CN110643635A - Application of gene silencing in Sema4D/PlexinB1 signaling pathway - Google Patents

Abstract

The invention belongs to the field of biomedicine, and specifically discloses the application of a Sema4D/PlexinB1 inhibitor in the preparation of medicines for treating and preventing fundus vascular diseases. The applicant finds that Sema4D is increased in the aqueous humor of patients with diabetic retinopathy, and the level of Sema4D is related to the patient's response to anti-VEGF On the one hand, the Sema4D/PlexinB1 signaling pathway can act on endothelial cells to promote their migration and lumen formation; It provides a new idea for retinal vascular proliferation and leakage diseases such as diabetic retinopathy.

Description

Application of gene silencing in Sema4D/PlexinB1 signaling pathway

This application is a divisional application of a Chinese invention patent application with application number 201811228364.X entitled "Application of Sema4D/PlexinB1 Inhibitor in the Preparation of Medicines for Treating and Preventing Fundus Vascular Diseases".

technical field

The invention belongs to the field of biomedicine, and in particular relates to the application of Sema4D/PlexinB1 signaling pathway gene silencing in preparing medicines for treating and preventing fundus vascular diseases.

Background technique

Diabetic retinopathy (DR), as the first cause of blindness in adults aged 20-75, has a high prevalence in patients with type 1 and type 2 diabetes. Globally: DR has a prevalence of 35.4% in the diabetic population. The prevalence of proliferative diabetic retinopathy (PDR) in the most severe stage is 7.5% (2017 Diabetes Care IF=13.397), and in China: the prevalence of DR in the diabetic population at different stages is also as high as 24.7%-37.5 (2014 guidelines), The World Health Organization (WHO) estimates that by 2025, the number of diabetic patients in the world will exceed 30 billion (2009EYE), which shows that the number of DR patients will further increase.

The treatment of DR has been mainly divided into three types of treatments since the Diabetic Retinopathy Vitrectomy Study (DRVS Study) was carried out in 1990: (1) laser photocoagulation, (2) vitrectomy, and (3) the application of anti-angiogenic drugs (mainly various forms of anti- VEGF therapy, 2004-present), laser photocoagulation therapy is the treatment method for abandoning the car and keeping the central visual field at the expense of the peripheral visual field, but only 30% of the patients receiving anti-VEGF therapy are effective. It can be seen that anti-VEGF therapy is resistant , repeated administration of infection, neuronal toxicity, and heavy economic burden all bring inconvenience and risks to treatment, and urgently require us to develop new treatment strategies.

The pathological process of DR is different from tumor angiogenesis, and its treatment has certain particularities. The initiating factor of DR is the slowing of fundus blood vessel flow rate, leading to local metabolic changes, activation of inflammation and related signaling pathways, resulting in vascular leakage, blood-retina The barrier is damaged, the parietal cells are lost, and the cell matrix is damaged, thereby causing the blood vessels to lose their function, forming a hypoxic area locally, leading to compensatory abnormal blood vessel proliferation, aggravating leakage and even dysfunctional abnormal new blood vessels, causing fundus hemorrhage and retinal detachment (CurrDiab Rep 2011; Diabetes. 2006). In tumors, angiogenesis originates from the induction of pro-angiogenic factors and hypoxia, through the activation of endothelial cells to protrude pseudopodia, the attachment of surrounding wall cells, and the fusion of adjacent neovascular lumens to form a vascular network. The purpose of angiogenesis in tumors is to support tumor growth. Therefore, the focus of angiogenesis treatment in tumors is to simply inhibit angiogenesis, but it is different in the fundus. The purpose of anti-angiogenesis therapy is to inhibit abnormal blood vessels and restore normal blood vessels. It can be seen that although the two diseases are both inhibiting angiogenesis, the treatment results can be quite different.

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 angiogenesis and angiogenesis. Among them, the single-pass transmembrane protein Se ma4D (Class4semaphorins) is hotly debated in angiogenesis. The extracellular free Sema4D fragment that it acts on is mainly derived from the cleavage of MMP-MT1 in various cells or the cleavage of ADAM17 in platelets. It is involved in tumor, bone, and trauma model angiogenesis, and its role is specific in different tissues and cells. In tumors, Sema4D promotes the phosphorylation of Met and Ron by binding PlexinB receptor to promote cell migration, angiogenesis leads to tumor cell metastasis (2009Valente), however, in lung cancer, Sema4D binds PlexinB1 to form a complex with ERBB2, which can inhibit Met by inhibiting Met. Inhibition of cell migration (Swiercz 2008), it can be seen that Se ma4D has different effects in different tissues and different diseases.

In the present invention, the applicant clarifies that inhibiting the Sema4D/PlexinB1 signaling pathway can effectively inhibit fundus angiogenesis and reduce fundus vascular leakage. More importantly, early inhibition of the Sema4D/PlexinB1 signaling pathway can effectively reduce the loss of pericytes, which is helpful for Protecting and restoring the function of fundus blood vessels can prolong the therapeutic effect to a certain extent, reduce the number of administrations, and provide a new strategy for the treatment of fundus angiogenesis and leakage.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of Sema4D/PlexinB1 inhibitor in the preparation of medicines for the treatment and prevention of fundus vascular diseases, including but not limited to fundus angiogenesis or fundus vascular leakage or pericyte loss.

In order to achieve the above object, the present invention adopts the following technical measures:

In view of the current limited treatment methods for fundus angiogenesis and leakage, the applicant obtained aqueous humor from patients with diabetic retinopathy, screened members closely related to angiogenesis, and found that Sema4D/PlexinB1 signaling pathway inhibitors can effectively inhibit fundus angiogenesis and leakage. leak.

Application of Sema4D/PlexinB1 signaling pathway inhibitor in the preparation of medicines for the treatment and prevention of fundus vascular diseases, the inhibitors include but are not limited to Sema4D/PlexinB1 signaling pathway inhibiting virus, or Sema4D/PlexinB1 signaling pathway neutralizing antibody, or Compounds that inhibit the function of the Sema4D/PlexinB1 signaling pathway.

In the above application, preferably, the angiogenesis and leakage of the fundus are caused by diabetes.

A method for treating ocular fundus vascular disease, which comprises injecting an inhibitor of Sema4D protein into the vitreous body;

A method for treating ocular fundus vascular disease, the method comprises injecting Sema4D protein inhibitor and anti-VEGF into vitreous body.

The fundus vascular diseases include but are not limited to fundus angiogenesis or fundus vascular leakage or pericyte loss.

In the above method, preferably, the inhibitor is a neutralizing antibody of Sema4D protein or a Sema4D/PlexinB1 signaling pathway inhibitor.

Compared with the prior art, the present invention has the following advantages:

In view of the current limited treatment methods for fundus angiogenesis and leakage, the existing main treatment methods are ① laser photocoagulation therapy ② vitrectomy ③ application of anti-angiogenesis drugs, among which anti-angiogenesis drugs are mainly anti-VEGF fundus Injection, but only 30% of patients who received anti-VEGF treatment were effective. In view of the limitations of current treatment, the applicant took aqueous humor from patients with diabetic retinopathy, screened Sema family members closely related to angiogenesis, and found the Sema4D/PlexinB1 signaling pathway The inhibitor can effectively inhibit fundus angiogenesis and leakage, and has demonstrated the effect of inhibiting Sema4D/P lexinB1 signaling pathway on fundus pathological angiogenesis and leakage in vivo, in vitro and in gene knockout mice, and the related mechanisms are clarified.

Description of drawings

Figure 1 is a schematic diagram of the close correlation between Sema4D and DR;

Among them, A: schematic diagram of optical coherence tomography (OCT);

The expression of Sema4D in the aqueous humor of B-C.DR patients was significantly increased (Western Blot)

The expression of Sema4D in the aqueous humor of D.DR patients was significantly increased (ELISA detection)

Free Sema4D in the aqueous humor of E-F.DR patients was negatively correlated with foveal thickness (CST) and macular volume (MV) after 3 months of anti-VEGF treatment (statistics obtained from Wuhan Union Medical College Hospital Ophthalmology Database)

Figure 2 is a schematic diagram of the elevated expression of Sema4D in mouse OIR and STZ models;

Among them, A: The mRNA expression of Sema4D in the retina of OIR model mice was increased (q-PCR);

B: Western Blot results show that the expression of Sema4D protein in retina of OIR model mice is increased;

C-D: Western Blot showed that the expression of Sema4D protein in the retina of STZ model mice increased at 3 months and 6 months.

Figure 3 is a schematic diagram of Sema4D gene knockout inhibiting fundus angiogenesis;

Among them, A-C: knockout mouse validation;

D-H: Immunofluorescence staining, Evans blue leakage test, HE staining results showed that Sema4D gene knockout can significantly inhibit the pathological angiogenesis and vascular leakage in the fundus.

Figure 4 is a schematic diagram showing that the Sema4D/PlexinB1 signaling pathway can promote endothelial cell lumen formation and leakage;

Among them, A-B: scratch experiments confirmed that Sema4D can promote endothelial cell migration;

C-D: Lumen formation experiments confirmed that Sema4D can promote endothelial cell lumen formation;

E: TEER experiments confirmed that Sema4D can promote endothelial cell leakage;

F: Fluorescein leakage experiments confirmed that Sema4D can promote endothelial cell leakage;

G: PlexinB1 down-regulation verification;

H-I: Scratch experiments confirmed that blocking and silencing the PlexinB1 receptor can block the effect of Sema4D on promoting endothelial cell migration;

J-K: Lumen formation experiments confirmed that silencing the PlexinB1 receptor can block the effect of Sema4D on promoting endothelial cell lumen formation;

L-M: TEER and fluorescein leakage experiments confirmed that silencing PlexinB1 receptor can block Sema4D promoting endothelial cell leakage;

Figure 5 is a schematic diagram showing that the Sema4D/PlexinB1 signaling pathway increases the leakage of endothelial and pericyte co-culture models;

Among them, A-B: co-cultured pericytes and endothelial cells TEER experiments and fluorescein leakage experiments confirmed that Se ma4D signaling promotes leakage;

C-D: In co-cultured pericyte and endothelial cell models, TEER assay and fluorescein leakage assay confirmed that silencing PlexinB1 receptor blocked Sema4D-induced leakage;

Figure 6 is a schematic diagram of anti-Sema4D inhibiting fundus angiogenesis in OIR model mice and fundus vascular leakage in STZ model mice; A: anti-Sema4D treatment can inhibit fundus angiogenesis in OIR model mice in a concentration-dependent manner;

B: Evans blue experiment confirmed that anti-Sema4D treatment can inhibit the leakage of fundus blood vessels in OIR model mice, in a concentration-dependent manner. C-D: Anti-Sema4D antibody specificity verification;

E-I: In the OIR model, anti-Sema4D treatment can inhibit angiogenesis and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D or anti-VEGF treatment alone;

J-K: A single dose of anti-Sema4D treatment in the STZ model can inhibit fundus vascular leakage, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D or anti-VEGF treatment alone.

Figure 7 is a schematic diagram of the effect of multiple anti-Sema4D treatments;

Among them, A-B: multiple administrations, observed after 1 week, STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D or anti-VEGF treatment alone

C-F: STZ model fundus anti-Sema4D treatment can inhibit pericyte loss and improve pericyte coverage, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D or anti-VEGF treatment alone, more importantly, anti-Sema4D Inhibition of pericyte loss and improvement of pericyte coverage were superior to anti-VEGF F treatment (P<0.05)

G-H: STZ model fundus anti-Sema4D treatment can increase VE-cadherin continuity and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D or anti-VEGF treatment alone

I-J: STZ model fundus anti-Sema4D treatment can increase the coverage of N-cadherin and the combined anti-VEGF treatment is significantly better than anti-VEGF treatment alone

K-L: After multiple administration, observed after 2 weeks, STZ model fundus anti-Sema4D treatment can inhibit fundus vascular leakage, and the combined anti-VEGF treatment effect is significantly better than anti-Sema4D alone or anti-VEGF treatment alone, more importantly, Multiple anti-Sema4D treatment for fundus vascular leakage was superior to multiple anti-VEGF treatment (P<0.05).

Detailed ways

The present invention will be further described below with reference to specific embodiments. The technical solutions of the present invention, unless otherwise specified, are conventional solutions in the field. The reagents or materials, unless otherwise specified, were obtained from commercial sources.

Example 1:

Findings that Sema4D is closely related to DR:

1) Retinal tomography

Comparing the OCT tomograms of patients with diabetic retinopathy and normal people, it was found that the thickness of the fovea was thickened in the patients with diabetic retinopathy (Fig. 1, A)

2) Detection of Sema4D expression in aqueous humor

The experimental group was the aqueous humor of the patients with diabetic retinopathy, and the control group was the aqueous humor of the cataract patients, which was extracted with a sterile syringe.

The expression of Sema4D protein in aqueous humor was detected by Western Blot, and the results showed that the expression of Sema4D in aqueous humor of DR patients was significantly increased (B-C in Figure 1).

ELISA kit (MyBioSource) was used to detect the expression of Sema4D protein in the aqueous humor. The results showed that the expression of Sema4D in the aqueous humor of DR patients was significantly increased, and n was the sample size.

3) Retinal tomography and detection of Sema4D protein in aqueous humor of DR patients after treatment

Figures E-F show that free Sema4D in the aqueous humor of DR patients was negatively correlated with foveal thickness (CST) and macular volume (MV) after 3 months of anti-VEGF treatment, and n represents the sample size.

Example 2:

Increased Sema4D expression in mouse OIR and STZ models:

1) Establish a mouse OIR model

Experimental group (OIR): C57BL/6 female mice from March to April were placed in an oxygen chamber containing 75% oxygen together with the pups on the 7th day of life, and the pups and mother mice were placed on the 12th day of life. Removed and then placed in normal air for 5 days. The above-treated young mice were anesthetized on the 12th, 14th, and 17th days after birth. After cardiac perfusion with normal saline, the eyeballs were removed, the retinas were peeled off, and RNA was extracted and reverse transcribed into cDNA or protein.

Control group (Normal): postnatal young mice, grown under normoxic conditions, anesthetized the mice on the 12th, 14th, and 17th days of life, after cardiac perfusion with normal saline, the eyeballs were taken out, the retina was peeled off, RNA was extracted and reverse transcribed. cDNA or extracted protein.

2) q-PCR detection of Sema4D mRNA expression

Using primers (CCTGGTGGTAGTGTTGAGAAC and GCAAGGCCGAGTAGTT AAAGAT), the cDNA in step 1) was used as the template to detect the expression of Sema4D. The results showed that in the OIR group, the mRNA expression of Sema4D was significantly increased (A in Figure 2). (P12 in Figure 2 refers to the mouse sample on the 12th day of birth, and so on, the same below)

3) Western Blot detection of Sema4D protein in experimental group (OIR) and control group

The protein extracted in step 1) was used as the antigen, and the sheepanti-SEMA4Dantibody (source: R&D Systems) was used as the antibody for Western Blot detection, and β-action was used as the control. The results showed that the expression of Sema4D protein in the retina of OIR model mice was significantly increased (B in Figure 2).

4) Establish a mouse STZ model

Experimental group (STZ): 8-week-old C57BL/6 mice were starved for 4 hours and then injected with STZ (streptozotocin, 50 mg/kg) intraperitoneally for 5 consecutive days, once a day, and blood glucose was detected on the 7th day after the injection. The blood sugar greater than 15mmol/L was regarded as successful modeling. The mice were anesthetized at the 3rd and 6th month after modeling, and after cardiac perfusion with normal saline, the eyeballs were removed, the retina was peeled off, and the protein was extracted.

Control group (Vehicle): The streptozotocin in the experimental group was replaced with normal saline, and the rest of the operations were the same.

5) Western Blot detection of Sema4D protein in experimental group (STZ) and control group (Vehicle)

The protein extracted in step 4) was used as the antigen, and the sheep anti-SEMA4D antibody (R&D Systems) was used as the antibody for Western Blot detection, and β-action was used as the control. The results showed that the expression of Sema4D protein in the retina of STZ model mice was significantly increased at 3 and 6 months after modeling (C and D in Figure 2).

Example 3:

Sema4D knockout significantly inhibits fundus angiogenesis and leakage in mice

The Sema4D gene knockout mouse used in this example was purchased from Huazhong Agricultural University, and was obtained by designing sgRNA in the Sema4d exon region of the mouse to knock out the expression of sema4D.

1) Validation of successful knockout of target gene (sema4D) in knockout mice:

DNA was extracted from the mouse tail to verify that the mouse target gene (sema4D) was successfully knocked out, and the wild-type mouse was used as the control: 3-5cm mouse tail was taken and placed in DNA lysis solution at 56°C for 10h, after isopropanol precipitation, 95% Washed with alcohol, configured into the following system, amplified, and electrophoresed in agarose gel.

Sema4d-GT-F1 TCTGGGGCTCTAAGAGGTCCTT Sema4d-GT-R1 AGCCACTGAGGTCACATACACC

A in Figure 3 shows the pattern of the sgRNA targeting area, and 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 of the wild group, indicating that the knockout is successful.

The protein extracted from mouse retina was used for Western Blot to detect Sema4D protein. The mouse retinal tissue was taken, and the extraction procedure was the same as that of general tissue extraction. There were 6 mice in each group, and β-action was used as the control; the Se ma4D protein antibody was SEMA4D antibody (R&D Systems).

C in Figure 3 shows that sema4D knockout homozygous mice have no sema4D protein expression, and knockout heterozygous mice have less sema4D protein expression than wild type. In Figure 3 C, from left to right are wild type, knockout pure Zygotes, knockout heterozygotes.

2) IB4 immunofluorescence staining showed that Sema4D knockout mice could significantly inhibit fundus angiogenesis in the OIR model

The OIR model was established in Sema4D knockout mice. The mice were anesthetized on the 17th day after birth. After cardiac perfusion with normal saline, the eyeballs were fixed in 4% paraformaldehyde at 4°C overnight, and the retinas were removed. B4 was stained overnight at 4°C, and the slides were photographed with Isolectin B4 (1:100, Vector Labo ratios). The OIR model established by wild-type mice was used as a control, with 6 mice in each group.

D in Fig. 3 shows the reduction of abnormal new blood vessels after OIR modeling of sema4D knockout mice, and E in Fig. 3 is a statistical schematic diagram of the proportion of abnormal blood vessels in the total blood vessel area using ImageJ.

3) The Evans blue extraction experiment confirmed that the Sema4D knockout mice can significantly inhibit the fundus vascular leakage in the OIR model. The experiment was divided into wild-type OIR model mice and sema4D knockout OIR model mice, with 6 mice in each group, as follows:

The OIR model mice were injected intraperitoneally with Evans blue dye (200 mg/kg) on the 17th day of life, and the mice were anesthetized after circulating for 4 hours. A microplate reader (wavelength 620-740mm) was used to measure the absorbance of the extraction liquid. The concentration is calculated according to the standard curve (with different concentrations of Evans blue dye as the abscissa, and the liquid absorbance measured by the microplate reader wavelength 620-740mm as the vertical axis to make a standard curve), and converted by retinal weight and retinal level in blood post statistics.

F in Figure 3 shows that Sema4D knockout mice can significantly inhibit fundus vascular leakage in the OIR model.

4) Sema4D knockout mice in OIR model can significantly inhibit the number of preretinal neovascular cells (HE staining)

The experiment was divided into four groups, namely: wild-type mouse group, sema4D knockout mouse group, wild-type OIR model mice and sema4D knockout OIR model mice, with 6 mice in each group. The specific steps are as follows:

The above-mentioned four groups of mice were all anesthetized on the 17th day of birth. After cardiac perfusion with normal saline, the eyeballs were taken and fixed in 4% paraformaldehyde. The paraffin-embedded sections were stained with HE, and the retinal inner boundary was observed and counted under a light microscope. The number of vascular endothelial cells in the membrane.

G in Figure 3 shows the number of cells in the preretinal neovascularization stained by HE. There was no neovascularization in the wild-type mouse group and the se ma4D knockout mouse group. The wild-type OIR model mice and the sema4D knockout OIR model All mice have neovascularization, but the number of new blood vessels in the sema4D knockout OIR model mice is significantly smaller than that in the wild-type OIR model mice. The arrows indicate preretinal neovascular cells. Figure 3 H shows the Sema4D gene knockout mice in the OIR model. Can significantly inhibit the number of cells in preretinal neovascularization.

Example 4:

Enhancement or inhibition of endothelial cell lumen formation and leakage by the Sema4D/PlexinB1 signaling pathway affects:

The cells or endothelial cells involved in this example all refer to mouse brain microvascular endothelial cells.

1) Sema4D can promote endothelial cell migration (scratch assay)

The culture wells were filled with mouse brain microvascular endothelial cells. After starvation with 0.5% FBS overnight, vertical lines were drawn on the cells with a 200ul pipette tip. After washing off the drawn cells, the cells were added to DMEM. The final concentration of sema4D protein was 400, 800, and 1600 ng/ml, and then added to the culture wells and incubated for 24 h. Micrographs were taken, and imageJ was used to count the cell migration.

The results showed that Sema4D could promote endothelial cell migration (A in Figure 4), and the migration ability of endothelial cells increased with the increase of se ma4D concentration (B in Figure 4).

2) Sema4D can promote endothelial cell lumen formation (lumen formation experiment)

150ul pre-cooled Matrigel/well (BD Biosciences) was added to a 48-well plate, placed in a cell incubator at 37°C for 30 minutes, and 2 × 104 mouse brain microvascular endothelial cells were grown in each well, and cultured for 3 days until the cells became full. Add sema4D with final concentrations of 400, 800, and 1600ng/ml to D MEM, respectively, and incubate for 24h after adding to the culture well. After 24h, take pictures with a microscope and image J for statistics. DMEM without sema4D was used as a control.

The results show that Sema4D can promote endothelial cell lumen formation, and it increases with the increase of sema4D concentration (C-D in Figure 4).

3) Sema4D can promote endothelial cell leakage (TEER assay)

A layer of fibronectin was first coated on the upper layer of a 24-well transwell chamber (0.4um), and incubated at room temperature for 1 h; after coating, 5×104 mouse brain microvascular endothelial cells were seeded, and cultured for 3 days until the cells became full. The sema4D protein with final concentrations of 400, 800, and 1600 ng/ml was added to the base, and DMEM without sema4D was used as a control. After 24 hours of incubation, the resistance value was measured by a cell transmembrane resistance meter.

E in Figure 4 shows that the TEER of endothelial cells decreased gradually after adding 400, 800, and 1600 ng/ml of sema4D, indicating that Sema4D can promote endothelial cell leakage, and the leakage aggravates as the concentration increases.

4) Sema4D can promote endothelial cell leakage (fluorescence leakage assay)

The upper layer of a 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, incubated at room temperature for 1 hour, and then seeded into mouse brain microvascular endothelial cells, and cultured for 3 days. The final concentrations of sema4D were 400, 800, and 1600 ng/ml, and DMEM without sema4D was used as a control. After 24 hours, fluorescently labeled dextran with a final concentration of 300 μg/ml was added to the upper DMEM of the chamber, and 30ul of the lower medium was extracted in 1 hour. 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 increased after the addition of sema4D at final concentrations of 400, 800, and 1600 ng/ml, indicating that Sema4D can aggravate endothelial cell leakage, and the leakage aggravates with increasing concentrations.

5) PlexinB1 in endothelial cells was down-regulated by lentiviral transfection, confirmed by protein level (Western Blot)

The lentiviruses involved in the examples of the present invention are all purchased from Jikai Company, wherein CRISPR-wt is an empty-loaded lentivirus with GFP, and CRISPR-plB1 is a lentivirus of CRISPR/Cas9 knocked out by PlexinB1, which is used to downregulate PlexinB1; The experiment was divided into 3 groups: 1. Normal group (Normal), that is, endothelial cell group without any substances; 2. Transfected empty lentivirus group with GFP (CRISPR-wt); 3. Transfected with CRISPR- The group of plB1, each group has 5 replicates, and the MOI value of the virus group is 50.

The expression of PlexinB1 was detected by Western Blot 48h after lentivirus transfection of mouse brain microvascular endothelial cells. The antibody used in the WB process was Plexin B1 antibody (1:500, Abcam).

The results show that: CRISPR-plB1 can significantly reduce the expression of PlexinB1 (G in Figure 4, the left picture is the WB picture, the right picture is the WB statistical picture).

6) The sema4D-mediated endothelial cell migration is attenuated after down-regulation of PlexinB1 (scratch assay)

Experimental steps: mouse brain microvascular endothelial cells were transfected with lentivirus (CRISPR-wt or CRISPR-plB1) for 48 hours, digested and seeded in cell plates, and the culture wells were filled with mouse brain microvascular endothelial cells, and the cells were filled with 0.5% F BS. After starvation overnight, use a 200ul pipette tip to draw vertical lines on the cells, wash off the drawn cells, incubate directly for 24h or add sema4D protein with a final concentration of 1600ng/ml and then incubate for 24h, 6 replicates per group , using micrographs, imageJ to count cell migration.

The results showed that sema4D protein promoted the increase of endothelial cell migration number (H-I in Figure 4), and down-regulation of PlexinB1 could inhibit this process.

7) After down-regulation of PlexinB1, the effect of sema4D-mediated lumen formation on endothelial cells is weakened (luminal formation assay)

Add 150ul pre-cooled Matrigel/well to a 48-well plate, place it in a cell incubator at 37°C for 30 minutes, and plant 2 × 104 mouse brain microvascular endothelium transfected with lentivirus (CRISPR-wt or CRISPR-plB1) in each well. Cells; after culturing for 3 days until the cells are full, incubate directly for 24h or add sema4D protein with a final concentration of 1600ng/ml and then incubate for 24h, with 6 replicates in each group.

Figure 4, J-K, shows that sema4D-mediated endothelial cell lumen formation is attenuated after downregulation of PlexinB1.

8) Reduced endothelial cell leakage mediated by sema4D after down-regulation of PlexinB1 (TEER assay)

The upper layer of a 24-well transwell chamber (0.4um) was first coated with a layer of adhesion factor (incubated at room temperature for 1 h), and then seeded into mouse brain microvascular endothelial cells transfected with lentivirus (CRISPR-wt or CRISPR-plB1). The cells were cultured for 3 days and then incubated directly for 24h after the cells were fully confluent or incubated with sema4D protein at a final concentration of 1600ng/ml for 24h, with 6 replicates in each group. After 24 hours of incubation, the resistance value was measured using a cell transmembrane resistance measuring instrument.

Panel L in Figure 4 shows that sema4D promotes increased leakage in endothelial cells, and downregulation of PlexinB1 inhibits this process.

9) Reduction of sema4D-mediated endothelial cell leakage after downregulation of PlexinB1 (fluorescence leakage assay)

The upper layer of a 24-well transwell chamber (0.4um) was first coated with a layer of adhesion factor, incubated at room temperature for 1 h, and then seeded into mouse brain microvascular endothelial cells transfected with lentivirus (CRISPR-wt or CRISPR-plB1). (5 × 104 cells), cultured for 3 days and waited for the cells to become full, incubate directly for 24h or add se ma4D protein with a final concentration of 1600ng/ml and then incubate for 24h, add 300μg/ml fluorescently labeled dextran to the upper DMEM of the chamber, After 1 h, 30 ul of the lower medium was extracted, and the absorbance at 625 nm was measured using a microplate reader.

M in Figure 4 shows that fluorescence leakage experiments show that sema4D promotes increased leakage of endothelial cells, and downregulation of Plexin B1 inhibits this process.

Example 5:

Sema4D/PlexinB1 signaling pathway can promote the leakage of endothelial and pericyte mixed culture model;

The endothelial cells used in this example are mouse brain microvascular endothelial cells, and the pericytes are mouse primary brain microvascular pericytes.

1) Sema4D can promote the leakage of endothelial cells and pericytes in co-culture system (TEER experiment).

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin and incubated at room temperature for 1 hour; mouse brain microvascular endothelial cells (5×104) were seeded in the lower layer of the chamber, and after 12 hours, microvascular endothelial cells (5×104) were seeded in the upper layer of the chamber. Mouse primary cerebral microvascular pericytes (2.5×104) were cultured for 3 days, the medium was changed, and sema4D protein with final concentrations of 400, 800, and 1600 ng/ml was added to the upper DMEM, and DMEM without sema4D was used as the control. After 24 hours of incubation, the resistance value was measured using a cell transmembrane resistance measuring instrument. A in Figure 5 shows that Sema4D can reduce the TEER value of the co-culture system of endothelial and pericytes, and the reduction value increases with the increase of Sema4D protein concentration.

2) Sema4D can promote the leakage of endothelial cells and pericytes in the co-culture system (fluorescence leakage experiment).

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, incubated at room temperature for 1 hour, and the brain microvascular endothelial cells were seeded in the lower layer of the chamber. After 12 hours, mouse primary brain microvascular pericytes were seeded in the upper layer of the chamber. After culturing for 3 days, the medium was changed, and sema4D protein with final concentrations of 400, 800, and 1600 ng/ml was added to the upper DMEM, and DMEM without sema4D was used as the control. After 24 hours, 300 μg/ml was added to the upper DMEM of the chamber. Fluorescently labeled dextran, with the cell sample without sema4D as the control, 30ul of the lower medium was extracted after 1 h, and the absorbance at 625nm wavelength was measured by a microplate reader.

Figure 5B shows that Sema4D can concentration-dependently promote the leakage of endothelial and pericyte co-culture systems.

3) In the co-culture model, silencing the PlexinB1 protein receptor in pericytes alone can reverse the effect of Sema4D to a greater extent than silencing PlexinB1 protein receptor in endothelial cells alone

A 24-well transwell chamber (0.4um) was first coated with a layer of fibronectin, incubated at room temperature for 1 hour, and the treated mouse brain microvascular endothelial cells (5×104) were seeded in the lower layer. After 12 hours, the treated mouse brain microvascular endothelial cells were seeded in the upper layer The mouse primary brain microvascular pericytes (2.5×104); after culturing for 3 days, the medium was replaced, and the final concentration of 1600ng/ml Sema4D protein was added to the upper layer of DMEM, with no Sema4D protein added as a control; then follow step 1) and 2), the TEER experiment and the fluorescence leakage experiment were carried out respectively;

The specific groups are as follows:

(1) CRISPR-wt group: endothelial cells transfected with empty lentivirus were implanted in the lower layer, and pericytes transfected with empty lentivirus were implanted in the upper layer of the chamber;

(2) PC-CRISPR-plB1 group: endothelial cells transfected with empty lentivirus were implanted in the lower layer, and pericytes transfected with CRISPR-plB1 were implanted in the upper layer of the chamber;

(3), EC-CRISPR-plB1 group: the lower layer was implanted with endothelial cells transfected with CRISPR-plB1, and the upper layer of the chamber was implanted with pericytes transfected with empty lentivirus.

The results showed that in the co-cultured pericyte and endothelial cell models, TEER assay and fluorescein leakage assay showed that pericyte PlexinB1 receptor silencing could more significantly block Se ma4D-induced leakage than endothelial cell PlexinB1 receptor silencing (Fig. 5 C-D)

Example 6:

A single injection of anti-Sema4D treatment can inhibit fundus angiogenesis and fundus vascular leakage in OIR model mice and STZ model mice

1) IB4 immunofluorescence staining experiment (single injection):

After birth, the C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the 12th day of life, the pups and mother mice were taken out. Different doses of Sema-4D neutralizing antibody (BMA-12) (0.5ug, 1ug, 2ug) were injected into the vitreous body, 2ug of IgG was used as a control, and an equal volume of PBS was used as a control, and continued to feed in normal air for 5 days; The young mice were anesthetized on the 17th day of life, and after cardiac perfusion with normal saline, the eyeballs were separated and fixed, and the retina was stripped for IB4 staining. The results showed that anti-Sema4D concentration-dependently reduced the percentage of abnormal blood vessels in the total retinal area (Figure 6, A). It indicated that anti-Sema4D treatment could inhibit the angiogenesis of the fundus of OIR model mice in a concentration-dependent manner.

2) Evans blue fluorescence experiment (single injection):

After birth, the C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the 12th day of life, the pups and mother mice were taken out. Different doses of sema4d neutralizing antibodies (0.5ug, 1ug, 2ug) were injected into the vitreous, (2ug of IgG was used as a control, an equal volume of PBS was used as a control, and the mice were fed in normal air for 5 days; young mice were born at 17 Evan's blue dye (200 mg/kg) was intraperitoneally injected at day time, and the young mice were anesthetized 4 hours later. After cardiac perfusion with normal saline, the retinas were taken and placed in a formamide (70 °C water bath) overnight, using a microplate reader (wavelength 620-740 mm). ) to determine the extraction liquid. The concentration was calculated according to the standard curve and calibrated by retinal weight and retinal Evans blue level in blood. The results showed that anti-Sema4D treatment could inhibit the fundus vascular leakage of OIR model mice in a concentration-dependent manner (Fig. 6 in B).

3) IB4 immunofluorescence staining experiment (wild-type mice and knockout mice, single injection):

wt group: C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After anesthesia, the mice were injected with Sema4D neutralizing antibody (2ug) or IgG (2ug) into the vitreous and continued to feed in normal air for 5 days; then the IB4 immunofluorescence staining experiment was performed;

Sema4D-KO group: C57BL/6 female mice with knockout of the Sema4D gene from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life, and the pups were placed on the 12th day after birth. The mice and mother mice were taken out, and then the young mice were anesthetized and injected with Sema4D neutralizing antibody (2ug) or IgG (2ug) in the vitreous, and continued to feed in normal air for 5 days; then the IB4 immunofluorescence staining experiment was performed;

The results showed that both anti-Sema4D treatment and Sema4D gene knockout could reduce fundus angiogenesis (IB4 immunofluorescence staining) caused by OIR model, and vitreous anti-Sema4D injection could not further inhibit abnormal angiogenesis in Sema4D gene knockout mice (Figure 6). middle C).

4) Evans blue fluorescence experiment (wild-type mice and knockout mice, single injection):

wt group: C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the mice were anesthetized, Sema4D neutralizing antibody (2ug) or IgG (2ug) was injected into the vitreous and continued to be fed in normal air for 5 days; according to the method in step 2), the Evans blue fluorescence experiment was performed;

Sema4D-KO group: C57BL/6 female mice with knockout of the Sema4D gene from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life, and the pups were placed on the 12th day after birth. The mice and mother mice were taken out, and then the young mice were anesthetized and injected with Sema4D neutralizing antibody (2ug) and IgG (2ug) into the vitreous respectively, and continued to feed in normal air for 5 days; follow the method in step 2), and then perform Evans blue fluorescence test ;

The results showed that both anti-Sema4D treatment and Sema4D gene knockout could reduce the fundus vascular leakage caused by OIR model (Evans blue fluorescence test), and vitreous anti-Sema4D injection could not further inhibit the vascular leakage of Sema4D gene knockout mice ( Figure 6 in D).

5) IB4 immunofluorescence staining experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After birth, the C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the 12th day of life, the pups and mother mice were taken out. Simultaneously inject sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) into the vitreous of mouse eyes; or inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug) separately , PBS (2ug); then carry out the IB4 immunofluorescence staining experiment according to the method in step 1).

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited fundus angiogenesis in OIR model mice, and the effect was significantly better than that of anti-Sema4D or anti-VEGF treatment alone (E-F in Figure 6).

6) Evans blue experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After birth, the C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the 12th day of life, the pups and mother mice were taken out. Simultaneously inject sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) into the vitreous of mouse eyes; or inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug) separately , PBS (1ul); then perform the Evans blue fluorescence experiment according to the method in step 2).

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited fundus vascular leakage in OIR model mice, and the effect was significantly better than anti-Sema4D or anti-VEGF treatment alone (Evans blue test) (Figure 6G)

7) HE staining experiment (anti-Sema4D combined with anti-VEGF treatment, single injection)

After birth, the C57BL/6 female mice from March to April were placed in an oxygen chamber with 75% oxygen together with the pups on the 7th day of life. After the 12th day of life, the pups and mother mice were taken out. Simultaneously inject sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) into the vitreous of mouse eyes; or inject anti-Se ma4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1 ul); mice were anesthetized on the 17th day of life, after cardiac perfusion with normal saline, the eyeballs were fixed in 4% paraformaldehyde, paraffin-embedded sections were stained with HE, and the retinal breakthrough was observed and counted under a light microscope. The number of vascular endothelial cells in the inner limiting membrane.

The results showed that anti-Sema4D combined with anti-VEGF treatment inhibited the number of preretinal neovascular cells in the OIR model mice, and the effect was significantly better than that of anti-Sema4D or anti-VEGF treatment alone (HE staining) (Figure 6H-I).

8) Evans blue fluorescence experiment in STZ model mice (anti-Sema4D combined with anti-VEGF treatment, single injection)

The STZ model mice were anesthetized at 5 months after model establishment, and sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) were simultaneously injected into the vitreous of the mouse eyes, or anti-Sema4D (2ug) was injected alone , anti-V EGF (2ug), IgG (2ug), PBS (1ul); one week after the injection, Evans blue dye (45mg/kg) was injected into the tail vein, and the mice were anesthetized 2 hours later. The retina was exposed to formamide (70°C water bath) overnight, and was measured using a microplate reader (wavelength 620-740mm). Concentrations were calculated according to the standard curve and calculated using retinal weight and retinal level in blood. At the same time, a part of the eyeballs were fixed in 4% PFA at room temperature for 2 hours, and then the retina was sliced and photographed by fluorescence.

The results showed that a single combination of anti-Sema4D and anti-VEGF treatment inhibited fundus vascular leakage in STZ model mice, and the effect was significantly better than anti-Sema4D or anti-VEGF treatment alone.

Example 7:

Multiple anti-Sema4D treatment can inhibit fundus vascular leakage in STZ model mice and is better than anti-VEGF in inhibiting fundus pericellular loss

1) Evans blue fluorescence experiment (multiple treatments of STZ model mice)

The STZ model mice were injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) at the same time in the vitreous body 4 months after modeling, or injected with anti-Sema4D (2ug), anti-VEGF ( 2ug), IgG (2ug), PBS (1ul), inject anti-Sema4D (2ug) or anti-VEGF (2ug) or IgG (2ug) or PBS (2ug) as control, once a week, the fifth injection One week later, Evan's blue dye (45 mg/kg) was injected into the tail vein, and the mice were anesthetized after 2 hours of circulation. After cardiac perfusion with normal saline, the retinas were taken and placed in formamide (70 °C water bath) overnight, using a microplate reader (wavelength 620- 740mm) measurement. Concentrations were calculated from a standard curve and calculated using retinal weights and retinal levels in blood. At the same time, a part of the eyeballs were taken and fixed in 4% PFA at room temperature for 2 hours, then the retinas were separated and sliced for fluorescence photography.

The results showed that: multiple anti-Sema4D treatments were injected once a week. One week after the fifth injection, it was found that it could inhibit the fundus vascular leakage of the STZ model. The multiple treatments combined with anti-Sema4D and anti-VEGF had significantly better effects than Multiple treatments with anti-Sema4D alone or anti-VEGF alone (Figure 7, A and B). (NODM in the figure is the normal control group, the control group refers to the intraperitoneal injection of STZ group, and the normal control group refers to the intraperitoneal injection of citrate group.

2) Glycogen staining experiment (multiple treatments of STZ model mice)

The STZ model mice were injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) at the same time in the vitreous body 4 months after modeling, or injected with anti-Sema4D (2ug), anti-VEGF ( 2ug), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after cardiac perfusion with normal saline, fresh eyeballs were fixed in 4% PFA After 24 hours, the retinas were then detached and digested with 3% trypsin (dissolved in pH.7.8 tris-hcl) at 37°C. When the retinas began to disintegrate, the retinas were then shaken until the vascular network was completely detached. The vascular network was transferred to a clean glass slide, dried and stained with glycogen, and photographed under a microscope to count the number of vascular non-irrigated areas. (NOD M in the figure is the normal control group)

The results showed that multiple treatments with anti-Sema4D could inhibit the formation of avascular areas in the fundus of the STZ model, and the effect of anti-Sema4D combined with anti-VEGF treatment was significantly better than multiple single anti-Sema4D or anti-VEGF treatments (Fig. D).

3) Desmin immunofluorescence staining (STZ model mice treated for multiple times)

The STZ model mice were injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) at the same time in the vitreous body 4 months after modeling, or injected with anti-Sema4D (2ug), anti-VEGF ( 2ug), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after cardiac perfusion with normal saline, fresh eyeballs were fixed in 4% PFA After 24 hours, the retinas were then detached and digested with 3% trypsin (dissolved in pH.7.8 tris-hcl) at 37°C. When the retinas began to disintegrate, the retinas were then shaken until the vascular network was completely detached. The vascular network was transferred to a clean glass slide for Desmin immunofluorescence staining, and photographed by a fluorescence microscope for statistics. Desmin (1:100, Ab cam).

The results showed that multiple anti-Sema4D treatments could inhibit the loss of pericytes on the surface of fundus vessels in STZ model, and it improved the pericyte coverage better than anti-VEGF treatment, and multiple anti-Sema4D combined with anti-VEGF treatment had significantly better effect than anti-VEGF treatment. Multiple anti-Sema4D or anti-VEGF treatments alone (Figure 7, E and F).

4) Immunofluorescence staining to examine the continuity of VE-cadherin in fundus blood vessels (STZ model mice treated for multiple times)

The STZ model mice were injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) at the same time in the vitreous body 4 months after modeling, or injected with anti-Sema4D (2ug), anti-VEGF ( 2ug), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after cardiac perfusion with normal saline, fresh eyeballs were taken and fixed in anhydrous methanol -20 ℃ for 2 hours , peeled off the retina, placed in blocking solution (1%BSA, 0.5%TritonX-100, PBS) overnight at 4°C, incubated with primary antibody (VE-cadherin/Collage nIV) for 5 days, incubated with secondary antibody for 3h at room temperature, and then spread the film and photographed . Antibodies: VE-cadherin (1:50, BD Biosciences), Collagen IV (1:100, Southern Biotech).

The results showed that multiple anti-Sema4D treatments could increase the continuity of VE-cadherin in the fundus blood vessels in the STZ model, and the multiple treatments of anti-Sema4D combined with anti-VEGF were significantly better than multiple single anti-Sema4D or multiple single treatments. anti-VEGF treatment (G-H in Figure 7).

5) Immunofluorescence staining to investigate the coverage of N-cadherin in fundus blood vessels

The STZ model mice were injected with sema4d neutralizing antibody (2ug/eye) and VEGF neutralizing antibody (2ug/eye) at the same time in the vitreous body 4 months after modeling, or injected with anti-Sema4D (2ug), anti-VEGF ( 2ug), IgG (2ug), PBS (1ul), injected once a week, a total of 5 injections, anesthetized after the 5th injection, after cardiac perfusion with normal saline, fresh eyeballs were taken and fixed in 4% PFA at 4°C for 2h , Peel off the retina, put it in blocking solution (1%BSA0.5%TritonX-100, PBS), after overnight at 4°C, incubate with primary antibody (N-cadherin/Collag enIV) for 5 days, incubate with secondary antibody at room temperature for 3h photo shoot. Antibodies: N-cadherin (1:50, LifeTechnol ogies), Collagen IV (1:100, Southern Biotech).

The results showed that multiple anti-Sema4D treatments could increase the coverage of N-cadherin in the fundus blood vessels in STZ model, and its effect was better than that of anti-VEGF treatment alone, and the effect of anti-Sema4D combined with anti-VEGF treatment was significantly better than that of anti-Sema4D alone. Or anti-VEGF treatment alone. (I-J in Figure 7)

6) Evans blue fluorescence experiment

According to the experimental scheme in step 1), the Evans blue fluorescence experiment was performed to prolong the observation time.

The results showed that multiple anti-Sema4D treatments extended the observation time, and after two weeks, it was confirmed that it could inhibit the fundus vascular leakage of the STZ model, and the effect was better than multiple anti-VEGF treatments, and the anti-Sema4D combined with anti-VEGF treatment had a significant effect. Better than anti-Sema4D or anti-VEGF treatment alone (K-L in Figure 7).

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. A lentiviral vector which is a PlexinB1 gene knockout CRISPR/Cas9 recombinant lentiviral vector.

2. A lentivirus comprising the lentiviral vector of claim 1.

Application of Sema4D gene knockout and Sema4D/PlexinB1 signal pathway lentivirus silencing in preparation of drugs for treating and preventing fundus vascular diseases.

4. Use according to claim 3, wherein the Sema4D/Plexin B1 signaling pathway lentiviral silencing is achieved using the lentiviral vector of claim 1 or the lentivirus of claim 2.

5. The use according to claim 3 or 4, wherein the medicament for treating and preventing ocular fundus vascular disease is a Plex inB1 inhibitor.

6. The use according to claim 3 or 4, the fundus vascular disease being fundus vascular neovascularization, fundus vascular leakage, or pericyte depletion.

7. The use according to claim 6, wherein the disease is caused by diabetes.

Images