly distributed throughout the bottom surface, and the CO ₂ constant temperature and humidity incubator is placed to continue the culture.

The second liquid change: the culture was changed for 10 to 13 days. Slightly tilt the flask, pipette a small amount of the old medium, and add an equal amount of fresh medium. The culture was continued by placing a CO ₂ incubator.

On the 14th to 18th day of the tissue block culture, when the area percentage of the cell clonal group reaches 70% to 80%, it is digested and harvested. The medium supernatant was removed and the cells were washed with 0.9% sodium chloride injection. Add appropriate amount of digestive enzyme to the culture flask until the bottom of the culture flask is infiltrated. After standing for 1 min, the culture flask was taken and observed under an inverted microscope. The cells were round, and most of the adherent tissue blocks and cells fell off, and the digestion was terminated (digestion time was less than 5 min). The cell suspension was transferred into a centrifuge tube, and a small amount of 0.9% sodium chloride injection was used to rinse the bottle wall. The washing liquid was transferred into a centrifuge tube, and the pipette was suspended and suspended for 30 seconds, filtered through a 100 μm sterile filter, and the filtrate was centrifuged, 300 g. , 10min. The washing supernatant was discarded and the cells were resuspended in 0.9% sodium chloride injection.

2. Inoculate the cell culture dish at a cell density of 60%, and incubate in a cell culture incubator overnight;

3. Carefully aspirate the medium in the cell culture dish and carefully add the culture dish to the α-MEM medium (containing 10% FBS) containing 8 μg/ml.

4. Add pCDH-seTRAIL and pCDH (empty vector control) lentiviral stocks to each cell culture dish, so that the MOI (the ratio of virus TU to the number of target cells) is about 10, and continue to culture for 24 hours in the cell culture incubator. ;

5. Carefully aspirate the medium in the cell culture dish, carefully add each cell culture dish with α-MEM medium containing 10% FBS, and continue to culture for 48 hours;

6. Observing MSC cells with green fluorescent signal by fluorescence microscope and collecting the cells on the culture clear. As shown in Figure 4, GFP is a photo under fluorescence, Light is a white light photo in the same field of view, and Merge is an integrated photo of two fields of view.

Example 6 Activity of MSC cells expressing and secreting TRAIL protein in a lentiviral vector infected with a lentiviral vector

The collected cell culture supernatant was analyzed for the TRAIL protein content in the supernatant using Abcam's TRAIL Human ELISA Kit (Catalog: ab46074), and the recombinant human TRAIL provided in the kit was accurately operated according to the kit instructions. Make a standard curve.

The results of the measurement are shown in Figure 5:

The TRAIL protein concentration in the supernatant of MSC medium infected with lentivirus pCDH-seTRAIL was approximately 634.93 pg/ml.

The TRAIL protein concentration in the MSC medium supernatant of the control empty vector lentiviral pCDH was approximately 32.96 pg/ml. This amount is the test error and the instrument reading background error range.

Example 7 Inhibition of human glioma cell line U87 cells by MSC cells secreting TRAIL protein

1. The collected cell culture supernatant of the MSC infected with the virus particles was centrifuged at 1,000 rpm for 10 minutes to remove the remaining cells, and the supernatant was aspirated as a conditioned medium;

2. The conditioned medium was serially diluted in a concentration gradient of 2% FBS in α-MEM medium, and the concentrations of 1, 0.5, 0.25, and 0.125 were set;

3. Human glioma cell line U87 cells (purchased from ATCC, Cat. No. HTB-14) cultured in α-MEM medium containing 10% FBS, seeded in 96-well plates at a density of 3,000 cells per well, placed in a 96-well plate. Incubate in a cell culture incubator overnight;

4. Carefully and cleanly remove the culture supernatant from each well;

5. Add 200 ul of each concentration of the conditioned medium to each well, and set a blank control well (adding α-MEM medium containing 10% FBS), and continue to culture for 72 hours;

6. Add 20ul of preset MTS solution (promega) to each well and continue to culture for 3 hours.

7. The absorbance values of the respective wells were measured at a wavelength of 490 nm and a reference wavelength of 690 nm using a Thermo Scientific Mulitiskan microplate reader;

8. Calculate the tumor inhibition rate by the following formula: tumor inhibition rate (%) = (blank hole absorbance value - conditioned medium treatment hole absorbance value) / blank hole absorbance value X100

Results As shown in Fig. 6, after the lentiviral vector expressing the secreted TRAIL protein of the present invention infects MSC cells, MSC can express and secrete a large amount of biologically active TRAIL protein into the cell culture medium, and has significant growth on U87 cells. Inhibition.

Example 8 Stability of TRAIL protein secreted by MSC cells infected with lentiviral vector

1. The supernatant of MSC conditioned medium infected with lentivirus pCDH-seTRAIL obtained in Example 6 was analyzed (the concentration of TRAIL protein in the supernatant was detected as: 634.93 pg/ml);

The MSC conditioned medium supernatant of the control empty vector lentiviral pCDH was infected as the first control medium;

The fresh medium supernatant was used as the second control medium (the concentration of TRAIL protein in the supernatant was detected to be about 0);

Commercially available recombinant human rhTRAIL protein (Pepro tech, Cat. No. 310-04) was used as a reference for research. Commercially available recombinant human rhTRAIL protein was separately dissolved in the first control medium and the second control medium to a final concentration of 650 pg/ml.

The MSC conditioned medium infected with lentivirus pCDH-seTRAIL was supernatantd, the supernatant of the first control medium in which rhTRAIL was dissolved, and the supernatant of the second control medium in which rhTRAIL was dissolved was placed in a 37 ° C warm bath at different times. (0, 3, 6, 12, 24, 48h) A small amount of supernatant was taken and quickly stored in a refrigerator at -70 °C.

After all the time points were sampled, each sample was determined by the ELISA method as in Example 6. TRAIL protein content.

The protein content at 0 h of each treatment group was 100%, and the TRAIL protein residual rate at each time point of each treatment group was calculated according to the following formula:

Residual rate = (TRAIL concentration at each time point / TRAIL concentration at 0h time point) x100%

The result is shown in Figure 7. As a result, it was confirmed that the TRAIL protein fragment prepared and secreted by the MSC cells carrying the lentiviral vector capable of secreting the TRAIL protein insert of the present invention has higher stability than the commercially available recombinant human rhTRAIL protein.

Example 9 Inhibition of human glioma cells by MSC cells secreting TRAIL protein in an animal model in nude mice

The experimental animals were 4-6 weeks old male nude mice (Chinese University of Hong Kong Experimental Animal Center, BALB/C nude mice). Breeding under standard experimental conditions: 12 hours light - 12 hours dark cycle, free access to water and food.

MSCs infected with lentivirus pCDH-seTRAIL, MSCs infected with control vector lentivirus pCDH, and human glioma cell line U87 cells were respectively in α-MEM medium containing 10% FBS, 37 ° C, 5% CO 2 Culture under saturated humidity conditions. After the animals were acclimated to the culture environment for 4 days, U87 cells were injected subcutaneously into nude mice, and each animal was inoculated into two positions, which were the left side of the back of the nude mouse and the right side symmetric thereto, and the inoculation of U87 cells at each locus. The amount was 2 x 10 ⁶ cells and the injection volume was 100 ul. The entire operation is done in a clean bench. The health status of the mice and the growth of the tumor were monitored after the completion of the vaccination. After all the mice developed obvious tumor entities, 8 mice with uniform tumor size on the left and right sides (the diameter of the vernier caliper was about 5 mm) were selected according to the tumor volume of the animals, and 2×106 infection controls were injected into the left tumor block. The MSCs of the vector lentivirus pCDH were injected with 2x106 MSCs infected with lentivirus pCDH-seTRAIL at the right tumor site, and the injection volume was 100 ul. The tumor size was measured with a vernier caliper every 4 days, and the tumor volume was calculated according to the following formula: tumor volume (mm ³ ) = 1/2 ab ² , a is the tumor long diameter, and b is the tumor short diameter.

Data analysis between groups was statistically analyzed by ANOVA statistical method, and P < 0.05 was considered to be significantly different.

Figure 8 shows the average tumor volume measurement results (volume unit mm ³ ) between the two groups during the experiment.

After injection of lentivirus-infected MSCs, the tumor volume of the two groups began to differentiate. The tumor volume of the MSCs injected with lentivirus pCDH-seTRAIL was gradually lower than that of the control group injected with MSCs infected with the control vector lentiviral pCDH. And on the 12th day and the 16th day showed a significant difference (P <0.05). The experimental results demonstrate that the MSC cells of the present invention infected with a lentiviral vector capable of secreting a TRAIL protein insert have a significant therapeutic effect on human glioma in animals.

The inventors of the present invention have unexpectedly found that umbilical cord mesenchymal stem cells prepared by the method of the present invention are effective in inhibiting brain tumors, particularly gliomas, in animals. One of the reasons for this unexpected discovery is a secreted TRAIL protein soluble fragment construct constructed based on the present invention and a lentiviral expression vector containing the construct.

The amino acid sequence of the soluble fragment coding region of the TRAIL protein of the present invention is added to promote the formation of a trimeric structure, which contributes to the stability and activity of the soluble fragment of the expressed TRAIL protein. Expression of a secreted TRAIL protein soluble fragment molecule in mammalian cells. In addition, the secreted TRAIL protein soluble fragment expression vector constructed by the present invention is added to the upstream region of the TRAIL protein soluble fragment coding gene in the downstream region of the promoter, and a highly efficient secretion signal peptide sequence is added, so that the TRAIL protein cloned downstream can be soluble. The TRAIL protein soluble fragment molecule expressed by the fragment encoding gene is secreted into the extracellular medium, which significantly increases the efficiency of protein expression, and the stability of the soluble fragment of the produced TRAIL protein is unexpectedly increased. Particularly in the case of using a lentiviral vector, the coding gene encoding a soluble fragment of the secreted TRAIL protein can be stably integrated into the genome of the host cell, which is not easy Lost, and the risk of insertional tumor formation is greatly reduced, more secure and reliable.

The present invention may be practiced in other ways, except as specifically described in the above description and examples, unless otherwise indicated. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art. Many variations and modifications are possible in accordance with the teachings of the present invention and are therefore within the scope of the invention. All patents, patent applications, and scientific papers referred to herein are hereby incorporated by reference.

Claims (12)

1. An isolated construct, wherein the construct comprises (1) a secretion signal peptide coding region; (2) a TRAIL protein trimer stable structural coding region; and (3) a TRAIL protein fragment coding region,

   Wherein the TRAIL protein fragment has an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

   (a) 114 to 281 amino acid residues of TRAIL;

   (b) has at least 90% identity with the amino acid sequence of (a), more preferably at least 95% identity, such as at least 99% identity, and has TRAIL protein activity.
2. The construct of claim 1, wherein said secretion signal peptide is human fibrin secretion signal peptide, human growth hormone secretion signal peptide, human immunoglobulin signal peptide, human interleukin 2 signal peptide, preferably human fibrillin secretion The signal peptide, for example, the secretion signal peptide coding region has the sequence shown in SEQ ID NO: 2.
3. The construct of claim 1 or 2, wherein the TRAIL protein trimer stable structural sequence is an isoleucine zipper structure, a leucine zipper structure, for example, the TRAIL protein trimer stable structural coding region has SEQ ID NO The sequence shown in :3.
4. The construct of any one of claims 1 to 3, wherein the TRAIL protein fragment coding region has the sequence set forth in SEQ ID NO: 1.
5. The construct of any one of claims 1 to 4 which has the sequence of SEQ ID NO:4.
6. A vector comprising the construct of any one of claims 1 to 5, preferably the vector is an animal cell expression vector, preferably a lentiviral vector, such as pCDH.
7. A mammalian cell genetically integrating the construct of any one of claims 1-5 And expressing and secreting the TRAIL protein or a fragment thereof, preferably a stem cell of a human, mouse, rat, pig or monkey.
8. The mammalian cell of claim 7, which is a mesenchymal stem cell, such as umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose-derived mesenchymal stem cells.
9. The mammalian cell of claim 7 or 8, which is produced by the following method:

   a construct constructing according to any one of claims 1 to 5, comprising: (1) a secretion signal peptide coding region; (2) a TRAIL protein trimer stable structural coding region; and (3) a TRAIL protein Fragment coding area,

   Wherein the TRAIL protein fragment has an amino acid sequence identical or substantially identical to the amino acid sequence of (a) or (b) below:

   (a) 114 to 281 amino acid residues of TRAIL;

   (b) having at least 90% identity with the amino acid sequence of (a), more preferably at least 95% identity, such as at least 99% identity, and having TRAIL protein activity;

   b. preparing a vector comprising the above construct, preferably, the vector is an animal cell expression vector, preferably a lentiviral vector, such as pCDH;

   c. Infecting mammalian cells with the vector, preferably a stem cell, such as a mesenchymal stem cell.
10. A pharmaceutical composition for treating a brain tumor, such as a glioma, comprising the construct of any one of claims 1 to 5 or the vector of claim 6 or the cell of claim 7.
11. The construct of any one of claims 1 to 5 or the vector of claim 6 or the cell of any one of claims 7 to 9 or the pharmaceutical composition of claim 10 in the treatment of a brain tumor, such as a glioma, Applications.
12. The construct of any one of claims 1 to 5 or the vector of claim 6 or claims 7-9 Use of the cell of any of the above or the pharmaceutical composition of claim 10 for the manufacture of a medicament for treating a brain tumor, such as a glioma.

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