Application of GDF11 in treatment of corneal endothelial injury

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a growth differentiation factor 11 (GDF11) in preparation of a medicine for promoting corneal endothelial injury repair. The invention reveals that the GDF11 can effectively promote the repair of corneal tissues by enhancing the proliferation activity and migration ability of corneal endothelial cells and inhibiting cell apoptosis. Based on the discovery, the invention provides a new strategy and thought for the treatment of corneal endothelial related diseases, and has important clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of growth factor GDF11 in the treatment of corneal endothelial injury. Background Art

[0002] As a key barrier for maintaining corneal transparency, the corneal endothelial tissue precisely regulates the hydration state of the corneal stroma through its unique "pump-leak" mechanism, keeping the cornea transparent to maintain vision. It is worth noting that human corneal endothelial cells have significant terminal differentiation characteristics and enter the cell cycle quiescence state in the late stage of embryonic development. Their limited regenerative ability leads to an extremely easy occurrence of irreversible reduction in cell number in adult individuals when dealing with senile degeneration, pathological injury, or iatrogenic trauma. When the endothelial cell density is lower than the critical threshold, it will trigger decompensated pathological changes such as corneal edema and loss of transparency, ultimately resulting in severely impaired vision. At present, clinical practice mainly relies on penetrating keratoplasty for intervention. However, problems such as the severe shortage of donor corneas and the risk of transplant rejection globally make this treatment plan have significant limitations. Although the recently emerging stem cell induction and differentiation technology and in vitro expansion and transplantation therapy have shown certain application prospects, key technical indicators such as the functional integration efficiency, phenotypic stability, and long-term survival rate of transplanted cells in the corneal microenvironment still lack reliable data support.

[0003] Growth differentiation factor 11 (GDF11), as an important member of the TGF-β superfamily, is expressed in most tissues and organs. It shows extensive expression in multiple tissues during the embryonic development stage and participates in physiological activities such as injury repair, cell proliferation, and migration by activating the SMAD signaling pathway. In recent years, it has attracted extensive attention due to its dual roles in tissue regeneration and aging regulation.

[0004] However, there are still certain controversies about GDF11 at present, and its specific roles in some physiological activities vary depending on tissues and organs. For example, in terms of fibrosis, it shows pro-fibrotic activity in the renal fibrosis model, while it plays an anti-fibrotic role during myocardial repair, which can reduce myocardial fibrosis in mice with myocardial infarction and decrease the occurrence of ventricular hypertrophy. This contradictory functional characteristic suggests that GDF11 may participate in the homeostasis maintenance of different tissues through a differential signal regulation network.

[0005] Through bioinformatics analysis, the inventors found that (GSE41616 dataset) the expression level of GDF11 in fetal corneal endothelial tissue was significantly higher than that in adult individuals (P<0.01). This finding first revealed the potential association between the expression level of GDF11 and the proliferative potential of corneal endothelial cells, providing an important theoretical basis for exploring the application of GDF11 in corneal endothelial regenerative medicine.

[0006] The present invention clarifies that GDF11 can accelerate the repair of corneal endothelial injury by promoting corneal endothelial proliferation, migration and inhibiting its apoptosis, providing a new idea for the treatment of corneal endothelial diseases. Summary of the Invention

[0007] The present invention proves the role of GDF11 in corneal injury repair, indicating that GDF11 can become a new target for corneal injury repair.

[0008] To achieve the above invention object, the technical solutions provided by the present invention are as follows: The present invention provides the application of GDF11 in the preparation of a drug for promoting corneal endothelial injury repair.

[0009] In the present invention, the drug achieves corneal tissue repair by enhancing the proliferative activity, migration ability of corneal endothelial cells and / or inhibiting cell apoptosis.

[0010] In the present invention, the drug is applicable to the treatment of corneal diseases caused by corneal endothelial decompensation, Fuchs endothelial dystrophy or iatrogenic corneal endothelial injury.

[0011] In the present invention, the drug contains an effective dose of GDF11 protein or its functional active fragment, and a pharmaceutically acceptable carrier.

[0012] In the present invention, the administration concentration of the GDF11 protein is 10 - 200 ng / mL, and it is preferably administered to the affected area by intracameral injection or topical eye drop.

[0013] In the present invention, GDF11 promotes the repair of corneal endothelium by regulating the expression of SOX9.

[0014] In the present invention, the role of GDF11 also includes promoting the remodeling of the extracellular matrix.

[0015] In the present invention, the drug further contains an in vitro amplification and culture system for corneal endothelial cells, wherein GDF11 is used as a culture medium additive to enhance the survival rate and function of transplanted cells.

[0016] The present invention provides a pharmaceutical composition for the treatment of corneal endothelial regeneration, which is characterized by comprising a therapeutically effective amount of recombinant GDF11 protein, a corneal penetrant delivery vehicle, and a cell adhesion enhancer.

[0017] According to the present invention, the growth factor GDF11 protein can accelerate the repair of corneal endothelial injury by promoting corneal endothelial proliferation, migration and inhibiting its apoptosis, etc., providing a new idea for the treatment of corneal endothelial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Next, in the description of the drawings and embodiments, for convenience, the term "growth factor 11" is abbreviated as "GDF11"; the term "Adeno-associated Virus" is abbreviated as "AAV".

[0019] Figure 1 is a schematic diagram of corneal endothelial injury and GDF11 expression changes in mice according to an embodiment of the present invention; Figure 2 is a schematic diagram of overexpressing GDF11 by intravitreal injection of AAV according to an embodiment of the present invention; Figure 3 is a schematic diagram of the effect of GDF11 on corneal repair after cryoinjury according to an embodiment of the present invention; Figure 4 is a schematic diagram of the effect of GDF11 on cell proliferation, migration and apoptosis according to an embodiment of the present invention; Figure 5 is a schematic diagram of the effect of GDF11 on cell proliferation, migration and apoptosis according to another embodiment of the present invention; Figure 6 is a schematic diagram of the effect of GDF11 overexpression on the expression level of SOX9 protein according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Hereinafter, the present invention will be described in detail according to exemplary embodiments, but the present invention is not limited to these embodiments. The present invention is embodied in the following various forms, but should not be construed as limited to the exemplary embodiments set forth herein. Therefore, the detailed description and embodiments of the present invention will convey the scope of the present invention to those of ordinary skill in the art and be construed as being within the scope of the present invention.

[0021] According to the present invention, by preparing GDF11 protein as a drug for the treatment of corneal endothelial injury, specifically, the recombinant GDF11 protein can be expressed in Escherichia coli or mammalian cells by genetic engineering techniques and purified by methods such as affinity chromatography and gel filtration to finally obtain a high-purity GDF11 protein.

[0022] According to the present invention, the recombinant GDF11 protein can further be dissolved in physiological saline, and an appropriate amount of a pharmaceutically acceptable carrier, such as hypromellose, is added to prepare a drug solution with a concentration of 10 - 200 ng / mL, preferably 50 ng / mL. The drug is administered to the corneal injury model animals by intracameral injection or topical eye drops.

[0023] In addition, drug optimization is also feasible. For example, the recombinant GDF11 protein is mixed with a corneal penetrant delivery carrier (such as sodium hyaluronate) and a cell adhesion enhancer (such as fibronectin fragment) to prepare an optimized drug composition.

[0024] Example 1 Animal experiments have demonstrated that GDF11 can promote the repair of corneal endothelial injury.

[0025] Animal Experiment 1 Establishment of animal and corneal freezing model Establishment of animal and corneal freezing model: C57BL / 6J mice at 6 - 8 weeks of age were selected for the experiment and raised in the Medical Experimental Animal Center of Chinese PLA General Hospital. The culture conditions were temperature 23 ± 2°C, relative humidity 55 ± 5%, 12 - hour light cycle, and free access to food and water.

[0026] The corneal endothelial injury model was referred to the existing technology protocol.

[0027] The mice were intraperitoneally injected with tribromoethanol (concentration 2.5%, dose 15 μL / g) for general anesthesia, and then topical anesthesia was performed by instilling oxybuprocaine hydrochloride eye drops (concentration 0.4%) on the corneal surface.

[0028] A stainless - steel freezing probe with an outer diameter of 2 mm was selected. After being soaked in liquid nitrogen, the working end temperature was stabilized at - 80°C. Then, the pre - cooled probe was gently placed on the corneal surface of the mice without heavy pressure. After 4 s, the probe was removed from the corneal surface. The successful operation was marked by the formation and rapid disappearance of frost crystals on the corneal surface instantaneously. Antibiotic eye ointment (containing 0.3% ofloxacin) was applied to the corneal surface of the mice. After injury, the cornea was photographed using a slit lamp (SL - D7, Topcon) and the corneal thickness was quantitatively measured by anterior segment optical coherence tomography (OCT, Visante OCT Model 1000).

[0029] Animal Experiment 2: Over - expression of GDF11 in vitro Appendix Figure 2 A shows the process of intracameral injection of AAV for over - expression of GDF11.

[0030] Viral vector construction: An AAV2 / 9 serotype (titer 1×10^13 vg / mL) was used to construct a targeted gene delivery system, which was divided into: Experimental group / Overexpression of GDF11: AAV2 / 9-CMV-GDF11-3flag-Zsgreen (AAV-GDF11) Control group / Negative control: AAV2 / 9-CMV-Zsgreen empty vector (AAV-NC) After general anesthesia of the mice, compound tropicamide was instilled on the corneal surface to dilate the pupils. 2 μL was injected into the anterior chamber using a 36G ultra-fine needle microinjector. Four weeks after injection, detection was performed by in vivo imaging and immunoblotting.

[0031] Animal experiment 3: Corneal immunofluorescence After sacrificing the mice by cervical dislocation, the intact eyeballs were quickly removed and placed in pre-cooled PBS buffer (pH 7.4, containing 1% double antibiotics). The corneas were dissected from the eyeballs, and the intact limbal structure was retained. They were fixed at room temperature in 4% paraformaldehyde (PFA, Sigma-Aldrich) for 2 h (protected from light). They were permeabilized at room temperature with 0.2% Triton X-100 (containing 10% normal goat serum) by shaking for 2 h (shaking speed 50 rpm, TS-100, Beyotime). The corneas were placed in the corresponding antibodies and incubated overnight at 4°C. After washing the antibodies, they were incubated with the corresponding fluorescent secondary antibodies at room temperature for 2 h. The corneas were laid flat on glass slides and sealed with DAPI. After sample preparation, observation was performed using a laser confocal microscope.

[0032] Animal experiment 4: Quantitative PCR Total RNA was extracted using TRIzol™ Reagent and dissolved in enzyme-free water. It was reverse transcribed into cDNA using the iScript™ cDNA Synthesis kit. Quantitative PCR was performed in a PCR system using the iTaq™ Universal SYBR® Green Supermix kit. The mRNA expression of the samples was compared using method.

[0033] Results of animal experiments Appendix Figure 1 Shows the changes in mouse corneal endothelial injury and GDF11 expression. Figure 1 A Slit lamp microscopic imaging showed that the cornea presented characteristic pathological changes after cryointervention, including stromal layer edema and thickening and a significant decrease in transparency. Figure 1 B Immunofluorescence quantitative analysis confirmed that the expression intensity of GDF11 in corneal endothelial cells in the injury group was increased compared with the control group. Figure 1 C and 1D Immunofluorescence staining demonstrated that after corneal endothelial injury, the expression of the mesenchymalization markers α-smooth muscle actin (α-SMA) and fibronectin 1 (FN1) was upregulated.

[0034] Appendix Figure 2The situation of intravitreal injection of AAV overexpressing GDF11 is shown. Figure 2 B and C demonstrated that immunofluorescence staining was performed on samples taken 4 weeks after intravitreal injection of AAV. It was found that in the overexpression group injected with AAV-GDF11, compared with the control group AAV-NC, the expression of GDF11 increased, and there was no change in corneal transparency. Figure 2 D demonstrated the increase in mRNA expression of GDF11 in corneal endothelium of the overexpression group by quantitative PCR.

[0035] Appendix Figure 3 A and B demonstrated the frozen injury repair process. After intravitreal injection of AAV, a frozen injury model was established. In the overexpression group injected with AAV-GDF11, compared with the control group AAV-NC, the corneal transparency recovery time was significantly shortened. Figure 3 C and D measured corneal thickness using OCT and demonstrated that after modeling, due to endothelial injury, corneal edema occurred and corneal thickness increased. The corneal thickness in the overexpression group decreased faster. α-SMA positive cells are related to extracellular matrix remodeling. When the number of α-SMA positive cells decreases, cell migration and wound contraction during wound healing are inhibited. Figure 3 E and F demonstrated this by immunofluorescence staining. The expression of α-SMA in corneal endothelium of the overexpression group increased, which may indicate that GDF11 accelerated cell migration and the repair of damaged extracellular matrix. When GDF11 is overexpressed, the expression of α-SMA is upregulated, promoting extracellular matrix remodeling at the wound site, thereby accelerating wound contraction and cell migration, and thus accelerating the repair of corneal endothelial injury. When corneal transparency returns to normal, the expression of α-SMA in the endothelium begins to decrease. Figure 3 G and H demonstrated by immunofluorescence staining that the number of ki67+ cells in corneal endothelium of the overexpression group increased, indicating that GDF11 promoted the proliferation of corneal endothelial cells.

[0036] The above corneal freezing model experiments can show that by using AAV to overexpress GDF11 in the corneal endothelium of in vivo mice, it was found that GDF11 can promote the repair of corneal endothelial injury.

[0037] Example 2 Cell experiments demonstrated that GDF11 promotes corneal endothelial migration, proliferation and inhibits its apoptosis.

[0038] Cell experiment 1 Culture and transfection of corneal endothelial cell line The human corneal endothelial cell line B4G12 was cultured in high-glucose DMEM + 6% fetal bovine serum + 1% penicillin / streptomycin double antibody and placed in an incubator at 37°C and 5% CO2. The plasmid was used to overexpress GDF11 in cells by the transfection reagent Lipofectamine 3000, and GDF11 in cells was knocked down by siRNA using the transfection reagent RNAiMAX. PCR detection and immunofluorescence detection were performed 24 h after transfection, and protein expression verification (Western blot) was performed 48 h after transfection.

[0039] Cell experiment 2 Quantitative PCR The specific operation method was the same as that of animal experiment 4.

[0040] Cell experiment 3 Western Blot The proteins in the cells were extracted using ripa lysis buffer containing protease inhibitors. After quantifying the proteins using the BCA protein quantification kit, equal amounts of proteins were electrophoresed using a 10% SDS-PAGE gel, and the proteins on the gel were transferred to a nitrocellulose membrane using semi-dry transfer. Then the membrane was blocked with 1:15 casin solution at room temperature for 2 h. After diluting the primary antibody according to the instructions, the membrane was incubated overnight at 4°C. After washing off the excess primary antibody, the corresponding secondary antibody was used to incubate the membrane at room temperature for 2 h, and then developed and exposed using ECL developer.

[0041] Cell experiment 4 Cell scratch assay The cells were seeded in a six-well plate. After 24 h of transfection, a scratch was made in the center of the well plate using a 200 μL pipette tip. The scraped and fallen cells were washed away with PBS, and the cells were cultured in high-glucose DMEM + 1% FBS for 24 h and then photographed to observe the changes in the scratch.

[0042] Cell experiment 5 Edu cell proliferation assay The cells were seeded in a six-well plate. After 48 h of transfection, the cells were stained with the Edu cell proliferation kit and Dapi, and photographed using a fluorescence microscope to count the number of Edu-positive cells.

[0043] Results of cell experiments As Figure 4 shown in A, at the mRNA level, knocking down GDF11 could down-regulate the expression of cyclin D1 and MCL1, and at the same time up-regulate the expression of TP53; as Figure 4 shown in B and 4C, at the protein level, knocking down GDF11 could also down-regulate the expression of cyclin D1 and MCL1, and at the same time up-regulate the expression of TP53. These results indicate that GDF11 can promote cell proliferation and inhibit cell apoptosis.

[0044] As Figure 4As shown in Figures 4D and 4E, 24 hours after transfection, the migration rate of cells in the si-NC group was 0.90 ± 0.04, while that of cells in the si-GDF11 group was 0.54 ± 0.13, indicating that knockdown of GDF11 could significantly inhibit the migration ability of cells.

[0045] As Figure 4 shown in Figures 4F and 4G, 48 hours after transfection, cell proliferation was detected by EdU assay. The results showed that the proportion of positive cells in the si-NC group was 26.59 ± 4.13%, while that in the si-GDF11 group was 17.45 ± 1.86%, indicating that knockdown of GDF11 could significantly inhibit the proliferation ability of cells.

[0046] As Figure 5 shown in Figure 5A, at the mRNA level, overexpression of GDF11 by plasmid could up-regulate the expression of cyclin D1 and MCL1, and down-regulate the expression of TP53; as Figure 5 shown in Figures 5B and 5C, at the protein level, overexpression of GDF11 could also up-regulate the expression of cyclin D1 and MCL1, and down-regulate the expression of TP53. These results further confirmed that GDF11 could promote cell proliferation and inhibit apoptosis.

[0047] As Figure 5 shown in Figures 5D and 5E, 24 hours after transfection, the migration rate of cells in the OE-NC group was 0.34 ± 0.04, while that of cells in the OE-GDF11 group was 0.88 ± 0.11, indicating that overexpression of GDF11 could significantly promote the migration ability of cells.

[0048] As Figure 5 shown in Figures 5F and 5G, 48 hours after transfection, cell proliferation was detected by EdU assay. The results showed that the proportion of positive cells in the OE-NC group was 22.83 ± 2.25%, while that in the OE-GDF11 group was 33.90 ± 2.44%, indicating that overexpression of GDF11 could significantly promote the proliferation ability of cells.

[0049] In the above cell experiments, overexpression or knockdown of GDF11 proved that GDF11 could promote the migration and proliferation of corneal endothelium and inhibit apoptosis, thus promoting the repair of corneal endothelial injury.

[0050] Example 3 GDF11 promotes the repair of corneal endothelial injury by regulating SOX9 As shown in Figure 6 Figures 6A and 6C, it was found by Western Blot detection that when GDF11 in cells was knocked down, the expression level of SOX9 protein was significantly reduced.

[0051] Furthermore, as shown in Figure 6As shown in FIGS. B and 6D, after overexpressing GDF11 in cells, the expression level of SOX9 protein increased significantly.

[0052] In addition, as shown in Figure 6 FIG. E, after cryopreserving the corneas of mice 4 weeks after intravitreal injection of AAV by immunofluorescence detection, the results showed that the expression level of SOX9 protein increased in corneal endothelial cells overexpressing GDF11, and its expression time sequence was extended. Given that previous studies have shown that SOX9 is closely related to cell proliferation.

[0053] Therefore, GDF11 promotes cell proliferation by regulating the expression of SOX9 protein, thereby accelerating the repair process of corneal endothelial injury.

[0054] It should be understood that the exemplary embodiments described herein should be considered only in a descriptive sense and not as a limitation on the scope of the present invention. The description of the technical solutions and embodiments of the present invention is intended to provide a comprehensive understanding of the present invention, but should not be construed as limiting the present invention to these specific embodiments. Those skilled in the art can make appropriate modifications or adjustments to the described embodiments without departing from the spirit and scope of the present invention.

[0055] The description of the features or aspects in each embodiment should generally be considered applicable to similar features or aspects in other embodiments as well, unless it is explicitly stated that it is not feasible. The specific technical means, parameter selections or experimental conditions mentioned in the present invention are only for illustrative purposes and should not be regarded as the sole limitation of the technical solutions of the present invention. Without departing from the basic principles of the present invention, those skilled in the art can replace, combine or optimize these features or aspects to achieve other specific embodiments of the present invention.

[0056] In addition, some technical features or operation steps mentioned in the present invention may not be described in detail in some embodiments, but this does not mean that these features or steps are not available or inapplicable in other embodiments. On the contrary, those skilled in the art can flexibly select and apply these technical features or operation steps according to specific needs and technical backgrounds to achieve the intended purpose of the present invention.

Claims

1. Application of growth differentiation factor 11 (GDF11) in the preparation of a drug for promoting the repair of corneal endothelial damage.

2. The use according to claim 1, characterized in that The drug achieves corneal tissue repair by improving the proliferation activity and migration ability of corneal endothelial cells and / or inhibiting cell apoptosis.

3. The use according to claim 1, characterized in that The drug is suitable for treating corneal diseases caused by corneal endothelial decompensation, Fuchs endothelial dystrophy or iatrogenic corneal endothelial damage.

4. The use according to claim 1, characterized in that The medicine comprises an effective dose of GDF11 protein or a functionally active fragment thereof, and a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that The GDF11 protein is administered at a concentration of 10-200 ng / mL, and is preferably administered to the affected area by intracameral injection or local eye drops. 6 . The use according to claim 1 , wherein the GDF11 promotes the repair of corneal endothelium by regulating the expression of SOX9.

7. The use according to claim 1, characterized in that: The role of GDF11 also includes promoting the remodeling of the extracellular matrix.

8. The use according to claim 1, characterized in that The drug further comprises an in vitro expansion culture system for corneal endothelial cells, wherein GDF11 is used as a culture medium additive to enhance the survival rate and function of transplanted cells.

9. A pharmaceutical composition for corneal endothelial regeneration treatment, characterized in that The invention comprises a therapeutically effective amount of a recombinant GDF11 protein, a cornea-penetrating delivery vehicle and a cell adhesion enhancer.

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