WO2020083007A1 - Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用 - Google Patents

Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用 Download PDF

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WO2020083007A1
WO2020083007A1 PCT/CN2019/109213 CN2019109213W WO2020083007A1 WO 2020083007 A1 WO2020083007 A1 WO 2020083007A1 CN 2019109213 W CN2019109213 W CN 2019109213W WO 2020083007 A1 WO2020083007 A1 WO 2020083007A1
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sema4d
fundus
plexinb1
leakage
vegf
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French (fr)
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胡波
李亚男
吴介洪
陈安琪
洪灿东
岳振宇
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华中科技大学同济医学院附属协和医院
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Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to the application of gene silencing of the Sema4D / PlexinB1 signaling pathway in the preparation of drugs for treating and preventing ocular fundus diseases.
  • PDR proliferative diabetic retinopathy
  • the pathological process of DR is different from tumor angiogenesis, and its treatment has certain specificities.
  • the initiating factor of DR is the slowing of the flow rate of the fundus blood vessels, resulting in local metabolic changes, activation of inflammation and related signaling pathways, leading to blood vessel leakage and blood-retina Barrier destruction, loss of parietal cells, and destruction of the cell matrix, resulting in the loss of function of blood vessels and the formation of hypoxic areas locally, leading to compensatory abnormal vascular hyperplasia, aggravating leakage and even dysfunctional abnormal new blood vessels causing fundus hemorrhage and retinal detachment (Curr Diab Rep2011; Diabetes. 2006).
  • angiogenesis begins with the induction of proangiogenic factors and hypoxia.
  • angiogenesis in the tumor is to support the growth of the tumor. Therefore, the treatment of angiogenesis in the tumor is to simply inhibit angiogenesis, but it is different in the fundus.
  • the purpose of the angiogenesis inhibition therapy is to suppress abnormal blood vessels and restore normal blood vessels. Function, provide blood supply in this area and reduce leakage, it can be seen that although both diseases inhibit angiogenesis, the treatment results can be completely different.
  • Semaphorin protein family As a protein that plays an important role in the development of the nervous system, the Semaphorin protein family is currently considered to play an important role in the mechanism of angiogenesis and vascular development.
  • Sema4D the single transmembrane protein Sema4D (Class4semaphorins) is hotly discussed in angiogenesis.
  • the active extracellular free Sema4D fragments are mainly derived from MMP-MT1 cleavage of a variety of cells or ADAM17 cleavage in platelets. They are involved in angiogenesis in tumor, bone and trauma models. Their role is specific in different tissues and cells.
  • Sema4D In tumors, Sema4D promotes Met and Ron phosphorylation by binding to PlexinB receptors to promote cell migration and angiogenesis leading to tumor cell metastasis (2009 Valente).
  • Sema4D combined with PlexinB1 and ERBB2 to form a complex can inhibit cells by inhibiting Met Migration (Swiercz 2008), it can be seen that Sema4D plays different roles in different diseases in different tissues.
  • the applicant has clarified that inhibiting the Sema4D / PlexinB1 signaling pathway can effectively inhibit fundus angiogenesis and reduce fundus vascular leakage, and more importantly, early inhibition of the Sema4D / PlexinB1 signaling pathway can effectively reduce pericyte loss and help Protecting and restoring the function of the fundus vessels can prolong the therapeutic effect to a certain extent, reduce the number of administrations, and provide new strategies for the treatment of fundus angiogenesis and leakage.
  • the purpose of the present invention is to provide the use of Sema4D / PlexinB1 inhibitors in the preparation of drugs for the treatment and prevention of fundus vascular diseases, including but not limited to fundus angiogenesis or fundus vascular leakage or pericytes.
  • the present invention adopts the following technical measures:
  • Sema4D / PlexinB1 signaling pathway inhibitors in the preparation of drugs for the treatment and prevention of fundus vascular diseases.
  • the inhibitors include but are not limited to Sema4D / PlexinB1 signaling pathway inhibitory viruses, or Sema4D / PlexinB1 signaling pathway neutralizing antibodies, or Compounds that inhibit the function of Sema4D / PlexinB1 signaling pathway.
  • the fundus angiogenesis and leakage are caused by diabetes.
  • a method for treating vascular diseases of the fundus is to inject an inhibitor of Sema4D protein into the vitreous of the eye;
  • a method for treating vascular diseases of the fundus is to inject an inhibitor of Sema4D protein and anti-VEGF into the vitreous of the eye.
  • the fundus vascular diseases include but are not limited to fundus angiogenesis or fundus blood vessel leakage or pericytes.
  • the inhibitor is a neutralizing antibody to Sema4D protein or an inhibitor of Sema4D / PlexinB1 signaling pathway.
  • the present invention also provides methods for treating or preventing fundus vascular diseases through gene therapy methods involving Sema4D / PlexinB1 signaling pathway inhibition, such as antisense oligonucleotides through Sema4D / PlexinB1 gene silencing, gene knockout or inhibition of Sema4D / PlexinB1 signaling pathway Treat or prevent fundus vascular disease.
  • gene therapy methods involving Sema4D / PlexinB1 signaling pathway inhibition, such as antisense oligonucleotides through Sema4D / PlexinB1 gene silencing, gene knockout or inhibition of Sema4D / PlexinB1 signaling pathway Treat or prevent fundus vascular disease.
  • the invention provides a lentiviral vector with Sema4D / PlexinB1 gene silencing, a Sema4D / PlexinB1 gene knockout vector or antisense oligonucleotides (ASO, antisense oligodeoxynucleotides) that can inhibit the Sema4D / PlexinB1 signal pathway.
  • ASO antisense oligonucleotides
  • the present invention has the following advantages:
  • the existing main treatment methods are 1 laser photocoagulation therapy 2 vitrectomy 3 application of anti-angiogenesis drugs, of which anti-angiogenesis drugs are mainly anti-VEGF fundus Injection, but only 30% of patients receiving anti-VEGF treatment were effective.
  • Figure 1 is a schematic diagram of the closely related results of Sema4D and DR;
  • OCT optical coherence tomography
  • the free Sema4D in the aqueous humor of E-F.DR patients was negatively correlated with the thickness of the foveal retinal fovea (CST) and the change of retinal macular volume (MV) after 3 months of treatment with anti-VEGF (statistics obtained from Wuhan Union Hospital Ophthalmology Database)
  • Figure 2 is a schematic diagram of increased expression of Sema4D in mouse OIR and STZ models
  • A the expression of mRAN in the retina Sema4D of OIR model mice is increased (q-PCR);
  • C-D Western Blot showed that the expression of Sema4D protein in the retina of STZ model mice increased at 3 and 6 months.
  • FIG. 3 is a schematic diagram of Sema4D gene knockout inhibiting fundus angiogenesis
  • A-C gene knockout mouse verification
  • FIG. 4 is a schematic diagram of the Sema4D / PlexinB1 signaling pathway can promote the formation and leakage of endothelial cells
  • A-B scratch experiments confirmed that Sema4D can promote endothelial cell migration
  • Figure 5 is a schematic diagram of Sema4D / PlexinB1 signaling pathway increasing leakage of endothelial and pericyte co-culture model
  • A-B TEER experiment and fluorescein leakage experiment of co-cultured pericytes and endothelial cells confirmed that Sema4D signal promoted leakage
  • Fig. 6 is a schematic diagram of anti-Sema4D inhibiting fundus angiogenesis in OIR model mice and STZ model fundus vascular leakage; among them, A: anti-Sema4D treatment can inhibit fundus angiogenesis in OIR model mice in a concentration-dependent manner;
  • anti-Sema4D treatment can inhibit angiogenesis and the effect of combined anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone;
  • J-K single administration, STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage and combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone.
  • Figure 7 is a schematic diagram of the effect of multiple anti-Sema4D treatments
  • A-B multiple administrations, observed after 1 week, STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage and combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone
  • STZ model fundus anti-Sema4D treatment can inhibit the loss of pericytes and improve the coverage of pericytes, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone, and more importantly, anti-Sema4D Inhibiting pericyte loss and improving pericyte coverage is better than anti-VEGF treatment (P < 0.05)
  • G-H STZ model fundus anti-Sema4D treatment can increase VE-cadherin continuity and combined anti-VEGF treatment is significantly better than anti-Sema4D alone or anti-VEGF treatment alone
  • STZ model fundus anti-Sema4D treatment can increase N-cadherin coverage ratio and combined anti-VEGF treatment is significantly better than anti-VEGF treatment alone
  • STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage and combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone, more importantly Multiple anti-Sema4D treatment of fundus vascular leakage is superior to multiple anti-VEGF treatments (P < 0.05).
  • the experimental group was the aqueous humor of patients with diabetic retinopathy, and the control group was the aqueous humor of cataract patients, which was extracted with a sterile syringe.
  • ELISA kit (MyBioSource) was used to detect the expression of Sema4D protein in aqueous humor. The results showed that the expression of Sema4D in humoral water of DR patients was significantly increased, and n was the sample size.
  • Figures E-F show that the free Sema4D in DR patients' aqueous humor is negatively correlated with the thickness of the foveal fovea (CST) and the volume change of the retina (MV) after 3 months of anti-VEGF treatment.
  • Control group young rats after birth, grown under normoxic conditions, anesthetized mice on the 12th, 14th, and 17th day of birth. After saline heart perfusion, the eyeballs were removed, the retina was stripped, RNA was extracted and reverse transcribed into cDNA or protein extraction.
  • mice 8-week-old C57BL / 6 mice were injected STZ (streptozotocin, 50 mg / kg) intraperitoneally 4 hours after starvation for 5 consecutive days, once a day, and blood glucose was measured on the 7th day after the injection Blood glucose greater than 15mmol / L is regarded as successful modeling.
  • STZ streptozotocin, 50 mg / kg
  • Blood glucose was measured on the 7th day after the injection
  • Blood glucose greater than 15mmol / L is regarded as successful modeling.
  • the mice were anesthetized at the 3rd and 6th month after modeling. After the heart was perfused with physiological saline, the eyeballs were removed, the retina was peeled off, and the protein was extracted.
  • Control group (Vehicle): The streptozotocin of the experimental group was replaced with normal saline, and the remaining operations were the same.
  • Sema4D gene knockout can significantly inhibit fundus angiogenesis and leakage in mice
  • Sema4D gene knockout mice used in this example were purchased from Huazhong Agricultural University, and were obtained by designing sgRNA for the Sema4d exon region of the mice to knock out the expression of sema4D.
  • the wild-type mouse was used as a control: 3-5cm mouse tail was placed in DNA lysate at 56 °C for 10h. After isopropanol precipitation, 95% After washing with alcohol, it is amplified by the following system, and then electrophoresed on agarose gel.
  • Sema4d-GT-F1 TCTGGGGCTCTAAGAGGTCCTT Sema4d-GT-R1 AGCCACTGAGGTCACATACACC
  • a in Figure 3 is a schematic diagram of the sgRNA target region
  • B in Figure 3 shows that the sema4D gene in the knockout group is truncated (Sema4D-KO), so the molecular weight is lower than that in the wild group, indicating successful knockout.
  • Sema4D protein antibody is SEMA4D antibody (R & D Systems) ).
  • FIG 3 C in Figure 3 shows that sema4D knockout homozygous mice have no expression of sema4D protein, and the expression of sema4D protein in heterozygous knockout mice is lower than that of wild type.
  • Figure 3 C from left to right are wild type and knockout Zygote, knock out heterozygotes.
  • mice were anesthetized on the 17th day after the mice were born. After cardiac perfusion with normal saline, their eyeballs were fixed in 4% paraformaldehyde at 4 ° C overnight. B4 staining overnight at 4 °C, the slices are photographed, Isolectin B4 (1: 100, Vector Laboratories). Using the OIR model established by wild-type mice as a control, 6 mice per group.
  • D in Fig. 3 shows the decrease of abnormal neovascularization after OIR modeling of sema4D knockout mice.
  • E in Fig. 3 is a statistical schematic diagram of the ratio of abnormal blood vessels to the total blood vessel area using ImageJ.
  • OIR model rats were intraperitoneally injected with Evans blue dye (200 mg / kg) on the 17th day of birth. After circulating for 4 hours, the mice were anesthetized. After cardiac perfusion with saline, the retinas were taken to formamide (70 ° C water bath) overnight. Microplate reader (wavelength 620-740mm) to measure the absorbance of the extracted liquid. The concentration is calculated according to the standard curve (using the Evan's blue dye of different concentrations as the abscissa, and the absorbance of the liquid measured by the microplate reader at a wavelength of 620-740mm as the vertical axis to make a standard curve), and using the retina weight and blood retina level conversion Post statistics.
  • Sema4D knockout mice in the OIR model can significantly inhibit the number of cells in the pre-retinal neovascularization (HE staining)
  • the experiment was divided into four groups, namely: wild-type mouse group, sema4D knockout mouse group, wild-type OIR model mouse and sema4D knockout OIR model mouse, each group of six, the specific steps are as follows:
  • mice were anesthetized on the 17th day of birth. After cardiac perfusion with normal saline, their eyes were fixed in 4% paraformaldehyde, paraffin-embedded sections were stained with HE, observed under an optical microscope and counted to break through the inner boundary of the retina The number of vascular endothelial cells in the membrane.
  • Figure 3G shows the number of HE-stained pre-retinal neovascular cells. No wild blood vessels were generated in the wild-type mouse group and the sema4D knockout mouse group. Wild-type OIR model mice and sema4D knock-out OIR model mice There is neovascularization, but the number of new blood vessels in sema4D knock-out OIR model mice is significantly smaller than that in wild-type OIR model mice. Arrows indicate the cells of pre-retinal neovascularization. H in Figure 3 shows that Sema4D knockout mice in the OIR model can be significant Inhibits the number of cells in the pre-retinal neovascularization.
  • Sema4D / PlexinB1 signaling pathway enhances or inhibits endothelial cell lumen formation and leakage:
  • mice brain microvascular endothelial cells The cells or endothelial cells involved in this embodiment are all referred to as mouse brain microvascular endothelial cells.
  • Sema4D can promote endothelial cell migration (scratch test)
  • the culture wells were planted with mouse brain microvascular endothelial cells. After starvation with 0.5% FBS overnight, a vertical vertical line was drawn on the cells with a 200ul pipette tip. After washing away the scratched cells, they were added to DMEM and added separately. The final concentration of sema4D protein was 400, 800, and 1600 ng / ml, and then added to the culture wells and incubated for 24 hours. Using microscopy, imageJ counted cell migration.
  • Sema4D can promote the migration of endothelial cells (A in Figure 4), and the migration capacity of endothelial cells increases as the concentration of sema4D increases (B in Figure 4).
  • Sema4D can promote endothelial cell lumen formation (lumen formation experiment)
  • Sema4D can promote the formation of endothelial cells, and increase with the increase of sema4D concentration (C-D in Figure 4).
  • Sema4D can promote endothelial cell leakage (TEER experiment)
  • a layer of fibronectin was first coated on the upper layer of the 24-well transwell chamber (0.4um) and incubated at room temperature for 1 hour; after coating, 5 ⁇ 104 mouse brain microvascular endothelial cells were implanted and cultured for 3 days.
  • E in Figure 4 shows that TEER of endothelial cells gradually decreases after adding 400, 800, and 1600 ng / ml of sema4D, indicating that Sema4D can promote endothelial cell leakage, and the leakage increases with the increase of concentration.
  • Sema4D can promote endothelial cell leakage (fluorescence leakage experiment)
  • a layer of fibronectin was first coated on the upper layer of the 24-well transwell chamber (0.4um), incubated for 1 hour at room temperature, coated and planted into mouse brain microvascular endothelial cells, cultured for 3 days, and added to the medium after the cells were overgrown.
  • Sema4D with a final concentration of 400, 800, and 1600 ng / ml
  • DMEM without sema4D as a control
  • add a fluorescently labeled dextran with a final concentration of 300 ⁇ g / ml to the upper DMEM of the chamber after 24 hours, and extract 30 ul of the lower medium at 1 hour, using The microplate reader measures the absorbance at a wavelength of 625 nm.
  • F in Figure 4 shows that the dextran fluorescence value of endothelial cell leakage increases after the addition of sema4D with final concentrations of 400, 800, and 1600 ng / ml, indicating that Sema4D can aggravate the leakage of endothelial cells, and the leakage increases as the concentration increases.
  • the lentiviruses involved in the embodiments of the present invention are all purchased from Jikai Company, where CRISPR-wt is an empty lentivirus with GFP, and CRISPR-plB1 is a CRISPR / Cas9 lentivirus knocked out by PlexinB1, which is used to down-regulate PlexinB1; experiment Divided into 3 groups: 1. Normal group, which is the endothelial cell group without any substance; 2. Transfected with GFP-loaded lentivirus group (CRISPR-wt); 3. Transfected with CRISPR-plB1 5 repeats in each group, and the MOI value of the virus group is 50.
  • Lentivirus was transfected into mouse brain microvascular endothelial cells 48 hours later to extract protein for Western Blot to detect the expression of PlexinB1, with ⁇ -action as a control, the antibody used in the WB process was PlexinB1antibody (1: 500, Abcam).
  • CRISPR-plB1 can significantly reduce the expression of PlexinB1 (G in Figure 4, left panel is WB panel, right panel is WB statistical graph).
  • Lentivirus (CRISPR-wt or CRISPR-plB1) was transfected into mouse brain microvascular endothelial cells for 48 hours, then digested and planted in the cell plate, and the culture wells were filled with mouse brain microvascular endothelial cells and starved with 0.5% FBS After overnight, draw a vertical line on the cells with a 200ul pipette tip. After washing away the underlined cells, directly incubate for 24h or add sema4D protein with a final concentration of 1600ng / ml and then incubate for 24h. 6 replicates per group, Using photomicrographs, imageJ counts cell migration.
  • J-K in Figure 4 shows that sema4D-mediated endothelial cell formation is less effective after downregulating PlexinB1.
  • the upper layer of the 24-well transwell chamber (0.4um) was first coated with a layer of adhesion factor (incubated at room temperature for 1h), and then coated with mouse brain microvascular endothelium transfected with lentivirus (CRISPR-wt or CRISPR-plB1) Cells were cultured for 3 days. After the cells were overgrown, they were directly incubated for 24 hours or added with sema4D protein at a final concentration of 1600 ng / ml and then incubated for another 24 hours, with 6 replicates per group. After incubation for 24h, the cell transmembrane resistance measuring instrument was used to measure its resistance value.
  • CRISPR-wt or CRISPR-plB1 mouse brain microvascular endothelium transfected with lentivirus
  • the upper layer of the 24-well transwell chamber (0.4um) was first coated with a layer of adhesion factor, incubated at room temperature for 1h, and then coated with mouse brain microvascular endothelial cells transfected with lentivirus (CRISPR-wt or CRISPR-plB1) (5 ⁇ 104 cells), after 3 days of culture, after the cells are full, incubate directly for 24h or add sema4D protein with a final concentration of 1600ng / ml and then incubate for 24h. Add 300 ⁇ g / ml fluorescently labeled dextran to the upper layer of DMEM After 1h, extract 30ul of the lower medium and measure the absorbance at 625nm with a microplate reader.
  • CRISPR-wt or CRISPR-plB1 mouse brain microvascular endothelial cells transfected with lentivirus
  • M in Figure 4 shows that the fluorescent leak experiment shows that sema4D promotes increased endothelial cell leakage, and PlexinB1 down-regulation can inhibit this process.
  • Sema4D / PlexinB1 signaling pathway can promote endothelial and pericyte mixed culture model leakage
  • the endothelial cells used in this example are mouse brain microvascular endothelial cells, and the pericytes are mouse primary brain microvascular pericytes.
  • Sema4D can promote the leakage of endothelial cells and pericyte co-culture system (TEER experiment).
  • a layer of fibronectin was first coated in a 24-well transwell chamber (0.4um) and incubated for 1 hour at room temperature; and mouse brain microvascular endothelial cells (5 ⁇ 104) were implanted in the lower layer of the chamber. After 12 hours, the cells were implanted in the upper layer of the chamber Primary rat brain microvascular pericytes (2.5 ⁇ 104), after 3 days of culture, change the medium, add sema4D protein with a final concentration of 400, 800, and 1600ng / ml to the upper layer of DMEM, with DMEM without sema4D as a control, After incubation for 24h, the cell transmembrane resistance measuring instrument was used to measure its resistance value.
  • a in Figure 5 shows that Sema4D can reduce the TEER value of the endothelial and pericyte co-culture system, and the decrease value increases with the increase of Sema4D protein concentration.
  • Sema4D can promote the leakage of endothelial cells and pericytes co-culture system (fluorescence leakage experiment).
  • a layer of fibronectin was first coated in a 24-well transwell chamber (0.4um), incubated at room temperature for 1 hour, and brain microvascular endothelial cells were implanted in the lower layer of the chamber. After 12 hours, mouse primary brain microvascular endothelial cells were implanted in the upper layer of the chamber. After culturing for 3 days, change the medium, add sema4D protein with a final concentration of 400, 800, and 1600 ng / ml to the upper layer of DMEM respectively. Take DMEM without sema4D as a control, add 300 ⁇ g / ml fluorescent label to the upper layer of DMEM after 24 hours For the dextran solution, the cell sample without sema4D was added as a control. After 1 h, 30 ul of the lower culture medium was extracted, and the absorbance at a wavelength of 625 nm was measured using a microplate reader.
  • Figure 5 B shows that Sema4D can promote the leakage of endothelial and pericyte co-culture system in a concentration-dependent manner.
  • the PlexinB1 protein receptor that silences pericytes alone can reverse the effect of Sema4D to a greater extent than the PlexinB1 protein receptor that silences endothelial cells alone.
  • a layer of fibronectin was first coated in a 24-well transwell chamber (0.4um), incubated at room temperature for 1 hour, and the lower layer was implanted with mouse brain microvascular endothelial cells (5 ⁇ 104). After 12 hours, the treatment was implanted on the upper layer of the chamber Primary brain microvascular pericytes (2.5 ⁇ 104) in the post-mice; after 3 days of culture, change the medium, add Sema4D protein at a final concentration of 1600ng / ml to the upper layer of DMEM, without Sema4D protein as a control; then follow step 1) And the method in 2), perform TEER experiment and fluorescence leakage experiment respectively;
  • CRISPR-wt group endothelial cells transfected with airborne lentivirus are seeded in the lower layer, and pericytes transfected with airborne lentivirus are seeded in the upper layer of the chamber;
  • PC-CRISPR-plB1 group endothelial cells transfected with empty lentivirus were seeded into the lower layer, and CRISPR-plB1 transfected cells were seeded into the upper layer of the chamber;
  • EC-CRISPR-plB1 group endothelial cells transfected with CRISPR-plB1 were seeded in the lower layer, and pericytes transfected with empty lentivirus were seeded in the upper layer of the chamber.
  • Single injection of anti-Sema4D treatment can inhibit fundus angiogenesis and fundus vascular leakage in OIR model mice and STZ model mice
  • mice After the birth of C57BL / 6 female mice from March to April, they are placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of birth. After the 12th day of birth, the pups and the females are taken out. Inject different doses of Sema-4D neutralizing antibody (BMA-12) (0.5ug, 1ug, 2ug) into the vitreous, using 2ug of IgG as a control and an equal volume of PBS as a control, and continue to feed in normal air for 5 days; The young mice were anesthetized on the 17th day of birth. After cardiac perfusion with normal saline, the eyeballs were separated and fixed, and the retinal IB4 staining was removed.
  • BMA-12 Sema-4D neutralizing antibody
  • Wt group After birth in C57BL / 6 female rats from March to April, they are placed in an oxygen chamber with 75% oxygen together with the baby rats on the 7th day of birth. After the 12th day of birth, the baby rats and the female rats are taken out, and then they are young After the rats were anesthetized, Sema4D neutralizing antibody (2ug) or IgG (2ug) was injected into the vitreous to continue feeding in normal air for 5 days; then IB4 immunofluorescence staining experiment was performed;
  • Sema4D-KO group After birth of C57BL / 6 female mice after Sema4D gene knockout from March to April, they are placed in an oxygen chamber with 75% oxygen together with pups on the 7th day of birth. Remove the mice and the female rats, and then inject vitiligo with Sema4D neutralizing antibody (2ug) or IgG (2ug) into the vitreous after anesthetization, and continue to feed in normal air for 5 days; then perform IB4 immunofluorescence staining experiment;
  • Wt group After birth in C57BL / 6 female rats from March to April, they are placed in an oxygen chamber with 75% oxygen together with the baby rats on the 7th day of birth. After the 12th day of birth, the baby rats and the female rats are taken out, and then they are young After the mouse is anesthetized, Sema4D neutralizing antibody (2ug) or IgG (2ug) is injected into the vitreous to continue feeding in normal air for 5 days; follow the method in step 2), and then perform Evan's blue fluorescence experiment;
  • Sema4D-KO group After birth of C57BL / 6 female mice after Sema4D gene knockout from March to April, they are placed in an oxygen chamber with 75% oxygen together with pups on the 7th day of birth. Remove the mice and the females, and then immediately inject viscous Sema4D neutralizing antibody (2ug) and IgG (2ug) into the vitreous for 5 days after anesthetization; follow the method in step 2), and then perform the Evans blue fluorescence experiment ;
  • mice After the birth of C57BL / 6 female mice from March to April, they are placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of birth. After the 12th day of birth, the pups and the females are taken out. Simultaneous injection of sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) in the vitreous of mouse eyes; or injection of anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul); mice were anesthetized on the 17th day of birth.
  • the STZ model mice were anesthetized 5 months after the model was injected, and sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) were injected into the vitreous body of the mouse eye at the same time, or anti-Sema4D (2ug) was injected separately , Anti-VEGF (2ug), IgG (2ug), PBS (1ul); after one week of injection, the tail vein was injected with Evan's blue dye (45mg / kg), anesthetized mice 2 hours later, after saline heart perfusion, take it The retina was placed in formamide (70 ° C water bath) overnight and measured using a microplate reader (wavelength 620-740mm).
  • the concentration is calculated according to the standard curve, and the statistics are calculated using the conversion of retinal weight and blood retinal level.
  • a part of the eyeballs were fixed at 4% PFA for 2 hours at room temperature, and then taken fluorescence pictures after retinal placement.
  • Multiple anti-Sema4D treatments can inhibit the leakage of fundus blood vessels in STZ model mice and are superior to anti-VEGF in inhibiting the loss of perivascular cells in the fundus
  • STZ model mice were injected with sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) simultaneously in the vitreous of the eye after 4 months of modeling, or injection of anti-Sema4D (2ug) and anti-VEGF (2ug) alone ), IgG (2ug), PBS (1ul) is injected with anti-Sema4D (2ug) or anti-VEGF (2ug) or IgG (2ug) or PBS (2ug) as a control, injected once a week, one week after the fifth injection Evans blue dye (45mg / kg) was injected into the tail vein, and the mice were anesthetized after 2 hours of circulation.
  • the retina was taken to formamide (70 ° C water bath) overnight, using a microplate reader (wavelength 620-740mm) Determination.
  • the concentration is calculated according to the standard curve, and is calculated using the conversion of retinal weight and blood retinal level.
  • a part of the eyeball was fixed at 4% PFA for 2 hours at room temperature, the retina was separated, and the plate was taken for fluorescence photography.
  • STZ model mice were injected with sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) simultaneously in the vitreous of the eye after 4 months of modeling, or injection of anti-Sema4D (2ug) and anti-VEGF (2ug) alone ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, after saline heart perfusion, take their fresh eyeballs were fixed in 4% PFA for 24 hours Then, the retina was separated and digested with 3% trypsin (dissolved in pH.7.8tris-hcl) at 37 degrees Celsius.
  • STZ model mice were injected with sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) simultaneously in the vitreous of the eye after 4 months of modeling, or injection of anti-Sema4D (2ug) and anti-VEGF (2ug) alone ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, after saline heart perfusion, take their fresh eyeballs were fixed in 4% PFA for 24 hours Then, the retina was separated and digested with 3% trypsin (dissolved in pH.7.8tris-hcl) at 37 degrees Celsius.
  • STZ model mice were injected with sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) simultaneously in the vitreous of the eye after 4 months of modeling, or injection of anti-Sema4D (2ug) and anti-VEGF (2ug) alone ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, after cardiac perfusion with saline, fresh eyeballs were fixed in anhydrous methanol at -20 °C for 2h, peeled off The retina was taken out and placed in a blocking solution (1% BSA, 0.5% TritonX-100, PBS) at 4 ° C overnight.
  • a blocking solution 1% BSA, 0.5% TritonX-100, PBS
  • VE-cadherin / CollagenIV After incubating the primary antibody (VE-cadherin / CollagenIV) for 5 days, the secondary antibody was incubated at room temperature for 3 hours and then photographed.
  • STZ model mice were injected with sema4d neutralizing antibody (2ug / eye) and VEGF neutralizing antibody (2ug / eye) simultaneously in the vitreous of the eye after 4 months of modeling, or injection of anti-Sema4D (2ug) and anti-VEGF (2ug) alone ), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the fifth injection, after cardiac perfusion with saline, fresh eyeballs were fixed in 4% PFA at 4 °C for 2h, peeled off The retina was taken out and placed in a blocking solution (1% BSA 0.5% TritonX-100, PBS).
  • N-cadherin / CollagenIV was incubated for 5 days, and the secondary antibody was incubated at room temperature for 3 hours.
  • step 1) carry out Evan's blue fluorescence experiment and prolong the observation time.

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Abstract

提供Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用，发现Sema4D在糖尿病视网膜病变患者眼房水中增加，Sema4D水平与患者对anti-VEGF的反应呈负相关，Sema4D/PlexinB1信号通路一方面可以作用于内皮细胞促进其迁移、管腔形成，一方面通过促进周细胞迁移加重渗漏，抑制Sema4D/PlexinB1信号通路，可有效抑制眼底血管新生及渗漏，为糖尿病视网膜病变等眼底血管增生、渗漏性疾病提供了新的思路。

Description

Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用
本申请要求申请号为201811228364X，发明名称为“Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用”的中国发明专利申请的优先权，前述申请的内容通过引用整体结合到本文中。
技术领域
本发明属于生物医疗领域，具体涉及Sema4D/PlexinB1信号通路基因沉默在制备治疗及预防眼底血管疾病药物中的应用。
背景技术
糖尿病视网膜病变(DR)作为20-75岁成年人致盲的第一位原因，在1型及2型糖尿病患者中患病率高，全球：DR在糖尿病人群中患病率达35.4％，其最严重阶段增殖性糖尿病视网膜病变(PDR)的患病率达7.5％(2017Diabetes Care IF＝13.397)，在中国：不同阶段DR在糖尿病人群中患病率也高达24.7％-37.5(2014指南)，世界卫生组织(WHO)估计，至2025年，全球将糖尿病患者将突破增值300亿(2009EYE)，可见DR患者的数目将进一步增加。
DR的治疗自1990年开展糖尿病视网膜病变玻璃体切割术研究(DRVS研究)至今主要为三类治疗：①激光光凝治疗 ②玻璃体切割术 ③抗血管新生药物的应用(主要为多种方式的抗-VEGF治疗,2004年-今)，激光光凝治疗为弃车保帅的治疗方法，牺牲周边视野保留中央视野，而接受抗-VEGF治疗的患者中仅30％有效，可见，anti-VEGF治疗抵抗、反复给药并发的感染、神经元毒性、沉重的经济负担都为治疗带来不便和风险，迫切要求我们研发新的治疗策略。
DR的病理过程不同于肿瘤血管新生，其治疗有一定的特殊性，DR的始发因素为眼底血管流速减慢，导致局部代谢改变，激活炎症及相关信号通路，导致血管渗漏、血-视网膜屏障破坏，壁细胞脱失，细胞基质破坏，从而使血管失去功能，局部形成缺氧区域，导致代偿性异常血管增生，加重渗漏甚至功能不全的异常新生血管造成眼底出血、视网膜脱离(Curr Diab Rep2011；Diabetes.2006)。而在肿瘤中血管新生始发于促血管新生因子及低氧的诱导，经过内皮细胞激活伸出伪足，周围壁细胞的贴附及与邻近新生管腔的融合，形成血管网。肿瘤中血管新生的目的是为肿瘤生长供养，因此肿瘤中针对血管新生的治疗重点为单纯的抑制血管新生，而在眼底却不同，其抑制血管新生治疗的目的在于抑制异常血管的同时恢复正常血管 功能，提供该区域血供并减轻渗漏，可见，虽然两种疾病同为抑制血管新生，但是治疗结果可截然不同。
Semaphorin蛋白家族作为在神经系统发育过程扮演重要角色的蛋白，目前被认为在血管新生及血管发育机制上同样发挥重要作用，其中单次跨膜蛋白Sema4D(Class4semaphorins)在血管新生中被热议，其起作用的胞外游离Sema4D片段主要来源于多种细胞的MMP-MT1切割或血小板中ADAM17切割，参与于肿瘤、骨、外伤模型血管新生，在不同的组织、细胞中其作用存在特异性，在肿瘤中，Sema4D通过结合PlexinB受体促进Met和Ron的磷酸化促进细胞迁移、血管新生导致肿瘤细胞转移(2009Valente)，然而，在肺癌中，Sema4D结合PlexinB1与ERBB2形成复合物可通过抑制Met抑制细胞迁移(Swiercz 2008)，可见Sema4D在不同组织不同疾病中作用不同。
在本发明中，申请人明确了抑制Sema4D/PlexinB1信号通路可有效抑制眼底血管新生并减轻眼底血管渗漏，更重要的是早期抑制Sema4D/PlexinB1信号通路可有效减轻周细胞脱失，有助于保护及恢复眼底血管功能，可一定程度延长治疗效果，减少给药次数，为眼底血管新生及渗漏的治疗提供新的策略。
发明内容
本发明的目的在于提供Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用，所述的眼底血管疾病包括但不限于眼底血管新生或眼底血管渗漏或周细胞脱失。
为了达到上述目的，本发明采取以下技术措施：
针对目前眼底血管新生及渗漏的治疗手段有限的情况，申请人取糖尿病视网膜病变患者房水，筛选与血管新生密切相关的成员，发现Sema4D/PlexinB1信号通路抑制剂可有效抑制眼底血管新生及渗漏。
Sema4D/PlexinB1信号通路抑制剂在制备治疗及预防眼底血管疾病药物中的应用，所述的抑制剂包括但不限于Sema4D/PlexinB1信号通路抑制病毒，或是Sema4D/PlexinB1信号通路中和抗体，或是抑制Sema4D/PlexinB1信号通路功能的化合物。
以上所述的应用中，优选的，所述的眼底血管新生及渗漏是由糖尿病引起的。
一种治疗眼底血管疾病的方法，所述的方法为向眼玻璃体中注射Sema4D蛋白的抑制剂；
一种治疗眼底血管疾病的方法，所述的方法为向眼玻璃体中注射Sema4D蛋白的抑制剂和anti-VEGF。
所述的的眼底血管疾病包括但不限于眼底血管新生或眼底血管渗漏或周细胞脱失。
以上所述的方法中，优选的，所述的抑制剂为Sema4D蛋白的中和抗体或 Sema4D/PlexinB1信号通路抑制剂。
本发明还提供通过涉及Sema4D/PlexinB1信号通路抑制的基因治疗方法治疗或预防眼底血管疾病的方法，例如通过Sema4D/PlexinB1基因沉默、基因敲除或抑制Sema4D/PlexinB1信号通路的反义寡核苷酸治疗或预防眼底血管疾病。本发明提供Sema4D/PlexinB1基因沉默的慢病毒载体，Sema4D/PlexinB1基因敲除载体或者可以抑制Sema4D/PlexinB1信号通路的反义寡核苷酸(ASO,antisense oligodeoxynucleotides)。
与现有技术相比，本发明具有以下优点：
针对目前眼底血管新生及渗漏的治疗手段有限的情况，现有的主要治疗手段为①激光光凝治疗 ②玻璃体切割术 ③抗血管新生药物的应用，其中抗血管新生药物主要为anti-VEGF眼底注射，但接受抗-VEGF治疗的患者中仅30％有效，申请人针对目前治疗的局限性，取糖尿病视网膜病变患者房水，筛选与血管新生密切相关的Sema家族成员，发现Sema4D/PlexinB1信号通路抑制剂可有效抑制眼底血管新生及渗漏，并在体内、外及基因敲除小鼠中证明了抑制Sema4D/PlexinB1信号通路对眼底病理性血管新生、渗漏的作用并明确相关机制。
附图说明
图1为Sema4D与DR密切相关结果示意图；
其中，A：光学相干断层扫描仪(OCT)示意图；
B-C.DR患者房水中Sema4D表达明显增高(Western Blot)
D.DR患者房水中Sema4D表达明显增高(ELISA检测)
E-F.DR患者房水中游离Sema4D与患者接受anti-VEGF治疗3月后视网膜黄斑中央凹厚度(CST)及视网膜黄斑体积改变(MV)呈负相关(由武汉协和医院眼科数据库获取后统计)
图2为小鼠OIR及STZ模型中Sema4D表达升高示意图；
其中，A：OIR模型小鼠视网膜Sema4D的mRAN表达升高(q-PCR)；
B：Western Blot结果显示OIR模型小鼠视网膜Sema4D蛋白表达升高；
C-D：Western Blot显示STZ模型小鼠视网膜3个月、6个月Sema4D蛋白表达升高。
图3为Sema4D基因敲除抑制眼底血管新生示意图；
其中，A-C：基因敲除小鼠验证；
D-H：免疫荧光染色，伊文氏蓝渗漏实验，HE染色结果显示Sema4D基因敲除可显著抑制眼底病理性血管新生、血管渗漏。
图4为Sema4D/PlexinB1信号通路可促进内皮细胞管腔形成及渗漏的示意图；
其中，A-B：划痕实验证实Sema4D可促进内皮细胞迁移；
C-D：管腔形成实验证实Sema4D可促进内皮细胞管腔形成；
E：TEER实验证实Sema4D可促进内皮细胞渗漏；
F：荧光素渗漏实验证实Sema4D可促进内皮细胞渗漏；
G：PlexinB1下调验证；
H-I：划痕实验证实阻断沉默PlexinB1受体可阻断Sema4D促内皮细胞迁移作用；
J-K：管腔形成实验证实沉默PlexinB1受体可阻断Sema4D促内皮细胞管腔形成作用；
L-M：TEER和荧光素渗漏实验证实沉默PlexinB1受体可阻断Sema4D促内皮细胞渗漏作用；
图5为Sema4D/PlexinB1信号通路增加内皮和周细胞共培养模型渗漏示意图；
其中，A-B：共培养周细胞及内皮细胞TEER实验及荧光素渗漏实验证实Sema4D信号促进渗漏；
C-D：共培养周细胞及内皮细胞模型中，TEER实验及荧光素渗漏实验证实沉默PlexinB1受体阻断Sema4D诱导的渗漏作用；
图6为anti-Sema4D抑制OIR模型小鼠眼底血管新生及STZ模型小鼠眼底血管渗漏示意图；其中，A：anti-Sema4D治疗可抑制OIR模型小鼠眼底血管新生，呈浓度依赖性；
B：伊文氏蓝实验证实anti-Sema4D治疗可抑制OIR模型小鼠眼底血管渗漏，呈浓度依赖性C-D：anti-Sema4D抗体特异性验证；
E-I：OIR模型中，anti-Sema4D治疗可抑制血管新生且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗；
J-K：单次给药，STZ模型眼底anti-Sema4D治疗可抑制眼底血管渗漏且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗。
图7为多次anti-Sema4D治疗的效果示意图；
其中，A-B：多次给药，1周后观察，STZ模型眼底anti-Sema4D治疗可抑制眼底血管渗漏且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗
C-F：STZ模型眼底anti-Sema4D治疗可抑制周细胞脱失，提高周细胞覆盖率，且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗，更重要的是anti-Sema4D抑制周细胞脱失和提高周细胞覆盖率优于anti-VEGF治疗(P&lt；0.05)
G-H：STZ模型眼底anti-Sema4D治疗可增加VE-cadherin连续性且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗
I-J：STZ模型眼底anti-Sema4D治疗可增加N-cadherin覆盖比例且联合anti-VEGF治 疗效果显著优于单独anti-VEGF治疗
K-L：多次给药，2周后观察，STZ模型眼底anti-Sema4D治疗可抑制眼底血管渗漏且联合anti-VEGF治疗效果显著优于单独anti-Sema4D或单独anti-VEGF治疗，更重要的是多次anti-Sema4D治疗眼底血管渗漏优于多次anti-VEGF治疗(P&lt；0.05)。
具体实施方式
下面结合具体实施例对本发明做进一步说明。本发明所述技术方案，如未特别说明为本领域的常规方案。所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
Sema4D与DR密切相关的发现：
1)视网膜断层扫描
比较糖尿病视网膜病变患者OCT断层扫描图和正常人OCT断层扫描图片，发现糖尿病视网膜病变患者中的黄斑中央凹厚度增厚(图1中A)
2)房水中Sema4D的表达量的检测
实验组为糖尿病视网膜病变患者眼房水，对照组为白内障患者眼房水，无菌注射器抽取。
Western Blot检测房水中Sema4D蛋白的表达量，结果显示DR患者房水中Sema4D表达明显增高(图1中B-C)。
ELISA试剂盒(MyBioSource)检测房水中Sema4D蛋白的表达量，结果显示DR患者房水中Sema4D表达明显增高，n表示样本量。
3)治疗后的DR患者的视网膜断层扫描和房水中Sema4D蛋白的检测
图E-F显示DR患者房水中游离Sema4D与患者接受anti-VEGF治疗3月后视网膜黄斑中央凹厚度(CST)及视网膜黄斑体积改变(MV)呈负相关，n表示样本量。
实施例2：
小鼠OIR及STZ模型中Sema4D表达升高：
1)建立小鼠OIR模型
实验组(OIR)：3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至含75％氧气的氧舱中，至出生第12天时将幼鼠与母鼠取出，之后放置正常空气中5天。上述处理的幼鼠在出生第12、14、17天时麻醉，生理盐水心脏灌注后，取其眼球，剥离出视网膜，提取RNA并反转录成cDNA或提取蛋白。
对照组(Normal)：为出生后的幼鼠，常氧条件下生长，在出生第12，14，17天麻醉老鼠，生理盐水心脏灌注后取眼球，剥离出视网膜，提取RNA并反转录成cDNA或提取蛋白。
2)q-PCR检测Sema4D的mRNA表达量
利用引物(CCTGGTGGTAGTGTTGAGAAC和GCAAGGCCGAGTAGTTAAAGAT)，以步骤1)中的cDNA为模板，进行Sema4D的表达量的检测，结果显示，OIR组中，Sema4D的mRNA表达量显著升高(图2中A)。(图2中P12指得是出生第12天的小鼠样本，以此类推，以下同)
3)Western Blot检测实验组(OIR)和对照组中Sema4D蛋白
以步骤1)中提取的蛋白为抗原，以sheepanti-SEMA4Dantibody(来源：R&DSystems)为抗体，进行Western Blot检测，以β-action为对照。结果显示，OIR模型小鼠视网膜Sema4D蛋白表达量显著升高(图2中B)
4)建立小鼠STZ模型
实验组(STZ)：8周大的C57BL/6小鼠饥饿4h后腹腔注射STZ(链脲佐菌素，50mg/kg)，连续5天，每天一次，注射完成后的第7天检测血糖，血糖大于15mmol/L视为造模成功。于造模后第3，6个月时麻醉老鼠，生理盐水心脏灌注后，取其眼球，剥离出视网膜，提取蛋白。
对照组(Vehicle)：将实验组的链脲佐菌素替换成生理盐水，其余操作相同。
5)Western Blot检测实验组(STZ)和对照组中(Vehicle)Sema4D蛋白
以步骤4)中提取的蛋白为抗原，以sheep anti-SEMA4D antibody(R&D Systems),为抗体，进行Western Blot检测，，以β-action为对照。结果显示，STZ模型小鼠视网膜在建模后第3个月、6个月Sema4D蛋白表达显著升高(图2中C和D)。
实施例3：
Sema4D基因敲除可显著抑制小鼠的眼底血管新生及渗漏
本实施例所用的Sema4D基因敲除小鼠购自华中农业大学，是通过对小鼠的Sema4d外显子区域设计sgRNA从而敲除sema4D的表达而获得。
1)基因敲除鼠的目的基因(sema4D)敲除成功的验证：
自鼠尾提取DNA验证小鼠目的基因(sema4D)敲除成功，以野生型的小鼠为对照：取3-5cm鼠尾放于DNA裂解液中56℃10h，异丙醇析出后，95％酒精洗涤，配置成下列体系后扩增，于琼脂糖凝胶中电泳。
Sema4d-GT-F1 TCTGGGGCTCTAAGAGGTCCTT
Sema4d-GT-R1 AGCCACTGAGGTCACATACACC
Figure PCTCN2019109213-appb-000001
图3中A为sgRNA打靶区域模式图，图3中B表明：敲除组的sema4D基因由于被截短(Sema4D-KO)，因此分子量较野生组较低，表明敲除成功。
利用小鼠视网膜提取蛋白做Western Blot以检测Sema4D蛋白，取小鼠视网膜组织，提取步骤同一般组织提取蛋白步骤，每组6只，以β-action为对照；Sema4D蛋白抗体为SEMA4D antibody(R&D Systems)。
图3中C表明sema4D敲除的纯合子小鼠无sema4D蛋白表达，敲除杂合子小鼠sema4D蛋白较野生型表达减少，图3中C中，从左至右分别为野生型，敲除纯合子，敲除杂合子。
2)IB4免疫荧光染色显示Sema4D基因敲除鼠可显著抑制OIR模型中眼底的血管新生
利用Sema4D基因敲除小鼠建立OIR模型，自小鼠出生起算后第17天麻醉老鼠，生理盐水心脏灌注后，取其眼球于4％多聚甲醛中4℃过夜固定，剥离出视网膜后，Isolectin B4染色4℃过夜，铺片拍照，Isolectin B4(1:100,Vector Laboratories)。以野生型小鼠建立的OIR模型为对照，每组6只老鼠。
图3中D显示敲除sema4D的鼠OIR造模后异常的新生血管减少，图3中E为是利用ImageJ对异常血管占总血管面积比例的统计示意图。
3)伊文氏蓝萃取实验证实Sema4D基因敲除鼠可显著抑制OIR模型中眼底血管渗漏实验分为野生型OIR模型鼠和sema4D敲除OIR模型鼠，每组6只，具体如下：
OIR模型鼠于出生第17天时腹腔注射伊文氏蓝染料(200毫克/公斤)，循环了4个小时后麻醉老鼠，生理盐水心脏灌注后，取其视网膜至于甲酰胺(70℃水浴)过夜，利用酶标仪(波长620-740mm)测定萃取液体吸光度。浓度是根据标准曲线计算的(以不同浓度的伊文氏蓝染料为横坐标，以酶标仪波长620-740mm测定的液体吸光度为纵轴制作标准曲线)，并利用视网膜重量及血液中视网膜水平换算后统计。
图3中F显示Sema4D基因敲除鼠可显著抑制OIR模型中眼底血管渗漏。
4)OIR模型中Sema4D基因敲除鼠可显著抑制视网膜前新生血管的细胞数目(HE染色)
实验分为四组，分别为：野生型小鼠组、sema4D敲除型小鼠组、野生型OIR模型鼠和sema4D敲除型OIR模型鼠，每组6只，具体步骤如下：
上述四组小鼠均于出生第17天麻醉，生理盐水心脏灌注后，取其眼球于4％多聚甲醛中固定，行石蜡包埋切片HE染色，在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞数目。
图3中G显示HE染色视网膜前新生血管的细胞数目，其中野生型小鼠组、sema4D敲除型小鼠组中均无新生血管的生成，野生型OIR模型鼠和sema4D敲除型OIR模型鼠均有新生血管生成，但sema4D敲除型OIR模型鼠的新生血管数目显著小于野生型OIR模型鼠，箭头表示视网膜前新生血管的细胞，图3中H显示OIR模型中Sema4D基因敲除鼠可显著抑制视网膜前新生血管的细胞数目。
实施例4：
Sema4D/PlexinB1信号通路的加强或抑制内皮细胞管腔形成及渗漏产生影响：
本实施例涉及到的细胞或内皮细胞均指得是小鼠脑微血管内皮细胞。
1)Sema4D可促进内皮细胞迁移(划痕实验)
在培养孔中种满小鼠脑微血管内皮细胞，用0.5％的FBS饥饿过夜后，用200ul枪头在细胞上划出垂直的竖线，洗去划下的细胞后，在DMEM中加入分别加入终浓度为400，800，1600ng/ml的sema4D蛋白，再加入培养孔后孵育24h，利用显微拍照，imageJ统计细胞迁移情况。
结果显示：Sema4D可促进内皮细胞迁移(图4中A)，内皮细胞迁移能力随sema4D浓度上升而增加(图4中B)。
2)Sema4D可促进内皮细胞管腔形成(管腔形成实验)
48孔板中加入150ul预冷基质胶/孔(BD Biosciences)，置于37℃的细胞培养箱30min后，每孔种2×104个小鼠脑微血管内皮细胞，培养3天待细胞长满后在DMEM中分别加入终浓度为400，800，1600ng/ml的sema4D，加入培养孔后孵育24h，24h后利用显微镜拍照image J统计，以不加sema4D的DMEM为对照。
结果显示Sema4D可促进内皮细胞管腔形成，且随sema4D浓度上升而增加(图4中C-D)。
3)Sema4D可促进内皮细胞渗漏(TEER实验)
在24孔的transwell小室(0.4um)上层先包被一层纤黏蛋白，室温孵育1h；包被后种入小鼠脑微血管内皮细胞5×104个，培养3天待细胞长满后在培养基内分别加入终浓度为400，800，1600ng/ml的sema4D蛋白，以不加sema4D的DMEM为对照，孵育24h后利用细胞跨膜电阻测量仪测量其电阻值。
图4中E显示加入400，800，1600ng/ml的sema4D后内皮细胞的TEER逐渐下降，表 明Sema4D可促进内皮细胞渗漏，且随浓度上升渗漏加重。
4)Sema4D可促进内皮细胞渗漏(荧光渗漏实验)
在24孔的transwell小室(0.4um)上层先包被一层纤黏蛋白，室温孵育1h，包被后种入小鼠脑微血管内皮细胞，培养3天待细胞长满后在培养基内分别加入终浓度为400，800，1600ng/ml的sema4D，以不加sema4D的DMEM为对照，24h后在小室上层DMEM中加入终浓度为300μg/ml荧光标记的右旋糖酐，于1h提取下层培养基30ul，利用酶标仪测量625nm波长的吸光度。
图4中F显示加入终浓度为400，800，1600ng/ml的sema4D后内皮细胞渗漏的dextran荧光值上升，表明Sema4D可加重内皮细胞渗漏，且随浓度上升渗漏加重。
5)慢病毒转染致内皮细胞PlexinB1下调，蛋白水平验证(Western Blot)
本发明实施例涉及到的慢病毒均购买自吉凯公司，其中CRISPR-wt为带GFP的空载慢病毒，CRISPR-plB1为PlexinB1敲除的CRISPR/Cas9的慢病毒，用于下调PlexinB1；实验分为3组：1、正常组(Normal)，即不加任何物质的内皮细胞组；2、转染了带GFP的空载慢病毒组(CRISPR-wt)；3、转染了CRISPR-plB1的组，每组5个重复，病毒组MOI值为50。
慢病毒转染小鼠脑微血管内皮细胞后48h提取蛋白进行Western Blot对PlexinB1的表达效果进行检测，以β-action为对照，WB过程中使用的抗体为PlexinB1antibody(1:500,Abcam)。
结果显示：CRISPR-plB1可显著降低PlexinB1的表达(图4中G，左图为WB图，右图为WB统计图)。
6)下调PlexinB1后sema4D介导的内皮细胞迁移作用减弱(划痕实验)
实验步骤：慢病毒(CRISPR-wt或CRISPR-plB1)转染小鼠脑微血管内皮细胞48h后消化种于细胞板中，在培养孔中种满小鼠脑微血管内皮细胞，用0.5％的FBS饥饿过夜后，用200ul枪头在细胞上划出垂直的竖线，洗去划下的细胞后，直接孵育24h或加终浓度为1600ng/ml的sema4D蛋白后再孵育24h，每组6个重复，利用显微拍照，imageJ统计细胞迁移情况。
结果显示：sema4D蛋白促进内皮细胞的迁移数目增加(图4中H-I)，PlexinB1下调可抑制该过程。
7)下调PlexinB1后sema4D介导的内皮细胞形成管腔作用减弱(管腔形成实验)
48孔板中加入150ul预冷基质胶/孔，置于37℃的细胞培养箱30min，每孔种2×104个转染了慢病毒(CRISPR-wt或CRISPR-plB1)的小鼠脑微血管内皮细胞；培养3天待细胞长满后，直接孵育24h或加终浓度为1600ng/ml的sema4D蛋白后再孵育24h，每组6个重复。
图4中J-K显示下调PlexinB1后sema4D介导的内皮细胞形成管腔作用减弱。
8)下调PlexinB1后sema4D介导的内皮细胞渗漏作用减少(TEER实验)
在24孔的transwell小室(0.4um)上层先包被一层粘附因子(室温孵育1h)，包被后种入转染了慢病毒(CRISPR-wt或CRISPR-plB1)的小鼠脑微血管内皮细胞，培养3天待细胞长满后直接孵育24h或加终浓度为1600ng/ml的sema4D蛋白后再孵育24h，每组6个重复。孵育24h后利用细胞跨膜电阻测量仪测量其电阻值。
图4中L显示显示sema4D促进内皮细胞的渗漏增加，PlexinB1下调可抑制该过程。
9)下调PlexinB1后sema4D介导的内皮细胞渗漏作用减少(荧光渗漏实验)
在24孔的transwell小室(0.4um)上层先包被一层粘附因子，室温孵育1h，包被后种入转染了慢病毒(CRISPR-wt或CRISPR-plB1)的小鼠脑微血管内皮细胞(5×104个)，培养3天待细胞长满后，直接孵育24h或加终浓度为1600ng/ml的sema4D蛋白后再孵育24h，在小室上层DMEM中加入300μg/ml荧光标记的右旋糖酐，于1h后提取下层培养基30ul，利用酶标仪测量625nm波长的吸光度。
图4中M显示，荧光渗漏实验显示sema4D促进内皮细胞的渗漏增加，PlexinB1下调可抑制该过程。
实施例5：
Sema4D/PlexinB1信号通路可促进内皮与周细胞混合培养模型渗漏；
本实施例所用的内皮细胞为小鼠脑微血管内皮细胞，周细胞为小鼠原代脑微血管周细胞。
1)Sema4D可促进内皮细胞与周细胞共培养体系的渗漏(TEER实验)。
在24孔的transwell小室(0.4um)先包被一层纤黏蛋白，室温孵育1h；，于小室下层种入小鼠脑微血管内皮细胞(5×104)，12h后，在小室上层种入小鼠原代脑微血管周细胞(2.5×104)，培养3天后，更换培养基，在上层DMEM中分别加入终浓度为400，800，1600ng/ml的sema4D蛋白，以不加sema4D的DMEM为对照，孵育24h后利用细胞跨膜电阻测量仪测量其电阻值。图5中A表示，Sema4D可降低内皮与周细胞共培养体系的TEER值，且其降低值随着Sema4D蛋白浓度的升高而增大。
2)Sema4D可促进内皮细胞与周细胞共培养体系的渗漏(荧光渗漏实验)。
在24孔的transwell小室(0.4um)先包被一层纤黏蛋白，室温孵育1h，于小室下层种入脑微血管内皮细胞，12h后，在小室上层种入小鼠原代脑微血管周细胞，培养3天后，更换培养基，在上层DMEM中分别加入终浓度为400，800，1600ng/ml的sema4D蛋白，以不加sema4D的DMEM为对照，24h后在小室上层DMEM中加入300μg/ml荧光标记的右旋糖 酐，以不加sema4D的细胞样本为对照，于1h后提取下层培养基30ul，利用酶标仪测量625nm波长的吸光度。
图5中B显示Sema4D可浓度依赖性促进内皮与周细胞共培养体系的渗漏。
3)共培养模型中，单独沉默周细胞的PlexinB1蛋白受体较之于单独沉默内皮细胞PlexinB1蛋白受体能更大程度上逆转Sema4D的作用
在24孔的transwell小室(0.4um)先包被一层纤黏蛋白，室温孵育1h，下层种入处理后的小鼠脑微血管内皮细胞(5×104)，12h后，在小室上层种入处理后的小鼠原代脑微血管周细胞(2.5×104)；培养3天后，更换培养基，在上层DMEM中加入终浓度为1600ng/mlSema4D蛋白，以不加Sema4D蛋白为对照；然后按照步骤1)和2)中的方法，分别进行TEER实验和荧光渗漏实验；
具体的分组如下：
(1)、CRISPR-wt组：下层种入转染了空载慢病毒的内皮细胞，小室上层种入转染了空载慢病毒的周细胞；
(2)、PC-CRISPR-plB1组：下层种入转染了空载慢病毒的内皮细胞、小室上层种入转染了CRISPR-plB1的周细胞；
(3)、EC-CRISPR-plB1组：下层种入转染了CRISPR-plB1的内皮细胞、小室上层种入转染了空载慢病毒的周细胞。
结果显示，共培养周细胞及内皮细胞模型中，TEER实验及荧光素渗漏实验显示周细胞PlexinB1受体沉默较内皮细胞PlexinB1受体沉默能更明显的阻断Sema4D诱导的渗漏作用(图5中C-D)
实施例6：
单次注射anti-Sema4D治疗可抑制OIR模型小鼠及STZ模型小鼠的眼底血管新生和眼底血管渗漏
1)IB4免疫荧光染色实验(单次注射)：
3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中注射不同剂量Sema-4D中和抗体(BMA-12)(0.5ug，1ug，2ug)，以注射2ug的IgG做为对照，等体积的PBS做为对照，继续正常空气中喂养5天；幼鼠在出生第17天时麻醉，生理盐水心脏灌注后，分离眼球并固定，剥离出视网膜IB4染色，结果显示anti-Sema4D浓度依赖性减轻异常血管占视网膜总面积的百分比(图6中A)，说明anti-Sema4D治疗可抑制OIR模型小鼠眼底血管新生，并呈浓度依 赖性。
2)伊文氏蓝荧光实验(单次注射)：
3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中注射不同剂量sema4d中和抗体(0.5ug，1ug，2ug)，(以注射2ug的IgG做为对照，等体积的PBS做为对照，继续正常空气中喂养5天；幼鼠在出生17天时腹腔注射伊文氏蓝染料(200毫克/公斤)，4小时后麻醉幼鼠，生理盐水心脏灌注后，，取其视网膜至于甲酰胺(70℃水浴)过夜，利用酶标仪(波长620-740mm)测定萃取液体。根据标准曲线计算浓度，并利用视网膜重量及血液中视网膜伊文氏蓝水平进行校准，结果显示anti-Sema4D治疗可抑制OIR模型小鼠眼底血管渗漏，并呈浓度依赖性(图6中B)。
3)IB4免疫荧光染色实验(野生型小鼠和基因敲除小鼠，单次注射)：
wt组：3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中注射Sema4D中和抗体(2ug)或IgG(2ug)继续正常空气中喂养5天；然后进行IB4免疫荧光染色实验；
Sema4D-KO组：3-4月的Sema4D基因敲除后的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中注射Sema4D中和抗体(2ug)或IgG(2ug)，继续正常空气中喂养5天；然后进行IB4免疫荧光染色实验；
结果显示：anti-Sema4D治疗及Sema4D基因敲除均可减少OIR模型导致的眼底血管新生(IB4免疫荧光染色)，玻璃体anti-Sema4D注射后不能够进一步抑制Sema4D基因敲除老鼠异常血管新生(图6中C)。
4)伊文氏蓝荧光实验(野生型小鼠和基因敲除小鼠，单次注射)：
wt组：3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中注射Sema4D中和抗体(2ug)或IgG(2ug)继续正常空气中喂养5天；按照步骤2)中的方法，然后进行伊文氏蓝荧光实验；
Sema4D-KO组：3-4月的Sema4D基因敲除后的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后玻璃体中分别注射Sema4D中和抗体(2ug)和IgG(2ug)继续正常空气中喂养5天；按照步骤2)中的方法，然后进行伊文氏蓝荧光实验；
结果显示：anti-Sema4D治疗及Sema4D基因敲除均可减少OIR模型导致的眼底血管渗 漏(伊文氏蓝荧光实验)，玻璃体anti-Sema4D注射后不能够进一步抑制Sema4D基因敲除老鼠血管渗漏(图6中D)。
5)IB4免疫荧光染色实验(anti-Sema4D联合anti-VEGF治疗，单次注射)
3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后于鼠眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)；或者分别单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(2ug)；然后按照步骤1)中的方法进行IB4免疫荧光染色实验。
结果显示：anti-Sema4D联合anti-VEGF治疗抑制OIR模型小鼠眼底血管新生，效果显著优于单独anti-Sema4D或单独anti-VEGF治疗(图6中E-F)。
6)伊文氏蓝实验(anti-Sema4D联合anti-VEGF治疗，单次注射)
3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后于鼠眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)；或者分别单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)；然后按照步骤2)中的方法进行伊文氏蓝荧光实验。
结果显示：anti-Sema4D联合anti-VEGF治疗抑制OIR模型小鼠眼底血管渗漏，效果显著优于单独anti-Sema4D或单独anti-VEGF治疗(伊文氏蓝实验)(图6中G)
7)HE染色实验(anti-Sema4D联合anti-VEGF治疗，单次注射)
3-4月的C57BL/6母鼠产子后，与出生第7天的幼鼠一起放至75％氧气的氧舱中，至出生第12天后将幼鼠与母鼠取出，随即幼鼠麻醉后于鼠眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)；或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)；小鼠均于出生第17天麻醉，生理盐水心脏灌注后，取其眼球于4％多聚甲醛中固定，行石蜡包埋切片HE染色，在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞数目。
结果显示：anti-Sema4D联合anti-VEGF治疗抑制OIR模型小鼠眼底视网膜前新生血管的细胞数目，效果显著优于单独anti-Sema4D或单独anti-VEGF治疗(HE染色)(图6H-I)。
8)STZ造模鼠伊文氏蓝荧光实验(anti-Sema4D联合anti-VEGF治疗，单次注射)
STZ造模鼠于造模后5月时被麻醉，于鼠眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)；注射一周后，尾静脉注射伊文氏蓝染料(45mg/kg)，2个小时后麻醉老鼠，生理盐水心脏灌注后，取其视网膜至于甲酰胺(70℃水浴)过夜，利用酶标仪(波长620-740mm)测定。 浓度根据标准曲线计算，并利用视网膜重量及血液中视网膜水平换算后统计。同时取一部分眼球于4％的PFA室温固定2h后，行视网膜铺片后荧光拍照。
结果显示：单次联合anti-Sema4D和anti-VEGF治疗抑制STZ模型小鼠眼底血管渗漏，效果显著优于单独anti-Sema4D或单独anti-VEGF治疗。
实施例7：
多次anti-Sema4D治疗可抑制STZ模型小鼠眼底血管渗漏并在抑制眼底血管周细胞脱失上优于anti-VEGF
1)伊文氏蓝荧光实验(STZ造模鼠多次治疗)
STZ造模鼠于造模4月后眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)以注射anti-Sema4D(2ug)或anti-VEGF(2ug)或IgG(2ug)或PBS(2ug)为对照，每周注射一次，第5次注射后一周尾静脉注射伊文氏蓝染料(45mg/kg)，循环2个小时后麻醉老鼠，生理盐水心脏灌注后，取其视网膜至于甲酰胺(70℃水浴)过夜，利用酶标仪(波长620-740mm)测定。浓度是根据标准曲线计算的，并利用视网膜重量及血液中视网膜水平换算后统计。同时取一部分眼球于4％的PFA室温固定2h后，分离视网膜，铺片行荧光拍照。
结果显示：多次anti-Sema4D治疗，每周注射一次，第5次注射后一周后检测发现其可抑制STZ模型眼底血管渗漏，联合anti-Sema4D及anti-VEGF的多次治疗效果显著优于单独anti-Sema4D或单独anti-VEGF的多次治疗(图7中A和B)。(图中NODM为正常对照组，照模组是指腹腔注射STZ组，正常对照组指腹腔注射柠檬酸盐组。
2)糖原染色实验(STZ造模鼠多次治疗)
STZ造模鼠于造模4月后眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)，每周注射一次，共注射5次，第5次注射后麻醉，生理盐水心脏灌注后，取其新鲜的眼球被固定在4％的PFA中24小时，然后视网膜被分离并以3％的胰蛋白酶(溶解在pH.7.8tris-hcl)在37摄氏度的状态下消化.当视网膜开始瓦解时，然后摇晃视网膜，直到血管网络完全分离出。将血管网络转移至于干净玻片上，干燥后行糖原染色，显微镜下拍照统计血管无灌区数目。(图中NODM为正常对照组)
结果显示：anti-Sema4D多次治疗可抑制STZ模型眼底血管无血管区形成，且anti-Sema4D联合anti-VEGF治疗效果显著优于多次单独anti-Sema4D或anti-VEGF治疗(图7中C和D)。
3)Desmin免疫荧光染色(STZ造模鼠多次治疗)
STZ造模鼠于造模4月后眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)，每周注射一次，共注射5次，第5次注射后麻醉，生理盐水心脏灌注后，取其新鲜的眼球被固定在4％的PFA中24小时，然后视网膜被分离并以3％的胰蛋白酶(溶解在pH.7.8tris-hcl)在37摄氏度的状态下消化.当视网膜开始瓦解时，然后摇晃视网膜，直到血管网络完全分离出。将血管网络转移至于干净玻片上行Desmin免疫荧光染色，利用荧光显微镜拍照统计。Desmin(1:100,Abcam)。
结果显示：多次anti-Sema4D治疗可抑制STZ模型眼底血管表面周细胞脱失，其提高周细胞覆盖率优于anti-VEGF治疗，且多次anti-Sema4D联合anti-VEGF治疗效果显著优于多次单独anti-Sema4D或anti-VEGF治疗(图7中E和F)。
4)免疫荧光染色考察眼底血管VE-cadherin的连续性(STZ造模鼠多次治疗)
STZ造模鼠于造模4月后眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)，每周注射一次，共注射5次，第5次注射后麻醉，生理盐水心脏灌注后，取新鲜眼球于无水甲醇-20℃固定2h后，剥离出视网膜，置于封闭液中(1％BSA，0.5％TritonX-100，PBS)4℃过夜，孵育一抗(VE-cadherin/CollagenIV)5天后，室温孵育二抗3h后铺片拍照。抗体：VE-cadherin(1:50,BDBiosciences)，CollagenIV(1:100,SouthernBiotech)。
结果显示：多次anti-Sema4D治疗可增加STZ模型中眼底血管VE-cadherin的连续性，且anti-Sema4D联合anti-VEGF的多次治疗效果显著优于多次单独anti-Sema4D或多次单独anti-VEGF治疗(图7中G-H)。
5)免疫荧光染色考察眼底血管N-cadherin的覆盖率
STZ造模鼠于造模4月后眼玻璃体中同时注射sema4d中和抗体(2ug/眼)和VEGF中和抗体(2ug/眼)，或者单独注射anti-Sema4D(2ug)、anti-VEGF(2ug)、IgG(2ug)、PBS(1ul)，每周注射一次，共注射5次，第5次注射后麻醉，生理盐水心脏灌注后，取新鲜眼球于4％的PFA中4℃固定2h，剥离出视网膜，置于封闭液中(1％BSA0.5％TritonX-100，PBS)，4℃过夜后，孵育一抗(N-cadherin/CollagenIV)5天后，室温孵育二抗3h后铺片拍照。抗体：N-cadherin(1:50,LifeTechnologies)，CollagenIV(1:100,SouthernBiotech)。
结果显示：多次anti-Sema4D治疗可增加STZ模型中眼底血管N-cadherin的覆盖率，其效果优于单独anti-VEGF治疗，且anti-Sema4D联合anti-VEGF治疗效果显著优于单独 anti-Sema4D或单独anti-VEGF治疗(图7中I-J)。
6)伊文氏蓝荧光实验
按照步骤1)中的实验方案，进行伊文氏蓝荧光实验，延长观察时间。
结果显示多次anti-Sema4D治疗，延长观察时间，两周后证实其可抑制STZ模型眼底血管渗漏，且效果优于多次anti-VEGF治疗，同时anti-Sema4D联合anti-VEGF治疗效果显著优于单独anti-Sema4D或anti-VEGF治疗(图7中K-L)。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (15)

  1. Sema4D或PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用。
  2. 根据权利要求1所述的应用，所述的眼底血管疾病为眼底血管新生、眼底血管渗漏或周细胞脱失。
  3. 根据权利要求1所述的应用，所述的抑制剂为Sema4D的抗体、抑制Sema4D或PlexinB1基因表达的病毒或Sema4D/PlexinB1信号通路抑制剂。
  4. 根据权利要求1所述的应用，所述的眼底血管疾病是由糖尿病引起的。
  5. 一种治疗眼底血管疾病的方法，所述的方法为向眼玻璃体中注射Sema4D蛋白的抑制剂。
  6. 一种治疗眼底血管疾病的方法，所述的方法为向眼玻璃体中注射Sema4D蛋白的抑制剂和anti-VEGF。
  7. 根据权利要求5或6所述的方法，所述的的眼底血管疾病包括但不限于眼底血管新生或眼底血管渗漏或周细胞脱失。
  8. 根据权利要求5或6所述的方法，所述的抑制剂为Sema4D蛋白的中和性抗体。
  9. 一种慢病毒载体，所述慢病毒载体是PlexinB1基因敲除的CRISPR/Cas9重组慢病毒载体。
  10. 含有权利要求9所述慢病毒载体的慢病毒。
  11. Sema4D基因敲除、Sema4D/PlexinB1基因沉默、抑制Sema4D/PlexinB1信号通路的反义寡核苷酸在制备治疗及预防眼底血管疾病药物中的应用。
  12. 根据权利要求11所述的应用，所述Sema4D/PlexinB1信号通路慢病毒沉默采用权利要求9所述的慢病毒载体或权利要求10所述的慢病毒实现。
  13. 根据权利要求11或12所述的应用，所述眼底血管疾病为眼底血管新生、眼底血管渗漏或周细胞脱失。
  14. 根据权利要求11所述的应用，所述疾病是由糖尿病引起的。
  15. 一种眼底血管疾病的基因治疗方法，所述方法为对对象施用Sema4D/PlexinB1基因敲除、基因沉默或者抑制Sema4D/PlexinB1信号通路的反义寡核苷酸的基因治疗手段。
PCT/CN2019/109213 2018-10-22 2019-09-29 Sema4D/PlexinB1抑制剂在制备治疗及预防眼底血管疾病药物中的应用 WO2020083007A1 (zh)

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CN115010790B (zh) * 2021-08-29 2024-03-08 湖北烛照生物科技有限公司 纳米小肽fg及其在制备治疗及预防眼底血管疾病药物中的应用
CN114933635B (zh) * 2021-08-29 2023-05-19 武汉夫图生物科技有限公司 纳米小肽fh及其在制备治疗及预防眼底血管疾病药物中的应用
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