CN109125731B - Application of the Sema4D/PlexinB1 inhibitor in preparation treatment and prevention optical fundus blood vessel disease medicament - Google Patents
Application of the Sema4D/PlexinB1 inhibitor in preparation treatment and prevention optical fundus blood vessel disease medicament Download PDFInfo
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- CN109125731B CN109125731B CN201811228364.XA CN201811228364A CN109125731B CN 109125731 B CN109125731 B CN 109125731B CN 201811228364 A CN201811228364 A CN 201811228364A CN 109125731 B CN109125731 B CN 109125731B
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Abstract
The invention belongs to biologic medical fields, specifically disclose application of the Sema4D/PlexinB1 inhibitor in preparation treatment and prevention optical fundus blood vessel disease medicament, it is found by the applicant that Sema4D increases in patients with diabetic retinopathy aqueous humor, Sema4D is horizontal negatively correlated to reacting for anti-VEGF with patient, on the one hand endothelial cell can be acted on promotes its migration to Sema4D/PlexinB1 signal path, segment dislocation, on the one hand by promoting pericyte migration to aggravate leakage, inhibit Sema4D/PlexinB1 signal path, optical fundus blood vessel new life and leakage can be effectively suppressed, for the optical fundus blood vessels hyperplasia such as diabetic retinopathy, leakage disease provides new thinking.
Description
Technical field
The invention belongs to biologic medical fields, and in particular to Sema4D/PlexinB1 inhibitor is in preparation treatment and prevention
Application in optical fundus blood vessel disease medicament.
Background technique
First reason of the diabetic retinopathy (DR) as 20-75 years old adult's blinding, in 1 type and 2 type glycosurias
Illness rate is high in patient, the whole world: illness rate is up to 35.4% in diabetic population by DR, most serious stage Proliferative diabetes
The illness rate of retinopathy (PDR) is up to 7.5% (2017Diabetes Care IF=13.397), in China: different phase
DR illness rate in diabetic population is also up to 24.7%-37.5 (2014 guide), the World Health Organization (WHO) estimation, until
2025, diabetic will be broken through 30,000,000,000 (2009EYE) that rise in value by the whole world, it is seen that the number of DR patient will be further increased.
The treatment of DR is carried out diabetic retinopathy vitrectomy research (DRVS research) from nineteen ninety and is led so far
It to be treated for three classes: 1. application (predominantly a variety of sides of laser photocoagulation treatment 2. vitrectomy 3. anti-angiogenic drugs
The anti-VEGF of formula is treated, and 2004-modern), laser photocoagulation treatment is the treatment method sacrificed the knights in order to save the queen, and sacrifices peripheral visual field and retains
Central vision field, and receive anti-VEGF treatment patient in only 30% effectively, it is seen then that anti-VEGF treatment resist, repetitively administered
Concurrent infection, neurotoxicity, heavy financial burden are all that treatment is made troubles and risk, an urgent demand we research and develop newly
Therapeutic strategy.
The pathologic process of DR is different from neonate tumour blood vessel, and treatment has certain particularity, and the factor that originates of DR is eye
Bottom vascular flow rate slows down, and local metabolic is caused to change, and activates inflammation and associated signal paths, leads to vascular leakage, blood-retina
Barrier breakdown, parietal cell depigmentation, cellular matrix destroy, so that blood vessel be made to lose function, are partially formed anoxic zones, cause compensatory
Sexual abnormality blood vessel hyperplasia, the aberrant nascent vessels for aggravating leakage even insufficiency cause fundus hemorrhage, detachment of retina
(CurrDiab Rep 2011；Diabetes.2006).And the angiogenesis promoting factor and low is originated from blood vessels in tumors new life
The induction of oxygen stretches out pseudopodium by vascular remodeling, the attaching of surrounding parietal cell and merging with neighbouring newborn lumen, is formed
Rete vasculosum.The purpose of blood vessels in tumors new life is to support for tumour growth, therefore the treatment emphasis of angiogenesis is directed in tumour
For simple angiogenesis inhibiting, and it is different on eyeground, and the purpose of angiogenesis inhibiting treatment is to inhibit abnormal vascular
While restore normal blood vessels function, which is provided and mitigates leakage, it is seen then that although two kinds of diseases are all inhibition blood vessel
New life, but treatment results can be completely different.
Semaphorin protein family is presently considered to as the albumen played an important role in nervous system development process
It equally plays a significant role on angiogenesis and vascular development mechanism, wherein single pass transmembrane Protein S ema4D (Class 4
Semaph orins) it is discussed warmly in angiogenesis, the extracellular free Sema4D segment to work is mainly derived from a variety of thin
ADAM17 cutting, participates in tumour, bone, surgical wound model angiogenesis, in different groups in the MMP-MT1 cutting of born of the same parents or blood platelet
Knit, in cell its effect exist specificity, in tumour, Sema4D pass through combine PlexinB receptor promote Met and Ron phosphorus
Acidification promotes cell migration, angiogenesis to lead to Nasopharyngeal neoplasms (2009Valente), however, in lung cancer, Sema4D knot
Closing PlexinB1 and ERBB2 formation compound can be by inhibiting Met to inhibit cell migration (Swiercz 2008), it is seen that Sema4D
It is acted in different tissues various disease different.
In the present invention, applicant, which specifies, inhibits Sema4D/PlexinB1 signal path that optical fundus blood vessel can be effectively suppressed
New life simultaneously mitigates optical fundus blood vessel leakage, it is often more important that early stage inhibits Sema4D/PlexinB1 signal path that can effectively mitigate week
Cell depigmentation helps to protect and restore optical fundus blood vessel function, can extended treatment effect, reduction administration number of times be to a certain degree
Optical fundus blood vessel is newborn and treating for leakage provides new strategy.
Summary of the invention
The purpose of the present invention is to provide Sema4D/PlexinB1 inhibitor in preparation treatment and prevention optical fundus blood vessel disease
Application in drug, the optical fundus blood vessel disease include but is not limited to newborn optical fundus blood vessel or optical fundus blood vessel leakage or pericyte
Depigmentation.
In order to achieve the above object, the present invention takes following technical measures:
For the situation that the treatment means that current optical fundus blood vessel is newborn and leaks are limited, applicant takes diabetic retinopathy
Become patient's aqueous humor, screening and the closely related member of angiogenesis, discovery Sema4D/PlexinB1 signal pathway inhibitor can have
Effect inhibits optical fundus blood vessel newborn and leakage.
Sema4D/PlexinB1 signal pathway inhibitor answering in preparation treatment and prevention optical fundus blood vessel disease medicament
With the inhibitor includes but is not limited to that Sema4D/PlexinB1 signal path inhibits virus or Sema4D/
PlexinB1 signal path neutralizing antibody, or inhibit the compound of Sema4D/PlexinB1 signal path function.
In above-described application, it is preferred that the optical fundus blood vessel is newborn and leakage is as caused by diabetes.
A method of optical fundus blood vessel disease being treated, the method is that the suppression of Sema4D albumen is injected into vitreum
Preparation；
A method of optical fundus blood vessel disease being treated, the method is that the suppression of Sema4D albumen is injected into vitreum
Preparation and anti-VEGF.
The optical fundus blood vessel disease includes but is not limited to that newborn optical fundus blood vessel or optical fundus blood vessel leakage or pericyte are de-
It loses.
In the process described above, it is preferred that the inhibitor is the neutralizing antibody or Sema4D/ of Sema4D albumen
PlexinB1 signal pathway inhibitor.
Compared with prior art, the invention has the following advantages that
For the situation that the treatment means that current optical fundus blood vessel is newborn and leaks are limited, 1. existing essential therapeutic arsenals are
The application of laser photocoagulation treatment 2. vitrectomy 3. anti-angiogenic drugs, wherein anti-angiogenic drugs be mainly
The injection of the eyeground anti-VEGF, but receive in the patient of anti-VEGF treatment only 30% effectively, applicant is directed to the office treated at present
It is sex-limited, take patients with diabetic retinopathy aqueous humor, screening and the closely related Sema family member of angiogenesis, discovery
Sema4D/PlexinB1 signal pathway inhibitor can be effectively suppressed that optical fundus blood vessel is newborn and leakage, and in vivo, outside and clpp gene
Inhibit Sema4D/PlexinB1 signal path to the effect of eyeground pathological, leakage and bright except demonstrating in mouse
True related mechanism.
Detailed description of the invention
Fig. 1 is Sema4D and the closely related result schematic diagram of DR；
Wherein, A: optical coherence tomography scanner (OCT) schematic diagram；
(Western Blot) is obviously increased in Sema4D expression in B-C.DR patient's aqueous humor
(ELISA detection) is obviously increased in Sema4D expression in D.DR patient's aqueous humor
It is central to receive macula retinae after anti-VEGF treats March by free Sema4D and patient in E-F.DR patient's aqueous humor
Recessed thickness (CST) and macula retinae stereomutation (MV) are negatively correlated (by uniting after the acquisition of Wuhan Concord Hospital ophthalmic data library
Meter)
Fig. 2 is that Sema4D expresses elevated view in mouse OIR and STZ model；
Wherein, the mRAN expression of A:OIR model mice retina Sema4D increases (q-PCR)；
OIR model mice retina Sema4D protein expression increases B:Western Blot as the result is shown；
C-D:Western Blot shows STZ model mice retina 3 months, 6 months Sema4D protein expressions increase.
Fig. 3 is that Sema4D gene knockout inhibits optical fundus blood vessel new life schematic diagram；
Wherein, A-C: knock out mice verifying；
D-H: immunofluorescence dyeing, Azo-Blue leakage experiment, HE coloration result show that Sema4D gene knockout can be significant
Inhibit eyeground pathological, vascular leakage.
Fig. 4 is the schematic diagram that Sema4D/PlexinB1 signal path can promote that endothelial cell lumen is formed and leaked；
Wherein, A-B: scratch experiment confirms that Sema4D can promote endothelial cell migration；
C-D: segment dislocation experiment confirms that Sema4D can promote endothelial cell lumen and be formed；
E:TEER experiment confirms that Sema4D can promote endothelial cell leakage；
F: Fluorescein Leakage experiment confirms that Sema4D can promote endothelial cell leakage；
G:PlexinB1 lowers verifying；
H-I: scratch experiment confirms to block silencing PlexinB1 receptor that Sema4D can be blocked to promote endothelial cell migration effect；
J-K: segment dislocation experiment confirms that silencing PlexinB1 receptor can block Sema4D to promote endothelial cell lumen and form work
With；
L-M:TEER and Fluorescein Leakage experiment confirm that silencing PlexinB1 receptor can block Sema4D to promote endothelial cell and seep
Leakage effect；
Fig. 5 is that Sema4D/PlexinB1 signal path increases endothelium and pericyte co-culture model leaks schematic diagram；
Wherein, A-B: co-culturing pericyte and endothelial cell TEER experiment and Fluorescein Leakage experiment confirms Sema4D signal
Promote leakage；
C-D: co-culturing in pericyte and endothelial cell model, and TEER experiment and Fluorescein Leakage experiment confirm silencing
The leakage effect of PlexinB1 receptor blocking Sema4D induction；
Fig. 6 is that anti-Sema4D inhibits OIR model mice optical fundus blood vessel newborn and STZ model mice optical fundus blood vessel leakage
Schematic diagram；Wherein, A:anti-Sema4D treatment can inhibit OIR model mice optical fundus blood vessel new life, be in concentration dependent；
B: Azo-Blue experiment confirms that anti-Sema4D treatment can inhibit OIR model mice optical fundus blood vessel leakage, is in concentration
The verifying of dependence C-D:anti-Sema4D antibody specificity；
In E-I:OIR model, anti-Sema4D treatment can inhibit angiogenesis and joint anti-VEGF therapeutic effect is aobvious
It writes and is treated better than independent anti-Sema4D or independent anti-VEGF；
J-K: single-dose, STZ model eyeground anti-Sema4D treatment can inhibit optical fundus blood vessel leakage and joint anti-
VEGF therapeutic effect is significantly better than independent anti-Sema4D or independent anti-VEGF treatment.
Fig. 7 is the effect diagram of multiple anti-Sema4D treatment；
Wherein, A-B: multiple dosing is observed after 1 week, and STZ model eyeground anti-Sema4D treatment can inhibit optical fundus blood vessel
Leakage and joint anti-VEGF therapeutic effect are significantly better than independent anti-Sema4D or independent anti-VEGF and treat
C-F:STZ model eyeground anti-Sema4D treatment can inhibit pericyte depigmentation, improve pericyte coverage rate, and join
It closes anti-VEGF therapeutic effect and is significantly better than independent anti-Sema4D or independent anti-VEGF treatment, it is often more important that anti-
Sema4D inhibits pericyte depigmentation and improves pericyte coverage rate better than anti-VEGF treatment (P < 0.05)
G-H:STZ model eyeground anti-Sema4D treatment can increase VE-cadherin continuity and joint anti-VEGF
Therapeutic effect is significantly better than independent anti-Sema4D or independent anti-VEGF treatment
I-J:STZ model eyeground anti-Sema4D treatment can increase N-cadherin coating ratio and joint anti-VEGF
Therapeutic effect is significantly better than independent anti-VEGF treatment
K-L: multiple dosing is observed after 2 weeks, STZ model eyeground anti-Sema4D treatment can inhibit optical fundus blood vessel leakage and
Joint anti-VEGF therapeutic effect is significantly better than independent anti-Sema4D or independent anti-VEGF treatment, it is often more important that more
Secondary anti-Sema4D treatment optical fundus blood vessel leakage is better than multiple anti-VEGF treatment (P < 0.05).
Specific embodiment
The present invention will be further described combined with specific embodiments below.Technical solution of the present invention is not said especially such as
The bright conventional scheme for this field.The reagent or material derive from commercial channel if not otherwise specified.
Embodiment 1:
Sema4D and DR closely related discovery:
1) Heidelberg retina tomography
Compare patients with diabetic retinopathy OCT faulted scanning pattern and normal person's OCT tomoscan picture, finds glycosuria
Macula lutea central fovea thickness in sick patient with retinopathy thickens (A in Fig. 1)
2) in aqueous humor the expression quantity of Sema4D detection
Experimental group is patients with diabetic retinopathy aqueous humor, and control group is cataract patient aqueous humor, aseptic injection
Device extracts.
Western Blot detects the expression quantity of Sema4D albumen in aqueous humor, as the result is shown Sema4D table in DR patient's aqueous humor
Up to obviously increasing (B-C in Fig. 1).
ELISA kit (MyBioSource) detects the expression quantity of Sema4D albumen in aqueous humor, as the result is shown DR patient room
Sema4D expression is obviously increased in water, and n indicates sample size.
3) detection of Sema4D albumen in the Heidelberg retina tomography and aqueous humor of the DR patient after treating
Free Sema4D and patient receive anti-VEGF and treat macula retinae after March in figure E-F display DR patient's aqueous humor
Central fovea thickness (CST) and macula retinae stereomutation (MV) are negatively correlated, and n indicates sample size.
Embodiment 2:
Sema4D expression increases in mouse OIR and STZ model:
1) mouse OIR model is established
Experimental group (OIR): it after the C57BL/6 female rat produce surviving of son of the 3-4 month, is put together with the young rat of birth the 7th day to containing 75%
In the oxygen cabin of oxygen, until taking out young rat and female rat when being born the 12nd day, place in normal air 5 days later.Above-mentioned processing
Young rat is anaesthetized when being born the 12nd, 14,17 day, after physiological saline heart perfusion, takes its eyeball, separates retina, extracts RNA
And reverse transcription at cDNA or extracts albumen.
Control group (Normal): for postnatal young rat, growing under normoxic condition, old in birth anesthesia in the 12nd, 14,17 day
Mouse takes eyeball after physiological saline heart perfusion, separates retina, extracts RNA and reverse transcription at cDNA or extracts albumen.
2) q-PCR detects the mrna expression amount of Sema4D
Using primer (CCTGGTGGTAGTGTTGAGAAC and GCAAGGCCGAGTAGTTAAAGAT), in step 1)
CDNA is template, carries out the detection of the expression quantity of Sema4D, the results show that the mrna expression amount of Sema4D significantly rises in OIR group
High (A in Fig. 2).(P12 refers to be the mouse sample of birth the 12nd day in Fig. 2, and so on, it is the same below)
3) Sema4D albumen in Western Blot test experience group (OIR) and control group
Using the albumen extracted in step 1) as antigen, with (the source: R&D sheep anti-SEMA4Dantibody
Systems) it is antibody, carries out Western Blot detection, is control with β-action.The results show that OIR model mice view
Film Sema4D expressing quantity significantly increases (B in Fig. 2)
4) mouse STZ model is established
Experimental group (STZ): STZ (streptozotocin, 50mg/ are injected intraperitoneally after 8 weeks big C57BL/6 mouse starvation 4h
Kg), continuous 5 days, once a day, the 7th day detection blood glucose after the completion of injecting, blood glucose was greater than 15mmol/L and is considered as modeling success.
It anaesthetizes mouse at the 3rd, 6 month after modeling, after physiological saline heart perfusion, takes its eyeball, separate retina, extract egg
It is white.
Control group (Vehicle): being substituted for physiological saline for the streptozotocin of experimental group, remaining operation is identical.
5) (Vehicle) Sema4D albumen in Western Blot test experience group (STZ) and control group
Using the albumen extracted in step 4) as antigen, with sheep anti-SEMA4D antibody (R&D Systems),
For antibody, Western Blot detection is carried out, it is control with β-action.The results show that STZ model mice retina is being built
3rd month after mould, 6 months Sema4D protein expressions significantly increase (C and D in Fig. 2).
Embodiment 3:
Sema4D gene knockout can significantly inhibit the optical fundus blood vessel new life and leakage of mouse
Sema4D knock out mice used in the present embodiment is purchased from Hua Zhong Agriculture University, is by the Sema4d to mouse
Exon region design sgRNA is to knock out the expression of sema4D and obtain.
1) target gene (sema4D) of clpp gene deratization knocks out successfully verifying:
DNA verifying mouse target gene (sema4D) is extracted from rat-tail to knock out successfully, is control with the mouse of wild type: being taken
3-5cm rat-tail is put in 56 DEG C of 10h in DNA lysate, and after isopropanol is precipitated, 95% ethanol wash expands after being configured to following system
Increase, the electrophoresis in Ago-Gel.
| Sema4d-GT-F1 | TCTGGGGCTCTAAGAGGTCCTT |
| Sema4d-GT-R1 | AGCCACTGAGGTCACATACACC |
A is sgRNA target practice region mode figure in Fig. 3, and B shows in Fig. 3: the sema4D gene of knockout group is due to being truncated
(Sema4D-KO), therefore wilder group of molecular weight lower, shows to knock out successfully.
Albumen is extracted using Mouse Retina and is Western Blot to detect Sema4D albumen, takes Mouse Retina group
It knits, for extraction step with general tissue extraction albumen step, every group 6, be control with β-action；Sema4D protein antibodies are
SEM A4D antibody(R&D Systems)。
The homozygote mouse that C shows sema4D knockout in Fig. 3 knocks out hybrid mice sema4D without sema4D protein expression
Albumen is expressed compared with wild type and is reduced, and in Fig. 3 in C, respectively wild type, knockout homozygote knock out heterozygote from left to right.
2) IB4 immunofluorescence dyeing show the deratization of Sema4D clpp gene can significantly inhibit eyeground in OIR model blood vessel it is new
It is raw
OIR model is established using Sema4D knock out mice, from the 17th day anesthesia mouse, physiology after mouse birth start of calculation
After salt water heart perfusion, its eyeball is taken to fix overnight for 4 DEG C in 4% paraformaldehyde, after separating retina, Isolectin B4
Overnight, tile is taken pictures, Isolectin B4 (1:100, Vector Laboratories) for 4 DEG C of dyeing.It is established with wild-type mice
OIR model be control, every group of 6 mouse.
Abnormal new vessels are reduced after D display knocks out the mouse OIR modeling of sema4D in Fig. 3, in Fig. 3 E be to utilize
Statistics schematic diagram of the ImageJ to the total vessel area ratio of abnormal vascular Zhan.
3) Azo-Blue extraction experiments confirm that the deratization of Sema4D clpp gene can significantly inhibit optical fundus blood vessel in OIR model and leak
Experiment is divided into wild type OIR model mouse and sema4D knocks out OIR model mouse, every group 6, specific as follows:
Evans Blue dye (200 mgs/kg) are injected intraperitoneally when being born the 17th day in OIR model mouse, have recycled 4 small
When after anaesthetize mouse, after physiological saline heart perfusion, its retina is taken overnight, to utilize microplate reader as formamide (70 DEG C of water-baths)
(wavelength 620-740mm) measures extraction liquids absorbance.Concentration is calculated according to standard curve (with her Wen of various concentration
Blue dyestuff is abscissa, makes standard curve as the longitudinal axis using the liquid absorbance of microplate reader wavelength 620-740mm measurement), and benefit
It is counted after being converted with retinal levels in retina weight and blood.
F shows that the deratization of Sema4D clpp gene can significantly inhibit optical fundus blood vessel in OIR model and leak in Fig. 3.
4) deratization of Sema4D clpp gene can significantly inhibit cell number (the HE dye of new vessels before retina in OIR model
Color)
Experiment be divided into four groups, be respectively as follows: wild-type mice group, sema4D knockout type mouse group, wild type OIR model mouse and
Sema4D knockout type OIR model mouse, every group 6, the specific steps are as follows:
Above-mentioned four groups of mouse were anaesthetized in birth the 17th day, after physiological saline heart perfusion, took its eyeball in 4% poly first
Fixed in aldehyde, row specimens paraffin embedding slices HE dyeing is observed under an optical microscope and counts the blood vessel for breaking through layer of retina,limiting,internal
Endothelial cell number.
G shows the cell number of new vessels before HE dyeing retina in Fig. 3, and wherein wild-type mice group, sema4D strike
Except having new life without the generation of new vessels, wild type OIR model mouse and sema4D knockout type OIR model mouse in type mouse group
Angiogenesis, but the new vessels number of sema4D knockout type OIR model mouse is significantly less than wild type OIR model mouse, arrow table
Show the cell of new vessels before retina, H shows that the deratization of Sema4D clpp gene can significantly inhibit retina in OIR model in Fig. 3
The cell number of preceding new vessels.
Embodiment 4:
The reinforcement of Sema4D/PlexinB1 signal path inhibits the formation of endothelial cell lumen and leakage to have an impact:
The cell or endothelial cell that the present embodiment is related to refer both to be mouse brain CMEC.
1) Sema4D can promote endothelial cell migration (scratch experiment)
Full mouse brain CMEC is planted in culture hole, after 0.5% FBS overnight starvation, with 200ul pipette tips
Vertical vertical line is marked on cell, after washing away the cell under drawing, is added in DMEM and is separately added into final concentration of 400,800,
The sema4D albumen of 1600ng/ml is incubated for for 24 hours after adding culture hole, and using photomicrograph, image J counts cell migration
Situation.
As the result is shown: Sema4D can promote endothelial cell migration (A in Fig. 4), and endothelial cell migration ability is dense with sema4D
Degree rises and increases (B in Fig. 4).
2) Sema4D can promote endothelial cell lumen and form (segment dislocation experiment)
150ul pre-cooling matrigel/hole (BD Biosciences) is added in 48 orifice plates, is placed in 37 DEG C of cell incubator
After 30min, every hole kind 2 × 104A mouse brain CMEC is cultivated 3 days and is added respectively in DMEM after cell covers with
Enter final concentration of 400,800,1600ng/ml sema4D, is incubated for for 24 hours after culture hole is added, is taken pictures afterwards using microscope for 24 hours
Image J statistics, the DMEM of sema4D is not added as control.
Sema4D can promote endothelial cell lumen and be formed as the result is shown, and rises with sema4D concentration and increase (C- in Fig. 4
D)。
3) Sema4D can promote endothelial cell leakage (TEER experiment)
It is first coated with one layer of fine mucin on the upper layer the cell transwell (0.4um) in 24 holes, is incubated at room temperature 1h；After coating
Kind enters mouse brain CMEC 5 × 104A, culture is separately added into final concentration after cell covers in 3 days in culture medium
The sema4D albumen for being 400,800,1600ng/ml is incubated for so that the DMEM of sema4D is not added as control and utilizes cell cross afterwards for 24 hours
Film resistance measuring instrument measures its resistance value.
The TEER of endothelial cell is gradually reduced after the sema4D of E display addition 400,800,1600ng/ml in Fig. 4, is shown
Sema4D can promote endothelial cell leakage, and rises leakage with concentration and aggravate.
4) Sema4D can promote endothelial cell leakage (fluorescence leakage experiment)
It is first coated with one layer of fine mucin on the upper layer the cell transwell (0.4um) in 24 holes, is incubated at room temperature 1h, after coating
Kind enters mouse brain CMEC, and culture is separately added into final concentration of 400 in 3 days after cell covers in culture medium,
800,1600ng/ml sema4D, the DMEM of sema4D is not added as control, for 24 hours after be added in the DMEM of cell upper layer it is dense eventually
Degree is the dextran of 300 μ g/ml fluorescent markers, extracts lower layer's culture medium 30ul in 1h, measures 625nm wavelength using microplate reader
Absorbance.
Endothelial cell leakage after final concentration of 400,800, the 1600ng/ml sema4D of F display addition in Fig. 4
Dextran fluorescent value rises, and shows that Sema4D can aggravate endothelial cell leakage, and rise leakage with concentration and aggravate.
5) slow-virus transfection causes endothelial cell PlexinB1 to lower, and protein level verifies (Western Blot)
The present embodiments relate to slow virus be purchased from Ji Kai company, wherein CRISPR-wt be the zero load with GFP
Slow virus, CRISPR-plB1 is the slow virus for the CRISPR/Cas9 that PlexinB1 is knocked out, for lowering PlexinB1；Experiment point
It is 3 groups: 1, normal group (Normal), that is, the endothelial cell group of any substance is not added；2, the unloaded slow virus group with GFP has been transfected
(CRISPR-wt)；3, the group of CRISPR-plB1 has been transfected, every group of 5 repetitions, virus group MOI value is 50.
48h extracts albumen and carries out Western Blot to PlexinB1 after slow-virus transfection mouse brain CMEC
Expression effect detected, be control with β-action, antibody used in WB process be PlexinB1 antibody (1:
500,Abcam)。
As the result is shown: CRISPR-plB1 can significantly reduce the expression of PlexinB1, and (G in Fig. 4, left figure are WB figure, and right figure is
WB statistical chart).
6) the endothelial cell migration declines (scratch experiment) that sema4D is mediated after downward PlexinB1
Experimental procedure: after slow virus (CRISPR-wt or CRISPR-plB1) transfects mouse brain CMEC 48h
Digestion kind plants full mouse brain CMEC in cell plates in culture hole, after 0.5% FBS overnight starvation, uses
200ul pipette tips mark vertical vertical line on cell, after washing away the cell under drawing, are directly incubated for for 24 hours or add final concentration of
It is incubated for again after the sema4D albumen of 1600ng/ml for 24 hours, every group of 6 repetitions, using photomicrograph, image J counts cell migration
Situation.
As the result is shown: sema4D albumen promotes the migration number of endothelial cell to increase (H-I in Fig. 4), and PlexinB1 is lowered
It can inhibit the process.
7) endothelial cell that sema4D is mediated after downward PlexinB1 forms lumen declines (segment dislocation experiment)
150ul is added in 48 orifice plates, matrigel/hole is pre-chilled, is placed in 37 DEG C of cell incubator 30min, every hole kind 2 × 104
A mouse brain CMEC for having transfected slow virus (CRISPR-wt or CRISPR-plB1)；It cultivates 3 days long to cell
Man Hou, directly be incubated for for 24 hours or add final concentration of 1600ng/ml sema4D albumen after be incubated for again for 24 hours, every group of 6 repetitions.
The endothelial cell formation lumen declines of sema4D mediation after PlexinB1 are lowered in J-K display in Fig. 4.
8) the endothelial cell leakage effect that sema4D is mediated after downward PlexinB1 reduces (TEER experiment)
It is first coated with one layer of adhesion factor (incubation at room temperature 1h) on the upper layer the cell transwell (0.4um) in 24 holes, after coating
Kind enters to have transfected the mouse brain CMEC of slow virus (CRISPR-wt or CRISPR-plB1), cultivates 3 days to cell
It is incubated for again for 24 hours after covering with the rear sema4D albumen for being directly incubated for for 24 hours or adding final concentration of 1600ng/ml, every group of 6 repetitions.It incubates
It educates and measures its resistance value using cell transmembrane resistance measuring instrument afterwards for 24 hours.
L display display sema4D promotes the leakage of endothelial cell to increase in Fig. 4, and PlexinB1 downward can inhibit the process.
9) the endothelial cell leakage effect that sema4D is mediated after downward PlexinB1 reduces (fluorescence leakage experiment)
One layer of adhesion factor is first coated on the upper layer the cell transwell (0.4um) in 24 holes, is incubated at room temperature 1h, after coating
Kind enters to have transfected the mouse brain CMEC (5 × 10 of slow virus (CRISPR-wt or CRISPR-plB1)4It is a), culture
It after cell covers with, is incubated for again after the direct sema4D albumen for being incubated for for 24 hours or adding final concentration of 1600ng/ml for 24 hours, small within 3 days
The dextran of 300 μ g/ml fluorescent markers is added in the DMEM of room upper layer, lower layer's culture medium 30ul is extracted after 1h, utilizes enzyme mark
The absorbance of instrument measurement 625nm wavelength.
M is shown in Fig. 4, and fluorescence leakage experiment display sema4D promotes the leakage of endothelial cell to increase, and PlexinB1 is lowered
It can inhibit the process.
Embodiment 5:
Sema4D/PlexinB1 signal path can promote endothelium and pericyte mixed cultivating model leaks；
Endothelial cell used in the present embodiment is mouse brain CMEC, and pericyte is mouse primary cerebral microvascular
Pericyte.
1) Sema4D can promote the leakage (TEER experiment) of endothelial cell Yu pericyte co-culture system.
It is first coated with one layer of fine mucin in the cell transwell (0.4um) in 24 holes, is incubated at room temperature 1h；, in cell lower layer
Kind enters mouse brain CMEC (5 × 104), after 12h, on cell upper layer, kind enters mouse primary cerebral microvascular pericyte
(2.5×104), after culture 3 days, culture medium is replaced, final concentration of 400,800,1600ng/ml are separately added into the DMEM of upper layer
Sema4D albumen be incubated for so that the DMEM of sema4D is not added as control and measure its electricity using cell transmembrane resistance measuring instrument afterwards for 24 hours
Resistance value.A is indicated in Fig. 5, and Sema4D can reduce the TEER value of endothelium Yu pericyte co-culture system, and its decreasing value with
The raising of Sema4D protein concentration and increase.
2) Sema4D can promote the leakage (fluorescence leakage experiment) of endothelial cell Yu pericyte co-culture system.
It is first coated with one layer of fine mucin in the cell transwell (0.4um) in 24 holes, 1h is incubated at room temperature, in cell lower layer
Kind enter brain microvessel endothelial cells in vitro, after 12h, kind enters mouse primary cerebral microvascular pericyte on cell upper layer, after culture 3 days, more
Culture medium is changed, is separately added into final concentration of 400,800,1600ng/ml sema4D albumen, in the DMEM of upper layer to be not added
The DMEM of sema4D is control, for 24 hours after the dextrans of 300 μ g/ml fluorescent markers is added in the DMEM of cell upper layer, to be not added
The cell sample of sema4D is control, and lower layer's culture medium 30ul is extracted after 1h, utilizes the extinction of microplate reader measurement 625nm wavelength
Degree.
In Fig. 5 B show Sema4D can concentration dependent promote endothelium and pericyte co-culture system leakage.
3) in co-culture model, the PlexinB1 protein receptor of independent silencing pericyte is compared to independent silencing endothelial cell
PlexinB1 protein receptor can reverse the effect of Sema4D to a greater extent
It is first coated with one layer of fine mucin in the cell transwell (0.4um) in 24 holes, is incubated at room temperature 1h, lower layer plants into place
Mouse brain CMEC (5 × 10 after reason4), after 12h, the kind micro- blood of mouse primary brain that enters that treated on cell upper layer
Peritubular cell (2.5 × 104)；After culture 3 days, culture medium is replaced, final concentration of 1600ng/ml is added in the DMEM of upper layer
Sema4D albumen, Sema4D albumen is not added as control；Then according to step 1) and 2) in method, respectively carry out TEER experiment
It is tested with fluorescence leakage；
Specifically it is grouped as follows:
(1), CRISPR-wt group: lower layer plants into the endothelial cell for having transfected unloaded slow virus, and cell upper layer kind enters transfection
The pericyte of unloaded slow virus；
(2), PC-CRISPR-plB1 group: lower layer plant into transfected the endothelial cell of unloaded slow virus, cell upper layer kind enters
The pericyte of CRISPR-plB1 is transfected；
(3), EC-CRISPR-plB1 group: lower layer plant into transfected the endothelial cell of CRISPR-plB1, cell upper layer kind enters
The pericyte of unloaded slow virus is transfected.
The results show that co-culturing in pericyte and endothelial cell model, TEER experiment and Fluorescein Leakage experiment display week
Cell PlexinB1 receptor silencing is compared with the leakage that endothelial cell PlexinB1 receptor silencing can more obviously block Sema4D to induce
It acts on (C-D in Fig. 5)
Embodiment 6:
Single injection anti-Sema4D treatment can inhibit OIR model mice and STZ model mice optical fundus blood vessel new life and
Optical fundus blood vessel leakage
1) IB4 immunofluorescence dyeing experiment (single injection):
After the C57BL/6 female rat produce surviving of son of the 3-4 month, put together with the young rat of birth the 7th day into the oxygen cabin of 75% oxygen, until
Young rat and female rat are taken out after being born the 12nd day, injected in the rear vitreous body of young rat anesthesia immediately in various dose Sema-4D and anti-
Body (BMA-12) (0.5ug, 1ug, 2ug), to inject the IgG of 2ug as control, isometric PBS continues normal as control
It is fed 5 days in air；Young rat is anaesthetized when being born the 17th day, after physiological saline heart perfusion, is separated eyeball and fixation, is separated
Retina IB4 dyeing, anti-Sema4D concentration dependent mitigates the percentage that abnormal vascular accounts for the retina gross area as the result is shown
(A in Fig. 6) it is newborn to illustrate that anti-Sema4D treatment can inhibit OIR model mice optical fundus blood vessel, and is in concentration dependent.
2) Azo-Blue fluorescence experiments (single injection):
After the C57BL/6 female rat produce surviving of son of the 3-4 month, put together with the young rat of birth the 7th day into the oxygen cabin of 75% oxygen, until
Young rat and female rat are taken out after being born the 12nd day, inject various dose sema4d neutralizing antibody in the rear vitreous body of young rat anesthesia immediately
(0.5ug, 1ug, 2ug), (to inject the IgG of 2ug as control, isometric PBS continues to feed in normal air as control
It supports 5 days；Evans Blue dye (200 mgs/kg) are injected intraperitoneally when being born 17 days in young rat, anaesthetize young rat, physiology after 4 hours
After salt water heart perfusion, take its retina overnight, to utilize microplate reader (wavelength 620-740mm) as formamide (70 DEG C of water-baths)
Measure extraction liquids.Concentration is calculated according to standard curve, and horizontal using retina Azo-Blue in retina weight and blood
It is calibrated, anti-Sema4D treatment can inhibit OIR model mice optical fundus blood vessel leakage as the result is shown, and be in concentration dependent
(B in Fig. 6).
3) IB4 immunofluorescence dyeing experiment (wild-type mice and knock out mice, single injection):
Wt group: it after the C57BL/6 female rat produce surviving of son of the 3-4 month, puts together with the young rat of birth the 7th day to the oxygen cabin of 75% oxygen
In, until taking out young rat and female rat after birth the 12nd day, Sema4D neutralizing antibody is injected in the rear vitreous body of young rat anesthesia immediately
(2ug) or IgG (2ug) continue to feed 5 days in normal air；Then the experiment of IB4 immunofluorescence dyeing is carried out；
Sema4D-KO group: after the C57BL/6 female rat produce surviving of son after the Sema4D gene knockout of the 3-4 month, with birth the 7th day
Young rat is put together into the oxygen cabin of 75% oxygen, until taking out young rat and female rat after birth the 12nd day, glass after young rat anesthesia immediately
Sema4D neutralizing antibody (2ug) or IgG (2ug) are injected in glass body, continue to feed 5 days in normal air；Then it is immune to carry out IB4
Fluorescent staining experiment；
As the result is shown: anti-Sema4D treatment and Sema4D gene knockout can reduce optical fundus blood vessel caused by OIR model
Newborn (IB4 immunofluorescence dyeing) it is old can not to further suppress Sema4D gene knockout after vitreum anti-Sema4D injection
Mouse abnormal vascular is newborn (C in Fig. 6).
4) Azo-Blue fluorescence experiments (wild-type mice and knock out mice, single injection):
Wt group: it after the C57BL/6 female rat produce surviving of son of the 3-4 month, puts together with the young rat of birth the 7th day to the oxygen cabin of 75% oxygen
In, until taking out young rat and female rat after birth the 12nd day, Sema4D neutralizing antibody is injected in the rear vitreous body of young rat anesthesia immediately
(2ug) or IgG (2ug) continue to feed 5 days in normal air；According to the method in step 2), Azo-Blue fluorescence is then carried out
Experiment；
Sema4D-KO group: after the C57BL/6 female rat produce surviving of son after the Sema4D gene knockout of the 3-4 month, with birth the 7th day
Young rat is put together into the oxygen cabin of 75% oxygen, until taking out young rat and female rat after birth the 12nd day, glass after young rat anesthesia immediately
Sema4D neutralizing antibody (2ug) is injected in glass body respectively and IgG (2ug) continues to feed 5 days in normal air；According in step 2)
Method, then carry out Azo-Blue fluorescence experiments；
As the result is shown: anti-Sema4D treatment and Sema4D gene knockout can reduce optical fundus blood vessel caused by OIR model
It leaks (Azo-Blue fluorescence experiments), Sema4D gene knockout can not be further suppressed after vitreum anti-Sema4D injection
Mouse vascular leakage (D in Fig. 6).
5) IB4 immunofluorescence dyeing experiment (anti-Sema4D combines anti-VEGF treatment, single injection)
After the C57BL/6 female rat produce surviving of son of the 3-4 month, put together with the young rat of birth the 7th day into the oxygen cabin of 75% oxygen, until
Young rat and female rat are taken out after being born the 12nd day, injected simultaneously in rathole vitreum after young rat anesthesia immediately in sema4d and anti-
Body (2ug/) and VEGF neutralizing antibody (2ug/)；Or individually inject anti-Sema4D (2ug), anti-VEGF
(2ug),IgG(2ug),PBS(2ug)；Then the experiment of IB4 immunofluorescence dyeing is carried out according to the method in step 1).
As the result is shown: anti-Sema4D combines anti-VEGF treatment and inhibits OIR model mice optical fundus blood vessel newborn, effect
It is significantly better than independent anti-Sema4D or independent anti-VEGF treatment (E-F in Fig. 6).
6) Azo-Blue experiment (anti-Sema4D combines anti-VEGF treatment, single injection)
After the C57BL/6 female rat produce surviving of son of the 3-4 month, put together with the young rat of birth the 7th day into the oxygen cabin of 75% oxygen, until
Young rat and female rat are taken out after being born the 12nd day, injected simultaneously in rathole vitreum after young rat anesthesia immediately in sema4d and anti-
Body (2ug/) and VEGF neutralizing antibody (2ug/)；Or individually inject anti-Sema4D (2ug), anti-VEGF
(2ug),IgG(2ug),PBS(1ul)；Then Azo-Blue fluorescence experiments are carried out according to the method in step 2).
As the result is shown: anti-Sema4D combines anti-VEGF treatment and inhibits OIR model mice optical fundus blood vessel leakage, effect
It is significantly better than independent anti-Sema4D or independent anti-VEGF treatment (Azo-Blue experiment) (G in Fig. 6)
7) HE Coloration experiment (anti-Sema4D combines anti-VEGF treatment, single injection)
After the C57BL/6 female rat produce surviving of son of the 3-4 month, put together with the young rat of birth the 7th day into the oxygen cabin of 75% oxygen, until
Young rat and female rat are taken out after being born the 12nd day, injected simultaneously in rathole vitreum after young rat anesthesia immediately in sema4d and anti-
Body (2ug/) and VEGF neutralizing antibody (2ug/)；Or individually inject anti-Sema4D (2ug), anti-VEGF (2ug),
IgG(2ug),PBS(1ul)；Mouse was anaesthetized in birth the 17th day, after physiological saline heart perfusion, took its eyeball in 4% poly
Fixed in formaldehyde, row specimens paraffin embedding slices HE dyeing is observed under an optical microscope and counts the blood for breaking through layer of retina,limiting,internal
Endothelial cell number.
As the result is shown: anti-Sema4D combines anti-VEGF treatment and inhibits new life before OIR model mice eye ground
The cell number of blood vessel, significant effect treat (HE dyeing) (Fig. 6 H- better than independent anti-Sema4D or independent anti-VEGF
I)。
8) STZ modeling mouse Azo-Blue fluorescence experiments (anti-Sema4D combines anti-VEGF treatment, single injection)
STZ modeling mouse is anesthetized when May after modeling, injects sema4d neutralizing antibody simultaneously in rathole vitreum
(2ug/) and VEGF neutralizing antibody (2ug/), or individually inject anti-Sema4D (2ug), anti-VEGF (2ug),
IgG(2ug),PBS(1ul)；After a week, tail vein injection Evans Blue dye (45mg/kg) is anaesthetized old after 2 hours for injection
Mouse after physiological saline heart perfusion, takes its retina overnight, to utilize microplate reader (wavelength 620- as formamide (70 DEG C of water-baths)
740mm) measure.Concentration is calculated according to standard curve, and is counted after being converted using retinal levels in retina weight and blood.
Take a part of eyeball after the 4% fixed 2h of PFA room temperature simultaneously, fluorescence is taken pictures after row inner nuclear layer retina.
As the result is shown: single combines anti-Sema4D and anti-VEGF treatment and STZ model mice optical fundus blood vessel is inhibited to seep
Leakage, significant effect are treated better than independent anti-Sema4D or independent anti-VEGF.
Embodiment 7:
Multiple anti-Sema4D treatment can inhibit STZ model mice optical fundus blood vessel leakage and inhibit optical fundus blood vessel week thin
It is better than anti-VEGF in born of the same parents' depigmentation
1) Azo-Blue fluorescence experiments (STZ modeling mouse is repeatedly treated)
STZ modeling mouse is injected in sema4d neutralizing antibody (2ug/) and VEGF in vitreum simultaneously after modeling April
With antibody (2ug/), or individually injection anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul)
To inject anti-Sema4D (2ug) or anti-VEGF (2ug) or IgG (2ug) or PBS (2ug) as control, one is injected weekly
Secondary, the latter all tail vein injection Evans Blue dyes (45mg/kg) of the 5th injection anaesthetize mouse, physiology after recycling 2 hours
After salt water heart perfusion, its retina is taken overnight, to survey using microplate reader (wavelength 620-740mm) as formamide (70 DEG C of water-baths)
It is fixed.Concentration is to be calculated according to standard curve, and count after being converted using retinal levels in retina weight and blood.Simultaneously
It takes a part of eyeball after the 4% fixed 2h of PFA room temperature, separates retina, tile row fluorescence is taken pictures.
As the result is shown: multiple anti-Sema4D is treated, and injection is primary weekly, and detection finds it after a week after the 5th injection
It can inhibit STZ model optical fundus blood vessel leakage, the multiple therapeutic effect of joint anti-Sema4D and anti-VEGF is significantly better than list
The multiple treatment (A and B in Fig. 7) of only anti-Sema4D or independent anti-VEGF.(NO DM is Normal group in figure, according to mould
Group refers to intraperitoneal injection STZ group, and Normal group refers to intraperitoneal injection citrate group.
2) staining for glycogen experiment (STZ modeling mouse is repeatedly treated)
STZ modeling mouse is injected in sema4d neutralizing antibody (2ug/) and VEGF in vitreum simultaneously after modeling April
With antibody (2ug/), or individually inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul),
Injection is primary weekly, co-injection 5 times, anaesthetizes after the 5th injection, after physiological saline heart perfusion, the eyeball for taking its fresh is consolidated
It is scheduled in 4% PFA 24 hours, then retina is separated and with 3% trypsase (being dissolved in pH.7.8tris-hcl)
Is digested in the state of 37 degrees Celsius when retina starts to disintegrate, and retina is then rocked, until blood vessel network is kept completely separate
Out.By blood vessel network transfer as on clean slide, it is dry after row staining for glycogen, take pictures under microscope and count blood vessel without irrigated area number
Mesh.(NO DM is Normal group in figure)
As the result is shown: anti-Sema4D more times treatments can inhibit STZ model optical fundus blood vessel avascular area and be formed, and anti-
Sema4D joint anti-VEGF therapeutic effect is significantly better than repeatedly individually anti-Sema4D or anti-VEGF treatment (C in Fig. 7
And D).
3) Desmin immunofluorescence dyeing (STZ modeling mouse is repeatedly treated)
STZ modeling mouse is injected in sema4d neutralizing antibody (2ug/) and VEGF in vitreum simultaneously after modeling April
With antibody (2ug/), or individually inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul),
Injection is primary weekly, co-injection 5 times, anaesthetizes after the 5th injection, after physiological saline heart perfusion, the eyeball for taking its fresh is consolidated
It is scheduled in 4% PFA 24 hours, then retina is separated and with 3% trypsase (being dissolved in pH.7.8tris-hcl)
Is digested in the state of 37 degrees Celsius when retina starts to disintegrate, and retina is then rocked, until blood vessel network is kept completely separate
Out.By blood vessel network transfer as clean slide uplink Desmin immunofluorescence dyeing, taken pictures statistics using fluorescence microscope.
Desmin(1:100,Abcam)。
As the result is shown: multiple anti-Sema4D treatment can inhibit STZ model optical fundus blood vessel surface pericyte depigmentation, mention
High pericyte coverage rate is treated better than anti-VEGF, and repeatedly anti-Sema4D joint anti-VEGF therapeutic effect is significantly excellent
In multiple individually anti-Sema4D or anti-VEGF treatment (E and F in Fig. 7).
4) immunofluorescence dyeing investigates the continuity of optical fundus blood vessel VE-cadherin (STZ modeling mouse is repeatedly treated)
STZ modeling mouse is injected in sema4d neutralizing antibody (2ug/) and VEGF in vitreum simultaneously after modeling April
With antibody (2ug/), or individually inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul),
Injection is primary weekly, co-injection 5 times, anaesthetizes after the 5th injection, after physiological saline heart perfusion, takes fresh eyeball in no water beetle
After -20 DEG C of alcohol fixed 2h, retina is separated, is placed in confining liquid (1%BSA, 0.5%Triton X-100, PBS) 4 DEG C of mistakes
Night was incubated for primary antibody (VE-cadherin/Collagen IV) after 5 days, and tile is taken pictures after being incubated at room temperature secondary antibody 3h.Antibody: VE-
Cadherin (1:50, BD Biosciences), Collagen IV (1:100, Southern Biotech).
As the result is shown: multiple anti-Sema4D treatment can increase the continuous of optical fundus blood vessel VE-cadherin in STZ model
Property, and the multiple therapeutic effect of anti-Sema4D joint anti-VEGF is significantly better than repeatedly individually anti-Sema4D or multiple
Independent anti-VEGF treatment (G-H in Fig. 7).
5) immunofluorescence dyeing investigates the coverage rate of optical fundus blood vessel N-cadherin
STZ modeling mouse is injected in sema4d neutralizing antibody (2ug/) and VEGF in vitreum simultaneously after modeling April
With antibody (2ug/), or individually inject anti-Sema4D (2ug), anti-VEGF (2ug), IgG (2ug), PBS (1ul),
Injection is primary weekly, co-injection 5 times, anaesthetizes after the 5th injection, after physiological saline heart perfusion, takes fresh eyeball in 4%
4 DEG C of fixed 2h, separate retina in PFA, are placed in confining liquid (1%BSA 0.5%Triton X-100, PBS), 4 DEG C of mistakes
After night, primary antibody (N-cadherin/Collagen IV) is incubated for after 5 days, tile is taken pictures after being incubated at room temperature secondary antibody 3h.Antibody: N-
Cadherin (1:50, Life Technologies), Collagen IV (1:100, Southern Biotech).
As the result is shown: multiple anti-Sema4D treatment can increase the covering of optical fundus blood vessel N-cadherin in STZ model
Rate, effect are treated better than independent anti-VEGF, and anti-Sema4D joint anti-VEGF therapeutic effect is significantly better than individually
Anti-Sema4D or independent anti-VEGF treatment.(I-J in Fig. 7)
6) Azo-Blue fluorescence experiments
According to the experimental program in step 1), Azo-Blue fluorescence experiments are carried out, extend observing time.
Multiple anti-Sema4D treatment as the result is shown, extends observing time, confirms that it can inhibit STZ model eye after two weeks
Bottom vascular leakage, and effect is treated better than multiple anti-VEGF, while anti-Sema4D joint anti-VEGF therapeutic effect is aobvious
It writes better than independent anti-Sema4D or anti-VEGF treatment (K-L in Fig. 7).
Claims (3)
1.Sema4D inhibitor treats or prevents the application in optical fundus blood vessel leakage or pericyte depigmentation drug in preparation, described
Sema4D inhibitor is the Sema4D neutralizing antibody that clone number is BMA-12.
2. application according to claim 1, the optical fundus blood vessel leakage or pericyte depigmentation are as caused by diabetes.
3. application according to claim 1 or 2, the drug contains the Sema4D inhibitor and anti-VEGF.
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| CN201811228364.XA CN109125731B (en) | 2018-10-22 | 2018-10-22 | Application of the Sema4D/PlexinB1 inhibitor in preparation treatment and prevention optical fundus blood vessel disease medicament |
| CN201910867385.4A CN110643635A (en) | 2018-10-22 | 2018-10-22 | Application of Sema4D/PlexinB1 signal channel gene silencing |
| PCT/CN2019/109213 WO2020083007A1 (en) | 2018-10-22 | 2019-09-29 | Application of sema4d/plexinb1 inhibitor in preparation of drugs for treating and preventing fundus vascular diseases |
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| CN115010790B (en) * | 2021-08-29 | 2024-03-08 | 湖北烛照生物科技有限公司 | Nanometer small peptide FG and application thereof in preparing medicament for treating and preventing fundus blood vessel diseases |
| CN114933635B (en) * | 2021-08-29 | 2023-05-19 | 武汉夫图生物科技有限公司 | Nano small peptide FH and application thereof in preparation of drugs for treating and preventing fundus vascular diseases |
| CN114359262B (en) * | 2022-02-28 | 2022-06-07 | 华中科技大学同济医学院附属协和医院 | Device and method for obtaining outflow effect parameters based on perfusion images |
| CN116949097B (en) * | 2023-09-20 | 2023-12-12 | 江苏集萃药康生物科技股份有限公司 | Construction method and application of SEMA4D humanized mouse model |
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| CN110643635A (en) * | 2018-10-22 | 2020-01-03 | 华中科技大学同济医学院附属协和医院 | Application of Sema4D/PlexinB1 signal channel gene silencing |
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