CN104721836B

CN104721836B - Applications of the chaperonin CCT γ in tumour diagnostic reagent is prepared

Info

Publication number: CN104721836B
Application number: CN201510075918.7A
Authority: CN
Inventors: 杨承刚; 崔双双; 边洋; 孙耀兰
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2015-02-13
Filing date: 2015-02-13
Publication date: 2018-03-23
Anticipated expiration: 2035-02-13
Also published as: CN104721836A

Abstract

The present invention relates to applications of the chaperonin CCT γ in tumour diagnostic reagent is prepared, the specific new application the present invention relates to chaperonin CCT γ in osteosarcoma diagnostic reagent is prepared.Inventor is based on high-flux sequence result and carries out genescreen using bioinformatics method analysis, pick out candidate gene CCT γ, further, confirm that CCT γ and osteosarcoma have good correlation by molecular cytobiology experiment, available for osteosarcoma assisting in diagnosis and treatment preparation is prepared, there is important clinical value.

Description

Applications of the chaperonin CCT γ in tumour diagnostic reagent is prepared

Technical field

The present invention relates to biomedicine field, applications of the chaperonin CCT γ in tumour diagnostic reagent is prepared, specific sheet Invention is related to new applications of the chaperonin CCT γ in osteosarcoma diagnostic reagent is prepared.

Background technology

The cyclic oligomer complex that chaperonin (chaperonins) is made up of about 60kDa subunits, it is wide in cell General presence, great expression and sequence are related, and the folding of more skin chains is mediated with a kind of ATP dependent reactions, and monomer and oligomer is sub- Unit is changed into the complete native protein of function.Eucaryon companion is known as TCP-1ring complex (TRiC), cytosolic Several calls such as chaperonin (c-cpn) or cpn-containing TCP-1 (CCT).Chaperonin CCT is a big circle The multi-subunit complex of tubular, has a central cavity structure being used for reference to non-collapsible and denatured polypeptide, CCT each ring be by Eight differences but homologous subunit of the molecular weight between 52-65kDa form.The CCT subunits of mammal are named as CCT α (CCT1), CCT β (CCT2), CCT γ (CCT3), CCT δ (CCT4), CCT ε (CCT5), CCT ζ (CCT6), CCT η (CCT7) and CCTθ(CCT8).From the point of view of existing research, the cooperation between different chaperones is the central tenet of protein folding process. However, what cooperation was perhaps not required for each new synthesis peptide chain.There is result to show, once polypeptide chain occurs, many companions Companion's molecule is just recruited to protein synthesis machine, and they are protected by these chaperones including CCT.Have confirmed in protokaryon and In eucaryote, chaperone can combine the polypeptide chain that ribosomes combines.Cell protein seemingly relies on according to chaperone Its Biological Functional three-dimensional structure is realized with non-dependent path.Albumen on chaperone dependent/non-dependent path is probably Little albumen, and they can not need any chaperone and fold.Chaperone dependence folding process is according to albumen sheet The difference of matter follows three kinds of different strategies：First, new synthetic proteins will be with Hsp70, the small chaperones of Hsp90 or other The of short duration hydrophobic region in the exposure of non-native protein surface combines, and protected protein is from assembling and will fold；Secondly, some albumen Combined successively by Hsp70 and Hsp90, and carry out folding process；3rd, sub-fraction newly synthesizes more skin chains and combines Hsp70 successively, Hsp90 and prefoldin/phosducin sample albumen, then CCT is transferred to complete finally to fold.The molecular chaperones of enormous amount Collection neutralize cooperation and result in the generation of three-dimensional structure and Biological Functional protein molecular.The failure of the process can cause many The aggregation of necessary albumen and false folding, and the allomeric function of cell may be produced and had a strong impact on.Recognize well at present It is many basic reasons for being referred to as " protein misfolding " or " protein conformation " disease to know the aggregation of albumen and false folding.I types It is found to take part in the regulation of false folding disease with II types chaperonin.

Osteosarcoma (Oseteosarcoma) is most common pernicious bone primary tumor, its histology in addition to plasmacytoma Feature is that tumour cell produces bone sample or immature bone as a rule.Osteosarcoma is easy to invade and grows rapid dry marrow end, Its typical position is in long bone, and distal femur and shin bone, the near-end of moon bright bone are most common site of pathological change, whole patients 50%-70% lesions occur in knee joint peripheral.Without obvious clinical symptoms during osteosarcoma onset, easily obscure with wound, And Lung metastases may occur for grade malignancy high early stage, therefore the big death rate of its harmfulness is very high.Clinical orthopaedicses worker causes always Power is in the research prevented and treated it, it is desirable to searches out the target spot that osteosarcoma is accurately effectively treated.

Inventor carries out high-flux sequence to 5 osteosarcoma case samples and 3 normal controls, with reference to bioinformatics side Method carries out genescreen, picks out candidate gene CCT γ.Existing research shows exist between CCT γ and its β-tubulin Interaction；CCT γ and D type retrovirus Mason-Pfizer monkey diseases poison Gag polyprotein interact, and take part in Gag Folding and/or Mouth Disease Virus Proteins.But not there are some researches show the relation between CCT γ and osteosarcoma, further, the present invention enters Molecular cytobiology method of having gone confirms the relation of CCT γ and osteosarcoma：CCT γ have related well to osteosarcoma Property, have the function that in the propagation of osteosarcoma cell and invasion and attack important, available for osteosarcoma assisting in diagnosis and treatment preparation is prepared, there is weight The clinical value wanted.

The content of the invention

It is an object of the invention to provide CCT γ genes and/or protein inhibitor answering in anti-osteosarcoma preparation is prepared With.

To achieve the above object, the present invention screens candidate's base by high-flux sequence combination bioinformatics method first Because of CCT γ, CCT γ and osteosarcoma relation by molecular cytobiology method validation today：CCT γ have with osteosarcoma Good correlation, CCT γ have the function that in the propagation of osteosarcoma cell and invasion and attack it is important, available for prepare osteosarcoma it is auxiliary Diagnosis and treatment preparation is helped, there is important clinical value.

Further, the anti-osteosarcoma preparation refers to suppress the preparation of the expression of CCT γ genes in osteosarcoma cell. The expression of the known suppressor of those skilled in the art can generally use one kind in following methods and/or several：By activating mesh The suppressor of gene, activating genes of interest inhibition of gene expression albumen, target gene suppressed using RNA perturbation techniques Expression, activation promote the microRNA of target gene mRNA degradeds, import the molecule for promoting the degraded of target gene encoding proteins, suppression System promotes the factor of destination gene expression and the expression of albumen.I.e. by activating the suppressor of CCT γ genes, activation suppresses The albumen of CCT γ gene expressions, the siRNA for importing suppression CCT γ gene expressions, activation promote CCT γ mRNA degradeds MicroRNA, the expression for importing the molecule for promoting CCT γ protein degradations, the factor of suppression promotion CCT γ gene expressions and albumen.

RNA interference (RNAi) refers to that exogenous and endogenous double-stranded RNA induces the mRNA of homologous target gene in vivo Selective degradation, cause the phenomenon of PTGS, be it is a kind of efficiently, specifically blocked using small double-stranded RNA it is internal The expression of certain specific gene, promotes mRNA to degrade, and cells show is gone out the technology of specific gene missing phenotype.SiRNA is designed After the completion of can use direct synthesis technique or structure SiRNA expression vector, the siRNA prepared can be coprecipitated by calcium phosphate Mechanical Method, the cationic-liposome such as shallow lake method, electroporation, DEAE- glucans and polybrene methods, microinjection or particle gun The approach transfectional cell such as reagent method.

Further, one kind in one sequence of the siRNA sequence of the suppression CCT γ gene expressions and/or several： SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6.It is preferred that SiRNA sequence is SEQ ID NO.1 and SEQ ID NO.2.

It is an object of the invention to provide a kind of anti-osteosarcoma preparation, the anti-osteosarcoma preparation suppresses in osteosarcoma cell The expression of CCT γ genes.Further, the siRNA for suppressing CCT γ gene expressions is contained in described anti-osteosarcoma preparation.

It is an object of the invention to provide application of the CCT γ genes in osteosarcoma diagnosis and treatment preparation is prepared.

Further, the diagnosis and treatment preparation of described osteosarcoma is included with fluorescence quantifying PCR method, method for gene chip detection bone The expression of CCT γ genes in sarcoma tissue.

Fluorescence quantitative PCR method is the specific probe by fluorescent dye or fluorescence labeling, and PCR primer is marked Tracking, real time and on line monitoring course of reaction, can be analyzed product with reference to corresponding software, calculate testing sample template Initial concentration.The appearance of quantitative fluorescent PCR, greatly simplifies the process of quantitative detection, and is truly realized absolute quantitation. The appearance of a variety of detecting systems, make the selectivity of experiment stronger.Automation mechanized operation improves operating efficiency, rapid reaction, repetition The good, high sensitivity of property, high specificity, result are clear.

Genetic chip is also known as DNA microarray (DNA microarray), can be divided into three kinds of main Types：1) it is fixed on poly- Nucleic acid probe or cDNA fragments on compound substrate (nylon membrane, nitrocellulose membrane etc.) surface, generally use the target of isotope marks Gene is hybrid with it, and is detected by radiography technology.2) DNA probe array on a glass is fixed with point sample method, By being detected with the hybridization of the target gene of fluorescence labeling.3) oligonucleotide probe directly synthesized on the hard surfaces such as glass Array, the target gene hybridization with fluorescence labeling are detected.Genetic chip is as a kind of advanced, extensive, high flux detection Technology, applied to the diagnosis of disease, its advantage has the following aspects：First, the sensitivity and accuracy of height；It is second, quick It is easy；Third, a variety of diseases can be detected simultaneously.

The described product for being used for CCT γ genes in fluorescence quantifying PCR method detection osteosarcoma contains a pair of specificity and expanded Increase the primer of CCT γ genes；Described genetic chip includes the probe with the nucleic acid array hybridizing of CCT γ genes.

Further, the diagnosis and treatment preparation of described osteosarcoma includes the table that CCT γ albumen in adenocarcinoma of lung is detected with immunization method Reach.It is preferred that in the immunologic detection method detection osteosarcoma CCT γ protein expressions for western blot and/or ELISA and/ Collaurum detection method.

Enzyme-linked immunosorbent assay (ELISA) will known antigen or antibody absorption in surface of solid phase carriers, make enzyme mark The technology that the antigen-antibody reaction of note is carried out in solid phase surface.The technology can be used for detection macromolecular antigen and specific antibody Deng, have the advantages that quick, sensitive, easy, carrier be easy to standardization.ELISA detection kit is according to testing goal and operation Step can be divided into indirect method, double-antibody method, competition law, double site one-step method, prize law survey IgM antibody, using Avidin and The ELISA of biotin.Horseradish peroxidase (HRP) or alkaline phosphatase may be selected in chromogenic substrate in ELISA detection kit Enzyme (AP).

Conventional immune colloid gold detection technique：(1) immune colloid gold light microscopic decoration method cell suspension smear or tissue are cut It piece, can be dyed with the antibody of colloid gold label, can also be strengthened with silver-colored developer solution and marked on the basis of colloid gold label, The silver atoms for making to be reduced are deposited on marked gold grain surface, can be remarkably reinforced the sensitiveness of colloid gold label.(2) it is immunized Collaurum staining method for electron microscopy can use the antibody of colloid gold label or antiantibody to be combined with negative staining Virus Sample or tissue ultra-thin section, Then negative staining is carried out.Observation and Viral diagnosis available for morphology of virus.(3) dot immunogold filtration assay application miillpore filter is made Carrier, first antigen or antibody point are added into sample to be checked on film after closing, it is corresponding with the antibody test of colloid gold label after washing Antigen or antibody.(4) specific antigen or antibody are fixed on film by colloidal gold immunity chromatography with ribbon, collaurum Labelled reagent (antibody or monoclonal antibody) is adsorbed on pad, when sample to be checked is added in the sample pad of test strips one end Afterwards, moved forward by capillarity, reacted to each other after dissolving the colloid gold label reagent on pad, it is fixed when being moved to During the region of antigen or antibody, the conjugate of thing and gold marked reagent to be checked occurs specific binding therewith again and is trapped, and assembles In detection band, colour developing result can be observed by the naked eye.The method has developed into diagnosis test paper, and use is very convenient.

Further, the ELISA method of the detection CCT γ albumen is to use ELISA detection kit.In the kit Antibody can use commercially available CCT γ monoclonal antibodies.Further, described kit includes：It is coated with CCT γ monoclonal antibodies Solid phase carrier, ELIAS secondary antibody, the substrate of enzyme, protein standard substance, negative controls, dilution, cleaning solution, enzyme reaction terminate liquid Deng.

Further, the colloidal gold method of the detection CCT γ albumen is that can use city using detection kit, described antibody The CCT γ monoclonal antibodies sold.Further, described gold-immunochromatographyreagent reagent for assay box uses colloidal gold immunochromatographimethod technology or colloid Golden percolation.Further, detection zone (T) specking on described gold-immunochromatographyreagent reagent for assay box nitrocellulose filter has anti-CCT γ Monoclonal antibody, quality control region (C) specking have Immunoglobulin IgG.

It is an object of the invention to provide a kind of PCR kit for fluorescence quantitative for detecting osteosarcoma, it is characterised in that described Kit detects gene C CT γ, and using special sense primer and anti-sense primer, upstream primer sequence is SEQ ID NO.9, under Trip primer sequence is SEQ ID NO.10.

Further, the PCR kit is suitable for presently, there are all types fluorescence quantitative gene extender of in the market, spirit Sensitivity is high, it is quantitative quick and precisely, stability it is good, have a good application prospect.

Further, above-mentioned PCR kit for fluorescence quantitative component includes：Specific primer, internal control primer, quantitative fluorescent PCR Reaction solution.Wherein described specific primer includes sense primer and anti-sense primer, and upstream primer sequence is SEQ ID NO.9, Downstream primer sequence is SEQ ID NO.10.The internal control primer is β-actin internal control primers, and upstream primer sequence is SEQ ID NO.11, downstream primer sequence are SEQ ID NO.12.

Described kit also includes RNA extraction agents.It is preferred thatReagent (invitrogen, article No. 15596-018) carry out sample rna extraction.

The present invention also have detected this kit sensitivity, and it is 10 as a result to show this kit detection range⁶-10²copies/μ L, minimum concentrations are 100copies/ μ l.

It is an object of the present invention to provide a kind of osteosarcoma detection kit, described detection kit detection CCT γ albumen. Further, described kit also includes other detection reagents.

It is an object of the present invention to provide a kind of genetic chip for detecting osteosarcoma, described genetic chip includes and CCT γ The probe of the nucleic acid array hybridizing of gene.

Brief description of the drawings

Fig. 1 CCT γ genes relative expression's spirogram in cancerous tissue and normal structure

Each group CCT γ mRNA expressions after Fig. 2 RNA interference

MG-63 cells propagation change before and after Fig. 3 RNA interference

Embodiment

With reference to specific embodiment, the present invention is expanded on further, is only used for explaining the present invention, and it is not intended that to this The limitation of invention.It will be understood by those skilled in the art that：Can in the case where not departing from the principle and objective of the present invention So that these embodiments are carried out with a variety of change, modification, replacement and modification, the scope of the present invention is limited by claim and its equivalent It is fixed.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition or according to the bar proposed by manufacturer Part examinations.

The high-flux sequence of embodiment 1 and analysis

5 osteosarcoma case samples and 3 normal control tissues are collected respectively, carry out RNA extractions, agar after RNA extractions Whether up-to-standard sugared gel electrophoresis, the RNA sample that can be extracted from electrophoresis result with preliminary judgement be, if can be used for further Transcriptome analysis.And then the extraction situation of RNA sample, RNA-seq sequencings are detected by NanoDrop1000 spectrophotometers Sample requirement：OD260/OD280 is 1.8-2.2.

Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, carries out high flux transcript profile depth Sequencing, we use Fast-QC (http after sequencing：//www.bioinformatics.babraham.ac.uk/projects/ Fastqc/) software carries out total evaluation to the quality of sequencing data, includes the quality Distribution value of base, the position point of mass value Cloth, G/C content, PCR duplication contents, kmer frequency etc..In differential genes expression analysis, according to obtaining FPKM values, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, during screening, LOG2FC ＞ 1 or ＜ -1, FDR ＜ 0.05.In order to be better understood from the function of difference expression gene, we have carried out Gene Onlogy and letter to difference expression gene Number path analysis, and functional annotation and protein interaction network analysis are carried out to difference expression gene, in view of data above The result of analysis, with reference to document, we have screened difference expression gene CCT γ.

The osteosarcoma tissue of embodiment 2 and normal structure CCT γ expression conditions

One material and method

1st, material

65 osteosarcoma tissues and 25 normal structures are collected, it is grouped and numbered.

2nd, method

The extraction of 2.1 osteosarcoma tissues and normal structure total serum IgE

UsingReagent (invitrogen, article No. 15596-018) carries out sample rna extraction, experimental implementation Carried out by product description, concrete operations are as follows：

Freeze to be put into the mortar of precooling tissue after liquid nitrogen, taking-up after collection sample and be ground, treat tissue sample This is into after powdered：

1. Trizol is added, room temperature preservation 5 minutes；

2. chlorination imitates 0.2ml, with forced oscillation centrifuge tube, fully mix, place -10 minutes 5 minutes at room temperature；

3. 12000rpm high speed centrifugations draw upper strata aqueous phase (inhaling 70%) into another new centrifuge tube pipe after 15 minutes, pay attention to The protein substance that be not drawn onto between two layers of aqueous phase.New pipe is moved into, adds the pre- cold isopropanol of isometric -20 DEG C, it is fully reverse Mix, be placed in 10 minutes on ice；

4. 12000rpm at a high speed from 15 minutes after carefully discard supernatant, in I ml/ml Trizol ratio add 75% DEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint sediment, and vibration mixes, 12000rpm high speed centrifugations 5 minutes at 4 DEG C；

5. discarding ethanol liquid, 5 minutes are placed at room temperature fully to dry precipitation, and it is heavy to add the treated water dissolvings of DEPC Form sediment；

6. measuring RNA purity and concentration with Nanodrop2000 ultraviolet specrophotometers, freeze in -70 DEG C.RNA mass is sentenced Calibration is accurate：The OD260/OD280 values of RNA samples are between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S band； 70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern and the collection of illustrative plates no significant difference before water-bath insulation.

2.2 reverse transcriptions synthesize cDNA

UsingIII Reverse Transcriptase (invitrogen, article No. 18080-044) enter Row cDNA reverse transcriptions, experimental implementation are carried out by product description, and concrete operations are as follows：

Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to 1 μ g total serum IgEs with RT Buffer.Using 25 μ l Reaction system, each sample take 1 μ g total serum IgEs to be separately added into following components in PCR pipe as template ribonucleic acid：

5 × RT Buffer, 5 μ l, 10mmol/l dNTP, 1.25 μ l, 0.1mmol/l DTT 2.5 μ l, 30 μm of mol/ 2 μ l, 200U/ μ l MMLV of lOligodT 1.25 μ l, the μ g of template ribonucleic acid 1, aqua sterilisa is added to the μ l of total system 25.42 DEG C are incubated 1 Hour, 72 DEG C 10 minutes, of short duration centrifugation.It is standby that -20 DEG C of refrigerators are put in cDNA preservations.

2.3 Real-Time PCR

2.3.1 instrument and analysis method

With the type quantitative real time PCR Instruments of ABI 7500, the relative quantitative assay of data is carried out using 2- Δ Δ CT methods.

2.3.2 design of primers

Using online primer-design software, synthesized after design of primers by invitrogen companies.Specific primer sequence is as follows：

The primer sequence of table 1

Operating process is as follows：

(1) reaction system：Use PowerGreen PCR Master Mix (invitrogen, article No. 4367659) expanded, experimental implementation is carried out by product description.Amplification program is：95 ° of 10min, (95 DEG C of 15sec, 60 DEG C 60sec) × 45 circulation.

The RealTime reaction systems of table 2

Component	Addition
		2×mix	10μl
Sense primer (10uM)	0.5μl
		Anti-sense primer (10uM)	0.5μl
Template	2μl
		Add sterile purified water	To 25 μ l

(2) primer screening

After each sample cDNA is mixed, 5 times of gradient dilutions are carried out as template, sample respectively takes 2 μ l to make template after dilution, Expanded respectively with target gene primer and reference gene primer, while melt curve analysis analysis is carried out at 60-95 DEG C, according to expansion Increasing Efficiency is high and the unimodal principle of solubility curve carries out primer screening.

(3) sample RealTimePCR is detected

After 10 times of dilutions of each sample cDNA 2 μ l will be taken to make template, entered respectively with target gene primer and reference gene primer Row amplification.Simultaneously solubility curve analysis is carried out at 60-95 DEG C.

Two experimental results

Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tube Rate is close, and the limit is flat and present without raising up, and exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is molten Solution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to qRT-PCR relative quantification formula：2- Δ Ct × 100%, compare expression of the CCT γ genes in osteosarcoma tissue and normal structure.As a result display (is specifically shown in figure 1)：QRT-PCR stable amplification results, wherein expressions of the CCT γ in cancerous tissue are higher than normal structure, result above checking The result of the confluence analysis CCT γ high expression in osteosarcoma tissue of high flux transcript profile expression data.

The osteosarcoma cell line MG-63 of embodiment 3 culture

First, material

(1) Specimen origin

Human osteosarcoma cell line MG-63 is purchased from Shanghai cell research institute of the Chinese Academy of Sciences.

(2) main agents

DMEM/HIGH Glucose (1 ×) (silent winged generation that biochemistry product (Beijing) Co., Ltd of match)

(3) main solution

1st, cell culture fluid

The standard hyclone of DMEM culture mediums+10%.

2nd, PBS (balanced salt solution)

8g NaCl, 0.25g KCl, 1.44g Na are dissolved in 800ml distilled water₂HPO₄With 0.24g KH₂PO₄Use HCl The pH value of solution is adjusted to 7.4, adds water to be settled to 1L, autoclaving, room temperature preservation.

3rd, 0.25% tryptic digestive juice

0.25g trypsase is added in 100ml deionized waters, filter filtration sterilization, and packing is standby.

2nd, experimental method

(1) cell culture

1st, passage

(1) nutrient solution original in the blake bottle that will cover with cell discards, and adds 0.25% trypsin solution 1ml, covering Cellular layer, bottleneck sterilization, capping；

(2) cellular change is observed under inverted microscope, over time, former adherent cell gradually tends to be circular, Cytoplasm bounces back, and space between cells increases, and discards pancreatin in also non-levitating, adds the culture that 5ml contains 10% hyclone Liquid terminates digestion；

(3) cell count：Above-mentioned cell suspension 0.5mI is taken, is instilled after appropriate dilution in blood cell counting plate, by leucocyte Counting method number corner four big lattice inner cell sum, nucleus and the complete cell of cytoplasm, cell in heaps are only counted during counting Calculated by a cell, the TCS in 4 block plaids is pressed into following formula scales into the cell number in every milliliter of cell suspension： Big lattice TCS/4 × 10 of TCS/ml=4⁴× extension rate；

(4) according to cell counts, further it is diluted to every milliliter with DMEM complete culture solutions and contains 3 × 10⁵Individual cell Concentration, (every bottle of 8ml/) is sub-packed in blake bottle, is positioned over 37C, 5%CO₂Cultivated in incubator.

2nd, cell cryopreservation

(1) vitellophag (method is same as above), cell suspension is collected into centrifuge tube, 1000rpm centrifugation 5min, abandons supernatant Liquid；

(2) nutrient solution of precipitation plus the liquid containing protection, counts, adjusts to 5 × 10⁶/ ml or so, by suspension point to cryopreservation tube In, every pipe 1ml；

(3) mouth of pipe will be frozen to obturage, will otherwise easily be burst during recovery, it is labelled, write cell category exactly, freeze day Phase；

(4) cool in the following order：4 DEG C of room temperature (20 minutes), freezer compartment of refrigerator (30 minutes), low temperature refrigerator (- 30 DEG C, 1 Hour), low temperature refrigerator (- 70C, overnight), liquid nitrogen.

3rd, cell recovery

(1) cryopreservation tube is taken out from liquid nitrogen, is immediately placed in 37 DEG C of warm water and constantly stirs.Make to freeze thing in cryopreservation tube Melted within 1 minute, pipe sterilization, enter platform；

(2) cryopreservation tube is opened, cell suspension is drawn onto in centrifuge tube, 1000rpm is centrifuged 10 minutes, abandoning supernatant；

(3) precipitation plus 10ml nutrient solutions, piping and druming is uniform, then centrifuges 10 minutes, abandons supernatant；

(4) add new nutrient solution suitably to dilute, gently mix, be inoculated in blake bottle and cultivate.

Embodiment 4RNAi suppresses CCT γ gene expressions and its influence to human osteosarcoma MG-63 cells

First, material

(1) Specimen origin

(2) main agents

Lipofectamine^TM2000Transfection Reagent (Invitrogen), MTT (Solarbio), Transwell cells (Coming), Matrigel glue (BD).

(3) siRNA structures and synthesis

According to (the http of Photographing On-line software siDirect version 2.0：//design.rnai.jp/), according to CCT γ genes are in GenBank (NCBI Reference Sequence：NM_001008800.2 the corresponding siRNA of sequences Design in). Synesis Company's synthesis is sent to after design.

2nd, experimental method

(1) RNA perturbation techniques specificity suppresses the expression of human osteosarcoma cell's CCT γ genes

1st, human osteosarcoma cell MG-63 culture

Method and step is the same as embodiment 3.

2nd, siRNA design and synthesis

SiRNA expression vector pSIREN-DNR contains neomycin resistance gene and GFP green fluorescent labels, can monitor in real time Transfection efficiency of the carrier in cell.According to purpose mRNA sequence, 3 RNA interference target sequences and negative control (table 3) are designed. For every selected siRNA target sequence, siRNA positive-sense strands and antisense strand are designed, is connected with loop (9nt), referred to as shRNA (shorthairpin RNA).Synthesize two of DNA profiling of every coding shRNA it is single-stranded, annealed dna is single-stranded to obtain shRNA DNA double chain template.Template strand stops site followed by RNA PoIyIII polymerase transcriptions, while both ends separately design BamHI and HindIII restriction enzyme sites, BamHI the and HindIII restriction enzyme sites of siRNA carrier multiple cloning sites can be cloned into Between.SiRNA empty carriers 1% agarose gel electrophoresis, reclaim linear carrier with after BamHI and HindIII double digestions.Moving back The DNA profiling double-strand of fire is connected in linear carrier.Using T4 ligases, the mol ratio of insertion and carrier is about 3：1.Even Thing of practicing midwifery converts DH5 α Escherichia coli, the coated plate on LB Amp culture mediums, 37 DEG C of overnight incubations.PCR is identified；Sequencing identification.Post Extract positive colony carrier and quantify.

The siRNA transcription templates sequences of table 3

3rd, cell packet and transfection

(1) cell is grouped

C groups：Blank control group；C1 groups：Transfect liposome group；C2 groups：Transfect nonspecific siRNA groups；S1, S2, S3 Group：Transfect specific siRNA groups.

(2) transfect

According to Lipofectamine^TMThe step of 2000Transfection Reagent are provided is carried out.

1. 24h before transfection, the cell pancreatin in growth period of taking the logarithm digest and counted, it is dense to adjust cell with DMEM culture mediums Spend for 1 × 10⁵/ ml, take 2ml to be inoculated in six orifice plates, be positioned over 37 DEG C, 5%CO₂Cultivate in incubator, merged in cell up to 80% When be used for transfect.The DMEM medium cultures 3-4h without serum is used before transfection.

2. prepare transfection liquid：

A liquid：250ul serum free mediums dilute 4.0ugDNA, gentle to mix；

B liquid：250ul serum free mediums dilute 10ul Lipofectamine, gentle to mix, and room temperature places 5min；

3. transfect：A liquid mixes with B liquid, incubation at room temperature 20min, directly compound is added in every hole, shakes Culture plate, gently mix.In CO₂37C is incubated 24-48h in incubator, changes liquid after 6h, adds the culture medium containing serum.

4th, the checking of transfection efficiency

(1) cellular morphology and transfected condition are observed under fluorescence inverted microscope

After transfecting 24h, culture plate is placed under fluorescence inverted microscope and observes cellular morphology and growth conditions, green fluorescence Lower observation transfected condition.

(2) using the change of CCT γ gene expressions before and after the detection transfection of Real-time PCR methods

1. the structure of standard curve：1 bottle of the osteosarcoma MG-63 cell normally cultivated in 50mI blake bottles is chosen at, is extracted RNA, determine RNA concentration and purity, carry out reverse transcription reaction, ten times of dilutions of DNA profiling of generation will be reacted, obtain equivalent to 10⁴- 10 ° of copies/ul DNA profiling, CCT γ primers and internal reference actin primers are separately added into, prepare 25ul reactants System, using Real-time PCR amplification instruments, carry out pcr amplification reaction.Obtain CCT γ and actin standard curve.

2. the change of CCT γ gene expressions before and after the detection transfection of Real-time PCR methods：The RNA of each group cell is extracted, RNA concentration and purity are determined, carries out reverse transcription reaction, every group of DNA profiling carries out CCT γ and actin Real-time simultaneously PCR reacts, and experiment is in triplicate.

3. row agarose gel electrophoresis are entered to PCR primer.

(2) RNAi suppresses influence of the human osteosarcoma MG-63 cell CCT γ gene expressions to its related biological behavior

1st, mtt assay surveys cell propagation

Succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to the bluish violet crystal of slightly solubility And be deposited in cell, and dead cell is without this function.Dimethyl sulfoxide (DMSO) (DMSO) can dissolve the purple crystal thing in cell, with purple Outer spectrophotometer determines its absorbance at 490nm, can indirect reaction living cells quantity.In the range of certain cell number, MTT The amount that crystal is formed is directly proportional to cell number.

Experimental procedure：

(1) MTT solution：250mg MTT are weighed, are put into small beaker, add 50mI PBS (0.01mol/L, pH7.4), 30min is stirred on electromagnetic force mixer, degerming with 0.22um miillpore filter, packing, 4C is preserved, in two weeks effectively.

(2) cell is grouped：Experiment is divided into normal cell group, negative control group and experimental group.Normal cell group is trained to be conventional Foster MG-63；Negative control group is 48h after non-specific siRNA transfections；Experimental group is 48h after siRNA transfection；Often Group sets 3 repetitions.

(3) inoculating cell：With 0.25% Trypsin Induced single-layer culturing cell, cultivated with containing 10% hyclone DMEM Liquid is made into individual cells suspension, and individual cell is inoculated in 96 well culture plates with every hole 1, per pore volume 100ul.

(4) cell is cultivated：Culture plate is put into CO₂Incubator, at 37 DEG C, 5%CO₂And cultivated under the conditions of saturated humidity.

(5) colour generation：0h, 24h, 48h, 72h, 96h after culture, per hole add MTT solution (5mg/ml) 20ul, 37 DEG C after It is continuous to be incubated 4h, culture is terminated, carefully discards in hole culture supernatant night.Supernatant is sucked, adds dimethyl sulfoxide (DMSO) 150ul, vibration 10min is completely dissolved crystallization.

(6) colorimetric：490nm wavelength is selected, each hole absorbance is determined on ultraviolet specrophotometer.It is if parallel with test hole It is not added with the blank control wells that cell only adds nutrient solution.It is repeated 3 times, records result, using the time as transverse axis, absorbance (A₄₉₀) it is vertical Axle draws cell growth curve.

2nd, vitro invasion is tested

(1) cell prepares

This Setup Experiments blank control group, negative control group and experimental group.Blank control group is the MG-63 of cellar culture； Negative control group is 48h after non-specific siRNA transfections；Experimental group is 48h after siRNA transfection.Each group cell is used 0.25% Trypsin Induced, terminate centrifugation after digesting and discard nutrient solution, washed 3 times, be resuspended in containing 0.1%BSA without blood with PBS In clear culture medium.

(2) structure and experimental procedure of vitro invasion model

Transwell cells are that conventional Matrigel glue rebuilds substrate membranous system, are in vitro study tumor cell invasions With the effective ways of displacement behavior.Matrigel is artificial reconstruction's basement membrane material, and main component is Laminin lens and w type glue Original, is a kind of extracellular matrix, is liquid at 4 DEG C, glue can be progressively solidified at 37 DEG C, irreversible.By Transwell cells It is put into 24 well culture plates, small interior deserves to be called room, and lower room is claimed in culture plate.

1. it is coated with basilar memebrane：Transwell cells are put into culture plate, upper indoor splendid attire culture supernatants, lower interior Lower floor's nutrient solution is contained, levels nutrient solution is separated by with polycarbonate membrane (8um apertures).Matrigel4 DEG C, overnight into liquid, is used The upper chamber face of the dilutions of 50mg/L Matrigel 1: 8 coating Transwell cells bottom film, each cell use 100ul, point Be coated on Transwell polycarbonate membrane for 3 times, for the first time with 50ul, second and third time respectively plus 25ul, each interval time For 10min, micropore all on film is covered by artificial basement's glue and is incubated 2h at 37 DEG C makes its gel, draws glue-line table The aqueous phase that face separates out, form artificial recombination basilar memebrane.10ul fibronectin splicing variants (fibronectin, FN) dilution is taken to be applied to The lower surface of cell, 4 DEG C air-dry.

2. inoculating cell：Each group cell is adjusted into density, takes 2 × 10⁵/ ml cells 200ul adds upper chamber, and lower room, which adds, to be made For the DMEM culture mediums 500ul containing 10%FBS of chemotactic thing.

3. cultivate cell：37 DEG C, 20h is incubated under the conditions of 5%CO2.

4. fixed and dyeing：Cell is taken out, indoor culture medium is discarded, film upper chamber face is carefully cleaned with cotton swab not

The cell of invasion and attack, after 10min is fixed in lower room face with methanol, 0.1% violet staining.Cut with pocket knife along edge, It is placed on slide, neutral gum mounting.

5. observation and counting：6 200 × visuals field are randomly selected under inverted microscope, theca cell number is worn in counting, is taken Value.Experiment is repeated 3 times.

3rd, experimental result

Real-time PCR detect transfection efficiency.Using Real-time PCR methods structure CCT γ and actin standard Curve, coefficient correlation are respectively 0.9987,0.9979, and linear relationship is good, meets the requirements.Compared with the method for double standard curves The expression of each group CCT γ genes.Blank control group, liposome transfection group, the expression of nonspecific transfection group gene are substantially similar, No significant difference.SiRNA1, SiRNA2, SiRNA3 play the role of to suppress CCT γ gene expressions, CCT γ-siRNA1 Effect become apparent from, suppress efficiency up to 89%, and CCT γ-siRNA2 and CCT γ-siRNA3 inhibitory action is respectively 35% With 29%, compared with blank control group, liposome transfection group, nonspecific transfection group, difference is statistically significant, P ＜ 0.05 (table 4, Fig. 2).

The each group CCT γ mRNA expressions of table 4

Group	CCT γ/actin relative concentration ratios (mean ± standard deviation)
		Blank control group	1.0
Liposome transfection group	1.0458±0.0772
		Nonspecific transfection group	0.9952±0.0821
1 group of CCT γ-siRNA	0.1148±0.0250
		CCT γ-siRNA2 groups	0.6568±0.0202
CCT γ-siRNA3 groups	0.7104±0.0353

Cell growth inhibition assay (MTT).Negative control group elects nonspecific transfection as, experimental group for transfection CCT γ- SiRNA1 groups.The blue colorimetric method of tetramethyl azo (MTT) experiment display, under identical primary condition, each group cell propagation speed Spend close, no difference of science of statistics (P ＜ 0.05).After 24h, siRNA transfection group (0.1599 ± 0.0193) cell propagation Speed slows down, normal cell group (0.2056 ± 0.0208), negative control group cell (0.2013 ± 0.0251) growth rate phase Closely, siRNA transfection group cell proliferation rate slows down, and difference is statistically significant (P ＜ 0.05).As observing time prolongs It is long, compare between preceding two groups of groups, growth rate is similar, no significant difference (P ＞ 0.05)；SiRNA transfection group with First two groups are compared, and growth rate substantially slows down, and have significant difference (P ＜ 0.05).MTT experiment result shows, specific siRNA The cell growth of transfection group is substantially suppressed (table 5, Fig. 3).

MG-63 cells propagation change before and after the RNA of table 5 interference

Time	Normal cell group	Negative control group	Experimental group
				0h	0.1255±0.0223	0.1204±0.0320	0.1274±0.0318
24h	0.2056±0.0208	0.2013±0.0251	0.1599±0.0193
				48h	0.3596±0.0311	0.3527±0.0198	0.2584±0.0289
72h	0.4482±0.0308	0.4451±0.0241	0.3301±0.0124
				96h	0.5580±0.0402	0.5397±0.0336	0.4013±0.0351

Vitro invasion is tested.Cell through Matrigel rebuild basilar memebrane number number reflect cell invasion ability Change.By counting experiments group and cellular control unit through the cell quantity of artificial basement membrane and compared with, it can be found that RNA The motion invasive ability of tumour cell is decreased obviously after interference CCT γ gene expressions, and the cell quantity through artificial basement membrane is bright Aobvious to reduce, blank control group (33.54 ± 1.85), negative control group (33.18 ± 2.74) and experimental group (19.62 ± 3.01) are invaded The relative number difference for attacking cell is statistically significant (P ＜ 0.05) (table 6).

Transwell values after the RNA of table 6 interference CCT γ genes

Group	Transwell values
		Blank control group	33.54±1.85
Negative control group	33.18±2.74
		Experimental group	19.62±3.01

The present invention filters out osteosarcoma pathogenic related gene CCT γ, binding molecule cell biology using high-flux sequence Experimental verification, it was confirmed that CCT γ have the function that important in the propagation of osteosarcoma cell and invasion and attack, suppress CCT γ genes Expression can reduce invasion and attack and the propagation of osteosarcoma cell.The present invention provides new target for osteosarcoma clinic diagnosis, has Good potential applicability in clinical practice.

Claims

The application of 1.CCT γ genes and/or protein inhibitor in anti-osteosarcoma preparation is prepared.
2. application according to claim 1, it is characterised in that the anti-osteosarcoma preparation uses one kind in following methods And/or several expression for suppressing CCT γ genes in osteosarcoma cell：By activating the suppressor of CCT γ genes, activation suppresses The albumen of CCT γ gene expressions, the siRNA for importing suppression CCT γ gene expressions, activation promote CCT γ mRNA degradeds MicroRNA, the expression for importing the molecule for promoting CCT γ protein degradations, the factor of suppression promotion CCT γ gene expressions and albumen.
3. application according to claim 2, it is characterised in that the siRNA sequence choosing of the suppression CCT γ gene expressions From：CCT γ-the siRNA1 being made up of SEQ ID NO.1 and SEQ ID NO.2；By SEQ ID NO.3 and SEQ ID NO.4 groups Into CCT γ-siRNA2；CCT γ-the siRNA3 being made up of SEQ ID NO.5 and SEQ ID NO.6.
Application of the 4.CCT γ genes in osteosarcoma diagnosis and treatment preparation is prepared.
5. application according to claim 4, it is characterised in that described osteosarcoma diagnosis and treatment preparation includes using fluorescent quantitation The expression of CCT γ genes in PCR method, method for gene chip detection osteosarcoma tissue.
6. application according to claim 5, it is characterised in that detect CCT γ bases in osteosarcoma with fluorescence quantifying PCR method The product of cause contains the primer of a pair of specific amplification CCT γ genes；Genetic chip includes miscellaneous with the nucleotide sequence of CCT γ genes The probe of friendship.
7. application according to claim 4, it is characterised in that described osteosarcoma diagnosis and treatment preparation includes being examined with immunization method Survey the expression of CCT γ albumen in osteosarcoma.
8. application according to claim 7, it is characterised in that CCT γ albumen tables in the immunization method detection osteosarcoma Reach for ELISA detection kit and/or gold-immunochromatographyreagent reagent for assay box.
9. application according to claim 6, it is characterised in that the Product checking gene C CT γ, using special upstream Primer and anti-sense primer, upstream primer sequence are SEQ ID NO.9, and downstream primer sequence is SEQ ID NO.10.