CN107308448A - Effect of the immunoglobulin in infant hemangioma course advancement - Google Patents

Effect of the immunoglobulin in infant hemangioma course advancement Download PDF

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CN107308448A
CN107308448A CN201710641447.0A CN201710641447A CN107308448A CN 107308448 A CN107308448 A CN 107308448A CN 201710641447 A CN201710641447 A CN 201710641447A CN 107308448 A CN107308448 A CN 107308448A
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igg
hemangioma
ivig
hemecs
cell
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刘少华
王逸飞
杨爱军
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Qilu Hospital of Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • G—PHYSICS
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

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Abstract

The invention discloses effect of the immunoglobulin in infant hemangioma course advancement.The present invention is studied for effect and mechanism of the immunoglobulin in infant hemangioma course advancement, by detecting the expression and distribution situation of antibody and complement membrane attack complex in different times IH, it is found that the regression of IgG C5b 9 co-deposition and IH has correlation；Tested by external pharmaceutical intervention, detect influences of the IVIg to HemECs propagation and secretion of VEGF situation, it was found that the IVIg that concentration is more than or equal to 10mg/mL can significantly inhibit HemECs propagation, laid the foundation to disclose antibody complement system with the relation that IH develops, while providing new thinking to explore treatment IH.

Description

Effect of the immunoglobulin in infant hemangioma course advancement
Technical field
The invention belongs to pharmaceutical technology field, it is related to diagnosis of the immunoglobulin in infant hemangioma course advancement With/therapeutic action.
Background technology
Infant hemangioma (Infantile hemangioma, IH) is the most common congenital benign tumour of infant, The incidence of disease is about 4%~10%, and is in raise trend year by year.The IH for betiding oromaxillo-facial region accounts for the 60% of whole body pathology, Neck surface skin, hypodermis are more common in, minority sees mucous membrane of mouth.About 10% IH growths are quick, such as not positive treatment, can There is respiratory tract obst ruction, visual impairment, ulcer, infection, bleeding, the multiple complications such as deformity, or even threat to life, to infant and Family causes serious social mentality to injure.A couple of days interior appearance after birth more than IH, Early manifestation is red fleck, with trouble The fast growing period of youngster, was typically presented fast-growth at 4 weeks and 4-5 months.Enter spontaneous regression process after 1 year old, can hold Continuous 3-8 or even longer time, majority have often left pigment deposition, scar, fibroplasia, skin not exclusively to disappear after regression Skin atrophy is sagging etc..
Advocate to carry out IH early stage active intervention in the world at present to reduce complication, conventional treatment method has a variety of, Such as surgery excision, laser therapy, hardening treating.Oral propranolol is chanced on from L é aut é-Labreze C in 2008 etc. (inderal) have treatment IH effect since, this method be increasingly becoming in recent years treat IH main method, but indication, Dispute is still suffered from terms of complication, the mechanism of action.
IH histologically show as with endothelial cells in infant hemangioma (Hemangioma endothelial cells, HemECs based on), the various kinds of cell such as fibroblast, fat cell, mast cell, pericyte and the vascular tumor deposited, HemECs directly decides IH various biological characteristicses as IH main cellular.Research shows, proliferation period IH with Based on HemECs, and cells show more active proliferation activity.And with the progress of lesion, apoptosis occurs for HemECs, and by Gradually replaced by fat cell.It is many using HemECs as research object for IH drug research at this stage, by studying medicine pair The regulatory mechanism of HemECs propagation or apoptosis, discloses IH pathogenesis, explores its potential for treating IH.
Humoral immunity is the important component of body immune system, is played a significant role in antineoplastic immune.Tumour Antigen can stimulate bone-marrow-derived lymphocyte or memory B lymphocytes Proliferation, Differentiation into thick liquid cell, and then secrete anti-tumour antibody, can be with Corresponding antigens are specifically bound, and pass through the CDCC (Complement-dependent of Complement Dependent Cytotoxity, CDC), Antibody -dependent cell cytotoxicity effect (Antibody-dependent cell-mediated Cytotoxicity, ADCC) and the opsonic action etc. of antibody play killing tumor cell effect.The research table of molecular level Bright, the molecular structure of antibody is globulin, i.e. immunoglobulin (Immunoglobulin, Ig).Humoral immunity is participated in human body Ig mainly have 5 kinds：IgG、IgA、IgM、IgE、IgD.Wherein IgG serum content highest, accounts for 75%-85%, is activation Complement system, plays the important antibody of antitumor action.IgG can pass through classical pathway (IgG1, IgG2, IgG3) or alternative route (IgG4) activating complement system, forms membrane attack complex (Membrane attack complex, MAC), plays bacteriolyze or molten The effect of cell.In tumour immunity, tumour antigen and antibody binding form antigen antibody complex, pass through complement activation Classical pathway, activates C1, C4, C2, C3, C5 successively, ultimately forms the MAC using C5b-9 as main component, forms transmembrane channel, Free in and out water and inorganic salts, K+It is excessive, a large amount of Ca2+To intracellular disperse, cell dissolving is ultimately resulted in and dead.
The research that child immune system is developed is shown, fetus an d neonate is without the B cell for producing IgG and IgA, and its is internal IgG be mainly derived from after parent, birth 3-5 months and minimize, then gradually recover, about 3-5 Sui close to adult levels.IH The course of disease develop and there is certain correlation with the maturation of infant's humoral immunity, the humoral immunity of pre-term low birth weight infant is developed more Imperfection, and its hemangioma incidence of disease is also higher.It is another there are some researches show, IH infants before 1 years old serum IgG and IgA levels compared with Normal child is relatively low.The development of complement system is relatively more complicated, and neonate is the ability that there is secretion complement, total benefit of children Body level typically can develop adult levels, but the various composition base how about one year old of complement system substantially at or so 6 monthly ages Originally reach maturity.
Human immunoglobulin(HIg) (Intravenous immunoglobulin, IVIg) used for intravenous injection is passed through by human normal plasma Isolate and purify, viral inactivation treatment is made, main component is IgG.Immunoglobulin can account for more than 95%, and each subtype distribution feelings Condition is substantially similar to internal situation, and major auxiliary burden is sorbierite or maltose.Can be clinically used for treatment primary Ig deficiency diseases, Secondary cases Ig defects are sick, spontaneous body epidemic disease etc..Recipient's serum's IgG levels can be significantly improved after venoclysis, enhancing is immune to adjust Save function.There are some researches show in Mice Body, IVIg can be by promoting NK cells to produce IL-12, and then suppresses the expansion of tumour Dissipate.By ELISA it is demonstrated experimentally that IVIg can mitigate VEGF (VEGF) mediation in lower limb ischemia mouse model Reperfusion injury.Found in the clinical research of Systemic Capillary Leak Syndrome, IVIg can reduce patients serum VEGF levels, and then improve the blood vessel endothelium high-permeability under morbid state, play therapeutic action.But be immunized in the prior art Effect and mechanism of the globulin in infant hemangioma course advancement not yet have been reported that.
The content of the invention
In view of the above-mentioned problems of the prior art, the present invention is directed to immunoglobulin in infant hemangioma course advancement In effect and mechanism studied, by detect antibody and complement membrane attack complex (MembraneAttack Complex, MAC) the expression and distribution situation in different times IH, it is found that the regression of IgG-C5b-9 co-deposition and IH has correlation；It is logical External pharmaceutical intervention experiment is crossed, influences of the detection IVIg to HemECs propagation and secretion of VEGF situation finds that concentration is more than HemECs propagation can be significantly inhibited in 10mg/mL IVIg, based on this, the present invention is inventors herein proposed.
An object of the present invention is to provide human immunoglobulin(HIg) used for intravenous injection (IVIg) in treatment infant's blood vessel Purposes in knurl.
The second object of the present invention is to provide applications of the IgG and C5b-9 in the diagnosis of infant hemangioma course advancement.
For foregoing invention purpose, specifically, the present invention provides following technical scheme：
First, treatment infant hemangioma is being prepared the invention discloses human immunoglobulin(HIg) used for intravenous injection (IVIg) Purposes in medicine.
Human immunoglobulin(HIg) (IVIg) used for intravenous injection suppresses the medicine of endothelial cells in infant hemangioma HemECs propagation preparing In purposes.
The medicine is preferred, in addition to pharmaceutic adjuvant；It is furthermore preferred that pharmaceutic adjuvant is solid-state or liquid, solid-state form Preparation include pulvis, tablet, discrete particles, capsule, pill；Wherein preferably, pulvis and tablet can include about 5% to about 95% Active component；Solid adjuvant material is selected from magnesium carbonate, magnesium stearate, talcum powder, sugar or lactose；
The preparation of liquid form includes solution, suspension and emulsion, it is preferred that liquid is selected from the parenteral injection aqueous solution Or water-propylene glycol solution, or add the oral administration solution of sweetener and contrast agent；
In addition, the medicine can be made into the small liquid drugs injection of injection, injection freeze-dried powder, big transfusion or primary infusion.
The valid density of the drug medication be 10mg/mL and more than, the valid density for reach focus internal medicine The concentration of thing.
Secondly, the invention discloses IgG and C5b-9 in the diagnostic reagent for infant hemangioma course advancement is prepared Application.
The diagnostic reagent is used for the expression for detecting IgG and C5b-9 in infantile hemangioma.
It is preferred that, the diagnostic reagent is the reagent for the co-deposition situation that immunofluorescence dyeing detects IgG and C5b-9.
The diagnosis of infant hemangioma course advancement includes：It is only a small number of intravascular red thin in propagation early stage (the 0-5 months) knurl body Cellular surface visible IgG and C5b-9 expression；Breeding visible most intravascular endotheliums in late period (the 5-12 months) knurl body, nearby IgG is positive Property expression；C5b-9 expression is less obvious；Visible IgG and the C5b-9 positive is co-deposited in paracmasis (more than December) knurl body, mainly Remaining near vessels are distributed in tumour；In all normal structures of knurl, obvious IgG and C5b-9 positive expressions are had no.
The present invention achieves following effect：
(1) in research at this stage, the research for effect of the immune system in the differentiation of the IH courses of disease is less, correlative study Mainly for immunocyte, particularly effect of the macrophage in IH angiogenesis, and have no on immunoglobulin with The research report of IH pathological development correlations.IH is combined by the present invention with immunoglobulin, by studying in different times IH IgG and complement membrane attack complex C5b-9 expression and distributions situation, effect of the research antibody-complement system in IH regression.Cross Immunofluorescence dyeing finds that C5b-9 is mainly expressed in paracmasis IH, and expresses unobvious in proliferation period IH, and IgG is disappearing Same substantially expression in phase IH, but in proliferation period IH, using 5 monthly ages for boundary, it is seen that IgG expression difference：Less than 5 Obvious IgG expression is had no in the patient of the moon, lesion；And it is more than the patient in May, in lesion near visible most intravascular endotheliums IgG is deposited；When carrying out interpretation of result, proliferation period IH is divided into propagation early stage (0-5 by expression of the present invention according to IgG again Month) and propagation late period (the 5-12 months), this causes the relation developed to IgG and C5b-9 expression with the IH courses of disease, had more Careful understanding.And the above results show, the IH course of disease develop and IgG and C5b-9 expression to there is correlation, i.e. IH quick During propagation (propagation early stage), IgG and C5b-9 expression be not obvious；Go out during IH proliferation slowed downs near (propagation late period), blood vessel endothelium Existing IgG deposition；And when IH starts to disappear, IgG and the C5b-9 positives are co-deposited.
(2) present invention further verifies IgG to endothelial cells in infant hemangioma (Hemangioma by In vitro cell experiment Endothelial cells, HemECs) effect, choose human immunoglobulin(HIg) (Intravenous used for intravenous injection Immunoglobulin, IVIg) as research object, tested and found by pharmaceutical intervention, concentration is more than or equal to 10mg/mL's IVIg can significantly inhibit HemECs propagation, while suppressing the IVIg that VEGF secretion, particularly concentration are 15mg/mL, its is right HemECs inhibited proliferation is more obvious with time lengthening, i.e., " negative growth " phenomenon occurs in cell.IVIg is treatment IH's There is provided new thinking.
(3) present invention is using IVIg as research object, and the medicine is a kind of clinically wide variety of people at this stage Immunoglobulin, the patient for being mainly used in treating immune deficiency disorder or severe infections, children security has also been obtained extensively It is general it was verified that compared with recombined human IgG, the medicine cost is relatively low, and is clinically widely used, by studying the medicine pair HemECs effect, can be to expand the potential applicability in clinical practice based theoretical wanted；Meanwhile, it is used as panimmunity globulin Mixture, actual conditions of the IVIg closer to immunity system；Meanwhile, as intravenously administrable formulation, IVIg security compared with Intramuscular injection type immunoglobulin is higher, especially for the lesion of this rich blood vessels of IH, either takes Formulations for systemic administration also It is that the local method being administered can ensure its security.
Brief description of the drawings
Fig. 1 different times IH organizes IgG, C5b-9 expression (bright place is positive expression) (a)：Breed early stage IH tissue IgG-TRITC dyes (2 months, 200 ×)；(b)：Breed the C5b-9-FITC dyeing (2 months, 200 ×) of early stage IH tissue；(c)：Increase Grow the IgG-TRITC dyeing (August, 200 ×) of late period IH tissues；(d)：Breed the C5b-9-FITC dyeing (8 of late period IH tissue Month, 200 ×)；(e)：The IgG-TRITC dyeing (1 years old August, 200 ×) of paracmasis IH tissues；(f)：Paracmasis IH tissues C5b-9-FITC dyes (1 years old August, 200 ×).It can be seen that propagation early stage IgG and C5b-9 express less, blood vessel is mainly distributed on Interior erythrocyte surface；Occur IgG deposition near propagation late period part blood vessel endothelium, but C5b-9 expression is not still obvious；Paracmasis There is IgG and C5b-9 positive expressions in IH tissues, be distributed in around remaining blood vessel.
Fig. 2 endothelial cells in infant hemangioma is separately cultured and Purification (a)：After tissue block inoculation (100 are seen under the 4th clear water surface ×), it is seen that begin with cell and climb out of adherent growth from tissue block edge, it is in irregular shape, based on polygonal, there is more projection. (b)：Tissue block is inoculated with 8 clear water surfaces and sees (100 ×), it is seen that tissue block peripheral cell dense growth, based on spindle shape, to organize To outgrowth centered on block, cell arrangement direction is consistent.(c)：Tissue block is inoculated with the 10th clear water surface and sees (100 ×)：Remove tissue Cell continued growth after block, based on spindle shape, orientation is consistent, and part cell is in polygonal.(d)CD31+Immunomagnetic beads (the 7th generation, 100 ×) is seen under cellular morphology mirror, it is seen that most cells shows are spindle shape, two ends are stretched out projection, relatively sieved after sorting Shortened before choosing, become flat, volume diminishes, projection is reduced；(e) flow cytometry identification CD31+HemECs after immunological magnetic bead sorting Purity, it is seen that cell CD31 the positive expression rates are up to more than 99% after magnetic bead sorting.
Change (a) the CCK-8 methods detection of the lower HemECs of Fig. 3 various concentrations IVIg effects propagation and VEGF secretory volumes is different Concentration IVIg acts on the change (5 multiple holes of every group of setting) that cell is bred after HemECs 24h, 48h.It can be seen that OD values are with IVIg Concentration increases and reduced, i.e., cell quantity is reduced, and cell propagation is suppressed；When reaching 10mg/ml to IVIg concentration, no matter handle 24h or 48h, experimental group OD values have obvious significant difference (P ＜ 0.05) compared with control group (MAL)；When drug concentration is During 15mg/ml, extend action time, OD values reduction (P ＜ 0.05)；It is to increase with drug concentration to drug concentration up to 20mg/ml Plus, OD value changes are not obvious (P ＞ 0.05)；(b) ELISA detects that various concentrations IVIg acts on cell after HemECs 24h, 48h The change (3 multiple holes of every group of setting, similarly hereinafter) of VEGF concentration in culture supernatant, when IVIg concentration is 5mg/mL, cell training The VEGF concentration supported in supernatant (hereinafter referred to as " supernatant ") is higher than control group (P ＜ 0.05)；When IVIg concentration is 10mg/mL When, the VEGF concentration after 24h in supernatant is acted on compared with control group without obvious significant difference (P ＞ 0.05), is acted on after 48h VEGF concentration reduces (P ＜ 0.05)；When IVIg concentration is more than 10mg/mL, the VEGF concentration in supernatant is less than control group (P ＜ 0.05).
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the present invention.Unless another Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
Term " human immunoglobulin(HIg) (Intravenous immunoglobulin, IVIg) used for intravenous injection ", by health Human plasma, through isolating and purifying, viral inactivation treatment is made, and main component is IgG, and immunoglobulin can account for more than 95%, and respectively Subtype distribution situation is substantially similar to internal situation.
" endothelial cells in infant hemangioma HemECs ", different from normal vascular endothelia cell, it is a kind of tumour cell to term, tool There is very active multiplication characteristic；Morphologically, endothelial cells in infant hemangioma shows as spindle shape, or has the polygonal of more projection Cell, cell volume is larger；And normal vascular endothelia ability of cell proliferation is not strong and shows as polygonal, cell is smaller, typically Without projection；Functionally, endothelial cells in infant hemangioma may make up capillary structure, but the function without normal blood vessels, in it Blood stays cool.HemECs directly decides IH various biological characteristicses as IH main cellular, propagation Phase IH is based on HemECs, and cells show more active proliferation activity.
Human immunoglobulin(HIg) used for intravenous injection (IVIg) is disclosed in an embodiment of the invention and is preparing treatment baby Purposes in the medicine of child's hemangioma.
Human immunoglobulin(HIg) used for intravenous injection (IVIg) is disclosed in another specific embodiment of the present invention in preparation Suppress the purposes in the medicine of endothelial cells in infant hemangioma HemECs propagation.
The medicine is preferred, in addition to pharmaceutic adjuvant；It is furthermore preferred that pharmaceutic adjuvant is solid-state or liquid, solid-state form Preparation include pulvis, tablet, discrete particles, capsule, pill；Wherein preferably, pulvis and tablet can include about 5% to about 95% Active component；Solid adjuvant material is selected from magnesium carbonate, magnesium stearate, talcum powder, sugar or lactose；
The preparation of liquid form includes solution, suspension and emulsion, it is preferred that liquid is selected from the parenteral injection aqueous solution Or water-propylene glycol solution, or add the oral administration solution of sweetener and contrast agent；
In addition, the medicine can be made into the small liquid drugs injection of injection, injection freeze-dried powder, big transfusion or primary infusion.
The valid density of the drug medication be 10mg/mL and more than, the valid density for reach focus internal medicine The concentration of thing.
Prepared in addition, disclosing IgG and C5b-9 in specific embodiments of the present invention for the infant hemangioma course of disease Application in the diagnostic reagent of progress.
The diagnostic reagent is used for the expression for detecting IgG and C5b-9 in infantile hemangioma.
It is preferred that, the diagnostic reagent is the reagent for the co-deposition situation that immunofluorescence dyeing detects IgG and C5b-9.
The diagnosis of infant hemangioma course advancement includes：It is only a small number of intravascular red thin in propagation early stage (the 0-5 months) knurl body Cellular surface visible IgG and C5b-9 expression；Breeding visible most intravascular endotheliums in late period (the 5-12 months) knurl body, nearby IgG is positive Property expression；C5b-9 expression is less obvious；Visible IgG and the C5b-9 positive is co-deposited in paracmasis (more than December) knurl body, mainly Remaining near vessels are distributed in tumour；In all normal structures of knurl, obvious IgG and C5b-9 positive expressions are had no.
Material of the present invention and reagent are as follows, not and reagent and equipment be in the art routinely to use.
1. hemangioma is cut into slices：Ratify through Ethics Committee of Shandong Qilu Hospital and inform that infant guardian knows After agreement, in the infant of Shandong University's Shandong department of stomatology, Hospital or pediatric surgery's row surgery excision between selection 2010-2016 Hemangioma case.Proliferation period (0-1 Sui) and each 10 of paracmasis (more than 1 years old).Inclusive criteria includes：1. marked according to Mulliken Quasi- pathological diagnosis is infant hemangioma；2. any other capable treatment before operative treatment；3. lesion surface is without obvious ulceration, sense Dye.Case-data refers to table 1, and the step such as fixation, dehydration, transparent, embedding of histotomy is by Shandong Qilu Hospital's pathology Section completes.
The hemangioma pathological section clinical case data of table 1
Note：* this case carries out cell culture simultaneously
2. fresh hemangioma sample：Ratify through Ethics Committee of Shandong Qilu Hospital and inform that infant guardian knows After agreement, Shandong Qilu Hospital's Oral and Maxillofacial Surgery, infant's proliferation period hemangioma of pediatric surgical operation excision are collected Fresh specimens 1, case information：Women infant, age 18 day April, shoulder hemangioma；Lesion meets Mulliken standards；Infant Without other whole body system diseases, operation can be resistant to；Any other capable treatment before operative treatment；Lesion is without infection, ulceration.
3. main agents
3.1 tissue section strain：Rabbit-anti people Glut-1 antibody is purchased from Wuhan doctor's moral company；Mouse anti human IgG antibody is purchased From Abcam companies of Britain；Rabbit-anti people C5b-9 antibody is purchased from Abcam companies of Britain；FITC mark goat anti-rabbit igg antibodies are purchased from north Mountain gold bridge company in capital；TRITC mark goat anti-mouse igg antibodies are purchased from mountain gold bridge company in Beijing；Super quick two step method immune group Change detection kit and be purchased from mountain gold bridge company in Beijing；10% sheep blood serum working solution is purchased from mountain gold bridge company in Beijing；Modified form Haematoxylin dye liquor is purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd；Anti- fluorescent quenching mountant is purchased from the green skies in Guangzhou Company；
3.2HemECs cultures, screening and identification：CD31 immunomagnetic beadses are purchased from Miltenyi companies of Germany；CD31-FITC flows Formula antibody is purchased from Affymetrix companies of the U.S.；Rabbit-anti people CD31 antibody is purchased from Wuhan doctor's moral company；Rabbit-anti people vWF antibody is purchased From Wuhan doctor's moral company；
3.3 other：Human immunoglobulin(HIg) used for intravenous injection is purchased from the safe biological products company of Shandong nation；Maltose is purchased from the U.S. Sigma companies；CCK-8 kits are purchased from Nanjing En Jing bio tech ltd；People VEGF ELISA kits are purchased from Wuhan Doctor's moral company；
Embodiment
First, test method
1. IgG and C5b-9 co-deposition situation in immunofluorescence dyeing detection different times IH
1) section is placed in 60 DEG C of thermostatic drying chambers and heats 45min, be sequentially placed into immediately after taking-up in dimethylbenzene I, II Each 2min in each 10min, 100%, 90%, 80%, 70% ethanol, distilled water；
2) it is added dropwise the 1 of 3%H2O2 and methanol:1 mixed liquor, room temperature lucifuge incubation 10min, phosphate buffer (PBS, 0.01M pH 7.2~7.3, similarly hereinafter) wash 5min × 3 time；
3) section is put into citrate buffer (TBS), puts heating in micro-wave oven and boil 30min, carry out antigen and repair It is multiple；
4) colouration box is taken out, is opened the lid, room temperature cooling 45min takes out section, PBS washes 5min × 3 time；
5) section is dried, sample is not touched, the closing of 10% sheep blood serum is added dropwise, 10min is incubated at room temperature；
6) blotting paper sops up confining liquid, does not wash, and PBS 1 is added dropwise in every slide:The rabbit anti-human igg of 100 dilutions and mouse are anti- People's C5b-9 mixing primary antibody is simultaneously put into wet box, 4 DEG C of overnight incubations；
7) PBS washes 10min × 3 time, and blotting paper blots surplus liquid；
8) PBS 1 is added dropwise:The goat anti-mouse igg mixing of the goat anti-rabbit igg and FITC marks of the TRITC marks of 100 dilutions 37 DEG C of lucifuges are incubated 1h in secondary antibody, wet box, and PBS washes 10min × 3 time；
9) core is redyed：DAPI is added dropwise, lucifuge is incubated 5min, and PBS washes 10min × 3 time；
10) liquid is blotted with blotting paper, with the mounting fluid-tight piece containing anti-fluorescence quenching, is adopted in fluorescence microscopy Microscopic observation Collect image.
2. tissue mass cell culture separation, purifying, identification HemECs
2.1 tissue block methods separate HemECs
1) the fresh proliferative phase infant hemangioma knurl body that operation is cut, after normal saline flushing, is soaked in ECM cultures In liquid, preserved in 4 DEG C of ice chests, cell culture chamber is transported in 15min；
2) sample is rinsed with a large amount of PBS, while the non-vascular tumor tissues such as blood clot, slough are removed with scissors, tweezers, Until visually observe flushing liquor color it is limpid, without muddy and slough tissue；
3) knurl body tissue is selected, is cut into small pieces with scissors and tweezers, and be dipped in 0.25% trypsase, 37 DEG C of water Bath vibration digestion 10min；
4) postdigestive knurl body tissue block is trimmed to 1mm with scissors and tweezers3/ block, is inoculated in six orifice plates, 3 pieces/ (37 DEG C, 5%CO in hole, cell culture incubator2, similarly hereinafter) and it is inverted 10min；
5) just put after six orifice plates are taken out out of incubator, adding a small amount of ECM culture mediums (penicillin containing 100U/mL, 100 μ G/mL streptomysins, 10%FBS, similarly hereinafter), nutrient solution is just moistened tissue block, do not float it, be put into cell culture incubator Overnight；
6) enough complete culture solutions are supplemented after 24h, tissue block is slowly gently submerged during addition, are sure not to float it Rise, be put into cell culture incubator and continue to cultivate；
7) daily observation cell climbs out of situation after being inoculated with, and changes nutrient solution within every 2 days, treats there are enough many cells (cells in hole When 50%) density about climb out of, tissue block is removed, changes and continues to cultivate after nutrient solution.
2.2 immunomagnetic beads methods purify HemECs
1) buffer solution is configured：PH=7.2's is free of Ca2+、Mg2+PBS add 0.5%FBS, plus 2mM EDTA, filter screen is degerming Filtering, it is -20 DEG C long-term to preserve；
2) HemECs (without screening) cell suspension, cell count, every 10 is prepared7Individual cell is (less than 107By 107Meter, under 60 μ L buffer solutions are added to be resuspended together)；
3) 20 μ L FcR sealers and 20 μ L CD31 magnetic beads are sequentially added, are gently mixed, 4 DEG C of incubation 30min；
4) 3mL buffer solutions are added, 300g centrifugation 3min abandon supernatant；
5) MS sorting posts are placed on brandreth, 500 μ L buffer solutions wash post；
6) every 107Individual cell rushes outstanding with 500 μ L buffer solutions, after screen pack, flows into sorting post；
7) 500 μ L × 3 time wash buffer pillar, collects trickle；
8) sorting post is removed from brandreth, is placed on sky centrifuge tube, rapid promotion plug after 1mL buffer solutions is added, pushes away Go out all liq, collect cell；
9) 4) -7) step is repeated, the second enriching column is crossed, cell is collected, is incubated at 25cm2In blake bottle.
2.3 flow cytometries identify HemECs purity
1) the HemECs cell suspensions after screening, row cell count are prepared
2) take containing 2 × 105The cell suspension of individual cell, is centrifuged again, is resuspended with 400 μ L PBS；
3) 2 streaming pipes are taken, respectively add 5 μ L CD31-FITC antibody, then each to add 200 μ L cell suspensions, gently vibration, often Warm lucifuge is incubated 30min；
4) 2mL PBS, 800rpm centrifugation 7min are added, supernatant is abandoned；
5) repeat to wash once, residual liquid flows back after centrifugation, and now the residual solution scale of construction is about 100 μ L in streaming pipe, whereby By the mixed Uniform of cell concussion；
6) 100-200 μ L PBS (appropriate, it is ensured that cell concentration) are added, upper machine testing in half an hour；
7) FlowJo software analysis results, mapping are used.
3.IVIg intervenes the change detection of lower HemECs propagation and VEGF amounts
3.1CCK-8 methods detect HemECs proliferative conditions
1) medicine ordinance：Be 0 according to the drug ratio compound concentration of table 2,5,10,15,20,25mg/mL IVIg；0.5g Maltose powder is added in 10mL PBS solutions, is fully mixed, aseptic filtration, prepares 5% maltose solution (MAL), -20 DEG C of guarantors Deposit, 25mg/mL is diluted to isometric ECM using preceding, it is now with the current；
The various concentrations IVIg of table 2 preparation (unit：mL)
Note：This table is preparation each acute drug consumptions of 10mL
2) plate is planted：Exponential phase HemECs is collected, is seeded to the density in 500/hole in 96 orifice plates, every group of setting is answered Hole × 5, the μ L of ECM 100 are added per hole, and holes around adds isometric PBS, 37 DEG C of culture 24h；
3) dosing：Suction out culture medium, PBS washes 2 times, be separately added into 0,5,10,15,20,25mg/mLIVIg and 25mg/mL Maltose solution (150 μ L/ holes), is placed in incubator and continues to cultivate；
4) after 24h or 48h, original culture medium is absorbed, PBS is washed 2 times, 100 μ L ECM and 10 μ L CCK-8 is added per hole；
5) culture plate is placed in incubator and is incubated 4h；
6) absorbance at 450nm is determined with ELIASA.
3.2ELISA methods detect the changes of contents of HemECs secretion of VEGF
3.2.1 the preparation of reagent
1) take 1mL sample diluting liquids to add in standard QC (10ng recombinant human VEGFs), 10min is stood after covering, then Overturn repeatedly, rubbing；
2) taking 0.2mL 10000pg/mL standard items to add has in the Ep pipes of 0.8mL sample diluting liquids, mixes, makes marks；
3) prepare 6 Ep pipe, often pipe plus 0.3mL sample diluting liquids, mark upper 1000 respectively, 500,250,125,62.5, 31.2、15.6pg/mL.Take 0.3mL 2000pg/mL standard items to add in mark 1000pg/mL pipe, equally taken out after mixing 0.3mL is added in next pipe.By that analogy, to last sample cell.
4) 100 μ L are needed to calculate total consumption according to per hole.Add the μ L of antibody diluent 99 by the 1 anti-human VEGF of μ L biotin labelings Proportions working solution, gently mix.
5) 100 μ L are needed to calculate total consumption according to per hole.By 1 μ L Avidins-peroxydase complex (ABC) plus ABC The μ L of dilution 99 proportions working solution, is gently mixed.
3.2.2 operation sequence
1) medicine ordinance：Be 5 according to the drug ratio compound concentration of subordinate list 2,10,15,20mg/mL IVIg；25mg/mL Maltose solution compound method is with experiment 5；
2) plate is planted：Exponential phase HemECs is collected, is seeded to the density in 10000/hole in 12 orifice plates, is added per hole ECM 1mL, 37 DEG C of culture 24h；
3) dosing：Suction out culture medium, PBS washes 2 times, be separately added into 5,10,15,20mg/mL IVIg and 25mg/mL malt Sugared (500 μ L/ holes), is placed in incubator and continues to cultivate；
4) cell culture supernatant (hereinafter referred to as " supernatant ") is collected after 24h, 48h, 2500rpm centrifugation 10min take Clearly；
5) by after 1000,500,250,125,62.5,31.2,15.6pg/mL standard items and drug-treated 24h, 48h Supernatant (multiple holes × 3, random pair) is sequentially added in ELISA Plate, and 1 hole only adds sample diluting liquid as zero hole,.Per hole liquid feeding Amount is 100 μ L.ELISA Plate adds shrouding film, 37 DEG C of reaction 90min；
6) liquid in ELISA Plate is got rid of after reacting, under opposite blotting paper bat is several, is not washed；
7) biotin labeling anti-human VEGF antibody working solution is sequentially added by every μ L of hole 100 and (removed in TMB blank colour developing hole Outside).ELISA Plate adds shrouding film, 37 DEG C of reaction 60min；
8) 1 × lavation buffer solution is washed 3 times, every time immersion 1min or so (per hole washing lotion at least 300 μ L).
9) ready ABC working solutions are sequentially added (TMB develops the color except hole) by every μ L of hole 100.ELISA Plate is plus envelope Plate film, 37 DEG C of reaction 30min；
10) 1 × lavation buffer solution is washed 5 times, every time immersion 1-2min or so (per hole washing lotion at least 300 μ L)；
11) the TMB nitrite ions for balancing 30min at 37 DEG C are sequentially added by every μ L of hole 90,37 DEG C of lucifuges react 20- 25min (there is obvious gradient blueness the time with the preceding 3-4 holes of naked eyes visual standard product, and rear 3-4 holes difference is not substantially defined)；
12) TMB terminate liquids are sequentially added by every μ L of hole 100, order is the same, and now blueness switchs to yellow；
13) OD values are determined in 450nm with ELIASA, it is final result to subtract TMB colour developing hole OD values；
14) Microsoft Excel draw standard curve, calculate regression equation, are calculated according to OD values in respective sample VEGF concentration；
15) amount (the Δ C of the lower HemECs 24h secretion of VEGF of various concentrations IVIg effects is calculatedVEGF=CVEGF-48- CVEGF-24).
Table 3ELISA standard curves
Standard curve regression equation：Y=181x2+ 231.6x+2.939, R2=0.998
2nd, result of the test
1. immunofluorescence dyeing is found：
Only a small number of intravascular erythrocyte surface visible IgG and C5b-9 expression is (attached in 1.1 propagation early stage (the 0-5 months) knurl bodies Fig. 1 a, b).
IgG positive expressions near visible most intravascular endotheliums in 1.2 propagation late period (the 5-12 months) knurl bodies；C5b-9 is expressed Less obvious, only a small number of blood vessels are visible positive (accompanying drawing 1c, d).
Visible IgG and the C5b-9 positive is co-deposited in 1.3 paracmasises (more than December) knurl body, is mainly distributed on residual in tumour Deposit near vessels (accompanying drawing 1e, f).
2. tissue mass cell culture separation, purifying, identification HemECs：
2.1 tissue block methods separate HemECs：After hemangioma block adhere-wall culture, typically begun with the 3rd day cell from Tissue block edge climbs out of adherent growth, in irregular shape, more in polygonal, there is more projection (accompanying drawing 2a)；Increase with cell density Plus, cell elongation, cell density can reach 50% after about 7 days, and mobile (accompanying drawing 2b) towards the direction propagation away from tissue block； Remove cell near tissue block, cell continued growth, about 10 days or so basic stitch blocks and be paved with bottom hole, cell is in spindle shape, growth Direction is consistent, is passed on (accompanying drawing 2c).2.2 immunomagnetic beads methods purify HemSCs：, immunomagnetic beads method be successfully separated CD31+ HemECs, the cell isolated is in polygonal or spindle shape, has longer projection to stretch out, and propagation is rapid (accompanying drawing 2d).Fluidic cell Art identifies HemECs purity up to more than 99% (accompanying drawing 2e).
3.IVIg intervenes the change detection of lower HemECs propagation and VEGF amounts
3.1CCK-8 methods detection IVIg intervenes lower HemECs proliferative conditions：OD values increase and reduced with IVIg concentration.Extremely IVIg concentration is more than or equal to 10mg/ml, and OD values have obvious significant difference (P ＜ 0.05) compared with control group (MAL)；Work as medicine When concentration is 15mg/ml, OD values are reduced (P ＜ 0.05) with extended durations of action；To drug concentration reach 20mg/ml, OD values with Drug concentration increase reduction is not obvious (P ＞ 0.05).
3.2ELISA methods detection IVIg intervenes the change of lower HemECs secretion of VEGF：IVIg act on HemECs 24h or After 48h, increase with drug concentration, VEGF concentration reduction (P ＜ 0.05) in cell culture supernatant (hereinafter referred to as " supernatant "). When IVIg concentration be less than 10mg/mL when, in VEGF concentration be higher than control group (P ＜ 0.05)；When IVIg concentration is more than 10mg/mL When, the VEGF concentration in supernatant is less than control group (P ＜ 0.05)；When IVIg concentration is 10mg/mL, supernatant after effect 24h VEGF concentration in liquid, without obvious significant difference (P ＞ 0.05), acts on VEGF concentration reduction (P ＜ after 48h compared with control group 0.05)。
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (10)

1. purposes of the human immunoglobulin(HIg) (IVIg) used for intravenous injection in the medicine for preparing treatment infant hemangioma.
2. human immunoglobulin(HIg) (IVIg) used for intravenous injection is in the medicine for suppressing endothelial cells in infant hemangioma HemECs propagation is prepared Purposes.
3. purposes according to claim 1 or 2, it is characterised in that the medicine also includes pharmaceutic adjuvant；
It is preferred that, pharmaceutic adjuvant is solid-state or liquid；
The preparation of solid-state form is selected from pulvis, tablet, discrete particles, capsule, pill；Wherein preferably, pulvis and tablet can be included The active component of about 5% to about 95%；
Solid adjuvant material is selected from magnesium carbonate, magnesium stearate, talcum powder, sugar or lactose；
The preparation of liquid form is selected from solution, suspension and emulsion；
It is preferred that, liquid is selected from the parenteral injection aqueous solution or water-propylene glycol solution, or adds the mouth of sweetener and contrast agent Take solution；
The small liquid drugs injection of injection, injection freeze-dried powder, big transfusion or primary infusion is made in the medicine.
4. purposes according to claim 1 or 2, it is characterised in that the valid density of the drug medication be 10mg/mL and More than；It is preferred that, the valid density of the drug medication for 15mg/mL and more than.
Applications of the 5.IgG and C5b-9 in the diagnostic reagent for infant hemangioma course advancement is prepared.
6. application according to claim 5, it is characterised in that the diagnostic reagent is used to detect infantile hemangioma Middle IgG and C5b-9 expression.
7. application according to claim 6, it is characterised in that the diagnostic reagent be immunofluorescence dyeing detection IgG and The reagent of C5b-9 co-deposition situation.
8. the application according to claim 5 or 6, it is characterised in that the diagnosis of infant hemangioma course advancement includes：Increase Grow month early stage 0-5, only a small number of intravascular erythrocyte surface visible IgG and C5b-9 expression in knurl body；In all normal structures of knurl, Have no obvious IgG and C5b-9 positive expressions.
9. the application according to claim 5 or 6, it is characterised in that the diagnosis of infant hemangioma course advancement includes：Increase Grow the month in late period 5-12, IgG positive expressions near visible most intravascular endotheliums in knurl body；C5b-9 expression is less obvious；Knurl is straight Often in tissue, obvious IgG and C5b-9 positive expressions are had no.
10. the application according to claim 5 or 6, it is characterised in that the diagnosis of infant hemangioma course advancement includes： More than December paracmasis, visible IgG and the C5b-9 positive is co-deposited in knurl body, is mainly distributed in tumour remaining near vessels；Knurl In all normal structures, obvious IgG and C5b-9 positive expressions are had no.
CN201710641447.0A 2017-07-31 2017-07-31 Effect of the immunoglobulin in infant hemangioma course advancement Pending CN107308448A (en)

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