CN103550231B

CN103550231B - Application of grincamycin B in preparing anti-glioma drugs

Info

Publication number: CN103550231B
Application number: CN201310582666.8A
Authority: CN
Inventors: 姚月良; 平轶芳; 卞修武
Original assignee: First Affiliated Hospital of TMMU
Current assignee: Third Military Medical University TMMU; First Affiliated Hospital of TMMU
Priority date: 2013-11-18
Filing date: 2013-11-18
Publication date: 2015-07-15
Anticipated expiration: 2033-11-18
Also published as: CN103550231A

Abstract

The invention belongs to the medical field and discloses an application of a grincamycin B compound in preparing anti-glioma drugs. According to the invention, the grincamycin B has selective action on glioblastoma cells, and also has very good biological activity; the grincamycin B acts in inducing the apoptosis of tumor cells and tumor stem cells and blocking cells at the stages G0-G1; the grincamycin B effectively inhibits glioma cloning; the grincamycin B effectively inhibits the formation of glioma stem cells; the grincamycin B effectively inhibits the expression of the dry gene of the glioma stem cells, and further is capable of effectively inhibiting the self-updating effect of the glioma stem cells; therefore, the grincamycin B is a potential anti-cancer drug and provides a new scheme and a new drug for oncotherapy of targeted cancer stem cells (CSC); and furthermore, the grincamycin B plays an important role in improving the oncotherapy effect and improving the life quality of a patient.

Description

The application of lattice Rake mycin B in the medicine of the anti-glioma of preparation

Technical field

The invention belongs to medical domain, relate to the new opplication of Ge Ruike mycin B compound, the particularly application of Ge Ruike mycin B compound in the medicine of the anti-glioma of preparation.

Background technology

Malignant tumor is the high incidence of serious harm human health and high fatality rate disease.Glioma

(glioma) be the tumor that the glial cell (astrocyte, oligodendrocyte and ependyma etc.) differentiated by neuroderm causes, in intracranial tumor, sickness rate ranks first.Classify according to WHO2007 central nerve neuroma, glioblastoma is defined as the glioma greater than or equal to WHO III grade, specifically comprise human anaplastic astrocytoma (III grade), anaplastic oligodendroglioma (III grade), anaplastic prominent astrocytoma (III grade), glioblastoma multiforme (IV grade) and giant cellular glioblastomas (IV grade) less, wherein former three can be referred to as " anaplastic glioma ".Glioblastoma is the modal malignant tumor of intracranial, and the sickness rate of glioblastoma is about 5 ~ 8,/10 ten thousand, accounts for 50% of all gliomas.In the U.S., annual new cases has 14000 examples, and over nearly 20 years, sickness rate is in rising trend.And, because glioblastoma is the unrestricted hypertrophy of wellability, is rich in blood vessel with normal cerebral tissue without obvious boundary, also there is high relapse rate and low cure rate.Though through making great efforts for many years, its mortality rate and disability rate are still very high, and the median survival interval of a modification astrocytoma is 2 ~ 5 years, and glioblastoma multiforme is only 12 ~ 15 months.

The treatment of current glioblastoma has developed into the Colligation Therapy Mode of operation, radiation and chemotherapy associating.Each Therapeutic Method is improving survival of patients, is improving collaborative in patients ' life quality playing a role, because glioblastoma has the strong feature of aggressive, neurosurgery is caused to excise completely, and the follow-up radiation and chemotherapy carried out is difficult to eliminate residual tumor cell up hill and dale, these cells finally cause the recurrence of tumor, cause patient's poor prognosis, mortality rate is high.Glioblastoma is insensitive to traditional radiation and chemotherapy, even if through years development, current therapeutic effect still can not be satisfactory, and Post operation is once recurrence, and poorer, the mean survival time only has 12 ~ 15 months.

Research in recent years finds, in multiple solid tumor, all there is cell few in number to have the feature of the similar stem cell such as infinite multiplication, self renewal, Multidirectional Differentiation, and thus propose " tumor stem cell hypothesis ", this hypothesis thinks most cells apoptosis after of short duration proliferation and differentiation in tumor, sub-fraction cell is only had to have the ability of recurrent tumors, itself and other stem cell of body are compared, is referred to as tumor stem cell (cancer stemcells, CSCs).The proposition of tumor stem cell theory, impels researcher re-examine glioblastoma and biological characteristics thereof.Research shows, there is glioma stem cells (glioma stem cells, GSCs) in glioblastoma, this group of cells content in glioma is very micro-, but there are very strong self renewal and Multidirectional Differentiation ability, in the generation, development of tumor, all play pivotal role.People are successively successfully separated and confirm the existence of brain Tumor Stem Cells from glioblastoma tumor tissue, and stem cell is successfully separated, identifies people's glioblastoma.Research display, this part cell has the propagation and self-renewal capacity that are different from other cell subset in tumor, people's glioma stem cells can at serum-free, culture fluid (serum-free medium without exogenous grow th factor without exogenous growth factor, SGFF-M) in, propagation, renewal form neural ball like cell ball, and these cell ball cells have the feature of glioma stem cells.The self renewal of glioblastoma stem cell does not rely on exogenous cytokines, and it may have the autoactivation mechanism of self renewal.Tumor stem cell is that tumor occurs, recurs, attacks, shifts " root ".The self renewal effect of Tumor suppression stem cell can eliminate the malignant nature such as recurrence, invasion and attack of tumor, and therefore, the self renewal of Tumor suppression stem cell cures the effective New Policy of tumor, has important clinical meaning.

At present from marine microbial technology, found that a few compounds novel structure, antitumor cell biological activity are strong, marine microbial technology has become the focus of domestic and international cancer therapy drug research and development.

Marine actinomycete comprises streptomyces (Streptomyces), micromonospora (Micromonosporas), Rhod (Rhodococcus), Nocardia (Nocardias), actinoplanes (Actinoplanes) etc., in recent years a lot of new Pseudomonas is also found, as Salinisporas genus, Verrucosisporas belong to.In recent years, the active substance found from marine actinomycete has a variety of, as nitrogen-containing hetero lopps, Macrolide, peptide class, aminoglycoside, ethers, ketone, terpenoid, esters, quinones etc.They have biological activity widely.Due to the living environment that self is special, marine actinomycete has complicated unique metabolic pathway, create many novel structures, the significant secondary metabolite of biological activity, the discovery for novel drugs provides abundant lead compound, and wherein some enters preclinical study.

Lattice Rake mycin series compound is deep-sea actinomycetes secondary metabolite, starts most to find that Ge Ruike mycin is at 1987(Hayakawa, Y.; Iwakiri, T.; Imamura, K.; Seto, H.; Otake, N.J.Antibiot.1987,40,1785-1787), find that Ge Ruike mycin can suppress the growth of mouse leukemia cell P388.Studied at present confirm Ge Ruike mycin series compound can inhibition tumor cell propagation and induce its apoptosis, there is anti-HepG2 hepatocarcinoma, SW-1990 cancer of pancreas, HeLa cervical cancer, the effect of NCI-H460 pulmonary carcinoma and MCF-7 breast carcinoma.But up to now, whether Ge Ruike mycin series compound has the effect of resisting tumour stem cells and whether has the effect suppressing glioblastoma tumor stem cell there is not been reported.

Summary of the invention

Present inventor passes through deep-sea actinomycetes secondary metabolite antitumor cell strain bioactivity screening, find that deep-sea actinomycetes secondary metabolite Ge Ruike mycin B has extraordinary biological activity to Malignant glioma cells strain, the chemical structural formula of Ge Ruike mycin B is as shown in formula I:

The object of the present invention is to provide the new opplication of Ge Ruike mycin B compound, find new antitumor lead compound.

For achieving the above object, technical scheme of the present invention is:

The application of lattice Rake mycin B in the medicine of preparation treatment glioma.

Further, described glioma is glioblastoma.

Further, the medicine of described treatment glioma is the medicine of anti-glioma stem cells.

Further, the medicine of described treatment glioma is the medicine of induction gum oncocyte and/or glioma stem cells apoptosis.

Further, the medicine of described treatment glioma is the medicine of retardance glioma cell and/or glioma stem cells cell division cycle.

Further, the medicine of described treatment glioma is the medicine suppressing glioma clone to be formed.

Further, the medicine of described treatment glioma is the medicine suppressing glioma stem cells to be formed.

Further, the medicine of described treatment glioma is the medicine suppressing the gene expression of glioma stem cells dryness.

Lattice Rake mycin B is preparing the application in anti-tumor drug, and described anti-tumor drug is the medicine of resisting tumour stem cells.

Beneficial effect of the present invention: the invention provides the application of Ge Ruike mycin B in the medicine of the anti-glioma of preparation, lattice Rake mycin B effectively suppresses the self renewal effect of glioma stem cells, it is a potential antitumor drug, for the oncotherapy of targeting tumor stem cells (CSC) provides new scheme and medicine, to raising oncotherapy effect, improve life in patients significant.On the other hand, Ge Ruike mycin B is deep-sea actinomycetes secondary metabolite, and extraction process is simple; Microorganism fermentation process is ripe and simple, free from environmental pollution, low production cost and without raw material trouble and worry.

Accompanying drawing explanation

In order to make the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the present invention is described in further detail, wherein:

Fig. 1 matched group U251 tumor cell line apoptosis detects Annexin-V FITC ⁺pI ⁺result.

Fig. 2 Ge Ruike mycin B process experimental group U251 tumor cell line apoptosis detects Annexin-VFITC ⁺pI ⁺result.

Fig. 3 matched group dative Rake mycin B experimental group process U251 tumor cell line cell cycle testing result.

Fig. 4 glioma clone forms figure.

Fig. 5 glioma clone forms result.

Fig. 6 Ge Ruike mycin B suppresses the formation of glioma stem cells to detect figure.

Fig. 7 Ge Ruike mycin B suppresses glioma stem cells to form result.

Fig. 8 glioma stem cells dryness gene expression results.

Detailed description of the invention

Below in conjunction with embodiment, the invention will be further described.Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.

The experimental technique of unreceipted actual conditions in illustrated embodiment of the present invention, usually conveniently condition, such as the Molecular Cloning: A Laboratory guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or according to the condition that manufacturer advises.

Statistical analysis: quantitative data all represents with means standard deviation, adopts SPSS12.0 statistical software to analyze, and use independent samples t test analyzes the difference between two groups, the difference between one factor analysis of variance more multiple groups.

Human malignant glioma cell line U251, U87 and glioma primary cell strain 091214,9944 and huve cell HUVECs-12 cultivate the DMEM culture medium (article No.: 12800-082 with the Gibco company containing 10% hyclone, configuration: to specifications, use the two pure water dilution of fresh Millipore, add NaHCO ₃2.0g/L (Chengdu Ke Long chemical reagent factory, analytical pure), HEPES2.38g/L(Scientificresearch special, article No.: 7365-45-9-5009), adjust pH 7.26, degerming with 0.22 μm of membrane filtration, with the dual anti-penicillin and the streptomycin that front add Hyclone, be all 100U/mL (article No.: SV30010), be positioned over 37 DEG C, CO ₂gas volume fraction is cultivate in 5% incubator.Cell is often crossed and is changed liquid in 2-3 days, with PBS(0.01M, pH7.2-7.4) (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces, article No.: ZLI-9062, configuration: to specifications, use after distilled water diluting) washing, pancreatin (the ScientificResearch Special of 0.25%, article No.: 0458-10g, configuration: to specifications, dilute with above-mentioned PBS, then add EDTA0.8g (Solarbio, article No.: E8030.)) had digestive transfer culture, 3-10 substitute is in testing.

Human malignant glioma cell line U251, U87 and huve cell HUVECs-12 buy in American ACT T company; Glioma primary cell strain 091214 and 9944 comes from the clinical samples that our hospital surgical has just excised, and obtains informed consent.Ge Ruike mycin series compound used in experiment provided by Guangzhou Chinese Academy of Sciences South Sea institute of oceanography Ju Jianhua researcher, and Ge Ruike mycin series compound is deep-sea actinomycetes secondary metabolite.

The suppression ratio of the Ge Ruike mycin series compound Malignant glioma cells strain of embodiment 1 variable concentrations

Adopt CCK8(purchased from the green skies, article No.: C0038) detect the Ge Ruike mycin series compound of variable concentrations to the suppression ratio of Malignant glioma cells strain, carry out in accordance with the following steps:

1, get 3-10 for the good U251 cell strain of growth conditions, be adjusted to 5 × 10 ⁴the cell of/mL concentration, every hole 100 μ L, is incubated at 96 orifice plates, spends the night adherent;

2, experimental group adds and accords with regular rules that (its final concentration is respectively 8 to Rake mycin series compound, 4,2,1,0.5 and 0.25 μMs) (Ge Ruike mycin series compound mother liquid concentration 10mM, be dissolved in dimethyl sulfoxide (DMSO) (Amresco, article No.: D8370) ,-20 DEG C of preservations), matched group is the culture fluid of final concentration 0.05% (v/v) DMSO, blank group only adds equivalent culture medium and does not add cell, and often group arranges 3 and repeats experimental port;

3,37 DEG C, CO is positioned over ₂gas volume fraction is cultivate in 5% incubator, and after 48 hours, every hole adds CCK810 μ L, cultivates after 2 hours, then uses microplate reader (Thermo company) to measure the absorbance (OD value) of 450nm wavelength.

The computing formula of suppression ratio is as follows:

Suppression ratio=(matched group OD value-processed group OD value-2 × blank group OD value)/(matched group OD value-blank group OD value) × 100%

Ge Ruike mycin series compound is obtained to Malignant glioma cells strain half-inhibition concentration (IC with medicine inhibition concentration software for calculation 1.0.0 version (LOGIT method) software ₅₀), result is as table 1.

Table 1 lattice Rake mycin (GCN) series compound suppresses the IC of glioma cell and huve cell HUVECs-12 ₅₀value

As can be seen from Table 1, Ge Ruike mycin B is to the IC of glioma cell line U251, U87 and glioma primary cell strain 091214,9944 ₅₀value is 1.9 μMs-3.3 μMs, to the IC of huve cell HUVECs-12 ₅₀value is 10.6 μMs, and Ge Ruike mycin B is to the selective effect of Malignant glioma cells.

Embodiment 2 lattice Rake mycin B is to the apoptosis-induced situation of glioblastoma U251 cell strain

Detect Ge Ruike mycin B to the apoptosis-induced situation of glioblastoma U251 cell strain by FACS Calibur type flow cytometer (U.S. company BD), carry out as follows:

1, get 3-10 for the good U251 cell strain of growth conditions, be adjusted to 5 × 10 ⁴the cell of/mL concentration, is incubated at 6 orifice plates, spends the night adherent in 2mL/ hole;

2, change culture fluid, experimental group be final concentration be respectively 1.0 μMs with the culture fluid of the Ge Ruike mycin B compound of 2.0 μMs, matched group is the culture fluid of the DMSO of final concentration 0.05% (v/v);

3,37 DEG C, CO is positioned over ₂gas volume fraction is cultivate in 5% incubator, and after 48 hours, collected by each group of cell dissociation and carry out flow cytomery Ge Ruike mycin B to the apoptosis-induced situation of glioblastoma U251 cell strain, result as depicted in figs. 1 and 2.

As depicted in figs. 1 and 2, DMSO matched group U251 tumor cell line apoptosis rate is 1.94%, 1.0 μMs of dosage Ge Ruike mycin B compound experimental group U251 tumor cell line apoptosis rates are 8.18%, 2.0 μMs of dosage Ge Ruike mycin B compound experimental group U251 tumor cell line apoptosis rates are 13.96%, show Ge Ruike mycin B compound inducing malignant U 251 glioma cell line apoptosis.

Embodiment 3 lattice Rake mycin B is to glioblastoma U251 cell strain Cell cycle influences situation

Adopt FACS Calibur type flow cytomery Ge Ruike mycin B to glioblastoma U251 cell strain Cell cycle influences situation, method is as follows:

2, change culture fluid add final concentration be respectively 1.0 μMs with the Ge Ruike mycin B compound of 2.0 μMs, matched group is the culture fluid of the DMSO of final concentration 0.05% (v/v);

3,37 DEG C, CO is positioned over ₂gas volume fraction is cultivate in 5% incubator, and after 48 hours, collected by each group of cell dissociation and carry out flow cytomery Ge Ruike mycin B to the situation of glioblastoma U251 cell strain Cell cycle influences, result as shown in Table 2 and Figure 3.

Table 2 matched group dative Rake mycin B experimental group process U251 tumor cell line cell cycle testing result

Embodiment 4 lattice Rake mycin B suppresses clonality to detect

1, get 3-10 for the good U251 cell strain of growth conditions, be adjusted to the cell of 100/mL concentration, be incubated at 24 orifice plates, 1mL/ hole, spend the night adherent;

2, change culture fluid, experimental group be final concentration be respectively 0.25 μM, 0.5 μM with the culture fluid of the Ge Ruike mycin B compound of 1.0 μMs, matched group is the culture fluid of the DMSO of final concentration 0.05% (v/v);

3,37 DEG C, CO is positioned over ₂gas volume fraction is cultivate in 5% incubator, changes once new culture fluid after 72 hours;

4, the 12nd day culture medium abandons, and 4% paraformaldehyde fixes 15 minutes, then cleans once with PBS, violet staining liquid (the green skies, article No.: C0121) dyes 10 minutes, is greater than 50 cells and calculates a clone, calculate each group of clone's number formed, result is as shown in table 3, Fig. 4 and Fig. 5.

Table 3 glioma clone forms result

Embodiment 5 lattice Rake mycin B suppresses the formation of glioma stem cells to detect

Nerve stem cell culture medium D-MEM/F-12(Gibco company in the present embodiment, article No.: 12500-039) collocation method: to specifications, use the two pure water dilution of fresh Millipore, add NaHCO ₃2.0g/L, HEPES2.38g/L, adjust pH 7.26, degerming with 0.22 μm of membrane filtration, with the dual anti-penicillin and the streptomycin that front add Hyclone, be all 100U/mL.

Nerve growth additive (B27): 50 ×, purchased from American Gibco company;

Recombinant human epidermal growth factor (recombinant human epidermal growth factor, rhEGF): 20ng/ml, purchased from American Sigma company;

Basic fibroblast growth factor (basic fibroblast growth factor, bFGF): 20ng/ml, purchased from American Upstate company;

D-MEM/F-12 adds B27(1 × before using), rhEGF(20ng/ml) and bFGF(20ng/ml).

Cell dissociation buffer (Accutase) (Millipore company).

Get 3-10 for the good U251 cell strain of growth conditions, be adjusted to 1 × 10 ⁴the cell of/mL concentration, cultivates with nerve stem cell culture medium D-MEM/F-12, within the 3rd day, adds the D-MEM/F-12 culture medium of 1/2, within 5-6 days, just can form cell ball and glioma stem cells; To the glioma stem cells Accutase of balling-up, 37 DEG C, after digestion 5-10min, blow and beat into unicellular gently, be adjusted to 1 × 10 ⁴the cell of/mL concentration, uses nerve stem cell culture medium D-MEM/F-12.Experimental group add lattice Rake mycin B final compound concentration be respectively 0.5 μM, 1.0 μMs with the culture fluid of 2.0 μMs, matched group is the culture fluid of the DMSO of final concentration 0.05% (v/v); Within 3rd day, add 1/2 amount containing corresponding concentration Compound D-MEM/F-12 culture medium, within the 5th day, detect that glioma stem cells formed number, result is as shown in table 4, Fig. 6 and Fig. 7.

Table 4 stem cell ball number

Embodiment 6 lattice Rake mycin B suppresses the detection of expression of glioma stem cells dryness gene

Nerve stem cell culture medium D-MEM/F-12(Gibco company in the present embodiment, article No.: 12500-039).Collocation method: to specifications, uses the two pure water dilution of fresh Millipore, adds NaHCO ₃2.0g/L, HEPES2.38g/L, adjust pH 7.26, degerming with 0.22 μm of membrane filtration, with the dual anti-penicillin and the streptomycin that front add Hyclone, be all 100U/mL.

Cell dissociation buffer (Accutase) (Millipore company);

Total RNA extracts reagent RNAiso plus (TaKaRa, article No.: 9109);

Reverse Transcription box PrimeScriptTM RT Master Mix (TaKaRa, article No.: RR036A);

PCR kit for fluorescence quantitative premix Ex TaqTM II (TaKaRa, article No.: RR820A).

1 experiment process

(1) get 3-10 for the good U251 cell strain of growth conditions, be adjusted to 1 × 10 ⁴the cell of/mL concentration, cultivates with nerve stem cell culture medium D-MEM/F-12, within the 3rd day, adds the D-MEM/F-12 culture medium of 1/2, within 5-6 days, just can form cell ball and glioma stem cells;

(2) to the glioma stem cells Accutase of balling-up, 37 DEG C, after digestion 5-10min, blow and beat into unicellular gently, be adjusted to 1 × 10 ⁴the cell of/mL concentration, cultivates with nerve stem cell culture medium D-MEM/F-12.Experimental group adds the culture fluid of the Ge Ruike mycin B compound of 2.0 μMs of final concentrations, and matched group is the culture fluid of the DMSO of final concentration 0.05% (v/v); Within 3rd day, add the 1/2D-MEM/F-12 culture medium containing corresponding concentration compound, the 5th day collecting cell;

The extraction step of 2RNA

After the cell 2 times of the above-mentioned collection of PBS buffer solution for cleaning, add RNAisoplus cell pyrolysis liquid, repeatedly blow and beat lysate, then sucked in the EP pipe without RNAase, room temperature leaves standstill 5min; Often pipe adds the chloroform of 1/5 cracking liquid measure, then shakes mixed liquor and become pink colour; 1.5 × 10 ⁴rpm4 DEG C centrifugal 15 minutes; Aspirate supernatant is to new without in the EP pipe of RNAase, and often add the isopropyl alcohol of equivalent in pipe, put upside down mixing, room temperature leaves standstill 10min; Again 1.5 × 10 ⁴rpm4 DEG C centrifugal 10 minutes; Supernatant discarded, just can see RNA and be deposited at the bottom of pipe, re-use DEPC(Beijing Suo Laibao Science and Technology Ltd.) water preparation 75% ethanol 1ml, add in EP pipe, repeatedly put upside down EP pipe; Again 1.5 × 10 ⁴rpm4 DEG C centrifugal 5 minutes; Supernatant discarded, after natural drying RNA, surveys its concentration and OD260/OD280 ratio with appropriate DEPC process water dissolution RNA, carries out cDNA reverse transcription after meeting the requirements.

3 reverse transcription steps

Carry out reverse transcription synthesis cDNA with Reverse Transcription box PrimeScriptTM RT Master Mix, step is as follows:

(1) by following component preparation RT reactant liquor (reactant liquor preparation is please carried out on ice)

Reagent	Use amount	Final concentration
			5×PrimeScript RT Master Mix（Perfect Real Time）	2μl	1×
Total RNA	*
			RNase Free dH2O	up to10μl

* reaction system can by the corresponding amplification of demand, and 10 μ l reaction systems can the maximum Total RNA using 500ng.

(2) RT reactant liquor has prepared rear soft mixing, carries out reverse transcription reaction, 37 DEG C of reverse transcription reaction 15min, and the inactivation reaction 5sec of 85 DEG C of reverse transcription preserves under 4 DEG C of conditions.

4 quantitative fluorescent PCRs

(1) synthesis of fluorescence quantification PCR primer and dilution

Quantitative fluorescent PCR the primer is synthesized by invitrogen company, brief centrifugation before dilution primer, then to specifications, uses the storage liquid without RNAase water, primer being diluted to 100nM of sterilizing, then from storage liquid, draw 5 μ l, add 45 μ l without RNAase water and primer working solution.Primer sequence is as follows:

(2) preparation of PCR reactant liquor and reaction condition:

According to PCR kit for fluorescence quantitative in Premix Ex TaqTM II, description operates, specific as follows:

Component	Volume
		Maxima SYBR Green/Rox qPCR Master Mix(2×)	10μl
Forward primer	0.3μmol/L
		Reverse primer	0.3μmol/L
Template DNA	≤500ng(1μl)
		Water,nuclease-free	To20μl
Total	20μl

Reaction condition carries out according to following table:

After loop ends, carry out melt curve analysis mensuration immediately, detected temperatures is 65 DEG C-95 DEG C, and heating rate is 0.5/ DEG C time, and constant temperature time is 1sec/ time.

PCR kit for fluorescence quantitative premix Ex TaqTM II detects dryness gene Nestin, and the expression of C-Kit, Sox2, Nanog, Oct4, result is as shown in table 5 and Fig. 8.

Table 5 lattice Rake mycin B suppresses the detection of expression of glioma stem cells dryness gene

As can be seen from table 5 and Fig. 7, the expression of Nestin matched group is 1.75 times of experimental group, the expression of C-Kit matched group is 1.76 times of experimental group, the expression of Sox2 matched group is 1.85 times of experimental group, the expression of Nanog matched group is 2.32 times of experimental group, and the expression of Oct4 matched group is 2.13 times of experimental group.

What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.

Claims

1. the application of lattice Rake mycin B in the medicine of preparation treatment glioma.

2. application according to claim 1, is characterized in that, described glioma is glioblastoma.

3. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine of anti-glioma stem cells.

4. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine of induction gum oncocyte and/or glioma stem cells apoptosis.

5. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine of retardance glioma cell and/or glioma stem cells cell division cycle.

6. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine suppressing glioma clone to be formed.

7. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine suppressing glioma stem cells to be formed.

8. application according to claim 1 and 2, is characterized in that, the medicine of described treatment glioma is the medicine suppressing the gene expression of glioma stem cells dryness.

9. the application of lattice Rake mycin B in the medicine preparing resisting tumour stem cells.