CN104800231B - Application of the Amifostine in Cyclin D_1 gene height expression tumor types - Google Patents

Abstract

The invention discloses a kind of Amifostine（Chemical name：YM-038310 Ed, English name：Amifostine, referred to as：AMF）In treatment Cyclin D_1 gene（CyclinD1）Application in height expression tumor types.The present inventor has found the effect for the suppression growth of tumour cell that there is Amifostine targeted inhibition oncogene CyclinD1 to express by studying and by suppressing the antitumor action of CyclinD1 expression, and is successfully authenticated this effect by MEG cell lines Dami；In clinical practice, the lymphoma mantle cell being characterized with the expression of CyclinD1 height and leucocyte height expression and is successfully cured.

Description

Application of the Amifostine in Cyclin D_1 gene height expression tumor types

Technical field

Field of medicaments is learned the invention belongs to raw, is related to a kind of Amifostine in Cyclin D_1 gene height expression tumor types Using.

Background technology

Cyclin D_1 gene（CyclinD1）The high expression in kinds of tumor cells, including it is lymphoma mantle cell, acute and chronic Leukaemia, breast cancer, the cancer of the esophagus, lung cancer, prostate cancer, thyroid cancer, myeloma, head and neck neoplasm, sarcoma, stomach cancer, uterine neck Cancer, colon cancer, glioma etc., are a preferable cancer targets.But clinical shortage targetedly control by targeting at present Treat medicine.

Cyclin D_1 gene（CyclinD1）Carcinogenic molecule mechanism is as follows：

1. CyclinD1, p16 biological function and its regulatory mechanism of cell cycle

The division of eukaryotic must be changed by G1 → S phases proceeds by DNA synthesis, by G2 → M phases change into Enter m period, the wherein CyclinD1 and G1 phases are closely related, CyclinD1 can be combined to form with CDK4 and CDK6 Complex, is activated, the complex of activation makes together with CyclinE, CDK2 complex as CDK activated protein kinases are activated Rb phosphorylations, discharge transcription factor E2F, and driving cell enters the S phases by the G1 phases, promote cell propagation.The G1 phases are in The pRb and transcription factor E2F of low phosphorylation state (functional status) are combined and are suppressed its activity, enter S so as to inhibit The transcription of the related gene of albumen needed for phase.When exogenous multiple fission signal (such as growth factor) is combined with corresponding acceptor Afterwards, CyclinD1 gene expressions are promoted by signal transmission.CyclinD1 albumen is combined with CDK4/6, the activation of rush. CyclinD1 overexpression can cause the phosphorylation modification of pRb albumen to be realized in advance, and accelerate cell to change from G1 → S phases Process.P16 albumen specific can suppress CDK4 activity with CyclinD1 competition bindings CDK, p16 albumen, protect pRb Dephosphorylation state is held, so as to prevent cell from entering the S phases from the G1 phases, suppresses cell propagation, when p16 genes are due to missing Or mutation is when can not express, CyclinD1 dominances are combined with CDK4, overstimulation cell division, and it is in out of control to make cell Property growth, and form tumour.These regulations centered on pRb because in being constituted a complicated regulatory pathway, i.e. p16- CyclinD1/CDK4/6, RB-Pi and E2F path.In a word, CyclinD1 overexpression, can lead oncogenic generation, suppress CyclinD1 overexpression, is to treat one of effective way of tumour.

2. CyclinD1, p16 gene and tumour

（1）CyclinD1, p16 and gynecological tumor

CyclinD1 was initially confirmed as proto-oncogene in 1991 in human thyroid knurl, reported in succession afterwards CyclinD1 and the relation of other tumours are also more close.The immunohistochemical techniques such as Lisui are detected in 79 ovarian neoplasms CyclinD1 expressions are shown, malignant tumour are higher than in benign tumour and borderline tumor, expression rate is respectively 72. 7% 、69. 6%、32. 4%.Prompting CyclinD1 overexpressions are the earliest events during ovary canceration.And differentiation ovary can be used as The index of innocent and malignant tumour.28 normal cervix of the application SABC such as Duk-soo Bae and the detection of Western traces, CyclinD1 expression in 31 CIN, 32 cervical carcinomas, expression rate is respectively 100%, 3%, 28%, is concluded Detect that CyclinD1 can be as the prognosis for predicting early cervical carcinoma using SABC.Andrzej etc. applies SABC skill Art detects the expression of cell cycle related proteins pRb, CyclinD1, p16 and CDK4 in 48 cases of endometrial carcinoma, knot Fruit is respectively 2%, 50%, 6% and 25%.There is at least a kind of table of Rb pathway proteins in 69% (33/48) carcinoma of endometrium Up to exception, p16, CyclinD1, pRb unconventionality expression clinical pathologic characteristic non-correlation different from carcinoma of endometrium, and CDK4 unconventionality expression is negatively correlated with patient age, draws a conclusion, and Rb pathway associated proteins are abnormal especially CyclinD1, CDK4, which take part in carcinoma of endometrium, to be developed.

（2）CyclinD1, p16 and tumor in digestive tract

Li etc. have detected 10 normal pancreatic tissues, 12 pancreas epithelium knurls, 47 cancers of pancreas using SABC CyclinD1 and β-catenin expression in tissue, as a result respectively 0. 0% (0/ 10), 50. 0% (6/ 12), 74. 5% (35/ 47) and 100% (10/10), 50. 0% (6/ 12), 68. 0% (32/ 47).Analysis result, CyclinD1 is related to cell proliferation and differentiation degree in cancer of pancreas, with tumor size, whether shift and 1 year survival period without Close.Expression of the β-catenin unconventionality expression by raising CyclinD1 plays an important role in cancer of pancreas develops. Detection is carried out to CyclinD1 using ImmunohistochemistryMethods Methods and finds that prognosis meaning is different in different tumor in digestive tract, The detection to 25 hepatocellular carcinoma CyclinD1 such as Yukihiko, two of which coloured differently pattern be nuclear targeting with Cytoplasm dyeing CyclinD1 positive rate is respectively 8% and 32%, in two positive samples of nuclear targeting, is not had The pathological diagnosis feature of characteristic, and in the sample of 8 cytoplasm stained positives, CyclinD1 expression and portal vein cancer Bolt, Intrahepatic metastasis have Close relation.This result shows that cytoplasm CyclinD1 expresses the prognosis phase with hepatocellular carcinoma Close.Different from Yukihiko reports, Takao Ito etc. are to 25 chronic cholecystitis, 5 adenoma of gallbladder, 6 adenoma cancers Become, 42 gallbladder cancer (20 intra-mucosal carcinomas, 22 infiltrating carcinomas), carry out SABC detection and find, CyclinD1 Nuclear expression be respectively 40%, 54% in intra-mucosal carcinoma and infiltrating carcinoma, and in Non-cancerous infringement, adenoma and adenoma canceration mark It can be examined in this without nuclear expression, cytoplasm CyclinD1 overexpression in tumprigenicity and a variety of Non-cancerous abnormal cystic epitheliums Measure, and overexpression rate is only 15%.This result shows that CyclinD1 nuclear targetings, the prognosis to gallbladder cancer is related. He thinks that overexpressions of the CyclinD1 in gallbladder cancer is largely focused on karyon simultaneously, seldom in endochylema or endochylema, born of the same parents Core exists simultaneously, is expressed simultaneously in endochylema and karyon, it is more likely that be caused by karyon CyclinD1 spills endochylema.Whether will P16, CyclinD1, pRb regulate and control problem as tumor cell proliferation simultaneously, are current cell biology, science of heredity and molecule Common study hotspot in the different fields such as biology, mainly includes two aspect contents:On the one hand research cell is how to open Biological event that is dynamic and completing relevant cell propagation;On the other hand how research cell ensures these events accurately in due order Sequence is carried out.CyclinD1 genes are a kind of non-dominant oncogenes to be cooperateed with ras the or myc genes activated can cause cell Conversion, and Tumor suppressor gene p16 and p53 also have adjustment effect to CyclinD1 activity, most of tumor types have CyclinD1 gene magnifications and/or expression enhancing, although clinical meaning waits further research, use antisense The Human colon cancer and esophageal cancer cell of CyclinD1cDNA transfections show doubling time extension or the funeral of oncogenicity really Lose, therefore, CyclinD1 is expected to a novel targets as therapy of tumor.

The current clinical practice of Amifostine

Amifostine is the wide spectrum cell-protecting developed by U.S.Army research institute, for nuclear radiation and chemical weapons The protection of damage.Its molecular formula is C5H15N2O3SP, and molecular weight is 214.22, and chemical constitution is 2- [(3- aminopropyls) ammonia Base] ethylenebis dithiocarbamate dihydrogen phosphoric acid ester.English alias Ethyol, Ethiofos, Fosteamine, Gammaphos, WR-2721 and YM-08310, structural formula is as follows：

Amifostine is amineothiot small-molecule substance, is a kind of pro-drug of nucleophilicity sulfur-bearing, in the alkaline phosphorus of film combination Dephosphorylation under acid esters enzyme effect, generates active metabolin WR-1065 (free mercaptan), its free sulfhydryl groups contained （thiol-）, it is the principal mode that Amifostine plays cytoprotection in vivo.WR-1065 can also further be metabolized to half Guang Amine, sulfinic acid and WR-33278, these three metabolites also have weaker cytoprotection in vivo.Due to tumour cell table The activity of face alkaline phosphatase it is low far beyond normal cell or it is scarce such as, and activity is most strong in the basic conditions for the enzyme, and tumour cell Usually sour environment, normal structure is in neutral environment, and therefore, the protective effect of Amifostine normal tissue and cell is long-range In tumour cell, has effective relative selectivity, i.e., it is generally acknowledged that the application of Amifostine will not lower the antitumor of chemicotherapy Effect.

U.S. FDA approval Amifostine is used for pre- preventing tumor Radiotherapy chemotherapy toxicity within 1996, and its tumour used almost covers All tumor types of the mankind, the Radiotherapy chemotherapy toxicity most often prevented includes marrow, heart, nerve, kidney and ototoxicity, dry Disease, mucositis, bone injury etc..Up to now, Amifostine is still mainly used in the Radiotherapy chemotherapy of tumour protecting normal structure, cell, Mitigate Radiotherapy chemotherapy toxicity.

In addition, with research deeply, Amifostine may also have the effect of class hematopoietic cell growth factor and induced tumor thin Born of the same parents' apoptosis, the antitumor action for suppressing cell cycle and anti-angiogenesis.But there is not yet about the latent of Amifostine antitumor action In the document report of target molecule or signal path.

The content of the invention

The technical problems to be solved by the invention are to provide a kind of Amifostine in Cyclin D_1 gene height expression tumor types In application, the present invention by Amifostine be used for treat tumour, can cure with CyclinD1 height expression and leucocyte rise for spy The tumor patient levied.

The technical scheme that the present invention is provided is：A kind of Amifostine is in treatment Cyclin D_1 gene height expression tumor types Using.

The tumour is lymphoma mantle cell, leukemia, breast cancer, the cancer of the esophagus, lung cancer, prostate cancer, first shape Gland cancer, myeloma, head and neck neoplasm, sarcoma, stomach cancer, cervical carcinoma, colon cancer, glioma etc..

Above-mentioned application, is to use Amifostine single therapy, dosage is 0.4g/ days, and drip-feed continuous 5 days weekly, stops 2 days, continuous use 4 weeks.

The invention has the advantages that：The present inventor had found by years of researches, a kind of clinical conventional wide spectrum Cell-protecting Amifostine has the effect of the growth of tumour cell of targeted inhibition oncogene CyclinD1 expression and by suppressing The antitumor action of CyclinD1 expression, this effect is successfully authenticated by mankind's MEG cell lines Dami； In clinical practice, 1 lymphoma mantle cell being characterized with the expression of CyclinD1 height and leucocyte rise and is successfully cured, is suffered from Person is disease-free to survive 5 years.

Figure of description

Fig. 1 each group internal reference β-actin amplification curve diagram（Abscissa is PCR reaction cycle numbers, and ordinate is believed for fluorescence Number amount）.

Fig. 2 each groups CyclinD1 amplification curve diagram（Abscissa is PCR reaction cycle numbers, and ordinate is fluorescence signal Amount）.

Fig. 3 each groups β-actin and CyclinD1 melting curve figure（Abscissa is PCR reaction temperatures, and ordinate is glimmering Optical signal amount）.

Fig. 4 Amifostines suppress the detection of Dami cell CyclinD1 protein expressions.

The Microscopic observation that Fig. 5 Amifostines influence on Dami cytomorphologies（Multiplication factor 10 × 40）.

The suppression that Fig. 6 Amifostines are bred to Dami cells.

Embodiment

Below by embodiment detailed description come the present invention is furture elucidated, but be not to the present invention limit System, is only illustrated.

Bioinformatics method prediction Amifostine has the effect for suppressing CyclinD1 height expression

（1）Bioinformatics method

1. data source：With " Amifostine（amifostine）" as keyword, the free gene table opened in internet Scanned for up in database, these databases include：Gene expression omnibus （GEO）, gene chip expression Database Affymetrix（Hu35KsubB、Hu35KsubD、HG-U95A、HG-U133A、 HG-U133B、HG-U95Av2、 HuGeneF、HG-Focus、HG-U-133 Plus）、Clontech（Atlas Human Toxicology 1.2 Array、 Atlas Plastic Human 8K Microarray, Atlas Glass Human 3.8 I Microarray and MWG Human 30k Array）；Gene expression data base SAGE, GeneCard, InterPro, ProtoNet, UniProt and BLOCKS。

2. the validity check of data：Initial data to acquisition is tested to judge whether to be adapted to further analysis. Method is：Using variance analysis, such as intra-class variance has notable significant difference, then this group of data are removed.In UHN SS- 19,207 groups of gene expression datas are had in Human 19Kv7 chip databases, every group includes blank control group（2 parallel Sample）With Amifostine treatment group（2 Duplicate Samples）, in 19,207 groups of all genes are organized（Same experiment condition）Data difference Different analysis.

3. the validation verification of gene：After the validation verification of data, the title to each gene confirms, such as Fruit RNA fragment of the system without known action or predicted gene, because its to function prediction without directive function, the title is removed.

4. Gene expression differential display：Blank control group and experimental group（Amifostine processing）Gene expression difference is using pairing T is examined, with P<0.01 is that difference is statistically significant.Relative expression's difference of gene represents that AMF represents ammonia phosphorus with " AMF/C " Spit of fland group, C represents control group.The intensity that AMF is acted on gene expression is with ± 2^|δ^|Represent,

δ= Log₂(AMF/C)

5. functional clustering is analyzed and gene annotation：Functional clustering is analyzed and gene annotation uses NIH DAVID2.1 software platforms carry out.Clustering is carried out to the significant gene of the expression that filters out, using human genome as Background, is carried out accurate according to gene expression abundance difference to the result of clusteringFisher’sExamine.

（2）Bioinformatic analysis prediction Amifostine regulation and control human gene express spectra

1. data screening：Amifostine is used only to filter out a qualified data in GEO databases for keyword Storehouse（accession: GSE3212）, all initial data are from UHN SS-Human 19Kv7 chips.

2. the validity check of data：Gene expression values in GSE3212 databases are binary channels color detection result, Totally 19,207 groups of gene expression datas, are blank control group per component（2 Duplicate Samples）With Amifostine treatment group（2 parallel Sample）, each group difference analysis P values are all higher than 0.05, and each group of data meets further analysis condition in the interior indifference of group.

3. the validation verification of gene：4 array datas of analysis are divided into, each array includes 19,207 genes, as a result sent out It is existing, respectively there are 5208 genes in each array without expression numerical value, these genes are removed from analysis, last each array is remaining 13,999 genes are used to further analyze.

4. gene expression difference：The gene expression profile of K562 groups of cells and blank control K562 groups of cells is handled Amifostine Paired t-test is carried out, is as a result shown, only 460 genes（2.14%）There is differential expression before and after Amifostine processing K562 cells （P<0.01）.

5. clustering and gene annotation description：Annotation analysis is carried out to 460 difference expression genes, GeneBank is utilized Database filters out gene known to function totally 139.Gene expression abundance difference after K562 cells is handled according to Amifostine, There are 77 gene expression up-regulations in 139 genes, including FBXL13, EDIL3, GPR26, GPR26, CD49c and MS4A4 etc., this A little genes are primarily involved in the important biomolecule process such as the proteolysis and hematopoietic regulation of ubiquitin mediation；62 down regulation of gene expression, Including TIE1, IL10RB, COL1A1, MCM4, cyclinD1, TIE1 and HYOU1 etc., these genes be primarily involved in cell function, Enzymatic activity, interferon-' alpha ' signal path and bone remoulding.Having 13 genes in these genes is newly expressed after Amifostine is handled , 5 genes are to express completely suppressed after handling（Table 1）.

Completely suppressed gene is expressed after the Amifostine of table 1. processing K562 cells

Gene code name	Gene Name	AMF acts on δ values	P values
				COL1A1	The type α 1 of collagen 1	-10.76	0.0025
MCM4	Minichromosomes maintain albumen 4	-6.80	0.0041
				CyclinD 1	Cyclin D_1 gene	-5.27	0.0037
TIE1	With immunoglobulin-like and epidermal growth factor-like domain protein 1	-3.89	0.0053
				HYOU1	Hypoxemia upregulated protein 1	-3.39	0.0024

"-" represents inhibitory action.

2. Amifostine suppresses Dami cells CyclinD1 expression and the experimental verification of cell propagation

（1）Amifostine suppresses the experimental verification of CyclinD1 expression

Part I：Fluorescence real-time quantitative PCR detection CyclinD1 mRNA expression

Experimental method：

Using 0.75mg/ml（Equivalent to 3.5mmol/L）The Amifostine processing Dami cell lines of concentration, processing mode is divided into Two kinds：（1）Amifostine continuous action, i.e., add Amifostine in cell culture system, and continuous action to harvesting is detected； （2）Amifostine interruption is acted on, i.e., add Amifostine in cell culture system, acts on 30 minutes, medicament elution is cultivated again Cell, daily processing 1 time.Two kinds of processing modes, respectively at cultivating the 24th hour, 48 hours, 72 hours harvestings, that is, are tested Component is six groups：Continuous action 24 hours groups, continuous action 48 hours groups, continuous action 72 hours groups, interruptions are acted on 24 hours Group, interruption act on 48 hours groups and interruption acts on 72 hours groups, and negative control group is set in addition（Medicine untreated fish group）And blank pair According to group, above-mentioned each group respectively sets 3 Duplicate Samples.Each group cell is harvested, Trizol freezes, extract RNA, reverse transcription does glimmering into cDNA Photoinitiator dye real-time quantitative PCR（SYBR^®Green Realtime PCR）Detect CyclinD1 expression change, reference gene be β- Actin, specific method is as follows：

1）Extract total serum IgE

1. culture cell suspension is collected by centrifugation（About 1 × 10⁶）In 1.5ml RNase-free EP pipes, 1ml is added

Trizolblue.Blow and beat repeatedly, cell is fully cracked, 10~20min is stood on ice.

2. 200 μ l chloroforms are added, 15sec is acutely shaken, 15min is stood on ice.12000rpm, 4 DEG C of centrifugations

10min。

3. upper strata colourless aqueous phase is drawn, is moved into another RNase-free EP pipes.Plus isometric isopropanol, stand 15min.12000rpm, 4 DEG C of centrifugation 15min.

4. supernatant, plus 75% ethanol 1ml are abandoned, 15sec is vibrated.7500rpm, 4 DEG C of centrifugation 5min.

5. repeat 4..

6. carefully outwell supernatant, take precipitation put superclean bench blow in machine drying.Again in the μ l DEPC water of Guan Zhongjia 20（Contain 0.1%DEPC）Dissolving.Above step is operated on ice.

Detect RNA integralities

Take the μ l of extract 2 to add 198 μ l deionized waters and survey OD₂₆₀/OD₂₈₀.The μ l of extract 2 are taken through 1% Ago-Gel electricity Swimming, 80V, 40min, 0.5 × TBE, ultraviolet transilluminator shows two RNA bands of 28s, 18s, illustrates RNA integralities.

Purity requirement OD₂₆₀/OD₂₈₀=1.8~2.0, -80 DEG C of preservations.

Reverse transcription synthesizes cDNA

1. reverse transcription reaction system is 20 μ l, including：Total mRNA 4 μ l, oligo（dT）Primer 1μl、RNase-free

H₂O 10μl、Primerscript RT Enzyme MixI 1μl、5×PrimerscriptBuffer（for Real Time）

4 μ l to 20 μ l.It is quick after mixing to centrifuge once.

2. reaction condition：37 DEG C, 15min；85 DEG C, 5s.The cDNA of acquisition enters performing PCR amplification or -80 DEG C at once

Refrigerator freezes.

5）RT-PCR reacts

1. primer：CyclinD1

Product sheet segment length：195bp

Sense primer：5’-CATCTACACCGACAACTCCATC-3’

Anti-sense primer：5’-GCACAGAGGGCAACGAAG-3’

Annealing temperature（Tm）：53.0 ℃

β -actin

Product sheet segment length：205 bp

Sense primer：5’-TGACGTGGACATCCGCAAAG-3’

Anti-sense primer：5’-CTGGAAGGTGGACAGCGAGG-3’

Annealing temperature（Tm）：56.0℃

Above-mentioned primer application NCBI primer blast are compared.

2. PCR reaction systems are 25 μ l, including the μ l of cDNA 2.5, each 1 μ l of upstream and downstream primer, SYBR GreenRealtime

PCR Master Mix12.5 μ l, distilled water complements to 25 μ l.

3. reaction condition：Pre-degeneration：95 DEG C, 5min × 1cycle；Amplification：95 DEG C, 15sec；Tm60 DEG C,

15sec；72 DEG C, 25sec × 40cycles；Solubility curve is analyzed：72~97 DEG C of 10sec.PCR reactions preceding 3~

The fluorescence signal of 15 circulations is used as autofluorescent background signal, regulation baseline to suitable place, each fluorescence curve and baseline

The period in crosspoint is Ct values.All test experiences include a blank control without template to exclude vacation

Positive findings.

4. real-time quantitative PCR result is by quantitative fluorescence analysis instrument automatic data collection and provides target gene and reference gene

Ct values, gene expression amount difference uses relative quantification method, is calculated as follows：

△Ct（Experimental group）=Ct（Target gene）- Ct（Reference gene）

△Ct（Control group）=Ct（Target gene）- Ct（Reference gene）

△△Ct=△Ct（Experimental group）- △Ct（Control group）

Relative change multiple=2^-△△Ct

Experimental result：See Fig. 1-3, table 2 shows that CyclinD1 mRNA expression is relatively compareed after Amifostine effect Dami cells Group is significantly suppressed, and in time dependence, further demonstrate the prediction of bioinformatics.

The each group Ct values of table 2., △ Ct, △ △ Ct and 2^{-△△ Ct}

Part II：Western blot (Western blot）Detect the expression of CyclinD1 albumen

Experimental method：

1）Prepare PAGE gel

1. PAGE gel glass guide channel is prepared：Two pads are put into (wherein one in the middle of two blocks of special glass plates

Block is spill）, both sides and base are obturaged with adhesive tape.It is determined that recording separation gel liquid surface mark line (away from sample Comb

At the cm of sub- bottom about 0.5-1.0）.

2. 10%SDS-PAGE separation gels (15ml is prepared）：

Deionized water 5.9ml

30% acrylic amine 5.0ml

1.5mol/L Tris-CIPH8.8 3.8ml

10% Ammonium Persulfate 98.5 0.15ml

10%SDS 0.15ml

Aforesaid liquid 1ml is taken, TEMED3 μ l is added, glass guide channel periphery is used with its envelope gel after mixing.

3. separation gel is recorded：After after edge sealing adhesive solidification, 7 μ l TEMED are added in remaining 14ml separation glues, are mixed

Close uniform, gently added in gel glass groove with suction pipe, make its liquid level to indicating that line position (avoids producing bubble）.

4. with 0.1%SDS liquid covering glue surface, (isolation air, contributes to gel polymerisation, gel face is formed straight immediately）,

Room temperature places about 40min to separation gel solidification.

5. preparing 5% concentration glue 4ml, (this step operation can simultaneously be carried out when preparing separation gel）：

Deionized water 2.7ml

30% acrylamide 0.67ml

1.0mol/L Tris-CIPH6.8 1.5ml

10% Ammonium Persulfate 98.5 0.04ml

10%SDS 0.04ml

TEMDE（Face before encapsulating and add again） 0.004ml

6. 0.1%SDS covering liquids are outwelled, with deionized water rinsing separation gel surface 3-4 times, to clean unpolymerized third

Acrylamide gel（Avoid leaving bubble at the top of glue or in glass plywood）, and sop up with blotting paper the liquid of residual Body.

7. gel slab is disposed vertically again, gently adds 5% lamination glue（Note avoiding producing bubble）, insert sample

Product are combed, and liquid level is indicated line position, room temperature gel about 40min to sample comb.

8. comb is gently taken out, envelope glass plywood adhesive tape is torn off, glass plywood " recessed " face is adjacent to electrophoresis tank,

Both sides folder one is extensively well fixed on electrophoresis tank.Electrophoretic buffer is drawn with detachable needle syringe gently to rinse

Loading wells, to remove unpolymerized gel.

Sample preparation and electrophoresis

1. 40 μ g total proteins, plus isometric 2 × LoadingBuffer are taken, 100 DEG C are boiled 5min, are placed on rapidly on ice.

2. after point sample, electrophoresis apparatus is arranged to voltage stabilizing state, switched on power, voltage is adjusted into 80V, and to pass through sample dense

Contracting glue（Voltage about 8V/cm）.After dyestuff enters separation gel, voltage is heightened into 12OV（About 12V/cm）,

Continuing electrophoresis makes dyestuff to separation gel appropriate location（Electrophoresis is carried out in 4 DEG C of refrigerators）, terminate electrophoresis.

Transferring film

1. the pretreatment of pvdf membrane and filter paper：Cut out with glue pvdf membrane of the same size, be dipped in methanol solution,

Soak 10sec（Quick activation pvdf membrane）, deionized water processing 5min, the slow liquid processing 10min of 1 × transfer：

Cut out with an equal amount of 8 metafiltration paper of glue, it is stand-by after being soaked with transfering buffering liquid.

2. electrophoresis plate is removed, by its horizontal（Make " recessed " surface glass plate under）, carefully take out the pad in clamping plate and remove

Upper glass plate, cuts off unnecessary gel, will contain sample glue and be rinsed once with 1 × transferring film liquid.

3. sample glue and film are fitted into the transferring film clamping plate for indicating positive and negative electrode：By cathode side, foam-rubber cushion is followed successively by

The metafiltration paper of the metafiltration paper of piece → 4 → sample glue → pvdf membrane → 4（Note：Exclude bubble）→ sponge pad, button

Tight transferring film clamping plate, is put into the electrophoretic blotting groove containing transferring film buffer solution.

4. correct connection electrophoretic blotting line, it is ensured that electric charge is from negative pole to anode flow.Switch on power, under pressure constant state,

80V transferring films 2h（This step operation is carried out preferably in 4 DEG C of refrigerators）.

5. it is careful to take out transfer membrane, rinsed twice in 1xTBST liquid.

Closing, antibody incubation and exposure

1. close：Film after transfer is put into confining liquid（5% skimmed milk power）In, it is slow in room temperature, shaking table to shake shape

Lh is closed under state.

2. primary antibody reacts：Primary antibody is diluted to proper proportion with 5% skimmed milk power, and the film after closing is directly placed into primary antibody

In working solution, 4 DEG C of reactions are stayed overnight.

3. film is washed：Reaction film is put into plate, with 1xTBST rinsings once, then washed with IXTBST 3 times,

（It is slow at room temperature to shake washing）Each 10min, cleans uncombined primary antibody.

4. secondary antibody reacts：Secondary antibody dilutes 2000 times with 1xTBST；Primary antibody reaction film after washing is put into secondary antibody working solution

In（Room temperature, lucifuge are slowly shaken）Act on 50min（No more than 60min）.

5. film is washed：Film is washed with 1 × TBST, method is with " 3）", wash away free secondary antibody.

6. expose and develop a film：By 1：l（v/v）Two kinds of liquid in ECL kits are mixed, mixed liquor is uniformly layered on

Pvdf membrane surface, room temperature effect 2min.Liquid on film is shaked off, is put it into the exposure box for being covered with preservative film,

Preservative film is folded again, to wrap pvdf membrane（Avoid occurring bubble above film）.In darkroom（It is red

Under light）X-ray film is directly pressed on the film after parcel, appropriate time is exposed in exposure box.X after exposing

Mating plate is put into developer solution and is fixed in development, clear water in rinsing, fixing solution.

Experimental result：CyclinD1 protein expressions are remarkably decreased after Amifostine processing Dami cells（Fig. 4）, further

Demonstrate the testing result of real-time quantitative PCR.

（2）Amifostine suppresses the experimental verification of Dami cells propagation

Experimental method：

1）Cell culture：Dami cells are suspended in the RPMI1640 culture mediums that volume fraction is 10% hyclone, Containing 5% CO₂37 DEG C of cultures that suspend in incubator, passage in every 2~3 days is once.Take the logarithm growth period cell carry out it is real Test.It is 1 × 10 to adjust cell density⁶Ml, extracts the trypan blue that 180 μ l add 20 μ l 0.4%, micro- Microscopic observation living cell rate Up to more than 98%（Living cell rate=total viable cell/viable count+dead cell number × 100%）.

2）Cultivate morphological observation：Dami cells are observed under inverted microscope, daily observation active somatic cell form, and Photograph in good time.

3）CCK-8 methods determine cell propagation：CCK-8（cell counting kit-8）It is MTT one kind containing wst-8 Upgrading substitute, can be orange-yellow by the reduction generation of some Intramitochondrial dehydrogenases in the presence of electronics coupled agent Formazan, its growing amount is directly proportional to number of viable cells, and the more much faster colors of cell propagation are deeper, the more colors of dead cell It is more shallow, determine the OD value at 450nm using enzyme-linked immunosorbent assay instrument（optical density, OD）, can between it is reversed Reflect the vigor of living cells.

4）Experiment packet：Dami cells are handled using continuous action method, experiment is divided into：Control group（Plus the culture of same volume Base, Amifostine 0.35mg/ml groups, Amifostine 0.75mg/ml groups and Amifostine 1.5mg/ml groups.

5）Administration：The Dami cells of the 4th generation exponential phase are taken, adjustment cell density is 1 × 10⁵/ ml is dense by each packet Degree adds medicine.96 well culture plate 12 is taken, the above-mentioned μ L of cell suspension 100 are added per hole, each acute drug group sets nothing simultaneously Cell blank hole, and set 3 parallel hole cultures.Cell is cultivated taken out after 0h, 24h, 48h, 72h respectively, and CCK-8 is added per hole 10 μ L, are cultivated for 3.5h.By culture plate slight oscillatory 5min, using 450nm as test wavelength on enzyme-linked immunosorbent assay instrument, Absorbance (A) value is read, cell viability is contrasted.Experiment is in triplicate.Cell proliferation inhibition rate calculation formula is as follows：

Cell proliferation inhibition rate（%）=（1- medications group mean OD value/control group mean OD value）×100%

6）Statistical analysis：Experimental data use SPSS17.0 software analysis, measurement data with（Means standard deviation）Table Show, multigroup is compared use One-way ANOVE and examined, and is compared between two groups and is examined with t, with P<0.05 is that difference has statistics meaning Justice.

Experimental result：

1）Morphological observation

The Dami cells of unused Amifostine processing are in suspended state, and cell is circular, and uniform in size, cell is complete, diopter It is good, 12h to 48h is cultivated, form is without significant change.After 0.35mg/ml Amifostine processing cell 24h, start aggregation it is agglomerating, Volume is not of uniform size, and refractivity is poor, and cytoplasmic granule sense is obvious, with drug exposure times extension and concentration increase, 0.75mg/ml After Amifostine processing 48h, cell starts shrinkage, and karyorrhexis is obvious（Fig. 5）.

The suppression that Amifostine is bred to Dami cells

Compared with control group, the Amifostine processing Dami cells 12h of 0.35mg/ml, 0.75mg/ml, 1.5mg/ml concentration, After 24h, 48h, the influence of cell proliferation is determined, 0.75mg/ml groups are as a result shown in, with the extension of Amifostine processing time （12h、24h、48h）, inhibitory rate of cell growth is respectively 31.1%, 48.9%, after 66.7%. Amifostines processing cell 24h, three Concentration group growth inhibition ratio is respectively that 15.8%, 48.9%, 95.8%, presses down with Amifostine concentration and time change cell propagation Processing procedure degree has significant difference（P<0.05）（Fig. 6）.

The preclinical phase result of the lymphoma mantle cell of Amifostine treatment CyclinD1 height expression

Lymphoma mantle cell has the gene phenotype of characteristic, i.e. CyclinD1 height expression.Some jacket cell lymph Knurl patient does not simultaneously appear as enlargement of lymph nodes, and with leucocyte（Based on lymphocyte）Exception increases, and is delivered headed by bone marrow involvement It is existing.But the lymphocyte of this some patients is monoclonal, expressed with abnormal CyclinD1 height.Clinical trials difficult, and suffer from Person is generally old, prognosis mala.

Early stage, the present inventor is in the case where obtaining patient's informed consent, using one 76 years old man of Amifostine single therapy Property by PBLC clonal expansion with CyclinD1 height expression with the characteristics of lymphoma mantle cell patient.Patient from

In September, 2009 starts to receive Amifostine drip-feed treatment, 0.4g/ days, continuous 5 days weekly, stops 2 days, repeats to use Medicine, after continuous 4 weeks, patient's leucocyte before treating 16 × 10⁹/ L, drops to 7.0 × 10⁹Marrow and outer after/L, and medication The CyclinD1 of all blood lymphocytes expression disappears.So far, patient is disease-free survives more than 5 years, and every half a year is once regular Monitoring, leucocyte is fluctuated in normal range (NR)（4.0-10×10⁹/L）, the immunophenotyping and genetic test of marrow and peripheral blood are not sent out The abnormal lymphocyte rise of existing phenotype and CyclinD1 unconventionality expression, lymph nodes of body as a whole ultrasound and PET/CT scannings have no different Normal enlarged lymph node and hepatosplenomegaly, all the time non-complete incidence graph state.Prompting Amifostine list medicine is by suppressing CyclinD1 table Up to can control even to cure lymphoma mantle cell.

In a word, confirmed from bioinformatics → cell and molecular biology → clinical study results：（1）Amifostine has The effect of targeted inhibition CyclinD1 expression；（2）Amifostine has the antitumor action by suppressing CyclinD1 expression.Ammonia phosphorus Spit of fland can be used for the tumour for treating CyclinD1 height expression, and effect is good.

Claims (2)

1. application of the Amifostine in the medicine for preparing treatment Cyclin D_1 gene height expression tumor types, it is characterised in that：Ammonia Phosphorus spit of fland is the sole active agent in the medicine, and the tumour is lymphoma mantle cell.

2. according to the application described in claim 1, it is characterised in that：The dosage of Amifostine is 0.4g/ days, drip-feed, weekly Continuous 5 days, stop 2 days, continuous use 4 weeks.