An extraction process and pain treatment preparation targeting the induction of mesenchymal stem cells to secrete a combination of TGF-beta and IL-10 anti-inflammatory factors

Abstract

The present application relates to a kind of target extraction process of inducing mesenchymal stem cell secretion TGF-β and IL-10 anti-inflammatory factor combination and its application in pain treatment preparation, the process is activated mesenchymal stem cell by VEGF-A-link-TNF-α fusion protein as shown in SEQ ID NO:4 Specific, significantly improve its TGF-β and IL-10 secretion level;The fusion protein is targeted with TNF-α inflammatory signal by VEGF receptor cooperation, realize the efficient induction of anti-inflammatory factor expression;Mesenchymal stem cell pretreated by VEGF-A-link-TNF-α fusion protein as shown in SEQ ID NO:4 can significantly relieve the hyperalgesia of neuropathic pain model, with significant clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and cell therapy technology, specifically relating to a process method for inducing mesenchymal stem cells to efficiently secrete anti-inflammatory factors TGF-β and IL-10 through targeted induction of fusion proteins, and its application in the preparation of pain treatment agents. Background Technology

[0002] Mesenchymal stem cells (MSCs) have become a research hotspot in cell therapy and tissue engineering due to their ease of isolation and expansion, low immunogenicity, and strong immunomodulatory capabilities. Their therapeutic effects mainly rely on paracrine mechanisms, releasing various bioactive factors, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), which are anti-inflammatory cytokines, to regulate the local immune microenvironment and promote tissue repair. However, in the resting state, the secretion levels of anti-inflammatory factors by MSCs are low, insufficient to produce significant therapeutic effects, which greatly limits their clinical translational applications.

[0003] To enhance the immunomodulatory function of MSCs, current techniques typically employ pretreatment with inflammatory factors, such as stimulating MSCs alone or in combination with tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), or interleukin-1β (IL-1β). While these methods can increase the secretion of anti-inflammatory factors to some extent, they still have significant limitations: First, cytokine stimulation lacks targeting, easily activating or damaging non-target cells, leading to off-target effects; second, single factors often only activate a limited number of signaling pathways, exhibiting a ceiling effect in induction efficiency, resulting in unstable and insignificant effects; furthermore, the use of high concentrations of inflammatory factors may induce MSC functional exhaustion or pro-inflammatory transformation, posing safety risks. Therefore, developing an induction strategy that can efficiently, safely, and specifically activate the anti-inflammatory potential of MSCs has become a crucial problem urgently needing to be solved in this field.

[0004] To address the shortcomings of existing technologies, this invention proposes a novel technical concept: a fusion protein is constructed using protein engineering techniques to fuse vascular endothelial growth factor A (VEGF-A), which has MSC-targeting function, with TNF-α, which has a strong stimulating signal, via a flexible linker peptide, thereby achieving dual and precise activation of MSCs. This design not only utilizes the internalization properties of VEGF-A binding to its receptor for targeted delivery but also effectively activates downstream signaling pathways such as NF-κB through TNF-α, with both significantly amplifying the expression efficiency of anti-inflammatory factors synergistically. This strategy overcomes the limitations of existing single-factor pretreatment methods, providing a new technical platform for preparing highly efficient MSC-derived anti-inflammatory factor compositions and developing novel pain treatment agents. Summary of the Invention

[0005] To address the problems of low anti-inflammatory factor secretion levels and lack of targeting and synergy in existing technologies for mesenchymal stem cells (MSCs), this invention aims to provide a novel solution by constructing a new fusion protein through protein engineering to efficiently and specifically induce MSCs to secrete high concentrations of a combination of anti-inflammatory factors (TGF-β and IL-10), and then applying it to pain treatment.

[0006] To address the aforementioned problems, the present invention first provides a VEGF-A-link-TNF-α fusion protein, characterized in that the amino acid sequence of the fusion protein comprises the sequence shown in SEQ ID NO:4;

[0007] In some embodiments, the VEGF-A-link-TNF-α fusion protein is used to induce mesenchymal stem cells to secrete a combination of TGF-β and IL-10 anti-inflammatory factors.

[0008] This invention provides a method for preparing a VEGF-A-link-TNF-α fusion protein;

[0009] In some embodiments, the fusion protein is prepared using a mammalian expression system and purified by Ni-NTA affinity chromatography and gel filtration chromatography.

[0010] The present invention also provides a method for inducing mesenchymal stem cells to secrete TGF-β and IL-10;

[0011] In some embodiments, the method includes co-culturing mesenchymal stem cells with VEGF-A-link-TNF-α fusion protein, collecting the culture supernatant, and extracting a combination of anti-inflammatory factors.

[0012] The present invention also provides a pain treatment preparation;

[0013] In some embodiments, the pain treatment formulation comprises a combination of TGF-β and IL-10 anti-inflammatory factors prepared by the method;

[0014] The present invention also provides a BMSC pretreated with VEGF-A-link-TNF-α fusion protein;

[0015] In some embodiments, the amino acid sequence of the fusion protein comprises that shown in SEQ ID NO:4.

[0016] This invention also provides its application in the preparation of pharmaceuticals for treating neuropathic pain;

[0017] In some embodiments, pain induced by a chronic compression injury (CCI) model of the sciatic nerve is preferred.

[0018] The core of this invention lies in providing a method for inducing MSCs to secrete a combination of anti-inflammatory factors using the fusion protein. A key step in this method is to co-culture MSCs with an effective dose of the VEGF-A-link-TN F-α fusion protein for 48 hours, thereby activating the cells and enabling them to efficiently secrete TGF-β and IL-10.

[0019] Compared with existing technologies, this invention has the following advantages: it is the first to design and construct a VEGF-A-link-TNF-α fusion protein with both targeting and activation functions; it establishes a new method for efficiently inducing MSCs to secrete a combination of anti-inflammatory factors; and it develops a corresponding extraction process and prepares a biological agent with significant analgesic effects, providing a novel strategy and means for treating chronic pain. Attached Figure Description

[0020] Figure 1 SDS-PAGE electrophoresis image of VEGF-A-link-TNF-α fusion protein.

[0021] Figure 2 ELISA results of TGF-β secretion levels after induction in each group.

[0022] Figure 3 ELISA results of IL-10 secretion levels after induction in each group.

[0023] Figure 4 Mechanical withdrawal threshold (MWT) of rats in each CCI model group changes over time.

[0024] Figure 5 The thermal shrinkage latency (TWL) curves of CCI model rats in each group as a function of time. Detailed Implementation

[0025] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0026] Example 1: Preparation of VEGF-A-link-TNF-α fusion protein

[0027] Human VEGF-A (NCBI Reference Sequence: NP_001273973.1): MAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR (SEQ ID NO:1);

[0028] Human TNF-α (GenBank: QCI55793.1):

[0029] MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHF

[0030] GVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWL

[0031] NRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTIS

[0032] RIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO:2);

[0033] linker (Gly4Ser)3: GGGGSGGGGSGGGGS (SEQ ID NO:3);

[0034] VEGF-A-link-TNF-α fusion protein:

[0035] MAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPL

[0036] MRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECR

[0037] PKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNE

[0038] RTCRCDKPRRGGGGSGGGGSGGGGSMSTESMIRDVELAEEALPKKTGGPQ

[0039] GSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSS

[0040] SRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEG

[0041] LYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPE

[0042] GAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIALHHHHHH(SEQ ID NO:4);

[0043] DNA fragments encoding human VEGF-A (as shown in SEQ ID NO:1) and human TNF-α (as shown in SEQ ID NO:2) were obtained using gene synthesis technology (gene synthesis technology services provided by GenScript Biotech). The two fragments were linked by a sequence encoding the flexible linker (Gly4Ser)3 (as shown in SEQ ID NO:3), and a 6×His tag sequence was introduced at its C-terminus. The complete fusion gene fragment was cloned into the mammalian expression vector pcDNA3.1(+) which had been digested with the same double restriction enzymes via NdeI and XhoI restriction endonuclease sites to construct the recombinant plasmid pcDNA3.1 / VEGF-TNF. The correctness of the inserted sequence and the accuracy of the reading frame were verified by DNA sequencing; Expi293F human embryonic kidney cells were used as the expression host. When the cell density reached 3.0 × 10⁶ cells / year... 6Transfection was performed when the cell count / mL and viability were higher than 95%. Linear polyethyleneimine (PEI) was used as the transfection reagent. The purified recombinant plasmid was mixed with PEI at a mass ratio of 1:2 in Opti-ME M medium and incubated for 20 minutes before being added to the cell culture system. Six hours after transfection, the corresponding enhancer and feed were added. The cells were cultured in a shaker at 37°C and 8% CO2. Ninety-six hours after transfection, the cell culture supernatant was harvested. The supernatant was centrifuged at 4°C and 8000 rpm for 20 minutes to remove cells and debris, and the clear supernatant was collected. The clear supernatant was filtered through a 0.45 μm filter at 4°C and loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Binding Buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4). The column was washed with 10 column volumes of Binding Buffer to remove unbound contaminants. Subsequently, a phased gradient elution was performed using Elution Buffer containing 500 mM imidazole, and the elution peaks were collected. The elution fractions rich in the target protein were combined and dialyzed in PBS (pH 7.4) buffer to remove imidazole and salt ions. To further improve purity, the dialyzed sample was subjected to HiLoad 26 / 600 Superdex 75 pg (Cytiva, catalog number: 28989334) gel filtration chromatography using PBS (pH 7.4) as the mobile phase. A single volume of elution peak C1 was collected, and its molecular weight was determined by SDS-PAGE, indicating high-purity VEGF-TNF fusion protein. (See [link to SDS-PAGE analysis]). Figure 1 .

[0044] Figure 1 The results showed that SDS-PAGE electrophoresis revealed a single, clear protein band, consistent with the theoretically calculated molecular weight of the fusion protein, 46.40 kDa, proving that high-purity VEGF-A-link-TNF-α fusion protein was successfully obtained.

[0045] Example 2: Experiment on the combined secretion of TGF-β and IL-10 anti-inflammatory factors by VEGF-A-link-TNF-α fusion protein in mesenchymal stem cells.

[0046] Group A: By We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6Cells were seeded at a density of 1:1 in 6-well plates. When the cell confluence reached 80%, the medium was replaced with serum-free medium, and VEGF-A-link-TNF-α fusion protein (working concentration 10 ng / mL) was added. The plates were then co-cultured for 48 hours. The culture supernatant was collected after stimulation, centrifuged at 1500×g for 10 minutes to remove cell debris, and then filtered through a 0.22 μm filter for sterilization. A protease inhibitor (1 mM PMSF) was added to prevent protein degradation. Pre-chilled acid / ethanol solution (95% ethanol + 1.5% concentrated hydrochloric acid) was added at a volume ratio of 4:1, and the mixture was incubated overnight at 4°C. The pH was adjusted to 3.0, and cold anhydrous ethanol and ether (1:1) were added for further precipitation. The protein precipitate was collected by centrifugation at 20000×g for 30 minutes.

[0047] Group B: By We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 10 ng / mL in 6-well plates. When the cell confluence reached 80%, the medium was replaced with serum-free medium, and VEG F165 protein (MedChemExpress, catalog: HY-P7110A), a subtype of vascular endothelial growth factor A (VEGF-A), was added at a concentration of 10 ng / mL. The plates were then co-cultured for 48 hours. The culture supernatant was collected after stimulation, centrifuged at 1500×g for 10 minutes to remove cell debris, and then filtered through a 0.22 μm filter for sterilization. A protease inhibitor (1 mM PMSF) was added to prevent protein degradation. Pre-chilled acid / ethanol solution (95% ethanol + 1.5% concentrated hydrochloric acid) was added at a volume ratio of 4:1, and the mixture was incubated overnight at 4°C. The pH was adjusted to 3.0, and cold anhydrous ethanol and ether (1:1) were added for further precipitation. The protein precipitate was collected by centrifugation at 20000×g for 30 minutes.

[0048] Group C: By We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6Cells were seeded at a density of 10 ng / mL in 6-well plates. When the cells reached 80% confluence, the medium was replaced with serum-free medium, and TNF-α (R&D Systems, catalog number: 210-TA-010 / CF) was added at a concentration of 10 ng / mL. The plates were then co-cultured for 48 hours. The culture supernatant was collected after stimulation, centrifuged at 1500×g for 10 minutes to remove cell debris, and then filtered through a 0.22 μm filter for sterilization. A protease inhibitor (1 mM PMSF) was added to prevent protein degradation. Pre-chilled acid / ethanol solution (95% ethanol + 1.5% concentrated hydrochloric acid) was added at a volume ratio of 4:1, and the mixture was incubated overnight at 4°C. The pH was adjusted to 3.0, and cold anhydrous ethanol and ether (1:1) were added for further precipitation. The protein precipitate was collected by centrifugation at 20000×g for 30 minutes.

[0049] Group D: By We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 1:1 in 6-well plates. When cell confluence reached 80%, the medium was replaced with serum-free medium, and an equal volume of PBS (AC histone) was added. The plates were then co-cultured for 48 hours. The culture supernatant was collected after stimulation, centrifuged at 1500×g for 10 minutes to remove cell debris, and then filtered through a 0.22μm filter for sterilization. A protease inhibitor (1 mM PMSF) was added to prevent protein degradation. Pre-chilled acid / ethanol solution (95% ethanol + 1.5% concentrated hydrochloric acid) was added at a volume ratio of 4:1, and the mixture was incubated overnight at 4°C. The pH was adjusted to 3.0, and cold anhydrous ethanol and ether (1:1) were added for further precipitation. The protein precipitate was collected by centrifugation at 20000×g for 30 minutes.

[0050] AD histone precipitates were collected, and the concentrations of TGF-β and IL-10 were measured using an ELISA kit (R&D Systems). See [link to ELISA kit]. Figure 2-3 .

[0051] Figure 2-3The results showed that unstimulated BMSCs secreted only trace amounts of TGF-β and IL-10. This confirmed that the cells themselves are in a "silent" immune state without inflammatory signal stimulation, with extremely low levels of anti-inflammatory factor secretion. Compared with group D, VEGF165 alone slightly increased the secretion of TGF-β and IL-10 (approximately 100 pg / mL), but this was not statistically significant. This indicates that the VEGF signaling pathway alone is insufficient to effectively activate the potent immune regulatory program of MSCs, and its main function may be more inclined to promote angiogenesis and cell proliferation. As a classic inflammatory stimulant, TNF-α in group C significantly induced the secretion of TGF-β and IL-10 by BMSCs, with concentrations significantly higher than those in group D (p<0.01) and group B. The concentrations of factors induced by the fusion protein in group A were significantly higher than those TNF-α in group C (p<0.01), with an increase of more than 60%. This study demonstrates that the VEGF-A-link-TNF-α fusion protein is not simply the sum of the functions of VEGF and TNF-α, but rather produces a synergistic effect of "1+1>2". The possible mechanism is that the VEGF-A portion preferentially targets and binds to VEGFR on the surface of MSCs, which may significantly enhance the cell's response efficiency to the subsequent TNF-α portion (activating the NF-κB / MAPK pathway) by internalizing or activating the auxiliary signaling pathway (PI3K / Akt), thereby greatly amplifying the expression of anti-inflammatory factors at the transcriptional level.

[0052] Example 3: Application of VEGF-A-link-TNF-α fusion protein-induced mesenchymal stem cells in a pain treatment model.

[0053] BMSCs pretreated with VEGF-A-link-TNF-α fusion protein: We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 10 ng / mL in 6-well plates. When the cell confluence reached 80%, the medium was replaced with serum-free medium, and VEGF-A-link-TN F-α fusion protein (working concentration 10 ng / mL) was added. The plates were co-cultured for 48 hours. The BMSCs pretreated with the fusion protein were washed twice with serum-free DMEM / F12 medium and resuspended at 5 × 10⁻⁶ cells / well. 6 Cell suspension with cells / mL;

[0054] Untreated BMSC suspension: by We offer bone marrow mesenchymal stem cells (BMSCs). Product name: Wistar rat bone marrow mesenchymal stem cells; catalog number: RAWMX-01001; cells were expanded to passage 3-5 using DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2; BMSCs were cultured at 1×10⁻⁶ cells / year. 6 Cells were seeded at a density of 1 / well in 6-well plates. When the cell confluence reached 80%, the medium was replaced with serum-free medium, and an equal volume of PBS (VEGF-A-link-TNF-α fusion protein) was added. The plates were co-cultured for 48 hours. The PBS-pretreated BMSCs were then washed twice with serum-free DMEM / F12 medium and resuspended at 5 × 10⁻⁶ cells / well. 6 Cell suspension with cells / mL;

[0055] Establishment of a chronic compression injury (CCI) pain model of the sciatic nerve: SPF-grade Wistar rats (male, weighing 220-250g) were selected and acclimatized for one week before surgery. Preoperative fasting was maintained for 12 hours, with free access to water. A 1% sodium pentobarbital injection (40mg / kg) was administered intraperitoneally, and the rats were fixed on the operating table after the corneal reflex disappeared. The left hind limb was shaved and disinfected. A 2cm incision was made along the course of the sciatic nerve to expose it (from the lower border of the piriformis muscle to the mid-femur). Two loose ligations were made 1mm apart near the proximal end of the nerve using 4-0 chromic catgut, ensuring that light contact with the nerve did not cause significant thinning. The muscles and skin were sutured layer by layer. In the sham surgery group, only the nerve was exposed without ligation. Rats were administered penicillin (100,000 U / kg) intramuscularly for 3 consecutive days, housed individually, and allowed free movement to observe postoperative recovery (to prevent incision infection). Seven days after establishing the CCI model (chronic inflammatory phase), cell transplantation was performed using a local sciatic nerve injection method: after anesthesia, the left sciatic nerve injury area was exposed, and 50 μL of cell suspension (total cell volume 5 × 10⁻⁶) was injected proximally and distally to the injury site using a microsyringe (25G needle). 5 BMSCs were injected at a rate of 5 μL / min, with the needle left in place for 30 seconds after injection to prevent leakage, followed by suturing the incision. The control group was injected with an equal volume of untreated BMSC suspension, fusion protein solution (10 ng / mL), or serum-free culture medium.

[0056] To clarify the therapeutic advantages of pretreatment with BMSCs, the following groups were set up (n=6 per group):

[0057] Sham surgery group: Only the sciatic nerve was exposed, and serum-free culture medium was injected, with an injection volume of 100 μL (50 μL each on the proximal and distal sides of the injury site);

[0058] Model control group: CCI model + injection of serum-free culture medium, injection volume of 100μL (50μL each on the proximal and distal sides of the injury site);

[0059] Simple fusion protein group: CCI model + injection of 10 ng / mL VE GF-A-link-TNF-α solution prepared in Example 1, with an injection volume of 100 μL (50 μL each on the proximal and distal sides of the injury site, and a total dose of fusion protein of 1 ng);

[0060] Untreated BMSC group: CCI model + BMSC suspension pretreated with PBS, cell suspension concentration was 5 × 10⁻⁶ 6 The injection volume was 100 μL (50 μL each on the proximal and distal sides of the injury site), and the total cell count was 5 × 10⁻⁶ cells / mL. 5 cells;

[0061] Pretreated BMSC group: CCI model + injection of BMSC suspension pretreated with VEGF-A-link-TNF-α, cell suspension concentration was 5×10⁻⁶ 6 The injection volume was 100 μL (50 μL each on the proximal and distal sides of the injury site), and the total cell count was 5 × 10⁻⁶ cells / mL. 5 cells.

[0062] Mechanical withdrawal threshold (MWT): The response threshold of the left hind limb to mechanical stimulation was measured using an electronic Von Frey analgesic device 1 day before transplantation, and 7, 14, and 21 days after transplantation. The intensity was initially measured starting at 2g. If a stimulus of this intensity did not elicit a positive response, a stimulus of a slightly higher intensity was applied; if a positive response occurred, a stimulus of a slightly lower intensity was applied, and so on, until the first positive and negative responses were observed. Four more measurements were then taken. The maximum intensity was 15g; any intensity greater than this was recorded as 15g. Each stimulus was spaced 30 seconds apart. At least five measurements were taken per group, and the average value was used. Results are shown below. Figure 4 .

[0063] Figure 4 The results showed that there were no significant differences among the groups 1 day before transplantation; 7 days after transplantation, the pretreated BMSC group was significantly higher than the model control group (P<0.01) and also higher than the untreated BMSC group (P<0.05); 14 days after transplantation, the pretreated BMSC group was significantly different from both the model control group and the untreated BMSC group (P<0.01); 21 days after transplantation, the pretreated BMSC group was close to the level of the sham surgery group and significantly higher than the model control group (P<0.01).

[0064] Heat-induced foot retraction latency (TWL): The latency of the left hind limb to thermal stimulation was measured using a thermal radiation analgesia meter to avoid tissue damage. The TWL was defined as the time from the start of irradiation until the rat lifted its leg to avoid the stimulation. The cut-off time was set to 20 seconds to prevent tissue damage. The intensity of the heat stimulation was kept constant throughout the experiment. Each animal was measured 5 times, with 3-minute intervals between each measurement. The average of the last 3 measurements was taken as the rat's TWL value. The measurement time points were the same as those for the mean time between heat stimulation (MWT). The results are shown in […]. Figure 5 .

[0065] Figure 5 The results showed that there were no significant differences among the groups 1 day before transplantation (P>0.05); 7 days after transplantation, the pretreated BMSC group was significantly higher than the model control group (P<0.05) and the untreated BMSC group (P<0.05); 14 days after transplantation, the pretreated BMSC group was significantly different from the model control group (P<0.01); 21 days after transplantation, the pretreated BMSC group was significantly higher than the untreated BMSC group (P<0.05) and close to the sham surgery group.

[0066] The above results indicate that mesenchymal stem cells induced by VEGF-A-link-TNF-α fusion protein can increase the mechanical withdrawal reflex threshold and prolong the thermal withdrawal latency in CCI rats; and can delay the formation of neuropathic pain in rats.

[0067] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A VEGF-A-link-TNF-α fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown as SEQ ID NO: 4, which is used to induce mesenchymal stem cells to secrete a combination of TGF-β and IL-10 anti-inflammatory factors.

2. The fusion protein of claim 1, wherein, The fusion protein is prepared by a mammalian expression system and purified by Ni-NTA affinity chromatography and gel filtration chromatography.

3. A method for inducing mesenchymal stem cells to secrete TGF-β and IL-10, comprising co-culturing mesenchymal stem cells with the fusion protein of claim 1 or 2, collecting the culture supernatant and extracting a combination of anti-inflammatory factors.

4. Bone marrow mesenchymal stem cells pretreated by VEGF-A-link-TNF-α fusion protein, characterized in that, The amino acid sequence of the fusion protein is shown as SEQ ID NO: 4, the pretreatment is to expand the cells to the 3rd-5th generation at 37℃, 5% CO2 using DMEM / F12 medium containing 10% fetal bovine serum (FBS); the bone marrow mesenchymal stem cells are inoculated in a 6-well plate at a density of 1×10 6 cells / well, when the cell confluence reaches 80%, the serum-free medium is replaced, the VEGF-A-link-TNF-α fusion protein of claim 1 is added at a working concentration of 10 ng / mL, and co-cultured for 48 hours; the fusion protein pretreated bone marrow mesenchymal stem cells are washed twice with serum-free DMEM / F12 medium and resuspended into a cell suspension of 5×10 6 cells / mL.

5. A pain treatment preparation comprising the bone marrow mesenchymal stem cells pretreated with the VEGF-A-link-TNF-α fusion protein of claim 4.

6. Use of the bone marrow mesenchymal stem cells of claim 4 or the preparation of claim 5 in the preparation of a pharmaceutical product for treating pain caused by chronic constriction injury (CCI) of the sciatic nerve.

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