CN113599390A - Application of reagent for promoting miR-21 expression in preparation of medicine for preventing and/or treating diabetic keratopathy - Google Patents
Application of reagent for promoting miR-21 expression in preparation of medicine for preventing and/or treating diabetic keratopathy Download PDFInfo
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Abstract
The invention provides application of a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy, and belongs to the technical field of biological medicines. According to the invention, a corneal epithelium injury model is established by using type I diabetes mice, miR-21agomir and NCagomir are injected subconjunctivally, and the dyeing result of corneal fluorescein sodium shows that miR-21 is increased in vivo to promote injury repair of corneal epithelium of diabetes mice. The specific staining result of the total corneal beta-tubulin 7 days after corneal epithelium scraping shows that the density of corneal central and peripheral nerve plexuses of the miR-21agomir group is obviously higher than that of the control group. The miR-21 is increased in vivo, so that the regeneration of the peripheral corneal nerve of the diabetic mouse can be promoted. Therefore, the invention provides application of miR-21 or a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy.
Background
Diabetes mellitus is a chronic metabolic disease caused by insufficient insulin secretion or insufficient insulin utilization, is one of the most common systemic diseases worldwide, and the incidence rate is increased year by year at present. Diabetic ocular complications are potentially vision-threatening diseases, with changes in the corneal epithelium (keratopathy) and the corneal sub-basal nerves (neuropathy) occurring in about 40% to 70% of diabetic patients.
The corneal epithelium is an important ocular surface defense system, providing a physical and immune barrier to external organic and inorganic substances, and when the content of glucose in the aqueous humor is excessively high for a long time, a series of pathological changes occur in the cornea. Mainly due to abnormalities in corneal epithelial gene expression in hyperglycemic states, alterations in basement membrane components, accumulation of glycosylation products, damage to corneal nerve endings, reduction in tear secretion and oxidative stress. Corneal wound healing is a highly structured process involving a variety of cellular processes, including migration and proliferation of epithelial cells, and interaction between the epithelium and stromal fibroblasts, as well as recruitment of various growth factors. Effective re-epithelialization within a defined time is critical to avoid potential blinding microbial superinfection and corneal haze, but the re-epithelialization process is relatively difficult in diabetic patients, possibly due to associated morphological changes in the epithelium in the diabetic state, including changes in the number of epithelial cells, reduction in the number of endothelial cells, fan thinning, polymorphisms, bullae, changes in the coefficient of cellular variation, and surface debris. The main clinical manifestations are persistent corneal epithelial erosion, dry eye, superficial punctate keratopathy, delayed healing of epithelial defects, reduced corneal sensitivity, neurotrophic ulcers, etc.
The incidence of diabetes continues to rise, the treatment of complications, especially delayed healing of diabetic corneal epithelial injury, remains a challenge, the treatment mainly passes through blood sugar control and symptoms related to hyperglycemia, and at present, the clinical conventional treatment methods comprise systemic medical treatment and local treatment of eyes, artificial tears, corneal contact lenses and more serious people can perform palpebral fissure suture, but the methods only provide a good environment for corneal re-epithelialization and cannot fundamentally accelerate corneal epithelialization, so the speed of corneal repair is accelerated, and more scholars are added into the current discussion. The aim of medical treatment is to achieve optimal blood sugar control, insulin becomes the first method for treating diabetes and complications thereof by reducing blood sugar, and researches report that subcutaneous injection of insulin can simulate the release and secretion of physiological insulin, maintain normal blood sugar and restore the circadian rhythm of mitosis of epithelial cells, thereby improving delayed healing of corneal epithelial injury caused by hyperglycemia. But insulin treatment requires long-term adherence to the patient and affects the quality of life of the patient. And the method for controlling blood sugar may cause that the insulin release is not regulated by the blood sugar, or the blood sugar release does not conform to the physiological rhythm, etc., the blood sugar of the patient often fluctuates greatly, and the method is easy to cause major adverse drug reactions such as hypoglycemia syncope, etc. The goal of topical treatment is to maintain a healthy tear film and ocular surface with intact epithelium, and to alleviate the symptoms associated with improved ocular surface comfort, current clinical treatments mainly include the following: the first preservative-free artificial tear helps maintain a moist environment of the ocular surface; secondary topical anti-inflammatory agents, such as non-steroidal anti-inflammatory drugs, steroids and cyclosporin a, help to reduce ocular surface inflammation, topical ointments maintain ocular surface moisture, are longer in retention than aqueous solutions, prolong tear break-up time, and form firm adhesions between the upper and lower eyelids, helping to prevent tear film evaporation. For diabetic keratopathy, topical ointments are often used to provide comfort in the presence of corneal erosion and ulceration, rather than actually treating the defect. The third bandage lens is used for treating corneal epithelium injury, and is often used in combination with topical anti-inflammatory drugs to prolong the contact time of the therapeutic drugs with the ocular surface, and can be used as a reservoir for drug absorption and release. The fourth autologous serum and amniotic membrane transplantation can ensure the visual quality of patients by promoting the repair of corneal epithelial cells, because they contain important components of tear film such as vitamin E, platelet-derived growth factor, nerve growth factor and other growth factors which can promote the proliferation and migration of corneal epithelial cells. And fifthly, the eyelids are sutured to reduce the interpalpebral fissure, so that the injury to healing epithelium caused by blinking can be eliminated, the tear evaporation loss is reduced, and the method is mainly used for treating the diabetic keratopathy in the severe stage. Topical application of insulin also promotes tear secretion and improves dry eye symptoms in diabetic patients, but these treatments have limited ability to repair damaged corneal tissue, provide a good environment for re-epithelialization of the cornea, and do not fundamentally accelerate corneal epithelialization. Because the current clinically applied treatment only provides a good environment for the re-epithelialization of the cornea, the re-epithelialization of the cornea cannot be fundamentally accelerated, and the repair capability of the re-epithelialization of the cornea tissue is very limited; therefore, experimental studies on how to accelerate corneal repair are reported in succession, including antioxidant stress therapy, growth factor and neuroprotective factor therapy, limbal stem cell transplantation therapy, gene therapy, and the like.
MicroRNAs (MiRNAs) are a group of non-coding single-stranded RNA molecules consisting of 18-24 nucleotides, and can be widely expressed in various tissues and organs of an organism by inhibiting translation or guiding degradation of mRNA, and negatively regulating expression of a target gene at a post-transcriptional level. They play important roles in a variety of physiological and pathological processes, such as development, cell differentiation, proliferation, migration, stress response, angiogenesis, apoptosis, and cancer, and are associated with a variety of diseases. Numerous research experiments show that miRNA plays an important role in many physiological and pathological processes, and miRNA is always concerned by extensive researchers, and the dynamic role of miRNA in the regulation of tissue homeostasis is more and more interesting, but the role of related miRNA in the research of diabetes and complications thereof is very small compared with cancer so far. In fact, many miRNAs have been suggested to have physiological roles in diabetic complication tissues, but whether these miRNAs are involved in the damage that occurs in diabetes has not been fully established; the study by K μ lkarni et al determined the expression profile of mirnas in limbal cells of normal and diabetic patients, found that the expression of limbal miR-21 was higher than the corneal center, and found that the differential expression of many mirnas was found in normal cornea and in the cornea of type 1 and type 2 diabetic patients. Type 1 and type 2 diabetes mellitus are also suggested to belong to different diseases although they have the same clinical manifestations and complications due to the differential expression of the subsets of miRNAs in type 1 and type 2 diabetes patients. In recent years, therapeutic studies of miRNA in DK have been continuously reported, and their importance in the maintenance of normal corneal function and the repair of corneal injury has also been confirmed. It has been reported that miRNA is probably an intracellular regulatory factor involved in the regulation of corneal nerve growth and affecting corneal nerve regeneration; wang et al report that miR-182 can protect trigeminal ganglion cells from peripheral nerve damage in a diabetic mouse experimental model, and the study shows that miR-182 can enhance axon growth of trigeminal ganglion sensory neurons and stimulate corneal nerve regeneration by reducing expression of target gene NOX4 thereof, and a plurality of miRNAs participate in corneal damage repair process, and the study finds that miR-146a and miR-424 inhibit epithelial damage repair by influencing factors related to corneal epithelial damage repair. In addition, miR-204-Sp is also found to participate in the repair process of diabetic corneal epithelial injury by regulating SIRT 1; miR-181a and miR-34c participate in regulating the growth of the corneal nerve and trigeminal ganglion of the diabetic patient by influencing autophagy, thereby influencing the epithelial wound healing process of the diabetic patient.
The MiR-21 plays an important regulation role in the differentiation, proliferation and apoptosis processes of cells, is over-expressed in most human tumor tissues and cancer cell lines at present, plays an important role in the occurrence and development of tumors, is considered as a biomarker of circulating malignant tumors, inhibits the expression of target genes such as PDCD4, Bax and Bcl-2 by participating in post-transcriptional gene regulation, targets miR-21 and can inhibit the progress of various tumors through various ways, and a pharmacological inhibitor developed by taking miR-21 as a target spot can relieve the malignant phenotype of certain cancers. In immune system diseases, miR-21 plays an important role in development and function of immune cells, for example, miR-21 is involved in asthma generation by regulating expression of IL-12p35 (a component of IL-12), while IL-12 is a key cytokine derived from macrophages and dendritic cells and is involved in acquired immune response of polarization of Th1 cells, and the fact that miR-21 possibly plays an important role in regulation of Th1 immune response except asthma is suggested. miR-21 also has a wide effect in STAT 3-mediated immunity, and the miR-21 expression is regulated to regulate the TLR4 activity for treatment, so that the miR-21 has guiding significance in the application of TLR4 in vaccine adjuvants or inflammatory diseases such as sepsis, rheumatoid arthritis and allergic asthma. In the occurrence and development of ocular diseases, recent researches show that miR-21 plays an important regulation role, and the targeting miR-21 can be a potential method for preventing or treating various ocular diseases. Research shows that miR-21 regulates the formation of new blood vessels after corneal alkali burn, and the inhibition of miR-21 can improve the formation of corneal new blood vessels by regulating the inactivation of Sprouty 2/4 mediated p-ERK; MiR-21 can down-regulate the gene expression of TGFBI by targeting the 3' UTR of the TGFBI, and has certain guiding significance in the treatment of corneal dystrophy; in glaucoma, miR-21 can inhibit apoptosis of trabecular meshwork cells through high expression of the miR-21 and inhibit activation of microglia through NF-B and ERK 1/2 signal pathways to slow down the development process of glaucoma; in diabetic retinopathy, miR-21 participates in regulation of retinal neovascularization generation, and expression of PEDF is reduced under an ischemic and anoxic environment, and the miR-21 inhibitor can remarkably reduce proliferation, migration and angiogenesis capacity of retinal microvascular endothelial cells, so that target miR-21 can be used as a potential therapy for preventing and treating retinal neovascularization.
In the process of wound repair and nerve injury regeneration, recent researches show that miR-21 has a certain correlation with wound repair and nerve regeneration, and in the research of healing of diabetic wounds, the research finds that miR-21 can participate in the up-regulation of MMP-1 and MMP-3 in fibroblasts in the wound healing process and inhibit the expression of TIMP3 and RECK, possibly through the regulation of Matrix Metalloproteinase (MMPs) to improve the functions of the fibroblasts, and the matrix metalloproteinase can participate in extracellular matrix (ECM) remodeling subsequently, so that the wound healing is accelerated. In addition, research reports that miR-21-5p promotes the proliferation and migration of keratinocytes in vitro through a Wnt/beta-catenin signal pathway and promotes diabetic wound healing in vivo through promoting re-epithelialization, collagen remodeling, angiogenesis and vascular maturation. On the other hand, in the nerve regeneration process, experimental research has proved that miR-21 participates in the regeneration and repair of peripheral nerves, and research finds that miR-21 can be used as a treatment drug to stimulate the regeneration of peripheral nerves after nerve injury. In vitro experimental studies report that miR-21 can regulate expression of a target gene PTEN, so that apoptosis of Schwann cells is reduced, which is probably one of key mechanisms of miR-21 playing a protective role in peripheral nerve injury repair, but the role and mechanism of miR-21 in occurrence and development of diabetic keratopathy are not clear at present.
Disclosure of Invention
In view of the above, the invention aims to provide an application of a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy.
The invention aims to provide a novel regeneration promoting medicine with small side effect, which has good treatment effect on diabetic keratopathy and corneal peripheral nerve regeneration.
The invention provides an application of miR-21 in preparation of a medicine for preventing and/or treating diabetic keratopathy.
The invention provides application of a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy.
Preferably, the nucleotide sequence of the miR-21 is shown in SEQ ID NO 1.
Preferably, the diabetic keratopathy includes diabetic corneal epithelial cell damage and diabetic peripheral corneal nerve reduction.
Preferably, the diabetes comprises type I diabetes.
Preferably, the agent for promoting miR-21 expression comprises a mimic of miR-21 or an analog of miR-21.
Preferably, the mimetic of miR-21 comprises a miR 21 agonist;
the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1;
the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2.
The invention provides a medicine for treating diabetic keratopathy, which comprises an agonist of miR-21 and pharmaceutically acceptable auxiliary materials;
the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1;
the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2.
Preferably, the medicament is in the form of injection;
the concentration of the miR-21 agonist in the injection is 180-250 nM.
The invention provides an application of miR-21 in preparation of a medicine for preventing and/or treating diabetic keratopathy. Firstly, a miR-21 mouse (miR-21KO) and a Wild Type (WT) mouse are knocked out of the whole body of the miR-21 mouse to induce a type I diabetes model, blood sugar and body weight are measured and recorded 4 months after the model is formed, and the blood sugar and body weight of the miR-21KO and WT diabetes mouse are shown to have no obvious difference, so that the miR-21 does not influence the severity of diabetes in the later stage of the type I diabetes mouse. A corneal epithelium damage model is established by using miR-21KO and WT diabetic mice, corneal fluorescein sodium is used for dyeing and observing epithelial defect conditions of 0h, 24h, 36h and 48h after corneal epithelium scraping, the result shows that the corneal epithelium repairing speed of the miR-21KO mice is obviously slower than that of the WT mice, and the specific dyeing result of the total corneal beta-tubulin after corneal epithelium scraping shows that the corneal peripheral nerve density of the miR-21KO mice is lower than that of the WT mice. Meanwhile, the beta-tubulin specific staining is carried out by using normal mice and diabetic mice of miR-21KO and WT under the condition of not damaging corneal epithelium, and the result shows that the corneal peripheral nerve density of the miR-21KO diabetic mice is obviously lower than that of the WT mice, which indicates that the loss of miR-21 inhibits the growth of corneal peripheral nerves of the diabetic mice. By utilizing a C57 mouse I-type diabetes model, miR-21agomir is given to an experimental group mouse for treatment 24h before and during corneal epithelium scraping, NC-agomir is given to a control group mouse, corneal fluorescein sodium staining is utilized to observe statistical epithelial defect conditions after corneal epithelium scraping, and the result shows that the corneal epithelium repairing speed of the diabetic mouse treated by miR-21agomir is obviously accelerated. The specific staining result of the total corneal beta-tubulin after corneal scraping shows that the corneal peripheral nerve regeneration density of the diabetes mice of the miR-21agomir treatment group is higher than that of the control mice. The results show that the miR-21 or the reagent for promoting miR-21 expression can promote corneal epithelium repair and corneal peripheral nerve regeneration of mice, can be applied to treatment of keratopathy diseases, and can be used for preparing medicaments for keratopathy.
The invention provides a medicine for treating diabetic keratopathy, which comprises an agonist of miR-21 and pharmaceutically acceptable auxiliary materials; the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1; the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2. The agonist of the miR-21 has the advantages of small molecular weight, simple structure and convenient administration mode; 3) the chemically modified miR-21agomir has high stability and can play a role stably in vivo.
Drawings
FIG. 1 shows that there is no significant difference between miR-21KO and WT diabetic mice in blood sugar and body weight; (A) the blood sugar of the miR-21KO and WT diabetic mice is obviously higher than that of the miR-21KO and WT normal mice, but the blood sugar level of the miR-21KO and WT diabetic mice is not obviously different (n is 9); (B) the body weights of the miR-21KO and WT diabetic mice are obviously lower than those of normal miR-21KO and WT normal mice, but the body weights of the miR-21KO and WT diabetic mice are not obviously different (n is 9); ns represents P > 0.05; represents P < 0.05;
FIG. 2 shows that the repairing speed of the corneal epithelium damage of the miR-21KO diabetic mouse is slower than that of the WT mouse, and the corneal nerve regeneration density is lower than that of the WT mouse; (A) repairing epithelia of WT and miR-21KO diabetic mice at different time points after the epithelia is scraped; (B) statistically analyzing the corneal epithelium defect rate (n is 4), wherein the corneal epithelium repairing speed of the miR-21KO diabetic mouse is obviously slower than that of the WT diabetic mouse; (C) corneal nerve staining at day 14 after corneal epithelium scraping; (D) statistically analyzing the density of the peripheral corneal nerves of each group (n is 4), wherein the peripheral corneal nerve plexus density of the miR-21KO diabetic mice is obviously lower than that of the WT diabetic mice; represents P < 0.05;
FIG. 3 shows that there is no significant difference between the corneal nerve density of miR-21KO and WT normal mice when the epithelium is not scraped, and the corneal nerve density of miR-21KO diabetic mice is lower than that of WT mice; (A) normal WT and miR-21KO mouse corneal nerve staining; (B) the nerve density (n is 3) in A is analyzed statistically, and the result shows that the corneal nerve density of two groups of mice has no obvious difference; (C) WT Diabetic Mice (DMWT) and miR-21KO diabetic mice (DM miR-21KO) undergo corneal nerve staining; (D) statistically analyzing the nerve density of each group (n is 4), and displaying that the nerve density of the miR-21KO diabetic mice is lower than that of the WT group; ns represents P > 0.05; represents P < 0.05;
FIG. 4 shows that miR-21agomir treatment promotes corneal epithelium repair and corneal nerve regeneration after diabetic mice are injured; (A) the expression of the posterior corneal epithelial tissue miR-21 after the subconjunctival injection of the miR-21agomir of the diabetic mouse is obviously up-regulated compared with that of a control group (NC); (B) injecting miR-21agomir and NC agomir under the conjunctiva of the diabetic mice, and carrying out fluorescein sodium staining at different time points after scraping the epithelium, wherein the result shows that the epithelium healing rate of the miR-21 group (miR-21agomir) is remarkably higher than that of the control group (NC); (C) analyzing the epithelial defect rate of each group by using Image J software; (D) the results of comparison of the regeneration density of the corneal peripheral nerve plexus of two groups of mice after corneal epithelium scraping show that the corneal epithelial nerve plexus density of the miR-21 group (miR-21agomir) is obviously higher than that of the control group (NC); (E) statistically analyzing each group of corneal epithelial plexus density (n-5); represents P < 0.05.
Detailed Description
The invention provides an application of miR-21 in preparation of a medicine for preventing and/or treating diabetic keratopathy. The nucleotide sequence of the miR-21 is preferably shown as SEQ ID NO. 1.
In the present invention, the corneal lesion preferably includes corneal epithelial lesions and corneal basal neuropathy. The corneal epithelial disorder is preferably corneal epithelial cell damage. The corneal basal neuropathy includes a corneal peripheral nerve decrease. The corneal lesions are mainly clinically manifested by loss of corneal sensitivity, repeated erosion of corneal epithelium, delayed healing of epithelial defects, dry eye, neuropathic corneal ulcer and the like.
In the invention, a miR-21 knockout mouse and a Wild Type (WT) mouse are taken as experimental objects to induce an I type diabetes model, and the blood sugar and the body weight of two groups of mice are determined to have no obvious difference, which indicates that miR-21 does not influence the severity of diabetes at the later stage of the type 1 diabetes mouse. Then, a miR-21 knockout mouse and a Wild Type (WT) mouse are used as experimental materials to construct a corneal epithelium damage model, the corneal epithelium repair speed of the mice in the two models is lower than that of the WT mice, and the specific staining result of beta-tubulin shows that the corneal peripheral nerve density of the miR-21KO diabetic mouse is obviously lower than that of the WT mouse, which indicates that the loss of miR-21 inhibits the growth of the corneal peripheral nerve of the diabetic mouse. It can be seen that the loss of miR-21 causes the damage repair speed of the diabetic corneal epithelial cells to be reduced and the regeneration of corneal peripheral nerves to be unfavorable. The invention achieves the purpose of treating diabetic keratopathy by improving the expression level of miR-21 in diseased tissues. In the present invention, the method for increasing the expression level of miR-21 in the diseased tissue is preferably a transgenic method well known in the art. Meanwhile, the invention also preferably provides application of the medicament taking miR-21 as a target point in treating diabetic keratopathy. The present invention is not particularly limited in the kind of the drug, and those known in the art can be used.
In the invention, in order to verify whether increasing miR-21 in corneal tissue can accelerate the corneal epithelial injury repair speed of a diabetic mouse and promote the regeneration of peripheral corneal nerves, a mode of injecting miR-21 agonist (miR-21agomir) under the conjunctiva of the diabetic mouse model is adopted for verification. The result shows that the corneal epithelium repair speed of the diabetic mice treated by the miR-21agomir is obviously accelerated. The specific staining result of the total corneal beta-tubulin after corneal scraping shows that the corneal peripheral nerve regeneration density of the diabetic mice treated by miR-21agomir is higher than that of the control mice. The results show that the expression of miR-21 in the corneal tissue of a diabetic mouse can be specifically increased to promote the corneal epithelium repair and nerve regeneration of the mouse. Therefore, the invention provides an application of a reagent for promoting miR-21 expression in preparation of a medicine for preventing and/or treating diabetic keratopathy. The diabetic keratopathy includes diabetic corneal epithelial cell damage and diabetic peripheral corneal nerve reduction. Preferably, the diabetes comprises type I diabetes. The reagent for promoting miR-21 expression preferably comprises a mimic of miR-21 or an analogue of miR-21. The mimetic of miR-21 preferably comprises a miR 21 agonist; the nucleotide sequence of the sense strand of the miR 21 agonist is preferably shown as SEQ ID NO. 1; the nucleotide sequence of the antisense strand of the miR 21 agonist is preferably shown as SEQ ID NO. 2.
The invention provides a medicine for treating diabetic keratopathy, which comprises an agonist of miR-21 and pharmaceutically acceptable auxiliary materials; the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1; the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2. The dosage form of the medicament is preferably injection. The method for preparing the drug is not particularly limited, and the method for preparing the drug well known in the field can be adopted. The concentration of the miR-21 agonist in the injection is preferably 180-250 nM, and more preferably 200 nM. The administration time of the drug is preferably 24 hours before and after the corneal epithelium scraping operation. The administration frequency of the medicine is 24h 1 times before operation and 1 time in operation. The dose of the drug administered was 2 times.
The following examples are provided to illustrate the application of the agent for promoting miR-21 expression in the preparation of a medicament for preventing and/or treating diabetic keratopathy in detail, but they should not be construed as limiting the scope of the present invention.
1. Sources of Experimental materials
(1) Laboratory animal
C57BL/6J mice (purchased from Weitongli, Beijing) of 6-8 weeks old, weighing 18-25g, healthy males;
at 6 weeks of age, healthy miR-21 knockout (miR-21KO) and wild-type (WT) mice (purchased from Shanghai, Nandina Biotech, Shanghai).
(2) Solution preparation:
1) preparing an anesthetic: adding 8% ketamine hydrochloride injection and 2% chlorpromazine hydrochloride injection into 0.9% sodium chloride injection water, mixing, and storing in 4 deg.C refrigerator.
2) STZ-citrate solution (7.5 mg/mL):
2.1) weighing of STZ: the standard of mouse intraperitoneal injection is STZ 60mg/kg, the daily STZ dosage is respectively subpackaged in an EP tube according to the quantity and the weight of mice, and the mice are externally wrapped and sealed by aluminum foil and stored at the temperature of-20 ℃.
2.2) citrate buffer: weighing 2.1g of citric acid, adding into 100m1 of double distilled water, and dissolving; 2.94g of sodium citrate is added into 100m1 of double distilled water for dissolution, then the two solutions are mixed according to the proportion of 1:1, and the pH value is adjusted to 4.2-4.5.
2.3) adding a proper amount of citrate buffer solution into an EP tube of the STZ to dissolve the STZ, finally, the solubility of the STZ-citrate solution is 7.5mg/ml, the mixed solution needs to be prepared immediately, the injection is completed within 15 minutes after the preparation as far as possible, and the whole process needs to be protected from light because the STZ is easy to degrade and inactivate.
3) Fluorescein sodium dye liquor: 1 piece of fluorescein sodium test paper (Tianjin Jingming New technology development Co., Ltd.) was put into a 1.5mL EP tube, and 1mL of sterile physiological saline was added, and the EP tube was wrapped with aluminum foil.
4) Preparation of MiR-21agomir and NC: miRNA agomir is miRNA mimic, and is transiently centrifuged and then treated with RNase-free ddH in a sterile super clean bench2Dissolving into 20 μ M stock solution, packaging each tube according to 20 μ l, and placing in refrigerator at-20 deg.C to-80 deg.C to avoid repeated freeze thawing.
miR-21agomir and negative control (NC control): synthesized by Roibo Biotech, Inc.
miR-21 agomir: sense strand: UAGCUUAUCAGACUGAUUGAUUGA (SEQ ID NO: 1);
antisense strand: AUCGAAUAGUCUGACUACAACU (SEQ ID NO: 2);
NC agomir control: sense strand: UCACAACCUCCUAGAAAGAGUAGA (SEQ ID NO: 3);
antisense strand: AGUGUUGGAGGAUCUUUCUCAUCU (SEQ ID NO: 4).
Example 1
Establishment of type I diabetes mouse model:
1) male C57 mice of 6-8 weeks are adopted, the mice are bred for one week after purchase, and the weight and the blood sugar of each mouse are recorded before injection; WT and miR-21KO male mice were raised in the same way until 6-8 weeks before the induction model was initiated.
2) Mice to be subjected to intraperitoneal STZ injection were fasted for 12 hours.
3) The STZ-citrate solution is prepared at present, the final concentration is 7.5mg/mL, the injection volume is calculated according to the dose of 60mg/kg for each mouse, l mL insulin injection is used for carrying out intraperitoneal injection on the mouse, attention is paid to the light-proof process during the injection, food is put into the mouse after the injection is finished, drinking water is replaced by 5% glucose, and the drinking water is replaced by normal water before fasting.
4) Control mice of the same age were injected intraperitoneally with the same volume of citrate buffer.
5) Intraperitoneal injection of STZ-citrate solution was performed according to the fasting and injection procedure described above for 5 consecutive days.
6) The next morning after completion of the 5 th injection, mice were switched from 5% glucose to normal water.
7) And weighing the weight of the mouse at the 3 rd month of the last injection, and measuring the blood sugar of the tail venous blood of the mouse three times by using a glucometer, wherein the blood sugar of the tail venous blood of the mouse is measured twice or more than 16.7mmol/L to judge that the induction of the type I diabetes model is successful (the blood sugar of the mouse still needs to be checked before the mouse is selected for the experiment every time).
Blood glucose and body weight were measured and recorded using a glucometer 4 months after the molding, while blood glucose and body weight were determined for WT normal mice (CONWT) and miR-21KO normal mice (CON miR-21 KO).
The results are shown in FIG. 1. The blood sugar of the diabetic mouse group is obviously increased compared with that of the normal mouse (A in figure 1), the body weight of the diabetic mouse group is obviously reduced compared with that of the normal mouse group (B in figure 1), but the blood sugar value and the body weight of the WT and miR-21KO diabetic mice have no obvious difference (A and B in figure 1). From the above, there is no obvious difference between the blood sugar and the body weight of the miR-21KO and WT diabetic mice, which indicates that miR-21 does not influence the severity of diabetes in the later stage of the type I diabetic mice.
Example 2
Establishing a mouse corneal epithelial injury repair model:
1) selecting the diabetic mice and the age-matched normal mice successfully induced in the embodiment 1, and recording blood sugar and weight; for WT and miR-21KO diabetic mice, mice with similar blood sugar and body weight were selected for the experiment.
2) General anesthesia of mice: 0.3ml of the prepared anesthetic was extracted and injected into the left lower abdomen of the mouse.
3) The right eye is the experimental eye, the left eye is not treated, after the mouse is anesthetized, the horns and the eyelashes are cut off by using the corneosclera, the peripheral part of the experimental eye is disinfected by using 0.5 percent of an iodine skin disinfectant, and 1 drop of an alkein eye drop is dropped into the conjunctival sac for surface anesthesia.
4) The trephine with a diameter of 2.5mm is gently pressed and rotated in the center of the mouse cornea to leave an impression on the surface, the strength is paid attention to avoid damaging the stroma, and the trephine edge cannot exceed the corneoscleral edge.
5) Gently scraping corneal epithelium along the trephine indentation in the area within the trephine indentation using a motorized epithelium scraper; note that the corneal epithelium in the indentation should be scraped off entirely, but not beyond the boundary of the indentation.
6) Staining with fluorescein sodium: at different time points after the epithelial injury is scraped, the experimental eye is dyed by fluorescein sodium test paper, the healing condition of the corneal epithelial injury of the mouse is observed by cobalt blue light under a slit lamp, and the picture is taken.
7) Measurement of epithelial defect area: and measuring and calculating the corneal epithelial defect area by using Image J software, calculating the defect rate of each time point, and analyzing and comparing the corneal epithelial defect rates of all groups.
The results are shown in FIG. 2. Establishing a corneal epithelial injury model by using a WTMR-21 KO diabetic mouse, wherein fluorescein sodium staining results at different time points show that the repairing speed of the corneal epithelium of the miR-21KO diabetic mouse is obviously slower than that of the WT diabetic mouse (A and B in a picture 2); on day 14 after corneal epithelial injury, miR-21KO diabetic mice showed significantly lower pericorneal plexus density than WT diabetic mice by whole-cornea β -tubulin specific staining results (C and D in fig. 2). The results show that the deletion of miR-21 inhibits the corneal epithelium repair and the corneal peripheral nerve regeneration of the diabetic mouse.
Example 3
Mouse whole cornea plating nerve staining
1) Taking miR-21KO normal mice, WT diabetic mouse models (DMWT) and miR-21KO diabetic mouse models (DM miR-21KO) as experimental objects respectively, killing the mice by a conventional method, taking out eyeballs according to groups, respectively putting the eyeballs into EP tubes filled with Zamboni stationary liquid, and fixing the eyeballs on ice for 1 h.
2) Cutting corneal tissue, taking attention to the fact that in the corneal membrane cutting process, fixing liquid completely submerges eyeballs and has a sclera edge of 0.5mm, removing redundant irises as far as possible, and continuing to fix the eyeballs on ice for 1h (a 24-hole plate is used, a 1ml gun is used for blowing and washing when the cornea is transferred, and endothelial side deposition and accumulation are prevented); the anterior half parts of the eyeball can be cut off together and then put into the fixative, and then the irises are cleaned one by one (cornea shrinkage can be avoided, and the irises are easy to clean).
3) The cornea was washed 3 times with PBS on a shaker for 5 minutes each time (24 well plates, note change wells and change solutions).
4) Placing the cornea into a 96-well plate containing 200 mul of sealing liquid in each well, placing 1 cornea in each well, soaking for 2h at room temperature, wherein the sealing liquid: TBS (6ml) + 5% BSA (2ml) + goat serum (2ml) + 40. mu.l 100% Triton-X100.
5) The Anti-Tubulin beta 3 antibody and the solution are prepared according to the proportion of 1:200, 200 mu l of the antibody is added into each hole, the mixture is kept overnight at 4 ℃, and the mixture is put into a wet box and protected from light.
6) The next morning, the 96-well plate was removed, rewarmed for 2 hours at room temperature, the corneas were placed in 24-well plates, one cornea per well, 400 μ l PBS per well, placed on a shaker at room temperature, washed 6 times in the dark, 10min each time.
7) Taking a clean glass slide, lightly clamping the cornea by using toothless forceps, dropping a drop of PBS to cut into 6 pieces, enabling the inner surface to face upwards, sucking the excess PBS at the edge of the cornea by using filter paper, dropping a drop of fluorescence quenching sealing tablet, lightly covering a glass slide along one side, paying attention to no bubbles, keeping the box in a dark wet state at 4 ℃, taking pictures and storing the pictures by using a confocal microscope within 1 day as far as possible, quantifying the corneal nerve density by using Image J Image software, analyzing and comparing the corneal nerve density of each group.
The results are shown in FIG. 3. Total corneal nerve staining of WT and miR-21KO mice in the normal, non-epithelialized and diabetic cases. The specific staining result of the whole cornea beta-tubulin shows that the corneal nerve density of WT and miR-21KO normal mice is not obviously different (A in figure 3); the results of corneal nerve staining of WT Diabetic Mice (DMWT) and miR-21KO diabetic mice showed that the nerve density of miR-21KO diabetic mice was lower than that of the WT mouse group (n ═ 4) (C in fig. 3). No obvious difference exists between miR-21KO and WT normal mice in the growth of the peripheral nerves of the cornea, but the peripheral nerve density of miR-21KO diabetic mice is obviously lower than that of WT mice, which indicates that the loss of miR-21 inhibits the growth of the peripheral nerves of the cornea of diabetic mice.
Example 4
A corneal epithelial injury model is established by using the type I diabetes mellitus mouse constructed in the example 1, miR-21agomir and NC agomir are injected subconjunctivally, and a mouse without corneal epithelial injury is used as a control. According to the experimental design requirements, according to different groups, medicines are respectively injected under conjunctiva 24h before operation, after operation or 24h after operation (the injection dose of miR-21agomir and NC agomir is 0.25nmol each time), and antibiotic eye ointment is coated after each operation to prevent the operation eye infection. miR-21agomir, sense strand: UAGCUUAUCAGACUGAUUGAUUGA (SEQ ID NO: 1); antisense strand: AUCGAAUAGUCUGACUACAACU (SEQ ID NO: 2). NC agomir: sense strand: UCACAACCUCCUAGAAAGAGUAGA (SEQ ID NO: 3); antisense strand: AGUGUUGGAGGAUCUUUCUCAUCU (SEQ ID NO: 4). The staining of sodium corneal fluorescein and the specific staining of whole cornea beta-tubulin were performed at 0h, 24h, 36h and 48h, respectively, as detailed in examples 2 and 3.
The results are shown in FIG. 4. A corneal epithelial injury model is established by using type I diabetes mice, miR-21agomir and NC agomir are injected subconjunctivally, and the corneal fluorescein sodium staining result shows that the epithelial repair speed of the miR-21agomir treatment group (miR-21agomir) is obviously higher than that of the control group (NC) (A and B in a figure 4), and the result shows that the in vivo increasing of miR-21 can promote the corneal epithelial injury repair of the diabetes mice. The results of specific staining of the whole cornea with beta-tubulin 7 days after corneal epithelium scraping showed that the density of corneal central and peripheral nerve plexuses was significantly higher in the miR-21agomir group (miR-21agomir) than in the control group (NC) (C and D in FIG. 4). The results show that miR-21agomir treatment can increase in-vivo miR-21 expression and can promote corneal epithelium repair and corneal nerve regeneration of diabetic mice after corneal epithelium injury.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shandong first medical university affiliated Qingdao ophthalmological Hospital (Shandong province institute of ophthalmology, Qingdao ophthalmological Hospital)
Application of miR-21 expression promoting reagent in preparation of medicine for preventing and/or treating diabetic keratopathy
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Claims (9)
- Application of miR-21 in preparation of medicines for preventing and/or treating diabetic keratopathy.
- 2. Application of a reagent for promoting miR-21 expression in preparation of medicines for preventing and/or treating diabetic keratopathy.
- 3. The use of claim 1 or 2, wherein the nucleotide sequence of miR-21 is shown as SEQ ID NO 1.
- 4. The use of claim 1 or 2, wherein the diabetic keratopathy includes diabetic corneal epithelial cell damage and diabetic peripheral corneal nerve reduction.
- 5. The use of claim 4, wherein the diabetes mellitus comprises type I diabetes mellitus.
- 6. The use of claim 2, wherein the agent that promotes miR-21 expression comprises a mimic of miR-21 or an analog of miR-21.
- 7. The use of claim 6, wherein the mimetic of miR-21 comprises an agonist of miR 21;the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1;the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2.
- 8. The medicine for treating diabetic keratopathy is characterized by comprising an agonist of miR-21 and pharmaceutically acceptable auxiliary materials;the nucleotide sequence of the sense strand of the miR 21 agonist is shown as SEQ ID NO 1;the nucleotide sequence of the antisense strand of the miR 21 agonist is shown in SEQ ID NO. 2.
- 9. The medicament of claim 8, wherein the medicament is in the form of an injection;the concentration of the miR-21 agonist in the injection is 180-250 nM.
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