Active polypeptide for inhibiting diabetic retina microangiogenesis

Abstract

The invention belongs to the technical field of protein engineering, and particularly relates to an active polypeptide for inhibiting diabetic retina microangiogenesis. The active polypeptide is SEQ ID NO: 1 formed by three core fragments through flexible linkage or a derivative sequence obtained by mutation in the core fragments. The group of active polypeptides can significantly inhibit the physiological processes of DNA synthesis, proliferation, migration, callus, tube formation and the like of retina microvascular endothelial cells in a high-glucose environment, thereby achieving the effect of inhibiting angiogenesis.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and particularly relates to an active polypeptide for inhibiting the formation of diabetic retinal microangiogenesis. Background Art

[0002] At present, with the rapid increase in the prevalence of diabetes worldwide, diabetic retinopathy, as one of the most important microvascular complications, has become a serious health challenge faced by people of all ages. In the progression of diabetic retinopathy, under the induction of persistent hyperglycemia and hypoxic microenvironment in the eye, especially the retina, typical pathological features of diabetic retinopathy appear, mainly including retinal neovascularization, vascular leakage, retinal fibrosis and gliosis, which can lead to vision loss, visual impairment and even complete blindness.

[0003] In recent years, the treatment mode targeting retinal neovascularization has become a classic principle in the clinical treatment of diabetic retinopathy. Generally, anti-compounds or VEGF antibodies, including Pegaptanib, Ranibizumab, Bevacizumab and Aflibercept, have been applied by intraocular injection to inhibit retinal vascular leakage and angiogenesis.

[0004] However, drug resistance to the above antibodies and side effects on normal blood vessels are always important issues faced in clinical applications. Therefore, it is necessary to explore new ideas and obtain new methods to inhibit angiogenesis in diabetic retinopathy from active peptides with smaller molecular weight, stronger permeability and easier metabolism. Summary of the invention

[0005] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a group of active polypeptides comprising three core fragments, which can effectively antagonize angiogenesis in the development of diabetic retinopathy in vitro and in vivo in experimental animals. The active polymorph of the present invention has a smaller molecular weight, in principle has better permeability, and is more conducive to synthesis.

[0006] In order to solve the above technical problems, the present invention provides a group of active polypeptides for inhibiting retinal microangiogenesis in the process of diabetic retinopathy, wherein the amino acid sequence of the active polypeptide is (a): comprising a polypeptide composed of three core fragments shown in SEQ ID NO: 1, which are flexibly linked in sequence by flexible linking peptides (GGG); the amino acid sequences of core fragment one, core fragment two and core fragment three are RKAADD, EEEFVEGI and TANDSGP, respectively.

[0007] Preferably, the amino acid sequence of the active polypeptide is (b): a derivative peptide segment having angiogenesis inhibition function shown by replacing one amino acid sequence in the amino acid sequence of the core fragment 2 in (a).

[0008] Preferably, after replacing one amino acid sequence in (b), the amino acid sequence of the core fragment 2 is EEEFVEGR.

[0009] Preferably, the amino acid sequence of the active polypeptide is (c): a derivative peptide segment having angiogenesis inhibition function shown by replacing one amino acid sequence in the amino acid sequence of the core fragment three in (a).

[0010] Preferably, after replacing one amino acid sequence in (c), the amino acid sequence of the core fragment three is TANRSGP.

[0011] Preferably, the amino acid sequence of the active polypeptide is (d): a derivative peptide segment having angiogenesis inhibition function shown by combining and replacing multiple amino acid sequences in the sequences of core fragments 2 and 3 in (a).

[0012] Preferably, after combining and replacing multiple amino acid sequences in (d), the amino acid sequences of the core fragment two and the core fragment three are EEEFVEGR and TANRSGP, respectively.

[0013] Preferably, a histidine tag His-tag (ie, HHHHHH) is further included, and the histidine tag His-tag is triple-linked to the core fragment through the flexible linking peptide.

[0014] Preferably, the concentration of the active polypeptide is 2 μmol / L to 8 μmol / L.

[0015] Preferably, the concentration of the active polypeptide is 4 μmol / L or 6 μmol / L.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a group of active polypeptides that can inhibit angiogenesis of retinal microvessels in a microenvironment simulating diabetic retinopathy. The active polypeptide is SEQ ID NO: 1 composed of three core fragments linked by flexible links or a derivative sequence thereof obtained by mutation in the core fragment. This group of active polypeptides can significantly inhibit physiological processes such as DNA synthesis, proliferation, migration, wound healing and tube formation of retinal microvascular endothelial cells in a high sugar environment, thereby achieving the effect of inhibiting angiogenesis. Moreover, the active polymorphism of the present invention has a smaller molecular weight, in principle has better permeability, and is more conducive to synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the cell proliferation trend determined by the CCK8 experiment in Example 1 of the present invention.

[0019] Figure 2 This is a schematic diagram of the cell proliferation trend determined by the CCK8 experiment in Example 2 of the present invention.

[0020] Figure 3 Schematic diagram of the cell migration rate trend in Example 3 of the present invention.

[0021] Figure 4 Schematic diagram of the cell migration rate trend in Example 4 of the present invention.

[0022] Figure 5 Schematic diagram of the relative cell density trend of scratches in Example 5 of the present invention.

[0023] Figure 6 Schematic diagram of the relative cell density trend of scratches in Example 6 of the present invention.

[0024] Figure 7 It is a schematic diagram of the length trend of cell tubes in Example 7 of the present invention.

[0025] Figure 8 It is a schematic diagram of the length trend of cell tubes in Example 8 of the present invention.

[0026] Fig. 9 It is a schematic diagram of the leakage trend of retinal blood vessels in Example 9 of the present invention.

[0027] Fig.10 It is a schematic diagram of the number trend of abnormal new blood vessels in Example 9 of the present invention.

[0028] Fig.11 It is a schematic diagram of the active polypeptide provided by the present invention. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The advantages and features of the present invention will become more apparent from the following description. It should be noted that the accompanying drawings are in very simplified form and in non-precise proportions, and are only used to conveniently and clearly assist in explaining the purpose of the embodiments of the present invention.

[0030] Example 1

[0031] The present invention provides an inhibitory effect of polypeptide (a) on the proliferation of retinal microvascular endothelial cells in a high-sugar environment; retinal microvascular endothelial cells (hRECs) were induced and cultured in DMEM medium with low sugar (5.5mM glucose), high sugar (25mM glucose), and high osmotic pressure control (5.5mM glucose + 20mM mannitol) for 72 hours, and then seeded in a 96-well plate at a density of 3000 cells / 100μL culture medium, respectively using the above corresponding culture medium, and in addition, 0, 2, 4, 6, 8μmol / L polypeptide (a) was added to the high-sugar culture medium. After incubation for 24 hours, the proliferation trend of hREC was detected using a cell counting kit-8 (CCK8, Dojindo Laboratories, Kumamoto, Japan).

[0032] OD was measured after 5 days 450nm ,The results showed that polypeptide (a) could effectively inhibit the proliferation of hRECs, and the ,added concentrations of 4μmol / L and 6μmol / L were optimal.

[0033] The following table 1 gives the Figure 1 Schematic diagram of cell proliferation trend determined by CCK8 assay. 450nm Measured values ​​(LG represents low sugar, HG represents high sugar, and LM represents LG + osmotic pressure control).

[0034] Table 1:

[0035]

[0036] Example 2

[0037] The present invention provides polypeptides (b), (c), and (d) that inhibit the proliferation of retinal microvascular endothelial cells in a high-sugar environment; after 72 hours of induction culture in a high-sugar (25 mM glucose) DMEM medium, retinal microvascular endothelial cells (hRECs) were seeded in a 96-well plate at a density of 3000 cells / 100 μL of medium, and 4 μmol / L of polypeptides (b), (c), and (d) were added to the high-sugar medium, respectively. After incubation for 24 hours, the proliferation trend of hRECs was detected using a cell counting kit-8 (CCK8, Dojindo Laboratories, Kumamoto, Japan).

[0038] OD was measured after 5 days 450nm The results showed that polypeptides (a), (b), (c), and (d) could effectively inhibit the proliferation of hRECs, and sequence (d) had the strongest inhibitory ability.

[0039] The following table 2 gives the Figure 2Schematic diagram of cell proliferation trend determined by CCK8 assay. 450nm Measured value.

[0040] Table 2:

[0041] HG HG+a(4μmol / L) HG+b(6μmol / L) HG+c (4 μmol / L) HG+d(4μmol / L) <![CDATA[OD 450nm ]]> 3.10±0.17 1.07±0.07 1.00±0.08 1.01±0.07 0.80±0.03

[0042] Example 3

[0043] The embodiment of the present invention provides the inhibitory effect of polypeptide (a) on the migration of retinal microvascular endothelial cells in a high-sugar environment; retinal microvascular endothelial cells (hRECs) were induced and cultured in DMEM medium with low sugar (5.5mM glucose), high sugar (25mM glucose), and high osmotic pressure control (5.5mM glucose + 20mM mannitol) for 72 hours, and then seeded in a 96-well plate at a density of 3000 cells / 100μL culture medium, respectively using the above corresponding culture medium, and in addition, 0, 2, 4, 6, 8μmol / L polypeptide (a) was added to the high-sugar culture medium. Thereafter, hRECs were placed in a transwell chamber with a pore size of 8.0μm, and 200μL of FBS-free culture medium containing 10×103 hRECs was added to the chamber and cultured for 48 hours. Aspirate the culture medium in the upper and lower chambers, wipe the cells in the upper chamber with a cotton swab, add 600 μL of 40g / L paraformaldehyde to the 24-well plate, fix at room temperature for 30 minutes, aspirate the paraformaldehyde, rinse the back of the chamber twice with PBS, add 600 μL of crystal violet to the 24-well plate, stain at room temperature for 20 minutes, aspirate and rinse the back of the chamber twice with PBS, observe and photograph under an optical microscope.

[0044] The results showed that polypeptide (a) could effectively inhibit the migration of hRECs, and the added concentrations of 4 and 6 μmol / L were optimal (the number of migrated cells in the high glucose environment was 100%).

[0045] The following table 3 gives Figure 3 Specific migration rates are shown as schematic diagrams of cell migration rate trends.

[0046] Table 3:

[0047]

[0048] Example 4

[0049] The present invention provides polypeptides (b), (c), and (d) that inhibit the migration of retinal microvascular endothelial cells in a high-sugar environment; after induced culture of retinal microvascular endothelial cells (hRECs) in a high-sugar (25 mM glucose) DMEM medium for 72 hours, the cells were seeded in a 96-well plate at a density of 3000 cells / 100 μL of medium, and 4 μmol / L of polypeptides (b), (c), and (d) were added to the high-sugar medium, respectively. Thereafter, the hRECs were placed in a transwell chamber with a pore size of 8.0 μm, and 200 μL of FBS-free medium containing 10×103 hRECs was added to the chamber and cultured for 48 hours. Aspirate the culture medium in the upper and lower chambers, wipe the cells in the upper chamber with a cotton swab, add 600 μL of 40g / L paraformaldehyde to the 24-well plate, fix at room temperature for 30 minutes, aspirate the paraformaldehyde, rinse the back of the chamber twice with PBS, add 600 μL of crystal violet to the 24-well plate, stain at room temperature for 20 minutes, aspirate and rinse the back of the chamber twice with PBS, observe and photograph under an optical microscope.

[0050] like Figure 4 The results shown show that polypeptide (a) can effectively inhibit the migration of hRECs, and the addition concentrations of 4 and 6 μmol / L are optimal (the number of migrated cells in the high glucose environment is 100%). Figure 4 The results show that polypeptides (a), (b), (c), and (d) can effectively inhibit the migration of hRECs, and the (d) sequence has the strongest inhibitory ability.

[0051] The following table 4 gives the Figure 4 Specific migration rates are shown as schematic diagrams of cell migration rate trends.

[0052] Table 4:

[0053]

[0054] Example 5

[0055] The present invention provides an inhibitory effect of polypeptide (a) on retinal microvascular endothelial cell healing in a high-sugar environment; after retinal microvascular endothelial cells (hRECs) were induced and cultured in a high-sugar (25mM glucose) DMEM medium for 72 hours, they were seeded in a 96-well plate at a density of 3000 cells / 100μL culture medium, and the above-mentioned corresponding culture medium was used respectively. In addition, 0, 2, 4, 6, and 8μmol / L polypeptide (a) were added to the high-sugar medium respectively. hRECs were seeded in an Ibidi 35mm culture dish (with wound healing plug-in Ibidi, 81176) at a cell density of 3000 cells / 100μL, and after culturing until the confluence reached 90%, they were observed using an optical microscope and photographed at 0h and 24h, respectively, and the images were scanned using IncuCyteZOOM (ESSEN BioScience, Ann Arbor, MI) software to calculate the scratch relative cell density (RWD).

[0056] like Figure 5 The results shown indicate that polypeptide (a) can effectively inhibit the healing of hRECs, and the added concentrations of 4 and 6 μmol / L are optimal.

[0057] The following table 5 gives the Figure 5 Relative cell density of specific scratches shown.

[0058] Table 5:

[0059]

[0060] Example 6

[0061] The present invention provides polypeptides (b), (c), and (d) that inhibit the healing of retinal microvascular endothelial cells in a high-sugar environment; after 72 hours of induction culture in a high-sugar (25 mM glucose) DMEM medium, retinal microvascular endothelial cells (hRECs) were seeded in a 96-well plate at a density of 3000 cells / 100 μL of medium, and 4 μmol / L of polypeptides (b), (c), and (d) were added to the high-sugar medium. hRECs were seeded in an Ibidi 35 mm culture dish (with wound healing plug-in Ibidi, 81176) at a cell density of 3000 cells / 100 μL, and after culturing until the confluence reached 90%, they were observed using an optical microscope and photographed at 0h and 24h, respectively, and the images were scanned using IncuCyteZOOM (ESSEN BioScience, Ann Arbor, MI) software to calculate the scratch relative cell density (RWD).

[0062] like Figure 6The results shown show that polypeptides (a), (b), (c), and (d) can effectively inhibit the healing of hRECs, and the (d) sequence has the strongest inhibitory ability.

[0063] The following table 6 gives the Figure 6 Relative cell density of specific scratches shown.

[0064] Table 6:

[0065]

[0066] Example 7

[0067] The embodiment of the present invention provides the inhibitory effect of polypeptide (a) on retinal microvascular endothelial cell tube formation in a high-sugar environment; after retinal microvascular endothelial cells (hRECs) were induced and cultured in a high-sugar (25mM glucose) DMEM medium for 72 hours, they were seeded in a 96-well plate at a density of 3000 cells / 100μL culture medium, and the above-mentioned corresponding culture medium was used respectively. In addition, 0, 2, 4, 6, and 8μmol / L polypeptide (a) were added to the high-sugar medium. 200μL of supporting gel was spread in a 48-well plate and solidified at 37°C for 30 minutes. About 3×104 hRECs cells were inoculated into each well, and the corresponding DMEM culture medium was added. After culturing for 3 hours, they were observed and photographed under an optical microscope, and the length of the cell tube was calculated using ImageJ software.

[0068] like Figure 7 The results shown indicate that polypeptide (a) can effectively inhibit the tube formation of hRECs, and the added concentrations of 4 and 6 μmol / L are optimal.

[0069] The following table 7 gives the Figure 7 Specific cell tube lengths shown.

[0070] Table 7:

[0071]

[0072] Example 8

[0073] The embodiment of the present invention provides the inhibitory effect of polypeptides (b), (c), and (d) on retinal microvascular endothelial cell tube formation in a high-sugar environment; after retinal microvascular endothelial cells (hRECs) were induced and cultured in a high-sugar (25mM glucose) DMEM medium for 72 hours, they were seeded in a 96-well plate at a density of 3000 cells / 100μL culture medium. In addition, 4μmol / L polypeptides (b), (c), and (d) were added to the high-sugar medium. 200μL of support gel was spread in a 48-well plate and solidified at 37°C for 30 minutes. About 3×104 hRECs cells were inoculated into each well, and the corresponding DMEM culture medium was added. After culturing for 3 hours, they were observed and photographed under an optical microscope, and the length of the cell tube was calculated using ImageJ software.

[0074] like Figure 8 The results shown show that polypeptides (a), (b), (c), and (d) can effectively inhibit the tube formation of hRECs, and the (d) sequence has the strongest inhibitory ability.

[0075] The following table 8 gives the Figure 8 Specific cell tube lengths shown.

[0076] Table 8:

[0077]

[0078] Example 9

[0079] The embodiment of the present invention provides the inhibitory effect of overexpression of polypeptide (d) in the eye on retinal microangiogenesis during diabetic retinopathy in STZ-induced diabetic rat model. The embodiment of the present invention uses 6 to 8 week old male Sprague-Dawley (SD) rats. Five days after intraperitoneal injection of STZ (60 mg / kg), blood glucose exceeds 16.7 mmol / L, indicating that the diabetic rat model has been established. The polypeptide (d) sequence is connected to the pAAV-MCS plasmid and packaged into the AAV9 virus. After intraperitoneal injection of ketamine (80 μg / g) and xylazine (4 μg / g) in rats, 5 μL AAV9 virus (1×1012 vg / mL, once every 8 weeks) is injected into the vitreous cavity, and sterile saline is used as a control. After 16 weeks, the rats were intravenously perfused with Evans blue, and after being killed, the retina was peeled off and the retina was photographed using an Evos fluorescence microscope and scanned with ImagJ software to calculate the retinal leakage area. At the same time, the rat retina was enzymatically hydrolyzed with shiyongtrypsin and stained with PAS dye, and photographed under an optical microscope to count the number of abnormal new blood vessels.

[0080] like Fig. 9 The results shown in the figure indicate that intraocular overexpression of polypeptide (d) can significantly reduce retinal vascular leakage in diabetic rats.

[0081] The following table 9 gives the Fig. 9 Leakage of specific retinal vessels shown (wherein STZ represents streptozotocin, STZ+NC represents STZ+sterile saline, and Norml represents the normal group).

[0082] Table 9:

[0083]

[0084] At the same time, intraocular overexpression of polypeptide (d) can significantly reduce the number of abnormal retinal neovascularization in diabetic rats.

[0085] The following table 10 gives the Fig.10 The number of abnormal retinal neovascularizations is shown.

[0086] Table 10:

[0087]

[0088] In summary, in the above embodiments, sequence (a) can significantly inhibit the proliferation of retinal microvascular endothelial cells under high glucose conditions; sequences (b), (c), and (d) can significantly inhibit the proliferation of retinal microvascular endothelial cells under high glucose conditions, and sequence (d) has the strongest inhibitory ability; sequence (a) can significantly inhibit the migration of retinal microvascular endothelial cells under high glucose conditions; sequences (b), (c), and (d) can significantly inhibit the migration of retinal microvascular endothelial cells under high glucose conditions, and sequence (d) has the strongest inhibitory ability; sequence (a) can significantly inhibit the migration of retinal microvascular endothelial cells under high glucose conditions. cells; (b), (c), and (d) sequences can significantly inhibit the healing of retinal microvascular endothelial cells under high glucose conditions, and (d) sequence has the strongest inhibitory ability; (a) sequence can significantly inhibit the tube formation of retinal microvascular endothelial cells under high glucose conditions; (b), (c), and (d) sequences can significantly inhibit the tube formation of retinal microvascular endothelial cells under high glucose conditions, and (d) sequence has the strongest inhibitory ability; in the STZ-induced diabetic rat model, overexpression of (d) sequence in the vitreous cavity can significantly inhibit retinal microvascular angiogenesis and retinal vascular leakage in the process of diabetic retinopathy.

[0089] The above description is only a description of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. Any changes or modifications made by a person skilled in the art in the field of the present invention based on the above disclosure shall fall within the scope of protection of the claims.

Claims

1. An active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy, characterized in that: The amino acid sequence of the active polypeptide is (a): comprising a polypeptide composed of three core fragments shown in SEQ ID NO: 1, which are flexibly linked in sequence by flexible linking peptides; the amino acid sequences of core fragment one, core fragment two and core fragment three are RKAADD, EEEFVEGI and TANDSGP in sequence.

2. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 1, characterized in that: The amino acid sequence of the active polypeptide is (b): a derivative peptide segment having angiogenesis inhibition function shown by replacing one amino acid sequence in the amino acid sequence of the core fragment 2 in (a).

3. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 2, characterized in that: After replacing one amino acid sequence in (b), the amino acid sequence of the core fragment 2 is EEEFVEGR.

4. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 1, characterized in that: The amino acid sequence of the active polypeptide is (c): a derivative peptide segment having angiogenesis inhibition function shown by replacing one amino acid sequence in the amino acid sequence of the core fragment three in (a).

5. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 4, characterized in that: After replacing one amino acid sequence in (c), the amino acid sequence of the core fragment three is TANRSGP.

6. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 1, characterized in that: The amino acid sequence of the active polypeptide is (d): a derivative peptide segment having angiogenesis inhibition function shown by combining and replacing multiple amino acid sequences in the sequences of the core fragments 2 and 3 in (a).

7. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 6, characterized in that: After combining and replacing multiple amino acid sequences in (d), the amino acid sequences of the core fragment two and the core fragment three are EEEFVEGR and TANRSGP, respectively.

8. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to any one of claims 1 to 7, characterized in that: It also includes a histidine tag His-tag, and the histidine tag His-tag is triple-linked to the core fragment through the flexible linking peptide.

9. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to any one of claims 1 to 7, characterized in that: The concentration of the active polypeptide is 2 μmol / L to 8 μmol / L.

10. The active polypeptide for inhibiting retinal microangiogenesis in the process of diabetic retinopathy according to claim 9, characterized in that: The concentration of the active polypeptide is 4 μmol / L or 6 μmol / L.

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