CN110478358A - Sphingosine 1-phosphate is preparing the application in the drug for protecting salivary gland radiation insult - Google Patents
Sphingosine 1-phosphate is preparing the application in the drug for protecting salivary gland radiation insult Download PDFInfo
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- CN110478358A CN110478358A CN201910848219.XA CN201910848219A CN110478358A CN 110478358 A CN110478358 A CN 110478358A CN 201910848219 A CN201910848219 A CN 201910848219A CN 110478358 A CN110478358 A CN 110478358A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The present invention relates to pharmaceutical technology fields, and in particular to sphingosine 1-phosphate is preparing the application in the drug for protecting salivary gland radiation insult.Present invention discover that sphingosine 1-phosphate has the function of that protecting or mitigate salivary gland caused by radioactive ray damages, by the way that sphingosine 1-phosphate is administered before radiation treatment, it can effectively reduce damage of the radioactive ray for salivary gland cell and salivary gland vascular endothelial cell, reduce the apoptosis of salivary gland cell and salivary gland vascular endothelial cell, the reduction of salivary gland microvessel density is reduced, salivary gland function obstacle caused by radiotherapy is fundamentally mitigated.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular to sphingosine 1-phosphate is in preparation for protecting salivary gland radiation damage
Application in the drug of wound.
Background technique
Cancer has become the principal risk and focus of Chinese public health, according to statistics, global lips in 2018 and oral cavity
Cancer new cases are 354,864, are the sixth-largest reasons of global cancer related mortality.It is pernicious swollen that radiotherapy has become incidence
One of the treatment means that knurl weight is wanted, the whole world need to receive radiotherapy per year over 50000 head and neck neoplasm patients.Incidence
Salivary gland is frequently included in radiation Yezhong when radiotherapy in the treatment, often results in the damage of salivary gland acute radiation and later period dysfunctional,
Cause the complication such as xerostomia, dysphagia, dysgeusia, saprodontia, further cause gastrointestinal disease, severely impacts patient
Quality of life.
Early in 1911, salivary gland radiation insult was it has been reported that research recently finds radioactive ray to the damage of salivary organization
Wound be it is irreversible, pathological change shows as acinar cells gradually atrophy or even disappearance, and body of gland essence is by a large amount of fibrous connective
Tissue replaces, and the function of salivary gland is caused to change, and the secretion of patient's saliva of buccal cavity is reduced, meanwhile, saliva permeability, viscosity, newborn iron
Albumen, protein, sodium, chloride and saliva potassium concn can obviously increase, and saliva calcium and amylase can reduce.Research card at present
Cause these change may be with acinar cells normal configuration and salivary gland vascular endothelial cell damage apoptosis, microvessel density in fact
Lower related.
The prevention of salivary gland radiation insult, clinical treatment status are less desirable.In order to preferably protect and restore saliva
Gland function, researcher have done very in terms of improving radiotherapy technology, exploitation radioactivity protection drug and gene transfer
It is attempt more.Radiation therapy technology improvement, which can effectively improve target dose and reduce normal surrounding tissue, is measured, but still cannot be protected
Salivary gland dosage is demonstrate,proved in damage threshold hereinafter, patient still will appear different degrees of complication, such as dysphagia, dysgeusia, dental caries
Tooth, gastrointestinal disease.The method that clinically there is no effective treatment salivary gland radiation insult at present, either amifostine, comospore
The treatment means such as the chemicals such as graveoline or artificial saliva, only can temporary relief of symptoms.Drug application (rapamycin)
And the means such as gene therapy (AdRat-Shh, AdAQP1) are protected and treatment miniature pig parotid gland radiation insult vessel density is obvious
It reduces, salivary gland function is not restored effectively, and action time is relatively short, and long-term effect is bad.
Therefore, the method that exploitation effectively can mitigate or prevent salivary gland radiation insult in radiation therapy process has weight
Want meaning.
Summary of the invention
The technical issues of to solve in the prior art, the purpose of the present invention is to provide sphingosine 1-phosphates (S1P) to make
It is ready for use on the application in the drug of protection salivary gland radiation insult and the drug with protection salivary gland radiation insult function.
To achieve the above object, technical scheme is as follows:
In a first aspect, the present invention, which provides sphingosine 1-phosphate, is preparing the medicine for preventing or mitigating salivary gland radiation insult
Application in object.
Second aspect, it is intravascular for reducing salivary gland cell or salivary gland in preparation that the present invention provides sphingosine 1-phosphate
Chrotoplast is to the application in the drug of the sensibility of radioactive ray.
The third aspect, the present invention provide drug of the sphingosine 1-phosphate in preparation for the protection of head and neck neoplasm radiotherapy
In application.
The present invention is analyzed by the protection salivary gland radiation insult function to multiple compounds, and screening has excellent
The substance of anti-salivary gland radiation insult function, discovery sphingosine 1-phosphate can effectively mitigate salivary gland function caused by radiotherapy
It can obstacle.
Preferably, in above-mentioned application, the unit dose of the drug at least contains sphingosine 1-phosphate 0.6mg.
It is highly preferred that the unit dose of the drug contains 0.6~0.8mg of sphingosine 1-phosphate.
When unit dose of the present invention is taking human as object, the drug used when primary emission line irradiation treatment is carried out
Dosage.
Preferably, in above-mentioned application, the dosage form of the drug is suspension, in injection, powder-injection, freeze drying powder injection
It is a kind of.
Fourth aspect, the present invention provide a kind of drug, contain sphingosine 1-phosphate；The drug is unit dosage shape
Formula, per unit dose at least contain sphingosine 1-phosphate 0.6mg.
Preferably, per unit dose contains 0.6~0.8mg of sphingosine 1-phosphate.
The drug provided by the invention has following any function:
(1) prevent or mitigate shield salivary gland radiation insult；
(2) salivary gland cell or salivary gland vascular endothelial cell are reduced to the sensibility of radioactive ray；
(3) protection of head and neck neoplasm radiotherapy.
Preferably, the drug is sphingosine 1-phosphate and other combination of active principles or exclusive use, and pharmacy is added
Suspension that the auxiliary material that field allows is prepared, injection, powder-injection, one of freeze drying powder injection.
The prior art mainly adopts methanol to dissolve S1P, for the bio-toxicity for overcoming the problems, such as solvent methanol, present invention discover that with
PBS solution containing polyethylene glycol, ethyl alcohol and Tween-80 is solvent, while being ultrasonically treated during the preparation process, can be incited somebody to action
S1P is uniformly suspended in solvent, and the suspension of the S1P of preparation can better ensure that the biological safety and validity of preparation.
It is highly preferred that the dosage form of the drug is suspension；The suspension is to contain polyethylene glycol, ethyl alcohol and spit
The PBS solution of temperature -80 is that solvent is prepared through Ultrasonic Pulverization.
As a preferred solution of the present invention, the dosage form of the drug is suspension；It is described poly- in the solvent of the suspension
The content of ethylene glycol is 4~5%；The content of ethyl alcohol is 1.5~2.5%；The content of Tween 80 is 0.8~1%.
Said medicine is for when protecting salivary gland radiation insult, being to be given before radiation treatment containing sphingosine 1-phosphate
Drug.It is administered within preferably before radiation treatment 0.5~5 hour, is administered within more preferably before radiation treatment 1~3 hour.
Said medicine is for when protecting salivary gland radiation insult, administration mode to be preferably salivary organization's local administration.
When the salivary organization is the parotid gland, can be administered by parotid duct.
Said medicine is for when protecting salivary gland radiation insult, taking human as object, dosage to be preferably 10~15 μ g/
kg。
5th aspect, the present invention provides the method using sphingosine 1-phosphate protection salivary gland radiation insult, to put
The drug containing sphingosine 1-phosphate is given before penetrating treatment.
Preferably, the drug containing sphingosine 1-phosphate is given within 0.5~5 hour before radiation treatment.
It is highly preferred that giving the drug containing sphingosine 1-phosphate within 1~3 hour before radiation treatment.
The above-mentioned administration mode for giving the drug containing sphingosine 1-phosphate before radiation treatment is preferably salivary organization
Local administration.When the salivary organization is the parotid gland, can be administered by parotid duct.
Above-mentioned to give the drug containing sphingosine 1-phosphate before radiation treatment, taking human as object, dosage is preferably
10~15 μ g/kg.
As a preferred solution of the present invention, the method for sphingosine 1-phosphate protection salivary gland radiation insult is utilized are as follows: putting
First 2 hours for the treatment of is penetrated, 10~15 μ g/kg are administered in salivary organization's locally injecting.
The beneficial effects of the present invention are: present invention firstly discovers that sphingosine 1-phosphate, which has, prevents and mitigates radiotherapy
The function of salivary gland radiation insult in the process can effectively reduce radiation by the way that sphingosine 1-phosphate is administered before radiation treatment
Damage of the line for salivary gland cell and salivary gland vascular endothelial cell reduces salivary gland cell and salivary gland vascular endothelial cell
Apoptosis, reduce salivary gland microvessel density reduction, fundamentally mitigate radiotherapy caused by salivary gland function obstacle；And
And sphingosine 1-phosphate has longer effective acting time for the protection and mitigation of salivary gland radiation insult.Hair of the invention
Now clinically to solve because of neck radiotherapy etc. caused by the prevention and treatment of salivary gland radiation insult and dysfunction provide
New idea and method.
Detailed description of the invention
Fig. 1 is the flow diagram of the middle-size and small-size pig unilateral side parotid gland radiation insult method for establishing model of the embodiment of the present invention 1；Its
In, A is that three-dimensional suitable-shape regulating is designed for that target area of the right parotid gland as radiotherapy is accurately positioned by force；B is radiotherapy the last week,
Miniature pig shoots incidence CT in prone position；C is based on the image guided radiation therapy for determining region.
Fig. 2 is the effect point of salivary gland function obstacle of the S1P for the parotid gland after radioactive ray process in the embodiment of the present invention 1
Analysis；Wherein, A is experimental design schematic diagram, and IR+S1P treatment indicates the time point of IR+S1P group injection S1P；B is
Saliva flow rate testing result；C is 20 weeks salivary organization's slice dyeing observation results after radiation；D is 20 weeks Masson after radiation
Dyeing observation result；E is the result of 20 weeks Western blotting detection AQP5 protein levels after radiation；NT represents blank pair
According to group, IR is represented radiation alone group (not giving S1P), and IR+S1P represents the experimental group that S1P is given before radiotherapy.
Fig. 3 is the side IR of the middle-size and small-size pig unilateral side parotid gland radiation insult model of the embodiment of the present invention 1 and the saliva stream of the non-side IR
Fast testing result；Wherein, A is that the saliva flow rate of the side IR detects；B is that the saliva flow rate of the non-side IR detects.
Fig. 4 is the group of multiple organs of 20 weeks NT groups, IR group and IR+S1P group after radiation treatment in the embodiment of the present invention 1
Knit observation result；Wherein, A is heart, and B liver, C is spleen, and D is lungs, and E is kidney.
Fig. 5 is S1P in the embodiment of the present invention 2 to the function analysis of 20 weeks parotid gland microvessel densitys after radiotherapy；Wherein,
A is regional flow's rate analysis；B is that Western blotting detects the result of CD31 expression (using Actin albumen in
Ginseng)；C is the result that IHC detects CD31 expression.
Fig. 6 is S1P in the embodiment of the present invention 2 for the function analysis of Apoptosis caused by parotid gland radiation insult；Wherein,
A is that early stage Level of Apoptosis after radiation treatment is detected using TUNEL；B is early stage after Western blotting detection radiation
The result of Bcl-2 and Bax protein level；C is the result for the protein level that IHC detects Caspase3；D is Western
The protein level of early stage Caspase3 after blotting detection radiation.
Fig. 7 is S1P in the embodiment of the present invention 2 for the function analysis of microvascular endothelial cells apoptosis after parotid gland radiation；Its
In, A is the detection of radiotherapy early stage blood flow rate；B is the protein level that Western blotting detects early stage CD31 after radiotherapy；C
For microvascular endothelial cells/acinar cells ratio of 1 Double immune fluorescent staining analysis apoptosis of parotid gland tissues TUNEL and AntiCD3 McAb.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation method of S1P suspension used in the following embodiment is as follows:
(1) preparation of PET solution: being basic solution with sterile PBS, prepare and contain 5% polyethylene glycol, 2.5% ethyl alcohol,
The PBS solution of 0.8% Tween-80 is as solvent.
(2) preparation of S1P suspension: the PET solution prepared using step (1) as solvent, every miniature pig according to weight,
The S1P pulvis for weighing 20 μ g/kg, is suspended into 4ml PET, uses Intelligent supersonic cell disruptor (the letter instrument in Shanghai
Co., Ltd's production), ultrasonic time 2min, interval time 2min, ultrasonic number 8 times, 4 DEG C of temperature, power adjustment 10.In order to
Heat production is avoided, centrifugation has used ice block cooling in tube wall week when ultrasound is suspended.In order to guarantee validity after preparation is suspended, in note
Same day configuration before penetrating, on the way 4 DEG C of refrigeration.
Polyethylene glycol, ethyl alcohol and Tween-80 used above is kept in dark place in room temperature；S1P pulvis freezes in -20 DEG C of ice
In case；All reagents same day configuration same day uses, and refrigerates, is kept in dark place on the way, shakes up before use.
Anesthesia in following embodiment is as follows: by free from worries and Su Mian Xin II by 1:1 mixing, by 0.1ml/kg dosage,
Posterior auricular muscle note.
It the experimental design of following embodiment and is grouped as follows: choosing August age, 25kg-30kg male Ba-Ma mini pig 30
(being purchased from China Agricultural University).9 miniature pigs are chosen first, are randomly divided into 3 groups, blank control group (NT), S1P combination radiotherapy group
(Irradiation+S1P, IR+S1P) and radiation alone group (Irradiation, IR), every group of 3 animals, with 20 weeks after radiotherapy
Time point be long-term point of observation, determine prevention effect of the S1P in salivary gland radiation insult.Miniature pig 21 are chosen again, with
Machine is divided into 7 groups, blank control group (NT), IR12h group, IR+S1P 12h group, IR group for 24 hours, IR+S1P group for 24 hours, IR 1week
Group and IR+S1P 1week group, every group of 3 animals determine with 12 hours, 24 hours and 7 days after radiotherapy for Short-term observation point
Prevention effect of the S1P in salivary gland radiation insult.The preceding animal fasting in 12 hours of experiment, taboo water every time.
In following embodiment, miniature pig collecting parotid sputum and saliva flow rate measuring method are as follows: full to miniature pig anesthesia
After meaning, dorsal position is taken, equivalent intramuscular injection pilocarpine nitrate 0.1mg/kg is (small-sized after bilateral ear according to every miniature pig weight
Pig saliva stimulating agent), about 10-20min after injection will connect vacuum extractor under being formed into stock, bead angular flux when limpid saliva
Lashley cup be adsorbed at bilateral parotid nipple, 15ml centrifuge tube negative pressure collect bilateral saliva 10min, record saliva flow rate
ml/10min.It is sub-packed in 1ml centrifuge tube after saliva centrifugation, the same day detects saliva biochemistry, stored refrigerated on the way.
In following embodiment, the collection method of small-sized pig blood is as follows: after miniature pig anesthesia is satisfied, dorsal position is taken, in
Blood 5ml is taken at vena cava anterior, wherein 2ml is sub-packed in anticoagulant blood-collecting pipe, and blood routine detects in 2 hours；3ml is placed in centrifuge tube
1600rpm is centrifuged 6min, collects serum, and biochemical index is used in 2 hours.It is stored refrigerated on the way.
Parotid gland local administration method in following embodiment is as follows: small according to every after bilateral ear after miniature pig general anesthesia
After type pig weight injecting atropine injection 0.1mg/kg, 15min, opening, by parotid gland master on the right side of salivary ducts insertion miniature pig
3cm in conduit, with 5ml syringe inject S1P suspension 4ml, keep opening and pig position, after retain be intubated and oppress the parotid gland
Conduit nipple 15min prevents liquid reflux, and suspension is enable effectively to be absorbed by the parotid gland.Conduit, silent is taken out, miniature pig lies on one's side quiet
Breath, fore side do not suffer oppression upper, and right side parotid gland radiation insult model foundation is carried out after 2 hours.
In following embodiment, the method for miniature pig unilateral side parotid gland radiation insult model foundation is as follows: radiotherapy the last week, small-sized
After pig general anesthesia is satisfied, prone position shoots head CT and carries out precise positioning；Parotid gland region on the right side of miniature pig delimited according to CT images, adopted
It is designed by force with three-dimensional suitable-shape regulating, central target region radiological dose reaches 95% or more, and clinical target dose is 100%, precise positioning
The right side parotid gland is target of prophylactic radiotherapy.After a week, the radiotherapy of image guidance is carried out according to the region of delimitation, miniature pig general anesthesia is satisfied
Prostrate is on station afterwards, and radioactive source is away from skin 95cm, ray average energy 6Mv, irradiation distance 100cm, dosage rate 3.2Gy/
Min, instrument are Elekta linear accelerator (Elekta AB, Stockholm, Sweden).
In following embodiment, parotid gland tissues sample collection and store method are as follows: 12 hours after radiotherapy, 24 hours, 7
It, 20 weeks this four time points when, miniature pig is injected into excessive anaesthetic respectively and is put to death, parotid gland region and periphery shaving are standby
Skin, sterile drape do notch at Mandibular Rumas rear 2cm and angle of mandible, successively cut skin, subcutaneous tissue, platysma,
It is being close to the platysma deep layer exposure parotid gland, entire parotid gland tissues is being removed, every miniature pig collects bilateral parotid, puts to death within 20 weeks
Group is additional to collect heart, liver, lung, kidney, spleen.It after tissue is cleaned using physiological saline, dries, is cut to 0.5cm*
0.5cm size tissue block, label.A part is organized in 4% paraformaldehyde and fixes 48 hours for 4 DEG C, stays overnight through bath, gradient wine
After smart dehydration, waxdip, paraffin embedding, slice thickness 5um bakes piece, carries out histopathology (HE dyeing) and immunohistochemistry
It detects (IHC)；A part of flesh tissue cutting is placed on 1ml centrifuge tube, saves in -80 DEG C of refrigerators, immune protein Blot experiment
It is spare；All loaded on 15ml centrifuge tube after the cutting of remaining flesh tissue, frozen in -80 DEG C of refrigerators.
In following embodiment, immunohistochemical staining method is as follows:
(1) conventional roasting piece, dimethylbenzene dewaxing；
(2) graded ethanol rehydration；
(3) PBST is washed 3 times, each 5min；
(4) hot high pressure repairs antigen: the sodium citrate of 0.01mol/L, pH 7.0-7.5 is put into pot, slide holding frame is put into it
In, lid lid be heated on pressure cooker after gas continue 2min, after be cooled to room temperature；5min × 3 time are washed in PBST；
(5) slice is placed in wet box, 3%H202 is added dropwise, is protected from light 30min, PBST is washed 3 times, each 5min；
(6) it draws a circle, 5%BSA serum closes 30min；
(7) it gets rid of BSA, is added dropwise the diluted primary antibody of 1%BSA, in moisture releasing box, 4 DEG C of refrigerator overnights；
(8) PBST is washed 3 times, each 5min；
(9) secondary antibody is added dropwise, 37 DEG C of oven 30min, PBST are washed 3 times, each 5min；
(10) DAB developing solution is added dropwise, positive staining occurs in microscopically observation, and rear distilled water terminates reaction；
(11) haematoxylin redyeing nucleus 3min, 0.5% hydrochloric acid-ethyl alcohol break up 3-5 seconds, and tap water returns blue 2min；
(12) graded ethanol is dehydrated, and dimethylbenzene is transparent, neutral gum mounting.
In following embodiment, the method that TUNEL and CD31 is dyed altogether is as follows:
(1) conventional section dewaxes to water, contains 0.005%X-100PBS solution embathes 5 minutes, and totally 3 times, microwave
Antigen retrieval；
(2) TUNEL apoptosis dyeing is illustrated to carry out by kit, and kit inner tissue Proteinase K is added dropwise, and 37 DEG C are incubated for 30 points
Clock, 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(3) TdT buffer is added dropwise, is incubated at room temperature 10 minutes, incline TdT buffer；
(4) end TdT labeled in situ liquid is added dropwise, 37 DEG C are incubated for 1 hour, and incline TdT marking fluid；
(5) Tris-borate buffer is added dropwise, is incubated at room temperature 2 minutes；
(6) 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(7) blocking agent is added dropwise, is incubated at room temperature 20 minutes；
(8) 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(9) FITC fluorescent color-developing agent is added dropwise, is protected from light 37 DEG C and is incubated for 30 minutes；
(10) it is protected from light 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(11) 10% normal rabbit serums (PBS dilution) closing, is protected from light incubation at room temperature 10 minutes, incline serum deprivation；
(12) CD31 antibody (1:100) is added dropwise, is protected from light 37 DEG C and is incubated for 1 hour；
(13) it is protected from light 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(14) the anti-sheep secondary antibody (1:200) of donkey is added dropwise, is protected from light 37 DEG C and is incubated for 30 minutes；
(15) it is protected from light 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(16) PI core at room temperature is protected from light to redye 1.5 minutes；
(17) it is protected from light 0.005%X-100PBS solution embathes 2 minutes, 3 times totally；
(18) hydrophilic anti-fluorescent quenching mountant mounting, fluorescence microscopy is under the microscope.
In following embodiment, the method for Western blotting detection is as follows:
1, total protein extraction
(1) PBS of tissue block pre-cooling is washed 2-3 times, is removed blood stains, is cut into small pieces and is placed in homogenizer.10 times of tissues are added
This reagent of volume is thoroughly homogenized on ice；
(2) homogenate is transferred in 1.5ml centrifuge tube, is vibrated.During which ice bath 30min is blown and beaten, really repeatedly with pipettor
Cell is protected to crack completely；
(3) 12000g is centrifuged 10min, collects supernatant, as total protein solution.
2, BCA method surveys protein concentration.
3, SDS-PAGE electrophoresis
(1) glass plate is cleaned；
(2) encapsulating and loading: being put into clamping in folder after glass plate is aligned, when operation to make two glass alignments in order to avoid leakage
Glue.It arranges to prepare separation gel by experiment, shaking up immediately after addition TEMED can encapsulating.Can remove photoresist upper water after about 45min
And remaining water is blotted with blotting paper.Match 5% concentration glue by above method, shaking up immediately after addition TEMED can encapsulating.It will
Remaining space fills concentration glue and then comb is inserted into concentration glue.
(3) plus enough electrophoresis liquids after loading electrophoresis.Sample is added in electrophoresis hole, electrophoresis.Concentrate glue voltage 75V, point
From glue 120V.Electrophoresis has just been run out of to bromophenol blue can terminate electrophoresis, carry out transferring film.
4, transferring film
(1) first will first be used before the use by preparing the filter paper of 67 × 9cm and a pvdf membrane being of moderate size, pvdf membrane
Alcohol activation；
(2) clip of transferring film, two blocks of foam-rubber cushions, a glass rod, filter paper and process are put into the basin added with transfer liquid
The pvdf membrane of activation；
(3) opening clip keeps black one side holding horizontal.Sponge, three layers of filter paper are padded on cushion；
(4) it carefully peels separation gel and is placed on filter paper, by membrane cover on glue, and bubble removing.Filter paper is opened simultaneously in film upper cover three
Remove bubble.Finally cover another foam-rubber cushion.
5, it is immunoreacted
(1) by the skim milk (0.5%TBST preparation) on the film to take a turn for the better at room temperature decolorization swinging table with 5%, 1h is closed；
(2) it dilutes primary antibody (5% skim milk of TBST dissolution, phosphorylated protein use the 5%BSA of TBST dissolution), 4 DEG C
Be incubated overnight (fast-turn construction) or 4 DEG C of incubation antibody 3h；
(3) it is washed three times on TBST at room temperature decolorization swinging table, each 5min；
(4) secondary antibody TBST is diluted 3000 times, after being incubated for 30min at room temperature, on TBST at room temperature decolorization swinging table
It washes three times, each 5min.
6, chemiluminescence: by two kinds of reagents of ECLA and ECLB in the medium volume mixture of centrifuge tube, by the protein powder of pvdf membrane
It is come into full contact with upward with this mixed liquor, after 1-2min, removes most raffinate, wrap, be put into camera obscura and expose.Finally with development, fixing
Reagent is developed and is fixed.Conditions of exposure is adjusted according to different luminous intensities.
7, image analysis.
In following embodiment, HE colouring method is as follows:
(1) glass frame is put into each 10min in dimethylbenzene (I, II, III) cylinder；
Each 5min in (2) 100% ethyl alcohol (I, II) cylinders；
Each 5min in (3) 95% ethyl alcohol (I, II) cylinders.
(4) it is put into haematoxylin dye liquor 4min；
(5) tap water rinses, differentiation liquid differentiation；
(6) it returns blue liquid and returns indigo plant；
(7) tap water impregnates 10min；
(8) distilled water flushing；
(9) enter eosin stain 40s；
(10) tap water rinses；
Each 5min in (11) 95% ethyl alcohol (I, II) cylinders；
Each 5min in (12) 100% ethyl alcohol (I, II) cylinders；
(13) each 5s in dimethylbenzene (I, II) cylinder；
(14) glass frame is put into dimethylbenzene (III) cylinder；
(15) resinene mounting.
In following embodiment, the method for Masson dyeing is as follows:
(1) it dewaxes: dewaxing 10-15 minutes in dimethylbenzene.Fresh dimethylbenzene is used instead again to dewax 10-15 minutes；
(2) 95% ethyl alcohol of piece after dewaxing is impregnated 2 minutes, then respectively each 2 minutes with 70%, 30% ethyl alcohol；
(3) it is hydrated: being impregnated 2 minutes in distilled water；
(4) rinse: 30-40 DEG C of water rinsing is twice, 30-60 seconds each；
(5) it is soaked slide 30-60 seconds with distilled water；
(6) core dye liquor dyes 60 seconds or so, and flushing liquor rinses 30 seconds；
(7) pulp liquid dyes 30-60 seconds, and flushing liquor rinses 30 seconds；
(8) color separation liquid color separation 6-8 minutes.Microscopically observation, collagenous fibres part takes off into light pink and is in sample
Preferably.If fading effect is bad, the time can be appropriately extended；
(9) it redyes liquid to dye 5 minutes, be rinsed well with dehydrated alcohol；
(10) after drying up, with mounting medium mounting；
(11) result is observed.
Embodiment 1S1P mitigates the salivary gland function obstacle after parotid gland radioactive ray process
The present embodiment analyzes effect of the S1P for the salivary gland function obstacle after parotid gland radioactive ray process, and specific method is such as
Under:
(1) miniature pig parotid sputum is collected first, detects its saliva flow rate.Vena cava anterior blood sampling, inspection are carried out to miniature pig
Look into the biochemistry of blood routine, serum.
(2) foundation of miniature pig unilateral side parotid gland radiation insult model: radiotherapy the last week, miniature pig shoot head CT, delimit
Parotid gland radioactive area reaches 95% or more using the strong design centre target area radiological dose of three-dimensional suitable-shape regulating, and clinical target dose is
100%.Image guided radiation therapy is carried out according to radiotherapy planning after a week, irradiation source is Elekata Synergy linear accelerating
Device, ray average energy 6Mv, irradiation distance 100cm, dosage rate 3.2Gy/min carry out 20Gy's to the parotid gland on the right side of radioactive area
Single radiotherapy.The Establishing process of unilateral parotid gland radiation insult model is as shown in Figure 1.
(3) S1P is administered
The screening of time is administered in conjunction with the analysis of the pharmacokinetics to S1P, be determined at before radiotherapy 2 hours to
Medicine.
S1P dosage is screened, determines that the dosage of every miniature pig is 20 μ g/kg；Comprehensively consider small-sized
Pig parotid gland liquid capacity determines that administered volume is 4ml.
It is driven in the wrong direction to IR+S1P group miniature pig by parotid duct and injects S1P suspension 4ml, the parotid gland is passed through to IR group miniature pig
Conduit, which drives in the wrong direction, injects PET 4ml.After radiotherapy at 12 hours, 24 hours, 7 days and 20 weeks, to NT group, IR group and IR+S1P
Three other miniature pigs of group of group detect blood flow rate, saliva flow rate, blood routine, blood biochemistry and the salivary component of the parotid gland respectively, and locate
Sample is extremely taken, analyzes S1P under ideal delivery time and dosage to the protective action of miniature pig parotid gland radiation insult, in fact
Design scheme is tested as shown in the A of Fig. 2.
The experimental result of above-mentioned detection is as follows:
(1) saliva flow rate is checked, the results show that 20 weeks after radiation, IR group miniature pig radiotherapy side saliva flow rate
20% or so of normal side is dropped to, and IR+S1P group can be restored to 40% or so of NT group (as shown in the B of Fig. 2).The side IR
As shown in figure 3, after radiotherapy, saliva flow rate is gradually reduced saliva flow rate with the non-side IR at any time, at the 18th week, the saliva of IR group
Flow rate maintains about 3ml/min, and the saliva volume of IR+S1P group maintains about 5ml/min (A of Fig. 3)；And non-IR after radiotherapy
The saliva flow rate of side does not have significant changes (B of Fig. 3).
(2) tissue section strain is observed as the result is shown (C of Fig. 2), the visible acinar cells number of the histotomy of IR+S1P group
Amount is apparently higher than IR group.Meanwhile the positive stained area Masson of IR+S1P group is significantly less than IR group.(D of Fig. 2).
(3) Western blotting testing result is shown, the protein level of the aquaporin 5 (AQP5) of IR+S1P group
It is significantly higher than IR group (E of Fig. 2).
The above result shows that S1P can significantly mitigate the function of parotid obstacle for radiating induction in Mini-pig model.
In order to assess S1P for the safety in the function of parotid obstacle Mini-pig model of radiation induction, radiating respectively
First 1 hour of processing, 1 weekly check Ca after 24 hours and radiation treatment after radiation treatment2+, K+, Na+, Cl-, ALP, ALT, AST,
The blood biochemical of LDH.Testing result is as shown in table 1, the results showed that, the blood biochemical between IR and IR+S1P group before and after radiation
Index does not detect significant difference.Further, after radiation treatment the multiple organs of 20 weekly checks (heart, liver, spleen,
Lung, kidney) histological observation, as a result as shown in Figure 4, the results showed that, the histological observation of IR group and IR+S1P group is not significant
Difference.
The Biochemical Indices In Serum testing result of Mini-pig model before and after 1 radiotherapy of table
N=3 in table 1, data are shown as average value ± SEM, and p < 0.05 represents variant.Abbreviation: ALP, alkaline phosphatase；
ALT, alanine aminotransferase；AST, aspartate aminotransferase；LDH, lactic dehydrogenase.
The above result shows that S1P safety with higher, for biochemical indicator and heart, liver, spleen, lung, kidney etc.
Tissue has no significant effect.
Embodiment 2S1P analyzes effect the present embodiment of radiation insult miniature pig parotid gland microvessel density and Apoptosis
Effect of the S1P to radiation insult miniature pig parotid gland microvessel density (MVD) and Apoptosis, the specific method is as follows:
12 hours, 24 hours and 7 days after selective emission processing are Early observation time point, put to death animal, utilize immune group
Change dyeing detection Caspase-3, tunel content, determines salivary gland caused by early stage is intravascular after whether S1P can inhibit radiotherapy
Endothelial apoptosis；Apoptosis executes albumen shadow after detecting radiotherapy by immune protein Blot experiment (Western blotting) simultaneously
The dynamic change of factor B ax and Bcl-2 content is rung, to determine that S1P inhibits the mechanism of salivary gland cell apoptosis.
20 weeks after selective emission processing are long-term point of observation, put to death animal, utilize immunohistochemical staining (IHC) and immune egg
White Blot experiment, using CD31 as the variable density of target detection capilary；Using AQP5 as target detection miniature pig salivary gland secretion
The variation of functional parameter.
The experimental result of above-mentioned detection is as follows:
According to reports, radiation treatment can significantly reduce local microvessel density and blood flow rate, to induce subsequent group
Knit damage.In the present embodiment, compared with IR group, at the 20th week, compared with NT group, nearly 70% can be maintained using S1P before IR
Regional flow lead, and IR group is only capable of maintaining 50% or so (A of Fig. 5).
The result expressed in conjunction with the Western blotting detection CD31 result (B of Fig. 5) expressed and IHC detection CD31
(C of Fig. 5) analyzes parotid gland microvessel density, and S1P is significantly restored using rear parotid gland microvessel density as the result is shown.
The experimental results showed that, S1P can effectively reduce the reduction of parotid gland microvessel density after radiation above.
To inquire into the mechanism that S1P prevention radiation induces function of parotid obstacle, using after TUNEL detection radiation treatment 12
Level of Apoptosis when hour, 24 hours and 1 week.The results show that IR group TUNEL positive cell is apparently higher than NT group, but IR+
The TUNEL positive cell of S1P group significantly reduces (A of Fig. 6).
The protein level of Bcl-2 and Bax and Level of Apoptosis are closely related, therefore, further use Western
The protein level of blotting detection Bcl-2 and Bax.The results show that Protein Bcl- 2/Bax proportional numerical value is high in IR+S1P group
In IR group (B of Fig. 6), show that S1P effectively inhibits Level of Apoptosis.After radiotherapy 7 days, each group of Bcl-2/Bax table
Up to having no notable difference (B of Fig. 6).
In apoptosis process, the caspase-3 albumen of activation is considered as that Apoptosis executes molecule.Therefore, into
One step detects the protein level of Caspase3 using Western blotting and IHC.The results show that the activation of IR+S1P group
IHC the and Western blotting testing result of Caspase3 is substantially less than IR group, it was confirmed that S1P has the work of anti-apoptotic
With.After radiotherapy 7 days, the Caspase-3 expression of each group has no notable difference (C and D of Fig. 6).
The above result shows that S1P has the function of Apoptosis caused by inhibiting radiation insult.
The final dysfunction of the apoptosis with the parotid gland of early stage microvascular endothelial cells is related after radiotherapy.In the present embodiment, In
12 hours after radiation treatment, compared with NT group, the local blood flow of IR+S1P group and IR group decrease beyond 12%；In radiation treatment
24 hours to 1 week afterwards, the blood flow rate of IR group continued to decline, and in IR+S1P group, blood flow rate maintains the pact of NT group
75% level (A of Fig. 7).
Further, it respectively 12 hours, 24 hours and 1 week after radiation treatment, is detected using Western blotting
The protein level for checking CD31, the results show that the CD31 level of S1P+IR group is significantly higher than IR group (B of Fig. 7).
Further, Double immune fluorescent was carried out to parotid gland tissues in 12 hours, 24 hours and 1 week after radiation treatment respectively
It dyes (TUNEL and AntiCD3 McAb 1), to analyze microvascular endothelial cells/acinar cells ratio of apoptosis.It is as follows to observe result: early stage
In the cell of apoptosis, the overwhelming majority is CD31+Vascular endothelial cell, prompt may be microvascular endothelial cells caused by IR damage
Cause salivary gland function obstacle.In addition, IR group microvascular endothelial cells apoptosis number is apparently higher than IR+S1P group and NT group (Fig. 7
C).
The above result shows that S1P mitigates microvascular lesions by the apoptosis of microvascular endothelial cells after reduction radiation.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1.1- phosphoric acid sphingol is preparing the application in the drug for preventing or mitigating salivary gland radiation insult.
2.1- phosphoric acid sphingol is in preparation for reducing salivary gland cell or salivary gland vascular endothelial cell to the sensitivity of radioactive ray
Application in the drug of property.
Application of the 3.1- phosphoric acid sphingol in drug of the preparation for the protection of head and neck neoplasm radiotherapy.
4. described in any item applications according to claim 1~3, which is characterized in that the unit dose of the drug at least contains
Sphingosine 1-phosphate 0.6mg.
5. application according to claim 4, which is characterized in that the unit dose of the drug contains sphingosine 1-phosphate
0.6~0.8mg.
6. described in any item applications according to claim 1~5, which is characterized in that the dosage form of the drug is suspension, injection
One of agent, powder-injection, freeze drying powder injection.
7. a kind of drug, which is characterized in that it contains sphingosine 1-phosphate；The drug is unit dosage form, per unit agent
Amount at least contains sphingosine 1-phosphate 0.6mg；Preferably, per unit dose contains 0.6~0.8mg of sphingosine 1-phosphate.
8. drug according to claim 7, which is characterized in that the drug has following any function:
(1) prevent or mitigate salivary gland radiation insult；
(2) salivary gland cell or salivary gland vascular endothelial cell are reduced to the sensibility of radioactive ray；
(3) protection of head and neck neoplasm radiotherapy.
9. drug according to claim 7 or 8, which is characterized in that the drug be sphingosine 1-phosphate and it is other effectively
At subassembly or exclusive use, and suspension, injection, powder-injection, the jelly that the auxiliary material that pharmaceutical field permission is added is prepared
One of dry powder injection.
10. drug according to claim 9, which is characterized in that the dosage form of the drug is suspension；The suspension is
Using the PBS solution containing polyethylene glycol, ethyl alcohol and Tween-80 as solvent, it is prepared through Ultrasonic Pulverization；
Preferably, in the solvent, the content of the polyethylene glycol is 4~5%；The content of ethyl alcohol is 1.5~2.5%；Tween
80 content is 0.8~1%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016183119A (en) * | 2015-03-25 | 2016-10-20 | 国立大学法人岐阜大学 | Non-injury site preparation containing sphingosine-1-phosphate receptor 2 activating compound |
| KR20180032540A (en) * | 2018-03-20 | 2018-03-30 | 주식회사 피토스 | Use of phytospingosine-1-phosphate or its derivatives for aduvant |
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2019
- 2019-09-09 CN CN201910848219.XA patent/CN110478358B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2016183119A (en) * | 2015-03-25 | 2016-10-20 | 国立大学法人岐阜大学 | Non-injury site preparation containing sphingosine-1-phosphate receptor 2 activating compound |
| KR20180032540A (en) * | 2018-03-20 | 2018-03-30 | 주식회사 피토스 | Use of phytospingosine-1-phosphate or its derivatives for aduvant |
Non-Patent Citations (2)
| Title |
|---|
| A. STESSIN等: "FTY720, Sphingosine 1-Phosphate Receptor Modulator, Attenuates Radiation-Induced Neurocognitive Deficits in Young Mice", 《INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS》 * |
| 段海峰等: "鞘氨醇-1磷酸盐的生理功能研究进展", 《生理科学进展》 * |
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