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

Abstract

The present invention relates to the field of stem cell technology and specifically discloses an application of mesenchymal stem cells (MSCs) for the treatment of lower extremity venous ischemia. This application involves pre-treating human mesenchymal stem cells (hMSCs) with the SDF1-FGF2 fusion protein. This pre-treatment significantly enhances the cell viability, blood flow recovery, and anti-inflammatory effects of hMSCs in a lower extremity venous ischemia model. When hMSCs pre-treated with SDF1-FGF2 were injected into mice modeling venous ischemia, they demonstrated superior effects compared to untreated cells in improving blood perfusion, reducing inflammatory responses, and restoring motor function. This method provides a highly effective stem cell therapy strategy for vascular diseases such as lower extremity venous 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 treating lower limb venous ischemia. Background Art

[0002] Lower extremity venous ischemia is caused by impaired venous return, resulting in inadequate tissue perfusion and often leading to tissue hypoxia, edema, pain, and functional impairment. Clinical treatments primarily rely on anticoagulation, circulatory improvement, or surgical intervention, but these approaches have limited efficacy and are difficult to reverse in patients with severe ischemia. Mesenchymal stem cells (MSCs) have become an important research focus in the treatment of vascular diseases due to their immunomodulatory and angiogenic abilities. However, untreated MSCs have limited survival and effector functions in the ischemic microenvironment, resulting in suboptimal therapeutic effects. Therefore, developing preconditioning strategies to enhance the therapeutic efficacy of MSCs is crucial.

[0003] Current studies have attempted to improve the survival and function of stem cells in ischemic tissues by locally injecting growth factors or genetically engineered stem cells, such as using single factors such as VEGF and bFGF to promote angiogenesis. However, single-factor stimulation often suffers from insufficient efficacy, short duration, or limited cellular response in complex ischemic environments. Furthermore, certain growth factors may induce abnormal angiogenesis at high concentrations, increasing safety risks. Therefore, there is an urgent need to develop more effective and stable multi-target activation strategies 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. Previous studies have shown that SDF1 activates the CXCR4 pathway to enhance stem cell migration and localization, while FGF2 enhances cell proliferation and differentiation through FGFR signaling. However, the combined construction of a fusion protein and its systematic application in stem cell preconditioning to enhance their therapeutic efficacy in a venous ischemia model is unprecedented, and the underlying mechanisms and potential applications remain understudied and unproven. Summary of the Invention

[0005] The present invention aims to solve the technical problems of insufficient biological activity and limited effect of mesenchymal stem cells in the treatment of lower limb venous ischemia in the prior art, and provides a new application method based on pre-treatment of mesenchymal stem cells with SDF1-FGF2 fusion protein to improve their efficacy in treating venous ischemia.

[0006] To achieve the above-mentioned object, the present invention provides a use of mesenchymal stem cells in the preparation of a drug for treating lower limb venous ischemia.

[0007] In a preferred embodiment, the mesenchymal stem cells are pre-treated with SDF1-FGF2 fusion protein before use.

[0008] The present invention also provides a method for constructing the fusion protein. The fusion protein is composed of human SDF1 and FGF2 repeatedly connected by a polypeptide linker (GGGGS), and has good biological stability and functional synergistic effects.

[0009] 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.

[0010] In a preferred embodiment, hMSCs pretreated with SDF1-FGF2 fusion protein showed higher cell activity, stronger blood flow recovery ability, lower inflammatory factor levels and better exercise endurance performance in a venous ischemia mouse model, as verified by in vitro CCK-8 detection, laser speckle blood flow imaging, ELISA inflammation detection and treadmill function evaluation, which was significantly better than the untreated group and the PBS-treated group.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects:

[0012] This invention constructs SDF1 and FGF2 into a fusion protein for the first time and uses it for hMSCs pretreatment, significantly improving the effect of stem cell treatment of 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, with good clinical translation potential, providing a new solution for stem cell treatment of severe lower limb venous diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 SDS-PAGE detection of SDF1-FGF2 fusion protein.

[0014] Figure 2 Effect of SDF1-FGF2 pretreatment on the cell activity of hMSCs.

[0015] Figure 3 Laser speckle flow imaging (LSCI) detection and analysis.

[0016] Figure 4 Serum inflammation levels were detected by ELISA. DETAILED DESCRIPTION

[0017] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0018] Example 1. Construction of SDF1-linker-FGF2 fusion protein

[0019] The amino acid sequence of stromal cell-derived factor 1 [Homo sapiens], NCBI Reference Sequence: NP_001264919.1, recorded in the NCBI database is as follows (truncated signal peptide):

[0020] KPVSLSYRCPCRFFESHYCTCLIRVSFHGATPLTQGSWVLYSLSCAGGETGLREPGPMVSPRVESHQEGRLGVPGPVNLGKA (SEQ ID NO:1);

[0021] The NCBI database records fibroblast growth factor 2 [Homo sapiens], NCBI Reference Sequence: NP_001997.5, and the corresponding amino acid sequence is as follows:

[0022] MVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGGRGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALP EDGGSGAFPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ IDNO:2);

[0023] The amino acid sequence of the SDF1-linker-FGF2 (abbreviated as SDF1-FGF2) fusion protein is as follows:

[0024] KPVSLSYRCPCRFFESHYCTCLIRVSFHGATPLTQGSWVLYSLSCAGGETGLREPGPMVSPRVESHQEGRLGVPGPVNLGKAGGGGSGGGGSGGGGSGGGGSMVGVGGGDVEDVTPRPGGCQISGRGARGCNGIPGAAAWEAALPRRRPRRHPSVNPRSRAAGSPRTRGRRTEERPSGSRLGDRGRGRALPGGRLGG RGRGRAPERVGGRGRGRGTAAPRAAPAARGSRPGPAGTMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (SEQ ID NO:3);

[0025] According to the amino acid sequence of the SDF1-linker-FGF2 fusion protein, the codons were optimized to obtain the nucleotide sequence:

[0026]

[0027] GenScript Biotech Co., Ltd. provided the HIS-tagged pET-28a-SDF1-FGF2 expression vector. The recombinant plasmid pET-28a-SDF1-FGF2, confirmed by sequencing, was transformed into competent Escherichia coli BL21(DE3) cells and inoculated into 20 mL of LB liquid medium containing kanamycin. The cells were cultured overnight at 37°C. The next day, the cells 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 the cells were induced overnight at 16°C. The cells were then harvested by centrifugation at 7500 rpm for 10 minutes, resuspended in PBS, and washed three times by centrifugation at 7500 rpm for 10 minutes. The cells were lysed by sonication and centrifuged at 7500 rpm for 10 minutes, and the supernatant was collected. The pellet was resuspended in Inclusion Body Binding Buffer, incubated at 4°C for 2 hours, lysed by sonication, and centrifuged at 7500 rpm for 10 minutes. The supernatant and pellet 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 volumes of Binding buffer (PBS, NaCl, 20 mmol / L imidazole, pH 8.0) were added to the column for equilibration and discarded after 30 min; the supernatant protein was added to the column, incubated at 4°C for 4 h, and passed through the column into a new tube; the liquid that passed through the column was re-applied to the column for binding, incubated at 4°C for 2 h, and the supernatant was discarded; 10 volumes of Binding buffer were added for washing; and 3 washes were performed with Eluction buffer (PBS, NaCl, imidazole, pH 8.0) for elution, 3 times with 3 mL each time. Finally, the eluted protein was passed through the column again, the protein was collected, and stored at -80°C. It was detected by SDS-PAGE, see. Figure 1 .

[0028] Figure 1 The results showed that the molecular weight of the SDF1-FGF2 fusion protein was 40.88 kDa, which was in line with expectations.

[0029] Example 2: Pretreatment of human mesenchymal stem cells with SDF1-GF2 fusion protein

[0030] The human mesenchymal stem cells (hMSCs) used in this example were purchased from Lonza (Lonza Group Ltd., Catalog No. PT-2501). The cells were stored and shipped in liquid nitrogen and revived according to the supplier's instructions. The cells were seeded in T-75 cell culture flasks in Mesenchymal Stem Cell Growth Medium (MSCGM™, Lonza, Catalog No. PT-3001) and cultured in a 37°C, 5% CO2 incubator for routine culture. When the cells reached 80% confluency, they were passaged at a 1:3 ratio and digested with 0.05% trypsin-EDTA (Gibco) for 5 minutes. Only cells from passages 3-5 were used in experiments to ensure the proliferation and differentiation capacity of the stem cells.

[0031] Experimental group: 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 6-well plates, with 1×10 5 After 24 hours of attachment, the medium was changed to serum-free MSC culture medium (MSCGM basal culture medium without FBS and other growth factors), 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.

[0032] Control group: Before pretreatment, hMSCs were seeded in 6-well plates at 1×10 5 After 24 hours of attachment, the medium was changed to serum-free MSC culture medium (MSCGM basal culture medium without FBS and other growth factors), 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.

[0033] hMSCs pretreated with SDF1-FGF2 and hMSCs pretreated with PBS were trypsinized and counted, and then resuspended in serum-free medium. The cells were seeded in a 96-well plate with 5,000 cells per well and 6 replicates were set up. 100 μL of serum-free culture medium was added to each well and incubated in a 37°C, 5% CO2 incubator 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 read at a wavelength of 450 nm using a microplate reader, which represents cell viability. Figure 2 .

[0034] Figure 2The results showed that SDF1-FGF2 pretreatment significantly increased the cell activity of hMSCs (p<0.05).

[0035] Example 3: Application of hMSCs after different pretreatments in the treatment of lower limb venous ischemia mouse model

[0036] 8-10 week old C57BL / 6 male mice weighing 20-25 g were used and divided into 3 groups:

[0037] Sham operation group: only the femoral vein was exposed without ligation.

[0038] Model group: No treatment was given after venous ischemia.

[0039] PBS pretreatment group: hMSCs (1×10 6 cells / cells).

[0040] SDF1-FGF2 pretreatment group: hMSCs (1×10 6 cells / cells).

[0041] Methods for establishing a lower limb venous ischemia model: Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg / kg). The surgical area was shaved and disinfected with iodine. A longitudinal skin incision was made along the medial thigh, and the femoral vein trunk and its branches (saphenous vein) were bluntly dissected. The proximal and distal ends of the femoral vein were double-ligated with 8-0 nylon suture, and the vein segment between the two ligatures (approximately 3 mm in length) was cut to completely block venous return. The muscle and skin were sutured layer by layer. Mice were placed on a 37°C warming pad for recovery, and penicillin (50,000 units / kg) was administered subcutaneously for 3 consecutive days to prevent infection. Within 24 hours after surgery, significant swelling and cyanosis of the affected limb appeared, indicating venous obstruction and confirming the successful establishment of a lower limb venous ischemia model.

[0042] Laser speckle blood flow imaging (LSCI) analysis: A PeriCam PSI HR (Perimed, Sweden) imaging instrument was used, with a laser wavelength of 785 nm. The imaging frequency was 20 Hz, with a single acquisition time of 5 seconds. The spatial resolution was 100 μm / pixel. The contrast algorithm used was temporal laser speckle contrast analysis (tLASCA). The ambient temperature was maintained at 25 ± 1°C, and the mouse imaging platform was preheated to 37°C. Postoperative anesthesia was performed using isoflurane inhalation (4% for induction and 1.5% for maintenance) to prevent residual sodium pentobarbital from interfering with blood flow. Mice were immobilized in the supine position, with the affected limb abducted and placed flat on a black background. The ischemic area (distal to the femoral vein ligation) and the control area (contralateral normal limb) were marked. The surgical area was shaved and depilatory cream (Veet) was applied. The area was left to rest for 5 minutes before being wiped with clean water to ensure that the skin surface was free of hair. The laser probe was held 15 cm vertically from the limb, and the focus was adjusted to ensure clear vascular texture. Three sets of dynamic images were acquired continuously (5 seconds per set). The software automatically removed frames with motion artifacts and averaged the blood perfusion value (PU). Testing time: 0 hour (baseline), 7 days, and 14 days after surgery. Inter-group comparisons were performed using one-way analysis of variance (ANOVA) and Tukey's multiple-test. Significance marks: *p<0.05 (vs. model group), #p<0.05 (vs. PBS pretreatment group). Figure 3 The blood recovery rate is calculated as follows:

[0043]

[0044] Figure 3 Results showed that 14 days after surgery, the blood flow recovery rate in the model group was only 25.7%, significantly lower than that in the sham-operated group, indicating that the venous ischemia model was successful but that autologous repair capacity was limited. The 14-day recovery rate in the PBS pretreatment group reached 49.2%, a 23.5% increase compared to the model group (p<0.05), suggesting that hMSC-based therapy is effective. The 7-day recovery rate in the SDF1-FGF2 pretreatment group (57.9%) exceeded the 14-day recovery rate in the PBS group (49.2%), and the 14-day recovery rate reached 83.5%, approaching normal levels and a 34.3% increase compared to the PBS group (#p<0.05). This suggests that SDF1-FGF2 pretreatment enhances the angiogenesis efficiency of hMSCs, providing evidence for shortening the treatment period for venous ischemia.

[0045] ELISA method for detecting serum inflammation levels: 3 days after surgery (peak inflammation), after anesthesia, 1 mL of whole blood was collected by cardiac puncture from mice in each group. The blood was allowed to stand at room temperature for 30 minutes and centrifuged at 3000 rpm for 15 minutes (4°C). The serum was separated, aliquoted, and stored at -80°C to avoid repeated freezing and thawing. The TNF-α detection kit (Invitrogen, Mouse TNF alpha ELISA Kit, Catalog No. BMS607-3) and IL-6 (Invitrogen™ Mouse IL-6 ELISA Kit, Invitrogen™ KMC0062) were used for detection according to the instructions of the detection kits, see [see TNF-α, Mouse TNF alpha ELISA Kit, Catalog No. BMS607-3]. Figure 4 .

[0046] Figure 4 Results showed that TNF-α and IL-6 levels in the model group were significantly higher than those in the sham group (*p<0.05), indicating that venous ischemia triggered a systemic inflammatory response. TNF-α and IL-6 levels 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 possess a basal anti-inflammatory effect. TNF-α and IL-6 levels in the SDF1-FGF2 pretreatment group further decreased by 49.9% and 53.3% compared with the PBS group (##p<0.01), indicating that SDF1-FGF2 fusion protein pretreatment significantly enhanced the inflammatory regulation capacity of hMSCs. SDF1-FGF2 may upregulate the secretion of anti-inflammatory cytokines TNF-α and IL-6 by activating the CXCR4 / FGFR1 pathway in hMSCs.

[0047] Treadmill endurance testing was performed on an animal-specific motorized treadmill (Columbus Instruments, model EXER-6). The initial speed was 5 cm / s, increasing by 1 cm / s every minute to a maximum speed of 25 cm / s. An electrical tail stimulation barrier (0.3 mA, 10-second intervals) was applied only for provocation and not the primary source of power. The room temperature was 25 ± 1°C, the light intensity was 50 lux, and the mice were fasted for 4 hours before testing. Pre-test acclimatization training was performed daily for 10 minutes at a low intensity (5 cm / s, no electrical stimulation) from days 10 to 13 after surgery to eliminate fear of the novel environment. Testing procedure (post-operative day 14): Mice were placed on a stationary track for 5 minutes to acclimate. The starting speed was 5 cm / s, increasing by 1 cm / s every minute. The following parameters were recorded: Maximum tolerable speed: the speed at which the mouse failed to return to the track three times in a row (accurate to the nearest 1 cm / s). Total duration: the time from the start of the test to exhaustion (minutes). Exhaustion criterion: the mouse was unable to continue running after its tail contacted the electrical stimulation barrier for more than 5 seconds. Termination criteria: reaching a maximum speed of 25 cm / s and maintaining it for 2 minutes, or reaching exhaustion; glucose solution (5%) was immediately provided for energy replenishment after the test. Statistical methods: One-way analysis of variance (ANOVA) with Tukey's multiple-test (TUT); significance thresholds: *p < 0.05 (vs. model group), ##p < 0.01 (vs. PBS pretreatment group); Data normalization: Data from mice that actively jumped off the runway during the test were excluded (≤ 1 mouse per group). See Table 1.

[0048] Table 1 Treadmill endurance test results of mice in each group

[0049]

[0050] The results in Table 1 show that the maximum running speed and duration of the model group were significantly lower than those of the sham operation group, indicating that venous ischemia caused severe motor dysfunction; the maximum speed and duration of the PBS pretreatment group were improved compared with the model group (*p<0.05), proving that hMSCs promoted muscle function recovery; the maximum speed and duration of the SDF1-FGF2 pretreatment group were close to the levels of the sham operation group and were improved compared with the PBS group ( ## The SDF1-FGF2 fusion protein pretreatment significantly enhanced the repair efficacy of hMSCs (p<0.01). Treadmill testing quantified the transition from ischemic compensation to functional recovery, providing a dynamic indicator for evaluating the clinical value of cell therapy.

[0051] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of mesenchymal stem cells in the preparation of a medicament for treating lower extremity venous ischemia, characterized in that: The mesenchymal stem cells are pretreated with SDF1-FGF2 fusion protein before use; the amino acid sequence of the SDF1-FGF2 fusion protein is shown in SEQ ID NO:

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

3. The use according to claim 1 or 2, wherein the mesenchymal stem cells are human mesenchymal stem cells (hMSCs).

4. The use according to claim 1 or 2, wherein the drug is used for treating lower limb venous ischemia by intravenous injection.

5. The use according to claim 1 or 2, wherein the pretreated hMSCs can significantly increase blood perfusion rate, reduce inflammatory factor levels, and improve motor function.

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