Application of human multifunctional proteoglycan in preparation of medicine for promoting proliferation and / or migration and / or dryness maintenance of corneal limbal stem cells

Abstract

The invention provides application of human multifunctional proteoglycan in preparation of drugs for promoting proliferation and / or migration and / or dryness maintenance of corneal limbal stem cells. The human multifunctional proteoglycan has positive effects on proliferation, migration and dryness maintenance of corneal limbal stem cells, is used for preparing the eye drops for treating corneal epithelium injury, can effectively treat corneal limbal stem cell deficiency, promotes corneal epithelium regeneration, and effectively repairs corneal epithelium injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of human versican in the preparation of drugs for promoting the proliferation and / or migration and / or the maintenance of the stemness of limbal stem cells. Background Art

[0002] Limbal stem cell deficiency is an eye disease characterized by corneal conjunctivalization, which can lead to corneal neovascularization, corneal opacity, and even blindness. Currently, surgical treatment is the main treatment, but there are still problems such as insufficient donor sources and immune rejection. How to restore the function of limbal stem cells and rebuild the limbal stem cell microenvironment to repair corneal epithelial damage is a current research hotspot. Summary of the invention

[0003] In view of the defects in the prior art, the object of the present invention is to provide the use of human versican in the preparation of drugs for promoting the proliferation and / or migration and / or maintaining the stemness of limbal stem cells.

[0004] The present invention provides the use of human multifunctional proteoglycan in the preparation of a drug for promoting the proliferation and / or migration and / or the maintenance of the stemness of limbal stem cells.

[0005] In one embodiment, the application is specifically: use of human versican in the preparation of a drug for treating corneal epithelial damage.

[0006] In one embodiment, it is characterized in that the drug comprises human versican.

[0007] In one embodiment, the medicament further comprises a pharmaceutically acceptable excipient.

[0008] In one embodiment, the auxiliary material is PF-127 hydrogel.

[0009] In one embodiment, the concentration of human versican in the medicament is 0.5 μg / ml.

[0010] In one embodiment, the drug is eye drops.

[0011] In one embodiment, the eye drops are prepared by the following method: Excipient preparation: PF-127 powder and water were prepared into PF-127 hydrogel with a mass concentration of 18 wt%; Preparation of eye drops: human versican powder is added to the excipients to prepare a mixed solution with a human versican concentration of 0.5 μg / ml, and the mixed solution is shaken at 4° C. overnight until a colorless and transparent solution is obtained, which is the eye drops.

[0012] Compared with the prior art, the present invention has the following beneficial effects: due to the small sources of limbal stem cells and the lack of effective treatment methods for patients with limbal stem cell deficiency, new treatment methods are urgently needed to promote the proliferation of limbal stem cells and the repair of the ocular surface. Therefore, the present invention provides the use of human multifunctional proteoglycan in the preparation of drugs for treating corneal epithelial damage. Human multifunctional proteoglycan has a positive effect on the proliferation, migration and stemness maintenance of limbal stem cells. Human multifunctional proteoglycan is used to prepare drugs for treating corneal epithelial damage, which can effectively treat limbal stem cell deficiency and promote corneal epithelial regeneration, and effectively repair corneal epithelial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 To test the biocompatibility of versican and PF-127 hydrogel in vivo and in vitro; Figure 2 This is a diagram to test the effect of versican on the proliferation of limbal stem cells; Figure 3 This is a diagram to test the effect of versican on the migration of limbal stem cells; Figure 4 This is a diagram to test the effect of versican on maintaining the stemness of limbal stem cells; Figure 5 This is a diagram to test the effect of versican-loaded PF-127 hydrogel on corneal epithelial repair; Figure 6 To test the effect of versican-loaded PF-127 hydrogel on corneal epithelial and structural repair; DETAILED DESCRIPTION

[0014] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0015] Example 1 This embodiment provides an eye drop for treating corneal epithelial damage, the eye drop comprises human multifunctional proteoglycan, and the eye drop further comprises a pharmaceutically acceptable excipient, the excipient is PF-127 hydrogel, and the eye drop for treating corneal epithelial damage is prepared by the following method: PF-127 powder and water are prepared into PF-127 hydrogel with a mass concentration of 18 wt%; Human versican powder (purchased from Raybiotech) was added to the PF-127 hydrogel to prepare a mixture with a concentration of 0.5 μg / ml of human versican, and the mixture was shaken at 4° C. overnight until a colorless and transparent solution was obtained, which is the drug for treating corneal epithelial damage.

[0016] The obtained eye drops for treating corneal epithelial damage are in liquid state at a relatively low temperature.

[0017] The eye drops for treating corneal epithelial damage in this embodiment are directly dripped onto the eyes when used, and turn into solid hydrogel after about 15 seconds, which continuously releases human versican.

[0018] Experimental example 1. Isolation and culture of limbal stem cells All procedures were approved by the Medical Ethics Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Limbal stem cells were isolated from 10-12 week-old New Zealand white rabbits and cultured in cell suspension. The limbal rings were washed with phosphate-buffered saline containing 50 μg / mL penicillin-streptomycin. After removing the excess sclera, conjunctiva, iris, and central cornea, the limbal rings were placed in 10 mg / mL Dispase II (Sigma-Aldrich) at 4°C overnight. Subsequently, the epithelial cell sheets were separated from the underlying stroma and digested with Tryple at 37°C for 5 minutes. The obtained single cell suspension was seeded and cultured in DMEM / F12 medium (Invitrogen), and 10% serum and 1% penicillin-streptomycin antibiotics were added to the medium. When the cell confluence reached 80%-90%, the limbal stem cells were subcultured with Tryple, and the 1st to 3rd generation limbal stem cells were used for further experiments.

[0019] 2. Biocompatibility of drugs and hydrogels.

[0020] For qualitative cell viability assessment and biocompatibility, a live / dead viability / cytotoxicity kit (Thermo Fisher) was used according to the manufacturer's instructions. In short, the limbal stem cells cultured in Example 1 were incubated in PBS with 2 μM calcein acetoxymethyl ester and 4 μM ethylenediamine homodimers for 15 minutes in the dark at 37°C. After removing the stain with PBS, images were captured under a fluorescence microscope, with red for dead cells and green for live cells, and statistical analysis was performed. For in vivo biocompatibility, healthy New Zealand white rabbits were instilled with saline or versican-loaded PF-127 hydrogel twice a day for fourteen days to observe changes in corneal transparency and morphology.

[0021] The results are as follows Figure 1 As shown, by Figure 1 It can be seen that both versican and PF-127 hydrogels have good biocompatibility. Green represents the number of living cells, and red represents the number of dead cells. According to statistical analysis, the proportion of dead cells to the total number of cells is less than 5%, indicating good in vitro biocompatibility ( Figure 1 Middle AB). Figure 1 ( Figure 1 The same applies to Chinese CD). Figure 1 In Figure E, the rabbits were given normal saline or the invented eye drops twice a day for 14 consecutive days, and it was found that the rabbits' eyes had good transparency and no abnormal reactions, which proved good in vivo biocompatibility. In summary, versican and PF-127 hydrogels both have good biocompatibility.

[0022] 3. CCK8 proliferation assay The corneal limbal stem cells cultured in Example 1 were seeded on a 96-well plate at 2,000 cells per well, and different concentrations (0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL) of versican were added. At 24 hours, 48 ​​hours, and 72 hours, 10 μL of CCK-8 solution was added to each well of the 96-well plate, respectively. Be careful not to generate bubbles in the wells, which would affect the absorbance. Then, the culture plate was returned to the incubator for further incubation for 4 hours, and the absorbance at 450 nm was measured with an ELISA reader. Cell viability and proliferation were calculated and analyzed based on the measured absorbance.

[0023] The results are as follows Figure 2As shown, versican can promote the proliferation of limbal stem cells, and the concentration of 50ng / ml is the best. At 24, 48, and 72 hours, the absorbance at 450nm of the groups with different concentrations of versican was higher than that of the blank group (absorbance reflects the level of cell proliferation), and the absorbance of 50ng / ml was the highest, so the proliferation ability of 50ng / ml was the strongest. Therefore, it was concluded that versican can promote the proliferation of limbal stem cells, and the concentration of 50ng / ml is the best.

[0024] IV. Migration scratch test The limbal stem cells cultured in Example 1 were 1×10 5 The cells were cultured at a density of 100 μg / well in a 6-well plate. The cells were grown to 80%-90% confluence and then scratched using a 200 μl sterile pipette tip to create a cell-free area. The suspended cells were then washed three times with PBS (Gibco) to remove them and the cells were divided into a versican (50 ng / mL) group and a control group (without versican). The scratched area was imaged at the same position under a microscope at 0, 6, and 12 h, and analyzed using Image J software.

[0025] The results are as follows Figure 3 As shown, versican can promote the migration of limbal stem cells. The narrower the scratch area in the middle, the faster the cells on both sides migrate. At 6h and 12h, the migration speed of the versican group was significantly faster.

[0026] 5. Dryness test Western blotting Limbal stem cells (versican-treated and untreated groups) were lysed using RIPA lysis buffer (Beyotime). Protein extracts were then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore). Nonspecific sites on the membrane were then blocked with 5% bovine serum albumin (BSA) in TBS buffer at room temperature. Primary antibodies were incubated overnight with stemness markers ABCB5 (Santa Curz), p63 (Abcam), and differentiation marker CK12 (Proteintech). The next day, samples were treated with fluorescently labeled secondary antibodies for 1 h at room temperature and scanned to compare protein expression.

[0027] The results are as follows Figure 4 ( Figure 4 As shown in Figure AB), versican maintains the stemness of limbal stem cells to a certain extent. Figure 4 ( Figure 4AB in the vertebral canopy showed that the protein expression of ABCB5 and P63, which represent the stemness of limbal stem cells, was higher in the versican group (in terms of thickness, depth, etc.), while the protein expression of CK12, which represents differentiation, was lower in the versican group. This proves that versican maintains the stemness of limbal stem cells to a certain extent. Gene expression of stemness markers P63, ABCG2, CK15 and differentiation markers CK3, CK12 (qPCR) Total RNA was extracted from limbal stem cells grown on 6-well plates, and the relative expression of mRNA was analyzed using the Pfaffl method. Purified RNA was quantified using NanoDrop™2000 (Thermo Fisher Scientific). cDNA was synthesized from 2000 ng of total RNA using a high-capacity RTPCR reverse transcription kit. Real-time PCR was performed using an ABI Prism 7000 instrument with SYBRGreen PCR Master Mix (Yeasen, China) and 2 -△△ The relative expression of stemness indexes P63, ABCG2, CK15 and differentiation indexes CK3 and CK12 were determined by CT method. The primer sequences are shown in Table 1. Figure 4 ( Figure 4 As shown in Figure C, versican maintains the stemness of limbal stem cells to a certain extent. Through qPCR and statistical analysis, it was found that the gene expression levels of P63, ABCG2, and CK15, which represent the stemness, increased in the versican group, while the gene expression levels of CK3 and CK12, which represent the differentiation indicators, decreased in the versican group, further proving that versican maintains the stemness of limbal stem cells to a certain extent. Table 1 Immunofluorescence staining The samples were fixed with 4% PFA for 20 minutes at room temperature and then fixed three times with PBS. The cell membrane was permeabilized with 0.3% triton X-100 (Sigma Aldrich) for 15 minutes and then blocked with 5% goat serum for 1 hour at room temperature. The samples were stained with primary antibodies for the stemness indicator P63 (Proteintech), CK14 (Abcam), and the differentiation indicator CK12 (Proteintech) at 4°C overnight. The next day, the specimens were washed three times with PBS and incubated with Alexa fluor-labeled secondary antibodies for 1 hour at room temperature. The samples were washed with PBS and nuclear staining was performed with DAPI (4', 6-diamidino-2-phenylindole) for 20 minutes, and then images were acquired using a fluorescence microscope. The staining was repeated at least 3 times. The results are shown in Figure 2. Figure 4 ( Figure 4 As shown in DF in Figure 5, versican maintained the stemness of limbal stem cells to a certain extent. Immunofluorescence staining showed that the positive rates of stemness markers P63 and CK14 were higher in the versican group, while the positive rate of differentiation marker CK12 was lower in the versican group, further proving that versican maintained the stemness of limbal stem cells to a certain extent.

[0028] VI. Clinical Evaluation A limbal stem cell deficiency model was established using New Zealand white rabbits weighing approximately 2 kg. All animal experiments were approved by the Medical Ethics Committee of the Ninth People's Hospital affiliated to Shanghai Jiao Tong University School of Medicine. Anesthesia was performed by intramuscular injection of 0.3 ml / kg of Zotal, and tetracaine eye drops were applied topically. A round Whatman III filter paper ring (6 mm in diameter) pre-soaked in 1.0 N NaOH (Sigma-Aldrich, St. Louis, MO, USA) was placed on the limbus for 30 s, and then the wound was rinsed with 0.9% saline for 1 min, and the corneal epithelium was gently scraped. Twenty New Zealand white rabbits were randomly divided into PBS group, PF-127 hydrogel group, versican group, and PF-127 hydrogel group loaded with versican, with 5 rabbits in each group. The wound surface was photographed on days 0, 7, and 14 after surgery.

[0029] Corneal fluorescein staining was performed with 2% sodium fluorescein (Sigma-Aldrich) to monitor the area of ​​corneal epithelial defect. Eye manifestations were photographed and evaluated on days 0, 7, and 14 after surgery. The degree of corneal opacity was observed under a slit lamp microscope and graded: grade 0 = completely transparent; grade 1 = slightly blurred, iris and pupil are easily visible; grade 2 = slightly opaque, iris and pupil can still be detected; grade 3 = opaque, almost imperceptible; grade 4 = completely opaque, pupil is invisible. Results are as follows Figure 5As shown, PF-127 hydrogel loaded with versican promoted the repair of corneal epithelium and restored transparency. On the seventh and fourteenth days, the rabbit cornea was photographed with white light and stained with sodium fluorescein (green represents defects), and the repair area ( Figure 5 A in the figure) and transparency ( Figure 5 From the scores and statistical analysis in (B), it can be seen that the repair effect of PF-127 hydrogel loaded with versican was the best on the seventh and fourteenth days.

[0030] 7. Histology and structural testing All experimental animals were sacrificed on the 14th day and their eyeballs were removed. The tissues were then fixed in 4% PFA and embedded in paraffin for sectioning. Tissue morphology was assessed using hematoxylin-eosin (HE) and Masson-Goldner (MG) staining; images were captured and analyzed using a Nikon microscope system. Figure 6 As shown, PF-127 hydrogel loaded with versican promoted the repair of corneal epithelium and structure. By using hematoxylin-eosin (HE) and Masson staining on the eyeballs of rabbits 14 days after eye drops treatment, it can be seen that the corneal structure of the PF-127 hydrogel group loaded with versican was the best, with a multi-layered epithelial structure and regularly arranged collagen, which is closest to normal corneal tissue.

[0031] 8. Data Analysis All data in this study were processed using Prism 10 (GraphPad), and data are expressed as mean ± SD. All experiments were performed independently at least three times unless otherwise specified. Two-tailed Student's t test was used to determine the comparison of means between two groups. One-way ANOVA or two-way ANOVA was used to compare the means between multiple groups. P<0.05 was defined as statistically significant. * p<0.05; ** p<0.01; *** p<0.001; ns, not statistically significant.

[0032] The specific embodiments of the present invention are described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. Application of human multifunctional proteoglycan in the preparation of drugs for promoting the proliferation and / or migration and / or maintaining the stemness of limbal stem cells.

2. The use according to claim 1, characterized in that: The application is specifically: the human multifunctional proteoglycan is used in the preparation of a drug for treating corneal epithelial damage.

3. The use according to claim 2, characterized in that The drug includes human versican.

4. The use according to claim 3, characterized in that: The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that: The auxiliary material is PF-127 hydrogel.

6. The use according to claim 3, characterized in that: The concentration of human versican in the drug is 0.5 μg / ml.

7. The use according to any one of claims 2 to 6, characterized in that: The medicine is eye drops.

8. The use according to claim 7, characterized in that: The eye drops are prepared by the following method: Excipient preparation: PF-127 powder and water were prepared into PF-127 hydrogel with a mass concentration of 18 wt%; Preparation of eye drops: add human versican powder to the excipients to prepare a mixed solution with a concentration of 0.5 μg / / ml of human versican, and shake the mixed solution at 4° C. overnight until a colorless and transparent solution is obtained, which is the eye drops.

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