Application of mesenchymal stem cells (MSC) in treatment of lower limb vein ischemia

Abstract

The invention relates to the technical field of stem cells, and particularly discloses application of mesenchymal stem cells (MSC) in treatment of lower limb vein ischemia. According to the application, the SDF1-FGF2 fusion protein is used for pretreating human mesenchymal stem cells (hMSCs), and the cell activity, the blood flow recovery capacity and the anti-inflammatory effect of the hMSCs in a lower limb vein ischemia model can be remarkably improved through pretreatment. The hMSCs pretreated by SDF1-FGF2 are injected into the body of a venous ischemia model mouse, and compared with untreated cells, the hMSCs have better effects in the aspects of improving blood perfusion, reducing inflammatory response, recovering motor function and the like. The method provides an efficient stem cell treatment strategy for vascular diseases such as lower limb vein ischemia.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell bioengineering and regenerative medicine, and more specifically, to an application of mesenchymal stem cells (MSCs) in the treatment of lower limb venous ischemia. Background Art

[0002] Lower limb venous ischemia is tissue hypoperfusion caused by venous return disorders, often leading to tissue hypoxia, edema, pain, and dysfunction. Clinical treatment methods mainly include anticoagulation, improving circulation, or surgical intervention, but the curative effect is limited, and it is difficult to reverse tissue damage in patients with severe ischemia. Mesenchymal stem cells (MSCs) have become an important research direction for the treatment of vascular diseases due to their immunomodulatory and angiogenesis-promoting abilities. However, the survival ability and effector functions of untreated MSCs in the ischemic microenvironment are limited, and the treatment effect is not yet ideal. Therefore, it is of great significance to develop a pretreatment strategy to improve the therapeutic efficacy of MSCs.

[0003] Currently, there have been studies attempting to improve the survival rate and function of stem cells in ischemic tissues by local injection of growth factors or genetically engineered stem cells. For example, single factors such as VEGF and bFGF are used to promote angiogenesis. However, single-factor stimulation often has problems such as insufficient potency, short duration, or limited cell response in a complex ischemic environment. At the same time, certain growth factors may induce abnormal angiogenesis at high concentrations, increasing the safety risk. Therefore, there is an urgent need to develop a more effective and stable multi-target activation strategy to enhance the comprehensive role of stem cells in ischemic repair.

[0004] SDF1 (stromal cell-derived factor 1) and FGF2 (fibroblast growth factor 2) play key roles in inflammatory chemotaxis, stem cell homing, and angiogenesis, respectively. Existing literature has shown that SDF1 can activate the CXCR4 pathway to enhance stem cell migration and localization, while FGF2 enhances cell proliferation and differentiation potential through the FGFR signal. However, it is the first time to systematically use the fusion protein constructed by combining the two for stem cell pretreatment to enhance its therapeutic effect in a venous ischemia model, and the related mechanisms and application values have not been fully studied and confirmed. Summary of the Invention

[0005] The purpose of the present invention is to solve the technical problem of insufficient bioactivity and limited effect of mesenchymal stem cells in the treatment of lower limb venous ischemia in the prior art, and to provide a new application method based on SDF1-FGF2 fusion protein pretreatment of mesenchymal stem cells to improve its therapeutic effect on venous ischemia.

[0006] To achieve the above object, the present invention provides a use of mesenchymal stem cells in the preparation of a drug for treating lower limb venous ischemia. In a preferred embodiment, the mesenchymal stem cells are pretreated with the SDF1-FGF2 fusion protein before use.

[0007] The present invention also provides a method for constructing the fusion protein, which is composed of human SDF1 and FGF2 repeatedly linked by a polypeptide linker (GGGGS), and has good biological stability and functional synergistic effect.

[0008] In a preferred embodiment, the concentration of the SDF1-FGF2 fusion protein is 200 ng / mL, and the pretreatment time for hMSCs is 24 hours.

[0009] In a preferred embodiment, through in vitro CCK-8 detection, laser speckle flow imaging, ELISA inflammation detection, treadmill function evaluation and other means, it is verified that hMSCs pretreated with the SDF1-FGF2 fusion protein show higher cell activity, stronger blood flow recovery ability, lower inflammatory factor levels and better exercise endurance performance in a mouse model of venous ischemia, which is significantly better than the untreated group and the PBS-treated group.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention constructs SDF1 and FGF2 into a fusion protein for the first time and uses it for the pretreatment of hMSCs, significantly improving the effect of stem cell therapy for venous ischemia; this method can significantly enhance cell activity, promote blood perfusion and improve functional recovery, and is expected to shorten the treatment cycle; the application method is safe and simple, has good clinical transformation potential, and provides a new solution for the stem cell treatment of severe lower extremity venous diseases. Description of the Drawings

[0011] Figure 1 SDS-PAGE detection of the SDF1-FGF2 fusion protein.

[0012] Figure 2 Effect of SDF1-FGF2 pretreatment on the cell activity of hMSCs.

[0013] Figure 3 Laser speckle flow imaging (LSCI) detection and analysis.

[0014] Figure 4 ELISA detection of serum inflammation level. Detailed Description of the Invention

[0015] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0016] Example 1. Construction of the SDF1-linker-FGF2 fusion protein The amino acid sequence of stromal cell-derived factor 1 [Homo sapiens] recorded in the NCBI database, NCBI Reference Sequence: NP_001264919.1, is as follows (the signal peptide is truncated): KPVSLSYRCPCRFFESHYCTCLIRVSFHGATPLTQGSWVLYSLSCAGGETGLREPGPMVSPRVESHQEGRLGVPGPVNLGKA (SEQ ID NO:1); The amino acid sequence corresponding to fibroblast growth factor 2 [Homo sapiens] recorded in the NCBI database, NCBI Reference Sequence: NP_001997.5, is as follows: MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO:2); The amino acid sequence of the SDF1-linker-FGF2 (abbreviated as SDF1-FGF2) fusion protein is as follows: KPVSLSYRCPCRFFESHYCTCLIRVSFHGATPLTQGSWVLYSLSCAGGETGLREPGPMVSPRVESHQEGRLGVPGPVNLGKAGGGGSGGGGSGGGGSGGGGSMVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO:3); Based on the amino acid sequence of the SDF1-linker-FGF2 fusion protein, the nucleotide sequence was obtained by codon optimization: The expression vector of pET-28a-SDF1-FGF2 with HIS tag was provided by GenScript Biotech Corporation. The recombinant plasmid pET-28a-SDF1-FGF2 determined by sequencing was transferred into Escherichia coli BL21(DE3) competent cells, inoculated into LB liquid medium (20 mL) containing kanamycin, and cultured overnight at 37 °C. The next day, the bacteria were inoculated at a ratio of 1:100. When the OD600 nm reached 0.4 - 0.6, 1.0 mmol / L IPTG was added, and induction was carried out at low temperature overnight at 16 °C. The cells were collected by centrifugation at 7 500 r / min for 10 min; resuspended with PBS, centrifuged at 7 500 r / min for 10 min, and washed 3 times; the cells were lysed by ultrasound, and the supernatant was collected by centrifugation at 7 500 r / min for 10 min; the precipitate was resuspended with inclusion body Binding Buffer, left standing at 4 °C for 2 h, then the cells were lysed by ultrasound, centrifuged at 7 500 rpm for 10 min, and the supernatant and precipitate were collected. The supernatant protein was filtered through a 0.45 μM filter tube into a new tube; 2 mL of Ni-agarose was added to the affinity chromatography column, and 10 times the volume of Binding buffer (PBS, NaCl, 20 mmol / L imidazole, pH 8.0) was added for column equilibration. After 30 min, it was discarded; the supernatant protein was added to the column, placed at 4 °C for 4 h, and passed through the column into a new tube; the liquid passing through the column was loaded onto the column again for binding, left standing at 4 °C for 2 h, and then the supernatant was discarded; 10 times the volume of Binding buffer was added for washing; Eluction buffer (PBS, NaCl, imidazole, pH 8.0) was added for elution, 3 mL each time for 3 times, and the finally eluted protein was passed through the column again, the protein was collected, stored at -80 °C, and detected by SDS-PAGE, as shown in Figure 1 。

[0017] Figure 1 The results showed that the molecular weight of the SDF1-FGF2 fusion protein was about 40.88 kDa, which was in line with expectations.

[0018] Example 2. Pretreatment of human mesenchymal stem cells with SDF1-GF2 fusion protein The human mesenchymal stem cells (hMSCs) used in this example were purchased from Lonza Group Ltd. (Catalog No. PT-2501). The cells were stored and transported in liquid nitrogen and thawed according to the supplier's instructions. The cells were seeded in a T-75 cell culture flask with Mesenchymal Stem Cell Growth Medium (MSCGM™, Lonza, Catalog No. PT-3001) and cultured routinely in an incubator at 37°C with 5% CO2. When the cell confluence reached 80%, the cells were passaged at a ratio of 1:3 and digested with 0.05% trypsin-EDTA (Gibco) for 5 minutes. Only the cells of passages 3-5 were used for the experiment to ensure the proliferation and differentiation ability of the stem cells.

[0019] Experimental group: The SDF1-FGF2 fusion protein was prepared by the method described in Example 1. Before pretreatment, the fusion protein was diluted to 200 ng / mL. hMSCs were seeded in a 6-well plate at a density of 1×10 5 cells per well. After 24 hours of adhesion, the medium was replaced with serum-free MSC medium (FBS and other growth factors were removed from the MSCGM basal medium), and the corresponding concentration of SDF1-FGF2 fusion protein was added. The cells were incubated for another 24 hours. After the treatment, the cells were collected and labeled as SDF1-FGF2 pretreated hMSCs.

[0020] Control group: Before pretreatment, hMSCs were seeded in a 6-well plate at a density of 1×10 5 cells per well. After 24 hours of adhesion, the medium was replaced with serum-free MSC medium (FBS and other growth factors were removed from the MSCGM basal medium), and the corresponding volume of PBS was added. The cells were incubated for another 24 hours. After the treatment, the cells were collected and labeled as PBS pretreated hMSCs.

[0021] The SDF1-FGF2 pretreated hMSCs and PBS pretreated hMSCs were digested with trypsin and counted respectively, and then resuspended in serum-free medium. The cells were seeded in a 96-well plate at a density of 5000 cells per well with 6 replicates. 100 μL of serum-free culture medium was added to each well and incubated in an incubator at 37°C with 5% CO2 for 24 hours. 10 μL of CCK-8 reagent was added to each well and incubated for another 2 hours. The absorbance (OD450) value was measured at a wavelength of 450 nm using a microplate reader to represent cell viability, as shown in Figure 2 .

[0022] Figure 2The results showed that SDF1-FGF2 pretreatment significantly improved the cell viability of hMSCs (p<0.05).

[0023] Example 3. Application of hMSCs pretreated differently in a mouse model of lower limb venous ischemia Male C57BL / 6 mice, 8 - 10 weeks old, weighing 20 - 25 g, were divided into 3 groups: Sham group: Only expose the femoral vein without ligation.

[0024] Model group: After venous ischemia, no treatment was given.

[0025] PBS pretreatment group: After venous ischemia, hMSCs pretreated with PBS were injected via the tail vein (1×10 6 cells / mouse).

[0026] SDF1-FGF2 pretreatment group: After venous ischemia, hMSCs pretreated with SDF1-FGF2 were injected via the tail vein (1×10 6 cells / mouse).

[0027] Method for constructing the lower limb venous ischemia model: The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg). After shaving the hair in the operative area, it was disinfected with iodophor. The skin was incised longitudinally along the inner thigh, and the main trunk and branches (saphenous vein) of the femoral vein were bluntly dissected; the proximal and distal ends of the femoral vein were ligated doubly with 8-0 nylon thread, and the vein segment between the two ligation points (about 3 mm in length) was excised to completely block venous return. The muscle and skin were sutured layer by layer. The mice were placed on a 37°C warming pad for resuscitation, and penicillin (50,000 units / kg) was injected subcutaneously for 3 consecutive days to prevent infection. Obvious swelling and cyanosis of the affected limb within 24 h after surgery indicated venous return obstruction, suggesting successful construction of the lower limb venous ischemia model.

[0028] Laser speckle contrast imaging (LSCI) detection and analysis: Prepare the PeriCam PSI HR (Perimed, Sweden) imaging instrument and adjust the laser wavelength to 785 nm; the imaging frequency is 20 Hz, and the single acquisition time is 5 seconds; the spatial resolution is 100 μm / pixel; the contrast algorithm is time laser speckle contrast analysis (tLASCA); the environmental temperature is room temperature 25 ± 1 °C, and the mouse imaging platform is preheated to 37 °C; during postoperative detection, isoflurane inhalation anesthesia is used (induction 4%, maintenance 1.5%) to avoid the interference of pentobarbital sodium residue on blood flow; the mouse is fixed in the supine position, and the affected limb is abducted and laid flat on a black background board, and the ischemic area (distal to the femoral vein ligation) and the control area (contralateral normal limb) are marked. After shaving the surgical area, apply depilatory cream (Veet), wait for 5 minutes and then wipe with clean water to ensure that there is no hair interference on the skin surface; the laser probe is vertically 15 cm away from the limb, and the focal length is adjusted until the blood vessel texture is clear. Continuously acquire 3 groups of dynamic images (5 seconds for each group), and the software automatically eliminates the motion artifact frames, and the average blood perfusion value (PU) is taken. Detection time: 0 hours (baseline), 7 days, and 14 days after surgery. Among them, for between-group comparison: one-way analysis of variance (ANOVA) and Tukey multiple tests; significant markers: *p < 0.05 (vs. model group), #p < 0.05 (vs. PBS pretreatment group), see Figure 3 . The calculation method of blood recovery rate is as follows: Figure 3 The results showed that the blood flow recovery rate of the model group was only 25.7% at 14 days after surgery, which was significantly lower than that of the sham operation group, indicating that the venous ischemia model was successful and the self-repair ability was limited; the recovery rate of the PBS pretreatment group reached 49.2% at 14 days after surgery, which was 23.5% higher than that of the model group (p < 0.05), suggesting that the basic treatment with hMSCs was effective. The recovery rate of the SDF1-FGF2 pretreatment group at 7 days after surgery (57.9%) had exceeded the level of the PBS group at 14 days after surgery (49.2%), and the recovery rate at 14 days reached 83.5%, approaching the normal level, which was 34.3% higher than that of the PBS group (#p < 0.05); this indicated that the SDF1-FGF2 pretreatment improved the vascular regeneration efficiency of hMSCs, providing a basis for shortening the treatment cycle of venous ischemia.

[0029] Method for detecting serum inflammatory level by ELISA: On the 3rd day after surgery (inflammatory peak period), after anesthesia, 1 mL of whole blood was collected from each group of mice by cardiac puncture, left standing at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes (4°C), the serum was separated, aliquoted and stored at -80°C, avoiding repeated freezing and thawing. Detection was performed according to the instructions of the TNF-α detection kit (Invitrogen, Mouse TNF alpha ELISA Kit, catalog number BMS607-3) and the IL-6 (Invitrogen™ Mouse IL-6 ELISA Kit, Invitrogen™ KMC0062) detection kit, see Figure 4 .

[0030] Figure 4 The results showed that TNF-α and IL-6 in the model group were significantly higher than those in the sham operation group (*p < 0.05), indicating that venous ischemia induced a systemic inflammatory response. TNF-α and IL-6 in the PBS pretreatment group decreased by 25.8% and 35.2% compared with the model group (**p < 0.01 vs. model group), suggesting that hMSCs had a basic anti-inflammatory effect. TNF-α and IL-6 in the SDF1-FGF2 pretreatment group further decreased by 49.9% and 53.3% compared with the PBS group (##p < 0.01), indicating that pretreatment with the SDF1-FGF2 fusion protein significantly enhanced the inflammatory regulation ability of hMSCs. SDF1-FGF2 may upregulate the secretion of anti-inflammatory factors TNF-α and IL-6 by activating the CXCR4 / FGFR1 pathway of hMSCs.

[0031] Treadmill endurance test method: Electric treadmill for animals (Columbus Instruments, model EXER-6); initial speed 5 cm / s, increasing by 1 cm / s every 1 minute, maximum speed 25 cm / s; tail electrical stimulation fence (0.3 mA, stimulation interval 10 seconds), only used for driving, not the main power source; room temperature 25 ± 1 °C, light intensity 50 lux, fasting for 4 hours before the test; pre-test adaptation training: from the 10th to 13th day after surgery, perform 10-minute low-intensity running training every day (speed 5 cm / s, without electrical stimulation) to eliminate the fear of the new environment in mice. Test procedure (14th day after surgery): Place the mice on the stationary runway for 5 minutes to adapt to the environment. Start at a speed of 5 cm / s and increase by 1 cm / s every 1 minute, and record the following parameters: Maximum tolerable speed: The speed at which the mice cannot return to the runway continuously for 3 times (accurate to 1 cm / s). Total duration: The time from start to exhaustion of the mice (in minutes). Exhaustion criterion: The mice cannot continue running when their tails touch the electrical stimulation fence for more than 5 seconds. Termination condition: Reach the maximum speed of 25 cm / s and maintain it for 2 minutes, or the mice are exhausted; immediately provide glucose water (5%) to supplement energy after the test. Statistical method: One-way analysis of variance (ANOVA) and Tukey multiple tests, significance threshold: *p < 0.05 (vs. model group), ##p < 0.01 (vs. PBS pretreatment group); data standardization: Exclude the data of mice that actively jump and escape from the runway during the test (≤1 mouse is excluded from each group), see Table 1.

[0032] Table 1 Results of treadmill endurance test of mice in each group

[0033] Table 1 results show that the maximum running speed and duration of the model group are significantly lower than those of the sham operation group, indicating that venous ischemia leads to severe motor dysfunction; the maximum speed and duration of the PBS pretreatment group are increased compared with the model group (*p < 0.05), proving that hMSCs promote muscle function recovery; the maximum speed and duration of the SDF1-FGF2 pretreatment group are close to the level of the sham operation group and are increased compared with the PBS group ( ## p < 0.01), showing that pretreatment with SDF1-FGF2 fusion protein significantly enhances the repair efficacy of hMSCs. The treadmill test quantifies the conversion effect from "ischemic compensation" to "functional recovery" and provides a dynamic index for evaluating the clinical value of cell therapy.

[0034] The above is the preferred implementation mode of the present invention. It should be pointed out that for those of ordinary skill in the art in this technical field, without departing from the principle described in the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of mesenchymal stem cells in the preparation of a medicament for treating lower limb venous ischemia, characterized in that, The mesenchymal stem cells are pretreated with SDF1-FGF2 fusion protein before use.

2. The use according to claim 1, wherein the amino acid sequence of the SDF1-FGF2 fusion protein is as shown in SEQ ID NO:

3.

3. The use according to claim 1 or 2, wherein the pretreatment concentration of the SDF1-FGF2 fusion protein is 200 ng / mL and the treatment time is 24 hours.

4. The use according to any one of claims 1 to 3, wherein the mesenchymal stem cells are human mesenchymal stem cells (hMSCs).

5. The use according to any one of claims 1 to 4, wherein the drug is used to treat lower limb venous ischemia by intravenous injection.

6. The use according to any one of claims 1 to 5, wherein the pretreated hMSCs can significantly increase the blood perfusion rate, reduce the level of inflammatory factors, and improve motor function.

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