CN103341157A - Application of pingyangmycin combined sodium hyaluronate in medicine for treating venous malformation - Google Patents
Application of pingyangmycin combined sodium hyaluronate in medicine for treating venous malformation Download PDFInfo
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Abstract
The invention discloses application of pingyangmycin combined sodium hyaluronate in a medicine for treating venous malformation and a medicine for treating venous malformation, in particular relates to a medicine for treating venous malformation of head and neck. The molecular weight of the sodium hyaluronate is 600,000-1,500,000 Dalton; the concentrations of the pingyangmycin and the sodium hyaluronate are 10 mg/mL and 400 mug/mL respectively.
Description
Technical field
The present invention relates to the application of Bleomycin A5 associating hyaluronate sodium in treatment venous malformation medicine.
Background technology
(venous malformation VM), is called cavernous hemangioma to venous malformation in the past, belongs to a kind of low flow velocity vascular malformation, is made of the vein of differentially expanding, and pathological changes and health are the ratio growth, and lifelong asymptotic development can not disappeared voluntarily.About 40% betides incidence, not only influences face, if compressing, invade and adjacent tissue's structure, can influence language, swallow and function such as breathing, and severe patient has the danger of death by suffocation.Its Therapeutic Method is numerous, and modern medicine is advocated the concrete state of an illness according to the patient, selects suitable treatment means.
The pathogenesis of venous malformation is still indeterminate, and TIE2 receptor mutation, autosome 9P site mutation, hormonal readiness change etc. may be its pathogenic factors.According to the iconography characteristics of reflux veins, venous malformation is divided into 4 types: the I type does not have obvious reflux veins, and II type reflux veins is normal, and III type reflux veins increases slightly, the expansion of IV type reflux veins.Common Therapeutic Method comprise operation, sclerosis, laser, freezing, electricity coagulates and copper acupuncture treatment etc., these methods respectively have pluses and minuses.In principle, should be according to position, size, scope, back-flow velocity and the technical conditions of pathological changes, for the patient works out the individualized treatment scheme.Venous malformation is at first considered non-operative treatment, i.e. expectant treatment, most common form is injection in the local tumor body of sclerosing agent.Sclerotherapy can be used separately, also can with therapeutic modality couplings such as laser, operation.Sclerosing agent commonly used has Bleomycin A5,5% sodium morrhuate, polidocanol, dehydrated alcohol etc.
Bleomycin A5 (pingyangmycin, PYM) sclerotherapy is one of at present the most frequently used method, Bleomycin A5 is a kind of antibiotics antitumor drug of separating from the streptococcus of Pingyang, is a kind of of bleomycin A5, also is a kind of antitumor drug of maturation.Because it is safe, simple and efficient, the Bleomycin A5 injection has been widely used in treating the neck surface venous malformation in the tumor body.Generally believe that at present the main mechanism of PYM treatment venous malformation is: DNA is combined with vascular endothelial cell, causes the DNA chain break, thereby suppresses the metabolism of vascular endothelial cell, causes endotheliocyte atrophy, degeneration; Vascular endothelial cell injury and tube wall, induction of vascular smooth muscle cell and endotheli ocytosis make tube wall thickening, the narrow final locking of tube chamber stenosis.
Bleomycin A5 is that we wish its generation " favourable " effect to the destruction of venous malformation vascular endothelial cell, after this medicine is used in the part, it produces same effect to all various kinds of cell that touch beyond the vascular endothelial cell inevitably, i.e. " unfavorable " effect, or claim side effect.In addition, Bleomycin A5 can produce severe anaphylactic reaction by chance, even causes death.Heavy dose of Bleomycin A5 of using can cause pulmonary fibrosis.At the abundant position of blood flows such as incidence, use Bleomycin A5 merely because it is short in the lesions position time of staying, cause using dosage big, and the risk that has side effects also increase.
Summary of the invention
The objective of the invention is for overcoming above-mentioned the deficiencies in the prior art, the application of Bleomycin A5 associating hyaluronate sodium in treatment venous malformation medicine is provided, and a kind of medicine that is used for the treatment of venous malformation, especially for the medicine for the treatment of incidence venous malformation.
For achieving the above object, the present invention adopts following technical proposals:
The application of Bleomycin A5 (PYM) associating hyaluronate sodium (HA) in treatment venous malformation medicine, wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton.
The concentration of described Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.
A kind of medicine that is used for the treatment of venous malformation, it contains as the Bleomycin A5 of active component and hyaluronate sodium, and the medicine acceptable carrier; Wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton, and the concentration of Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.
Beneficial effect of the present invention:
The present invention adds hyaluronate sodium in order to slow down the fast Absorption of Bleomycin A5, and both associating makes Bleomycin A5 keep valid density in the injection focus in the long period, reduces the total amount of Bleomycin A5, reduces its side effect.In addition, both associatings can strengthen Bleomycin A5 to the venous malformation key component---and the selective therapy effect of vascular endothelial cell strengthens therapeutic effect.
Hyaluronic acid is a kind of biological active substances that extensively is present in animal and human's body, in application on human skin, synovium of joint liquid, umbilical cord, aqueous humor and vitreum, distribution is arranged all, but be a kind of Natural Degradation, absorbable biomedical material, it has rheological properties and the excellent biological compatibility of high viscoelasticity, plasticity, permeability, uniqueness.Simultaneously, it all plays an important role in processes such as cell proliferation, migration, angiogenesis, tumor generation.Hyaluronic acid and sodium salt thereof can promote drug absorption, improve bioavailability of medicament.According to hyaluronic molecular weight size, it can be divided into natural macromolecular hyaluronic acid and hyaluronic acid oligose fragment, the natural macromolecular amount that the mostly is hyaluronic acid that exists in the normal structure, hyaluronic acid oligose fragment are that the natural macromolecular hyaluronic acid decomposes to come under the pathological conditions.The effect that high molecular weight hyaluronic acid performance support structure and antiinflammatory, inhibition immunoreation and anti-new vessels form, low-molecular-weight hyaluronic acid have the inflammation of promotion immunity of organism and promotion angiogenesis take place, stimulate.Therefore, the present invention selects macromolecule hyaluronate sodium and Bleomycin A5 drug combination for use.
The endotheliocyte of different tissue sources there are differences at aspects such as form, function, antigen presentation and metabolic characteristicss, one aspect of the present invention is cultivated the method for primary cell by piece of tissue, be to cultivate the venous malformation endotheliocyte the venous malformation tissue from pathological diagnosis, thereby the physio-biochemical characteristics that reflect such disease endotheliocyte, and then the accurately objectively pharmacological action of reflection medicine, carry out clinical treatment by Bleomycin A5 and hyaluronate sodium with appropriate proportioning on the other hand, the effect of its treatment incidence venous malformation of preliminary observation.
The venous malformation endotheliocyte of cultivating is seeded in in-vitro simulated vascularization experiment on the Matrigel matrigel, similar matrix environment in the analogue body forms blood vessel sample network structure by endotheliocyte at Matrigel and how much judges that determinand promotes still to suppress angiogenesis.Cultivated 12h(hour) after just observe the venous malformation endotheliocyte and namely formed blood vessel sample network structure in Matrigel substrate, formed stable network structure during 24h, have only the blank group still to become pipe during 48h, and hyaluronate sodium processed group, Bleomycin A5 processed group, the formed tube chamber of Bleomycin A5 associating hyaluronate sodium processed group disappear.Become the situation of blood vessel to add up during to 24h, result's matter acid that shows transparency can suppress the angiogenesis function of venous malformation endotheliocyte, and when its associating Bleomycin A5 is used, suppress the venous malformation endotheliocyte and become the effect of pipe better than using the Bleomycin A5 effect separately.This has proved the inhibition angiogenesis function of macromole hyaluronate sodium, and its associating is during Bleomycin A5, macromole hyaluronate sodium bag is by the Bleomycin A5 molecule, combine with the receptor on the venous malformation endotheliocyte, thereby carry the Bleomycin A5 permeates cell membranes, allow more Bleomycin A5 can enter kytoplasm, and then both can unite interference signal transduction pathway wherein, and Bleomycin A5 also can act on the DNA in the nuclear more quickly, and the apoptosis result has proved this point.
Bleomycin A5 of the present invention and hyaluronate sodium are with suitable concentration (hyaluronate sodium 400 μ g/mL, Bleomycin A5 10 μ g/mL) act on the venous malformation endotheliocyte after, compare with independent application Bleomycin A5, the propagation of venous malformation endotheliocyte and one-tenth blood vessel ability reduce, and apoptosis strengthens.Use in conjunction Bleomycin A5 and hyaluronate sodium can suppress significantly the venous malformation endotheliocyte propagation, become the blood vessel ability, promote its apoptosis, bring out disappearing of venous malformation.Unite both medications, will bring new opportunity to the treatment of venous malformation clinically.
On the histology, venous malformation is made up of the countless blood sinus that are lined with endotheliocyte, thus to cultivate venous malformation endotheliocyte (the human vascular malformation endothelial cells from people's venous malformation tissue, HVMECs) as study model, by use in conjunction Bleomycin A5 and hyaluronic sodium salt (hyaluronate sodium) it is handled, observe its effect aspect cell proliferation, angiogenesis, apoptosis, observe Bleomycin A5 and hyaluronate sodium thus intuitively to the therapeutic effect of venous malformation.
Cultivate the venous malformation endotheliocyte with tissue mass cell culture, the growing state of observation post's cultured cell and morphological characteristic under the inverted phase contrast microscope are with the anti-people vWF(1:150 of endothelial cell specific antibodies rabbit) and rabbit antihuman CD 34 (1:150) identify.Trace the variation of cell growth curve with the MTT colorimetry, screening Bleomycin A5 and hyaluronate sodium act on the optium concentration of venous malformation endotheliocyte respectively.According to determined best concentration, institute's cultured cells is handled with hyaluronate sodium, Bleomycin A5, Bleomycin A5 associating hyaluronate sodium respectively, with the influence of MTT colorimetry detection of drugs to the venous malformation endothelial cell proliferation, the venous malformation endotheliocyte is become the influence of blood vessel ability with Tube formation method observation medicine, use the TUNEL test kit, detection of drugs is to the influence of venous malformation endothelial cell apoptosis.
The cultured cells vWF of tissue block method, CD34 dyeing expressions that be positive, confirmation institute cultured cell is the venous malformation endotheliocyte.When acting on the venous malformation endotheliocyte separately with hyaluronate sodium, Bleomycin A5, optimum effective concentration is respectively 400 μ g/mL, 10 μ g/mL.The MTT colorimetry matter acid sodium associating Bleomycin A5 that shows transparency can more effectively suppress the propagation of venous malformation endotheliocyte.Tube formation observation drug effect posterior vein deformity endotheliocyte becomes the blood vessel ability to change, the result shows that the effect of Bleomycin A5 associating hyaluronate sodium uses the Bleomycin A5 cell more separately and become the blood vessel ability to descend, and become the pipe persistent period to shorten, tube wall disappears soon, has compared notable difference with the blank group.Change with TUNEL test kit observation drug effect posterior vein deformity endothelial cell apoptosis ability, the result shows that the effect of Bleomycin A5 associating hyaluronate sodium uses the aggravation of Bleomycin A5 apoptosis more separately, compares with the blank group that there were significant differences.
Bleomycin A5 associating hyaluronate sodium is treated routine incidence venous malformation surplus 50, finds that clinical effectiveness is extremely remarkable.
Description of drawings
Fig. 1 a be different quality concentration Bleomycin A5 effect different time to the proliferation function effect of HVMECs, Fig. 1 b is that different quality concentration hyaluronate sodium effect different time is to the proliferation inhibiting effect of HVMECs;
Fig. 2 is the OD value curve behind the adding medicine;
Fig. 3 is the growth curve before and after cell disturbs;
Fig. 4 is the difference that cell became the blood vessel ability before and after medicine disturbed;
Fig. 5 is the difference of apoptosis ability before and after medicine disturbs;
Fig. 6 is that the venous malformation endotheliocyte is cultivated by tissue block method, and it is adherent to be the paving stone sample;
Fig. 7 is first generation venous malformation endotheliocyte, becomes the fiber-like outward appearance, and endochylema is evenly clear, examines circle or oval, and increase is in various degree arranged;
Fig. 8 is the venous malformation endotheliocyte, and the endotheliocyte mark vwf dyeing expression that is positive mainly is expressed in kytoplasm, and obvious expression is not seen on the nucleus surface;
Fig. 9 is the venous malformation endotheliocyte, and the endotheliocyte mark CD34 dyeing expression that is positive mainly is expressed in kytoplasm, and obvious expression is not seen on the nucleus surface;
Figure 10 handles 48h posterior vein deformity endothelial cell growth situation through different pharmaceutical, (a) control group, and cell is Exponential growth, a large amount of propagation, the iuntercellular contact surpasses 80%, reaches the density that goes down to posterity; (b) HA processed group, the cell poor growth, propagation is received certain influence; (c) PYM processed group, cell growth retardation, mortality, visible cracked cell relic; (d) PYM unites the HA processed group, remains the cell of a small amount of survival;
Figure 11 is that cell became vessel graph before and after medicine disturbed; (a) during 12h, the Control group has become the blood vessel rule, and tube chamber is complete; The HA group has become the blood vessel rule, and tube chamber is complete, PYM group and the mixed and disorderly random distribution still of HA associating PYM group cell; (b) during 24h, four groups have all become pipe, but Control group and HA form the blood vessel rule and tube chamber is more complete, and it is more thicker with tube wall that Control organizes formed nodal point number, and PYM group and the most of gathering of PYM+HA group cell are in heaps, and faint one-tenth blood vessel ability is only arranged; (c) during 48h, observe existing a large amount of cell deaths, become the tube wall of blood vessel to begin atrophy, NC group, HA group are still keeping certain shape of blood vessel, and PYM group, the atrophy of the poly-heap of PYM+HA group cell are remarkable, and established tube chamber also disappears obviously;
Figure 12 is that the TUNEL method detects apoptosis, and normal cell nuclear is dyed blueness, and it is yellow to brown that apoptotic nucleus is, (a) control group, and apoptotic state appears in small amounts of cells; (b) HA processed group, apoptotic state appears in about half cell; (c) PYM processed group, only small amounts of cells dyeing nuclear is normal; (d) PYM associating HA processed group, no normal cell exists.
The specific embodiment
The present invention will be further elaborated below in conjunction with drawings and Examples, should be noted that following explanation only is in order to explain the present invention, its content not to be limited.
Embodiment 1:
A kind of preparation that is used for the treatment of the medicine of venous malformation involved in the present invention:
The positive mycin 0.1mg that makes even, hyaluronate sodium 4mg adds the medicine acceptable carrier of preparation injection, and preparation obtains the 10mL injection.Owing to the liquid in the tumor body can not be released fully and is replaced as the medicine of this concentration, can suitably adjust the consumption of Bleomycin A5 and hyaluronate sodium, make the Bleomycin A5 that enters in the tumor body and the actual concentrations of hyaluronate sodium be about 10 μ g/mL and 400 μ g/mL.
Hyaluronate sodium is commercial sodium hyaluronate injection, Shandong Bausch ﹠ Lomb Freda Pharmaceutical Co., Ltd., authentication code: the accurate word H10960136 of traditional Chinese medicines.
Further specify its pharmaceutically active below by pharmacodynamic experiment.
The test example
1. key instrument equipment
Superclean bench (east, Harbin connection electronic technology development corporation, Ltd.);
Ultra-pure water and sterilization system (MILLI-Q, France);
Autoclave sterilizer (HIRAYAMA, Japan);
High speed low temperature centrifugal machine (THERMO, Germany);
Electric heating constant temperature tank (going up the grand experimental facilities company limited of Nereid);
Constant temperature CO
2Cell culture incubator (German Heraeus company);
Ice machine, cryogenic refrigerator (SANYO, Japan);
4 ℃ ,-20 ℃ of refrigerators (Hefei Meiling Co. Ltd.);
Inverted phase contrast microscope and photographic system (LEICA, Germany);
Fluorescence microscope and photographic system (LEICA, Germany);
Electronic analytical balance (METTLER TOLEDO, Switzerland);
Constant Temp. Oven (going up the grand experimental facilities company limited of Nereid);
Hypoxia constant temperature cell culture incubator (THERMO, the U.S.);
Micropipettor (GIBCO);
Weighing scale, precision balance, PH count (METTLER TOLEDO);
Ultraviolet spectrophotometer (U.S. Biochrom company);
STS-8A decolorization swinging table (QILINBEIER).
Conventional surgical apparatus: eye scissors, ophthalmology tweezer, blade, needle holder, mosquito forceps, probe, blade etc.;
Disposable culture dish, disposable culture bottle, culture plate, 15mL centrifuge tube, 50mL centrifuge tube (NUNC);
2. main agents and preparation
(1) ECM culture medium is available from ScienCell company.
(2) hyclone FBS is available from ScienCell company.
(3) mass concentration 0.25% trypsin is available from GIBCO company.
(4) PBS buffer is available from Sergene company.
(5) DMSO is available from SIGMA company.
(6) DAB colour developing liquid is available from gene tech.
(7) HRP labelling goat anti-rabbit igg, working concentration are 1:3000, available from gene tech.
(8) the anti-people's polyclonal antibody of vWF rabbit, ready to use is available from gene tech.
(9) the anti-people's polyclonal antibody of CD34 rabbit, ready to use is available from gene tech.
(10) TUNEL apoptosis detection kit is available from Roche company.
(11) MTT solution is with the solution that before is made into 5mg/mL, available from SIGMA company.
The Matrigel matrigel is available from U.S. Bi Di company.
3. experimental technique:
3.1 cell culture
A: tissue block method's cultured cell
1) with aseptic PBS buffer flushing excision venous malformation specimen (from the flesh tissue of incidence venous malformation excision) to limpid, do not have muddy and the courageous and upright tissue that comes off, remove unnecessary skin and fatty tissue with eye scissors and tweezers;
2) be trimmed to 1～2cm
3Piece of tissue is put into 37 ℃ mass concentration 0.25% trypsin solution, water-bath vibration digestion 10～20 minutes;
3) add the culture fluid that contains hyclone and stop digestion, and postdigestive piece of tissue is trimmed to 1mm
3
4) will prune good piece of tissue and be inoculated in the culture bottle, and culture bottle will be put in to be inverted in 37 ℃ of incubators cultivate 10 minutes;
5) carefully add culture medium in culture bottle, the jog culture bottle does not make piece of tissue floating, and liquid level just in time covered piece of tissue;
6) put into incubator (37 ℃, volume fraction 5%CO
2) the middle cultivation, changed liquid, removed piece of tissue, went down to posterity in 7～9 days in 5～6 days in 3～4 days.
B: passage
1) discard culture fluid in the culture bottle, with aseptic PBS flushing 2～3 times, add the 1mL0.25% trypsin solution and cultivate, place 37 ℃ about 1 minute, the metamorphosis of observation of cell under inverted microscope.When the cell process retraction, form change circle, intercellular substance increases, and has simultaneously when taking off parietal cell on a small quantity, stops digestion immediately, and the sucking-off Digestive system also adds the clean Digestive system of a little ECM complete medium, absorbs liquid, adds the ECM culture medium;
2) cell on the piping and druming bottle wall repeatedly successively from one side of culture bottle bottom to another side is made single cell suspension after making cell detachment bottle wall, during piping and druming action soft, do not produce bubble as far as possible;
3) single cell suspension that obtains is transferred in the centrifuge tube, carries out centrifugalize (800 rev/mins rotating speed centrifugal 7 minutes), abandon supernatant, add complete ECM culture medium, the cell mass sheet is blown and beaten gently be single cell suspension, do not produce bubble as far as possible; With 5 * 10
5/ mL is inoculated in culture bottle and the culture plate, goes down to posterity and tests.
C: cell cryopreservation and recovery:
(1) cell cryopreservation
1) preparation hyclone and DMSO volume ratio are the frozen culture fluid of 9:1;
2) the take the logarithm cell of trophophase gets off the cell dissociation of monolayer growth with trypsin, cell is moved in the 15mL centrifuge tube centrifugal 1000rpm, 5min;
3) remove trypsin and old culture fluid, add the frozen culture fluid for preparing in right amount, blow and beat gently with suction pipe and make cell evenly and counting, the final densities of regulating cell in the cryopreserving liquid is 5 * 10
6/ mL～1 * 10
7/ mL packs the cell branch in the frozen pipe into every pipe 1～1.5mL;
4) in the title of frozen pipe subscript clear-cells, frozen time and operator;
5) frozen pipe is put into the programmed cooling box, put into-80 ℃ of refrigerator overnight then, take out frozen pipe, move in the liquid nitrogen container.
(2) cell recovery
1) from liquid nitrogen container, takes out frozen pipe, directly immerse in 37 ℃ of warm water, and shake frequently and make it melt as early as possible;
2) take out frozen pipe from 37 ℃ of water-baths, uncap with suction pipe sucking-off cell suspension, is added to centrifuge tube and drips culture fluid more than 10 times, and is centrifugal behind the mixing, 1000rpm, 5min;
3) abandoning supernatant adds complete ECM culture medium re-suspended cell, and counting is adjusted cell density, the inoculated and cultured bottle, and 37 ℃ of incubators leave standstill cultivation;
4) changed a culture fluid in second day, continue to cultivate, passage (changed liquid, went down to posterity in 5～6 days in 2～3 days) is observed at any time and is taken pictures.
The morphological observation of D.HVMECs and evaluation
(1) morphological observation: observe growing state and the morphological characteristic of HVMECs under the inverted phase contrast microscope.
(2) SABC EnVision method is identified
1) former generation HVMECs is seeded in 24 orifice plates makes cell climbing sheet, treated cell density at about 70% o'clock, take out creep plate, PBS flushing 3 times;
2) fixing 10min under the mass concentration 4% paraformaldehyde room temperature, PBS flushing 3 times;
3) mass concentration 0.3%H
2O
2With hatch 10min under the methanol equal-volume mixed liquor room temperature, remove endogenous peroxydase, PBS flushing 3 times;
4) mass concentration 10% sheep blood serum sealing 10min adds the anti-people vWF(1:150 of rabbit respectively) and mouse-anti people CD34(1:150), hatch 2h under 37 ℃, PBS washes 3 times;
5) add the anti-rabbit igg of HRP labelled goat, hatch 1h under 37 ℃, after the PBS flushing, the DAB colour developing, haematoxylin is redyed, and volumetric concentration is 0.5% hydrochloride alcohol (concentrated hydrochloric acid proportioning volumetric concentration is than the ethanol that is 75%) differentiation, volumetric concentration is that 5% ammonia returns indigo plant, and mounting is taken pictures.
3.2 selected HA, PYM suppress the optium concentration of HVMECs
With HVMECs with 2 * 10
4Individual/mL concentration is inoculated in 96 orifice plates, and every hole 200 μ L change culture fluid behind the cultivation 48h.Process element is PYM processed group and HA processed group, and each processed group adds 5 variable concentrations gradients (PYM:1,5,10,20,50 μ g/mL; HA:10,100,200,500,1000 μ g/mL) medicine, and establish blank group (not adding medicine), after cultivating different time (0,12,24,48,72h), every hole adds MTT solution (5mg/mL) 20 μ L, continue to hatch 4h, culture fluid is abandoned in suction, add 150 μ L dimethyl sulfoxide (DMSO), vibration 10min, surveying wavelength on enzyme-linked immunosorbent assay instrument is absorbance (OD) value of 490nm, experiment repeats 3 times, gets the meansigma methods of 3 secondary data, chooses the concentration of suitable PYM and HA effect and HVMECs.
3.3MTT the method detection of drugs is handled multiplication capacity and the vigor of each group of back
A. experiment grouping
1) get the stable HVMECs of growth and experimentize, being divided into is 4 groups:
The 1st group: HVMECs cultivation group;
The 2nd group: HA disturbs HVMECs cultivation group;
The 3rd group: PYM disturbs HVMECs cultivation group;
The 4th group: HA associating PYM disturbs HVMECs cultivation group;
2) respectively organize cell all with 1 * 10
3/ hole is inoculated in 96 well culture plates, every group of 5 holes; Culture medium is complete ECM culture medium; Change a subculture in per 2～3 days, and cultivated after 3 days, began to get respectively 12h from the 4th day, 24h, 48h, the time period of 72h carries out MTT to three groups of cells and detects totally 4 times.
The B.MTT method detects multiplication capacity and the vigor thereof of respectively organizing cell
Draw old culture medium in the hole, after the ECM culture medium flushing that does not contain FBS, changing culture medium is the ECM culture medium that contains 5%FBS, simultaneously every hole adds the MTT20 μ L of mass concentration 0.5%, continuation is inhaled and is removed culture fluid in the hole after 37 ℃ of cell culture incubators are cultivated 4h, adds the DMSO of 150 μ L in every hole, concussion 10min measures light absorption value (OD value) under spectrophotometer 490nm wavelength.The OD value of surveying is represented with mean ± standard deviation, draws and respectively organizes cell growth curve, adopts the SPSS statistical software to handle, and carries out variance analysis and q check with SPSS software, and there is statistical significance P＜0.05 for difference.
3.4Tube Formation method:
1) the Matrigel matrigel is spent the night for 4 ℃;
With at the bottom of the Matrigel matrigel tiling hole, 200 μ L/ hole, totally 6 holes are divided 4 groups, 3 every group multiple holes under the low temperature.
The 1st group: HVMECs cultivation group;
The 2nd group: HA disturbs HVMECs cultivation group;
The 3rd group: PYM disturbs HVMECs cultivation group;
The 4th group: HA associating PYM disturbs HVMECs cultivation group;
2) put into 37 ℃ of constant temperature incubators 30～60 minutes;
3) 100,000 cells are inoculated in every hole, add culture medium 1mL;
4) 37 ℃ of cultivations, the 1st group is used for paired observation, is observing photo under the mirror at any time for the 2nd group and the 3rd group, and to carrying out statistical analysis after the quantification of 24h one-tenth pipe.
3.5TUNEL apoptosis detection method:
The experiment grouping:
The 1st group: HVMECs cultivation group;
The 2nd group: HA disturbs HVMECs cultivation group;
The 3rd group: PYM disturbs HVMECs cultivation group;
The 4th group: HA associating PYM disturbs HVMECs cultivation group;
1) carries kind plate the previous day, treat that cell confluency reaches at 50% o'clock and can carry out the apoptosis detection;
2) the fixing 1h of the paraformaldehyde of mass concentration 4%, PBS flushing 5min * 3 time;
3) mass concentration 3%H
2O
2Blocking-up liquid chamber temperature effect 10min, PBS wash 5min * 3 time;
4) add 4 ℃ of 20min of penetrating fluid (mass concentration 0.1%Triton-X100 sodium citrate buffer solution), PBS flushing 5min * 3 time;
5) add 50 μ L TUNEL reaction mixtures, add coverlay, 37 ℃ of effect 1h in the wet box, PBS flushing 5min * 3 time;
6) 37 ℃ of the bovin serum albumin of mass concentration 3% sealing 20min, PBS flushing 5min * 3 time;
7) add 50 μ L conversion POD solution, add coverlay, 37 ℃ of effect 30min in the wet box, PBS flushing 5min * 3 time;
8) add 50 μ L DAB substrates (Roche company), lucifuge colour developing 10min in 15 ℃ of-25 ℃ of wet boxes, PBS flushing 5min * 3 time;
9) routine is carried out haematoxylin and is redyed, the hydrochloride alcohol differentiation, and ammonia returns indigo plant.Mounting is taken pictures.
10) selection area calculates number and the ratio of apoptotic cell in the zone of certain area; 4 groups of apoptosis differences of ANOVA and q inspection.
4. experimental result
4.1 venous malformation endothelial cell morphology and evaluation:
The venous malformation endotheliocyte is cultivated by tissue block method, shows as paving stone sample (Fig. 6) behind the cell attachment, and endochylema is evenly clear, examines circle or oval, and increase is in various degree arranged, and collects cluster long (Fig. 7); Utilize the method for immunocytochemistry to detect two kinds of significant antigen vWF of endotheliocyte and CD34, the expression that all is positive (Fig. 8～9) confirms that institute's cultured cells is the venous malformation endotheliocyte.
4.2 selected HA, PYM suppress the optium concentration of HVMECs
Different quality concentration Bleomycin A5 effect different time is seen Fig. 1 (a) to the proliferation function effect of HVMECs.Under the effect of identical Bleomycin A5 mass concentration, along with the prolongation of action time, the proliferation inhibition rate of cell increases gradually; And in same action time of section, with the increase of mass concentration, the proliferation inhibition rate of cell also increases gradually, demonstrates dose-dependent effect and time-dependent effect, and the Bleomycin A5 group of this experiment has been verified this point.When Pinyangmycin concentration 〉=10 μ g/mL, the propagation of HVMECs is had the obvious suppression effect, so this experiment associating hyaluronate sodium when acting on HVMECs used Pinyangmycin concentration be 10 μ g/mL.
Different quality concentration hyaluronate sodium effect different time is seen Fig. 1 (b) to the proliferation inhibiting effect of HVMECs, demonstrate dose-dependent effect and time-dependent effect equally, when high concentration, can slightly suppress HVMEVCs propagation, inhibition occurs during since 400 μ g/mL, so the concentration of the HA that selects for use during drug combination is 400 μ g/mL.
4.3MTT method detects cell proliferation vigor and survival ability:
After adding medicine, pick up counting, detect 0h respectively, 12h, 24h, 48h, the OD value between four groups of 72h, and trace curve, as Fig. 2.
After adding medicine, pick up counting, detect 0h respectively, 12h, 24h, 48h, the OD value between four groups of 72h, and calculate it to the suppression ratio of HVMECs, as table 1.
The average suppression ratio that the different medicine of table 1. is bred HVMECs in time
Above result shows:
0h: do not have significant difference between each group.
12h:PYM+HA group, PYM group are compared obvious difference, * * * P＜0.001 with Control group, HA group; Difference not statistically significant between PYM+HA group and the PYM group.
24h:PYM+HA group, PYM group are compared obvious difference, * * * P＜0.001 with Control group, HA group; Significant difference is arranged, * P＜0.05 between PYM+HA group and the PYM group.
48h:PYM+HA group, PYM group are compared obvious difference, * * * P＜0.001 with Control group, HA group; Have between PYM+HA group and the PYM group more obvious than 24h difference, * P＜0.05.
72h:PYM+HA group, PYM group are compared obvious difference, * * * P＜0.001 with Control group, HA group; Have between PYM+HA group and the PYM group more obvious than 48h difference, * P＜0.05.
It is obvious further in time to the inhibition of venous malformation endotheliocyte that Bleomycin A5 associating hyaluronate sodium is used Bleomycin A5 more separately.
Trace the growth curve that cell disturbs front and back, as Fig. 3, cell growth condition is seen Figure 10.
In summary, added PYM in the cell culture fluid after, ability of cell proliferation is suppressed, cell viability and survival ability descend, effect is more obvious when PYM and HA use in conjunction, as time passes, demonstrates the slow release of HA.
4.4 cell became the difference of blood vessel ability before and after medicine disturbed:
We enumerate out the one-tenth pipe situation of two groups of 12h, 24h, 48h, and as Figure 11, during 12h, the Control group has become the blood vessel rule, and tube chamber is complete; The HA group has become the blood vessel rule, and tube chamber is complete, PYM group and the mixed and disorderly random distribution still of HA associating PYM group cell.
During 24h, four groups of groups have all become pipe, but Control group and HA composition blood vessel rule and official jargon are more complete, and it is more thicker with tube wall that Control organizes formed nodal point number, and PYM group and the most of gathering of PYM+HA group cell are in heaps, and faint one-tenth blood vessel ability is only arranged;
During 48h, observe existing a large amount of cell deaths, become the tube wall of blood vessel to begin atrophy, Control group, HA group are still keeping certain shape of blood vessel, and PYM organizes, the atrophy of the poly-heap of PYM+HA group cell is more severe, and established tube chamber also disappears obviously.
Carrying out independent repeated experiments three times, is digit to become pipe active node (three the crossing active node that is considered as of tube wall) among every figure, quantizes laggard row relatively and analyzes, and the t check has statistical significance; * P＜0.05 is as Fig. 4.
In summary, can slightly suppress it when HA acts on HVMECs separately and become blood vessel, when it is united with PYM, than using PYM separately, can obviously suppress the one-tenth blood vessel ability of HVMECs.
The difference of apoptosis ability before and after 4.5 medicine disturbs:
The TUNEL method detects apoptosis, and normal cell nuclear is dyed blueness, and it is yellow to brown that apoptotic nucleus is.Four groups of apoptosis pictures such as Figure 12.Compare the ratio that number/whole number of cells draw of nuclear xanthochromia cell under four groups of equal visuals field of cell, carry out statistical analysis.T check analysis result shows that the Control group is compared no significant difference with the HA group, does not have statistical significance P〉0.05.PYM group and HA associating PYM group obvious difference, * * * P＜0.001 is as Fig. 5.
By above-mentioned preliminary proof, after PYM associating HA acted on the venous malformation endotheliocyte, the venous malformation endothelial cell apoptosis increased, and cell viability descends.
5. conclusion
5.1 the Bleomycin A5 of suitable concn associating hyaluronic acid than independent Bleomycin A5 can suppress more effectively the venous malformation endotheliocyte multiplication capacity, become the blood vessel ability, promote the apoptosis of venous malformation endotheliocyte.
5.2 Bleomycin A5 associating hyaluronic acid acts on the mechanism of venous malformation endotheliocyte more effectively, may be that the hyaluronic acid bag is acted on venous malformation endothelial cell surface receptor CD44 by Bleomycin A5, make Bleomycin A5 more enter cell, thereby cause the decline of endothelial cell proliferation and one-tenth blood vessel ability, apoptosis capacity strengthens.
The applicant is example surplus Bleomycin A5 associating sodium hyaluronate treatment incidence vascular malformation 50, has obtained extremely significant clinical effectiveness.Compare with the simple Bleomycin A5 of using of past, the treatment number of times obviously reduces, and therapeutic effect significantly improves.
Being that example describes with two cases, is that the position comparison sheet selected the routine patient surplus 50 is shallow, easy macroscopic patient (most lesion locations exist deeply, and naked eyes are difficult to direct observation).
Case one: the man, 60 years old, left side headache was in recent years bowed and swelling aggravation when being engaged in physical work in about 15 years of left facial part swelling.The aggravation of carrying out property of swelling and headache.CT and MRI check and find that the facial vascular malformation in left side extensively destroys sclerotin and soft tissue that clinical diagnosis is the left facial part vascular malformation.Utilize the Bleomycin A5 associating sodium hyaluronate treatment of recipe ratio of the present invention once, method is as follows: earlier Pingyang mycin (8mg) is used the 8mL physiological saline solution, obtain the Bleomycin A5 solution of 1mg/mL, extract 0.1～0.2mL solution for later use.(2mL 20mg) extracts 0.4～0.8mL in the hyaluronate sodium liquid, extracts an amount of injection normal saline then, makes cumulative volume reach 10mL, fully mixing from one again.Be percutaneously advanced into the tumor intracavity with No. 7 half a head sword-shaped needles, take out its interior blood to the greatest extent as far as possible, keep scalp acupuncture at the invariant position of tumor intracavity, result according to NMR (Nuclear Magnetic Resonance)-imaging or CT imaging inspection before the operation, pathological changes cumulative volume according to a preliminary estimate, and with reference to the blood volume of extracting out, draw the lesion volume for the treatment of under the position.The above-mentioned sclerosing agent that configures is injected in the tumor body according to this a volume of.After one month, headache and swelling complete obiteration obtain clinical recovery.
Case two: the woman, 17 years old, tongue venous malformation, extent of disease accounted for tongue body 4/5, the difficulty of remaining silent, have a strong impact on swallow, language and social communication.Utilize the Bleomycin A5 of recipe ratio of the present invention to unite sodium hyaluronate treatment 2 times, 1 time every month, each Therapeutic Method is with case one.After 2 months, obtain recovery from illness.
Though above-mentionedly by reference to the accompanying drawings the specific embodiment of the present invention is described; but be not limiting the scope of the invention; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various modifications that creative work can make or distortion still in protection scope of the present invention.
Claims (3)
1. the application of Bleomycin A5 associating hyaluronate sodium in treatment venous malformation medicine is characterized in that wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton.
2. application according to claim 1 is characterized in that, the concentration of described Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.
3. a medicine that is used for the treatment of venous malformation is characterized in that, it contains as the Bleomycin A5 of active component and hyaluronate sodium, and the medicine acceptable carrier; Wherein, the molecular weight of described hyaluronate sodium is 600,000～1,500,000 dalton, and the concentration of Bleomycin A5 and hyaluronate sodium is respectively 10 μ g/mL and 400 μ g/mL.
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| CN103800278A (en) * | 2014-02-25 | 2014-05-21 | 山东大学齐鲁医院 | Application of lauromacrogol combined hyaluronic acid in preparation of medicine for treating venous malformed foam sclerosis |
| CN114558124A (en) * | 2022-01-27 | 2022-05-31 | 中山大学附属第一医院 | Compound preparation for treating vascular malformation and preparation method and application thereof |
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