Application of stem cell exosome composition in preparation of medicine for treating diabetic complications

Abstract

The invention relates to application of a stem cell exosome composition in preparation of a medicine for treating diabetic complications. Specifically, the VEGF monoclonal antibody 3C7 provided by the invention can effectively inhibit the activity of VEGF in cells so as to effectively treat proliferative diabetic retinopathy. Meanwhile, after the monoclonal antibody is combined with the stem cell exosome, the expression of VEGF can be effectively and synergistically reduced, the proliferative diabetic retinopathy can be effectively treated, and the application prospect is relatively good.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to the use of a stem cell exosome composition in the preparation of a drug for treating diabetic complications. Background Art

[0002] Diabetic retinopathy (DR) is a common complication of diabetes. It is a retinal microcirculatory disorder caused by long-term chronic hyperglycemia. In the late stage, it can develop into proliferative DR, which is often accompanied by serious complications such as proliferative neovascularization, such as vitreous hemorrhage and tractional retinal detachment, which can lead to irreversible vision loss. At present, the pathogenesis of DR has not been fully clarified. Endothelial cell dysfunction leads to microvascular abnormalities such as vascular occlusion and vascular leakage, which cause retinal dysfunction. Chronic inflammatory reactions such as vascular inflammatory cell infiltration and accumulation of proinflammatory factors aggravate the occurrence and progression of DR. At present, the main treatments for DR are laser photocoagulation, anti-vascular endothelial growth factor drugs and vitrectomy, but they may cause complications such as nerve damage and retinal atrophy.

[0003] VEGF is the most direct factor that promotes vascular endothelial cell division. The VEGF family includes placental growth factor (PIGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGFEU. Among them, VEGF-A has been studied most deeply, and is divided into VEGF206, VEGF189, VEGF183, VEGF165, VECF148, VEGF145 and VECF121 according to the length of its amino acids. VEGF performs its various functions by binding to its receptors. The key steps of VEGF-induced angiogenesis include protease production, enhanced vascular permeability and promotion of endothelial cell migration and proliferation. There are two main types of VECF receptors: KDR and FtKDR. FIt-1 is the main transducer of VEGF signals in endothelial cells, which promotes VEGF-induced tyrosine phosphorylation, promotes cell mitosis, chemotaxis, actin reorganization and mediates endothelial cell proliferation and differentiation. It has been confirmed that VEGF-1mRNA and VECF-2RNA are expressed in the conjunctiva, lens capsule, iris, retina and choroid pigment epithelium of normal monkey eyes, and the content is higher in the eye tissues of diabetic patients. Many studies have shown that VEGF plays an important role in the pathogenesis of diabetic macular edema (DME) and DR. In addition, the level of VECF in the vitreous of humans with proliferative diabetic retinopathy (PDR) is very high. Animal experiments and clinical observations have shown that VECF expression increases in both the proliferation and background stages of DR. Inhibiting VEGF binding to receptors, VEGF expression, and VEGF downstream signaling pathways can all be used as targets for the treatment of DR.

[0004] VEGF is a key factor in retinal neovascularization and vascular leakage in DR. It exacerbates retinal neovascularization by destroying and degrading VE-cadherin, a key endothelial cell adhesion factor, and destroys the integrity of the retinal barrier, promoting vascular leakage. However, MSC-Exos not only significantly inhibited VEGF mRNA expression and downregulated the activity of related proteins, but also increased the expression of VE-cadherin mRNA, which helped maintain the stability of the blood-retinal barrier, thereby reducing neovascularization and vascular leakage. MSC-Exos not only improved retinal function by delivering cytokines, but also reduced the immune activity of retinal ganglion cells and inner nuclear layer to inducible nitric oxide synthase and NF-κBp65, reduced the response of nerve fiber layer and ganglion cell layer to VEGF, and the response of microglia to glial fibrillary acid protein. All of the above effects help to reduce immune inflammatory response and inhibit the formation of new blood vessels.

[0005] Some recent studies have shown that combined laser therapy and bevacizumab injection can quickly prevent the formation of new blood vessels, and the therapeutic effect is better than single therapy. However, the monoclonal antibody in the method is expensive and requires the use of laser, which is not suitable for large-scale promotion and use. Therefore, the combined application therapy needs to be further improved and enhanced. Summary of the invention

[0006] The present invention provides a VEGF monoclonal antibody 3C7, wherein the light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region sequence is shown as SEQ ID NO: 2.

[0007] VEGF plays an important role in the pathogenesis of diabetic macular edema (DME) and DR. The level of VEGF in the vitreous of humans with proliferative diabetic retinopathy (PDR) is very high. The present invention effectively treats proliferative diabetic retinopathy by using VEGF monoclonal antibody 3C7 to inhibit the activity of intracellular VEGF.

[0008] Furthermore, the present invention provides a drug for treating diabetic complications, characterized in that it is composed of VEGF monoclonal antibody 3C7, the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0009] Furthermore, it is known from the prior art that MSC-Exos not only significantly inhibit the expression of VEGF mRNA and downregulate the activity of related proteins, but also increase the expression of VE-cadherin mRNA, which helps to maintain the stability of the blood-retinal barrier, thereby reducing neovascularization and vascular leakage, and can therefore be used to treat diabetic retinopathy.

[0010] Furthermore, the present invention provides a drug kit for treating diabetic complications, characterized in that it is composed of VEGF monoclonal antibody 3C7 and stem cell exosomes, the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

[0011] Furthermore, the medicine or medicine kit of the present invention also contains a pharmaceutically acceptable carrier.

[0012] In some embodiments, the light chain variable region of the invention

[0013] i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1; or

[0014] ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1; or

[0015] (ii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 1, preferably, the amino acid changes do not occur in the CDR regions.

[0016] In some embodiments, the heavy chain variable region of the invention

[0017] i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2; or

[0018] ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2; or

[0019] (ii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 2, preferably, the amino acid changes do not occur in the CDR regions.

[0020] Furthermore, the medicine or medicine kit of the present invention also contains a pharmaceutically acceptable excipient.

[0021] Examples of suitable pharmaceutically acceptable excipients include one or more polymers, wetting agents or surfactants, pH adjusters, isotonicity adjusters, preservatives, buffers, and chelating agents, or any combination thereof.

[0022] In addition, the pharmaceutical composition of the present disclosure may also include other pharmaceutical agents, carriers, adjuvants, diluents and excipients. In certain embodiments, carriers, excipients or excipients may promote the administration and delivery of the composition, and / or improve the preservation of the composition. In other embodiments, the one or more carriers include but are not limited to saline solutions, such as normal saline, Ringer's solution, PBS (phosphate buffered saline) and general mixtures of various salts, including potassium salts and phosphates, with or without sugar additives, such as glucose. The carrier may include aqueous and non-aqueous sterile injection solutions, which may include antioxidants, buffers, antibacterial agents, antibacterial antibiotics and solutes that make the preparation isotonic with the intended recipient's body fluids; and water-based and non-aqueous sterile suspensions, which may include suspensions and thickeners. In other embodiments, one or more excipients may include but are not limited to water, saline, glucose, glycerol, ethanol and combinations thereof. Nontoxic auxiliary substances such as wetting agents, buffers or emulsifiers may also be added to the composition. Oral formulations can include commonly used excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

[0023] Examples of suitable pharmaceutically acceptable pH adjusting agents include, but are not limited to, sodium hydroxide, citric acid, hydrochloric acid, boric acid, acetic acid, phosphoric acid, succinic acid, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium oxide, calcium carbonate, magnesium carbonate, magnesium aluminum silicate, malic acid, potassium citrate, sodium citrate, sodium phosphate, lactic acid, gluconic acid, tartaric acid, 1,2,3,4-butanetetracarboxylic acid, fumaric acid, diethanolamine, monoethanolamine, sodium carbonate, sodium bicarbonate, triethanolamine, or any combination thereof. In one embodiment, the pharmaceutically acceptable pH adjusting agent is present in an amount of about 0.01% to about 2.0% (weight / volume), preferably about 0.05% to about 1% (weight / volume).

[0024] Specifically, the stem cell exosomes of the present invention are exosomes secreted by any one of human umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, placental mesenchymal stem cells, liver mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, pancreatic mesenchymal stem cells and dental pulp mesenchymal stem cells.

[0025] In some examples, exosomes derived from human mesenchymal stem cells can be isolated after culturing stem cells with a culture medium. In some examples, the cultured supernatant can be filtered using a filter to obtain exosomes. In some examples, the pore size of the filter (i.e., the diameter of the pores) can be 0.22 μm (micrometer). Thus, the components with larger particle sizes in the supernatant can be removed, and the exosomes can be retained in the supernatant as much as possible. In some examples, the filtered supernatant can be centrifuged, and the precipitate after centrifugation is an exosome. In some examples, the filtered supernatant can be ultracentrifuged at a high speed for 70 minutes, and the precipitate after centrifugation is an exosome. Thus, exosomes derived from human umbilical cord mesenchymal stem cells can be prepared.

[0026] Beneficial Effects

[0027] The present invention provides a use of a stem cell exosome composition in the preparation of a drug for treating diabetic complications. Specifically, the VEGF monoclonal antibody 3C7 provided by the present invention can effectively inhibit the activity of intracellular VEGF to effectively treat proliferative diabetic retinopathy, which is better than the effect of the positive control. At the same time, the monoclonal antibody of the present invention can effectively and synergistically reduce the expression of VEGF after being used in combination with stem cell exosomes, effectively treating proliferative diabetic retinopathy, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 VEGF monoclonal antibody 3C7 Western blot test results

[0029] Figure 2 The results of the effects of different doses of VEGF monoclonal antibody 3C7 on the expression of VEGF protein. DETAILED DESCRIPTION

[0030] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0031] Example 1 Preparation of VEGF monoclonal antibody

[0032] Three 8-week-old SPF BALB / c mice were immunized with recombinant VEGF recombinant human protein (catalog number PHC939, Gibco). The recombinant protein was mixed with Freund's complete adjuvant in a 1:1 ratio, with a first immunization dose of 80 μg / mouse. A second immunization was performed at an interval of 14 days. The recombinant protein was mixed with incomplete adjuvant in a 1:1 ratio, with a second immunization dose of 100 μg / mouse. A third immunization was performed at an interval of 14 days at a dose of 100 μg / mouse. Blood was collected from the immunized mice at the tip of their tails 5 days later, and the serum titer was determined by indirect immunofluorescence. Mouse No. 2 with a higher serum titer was selected for booster immunization at a dose of 100 μg / mouse. On the third day, mouse spleen cells were taken and fused with myeloma cells SP2 / 0, and hybridoma cells were screened using HAT medium. The hybridoma supernatant was positively screened using a 96-well plate coated with VEGF recombinant human protein, and the positive wells were further screened using a 96-well plate coated with rat VEGF recombinant protein. The cells in the hybridoma wells that can bind to both human VEGF and rat VEGF were gently blown evenly, 10 μL was taken to a cell counting plate for counting and diluted to 1000 cells / mL with 10% HT growth solution, 100 μL of the diluted cells were aspirated into 10 mL of 10% HT growth solution, and after sufficient mixing, 100 μL / well of cells were plated in a 96-well cell plate, and then 10% HT was added to 150 μL / well. The 96-well cell plate was cultured at 37°C, and the supernatant in the monoclonal well was taken for ELISA detection on the 10th day. The above steps were repeated 4 times until the positive rate of all monoclonal cell wells reached 100%, and the positive hybridoma cells with the best growth status were selected for expansion and cryopreservation. The hybridoma cells were named 3C7.

[0033] Recombinant human VEGF protein, rat VEGF protein and mouse blood cell total protein were taken, and the supernatant of hybridoma cell 3C7 was used as the primary antibody and HRP-labeled goat anti-mouse IgG was used as the secondary antibody for Western blot analysis of the specificity of the monoclonal antibody. Figure 1 shown.

[0034] from Figure 1 The results showed that the VEGF monoclonal antibody 3C7 produced by hybridoma cell 3C7 could effectively bind to human VEGF and rat VEGF recombinant proteins, but did not react with mouse blood cells, and had good binding specificity.

[0035] The mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. After about 12 days, hybridoma cells 3C710 were inoculated intraperitoneally. 7 When the mice showed abdominal swelling, weight loss, rapid breathing, decreased activity and other signs, the ascites was collected and centrifuged at 2000r / min for 10min. The supernatant was collected and purified by column. The purity was above 98.5% as determined by SDS-PAGE. The protein concentration was adjusted to 5mg / mL and stored in aliquots at -70℃ for later use.

[0036] Example 2 Performance Evaluation of VEGF Monoclonal Antibody 3C7

[0037] The affinity of monoclonal antibodies was determined by indirect ELISA. The plate was coated with recombinant VEGF recombinant human protein at concentrations of 1, 0.5, 0.25, and 0.125 μg / mL, respectively. Blank wells and negative control wells were set up at the same time, and the plate was incubated at 4°C overnight. The plate was washed with 1‰ PBST solution and blocked with 1% BSA at 37°C for 1 hour. After washing, monoclonal antibody 3C7 (1 μg / mL) was added to each well, and the plate was shaken and placed at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse antibody (1:2000 dilution, 100 μL per well) was added, and the plate was incubated at 37°C for 40 minutes. The plate was washed with 1‰ PBST, and the optical density of each well [D(450)] was measured at 450nm using an enzyme reader after color development using the TMB color development system. Then, according to the formula Kd = (n-1) / 2(n[Ab']-[Ab]) (where n = [Ag] / [Ag']; [Ab] or [Ab'] represents the antibody concentration at half the maximum optical density in the indirect ELISA method), the relative affinity of the monoclonal antibody was calculated. The results showed that the affinity of the VEGF monoclonal antibody 3C7 reached 1.47×10 9 (L / mol), which indicates a good affinity.

[0038] The monoclonal antibody 3C7 was entrusted to Huijun Biotechnology to sequence the variable regions of the antibody. The results showed that the light chain variable region sequence of the monoclonal antibody was as shown in SEQ ID NO: 1, and the heavy chain variable region sequence was as shown in SEQ ID NO: 2.

[0039] Example 3 Identification of cytological activity of VEGF monoclonal antibody 3C7

[0040] Retinal cell line (R28) catalog number EUR201, Kerafast was used for proliferation culture. The experiment was divided into 5 groups in a 96-well plate, and the cell concentration in each group was adjusted to 1×10 4 / mL, corresponding to the final concentration of VEGF monoclonal antibody 3C7 added was 10μg / mL (Group A), 50μg / mL (Group B), 100μg / mL (Group C), 200μg / mL (Group D), 500μg / mL (Group E), and the blank control group was not treated with monoclonal antibody; the positive control group was bevacizumab at a concentration of 100μg / mL. After monoclonal antibody treatment for 24h, the cells were collected by centrifugation, washed 3 times with PBS, and lysed. After adjusting the protein concentration to the same, Western blot was performed to calculate the relative expression of VEGF protein as VEGF / β-actin, and the relative expression of the blank control group was calculated as 1. The relative expression results of each group are shown in the figure. Figure 1 shown.

[0041] from Figure 2 It can be seen that VEGF monoclonal antibody 3C7 has a dose-dependent inhibitory effect on the expression of VEGF protein in cells. Under the same concentration conditions, VEGF monoclonal antibody 3C7 also has a relatively better inhibitory activity on VEGF expression than the positive control group, and the effect is better.

[0042] Example 3 Preparation of mesenchymal stem cell exosomes

[0043] Human bone marrow mesenchymal stem cells (article number JY-J073) were purchased from Shanghai Jinyuan Biotechnology Co., Ltd., and 4 ml of DMEM-F12 complete medium was added and cultured in a 37°C, 5% CO2 incubator. MSCs were cultured at 5000 cm 2 The cells were inoculated at a density of 100 μg / mL in a 12-well plate and cultured in DMEM-LG complete medium without fetal bovine serum for 72 h. The culture medium was collected and centrifuged at 500 r / min for 10 min to collect the supernatant. The cells were centrifuged at 2000 r / min for 20 min to collect the supernatant. 5 r / min centrifugation for 70 min. After resuspending in 20 ml phosphate buffer (PBS), 1×10 5 The precipitate was diluted with PBS at a ratio of 1:20 and centrifuged at 2000 r / min for 200 min. 40% sucrose gradient 1×10 5 Centrifuge at r / min for 70 min; collect the supernatant. 1×10 5 The mixture was centrifuged at 400 rpm for 70 min, and the precipitate was collected and dissolved in PBS to adjust the concentration to 1 mg / mL to obtain mesenchymal stem cell exosomes.

[0044] The collected exosomes were made into a suspension with PBS, and 2 μl of the exosome suspension was dripped onto the formvar-carbon sample-loading copper grid, left to stand for 5 minutes, and stained with uranyl acetate in the dark for 5 minutes. The samples were washed with PBS 3 times, semi-dried at room temperature, and observed by transmission electron microscopy and photographed to show that high-purity exosome particles were obtained, with a main diameter distribution of about 80-130 nm. Western blotting was used to detect the high expression of CD63 protein in exosomes, indicating that relatively pure exosomes were prepared.

[0045] Example 4 Mouse model experiment

[0046] Ten rats in the control group were fed with standard feed, and ten rats in each of the model group, mesenchymal stem cell exosome group, VEGF monoclonal antibody 3C7 group, exosome combined with 3C7 group, and positive control group were fed with high-fat feed. After 8 weeks, the control group was given normal saline, and the rats in the model group, mesenchymal stem cell exosome group, VEGF monoclonal antibody 3C7 group, exosome combined with 3C7 group, and positive control group were intraperitoneally injected with 40 mg / kg streptozotocin. Blood glucose was measured by collecting tail vein blood of rats. If blood glucose was ≥16.7 mmol / L, the diabetic rat model was successfully established. The diet of rats in each group was the same as before. After continuing to feed for 4 weeks, fundus vascular fluorescence angiography was performed to confirm that diabetic retinopathy occurred in rats, and the model was successfully established. Specific administration groups: The control group was given 0.05 mL of normal saline intraperitoneally; the rats in the VEGF monoclonal antibody 3C7 group were given 0.05 mL of 3C7 monoclonal antibody (5 mg / mL) intravitreally; the rats in the positive control group were given 0.05 mL of bevacizumab (5 mg / mL) intravitreally; the mesenchymal stem cell exosome group was given 0.1 mL of exosomes (1 mg / mL) prepared in Example 3 intraperitoneally; the exosome combined with 3C7 group was given 0.05 mL of 3C7 monoclonal antibody (5 mg / mL) intravitreally and 0.1 mL of exosomes (1 mg / mL) prepared in Example 3 intraperitoneally. The control group and the model group were given an equal volume of normal saline intervention, and each group was given medication once a day for 2 consecutive weeks.

[0047] Color Doppler ultrasound (CDFI) was used to detect the blood flow parameters of the central retinal artery (CRA), and the peak blood flow velocity was determined based on the Doppler ultrasound blood flow index: EDV, PSV, RI and PI. The above operation was repeated 3 times, and the average value of each index was taken. The results are shown in Table 1.

[0048] Table 1 Results of central retinal artery (CRA) blood flow parameters in each group

[0049]

[0050] Retinal CRA is the main blood vessel supplying retinal internal reference, belongs to the terminal artery, is a branch of the ophthalmic artery, and plays an important role in maintaining visual function. Retinal artery parameters can be used to reflect the blood supply of the retina and the state of retinal microcirculation. As can be seen from Table 1, each treatment group of the present invention can improve the EDV and PSV levels of rats, reduce the RI and PI levels, thereby improving the hemodynamics of retinal CRA, especially after the exosomes are used together with the monoclonal antibody 3C7, this improvement has been significantly improved, and the synergistic effect is obvious, which is better than the effect of using exosomes alone or monoclonal antibody 3C7 alone.

[0051] 2 mL of tail venous blood was drawn from each group of rats, washed with PBS buffer, centrifuged at 2500 r / min for 8 min, the supernatant was discarded, and the VEGF level in the plasma of each group of rats was detected using a double antibody sandwich enzyme-linked immunosorbent assay kit. The results are shown in Table 2.

[0052] Table 2 VEGF content in plasma of rats in each group

[0053] Group VEGF content (pg / mL) Control group 40.89±3.54 Model Group 94.87±8.53 Exosome group 77.46±6.19 Monoclonal antibody 3C7 group 52.14±3.97 Exosome combined with 3C7 group 43.94±3.77 Positive control group 55.18±4.86

[0054] As shown in Table 2, compared with normal rats, the VEGF level in the model group was significantly increased, with significant differences (P<0.05); compared with the model group, the VEGF levels in each treatment group and the positive control group were significantly reduced, especially the monoclonal antibody 3C7, which was more significantly reduced, with significant differences (P<0.05). Exosomes combined with 3C7 have a synergistic effect, and the VEGF content is extremely significantly reduced.

[0055] The rats in each group were killed, and the left eyeball of each rat was removed and fixed with 10% formaldehyde for 24 hours. After decalcification, dehydration, transparency and embedding, paraffin blocks were made. The tissues were sliced ​​and stained with HE. Under the light microscope, it was found that the inner and outer segments of the cells in the control group were regular, and the cells of each layer of the retina were neatly arranged. The retinal blood vessels of the rats in the model group were dilated, the cell structures of each layer of the retina were disordered, and there was edema between the cells. After treatment with the monoclonal antibody 3C7 group, the positive control group, and the exosome combined with 3C7 group, the arrangement of the cells in each layer of the retina was relatively complete, especially the arrangement of the exosome combined with 3C7 group was very complete and basically similar to the control group, indicating that the treatment effect was good.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A VEGF monoclonal antibody 3C7, characterized in that The light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

2. A drug for treating diabetic retinopathy, a complication of diabetes, characterized in that The drug comprises VEGF monoclonal antibody 3C7 and a pharmaceutically acceptable carrier. The light chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO: 1, and the heavy chain variable region sequence is shown as SEQ ID NO:

2.

3. A medicine kit for treating diabetic complication diabetic retinopathy, characterized in that The invention is composed of VEGF monoclonal antibody 3C7 and bone marrow stem cell exosomes, wherein the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO:

2.

4. Use of VEGF monoclonal antibody 3C7 and bone marrow stem cell exosomes in the preparation of a drug kit for treating diabetic retinopathy, a complication of diabetes, wherein the light chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO: 1, and the heavy chain variable region sequence is shown in SEQ ID NO: 2.

Images