CN104188950B - The purposes of chlorogenic acid in the medicine of preparation treatment ependymoma - Google Patents
The purposes of chlorogenic acid in the medicine of preparation treatment ependymoma Download PDFInfo
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Abstract
The invention provides the purposes of chlorogenic acid in the medicine of preparation treatment ependymoma. Chlorogenic acid of the present invention has the effect that suppresses ependymoma, and can suppress the growth of brain Tumor Stem Cells, energy Substitute For Partial chemicotherapy, and alleviating patient can react because of the discomfort that chemicotherapy causes, is new class of medications and the selection for the treatment of ependymoma.
Description
Technical field
The present invention relates to the purposes of chlorogenic acid in the medicine of preparation treatment ependymoma.
Background technology
In recent years, the death rate of cancer has leapt to first of the domestic various cause of the death. May be old with smoking, populationAge, industrialized process etc. are relevant. Traditional be poorly off regional kinds of tumor as the cancer of the esophagus, cancer of the stomach,The incidences of disease such as liver cancer are still high, and the multiple tumours such as the lung cancer of rich countries, breast cancer, colon cancerBut rapid growth. Latest survey demonstration, the incidence of disease of developed country's brain tumor constantly rises, and this disease is alsoShow the trend that significantly becomes younger, approach gradually 40 years old following young people, and become threat children's healthOne of important tumour. According to U.S. statistics, in 20-40 year crowd tumor mortality cause of disease, brain tumor existsThe male sex is primary Death causes, ranked fifth position in women, and visible brain tumor is big to social harm.At present, brain tumor is still one of adult's tumour that case fatality rate is the highest, is also that child morbidity is the highest simultaneouslySolid tumor.
Glioma also claims spongiocytoma, is the common a kind of malignant tumour of central nervous system, comesCome from neural epithelium and account for 40%～50% of whole ICTs, there is high incidence, high relapse rate, high sickThe feature of dead rate and low cure rate. Be divided into astrocytoma, few prominent according to the differentiation situation of glioma cellGlioma, ependymoma etc., its growth characteristic is infiltrative growth, with normal cerebral tissue without obvious boundary,Majority is not limited to a cerebral lobe, is finger-like and deeply destroys brain tissue outside brain tissue. Current gliomaPrimary treatment measure is operation excision, but postoperative 5 annual survival rates are very low, all insensitive to radiation and chemotherapy,Therefore, be badly in need of the bio-pharmaceutical of exploitation treatment glioma.
The method of brain tumor classification is a lot, there is no at present unified sorting technique, and the group of various tumoursHair-weaving is raw different from pathological characters, and it is optimum and pernicious and thing characteristic is also different. And astrocyteKnurl and mesoglioma, ependymoma all there are differences in many-side, and concrete difference is as follows:
1. astrocytoma refers to the one of the glioma being made up of astroglia, is nervous systemModal intractable tumour, accounts for 13%～26% of ICT, account for 21.2% of neural epithelium tumour～51.6%. Wherein glioma glioblastoma is the modal primary brain tumors of adult, in its patientPosition only has about 15 months life cycle. Cutose cytoma presents infiltrative growth, can be to normal cerebral tissueExtensively infiltrate, cause operation excision difficulty; Secondly, glioblastoma has conventional Radiotherapy chemotherapyRepellence, becomes its fatal rate height and high major reason of recurrence rate. Owing to illustrating its morbidity not yet completelyMechanism, lacks the effective methods for the treatment of for Gliblastoma at present. Neuroastrocytoma is with fewProminent spongiocytoma there are differences at the expression of RTN4, and in oligodendroglioma, RTN4A expressesApparently higher than neuroastrocytoma, in order to differentiate specificity and the sensitivity of oligodendroglioma.
2. oligodendroglia tumour is a kind of more rare Neuro-epithelial tumor, and mesoglioma is mainly in intoYear people, average age of onset is about 40 years old. Past is because of the restriction of inspection technology and pathological diagnosis level,Often failed to pinpoint a disease in diagnosis or mistaken diagnosis is other types glioma, previous literature thinks that oligodendroglia tumour accounts for intracranial primary and sends out2%～5% of property tumour, accounts for 2%～12% of glioma, but in recent years along with clinical research deepens continuouslyAnd pathological diagnosis level improves constantly, oligodendroglia tumour recall rate obviously increases, and has bibliographical information fewProminent glia tumour accounts for 33% of Intracranial Gliomas. Oligodendroglioma is that encephalic originates from oligodendrogliaThe tumour of cell, is the independent type of one of glioma, accounts for 4.39% of ICT. In the last few years,The molecule genetics research of oligodendroglioma has had larger progress, and oligodendroglioma is the most commonScience of heredity change be No. 19 long-armed loss of heterozygosities of chromosome, second common hereditary change is 1The loss of heterozygosity of number the short arm of a chromosome. According to the up-to-date classification pathology of the World Health Organization (WHO) in 2007Classification, is divided into simple form oligodendroglioma and mixed type prominent astrocytoma less by oligodendroglia tumour,The former can be divided into again low level oligodendroglioma (II level), high-level oligodendroglioma (III level)And glioblastoma multiforme (IV level); It is prominent less that the latter is divided into less prominent astrocytoma (II level), a modificationAstrocytoma (III level). Research in recent years shows, low-level oligodendroglia tumour is more responsive to chemotherapy,And its Clinical Outcome of oligodendroglia tumour and the prognosis of different stage have notable difference.
3. to derive from the ependymocyte of the ventricles of the brain and myelocoele or brain white matter endyma thin for ependymomaThe central nerve neuroma of born of the same parents' nest. In glioma, account for 18.2%, man more than female, be more common in children andYouth, approximately 75% is positioned under curtain, on curtain, only accounts for 25%. Tumour is positioned at the ventricles of the brain mostly, and minority knurl main body existsIn brain tissue. Ependymoma is a kind of Neuro-epithelial tumor that originates from ependymal epithelium cell, can send outBe born in encephalic and canalis spinalis, and can send out along cavum subarachnoidale, be also difficult at present cure completely. Different sickThe glioma of reason type respectively has its age occurred frequently, and the age occurred frequently of ependymoma is before 10 years old. External systemMeter 96% spinal ependymoma occurs in adult, and age of onset mostly is 35～45 years old, be grown up the most commonTumor of spinal cord, account for greatly 34.5% of all central nervous system ependymomas, account for 60% of tumour in marrow,Account for 75% of adult's ependymoma. Research is found, in ependymoma, more than 50% is had chromosome No. 22The loss of fragment, and find that the relation of SV40 gene and ependymoma is comparatively close. WHO2000New classification is divided into Ependymoma tumor: (1) ependymoma: 1. cellular type of 4 hypotypes of classifying again; 2. nippleType; 3. clear cell type; 4. tanycytes type. (2) between, become or pernicious ependymoma: tumour cell causesClose cell and nuclear morphology are different in flakes, and Visible Core division phase and focal necrosis. (3) mucus nipple type chamberPeriosteum knurl: tumour cell mamillary is arranged, and often has mucoid around the connective tissue at [Dan centerBecome. (4) Subependymoma: the main cell that forms tumour is spongiocyte under endyma, visible false chrysanthemum shapeGroup's sample is arranged. Sometimes visible a small amount of ependymocyte and endyma mother cell are distributed between gtelatinous fibre.Ependymoma tumor is to the insensitive tumour of chemotherapy, in clinical testing, the independent use of various chemotherapeutic orCombine to use and produce little effect.
Gliomatous treatment is taking operation as main, but because its differentiation is poor, breed soon, aggressive is strong,Also tumor resection completely of current modus operandi, generally all advocates complex treatment. Hand postoperation radiotherapy andChemotherapy is generally accepted methods for the treatment of, but tumour cell may cause residual to the radiation tolerance of radiotherapyRemaining focus recurs again. In addition, brain tumor is all not too responsive to chemotherapy, and one of reason is to only have minority medicineThing can pass through blood-brain barrier. Because in blood-brain barrier, tumor tissues and periphery brain tissues of edema gap is quietThe more high factor of hydraulic pressure causes the valid density of Intratumoral chemotherapy medicine lower. In addition, drug resistance of tumorThe factors such as the bad reaction of generation, systemic administration all impact the result for the treatment of of chemotherapy. In order to improveThe result for the treatment of of glioma, people have done to new clinical treatment means from the Molecular pathogenesis of gliomaA large amount of work.
Chlorogenic acid is extensively present in various medicinal plants, in honeysuckle, at present its chemical constitution is groundStudy carefully clearly, existing people carries out medicinal research to it, has reported that chlorogenic acid can be applied to treatment tumourEtc. disease. At present the document that is used for the treatment of glioblastoma about chlorogenic acid of report is mainly from effectMechanism is set forth, as document: Thechemopreventivepropertiesofchlorogenicacidrevealapotentialnewroleforthemicrosomalglucose-6-phosphate(the chemoprophylaxis characteristic of chlorogenic acid has disclosed translocaseinbraintumorprogressionIt is the new role to G-6-P transferase in brain tumor progress), report that glioma is thinThe adjustable intracellular signaling pathway of G-6-P transferase (G6PT) in born of the same parents' strain (U87), is high tableReach state, closely related with the wellability of tumour cell; Chlorogenic acid can resist the U87 cell of G6PT inductionMigration, chlorogenic acid can obviously suppress the U87 cell migration of sphingosine (SIP) induction. Confirm chlorogenic acidBy suppressing matrix metalloprotease transferase and G-6-P transferase active, reach inhibition tumour and turnThe effect of moving. Document: InhibitionofMMP-2secretionfrombraintumorcellssuggestschemopreventivepropertiesofafuranocoumaringlycosideandOfchalconesisolatedfromthetwigsofDorsteniaturbinata (furans perfume (or spice)Legumin glycosides and chalcone have the secretion activity that suppresses MMP-2 in brain tumor cell) ", reportChlorogenic acid can suppress the secretion of MMP-2 as the inhibitor of matrix metalloprotease transferase, reach inhibitionThe transfer of glioblastoma cells in brain tumor. Chlorogenic acid is G6PT inhibitor. Side light chlorogenic acidAnti-brain tumor effect. But the carcinogen that the G6PT that document is set forth is broad sense, the brain tumor relating toAlso be a generalized concept, the direct effect of chlorogenic acid to brain tumor can not be described. Wang Le, from acer truncatumThe technical study of extraction, separation and purification chlorogenic acid in leaf, Northwest University, master thesis 20100401,Report the anticancer anti-aging effects of chlorogenic acid, wherein disclose chlorogenic acid and can suppress gliomatous migrationAnd the secretion of metallothionein (MMP) in people Hep3B HCC, to colorectal cancer, liver cancer and laryngocarcinomaAll there is significant curative effect, be considered to effective protective agent of cancer.
In sum, the chlorogenic acid that existing document all discloses is confined to star on the one hand to the effect of brain tumorGlioblastoma (elaboration of mechanism of action aspect) in cytoma, does not have chlorogenic acid treatment brain colloid female thinIn the body of born of the same parents' knurl, the test of pesticide effectiveness proves, there is no the relevant report of chlorogenic acid treatment ependymoma, does not also have greenOrtho acid is about there being the relevant report that suppresses human brain glioma stem cells effect.
Summary of the invention
Technical scheme of the present invention has been to provide the new purposes of chlorogenic acid.
The purposes of chlorogenic acid of the present invention in the medicine of preparation treatment ependymoma.
Wherein, described medicine is to the inhibiting medicine of transplantability glioma.
Wherein, described medicine is the medicine that C6 glioma cell is had to growth inhibition effect.
Wherein, described medicine is the medicine that C6 glioma stem cells is had to growth inhibition effect.
Wherein, described medicine be chlorogenic acid taking effective dose as active component, add pharmaceutically acceptableAuxiliary material or the preparation that is prepared from of complementary composition.
Wherein, chlorogenic acid 1～3000mg is contained in described pharmaceutical preparation Zhong Mei preparation unit.
Further, the using dosage of described pharmaceutical preparation Content of Chlorogenic Acid is 10-40mg/kg.
The using dosage of still more preferably, described pharmaceutical preparation Content of Chlorogenic Acid is 20mg/kg.
Wherein, described medicament is oral formulations or injection.
Although bibliographical information the chlorogenic acid undue expression that can resist recombinant G6PT albumen in testing in vitroThe transfer of the U-87 glioblastoma of bringing out, only illustrated chlorogenic acid in brain tumor progress to PortugalThe effect of grape sugar-6-phosphotransferase, does not directly prove that chlorogenic acid has anti-U-87 glioblastomaEffect; And chlorogenic acid suppresses the transfer of U-87 people's glioblastoma, is to belong to glioblastoma, butGlioblastoma and oligodendroglioma, ependymoma are the dissimilar of glioma, are differentIndication. The chlorogenic acid that therefore, can not push away has the effect that suppresses oligodendroglioma and ependymoma.
Glioblastoma and oligodendroglioma, ependymoma is in teiology, histopathology and faceThere is obvious difference bed aspect. Ependymoma is a kind of neural epithelium that originates from ependymal epithelium cellProperty tumour, can betide in encephalic and canalis spinalis, and can send out along cavum subarachnoidale, be also difficult at present completelyCure. The spinal ependymoma of foreign statistic 96% occurs in adult, and age of onset mostly is 35～45 years old, isThe modal tumor of spinal cord of being grown up, accounts for greatly 34.5% of all central nervous system ependymomas, accounts in marrow60% of tumour, accounts for 75% of adult's ependymoma. The up-to-date classification of WHO2000 is divided into Ependymoma tumor:(1) ependymoma: 1. cellular type of 4 hypotypes of classifying again; 2. nipple type; 3. clear cell type; 4. ependymaCellular type. (2) between, become or pernicious ependymoma: tumour cell is in flakes fine and close, and cell and nuclear morphology are different,And Visible Core division phase and focal necrosis. (3) mucus nipple type ependymoma: tumour cell mamillary is arranged,Often there is mucoid to become around the connective tissue at [Dan center. (4) Subependymoma: formation tumourMain cell is spongiocyte under endyma, and visible pseudorosette sample is arranged. Sometimes visible a small amount of endymaCell and endyma mother cell are distributed between gtelatinous fibre. At present for the chamber pipe that does not have cerebrospinal fluid to send outThe therapeutic modality of film knurl is as far as possible entirely cut and is aided with after surgery local radiation treatment for operation can provide bestTreatment effect, but be that traditional combined chemotherapy or HDC seem all can not improve prognosis,Although part test shows that tumour is to chemosensitivity.
Oligodendroglia tumour is a kind of more rare Neuro-epithelial tumor, and previous literature is thought oligodendrogliaTumour accounts for 2%～5% of intracranial primary tumour, accounts for 2%～12% of glioma, but in recent years along with clinicalResearch deepens continuously and pathological diagnosis level improves constantly, and oligodendroglia tumour recall rate obviously increases,There is bibliographical information oligodendroglia tumour to account for 33% of Intracranial Gliomas. According to the World Health Organization in 2007(WHO) up-to-date classification pathological classification, is divided into simple form oligodendroglioma and mixing by oligodendroglia tumourType is prominent astrocytoma less, and the former can be divided into again low level oligodendroglioma (II level), high-level fewProminent spongiocytoma (III level) and glioblastoma multiforme (IV level); The latter is divided into less prominent astrocytoma(II level), a modification be prominent astrocytoma (III level) less. Research in recent years shows, oligodendroglia tumour is to changingTreat more responsively, and its Clinical Outcome of oligodendroglia tumour and the prognosis of different stage have notable difference.
Chlorogenic acid of the present invention has the effect that suppresses oligodendroglioma, and can suppress brain Tumor Stem CellsGrowth, can Substitute For Partial chemicotherapy, alleviate the discomfort reaction that patient can cause because of chemicotherapy, be to controlTreat new class of medications and the selection of ependymoma.
Brief description of the drawings
The dose-effect curve of Fig. 1 chlorogenic acid to human brain ependymoma
Detailed description of the invention
The experimental study of the thin apoptosis of test example 1 chlorogenic acid induction people's ependymoma
1. material
Cell: people's ependymoma cell is transplanted engineering and trnasplantion immunity weight by the Huaxi Hospital Attached to Sichuan Univ Ministry of Public HealthPoint laboratory provides.
Medicine and reagent: chlorogenic acid, by Sichuan, Jiu Zhang bio tech ltd provides; Glutamic acid is purchased from ZhejiangThe auspicious Chemical Co., Ltd. of the sky over the river; Tetramethyl azo azoles salt (MTT), 0.25% trypsase, RPMI1640 trainingNutrient solution and hyclone are all purchased from Sigma company of the U.S..
2. method
The former culture of 2.1 human brain ependymoma cells
(1) former culture
Fresh human brain ependymoma tumor tissue piece is rinsed well with the RPMI1640 nutrient solution that does not contain serum,Remove blood stains, cut into 1mm3The fritter of size, then wash 2～3 times with the nutrient solution of serum-free, extremelyLiquid clarification. Discard nutrient solution, add 0.25% trypsase of 5～10 times of volumes, 37 DEG C of water-bathsMiddle digestion 30min, 100 hole order steel meshes filter, and are prepared into tumour cell suspension, move into centrifuge tube with 1500～Supernatant discarded after the centrifugal 10min of 2000r/min. Not contain the resuspended precipitation of nutrient solution of calf serum, pressAfter centrifugal 2 times of same time of same speed, remove supernatant. Cultivate with the RPMI1640 containing 10% calf serumLiquid re-suspended cell, CO2In incubator, hatch for 37 DEG C.
(2) going down to posterity of primary cell
After at the bottom of cell covers with bottle, remove old nutrient solution, wash 1 time with the RPMI1640 nutrient solution of serum-free.Add 0.25% the about 5min of trypsinization, with stopping digestion containing serum free culture system liquid. Piping and druming repeatedly, collectsCell, counting, by 5 × 104Individual/cm2Density be inoculated in 25cm2The cultivation of going down to posterity in blake bottle.
(3) morphological observation of human brain ependymoma cell
The cell of former culture carries out morphological observation at inverted phase contrast microscope. Contain to adding in cultured cellThere is the 1mLHank ' s of 5 μ mol/LFluo-3/AM fluorescence probes and 5 μ mol/LPluronicF-127Liquid, 37 DEG C of load 40min. With not containing Hank ' the s liquid washing of fluorescence probe 3 times. Good thin of loadCytosol, at the state of laser confocal scanning microscope observation of cell, carries out with former surgery Pathology check resultContrast, confirms auxocyte behaviour ventricles of the brain periosteum oncocyte. Cultured cell is again through GFAPSABC detects to be confirmed.
(4) mtt assay detects the inhibitory action of chlorogenic acid to the cell proliferation of former culture human brain ependymoma
Cell inoculation: the cell in growth period of taking the logarithm, 0.25% Trypsin Induced, washs with basal mediumCentrifugal (twice of 1000rpm5min), with complete medium suspension cell and adjust cell concentration and be 6 ×104/ml, inoculating cell in 96 orifice plates, every hole 50 μ l (cell quantity 6 × 103/ holes), each medicine3 multiple holes of substrate concentration inoculation, and establish Normal group (tumour cell+complete medium) and blankGroup (complete medium), 3 every group multiple holes.
Dosing: after inoculation, press following grouping dosing next day after cell attachment, and chlorogenic acid group, with cultivating completelyBase is mixed with 6.4mg/ml chlorogenic acid storage solutions the solution of 256 μ g/ml, then uses complete mediumProgressively dilute by two-fold dilution's method, must contain the complete medium of chlorogenic acid 256,128,64 μ g/ml, standbyWith; Get the above-mentioned complete medium containing variable concentrations chlorogenic acid of 50 μ l in 96 orifice plates of inoculating cell,Make chlorogenic acid final concentration be respectively 128,64,32 μ g/ml, the multiple hole of each concentration 3. Leave standstill and cultivate 48Hour; Blank group, not inoculating cell of every hole, (l) complete medium of 100 μ, establishes only to add equivalent3 multiple holes are as blank.
Measure: after dosing, cell is cultivated 48 hours, in above-mentioned 3 experimental group and blank group hole, addsEnter 10 μ lWST-1 solution, put 37 DEG C of cell culture incubators and continue to hatch after 4 hours, micro-at μ-QuantOn orifice plate analyzer, measure the suction at different pharmaceutical group, Normal group and blank group 440nm wavelength placeReceipts value.
Data processing: the calculating of growth of tumour cell inhibiting rate: inhibiting rate=(control group A value-experimental group AValue)/control group A value × 100%. Experiment repeats 3 times.
3. experimental result
The external tumor-inhibiting action of 3.1 chlorogenic acids to human brain ependymoma
(1) cellular morphology observed result
The dosing of each group was processed after 48 hours, and observation of cell form under inverted microscope, with Normal group ratio, there is cell quantity compared with normal cell obviously to reduce when chlorogenic acid (32～128 μ g/ml) low concentration;There is cell fragment, produce apoptosis.
(2) inhibiting rate and the dose-effect curve of chlorogenic acid to human brain ependymoma
Chlorogenic acid is to the inhibiting rate of human brain ependymoma in table 1, and dose-effect curve is shown in Fig. 1.
The inhibiting rate of table 1 chlorogenic acid to human brain ependymoma
Chlorogenic acid after 32,64 and 128 μ g/ml effect 48h to the inhibiting rate curve of primary cultured cell asShown in Fig. 1. Along with the increase of chlorogenic acid activity, the suppressed effect of ependymoma cell is more remarkable.Visible, chlorogenic acid is obvious dose-effect relationship to the growth inhibition of former culture human brain ependymoma cell.
4. conclusion
The external tumor suppression of chlorogenic acid experiment showed, that chlorogenic acid suffers from brain ependymoma to people and have inhibitory action. Above-mentioned examinationVerify brightly, chlorogenic acid of the present invention has inhibition effect to human brain ependymoma, can Substitute For Partial chemicotherapy, subtractLight patient can react because of the discomfort that chemicotherapy causes, is the new tool for the treatment of human brain ependymoma.
Test example 2 chlorogenic acids are to the inhibiting research of transplantability glioma
1. material
Material: human glioma cell line, exempted from by Huaxi Hospital Attached to Sichuan Univ Ministry of Public Health transplanting engineering and transplantingEpidemic disease key lab provides. Chlorogenic acid, by Sichuan, Jiu Zhang bio tech ltd provides.
2. method
(1) 50 mouse of modeling are divided into 5 groups at random, 10 every group. Gliblastoma cell line is joinedThan suitable dilution, be inoculated in respectively in 5 groups of left cortex of temporal lobe of mouse.
(2) after administration intervention inoculation 24h, respectively 5 groups of mouse are carried out to intraperitoneal injection. Be respectively greenOrtho acid high (40mg/kg), in (20mg/kg), low (10mg/kg) dosage group, positive controls docetaxel5mg/kg, model control group is to the physiological saline of same volume, successive administration 15d.
(3) survey and last day of tumour inhibiting rate stop administration and all mouse are put to death, dissect mouse, get tumourOrganize and weigh. The heavy control group of inhibiting rate=(the average knurl weight of the average knurl weight-treatment group of control group)/knurl is flatAll knurls heavy × 100%.
(4) all data of statistical method all adopt mean ± standard deviationRepresent, data analysis adoptsStatistics program software kit (SPSS13.0forWindows) carries out variance analysis, taking P < 0.05 as differenceThere is conspicuousness.
3. result
Found that by analysis, administration group is organized its tumour inhibiting rate with respect to blank the situation of remarkable lifting, cardBright chlorogenic acid has effect of neoplasm growth, and its tumor killing effect is the most obvious with middle dosage group, high dose groupTake second place, concrete data are asked for an interview table 2.
The impact of table 2 chlorogenic acid on human glioma mouse tumor weight and tumour inhibiting rate
4. conclusion
Tumor suppression experiment showed, that chlorogenic acid suffers from glioma to people and have inhibitory action in chlorogenic acid body. Above-mentioned examinationVerify brightly, chlorogenic acid of the present invention has inhibition effect to human glioma, can Substitute For Partial chemicotherapy, alleviatePatient can react because of the discomfort that chemicotherapy causes, is the new tool for the treatment of human glioma.
The experiment in vitro research of test example 3 chlorogenic acids to the effect of C6 glioma stem cells
One, medicine and main agents consumptive material
1. chlorogenic acid powder-injection, purity 99%, by Sichuan, Jiu Zhang bio tech ltd provides, moleculeAmount 354.
2. key instrument: superclean bench, CO2Constant temperature and humidity incubator, all size micro sample adding appliance,Electrical distiller, quartzy redistilled water device, culture vessel, light microscope, ultra low temperature freezer, sizeCentrifuge, electric heating pressure steam sterilizer, constant temperature roaster, flow cytometer, micro-electronic balance etc.
3. cell line: C6 rat Glioma cells system for experiment, the Ministry of Public Health moves by Huaxi Hospital Attached to Sichuan UnivPlant engineering and trnasplantion immunity key lab provides.
Two, test procedure
(1) cell recovery and inoculation
1. the recovery of cell
(1) indoor ultraviolet disinfection; Culture medium, PBS are in 37 DEG C of pre-stand-by heats of water bath with thermostatic control.
(2) wear safety goggles and gloves, from liquid nitrogen container, take out the cryopreservation tube that C6 cell is housed, rapidlyPut into the enamel pot that fills 36 DEG C-37 DEG C, shake, thaws as early as possible frequently.
(3) cut off gauze pocket, take out peace and cut open, with after 70% alcohol wipe sterilization, on superclean bench, beatOpen cryopreservation tube lid, with suction pipe sucking-off cell suspension, pack in centrifuge tube, then the 8ml that adds pre-temperature is containing 10%The DMEM/F12 nutrient solution of hyclone, piping and druming suspends cell.
(4) low-speed centrifugal (500-1000 rev/min) 5 minutes, get repeat again after supernatant with PBS rinsing, fromThe heart.
(5) add nutrient solution suitably after dilution, then move and pack in blake bottle, put 37 DEG C, 5%CO2Incubator trainingSupport, record the recovery date, change next day after a nutrient solution and continue again to cultivate.
2. cell cryopreservation
(1) prepare cell: select exponential phase C6 glioma cell, before collection, change liquid 1 time. 0.25%After trypsin digestion cell, blow and beat, make single cell suspension, density reaches 1 × 105/ ml, 800 revs/min,Centrifugal 8 minutes.
(2) prepare cryopreserving liquid: add 1 part of dimethyl sulfoxide (DMSO) (DMSO) with 9 parts of nutrient solutions and make 10%DMSO and freezeLiquid storage.
(3) cryopreserving liquid is slowly added in the cell precipitation in centrifuge tube, blow and beat gently and make cell weight with suction pipeOutstanding.
(4) cell suspension is distributed in cryopreservation tube to sealing.
(5) frozen: cryopreservation tube is placed in-4 DEG C 2 hours ,-20 DEG C 2 hours ,-80 DEG C of refrigerator overnight, thenDrop in liquid nitrogen container and preserve.
The cultivation of 3.C6 cell with go down to posterity
(1) tight observation of cell growing state when about 80-90%, is gone down to posterity at the bottom of cell is paved with bottle.
(2) from 37 DEG C, 5%CO2Incubator takes out blake bottle. In superclean bench, will grow up to fine and close individual layerIn the cell bottle of cell, original nutrient solution discards;
(3) add 0.25% pancreatin solution, it is 0.5ml or more that the size of visual cell's bottle adds volume, withCell is fully infiltrated, and general digestion time is about 1 to 3 minute, visually observes bottle wall translucent thinBorn of the same parents' layer, when seeing while there is space, fine needle hole, discards pancreatin solution, and adds appropriate cell culture fluidStop digestion;
(4) low-speed centrifugal (500-1000 rev/min) 5 minutes, abandons supernatant, adds DMEM/F12 nutrient solution,Softly blow and beat into cell suspension, be sub-packed in 75cm in 1:3 ratio2In floor space blake bottle, continue to cultivate.
In 4.C6 clone the cultivation of glioma stem cells with go down to posterity
(1) first C6 cell is inoculated in to tradition containing in serum training base, in blake bottle, goes down to posterity. Treat cell placeIn exponential phase, with the cleaning of PBS liquid, pancreatin (0.25%) digestion. The piping and druming of self-control pasteur pipet machineryBecome unicellular, be resuspended in SFM, Trypan Blue counting. With 1 × 105/ hole is inoculated in 25cm2Bottom surfaceIn long-pending blake bottle, at 37 DEG C, 5%CO2, cultivate in saturated humidity incubator.
(2) inoculating cell, after SFM, according to the speed of C6GSC propagation and the size of C6GSC, is waited to cultivateMonoclonal cell group regular shape, the volume large rear (general 4-5 days) that in base, suspend, in ultra-clean workCell mass is proposed in vitro together with culture medium in platform, attached cell at the bottom of abandoning bottle, draws culture mediumAnd after centrifugal, discard the outmoded training base of half amount, with the centrifugal 5min of 800rpm/min, abandon supernatant. SFM is resuspended thinBorn of the same parents, the piping and druming gently repeatedly of self-control Pasteur point mouth suction pipe, resolves into cell mass unicellular, by 1:2 orThe ratio of person 1:3 is inoculated in 25cm2 floor space blake bottle, adds SFM to 6ml; Or with same concentrationsBe inoculated in standard 6 orifice plates, every hole adds SFM2.5ml, continues at 37 DEG C, 5%CO2, saturated humidity cultivateIn case, cultivate.
The induction of glioma stem cells differentiation in 5.C6 clone
In SFM, third generation C6GSC forms after 1d, the unicellular D-Hanks of using by C6GSC or after dispellingLiquid cleans, and is resuspended in SSM, and C6GSC is by 1 × 105/ hole is inoculated in 6 orifice plates. Observe every day its differentiation andGrowing state.
(2) mtt assay detects external tumor-inhibiting action (this of chlorogenic acid to C6 glioma and C6 glioma stem cellsExperiment repeats 3 times)
(1) be taken at containing the cell that is exponential phase in blood serum medium and serum free medium, pancreatin disappearsAfter consumption, make individual cells, tumour cell is still inoculated in containing in blood serum medium, and tumor stem cell is inoculated inIn serum-free stem cell media, all adjusting cell density is 2.0 × l05/ ml, is inoculated in 96 by cellOrifice plate, every hole 100 μ l, totally 5 groups, wherein 4 groups is experimental group, 1 group of negative contrast; And establish and do not containThe nutrient solution of cell is blank, establishes 5 multiple holes for every group. At 37 DEG C, 5%CO2, saturated humidity cultivateIn case, cultivate, spend the night. In the time being inoculated in 96 orifice plate, the culture hole of periphery is discarded, to avoid edgeEffect.
(2) after inoculation, press following grouping dosing next day after cell attachment, leaves standstill and cultivate 48 hours. With completelyCulture medium is mixed with 6.4mg/ml chlorogenic acid storage solutions the solution of 256 μ g/ml, then with training completelySupport base and progressively dilute by two-fold dilution's method, must be containing the training completely of chlorogenic acid 256,128,64 and 32 μ g/mlSupport base, for subsequent use; Get the above-mentioned complete medium containing variable concentrations chlorogenic acid of 50 μ l to inoculating cellIn 96 orifice plates, make chlorogenic acid final concentration be respectively 128,64,32 and 16 μ g/ml, each concentration 3 is multipleHole.
(3) establish tumour cell Normal group hole simultaneously, do not add drug-treated, (100 μ l) only to add equivalentComplete medium, synchronizes and tests with above-mentioned 3 groups.
(4)5％CO2, 37 DEG C are continued to hatch, and observe and take pictures after 48h under inverted microscope.
(5) under aseptic operating platform, lucifuge, every hole adds 20 μ L0.5%MTT, continues to cultivate 4h, in 37 DEG CPlacement is spent the night after (respectively organizing time consistency), surveys OD with ELIASA, and the light absorption value in each hole is measured at 440nm place.
(6) calculate inhibitory rate of cell growth
Formula: inhibitory rate of cell growth IR (%)=1-(experimental group cell OD value/cellular control unit OD value)×100％。
(3) statistical method
Mean ± standard deviation for this experimental resultRepresent, user's difference analysis is analyzed result.Complete the statistical analysis of data with SPSS13.O software kit. Inspection level: P < 0.05 is for there being statisticsDifference, P < 0.01 is for there being remarkable significant difference.
Three, result of the test
(1) cell cultivation results
The cultivation of 1.C6 glioma cell
Containing in blood serum medium, a few hours rear section cell be adherent growth, after 24h, can be observed thinKaryon is larger, and endochylema is abundant, and cell attachment growth, stretches out projection, and after 48h, cell grows up to projection and shapeReticulate, form monolayer adherence cellular layer, 3d, attached cell approximately covers culture plate bottom approximately 80%.
The cultivation of 2.C6 glioma stem cells
The C6 cell of choosing exponential phase is inoculated in serum free medium, and after 24h, it is single that cell isCell is uniformly distributed and is dispersed in adherently, has a little cell aggregation and start to produce to be suspended in spherical in culture mediumCell, can be observed part cell and grows up to cell ball sample after 48h, similar NSC ball, cell ballSuspension growth, spherical in shape or oval, not of uniform size, refractivity is strong; All the other cells are deposited in cultivationAt the bottom of plate, be unicellular growth. When 3d, dozens of cell is the visible globuli cell of spherical suspension growth group, 5d,When 7d, observe cell ball further growth, increase.
After the induction differentiation of 3.C6 glioma stem cells in the cultivation containing in blood serum medium
Suspension cell ball is inoculated in to 10% containing in hyclone culture medium, and when 24h, cell ball starts to occurAdherent phenomenon grows projection, and after 48h, cell quantity increases and form strip cell island, cell island when 3dExpand with the cell island of closing on and merge, cell attachment growth. When 7d, observe attached cell well-grown, pasteParietal cell approximately covers culture plate bottom approximately 60%.
(2) the external tumour inhibiting rate experimental result of chlorogenic acid
The chlorogenic acid that mtt assay is measured variable concentrations acts on after two kinds of cells, surveys OD value, calculates cell and presses downRate processed. Result shows the increase along with chlorogenic acid drug concentration, the inhibiting rate of each experimental group to two kinds of cellsAlso increase successively.
OD value and the inhibiting rate of table 3 chlorogenic acid to C6 glioma cell and the effect of C6 glioma stem cells
Four, conclusion
Chlorogenic acid is to experiment showed, in the body of C6 glioma stem cells effect that chlorogenic acid is to C6 glioma cell and C6Glioma stem cells has growth inhibition effect, less to the inhibitory action of tumor stem cell. Above-mentioned test cardBright, chlorogenic acid of the present invention has inhibition effect to human glioma, and can suppress brain Tumor Stem CellsGrowth is the new tool for the treatment of human glioma.
Claims (9)
- Chlorogenic acid as unique active component the purposes in the medicine of preparation treatment ependymoma.
- 2. purposes according to claim 1, is characterized in that: described medicine is to transplantability brainThe inhibiting medicine of glioma.
- 3. purposes according to claim 1, is characterized in that: described medicine is to C6 colloidOncocyte has the medicine of growth inhibition effect.
- 4. purposes according to claim 1, is characterized in that: described medicine is to C6 colloidKnurl stem cell has the medicine of growth inhibition effect.
- 5. according to the purposes described in claim 1-4 any one, it is characterized in that: described medicine isTaking the chlorogenic acid of effective dose as active component, add pharmaceutically acceptable auxiliary material or complementary composition systemThe standby preparation forming.
- 6. purposes according to claim 5, is characterized in that: every preparation in described pharmaceutical preparationUnit contains chlorogenic acid 1～3000mg.
- 7. purposes according to claim 5, is characterized in that: described pharmaceutical preparation Content of Chlorogenic AcidUsing dosage be 10-40mg/kg.
- 8. purposes according to claim 7, is characterized in that: described pharmaceutical preparation Content of Chlorogenic AcidUsing dosage be 20mg/kg.
- 9. according to the purposes described in claim 6-8 any one, it is characterized in that: described medicament isOral formulations or injection.
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