CN110051842A - Induction tumour cell is changed into neuron cell to inhibit the preparation of tumour growth - Google Patents

Abstract

The present invention relates to induction tumour cells to be changed into neuron cell to inhibit the preparation of tumour growth.The combination of small molecule compound is provided, which can induce tumour cell and be directly translated into non-tumorigenic cells, while inhibit tumour growth；The small molecule combinatorial can be prepared into clinical tumor therapeutic agent.The present invention also provides application, the medicine boxs etc. that are combined based on the small molecule compound.

Description

Induction tumour cell is changed into neuron cell to inhibit the preparation of tumour growth

Technical field

The invention belongs to oncologies, more particularly it relates to induce tumour cell (such as glioblastoma) It is changed into neuron cell to inhibit the preparation of tumour growth.

Background technique

Tumour is made of the abnormal cell in various different differentiation degrees, and has heterogeneous feature.Existing radiation is controlled The oncotherapies side such as killing after treatment (radiotherapy), chemotherapy (chemotherapy), targeted therapy, biological immune treatment, induction differentiation Method is to be directed to how to kill tumour cell, but in view of the intractable of tumour, current various therapies still have problems, There is an urgent need in the art to develop novel low-poison efficient antineoplastic medicine, to be clinical antineoplastic treatment, raising patient's survival Rate provides new approach.It is changed to direct transdifferentiation as can tumour cell will be killed it is normal or non-tumorigenic cells, then will be With great meaning.

Glioblastoma (Glioblastoma, GBM) is most universal, most pernicious tumour in Adult Human Brain, in experience hand After art excision, radiotherapy, chemotherapy, the average survival time median of glioblastoma patient still only has 15 months or so.Colloid Glioblastoma stem cell present in blastoma is believed to resist radiation and chemotherapy effect, in angiogenesis, swells Tumor migration and recurrence etc. play a significant role.In addition, glioblastoma tumour is real under conditions of radiotherapy or chemotherapy With glioblastoma stem cell phase co-conversion can occur for cell plastid, this makes exploitation that can target glioblastoma simultaneously The novel strategy of tumor parenchymal cells and glioblastoma stem cell becomes particularly important, to reach more ideal treatment effect Fruit.The fatal property of glioblastoma is inseparable with hyper-proliferative and invasive ability that it has.

One potential effective strategy is that tumour cell is changed into terminal differentiation, proliferation and invasive ability all obviously to subtract Weak cell.This strategy provides for realization for the birth of iPS technology and thus derivative cell reprogramming and transdifferentiation technology It may.For example, it is the cell with neuron property that transcription factor, which can induce glioblastoma cells, these inductions The growth of cell in vitro and in vivo after transformation is obviously inhibited, and the service life of glioblastoma model mouse is also shown It writes and extends, to assist the treatment of glioblastoma to provide new means.

The problems such as convenience that transcription factor faces and safety, limits their uses clinically, thus has energy Enough pass freely through cell membrane, immunogenicity are lower, be readily synthesized preservation standardization, biology effect it is reversible it is fine-tuning, do not destroy The small molecule compound of the features such as cellular genome, even clinically compound medicine currently in use, as can for inducing The transformation of cell fate is then more valuable.For example, existing research report is combined mouse using small molecule compound into fibre Tieing up cell reprogramming is iPS cell, it was demonstrated that small molecule compound can the case where being completely independent of external source transcription factor It is lower by reprogramming of somatic cells be totipotent cell.After this, have been reported that mouse successively using small molecule compound combination and The fibroblast difference transdifferentiation of people is neural progenitor cell and nerve cell, it was demonstrated that small molecule compound, which can induce, obtains it His cell type, also provides new thinking and strategy for the regeneration of nerve cell.The first studies have shown that of the present inventor is specific Small molecule compound combination the astroglia of mouse or people can be induced for nerve cell (Cheng, L., Gao, L.,Guan,W.,Mao,J.,Hu,W.,Qiu,B.,Zhao,J.,Yu,Y.,and Pei,G.(2015).Direct conversion of astrocytes into neuronal cells by drug cocktail.Cell Research 25,1269-1272；Gao,L.,Guan,W.,Wang,M.,Wang,H.,Yu,J.,Liu,Q.,Qiu,B.,Yu,Y.,Ping, Y.,and Bian,X.,et al.(2017).Direct Generation of Human Neuronal Cells from Adult Astrocytes by Small Molecules.Stem Cell Reports 8,538-547)。

Although having there is some research achievements as above, tumour higher for some grade malignancies, such as colloid Blastoma, if can reduce the cell that induction is both sexes or the cell that grade malignancy reduces, what kind of small molecule compound Has this ability, this field there is a need to carry out more in-depth study.

Summary of the invention

It is neuron cell to inhibit colloid the purpose of the present invention is to provide induction gum blastoma cells switch The preparation of blastoma growth.

In the first aspect of the present invention, a kind of composition for inhibiting tumour is provided, it is living that there are the following groups in the composition Property component:

(1) Temozolomide or its analog, isomers, salt, hydrate or precursor；With

(2) selected from following (a)~(b) the two or one of:

(a) compound selected from TGF beta inhibitor or aryl hydrocarbon receptor activator；

(b) Rho kinase inhibitor.

In another aspect of this invention, the purposes of TGF beta inhibitor or aryl hydrocarbon receptor activator is provided, be used for and is replaced not Azoles amine or its analog, isomers, salt, hydrate or precursor complex, preparation induction tumour cell are changed into neuron cell Or inhibit the composition of tumour；Preferably, the TGF beta inhibitor include: tranilast or its analog, isomers, salt, Hydrate or precursor.

In another aspect of this invention, the purposes of Rho kinase inhibitor is provided, for Temozolomide or its analog, Isomers, salt, hydrate or precursor complex, preparation induction tumour cell are changed into neuron cell or inhibit the combination of tumour Object；Preferably, the Rho kinase inhibitor includes: Fasudil or its analog, isomers, salt, hydrate or precursor.

In another aspect of this invention, the purposes of mentioned-above composition is provided, induction tumour cell is used to prepare and turns Become neuron cell or inhibits drug, cells transdifferentiate reagent, kit or the medicine box of tumour.

In another aspect of this invention, a kind of method for inducing tumour cell to be changed into neuron cell is provided, it is described Method includes: using mentioned-above compositions-treated tumour cell, so that tumour cell be made to be changed into neuron cell；Compared with Goodly, the tumour includes: astrocytoma, ependymoma, oligodendroglioma, brain stem glioma, mixed type colloid Tumor, medulloblastoma, pernicious primitive neuroectodermal tumor, liver malignancy, pancreatic neoplasm etc.；Preferably, the star Shape cytoma includes: glioblastoma, diffusivity astrocytoma, human anaplastic astrocytoma.

In a preferred embodiment, it is non-therapeutic that the induction tumour cell, which is changed into the method for neuron cell, Method.

In another aspect of this invention, it provides a kind of for inducing tumour cell to be changed into neuron cell or inhibiting swollen Medicine box/kit of tumor, including pharmaceutical composition of the present invention.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

Fig. 1: the glioblastoma cells that pharmaceutical composition induction is cultivated in the training liquid containing serum are neuron cell. Figure 1A shows the isolated primary glioblastoma cells from glioblastoma tissue, these cells are containing blood respectively It is cultivated in the culture solution of cleer and peaceful serum-free.Figure 1B be using cell streaming art detect stem cells film surface marker CD15 and A2B5.Fig. 1 C is the expression of stem cell labeling object NETSIN and SOX2 in Immunofluorescence test glioblastoma cells. Fig. 1 D be qPCR detect neuron generate have transcription factor ASCL1, BRN2 of critical function, MYT1L, NEUROD1, The expression of NEUROG2 etc., wherein for NESTIN as negative control, statistical difference is the benefit compared with the 0th day sample P value is obtained with double tail t checking computations.Fig. 1 E is Immunofluorescence test glioblastoma cells after TTF combined treatment 5 days The expression of interior NEUROD1.After Fig. 1 F shows that TTF is induced 5 days, glioblastoma cells express nerve cell marker DCX.Figure After 1G shows that TTF is induced 8 days, glioblastoma cells express nerve cell marker MAP2.Fig. 1 H, which is shown, utilizes patch-clamp The electrophysiological index of technology detection glioblastoma cells after TTF is handled 38 days.Fig. 1 I and Fig. 1 J show process respectively Glioblastoma cells can be induced action potential and sodium ion electric current after TTF processing.Fig. 1 K and Fig. 1 L show warp respectively The purity and induced efficiency of neuron cell after crossing TTF induction, wherein GBM3 and GBM4 respectively indicates two different patients The GBM cell in source.The data of Fig. 1 D to Fig. 1 L are all the glioblastomas using the amplification cultivation in the training liquid containing serum Cell (GBM-3) is done.Scale is 50 μm in figure.

Fig. 2: the glioblastoma cells cultivated in pharmaceutical composition induction serum-free medium are changed into neuron cell. Fig. 2A shows that the cellular morphology of the glioblastoma cells after TTF is handled 6 days is shown as neuron cell form.Fig. 2 B It detects TTF for qPCR to handle glioblastoma cells the 0th, 2,4,6 day, ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 Etc. transcription factors expression；Statistical difference is to obtain p value using double tail t checking computations compared with the 0th day sample.Figure After 2C and 2D show that Immunofluorescence test TTF is induced 6 days, glioblastoma cells express nerve cell marker DCX and MAP2.Fig. 2 E is the expression of Flow cytometry glioblastoma cells DCX after TTF is handled 6 days.Fig. 2 F is The expression of flow cytometer detection MAP2 in glioblastoma cells after TTF is handled 6 days.Fig. 2 G, which is shown, utilizes fluidic cell The cell of fluorescence sorting technology enrichment the CD15 positive and CD15 feminine gender.Fig. 2 H show TTF by the cell of CD15 positive of enrichment with The cell that the cell of CD15 feminine gender all induces as the MAP2 positive.Fig. 2 I, which is shown, is enriched with A2B5 using fluidic cell fluorescence sorting technology Positive and A2B5 feminine gender cell.Fig. 2 J shows that TTF induces the cell of the cell of the A2B5 positive of enrichment and A2B5 feminine gender For the cell of the MAP2 positive.Scale is 50 μm in figure.

Fig. 3: the tumour property of pharmaceutical composition inhibition glioblastoma cells.Fig. 3 A display control and TTF processing for The influence of the glioblastoma cells balling-up ability of free serum culture；Wherein GBM-387 and GBM-17 is that two batches colloid is female thin Born of the same parents' oncocyte culture.Scale is 1mm (black scale) or 50 μm (white scale).Fig. 3 B is gradient dilution experiment detection pair According to the clonality with the glioblastoma cells of free serum culture under TTF treatment conditions；Data are by online ELDA points Software (http://bioinf.wehi.edu.au/software/elda/) analysis is analysed, and is united with Chi-square Test therein Meter analysis.Fig. 3 C shows that the cell proportion of flow cytometry stem cell labeling object CD15 positive after TTF is handled 6 days is aobvious Write decline.Fig. 3 D is the statistical result of Fig. 3 C, utilizes double tail t checking computation p values.Fig. 3 E shows that flow cytometry is passed through The cell proportion of the stem cell labeling object A2B5 positive is remarkably decreased after TTF is handled 6 days.Fig. 3 F is the statistical result of Fig. 3 E, is utilized Double tail t checking computation p values.Fig. 3 G is the balling-up ability of control and the glioblastoma cells after TTF processing 6 days； Scale 1mm (black scale) or 50 μm (white scale).Fig. 3 H be gradient dilution experiment detection control and TTF handle 6 days it The clonality of glioblastoma cells afterwards；Data by online ELDA analysis software (http: // Bioinf.wehi.edu.au/software/elda/ it) analyzes, and for statistical analysis with Chi-square Test therein.Fig. 3 I is Experiment detection cell proliferative conditions are mixed using EdU.Fig. 3 J is the statistical result of Fig. 3 I, utilizes double tail t checking computation p values.Figure 3K is that qPCR detects Cell cycle-related genes and is enriched with the expression of the gene of expression in glioblastoma；Double tail t Inspection be used to calculate p value.After Fig. 3 L shows that TTF is handled 5 days, the ratio of the cell proliferation markers KI67 positive changes.Fig. 3 M It is the statistical result of Fig. 3 L, double tail t inspections be used to calculate p value.Fig. 3 N is that Transwell experiment and violet staining detection are thin The invasive ability of born of the same parents, scale is 50 μm in figure.Fig. 3 O is the statistical result of Fig. 3 N, and double tail t inspections be used to calculate p value.

Fig. 4: pharmaceutical composition inhibits tumour growth in vivo.Fig. 4 A is that diagram indicates experiment in vivo process.Fig. 4 B is immune The expression of fluorescence detection TUJ1.Fig. 4 C is the statistical result of Fig. 4 B, and double tail t inspections be used to calculate p value.Fig. 4 D is immunofluorescence It detects BrdU and mixes situation, double tail t inspections be used to calculate p value.Fig. 4 E is the statistical result of Fig. 4 D, and double tail t inspections are used for Calculate p value.Fig. 4 F is the Caspase-3's (caspase-3 activated form) on Immunofluorescence test mouse brain slices by shearing Expression.Fig. 4 G is the statistical result of Fig. 4 F, and double tail t inspections be used to calculate p value.Fig. 4 H be by GFP and hematoxylin she Red colouring experiment detects the size of intracerebral tumour, and scale is 1mm in figure.Fig. 4 I be Fig. 4 H statistical result, statistical difference by ANOVA and the analysis of Tukey ' s Multiple range test obtain.Fig. 4 J and 4M are the dynamic change that bioluminescence living imaging detects tumor size Change, the red boxes in Fig. 4 M indicate that the mouse is dead in corresponding number of days.Fig. 4 K be Fig. 4 J statistical result, Fig. 4 N and 4O is the statistical result of Fig. 4 M, and the statistics that Two-way ANOVA and Tukey ' s Multiple range test is used to calculate Fig. 4 K and 4N is poor Different, Two-way ANOVA and Bonferroni ' s Multiple range test is used to calculate the statistical difference of 4O.Fig. 4 L and 4P are small The survivorship curve of mouse.Statistical difference is obtained by Log-rank (Mantel-Cox) checking computation.In Fig. 4 J, 4K, 4L experiment, TMZ, Tranilast, Fasudil are gastric infusion；In order to meet application method (the clinically TMZ of clinically these drugs Be with Tranilast it is oral, Fasudil be intravenous injection) with by Ethics Committee examine progress clinical trial, Fig. 4 M, In the experiment of 4N, 4O, 4P, TMZ, Tranilast are gastric infusion, and Fasudil is intraperitoneal injection.Scale is in addition to 4H in figure 50 μm, scale is 1mm in 4H figure.

Fig. 5: it identifies containing the glioblastoma cells and screening pharmaceutical composition cultivated in serum training liquid.Fig. 5 A is glimmering to be immunized Light detection astroglia gene GFAP, S100B, stem cell gene SOX2, NESTIN, neuronal genes MAP2, The expression in glioblastoma cells that NEUROD1, DCX are cultivated in containing serum training liquid.Fig. 5 B is the statistics of Fig. 5 A As a result.Fig. 5 C is that neuron generates relevant transcription factor MYT1L, NEUROG2 etc. in non-tumour and glioblastoma Expression.Fig. 5 D shows that NEUROD1 or MAP2 express the life cycle of high patient significantly more compared with expressing low patient Long, the data of Fig. 5 C and Fig. 5 D are obtained by GlioVis (gliovis.bioinfo.cnio.es).Fig. 5 E is shown in Tranilast is added on the basis of TMZ can significantly raise the expression of NEUROD1, MYT1L, and double tail t, which are examined, is used for statistical difference It is different.Fig. 5 F shows that TMZ can substantially change cellular morphology plus Fasudil.Fig. 5 G, which is shown in TTF combination, can significantly raise mind The expression of important transcription factor ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 etc. are generated through member, and on the basis of TTF Remove any one drug all and can influence the up-regulation multiple for these transcription factors.Double tail t, which are examined, is used for statistical difference It is different.*, * *, * * * indicate the difference with control sample, and #, ##, ### indicate the difference with TMZ processing sample.Scale is 50 μ in figure m。

The glioblastoma cells that Fig. 6: pharmaceutical composition TTF induction is cultivated in containing serum training liquid are changed into neuron Cell.Fig. 6 A is to measure intracellular ATP levels using bioluminescence, to detect influence of the TTF to cell viability；Two-way ANOVA and Tukey ' s Multiple range test are used for computational statistics difference.Fig. 6 B is the cellular morphology under time-lapse shooting TTF processing.Figure 6C to Fig. 6 E is Immunofluorescence test glioblastoma cells (GBM-4) NEUROD1, DCX, MAP2 etc. after TTF is handled Expression.Fig. 6 F to Fig. 6 G display Immunofluorescence test glioblastoma cells are after TTF is handled 8 days, neural meta-tag The expression of object SYN1, VGLUT1.Scale is 50 μm in figure.

Fig. 7: the glioblastoma cells cultivated in pharmaceutical composition induction serum-free medium are changed into neuron cell. Fig. 7 A is Immunofluorescence test glioblastoma cells of the neuron marker TAU after TTF is handled 6 days as the result is shown (GBM-387) positive expression in.Fig. 7 B show compared with the control group, by TTF handle cell in the GFAP positive cell ratio Example is considerably lower.Fig. 7 C is the statistical result of Fig. 7 B, and double tail t, which are examined, is used for computational statistics difference.Fig. 7 D to Fig. 7 F shows TTF Glioblastoma cells (GBM-17) expression MAP2, DCX and TAU that another strain is cultivated in serum-free medium after processing 6 days. Fig. 7 G and Fig. 7 H are that glioblastoma cells (GBM-17) express DCX and MAP2 after Flow cytometry TTF is handled 6 days. Scale is 50 μm in figure.

Fig. 8: pharmaceutical composition inhibits the growth including tumour.Fig. 8 A, which is shown, gives TTF pharmaceutical composition for mouse weight It influences suitable with the influence of TMZ.Fig. 8 B is that hematoxylin eosin stain detects control group, TMZ administration group and TTF administration group mouse The slices of organs such as heart, kidney, liver, spleen and lung.Fig. 8 C to Fig. 8 E is that glioblastoma is transplanted to mouse is subcutaneous, is given Relative medicine processing is given, compared with TMZ, TTF inhibits the ability of tumour growth stronger.Fig. 8 D by Two-way ANOVA and Tukey ' s Multiple range test is used for computational statistics difference, and Fig. 8 E is by One-way ANOVA and Tukey ' s Multiple range test based on Calculate statistical difference.Scale is 50 μm in figure.

Specific embodiment

The present inventor passes through in-depth study, discloses a kind of composition of small molecule compound, and it is thin to can induce tumour Born of the same parents are directly translated into non-tumorigenic cells, while inhibiting tumour growth；The small molecule compositions can be prepared into clinical treatment tumour Drug.The present invention also provides application, the medicine boxs etc. that are combined based on the small molecule compound.The present invention is believed by regulating cell The variation of number access and epigenetic changes cell fate, without realizing tumour cell in the case where being transferred to foreign gene Transformation.

Associated with compound

After widely studying, the present inventor is put forward for the first time, and on the basis of using Temozolomide, is inhibited simultaneously TGF signal beta access (Transforming Growth Factor β signaling pathway) or activation aryl hydrocarbon receptor (Aryl Hydrocarbon Receptor, AHR), or inhibit the work of Rho kinases (Rho associated kinase, ROCK) Property, tumour cell can be induced to be changed into non-tumorigenic cells, such as (also referred to as neuron is thin for the cell with neuron property Born of the same parents), the purpose for inhibiting tumour may be implemented.On the basis of using Temozolomide, if inhibiting TGF signal beta access and Rho simultaneously The activity of kinases, or activate aryl hydrocarbon receptor simultaneously and inhibit activity then cells switch effect and the tumour suppression of Rho kinases Effect processed is even more ideal.

As used in the present invention, described " inhibiting tumour " includes: that tumour cell is changed into non-tumorigenic cells, by tumour Cells switch is neuron cell, and tumor stem cell is changed into non-stem cell, weakens the tumour property of tumour cell (as fastly Fast-growing is long, invades or shifts), it also include alleviation or treatment tumor disease.

In the present invention, the Temozolomide (Temozolomide, TMZ) chemical name: 3,4- dihydro -3- methyl -4- oxygen For imidazo [5, l-d]-l, 2,3,5- tetrazine -8- amides (preferably, its No. CAS is 85622-93-1), are imidazo tetrazine Class alkylating agent with anti-tumor activity, molecular structural formula is as shown in following formula (I):

It is generally believed that Temozolomide is DNA alkylating agent, cause DNA damage and cell death.Clinically, by conduct The chemotherapeutics of glioblastoma.But it clinically there is also the higher problem of side effect, curative effect still needs to improve.

As used in the present invention, " the TGF beta inhibitor " refers to the inhibition for being able to suppress TGF signal beta access in cell Agent, including tranilast.Other tool tranilast identical functions, or the same class TGF signal beta access of the identical target spot of induction inhibit Agent can also be included in the present invention, such as SB431542, pirfenidone (Pirfenidone) etc..Pirfenidone is in clinic On frequently as treatment idiopathic pulmonary fibrosis drug, be TGF beta inhibitor with SB431542.

" the aryl hydrocarbon receptor activator " is the compound for referring to activation aryl hydrocarbon receptor.Tranilast is also one Kind aryl hydrocarbon receptor activator.Other tool tranilast identical functions, or the same class aryl hydrocarbon receptor of the identical target spot of induction swash Agent living can also be included in the present invention, such as leflunomide (Leflunomide) etc..Leflunomide has activation fragrance The activity of hydrocarbon receptor is clinically a kind of antirheumatic drug.

As preferred embodiment of the invention, the TGF beta inhibitor is tranilast (Tranilast), be N- (3, 4- dimethoxycinnamoyl base) anthranilic acid (preferably, its No. CAS is 53902-12-8)；Its molecular structural formula such as following formula (II) shown in:

It is mainly used to prevent and treat allergic asthma and other anaphylactias (such as bronchial asthma and anaphylaxis by this field The prophylactic treatment of rhinitis, atopic dermatitis), also have and is applied to treatment allergic conjunctivitis, diabetic keratopathy scleredema, disease The report of rationality scar, pneumoconiosis, Crohn disease etc..But there has been no it is applied to antitumor report so far.

Although having carried out the research of emphasis to tranilast, in a specific embodiment of the present invention, it was demonstrated that SB431542, It is thin that Pirfenidone also can be changed into neuron applied to induction tumour cell with TMZ and Rho kinase inhibitor combination Born of the same parents.Meanwhile Leflunomide can also be applied to induction tumour cell with TMZ and Rho kinase inhibitor combination and be changed into nerve First like cell.

As preferred embodiment of the invention, the Rho kinase inhibitor is Fasudil (Fasudil), is a kind of Isoquinolin sulphone amide derivative, chemical name hexahydro -1- (5- isoquinolinesulfonylcompounds)-homopiperazine is (preferably, its No. CAS is 103745-39-7), molecular structural formula is as shown in following formula (III):

Fasudil is clinically used to improve and prevent the postoperative cerebral angiospasm of subarachnoid hemorrhage and causes therewith Cerebral ischemia disease.Fasudil has the biological effect for inhibiting Rho kinases (Rho kinase).Clinically commonly use its hydrochloric acid Salt.

In addition to above-mentioned preferably specific TGF signal beta pathway inhibitor, aryl hydrocarbon receptor activator, Rho kinase inhibitor, It is other to can inhibit TGF signal beta access, activation aryl hydrocarbon receptor, inhibit Rho kinase activity, the identical target spot of induction regulating controlling, or The same type inhibitor of tool identical function can also realize same technical effect, should also be included in the present invention.

The invention also includes the compound equivalent with above compound I, II or III, pharmaceutical product, analog and/or its Salt, hydrate or precursor；It also include that it is generated and artificial-synthetic compound naturally.

The analog of the compound includes but is not limited to: isomers, the racemic modification of the compound.Compound has One or more asymmetric centers.So these compounds can be used as racemic mixture, individual enantiomter, Individual diastereoisomer, non-enantiomer mixture, cis or trans isomers exist." isomers " includes: geometry Isomers, enantiomter, diastereoisomer (such as cis-trans-isomer, conformer).

" salt " includes but is not limited to: (1) salt formed with following inorganic acid: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid Deng；(2) salt formed with following organic acid, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid or arginine Deng.Other salt include the salt formed with alkali or alkaline earth metal (such as sodium, potassium, calcium or magnesium).

" precursor of compound " refers to that the precursor of the compound is being cultivated after being applied or being handled with method appropriate A kind of compound of any of the above-described compound or one kind of any of the above-described compound can be transformed into base or in animal or human body Salt or solution composed by compound.

Pharmaceutical composition and its application

As used herein, term " containing " or " comprising " include "comprising", " substantially by ... constitute " and " by ... constitute ".Term " substantially by ... constitute " refer in the composition, in addition to containing neccessary composition or necessary component it Outside, also containing submember and/or impurity a small amount of and that do not influence effective component.For example, can be containing sweetener to change Kind taste, antioxidant are to prevent block and other medicated premix commonly used in the art, carrier, excipient.

As used herein, the ingredient of term " pharmaceutically acceptable " is suitable for people and/or animal and without excessively bad Side reaction (such as toxicity, stimulation and allergy), that is, there is the substance of reasonable benefit/risk ratio；Such as medicine commonly used in the art Object carrier or excipient.

As used herein, term " effective quantity " refer to people and/or animal can be generated function or it is active and can by people and/ Or the amount that animal is received.

As used herein, term " pharmaceutically acceptable carrier or excipient ", wherein carrier, which refers to, can change drug entrance The mode of human body and the rate of release of distribution, control drug in vivo and the system for conducting drugs to target organs；Drug Carrier itself is not necessary active constituent, and does not have excessive toxicity after applying.Suitable carrier is the common skill in this field Known to art personnel, including but not limited to: water, salt water, phosphate buffer and other aqueous solvents；(dimethyl is sub- by DMSO Sulfone), glycerol and ethyl alcohol and other organic solvents；Microballoon, liposome, microemulsion, high molecular surfactant；Colloid type carries medicine System, novel high polymer drug-loading system, new drug carrier and other carriers pharmaceutically；Wherein excipient refers in drug system Additives in agent in addition to main ingredient, alternatively referred to as auxiliary material.Such as binder, filler, disintegrating agent, the lubricant in tablet；Half Base portion in solid pharmaceutical preparation ointment, creme；Preservative, antioxidant, corrigent, aromatic, hydrotropy in liquid preparation Agent, emulsifier, solubilizer, osmotic pressure regulator, colorant etc. can be described as excipient.

General requirement to excipient is that property is stablized, and with main ingredient without incompatibility, does not generate side effect, does not influence to treat Effect, is unlikely to deform, is dry and cracked, going mouldy, damaging by worms, is harmless, at normal temperature without physiological action, does not generate chemistry or object with main ingredient Reason effect, does not influence the assay etc. of main ingredient.Pass can be found in Remington ' s Pharmaceutical Sciences In discussing fully for pharmaceutically acceptable carrier or excipient.Its carrier or excipient include but is not limited to: water, salt water, phosphorus The aqueous solutions such as acid buffer；The organic solvents such as DMSO (dimethyl sulfoxide), glycerol and ethyl alcohol；Microballoon, liposome, microemulsion, height Molecular surface active agent；Colloid type drug-loading system, novel high polymer drug-loading system, new drug carrier and other pharmaceutically Carrier；Preservative, antioxidant in liquid preparation, corrigent, aromatic, cosolvent, emulsifier, pH buffer substance, in tablet Binder, filler, lubricant and other medicines excipient etc..

As used herein, the pharmaceutical dosage form in term " pharmaceutical dosage form that composition can be prepared " refers to: for adaptation treatment or in advance Anti- needs and the medicinal application form prepared, referred to as pharmaceutical dosage form；The pharmaceutical dosage form packet that any combination object of the present invention can be prepared Include but be not limited to: pulvis, powder, tablet, pill, capsule, sustained release agent, rate controlling release agent and other solid dosage forms；It is injection, defeated Liquor, suspension and other dosage forms such as other liquid dosage forms and gas formulation, semisolid dosage form.

As used herein, " parts by weight " or " parts by weight " are used interchangeably, and the parts by weight can be any one Fixed indicates weight (such as 1ug, 1mg, 1g, 2g, 5g or kg) with microgram, milligram, grams or a kilogram number.For example, one by The composition that 1 parts by weight of component a and 9 parts by weight of component b is constituted, can be a+9 grams of component b of 1 gram of component, is also possible to 10 grams of groups Divide the composition of the compositions such as a+90 grams of component b.In the composition, degree=(weight of the component of a certain component The sum of number/all components parts by weight) × 100%.Therefore, the group being made of 1 parts by weight of component a and 9 parts by weight of component b It closes in object, the content of component a is 10%, and component b is 90%.The unit of weight of the weight part ratio may is that kilogram (kg), milli Any unit of weights such as gram (mg), microgram (ug).

In solution state, above-mentioned " parts by weight ", which can also convert, becomes " molal quantity "；" weight part ratio " can also convert As " molar concentration rate ".The molal unit of mole (concentration) ratio, which may is that, to rub (M), mmoles (mM), micro- rubs that (uM) etc. is any to rub That concentration unit.

In a preferred embodiment, there are active component Temozolomides and TGF beta inhibitor/aromatic hydrocarbon in the composition The ratio of receptor activators tranilast, Temozolomide and tranilast is 1:(1~30 according to weight)；Preferably 1:(1~ 20)；It is more preferably 1:(1~10)；Such as 1:8,1:5,1:3,1:2.In another preferred example, there is activity in the composition The ratio of component Temozolomide and Rho kinase inhibitor Fasudil, Temozolomide and Fasudil is 1:(0.1 according to weight ~10)；Preferably 1:(0.2~5)；It is more preferably 1:(0.3~3)；Such as 1:0.5,1:0.8,1:1,1:1.2,1:1.5.

As more preferable mode of the invention, in the composition, including Temozolomide, TGF beta inhibitor/aromatic hydrocarbon Receptor activators tranilast and Rho kinase inhibitor Fasudil, also, Temozolomide, tranilast and Fasudil Ratio is 1:(1~30 according to weight): (0.1~10)；Preferably 1:(1~20): (0.2~5)；More preferably for 1:(1~ 10) (0.3~3)；Such as 1:5:0.5,1:4:0.8,1:3:1,1:2:1.5.

It should be understood that the effective dose of composition used can be with the mould of application when for developing preparation pharmaceutical composition The type and the severity of disease of formula and tumour to be treated and change.And in vivo in use, usually using " weight/kilogram (weight) " is used as dosage unit；When the small molecule compositions are applied to big animal and tumour patient, agent is used by toy Amount is by corresponding professional reduction formula, and effective dosage of the big animal conversed or people is (including solid-state or solution state Dose lonvestion), also belong to protection scope of the present invention.

The dosage form of composition of the present invention is not particularly limited, can be any suitable for mammal clothes Dosage form；The dosage form that can be prepared includes: that pulvis, powder, tablet, pill, capsule, sustained release agent, rate controlling release agent and other solid Body dosage form；Injection, infusion solution, suspension and other liquid dosage forms；And other dosage forms such as gas formulation, semisolid dosage form. Preferably, the dosage form can be but not limited to: the solid dosage forms such as powder agent, granule, capsule, sustained release agent, tablet or note Penetrate the liquid dosage forms such as agent, infusion solution, solution, suspension.

The preparation method of composition of the invention determines according to the dosage form of required preparation and administration route, this field skill Art personnel are after with reference to combination provided by the present invention and proportion, using the preparation method of conventional pharmaceutical composition Prepare composition of the invention.

Although should be understood that the present inventor lists several composition forms, those skilled in the art in a specific embodiment Can also thus derive: other any combining forms of the invention are also same with prominent effect.

The present inventor confirms that small molecule compositions of the invention can be used for developing preparation prevention, improve or treat swollen for the first time The drug or pharmaceutical formulation of tumor.When for when preventing, improving or treat tumour, the effective dose of composition used can be with application Mode and tumor type to be treated and the severity of disease and change.Concrete condition according to the individual instances of subject come It determines, this is in the range of skilled practitioners or pharmacists may determine that.

In the present invention, the tumour cell is a kind of cell with the potential for being divided into neuron cell.Tumour Cell includes but is not limited to: astrocytoma, ependymoma, oligodendroglioma, brain stem glioma, mixed type glioma, Medulloblastoma, pernicious primitive neuroectodermal tumor, liver malignancy, pancreatic neoplasm etc..Although in the present invention, with colloid Representative of the blastoma as tumour cell, but it is as a kind of astrocytoma, with above-mentioned other tumours (ependymoma, Oligodendroglioma, brain stem glioma, mixed type glioma, medulloblastoma, pernicious primitive neuroectodermal tumor, liver Malignant tumour, pancreatic neoplasm) all have the potential for being converted into nerve cell.Wherein, ependymoma, oligodendroglioma, brain Dry glioma, mixed type glioma belong to glioma as glioblastoma；Medulloblastoma, pernicious original nerve Ectoderm tumour etc. originates from the cell of mesoderm growing early stage, these cells are considered to have the potential of Multidirectional Differentiation, can be lured by drug It leads as neuron cell.The astrocytoma includes: glioblastoma, diffusivity astrocytoma, denaturation star Shape cytoma etc..

As preferred embodiment of the invention, pharmaceutical composition of the invention is applied to that the swollen of the state of an illness can be improved by Temozolomide Tumor reinforces the therapeutic effect of Temozolomide, such as glioma, glioblastoma multiforme or human anaplastic astrocytoma etc..

It is according to the embodiment as a result, small molecule compositions of the invention to be applied to glioblastoma effect distinguished.Colloid Blastoma is the highest tumour of grade malignancy in astrocytic tumor.By glioblastoma cells induction for neuron The cell of property inhibits the tumour property of glioblastoma cells, provides feasible side for the treatment of glioblastoma Case.

Medicine box/kit

The present invention provides a kind of medicines for being used to prepare induction tumour cell and being changed into neuron cell or inhibition tumour Box/kit, contains: container 1, and the Temozolomide being placed in container 1 or its analog, isomers, salt, hydrate or preceding Body；Container 2 and the TGF beta inhibitor or aryl hydrocarbon receptor activator being placed in container 2；It container 3 and is placed in container 3 Rho kinase inhibitor.

The present invention also provides a kind of are used to prepare to induce tumour cell to be changed into neuron cell or inhibit tumour Medicine box/kit contains present invention pharmaceutical composition above-mentioned.

In addition, the material of some adjuvant drugs, such as injection needle tubing can also be contained in the medicine box/kit Deng.

In addition, can also contain operation instructions in the medicine box/kit, illustrate to combine based on the compound of the present invention The method for treating tumour.

The beneficial effects of the present invention are:

1. in the present invention, the small molecule compound property of application is stablized, and time, dosage and the combination of effect are easy to control System, function and effect are reliable and stable.Small molecule compound is readily produced application.

2. method of the invention, no foreign gene is imported, and does not change the gene structure of cell, avoid foreign gene import or Structural gene change causes new carcinogenic risk, therefore comparatively safe reliable.

6. being neuron cell by tumor cell induction, and can be reduced the ratio of stem cell in tumour cell in the present invention Example, effectively improves tumor treatment efficiency.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

I. material and method

1, patient specimen and animal

The collection of primary glioblastoma tissue sample is ratified by Huashan Hospital Affiliated To Fudan Univ Ethics Committee.This The tissue used in invention is all that the glioma classification standard announced by pathologist according to the World Health Organization regards as Section IV The glioblastoma of grade.All zooperies and corresponding research approach all follow NIH management of laboratory animal and guide for use. BALB/c nude mice is purchased from Shanghai Slac Experimental Animal Co., Ltd., raises under illumination in 12 hours/dark cycle environment, and Food and water are freely absorbed in permission.

2, primary glioblastoma cells separation and culture

Adult glioblastoma sample is transferred to laboratory in operation excision 1 hour and is separated.Sample is first It is washed one time by homemade D-Hank ' s solution, is then shredded with scissors.It is female for the colloid cultivated in containing serum training liquid The separation of cell carcinoma cells, the sample shredded are digested 25 minutes in 37 DEG C using 0.125% pancreas enzyme -EDTA solution.Digestion Tissue is blown and beaten into unicellular, is filtered with 40 micron screens, red blood cell cracked by homemade erythrocyte cracked liquid, glioblast Oncocyte single cell suspension utilizes the culture of DMEM+10% fetal calf serum.When glioblastoma cells are long full to 80-90%, They are passed on by pancreatin digestion.In order to reduce influence of the Long Term Passages to cell, the cell quilt in 2nd generation and the 5th generation For subsequent experimental.In order to separate the glioblastoma cells cultivated in serum-free medium, the sample tissue quilt shredded Accutase digestive juice digests 15 minutes, is then blown up and beats as single cell suspension.Red blood cell is also by homemade erythrocyte cracked liquid Cracking.It is unicellular, in 37 DEG C of 1ug/ml Laminin coatings, 2 hours culture plates, to be trained using serum-free by cover plant in advance Liquid culture (formula are as follows: Neurobasal Medium, 1 × N2 additive, 1 × B27 additive, 1 × nonessential amino acid, 1 × Sodium Pyruvate, 1 × glutamine, 10ng/ml basic fibroblast growth factor and 10ng/ml epithelical cell growth factor). When isolated cell density reaches 50%, they are digested by Accutase, and the balling-up culture in low adsorption culture plate；It is right In maintenance culture of the glioblastoma cells in serum-free medium, cell every 3~4 days by Accutase had digestive transfer culture.Alkali Property fibroblast growth factor and epithelical cell growth factor all add in culture solution daily.

3, induction gum blastoma cell stem cells into neuron-like cells changes

For the glioblastoma cells cultivated in serum, cell is existed with the density cover plant every square centimeter of 50,000 cells On the coated sheet glass of poly-D-lysine, and liquid culture, after 24 hours, cell density are trained using DMEM+10% fetal calf serum Should be greater than 90%, at this time cell culture fluid be replaced by nerve cell induction training liquid (nerve cell induction training liquid by STEMCELL The BrainPhys training liquid of company adds B27 additive, N2 additive, 10ng/ml brain-derived neurotrophic factor, 10ng/ml glue Cell plastid derived neurotrophic factor, 10ng/ml insulin-like growth factor, 0.2 μM of ascorbic acid, 100 μM of two one ring of butyryl Adenylate (dibutyryl-cAMP)), the concentration of TTF pharmaceutical composition is 50 μM of TMZ, 100 μM Tranilast and 50 μM Fasudil.It is primary that nerve-inducing including induced drug trains replacement in liquid every 4 days.For the glioblastoma of free serum culture Cell, cell with the density cover plant every square centimeter of 50,000 cells on polylysine and the coated sheet glass of Laminin, with nothing Serum trains liquid culture.2nd day, change above-mentioned nerve-inducing training liquid into, and 1ug/ml Laminin and relative medicine combination is added.Packet The nerve-inducing for including induced drug and Laminin trains replacement in liquid every four days once.Zeiss microscope (Zeiss Observer.Z1microscope) it is used to the photograph via bright field of shooting respective sample.

4, RNA extracting and real-time quantitative PCR

Trizol (sigma) reagent is used to extracting cell total rna, the extractive process experiment subsidiary according to Trizol reagent Operating process carries out.1 microgram RNA is used to do reverse transcription, reverse transcription be M-MLV reverse transcriptase, random Hexanucleotide primer, Under the conditions of deoxynucleotide etc. according to reagent manufacturer illustrate carry out.Real-time quantitative PCR is in corresponding primer and in real time Quantitative PCR reaction mixture utilizes the progress of MX3000P quantitative PCR apparatus.The relative expression levels of corresponding gene compare with inherence Gene HP RT compares acquisition.The primer sequence of real-time quantitative PCR such as table 1.

Table 1

5, flow cytometry and FACS sorting

The cell sorted for flow cytometry and FACS is digested using Accutase, and utilizes the MACS of pre-cooling Buffer (contains 0.5% fetal calf serum, 2mM EDTA) in 1 × PBS buffer solution.For cell surface protein (CD15, A2B5) Dyeing, cell is incubated for 30 minutes on ice in primary antibody or corresponding isotype control Ab；Two are washed using MACS buffer After, cell is incubated for 30 minutes on ice in the secondary antibody of fluorochrome label.For intracellular protein dye, cell first by The penetrating liquid fixation of the fixation of eBioscience company is penetrating, then washes one time by the penetrating liquid of eBioscience company, primary antibody Or corresponding isotype control Ab is diluted using penetrating liquid, and is incubated for 30 minutes on ice；After washing twice using penetrating liquid, Cell is incubated for 30 minutes on ice in the secondary antibody of fluorochrome label.After cell after secondary antibody incubation washes twice with corresponding cleaning solution Upper machine analysis or sorting.Flow cytometry and FACS sorting experiment are utilized respectively the progress of BD LSRII and BD Influx instrument. In order to analyze sorting after cell purity, sorting before and sorting after cell divided at once using BD Influx after sorting Analysis.Flow cytometry and the data of sorting are analyzed using Flowjo software.

6, immunofluorescence dyeing

For the cell of culture, cell is washed one time by 1 × PBS first, then fixes 10 in room temperature with 4% paraformaldehyde Minute.For mouse brain slice, mouse is utilized respectively 1 × PBS and the successive perfusion of 4% paraformaldehyde after being anesthetized, then mouse Brain is removed, and 4 DEG C of fixations are stayed overnight in 4% paraformaldehyde.For the nerve ball of culture, nerve ball be collected into 15 milliliters from Heart pipe, is placed at room temperature for, and after natural subsidence, fixes 15 minutes using 4% paraformaldehyde room temperature.Fixed brain or nerve ball Then 4 DEG C dehydration 48 hours in 30% sucrose, are transferred to after spy and freeze in protection liquid, and liquid nitrogen cryopreservation.Mouse brain or Person's nerve ball carries out frozen section with 30 microns of thickness.Later, the frozen section of the cell, mouse or the nerve ball that fix The penetrating closing of room temperature 1 hour, is then incubated overnight in 4 DEG C of primary antibodies in penetrating confining liquid.After experience PBS is sufficiently washed, The secondary antibody greenhouse of fluorochrome label is incubated for 1 hour.Nucleus is dyed using DAPI, carries out mounting later.For brain piece Upper TUJ1 and MAP2 dyeing, brain piece carry out antigen retrieval first with the antigen retrieval buffers of green skies company before penetrating closing. For BrdU Coloration experiment, brain piece is first handled 30 minutes at 37 DEG C of hydrochloric acid of 1 mole every liter before penetrating closing, then with 1 × PBS is sufficiently washed.Immunofluorescence picture is shot using Leica Lycra SP8 Laser Scanning Confocal Microscope.

7, electro physiology detects

Whole-cell recording uses Multiclamp 700B amplifier.Cell maintains the mechanical brains of flowing always In spinal fluid, and it is continually fed into 95% oxygen/5% carbon dioxide, artificial cerebrospinal fluid formula is 93mM K-IAO, 16mM chlorination Potassium, 2mM magnesium chloride, 10mM 4- hydroxyethyl piperazineethanesulfonic acid, 4mM atriphos magnesium salts, 0.3mM guanosine triphosphate disodium salt, 10mM Creatine Phosphate Sodium, 488 dyestuff of 0.5mM Alexa Fluor, 0.4% neurobiotin, pH 7.25, osmotic pressure 290- 300 milliosmols.For induced action potential, cell membrane potential maintains -20 millivolts, the incremental electric current injection of 10 pico-amperes training Cell.10 millivolts of incremental voltage injects cell to induce inside sodium ion electric current.Data utilize 10 software of pClamp point Analysis.

8, hematoxylin eosin stain

Hematoxylin eosin stain is carried out using the hematoxylin eosin stain liquid in the green skies.Frozen section is in distillation washing one time Afterwards, it is dyed 10 minutes using hematoxylin solution greenhouse, (about 10 minutes) is then sufficiently washed using distilled water, then 95% ethanol washing 10 seconds is incubated for 10 seconds in eosin stains liquid later, finally twice with 70% ethanol washing, and carries out Mounting.Hematoxylin eosin stain picture is shot using Zeiss microscope (Observer.Z1), and using in ImageJ software Jigsaw puzzle plug (stitching plugin) carries out picture mosaic to obtain full mind map.

9, cell invasion is tested

The invasive ability of cell is detected using invasion plank (Corning).It first uses the bottom side of built-in film 0.1% 37 DEG C of gelatin is coated with 30 minutes.20000 cell cover plants are in the cell on film top, and with 100 microlitres of serum-frees DMEM/F12 culture solution culture, 500 microlitres of DMEM/F12 culture solutions containing serum are added in the hole of orifice plate (film bottom side).Carefully After born of the same parents cultivate 12 hours in 5% carbon dioxide, 37 DEG C of incubator, cell is fixed by 4% paraformaldehyde, and utilizes crystal violet Dyeing liquor dyeing.Violet staining picture utilizes Zeiss microscope (Observer.Z1).

10,5- bromodeoxyuridine nucleosides (5-Bromo-2-deoxyUridine, EdU) incorporation experiment

10 μM of EdU are added in corresponding cell supernatant, and it is small that 2 are cultivated in 5% carbon dioxide, 37 DEG C of incubator Shi Hou, is digested to unicellular using Accutase, is fixed 15 minutes with 4% paraformaldehyde room temperature, is contained later using penetrating liquid 1 × PBS progress of 1%Saponin is penetrating, and the penetrating reaction mixture of Click-iT after ten minutes is added directly into respective sample And be incubated for 30 minutes in room temperature dark place, it recycles DAPI to dye nucleus, undergoes upper machine flow cytometer detection after sufficiently washing. BD LSRII instrument and Flowjo are respectively used to obtain and analyze data.

11, nerve ball is formed and Method of Limited Dilution is tested

Nerve ball formation is that the glioblastoma of 100 serum-free medium cultures of cover plant in each hole of 96 orifice plates is thin Born of the same parents are unicellular, and cultivate in serum-free medium.Each hole is shot using Zeiss microscope (Observer.Z1) after 10 days Picture, and picture mosaic is carried out using the jigsaw puzzle plug (Stitching plugin) in ImageJ software to obtain each hole Figure.Method of Limited Dilution experiment is the glue that 1,3,10,30 and 100 serum-free medium culture of cover plant is distinguished in each hole of 96 orifice plates Matter blastoma cell is unicellular, and cultivates in serum-free medium.Record whether each hole has nerve ball to be formed after 10 days, And it is analyzed and is counted using Elda online software.

12, the tumour cell heterograft in patient source and bioluminescence living imaging

The BALB/c nude mice of the female of 6 week old is used as the receptor of heterograft.Primary glioblastoma is thin Born of the same parents' such as above method separates, and cultivates in serum-free medium, and is marked using the slow virus of expression GFP and luciferase Note.The glioblastoma cells of culture are digested to unicellular using Accutase, and are resuspended to 100,000 cells with PBS per micro- It rises, ready cell suspension is placed on ice, and every 5 minutes even with finger bullet.For encephalic orthotopic transplantation, mouse is utilized It is placed and secured in after yellow Jackets holonarcosis on locating injection instrument, 500,000 cells inject position by locating injection to intracerebral Set the coordinate relative to Bergma are as follows: AP axis :+1.0mm, ML axis :+2.0mm, DV axis: -2.5mm.The speed of injection is by digital pump Control, and it is maintained at 1 microlitre per minute.After having injected, injection needle is kept 2 minutes in situ, later just slowly extracts needle out, To prevent cell from flowing backwards.For subcutaneous transplantation, 2,000,000 cells are transplanted to subcutaneous on the left of mouse.It is swum at corresponding time point Mark the length and width of calliper to measure tumour, the calculation formula of gross tumor volume are as follows: V=(π/6) * L*W2, wherein L and W are respectively The length and width of tumour.According to Ethical Demand, when the weight loss of mouse be more than 20% or when mouse be unable to free water, When feed or action, mouse is carried out euthanasia.

Bioluminescence living imaging is realized using Xenogen IVIS Spectrum in-vivo imaging system.D type fluorescent Mouse is put into isoflurane gas Anesthesia machine is anaesthetized after five minutes by element with the dosage Intraperitoneal injection mouse of 150mg/kg weight, Injection carries out mouse living imaging after ten minutes.Imaging parameters are as follows: biodiversity resources: time for exposure: 1 minute；Binning: Medium；F/stop:1；Exciter filter: Block；Emit optical filter: Open；Photograph via bright field: time for exposure: 0.2 minute； Binning:Medium；F/stop:8.

13, in body administration and BrdU label

It is administered, is administered once a day corresponding number of days after transplanting, until dead mouse.0.5% carboxymethyl cellulose Plain sodium solution is used as drug administration carrier (Vehicle).Mouse weight, TMZ and Tranilast difference are all measured before daily administration Primary with the daily gastric infusion of the dosage of 30mg/kg and 120mg/kg weight, Fasudil is every with the dosage of (1) 30mg/kg weight Its intraperitoneal injection is primary；Or the daily gastric infusion of dosage of (2) 60mg/kg weight is primary.Data administration in 4J, 4K, 4L Mode is that TMZ, Tranilast and Fasudil are gastric infusion；Except 4J, outside this 3 small figures of 4K, 4L, other internal animals The administration mode of experimental data is that TMZ and Tranilast is gastric infusion, and Fasudil is intraperitoneal injection.BrdU is marked Experiment, BrdU are carried out after one hour using 1 × PBS and 4% paraformaldehyde with the dosage Intraperitoneal injection mouse of 50mg/kg weight Perfusion, and mouse brain is taken out, later the step of it is identical as mouse brain processing step in above-mentioned immunofluorescence dyeing.

14. cell viability detects

In order to detect cell viability, the cell of the training liquid culture containing serum is inoculated into 96 orifice plates by the density in 5000 every holes In, second day training liquid changes nerve-inducing training liquid into, and corresponding drug is added and is handled.CellTiter-Glo reagent (Promega) it is added in orifice plate according to the explanation of reagent manufacturer at corresponding time point, orifice plate is protected from light room temperature on shaking table It shakes after ten minutes, detects bioluminescence using SpectraMax M5 (Molecular Devices).

15, data are analyzed

All quantitative datas show all in the form of average value ± standard error, and for statistical analysis.T inspection, single factor test Variance analysis, two analysis of variance etc. are used for computational statistics p value, and specific method is shown in each figure explanation.P value is less than 0.05 quilt Think with statistical difference.* indicate that p indicates that p indicates that p is less than less than 0.01, * * * and ### less than 0.05, * * and ## with # 0.001。

II. embodiment

The glioblastoma cells that embodiment 1, pharmaceutical composition induction are cultivated in the training liquid containing serum are neuron Cell

These cells are being contained blood respectively by the isolated primary glioblastoma cells from glioblastoma tissue It is cultivated in the cleer and peaceful culture solution without serum, the result of cell culture is as shown in Figure 1A.

Stem cells film surface marker CD15 and A2B5 are detected using cell streaming art, as shown in Figure 1B, is containing blood The glioblastoma cells cultivated in clear training liquid there is no the cell of CD15 or the A2B5 positive, and in serum-free medium The cell for having 10~15% or so in the cell of middle culture is CD15 or A2B5 positive, they are deemed likely to be that colloid is female thin Born of the same parents tumor stem cell.

The expression of stem cell labeling object NETSIN and SOX2 in Immunofluorescence test glioblastoma cells.As a result Such as Fig. 1 C, the glioblastoma cells cultivated in containing serum training liquid there is no the cell of NETSIN or the SOX2 positive, And the cell cultivated in serum-free medium is then with the presence of apparent NETSIN or SOX2 positive cell.

Glioblastoma cells are handled with TTF pharmaceutical composition, generating using qPCR detection neuron has critical function Transcription factor ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 etc. expression.The results show that in TTF pharmaceutical composition In processing glioblastoma cells 5 days, the expression of transcription factor ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 have Different degrees of raising, such as Fig. 1 D.

TTF pharmaceutical composition and control treatment glioblastoma cells pass through TTF combined treatment using Immunofluorescence test After 5 days in glioblastoma cells NEUROD1 expression.As a result such as Fig. 1 E, treated that cell is rendered as by display TTF NEUROD1 is positive.

TTF pharmaceutical composition and control treatment glioblastoma cells detect the expression feelings of nerve cell marker DCX Condition.As a result such as Fig. 1 F, after display TTF is induced 5 days, glioblastoma cells express nerve cell marker DCX.

TTF pharmaceutical composition handles glioblastoma cells, detects the expression of nerve cell marker MAP2.As a result Such as Fig. 1 G, after showing that TTF is induced 8 days, glioblastoma cells express nerve cell marker MAP2.

TTF pharmaceutical composition handles glioblastoma cells, utilizes patch clamp technique detection glioblastoma cells Electrophysiological index.As a result as Fig. 1 H is shown.TTF pharmaceutical composition handles glioblastoma cells, measurement glue after TTF is handled Matter blastoma cell can be induced action potential and sodium ion electric current.As a result such as Fig. 1 I and Fig. 1 J is shown, is shown by TTF The property of pharmaceutical composition treated cell obtains nerve cell.

TTF pharmaceutical composition handles glioblastoma cells, measures the neuron cell after TTF is induced 8 days Purity and transformation efficiency (being counted based on MAP2 coloration result), as a result as Fig. 1 K and Fig. 1 L are shown, it is seen that TTF processing can be extremely It is significantly neuron cell by glioblastoma cells.

TTF pharmaceutical composition, TMZ and control treatment glioblastoma cells, detection are measured intracellular using bioluminescence ATP is horizontal, to detect influence of the TTF to cell viability.As a result as shown in Figure 6A, TTF reduces cell viability significantly.

The lower cellular morphology of time-lapse shooting TTF processing, such as Fig. 6 B, display glioblastoma cells cellular morphology is from flat Shape is changed into the cellular morphology of nerve cell.

The expression of Immunofluorescence test glioblastoma cells NEUROD1, DCX, MAP2 etc. after TTF is handled. Such as Fig. 6 C to Fig. 6 E, after TTF is handled 5 days or 8 days, glioblastoma cells express the nerves such as NEUROD1, DCX, MAP2 Cell marker.Such as Fig. 6 F to Fig. 6 G, after TTF is handled 8 days, the expression of neuron marker SYN1, VGLUT1 are rendered as sun Property.

According to the above results, the glioblastoma cells that the induction of TTF pharmaceutical composition is cultivated in the training liquid containing serum are Neuron cell.

The glioblastoma cells cultivated in embodiment 2, pharmaceutical composition induction serum-free medium are changed into neuron Cell

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), and measurement is handled 6 days by TTF (d6) cellular morphology of glioblastoma cells afterwards, not add glioblastoma cells (the serum-free medium training of TTF Support) it is control.As a result it shows such as Fig. 2A, by TTF, treated that cell is rendered as neuron cell form.

TTF pharmaceutical composition handle glioblastoma cells (serum-free medium culture), using qPCR detection SCL1, The expression of the transcription factors such as BRN2, MYT1L, NEUROD1, NEUROG2.As a result as Fig. 2 B, display TTF handle glioblastoma Cell the 0th, 2,4,6 day, the expression of the transcription factors such as ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 had significant up-regulation, And the expression of stem cell labeling object CD133 and CD15 are significantly lowered.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), using Immunofluorescence test mind Expression through cell marker DCX and MAP2, not add glioblastoma cells (the serum-free medium training of TTF Support) it is control.As a result as illustrated in figs. 2 c and 2d, after TTF is induced 6 days, glioblastoma cells express nerve cell marker DCX and MAP2.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), using Flow cytometry The expression of glioblastoma cells DCX, not add the glioblastoma cells (serum-free medium culture) of TTF For control.As a result it such as Fig. 2 E, is handled 6 days by TTF, there is a certain proportion of DCX positive cell in cell.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), passes through TTF using flow cytometer detection After processing 6 days in glioblastoma cells MAP2 expression, not add glioblastoma cells (no blood of TTF Clear culture solution culture) it is control.As a result such as Fig. 2 F, there is a certain proportion of MAP2 positive cell in TTF treated cell.

Using the cell of fluidic cell fluorescence sorting technology enrichment the CD15 positive and CD15 feminine gender, and examined with flow cytometry The purity of enrichment of cell is surveyed, as shown in Figure 2 G.Immunofluorescence assay such as Fig. 2 H shows, TTF is by the cell of the CD15 positive of enrichment The cell for the MAP2 positive is all induced with the cell of CD15 feminine gender.

Using the cell of fluidic cell fluorescence sorting technology enrichment the A2B5 positive and A2B5 feminine gender, and examined with flow cytometry The purity for surveying enrichment of cell, as Fig. 2 I is shown.Epidemic disease fluoremetry such as Fig. 2 J shows, TTF by the cell of the A2B5 positive of enrichment and The cell that the cell of A2B5 feminine gender all induces as the MAP2 positive.

According to the above results, the glioblastoma cells cultivated in TTF pharmaceutical composition induction serum-free medium are changed into Neuron cell.

Embodiment 3, pharmaceutical composition inhibit the tumour property of glioblastoma cells

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is control, and observation control and TTF handle the glioblastoma cells balling-up energy for cultivating in serum-free The influence of power.As a result such as Fig. 3 A is shown, TTF processing is substantially reduced the balling-up ability of glioblastoma cells.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is control, and the colloid cultivated in serum-free under gradient dilution experiment detection control and TTF treatment conditions is female thin The clonality of born of the same parents' oncocyte.As a result show such as Fig. 3 B, TTF processing significantly reduce in glioblastoma cells with gram The ratio of grand Forming ability cell.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is control, using flow cytometry after TTF is handled 6 days positive thin of stem cell labeling object CD15 Born of the same parents' ratio.As a result such as Fig. 3 C~D, the cell proportion of the stem cell labeling object CD15 positive are remarkably decreased.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is control, using flow cytometry after TTF is handled 6 days positive thin of stem cell labeling object A2B5 Born of the same parents' ratio.As a result such as Fig. 3 E~F, the cell proportion of the stem cell labeling object A2B5 positive are remarkably decreased.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is control, and observation compares the influence after handled 6 days with TTF for glioblastoma cells balling-up ability. As a result as the balling-up ability of Fig. 3 G, control and the glioblastoma cells after TTF processing 6 days obviously weakens.Fig. 3 G Be with the difference of Fig. 3 A: Fig. 3 A is to add DMSO (control) or TTF to handle while balling-up experiment, and Fig. 3 G is that cell first passes through It crosses after DMSO (control) or TTF is handled 6 days and carries out balling-up experiment again, all without drug-treated in globulation.

TTF pharmaceutical composition handles glioblastoma cells (serum-free medium culture), with the colloid handled without TTF Blastoma cell is to compare, gram of gradient dilution experiment detection control and the glioblastoma cells after TTF processing 6 days Grand Forming ability.As a result there is clonality cell in the glioblastoma cells after handling 6 days such as Fig. 3 H, TTF Ratio considerably less than control group.The difference of Fig. 3 H and Fig. 3 B is: Fig. 3 B is while detecting clonality experiment Add DMSO (control) perhaps TTF handles Fig. 3 H is that cell first passes through DMSO (control) or TTF and handles and detects again later within 6 days gram Grand Forming ability, detecting does not have drug-treated in the experiment of clonality.

TTF pharmaceutical composition handles glioblastoma cells, and (wherein GBM-17 is serum-free medium culture, and GBM-3 is Cell containing serum free culture system), it is thin to mix experiment detection using EdU for control for the glioblastoma cells to handle without TTF Born of the same parents' proliferative conditions.As a result such as Fig. 3 I~J, after being handled 24 hours by TTF, the cell proportion in proliferation period of the EdU positive It is decreased obviously.

For glioblastoma cells (cell containing serum free culture system) after TTF is handled 5 days and 10 days, qPCR detects cell Cycle associated genes and be enriched in glioblastoma expression gene expression.As a result it such as Fig. 3 K, is handled by TTF After 5 days and 10 days, the expression of most cells cycle associated genes has different degrees of downward.

Glioblastoma cells (cell containing serum free culture system) are handled 5 days by TTF, measure cell proliferation markers The ratio of the KI67 positive.As a result as shown in Fig. 3 L~M, the ratio of the cell proliferation markers KI67 positive is substantially reduced.

Glioblastoma cells (cell containing serum free culture system) are handled 5 days by TTF, Transwell experiment and crystallization The invasive ability of purple dyeing detection cell.As a result as shown in Fig. 3 N~O, after TTF is handled 5 days, glioblastoma cells are invaded Ability is attacked obviously to weaken.

Immunofluorescence test neuron marker TAU positive table in the glioblastoma cells after processing 6 days in TTF Up to situation, as shown in Figure 7 A, significant expression is presented in TAU.

Cell proportion of the Immunofluorescence test by the GFAP positive in TTF processing and the cell compareed.As a result such as Fig. 7 B~C Shown, compared with the control group, the cell proportion of the GFAP positive is considerably lower in the cell handled by TTF.

The Immunofluorescence test glioblastoma that another strain is cultivated in serum-free medium after TTF is handled 6 days is thin The case where born of the same parents (being the cell in different patient sources from the glioblastoma cells in Fig. 2) expression DCX, MAP2 and TAU, with not The glioblastoma cells (serum-free medium culture) for adding TTF are control.As a result as shown in Fig. 7 D~F, TTF induction 6 After it, glioblastoma cells express nerve cell marker DCX, MAP2, TAU.

Glioblastoma cells are (with the glioblastoma in Fig. 2 after being handled 6 days using Flow cytometry TTF Cell be different patient sources cell) expression DCX and MAP2 the case where, not add the glioblastoma cells (nothing of TTF The culture of serum free culture system liquid) it is control.As a result such as Fig. 7 G~H, there is a certain proportion of DCX and MAP2 sun in TTF treated cell Property cell.

According to the above results, the glioblastoma cells cultivated in TTF pharmaceutical composition induction serum-free medium are changed into Neuron cell, TTF pharmaceutical composition are able to suppress the tumour property of glioblastoma cells.

Embodiment 4 trains the glioblastoma cells cultivated in liquid containing serum, gives the effect measuring of different pharmaceutical combination

The glioblastoma cells cultivated in containing serum training liquid, identify the expression feelings of each gene under different cultivation conditions Condition.

Immunofluorescence test astroglia gene GFAP, S100B, stem cell gene SOX2, NESTIN, neuron base The expression such as Fig. 5 A~B in glioblastoma cells cultivated in containing serum training liquid by MAP2, NEUROD1, DCX. The results show that there are GFAP, S100B positive cells, it is positive thin but there is no SOX2, NESTIN, MAP2, NEUROD1, DCX Born of the same parents.

Detection neuron generates relevant transcription factor MYT1L, NEUROG2 etc. in non-tumour and glioblastoma Expression.As a result such as Fig. 5 C, show that the expression of these transcription factors in glioblastoma has significant decline.

The correlation of NEUROD1 or MAP2 expression height and disease prognosis in patient is observed respectively.As a result such as Fig. 5 D is shown, Compared with NEUROD1, perhaps low patient is expressed in MAP2 expression NEUROD1 or MAP2 express high patient life cycle it is significant It is longer.

Observation give glioblastoma cells different pharmaceutical and control treatment after, MYT1L, NEUROD1 therein and The differential expression of NESTIN.As a result as shown in fig. 5e, Tranilast being added on the basis of TMZ can significantly raise The expression of NEUROD1, MYT1L.

The cellular morphology after glioblastoma cells different pharmaceutical and control treatment is given in observation.As a result such as Fig. 5 F institute Show, TMZ can substantially change cellular morphology plus Fasudil, by the cellular morphology of glioblastoma cells by flat change For the cellular morphology as nerve cell.

Detection removes the changes in gene expression of any one drug on the basis of TTF.As a result as depicted in fig. 5g, TTF is combined The expression that neuron generates important transcription factor ASCL1, BRN2, MYT1L, NEUROD1, NEUROG2 etc. can be significantly raised, And any one drug is removed on the basis of TTF all can influence the up-regulation multiple for these transcription factors.

Embodiment 5, pharmaceutical composition inhibit tumour growth in vivo

In the present embodiment, the test that pharmaceutical composition of the invention inhibits tumour growth in vivo, experiment in vivo process are carried out Such as Fig. 4 A.The primary colloid marked by the slow virus of expressing green fluorescent protein (GFP) and luciferase (luciferase) Blastoma cell is transplanted in mouse brain, is started to give relative medicine processing after 4 days, is administered once a day until dead mouse Or it is condemned to death.In the 30th day progress hematoxylin eosin stain and immunofluorescence dyeing.BrdU label is previous in execution mouse Hour injection, it is injected intraperitoneally by the amount of 50 milligrams per kilogram of weight.Bioluminescence living imaging since the 4th day, weekly at As primary.Life cycle analysis is the natural death time for recording mouse, and the time put to death mouse due to ethical concerns.Point Group are as follows: Vehicle group gives 0.5% carboxymethylcellulose sodium solution；TMZ is singly administered in TMZ group；TTF group administration TMZ, Tranilast and Fasudil.

Obtain intracerebral tissue preparation brain piece, the expression of Immunofluorescence test TUJ1.As a result such as Fig. 4 B~C, TTF pharmaceutical composition The ratio of TUJ1 positive cell is increased significantly in the mouse brain of processing, prompts TTF combination that can promote colloid female thin in vivo The property of born of the same parents' oncocyte acquisition neuronal cell.

Intracerebral tissue preparation brain piece is obtained, Immunofluorescence test BrdU mixes situation.As a result such as Fig. 4 D~E, TTF medicine group The cell proportion for closing the BrdU positive in the mouse brain of processing in proliferation period is declined.

Intracerebral tissue preparation brain piece is obtained, the Caspase-3 of shearing is passed through on Immunofluorescence test mouse brain slices The expression of (caspase-3 activated form).As a result it such as Fig. 4 F~G, is activated in the mouse brain handled by TTF pharmaceutical composition The ratio of the Caspase-3 of form is raised.

Intracerebral tissue preparation brain piece is obtained, the size of GFP and hematoxylin eosin stain experiment detection intracerebral tumour are passed through.Knot Fruit such as Fig. 4 H~I, TTF handle the mouse that the tumor size in mouse brain is less than TMZ processing.

The dynamic change of bioluminescence living imaging detection tumor size.As a result it such as Fig. 4 J~K, shows compared with TMZ, The ability that TTF combination significantly inhibits tumour growth is stronger (TMZ, Tranilast, Fasudil are gastric infusion).

Make the survivorship curve of each group mouse.As a result such as Fig. 4 L, it is compared to control group, TTF can extend glue significantly The life cycle of matter blastoma model mice, and TMZ prolongation effect is obvious not as good as TTF that (TMZ, Tranilast, Fasudil are Gastric infusion).

The dynamic change of bioluminescence living imaging detection tumor size.As a result it such as Fig. 4 M~O, shows compared with TMZ, TTF combination significantly inhibits the ability of tumour growth, and stronger (TMZ, Tranilast are gastric infusion, and Fasudil is that abdominal cavity is infused It penetrates).

Make the survivorship curve of each group mouse.As a result such as Fig. 4 P, it is compared to TMZ, TTF can extend colloid mother significantly The life cycle of cytoma model mice (TMZ, Tranilast are gastric infusion, and Fasudil is intraperitoneal injection).

Give TTF pharmaceutical composition, within 31 day time, observe its influence for mouse weight, with give TMZ and Vehicle is control.As a result such as Fig. 8 A, give influenced caused by TTF pharmaceutical composition it is suitable with the influence for giving TMZ.

Utilize hematoxylin eosin stain detection control group, heart, the kidney, liver of TMZ administration group and TTF administration group mouse The slices of organs such as dirty, spleen and lung.As a result such as Fig. 8 B, TMZ or TTF is not observed, apparent device matter is generated to above-mentioned organ Property damage.

It is subcutaneous that glioblastoma is transplanted to mouse, gives relative medicine processing.As a result such as Fig. 8 C~F, with TMZ phase Than TTF inhibits the ability of tumour growth significantly stronger.

According to the above results, TTF pharmaceutical composition can inhibit tumour growth significantly in vivo.

The inducing action of embodiment 6, different compound combinations

The present inventor have detected TMZ and different TGF beta inhibitors, aryl hydrocarbon receptor activator, Rho kinase inhibitor into Inducing action after row combination, the induction of observation compound combination become the form of the glioblastoma cells containing serum free culture system Change, the results are shown in Table 2.

Table 2

Compound combination	Neuron cell (%)	Means neurite plays number/cell
			Ctrl	7.23±2.01	0.184±0.0384
TMZ+Leflunomide+Fasudil	31.78±2.35	0.843±0.0947
			TMZ+Pirfenidone+Fasudil	41.43±3.82	0.995±0.150
TMZ+SB431542+Fasudil	37.01±2.60	1.041±0.146
			TMZ+Tranilast+Fasudil	64.05±4.13	1.360±0.143

" neuron cell (Neuronal like cell) (%) ", which refers to after relative medicine combined treatment 4 days, to be had There are the cell percentages of neuronal cell morphology." means neurite plays number/cell (Average neurite number Per cell) " refer to the nervous process number that average each cell has after relative medicine combined treatment 4 days.

The result of table 2 illustrates, TMZ and TGF beta inhibitor not of the same race, aryl hydrocarbon receptor activator, and with Rho kinase inhibition Agent combined application, having induction tumour cell is the ability of neuron cell.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

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Claims (18)

1. a kind of composition for inhibiting tumour, which is characterized in that there are the following group active components in the composition:

(1) Temozolomide or its analog, isomers, salt, hydrate or precursor；With

(2) selected from following (a)~(b) the two or one of:

(a) compound selected from TGF beta inhibitor or aryl hydrocarbon receptor activator；

(b) Rho kinase inhibitor.

2. composition as described in claim 1, which is characterized in that the TGF beta inhibitor or aryl hydrocarbon receptor activator packet It includes: tranilast, leflunomide, pirfenidone, SB431542, Repsox or its analog, isomers, salt, hydrate or preceding Body.

3. composition as described in claim 1, which is characterized in that the Rho kinase inhibitor includes: Fasudil, Ripasudil or its analog, isomers, salt, hydrate or precursor.

4. composition as claimed in claim 2 or claim 3, which is characterized in that there are active components in the composition for not azoles The ratio of amine and tranilast, Temozolomide and tranilast is 1:(1~30 according to weight)；Preferably 1:(1~20)；More It goodly is 1:(1~10)；Such as 1:8,1:5,1:3,1:2.

5. composition as claimed in claim 2 or claim 3, which is characterized in that there are active components in the composition for not azoles Amine and Fasudil, wherein the ratio of Temozolomide and Fasudil is 1:(0.1~10 according to weight)；Preferably 1: (0.2~5)；It is more preferably 1:(0.3~3)；Such as 1:0.5,1:0.8,1:1,1:1.2,1:1.5.

6. composition as claimed in claim 2 or claim 3, which is characterized in that there are active components in the composition for not azoles Amine, tranilast and Fasudil, wherein the ratio of Temozolomide, tranilast and Fasudil according to weight be 1:(1~ 30): (0.1~10)；Preferably 1:(1~20): (0.2~5)；More preferably it is 1:(1~10) (0.3~3)；Such as 1:5:0.5, 1:4:0.8,1:3:1,1:2:1.5.

7. composition as described in claim 1, which is characterized in that the composition is by inducing tumour cell to be changed into mind Through first like cell, and/or the stem cell ratio in tumour cell is reduced, to inhibit tumour.

8. composition as described in claim 1, which is characterized in that the cell of the tumour, which has, is divided into neuron cell Potential；Preferably, the tumour includes: astrocytoma, ependymoma, oligodendroglioma, brain stem glioma mixed Mould assembly glioma, medulloblastoma, pernicious primitive neuroectodermal tumor, liver malignancy, pancreatic neoplasm；Preferably, institute The astrocytoma stated includes: glioblastoma, diffusivity astrocytoma, human anaplastic astrocytoma.

9. composition as described in claim 1, which is characterized in that further include pharmaceutically acceptable in the composition Carrier or excipient；Or

The dosage form of the composition include: pulvis, powder, tablet, pill, capsule, sustained release agent, rate controlling release agent, injection, Infusion solution, suspension.

The purposes of 10.TGF beta inhibitor or aryl hydrocarbon receptor activator, for Temozolomide or its analog, isomers, salt, Hydrate or precursor complex, preparation induction tumour cell are changed into neuron cell or inhibit the composition of tumour；Preferably, The TGF beta inhibitor includes: tranilast or its analog, isomers, salt, hydrate or precursor.

The purposes of 11.Rho kinase inhibitor, for matching with Temozolomide or its analog, isomers, salt, hydrate or precursor It closes, preparation induction tumour cell is changed into neuron cell or inhibits the composition of tumour；Preferably, the Rho kinases Inhibitor includes: Fasudil or its analog, isomers, salt, hydrate or precursor.

12. the purposes of any composition of claim 1~8, which is characterized in that be used to prepare induction tumour cell transformation For neuron cell or drug, cells transdifferentiate reagent, kit or the medicine box of inhibition tumour.

13. a kind of method that induction tumour cell is changed into neuron cell, which is characterized in that the described method includes: using Any compositions-treated tumour cell of claim 1~8, so that tumour cell be made to be changed into neuron cell；Compared with Goodly, the tumour includes: astrocytoma, ependymoma, oligodendroglioma, brain stem glioma, mixed type colloid Tumor, medulloblastoma, pernicious primitive neuroectodermal tumor, liver malignancy, pancreatic neoplasm etc.；Preferably, the star Shape cytoma includes: glioblastoma, diffusivity astrocytoma, human anaplastic astrocytoma.

14. method as claimed in claim 13, which is characterized in that the tumor cell culture is not in containing serum or containing In the culture medium of serum.

15. a kind of for inducing tumour cell to be changed into neuron cell or inhibiting medicine box/kit of tumour, feature exists In, comprising:

(1) Temozolomide or its analog, isomers, salt, hydrate or precursor；With

(2) selected from following (a)~(b) the two or one of:

(a) compound selected from TGF beta inhibitor or aryl hydrocarbon receptor activator；

(b) Rho kinase inhibitor.

16. composition as claimed in claim 15, which is characterized in that the TGF beta inhibitor or aryl hydrocarbon receptor activator Include: tranilast, leflunomide, pirfenidone, SB431542, Repsox or its analog, isomers, salt, hydrate or Precursor；Or

The Rho kinase inhibitor includes: Fasudil, Ripasudil or its analog, isomers, salt, hydrate or Precursor.

17. medicine box/kit as claimed in claim 16, which is characterized in that wherein there is active component Temozolomide and song Ni Site, wherein the ratio of Temozolomide and tranilast is 1:(1~30 according to weight)；Preferably 1:(1~20)；More It goodly is 1:(1~10)；Such as 1:8,1:5,1:3,1:2；Or

Wherein there is active component Temozolomide and Fasudil, wherein the ratio of Temozolomide and Fasudil is according to weight For 1:(0.1~10)；Preferably 1:(0.2~5)；It is more preferably 1:(0.3~3)；Such as 1:0.5,1:0.8,1:1,1:1.2, 1:1.5；Or

Wherein there is active component Temozolomide, tranilast and Fasudil, wherein Temozolomide, tranilast and method are relaxed Your ratio of ground is 1:(1~30 according to weight): (0.1~10)；Preferably 1:(1~20): (0.2~5)；More preferably it is 1: (1~10) (0.3~3)；Such as 1:5:0.5,1:4:0.8,1:3:1,1:2:1.5.

18. the medicine box as described in claim 15~17 is any, which is characterized in that also contain in the medicine box: operation instruction Book, the method for illustrating to treat neuroblastoma.