CN107827953A - It is a kind of to target cell-penetrating peptide, the medicine with it and application - Google Patents

Abstract

Cell-penetrating peptide, the medicine with it and application are targetted the present invention relates to a kind of.The targeting cell-penetrating peptide includes the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence shown in SEQ ID NO.1 is AVPIRRKRRRCNYYSNS.Medicine can be made in the targeting cell-penetrating peptide, applied in the preparation of anti-tumor drug.The vascular endothelial growth factor receptor that the present invention can be overexpressed with tumor cell surface is combined, targeting plays the antitumor activity for suppressing tumor cell proliferation and promoting apoptosis of tumor cells, the antitumor activity peptide of design have molecular weight is small, space structure not malleable, it is special it is good, dosage can be reduced, reduce toxic side effect, and synthesis is convenient, the advantages that reducing drug cost, it is a kind of anti-cancer agent for safely and effectively, with targeting wearing film ability.It is generalized among antitumor clinical practice, will has a tremendous social and economic benefits.

Description

It is a kind of to target cell-penetrating peptide, the medicine with it and application

Technical field

The invention belongs to biomedicine field, more particularly to a kind of targeting cell-penetrating peptide, the medicine with it and application.

Background technology

Cell membrane is made up of lipid bilayer, is made up of hydrophobic nonpolar molecule inside it.Cell membrane is material One of barrier of cell is passed in and out, many large biological molecules such as polypeptide, protein, DNA are can not freely to cross over cell membrane to carry out thing Matter and information transmission.Although this barrier has protective effect to body, also abandoned many medicines for having application prospect.

In recent years, foreign scholar is found that a kind of protein structure domain in succession in the research to some virus transfection characteristics, Such as：The trans activating transcription factor (TAT) of human immunodeficiency virus (human immunodeficiency virus, HIV-1), Herpes simplex virus I-type VP22 transcription factors etc., not only with cell-penetrating ability, and with mediation heterologous protein, oligomerization The macromoleculars such as nucleotides, liposome are directed through the function that cell membrane enters cell, are named as cell-penetrating peptide (cell- Penetrating peptides, CPPs), be also referred to as protein transduction domain (protein transduction domain, PTD).Cell-penetrating peptide (cell penetrating peptides, CPPs) is also known as protein transduction domain or film transduction peptide, is It is a kind of by 30 or the polypeptide for being capable of penetration cell film that less amino acid forms, many including medicine can be delivered Material.

At present, although having had many chemical synthesis series antineoplastic medicaments to come out and having entered clinic, this kind of medicine is deposited Side effect compared with it is strong, may to normal affecting cells, biologically active drug dosage is big, drug therapy is less efficient etc. Problem, limit the application of antineoplastic.Therefore, it is badly in need of the research and development of new targeting anti-tumor polypeptide drugs, not only Dosage, reduction toxic side effect can be reduced, and synthesizes convenient, reduction drug cost, is developed as a kind of safe and effective, tool There is the anti-cancer agent that film ability is worn in targeting.If be generalized among antitumor clinical practice, huge society will be produced Can benefit and economic benefit.

The content of the invention

In view of the problems of prior art, the present invention provides a kind of targeting cell-penetrating peptide, has its medicine and answer With.The targeting cell-penetrating peptide includes the amino acid sequence shown in SEQ ID NO.1, and the amino acid sequence shown in SEQ ID NO.1 is AVPIRRKRRRCNYYSNS.Medicine can be made in the targeting cell-penetrating peptide, applied in the preparation of anti-tumor drug.The present invention With that can reduce dosage, reduce toxic side effect, and synthesize convenient, reduce drug cost, be it is a kind of safely and effectively, tool There is the anti-cancer agent that film ability is worn in targeting.Be generalized among antitumor clinical practice, will produce huge social benefit and Economic benefit.

The technical scheme that the present invention solves above-mentioned technical problem is as follows：

The present invention provides a kind of targeting cell-penetrating peptide, and the targeting cell-penetrating peptide includes the amino acid sequence shown in SEQ ID NO.1, Amino acid sequence shown in SEQ ID NO.1 is AVPIRRKRRRCNYYSNS.

The vascular endothelial growth factor receptor that the present invention can be overexpressed with tumor cell surface is combined, and targeting, which plays, to be suppressed Tumor cell proliferation and the antitumor activity for promoting apoptosis of tumor cells, the antitumor activity peptide of design have molecular weight small, empty Between structure not malleable, it is special it is good, dosage can be reduced, reduce toxic side effect, and synthesize convenient, reduce drug cost The advantages that, it is a kind of anti-cancer agent for safely and effectively, with targeting wearing film ability.The peptide of tumor chalone antitumor activity peptide 7 CNYYSNS has β-bend, and it has certain Proliferation Ability and pro-apoptosis bioactivity to tumour cell, but it gives birth to tumor vessel Long effect is weaker, and with certain unstability, degradable.For this feature by Antineoplastic angiogenesis activity, targeting Peptide, cell-penetrating peptide sequence add, and ensure that the active region β-bend space structure of the peptide of C-terminal 7 is constant, the cell-penetrating peptide of synthesis only has 17 ammonia Base acid, improves targeting and wears film ability.

The present invention also provides a kind of targeting cell-penetrating peptide medicine, and the medicine includes above-mentioned targeting cell-penetrating peptide, and the medicine can be with Including other auxiliary materials, for preparing the formulation suitably applied.The medicine be easily soluble in water, molecular weight it is small, without antigenicity, example Such as：Injection can be used as to carry out subsequent applications.

The present invention also provides application of the above-mentioned targeting cell-penetrating peptide in antineoplastic is prepared.

Above-mentioned targeting cell-penetrating peptide suppresses the medicine of proliferative activity o f tumor preparing, promotes the medicine of apoptosis of tumor cells Application in thing, the medicine for influenceing tumour cell energetic supersession and/or the medicine for suppressing tumour cell glycolysis.

The present invention also provides application of the above-mentioned targeting cell-penetrating peptide in treatment and/or prevention gastric cancer medicament is prepared.

Above-mentioned targeting cell-penetrating peptide suppresses the medicine of proliferation of human gastric cancer cell activity preparing, promotes the medicine of apoptosis in gastric cancer Application in thing, the medicine for influenceing stomach cancer cell energetic supersession and/or the medicine for suppressing stomach cancer cell glycolysis.

The present invention also provides application of the above-mentioned targeting cell-penetrating peptide in treatment and/or the medicine that prevents liver cancer is prepared.

Above-mentioned targeting cell-penetrating peptide suppresses the medicine of hepatoma cell proliferation activity preparing, promotes the medicine of hepatoma cell apoptosis Application in thing, the medicine for influenceing liver cancer cells energetic supersession and/or the medicine for suppressing liver cancer cells glycolysis.

The present invention also provides a kind of nucleotide sequence for encoding above-mentioned targeting cell-penetrating peptide.

The beneficial effects of the invention are as follows：The present invention have it is special it is good, dosage can be reduced, reduce toxic side effect, and The advantages that reducing drug cost, it is a kind of anti-cancer agent that safely and effectively, there is targeting to wear film ability and synthesis is convenient.Push away Extensively arrive among antitumor clinical practice, will have a tremendous social and economic benefits.

Brief description of the drawings

Fig. 1 is the growth curve that T7-R-T polypeptides act on tri- kinds of cells of SGC-7901, HepG-2,293t.

Fig. 2 is that T7-R-T polypeptides act on tri- kinds of cell HE coloration results (40 × 10) of SGC-7901, HepG-2,293t.

Fig. 3 A are that T7-R-T polypeptides act on tri- kinds of cell flow cytometer detection apoptosis results of SGC-7901, HepG-2,293t；Fig. 3 B For the testing result of tri- kinds of apoptosis rates of SGC-7901, HepG-2,293t.

Fig. 4 is influence of the T7-R-T polypeptides to tri- kinds of cell cycles of SGC-7901, HepG-2,293t.

Fig. 5 is influence of the T7-R-T polypeptides to tri- kinds of cell cycles of SGC-7901, HepG-2,293t.

Fig. 6 is T7-R-T polypeptides to tri- kinds of cell HK effect of vigor of SGC-7901, HepG-2,293t.

Fig. 7 is T7-R-T polypeptides to tri- kinds of cell PK effect of vigor of SGC-7901, HepG-2,293t.

Fig. 8 is T7-R-T polypeptides to tri- kinds of cell SDH effect of vigor of SGC-7901, HepG-2,293t.

Fig. 9 is that T7-R-T polypeptides influence on tri- kinds of cell LD contents of SGC-7901, HepG-2,293t.

Figure 10 is that T7-R-T polypeptides influence on tri- kinds of cellular ATP contents of SGC-7901, HepG-2,293t.

Figure 11 is influence of the T7-R-T polypeptides to several protein expressions of SGC-7901 cells.

Figure 12 is influence of the T7-R-T polypeptides to several protein expressions of HepG-2 cells.

Figure 13 is influence of the T7-R-T polypeptides to several protein expressions of 293t cells.

Embodiment

The principle and feature of the present invention are described below in conjunction with accompanying drawing, the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the present invention.

The present invention has synthesized polypeptide first, and polypeptide has the amino acid sequence shown in SEQ ID NO.1, is specially AVPIRRKRRRCNYYSNS, it is named as T7-R-T.

It is anti-to gastric carcinoma cells (SGC-7901) and human liver cancer cell (HepG-2) swollen that the present invention have studied peptide T 7-R-T Tumor activity and the influence of energetic supersession, in the course of the study, comparative study is used as using people's kidney blastocyte (293t).

Proliferation inhibition rate (SGC-7901 cells, people liver of the T7-R-T to four kinds of tumour cells have detected by MTT Cancer cell HepG-2, human lung cancer cell A549, human breast cancer cell line Bcap-37), finally filter out two kinds of more sensitive cells Strain-human stomach cancer cell line (SGC-7901), human hepatoma cell strain (HepG-2), and people's kidney blastocyte strain (293t) is chosen as just Normal cell controls.The agent of mtt assay, growth curve method detection medicine to the inhibited proliferation, respectively detection effect of tumour cell Measure dependence and time dependence；The double dye method detection medicines of HE decoration methods, Anexin V-PI are made to the rush apoptosis of tumour cell With the influence of change and apoptosis rate of the detection medicine to tumour cell form respectively.Mono- dye method (flow cytometer) the detection medicines of PI Influence of the thing to tumour cell cycle.

Concrete operations have：1.MTT methods screen sensitive cells strain and administration concentration, increasings of the detection T7-R-T to tumour cell Inhibiting rate and IC50 are grown, research medicine acts on the concentration dependent of tumour cell；2. cell counting draws growth curve, grind Study carefully inhibited proliferations of the T7-R-T to tumour cell and the time dependence to tumour cell；3.HE decoration methods observe T7-R-T After effect, the metamorphosis of cell；The double dye methods of 4.Annexin V-PI, flow cytomery apoptosis rate；The mono- dyes of 5.PI, The influence of flow cytomery T7-R-T cell cycles；6. spectrophotometry cellular energy metabolism enzyme-hexokinase, Pyruvate kinase and Succinate Dehydrogenase Activity and energetic supersession product lactic acid and ATP changes of contents；7.Western blot methods Detect intracellular hypoxia inducible factor (HIF-1 α), mono carboxylic transporter (MCT-4) and glucose transporter (GLUT-1) expression Change.

Result of study shows：1. the two kind tumor cell line-SGC-7901s sensitive to T7-R-T are filtered out by MTT results And HepG-2,293t is selected else as control cell.T7-R-T is respectively 379 μ g/mL, 643 μ g/ to the IC50 of three kinds of cytosiies ML, 948 μ g/mL, cell proliferation inhibition rate increase and increased with dosage, and effect has concentration dependent.2. according to growth Dependence Results are drawn, SGC-7901, HepG-2,293t cell survival rate extend with administration time gradually to be reduced, the display time according to Lai Xing, first two inhibiting tumour cells are acted on stronger.3.HE coloration results show that medicine is bred to SGC-7901, HepG-2 Suppress significantly, metamorphosis it is obvious, show as cell rounding, volume-diminished, nuclear hyperchromatism and darkens at kytoplasm aggegation, cell Density significantly reduces.Change unobvious to 293t cellular morphologies, only cell density slightly reduces.The double dye methods of 4.Annexin V-PI Detect in apoptosis experiment, the apoptosis rate of SGC-7901, HepG-2 and 293t cell is respectively 25.8%, 22.6% and after administration 9.9%, there is significant difference compared with control group；5. flow cytometer detection cell cycle result is shown, SGC-7901 and HepG-2 After the effect of T7-R-T peptides, G0/G1 phases and S phase percentage raise cell, and G2/M percentage reduces, and cell is arrested in G0/ The G1 phases.And for 293t cells, after medication the cell cycle there is no significant change.It is and right 6. energetic supersession kit results are shown Compared according to group, HK vigor, ATP and the lactic acid content of two after medication kind tumour cell are remarkably decreased (P ＜ 0.01), SDH activity Declining obvious (P ＜ 0.05), HepG-2 declines obvious (P ＜ 0.01) compared with SGC-7901 to PK vigor, and to 293t HK and PK Vigor, ATP and lactic acid content have declined, and SDH vigor in 293t cells is had no significant effect (P ＞ 0.05)；7.Western Blot results are shown, compared with control group, in SGC-7901 cells HIF-1 α, MCT-4 and GLUT-1 expression all significantly reduce, Only have HIF-1 alpha expressions to reduce in HepG-2 cells, and three kinds of protein expressions do not have significant changes in 293t cells.Conclusion： T7-R-T peptides can suppress the expression of HIF-1 α in SGC-7901 cells and HepG-2 cells, suppress HK and PK in energetic supersession, SDH activity, so as to reduce lactic acid and ATP generations, glycolysis is had a great influence, tumour cell energy supply is hindered, is finally reached Suppress tumor cell proliferation, promote the effect of its apoptosis.Compared with two kinds of tumour cells, T7-R-T polypeptides are to 293t cells in egg Influenceed in terms of white expression, energy supply and cell state smaller.

The T7-R-T peptide antitumor activities of embodiment 1

The present invention has synthesized polypeptide first, and the polypeptide has the amino acid sequence shown in SEQ ID NO.1, is specially AVPIRRKRRRCNYYSNS, it is named as T7-R-T.T7-R-T is have detected by MTT to press down the propagation of four kinds of tumour cells Rate (SGC-7901 cells, human liver cancer cell HepG-2, human lung cancer cell A549, human breast cancer cell line Bcap-37) processed, most Two kinds of more sensitive cell line-human stomach cancer cell lines (SGC-7901), human hepatoma cell strains (HepG-2) are filtered out eventually, and are selected People's kidney blastocyte strain (293t) is taken to be used as normal cell controls.Mtt assay, growth curve method detect propagation of the medicine to tumour cell The dose dependent and time dependence of inhibitory action, respectively detection effect；The double dye method detections of HE decoration methods, Anexin V-PI Medicine detects the influence of change and apoptosis rate of the medicine to tumour cell form respectively to the apoptosis-promoting effect of tumour cell.PI Influence of single dye method (flow cytometer) detection medicine to tumour cell cycle.

Cell line human hepatoma cell strain (HepG-2), human stomach cancer cell line (SGC-7901) used in the present invention, people Kidney blastocyte strain (293t), human lung carcinoma cell line (A549), Breast cancer lines (MCF-7) public can be from Chinese Academy of Sciences Shanghai Cell biological research institute is obtained, and the biomaterial is only attached most importance to used in the related experiment of duplicate invention, other purposes can not be used as to make With.

T7-R-T peptides (purity ＞ 96%), are synthesized by Hefei Sai Mannuo bio tech ltd.According to a kind of in human body The endogenic peptide of tumor chalone antitumor activity 7, wear film sequence is added, promote apoptosis sequence, by sequence alignment, be attached son Connection obtains corresponding amino acid sequence, by applying Fmoc- solid phase synthesis process, the side of HPLC high-efficient liquid phase chromatogram purifications Formula, obtain the high polypeptide of purity.

T7-R-T peptides anti tumor activity in vitro detects

1. experimental method

1.1 Inhibit proliferatons activity

1.1.1 mtt assay

Sensitive Tumor Cells strain and optimal administration concentration scope are screened, detection T7-R-T peptides suppress tumor cell proliferation effect And dose dependent.

By several cell (SGC-7901 cells, human liver cancer cell HepG-2, human lung cancers in exponential phase Cell A549, human breast cancer cell line Bcap-37, people's kidney blastocyte 293t) add cell counting count board count up to cell concentration for 5 × 10⁴Individual/mL, 100 μ l cell suspensions are inoculated with per hole in 96 well culture plates.96 orifice plates are placed in 37 DEG C, 5%CO2 constant incubators Middle culture 24h.Original fluid is suctioned out after 24h, is administered by following concentration gradient, per the μ l of hole 100, final concentration is respectively：60μg/ mL、120μg/mL、240μg/mL、360μg/mL、480μg/mL、600μg/mL、720μg/mL、840μg/mL.Positive controls The μ g/mL of 5-Fluorouracil concentration 10 are given, negative control group adds isometric serum-free 1640 culture medium, every group of 3 multiple holes.It is put into 5%CO₂, the CO of 37 DEG C of saturated humidities₂48h is cultivated in constant incubator.10 μ lMTT (5mg/mL) are added to continue to cultivate 4h per hole.Abandon Stoste, dissolved after 150 μ lDMSO are added per hole with micro oscillator concussion 10min to purple precipitation, with ELIASA in 490nm wavelength Place's detection optical density (OD) value value.Inhibiting rate and IC50 values are calculated, filters out the cell line and optium concentration model sensitive to T7-R-T Enclose.Inhibiting rate (%)=(blank group mean OD value-administration group mean OD value)/blank group mean OD value × 100%.

1.1.2 growth curve method

Detect the time dependence that T7-R-T peptides suppress tumor cell proliferation.

By the SGC-7901 cells of exponential phase, human liver cancer cell HepG-2, people kidney blastocyte 293t PBS Buffer solution rinses 3 times, removes the cell for being suspended in nutrient solution upper strata, adds iuntercellular under 1mL Trypsin Induceds to microscope Gap increases, and edge is shinny, discards trypsin solution, adds serum-containing media and blows and beats repeatedly to formation single cell suspension, cytometer Number plate counts up to cell concentration as 5 × 10⁴Individual/mL.400 μ l cell suspension is inoculated with per hole to 24 well culture plates.By 24 orifice plates 37 DEG C are positioned over, 5%CO₂Continue to cultivate 24h in incubator.T7-R-T administration groups (final concentration of 300 μ g/mL), the positive are set Medicine group (the μ g/mL of 5-Fluorouracil concentration 10), three groups of negative control group (isometric serum-free 1640 culture medium).Former culture is abandoned in suction Liquid, 400 μ l decoctions are given per hole, negative control group adds the culture mediums of 400 μ l 1640.In 37 DEG C, 5%CO₂Continue to cultivate in incubator 24h。

Suction is rinsed 3 times after abandoning original fluid with PBS, and after trypsin solution fully digests, it is molten to add 0.4% trypan blue Liquid is to cell suspension and mixes, make its final concentration of 0.04%.Living cells and dead cell are carried out respectively under light microscopic in 3min Repeatedly count and average.The visible dead cell that blueness is dyed to because of membrane permeability increase under mirror, living cells is due to membrane structure Completely in colorless and transparent.Counted once every 24 hours, persistently count 6 days afterwards.Using cultivated days as abscissa, survivaling cell Number is ordinate, draws cell growth curve figure.

1.1.3 Flow cytometry cell cycle (PI)

(1) cell sample prepares：Take the logarithm the SGC-7901 cells in growth period, human liver cancer cell HepG-2, people Kidney blastocyte 293t, 37 DEG C are put into after normal passage, 5%CO₂Continue to cultivate in incubator, be administered after 24h, every kind of cell is divided into T7-R-T administration groups (final concentration of 300 μ g/mL), positive drug group (the μ g/mL of 5-Fluorouracil concentration 10), negative control group (etc. Volume serum-free 1640 culture medium) three groups, continue culture after administration 48 hours and collect sample, the collection of upper cell nutrient solution It is standby, blow and beat with the abundant vitellophag of pancreatin without EDTA and gently to single cell suspension is formed, cell counting count board counts, sample Product cell quantity is more than 10⁶, the centrifuge tube for being collected into 10mL, 800rpm centrifugations are merged with the cell culture fluid collected in advance 5min, supernatant discarding.Add the PBS 1mL of ice bag precooling and cell is resuspended, 800rpm centrifugations 5min.Repeat above-mentioned heavy Outstanding step twice, single cell suspension is prepared as after collection cell with the PBS of 250ul precoolings.

(2) cell is fixed：The absolute ethyl alcohol of 750ul-20 DEG C of precooling is slowly added to, gently piping and druming mixes, sealed membrane envelope Mouthful, -20 DEG C are fixed more than 10 hours.

(3) dye：1000rpm or so centrifuges 5min, careful supernatant discarding, adds 1mL precooling PBS on ice, washs cell Once.Centrifuge cell and remove supernatant again.With 200ul, cell is resuspended in precooling PBS on ice, adds Rnase A 20ul, 37 DEG C Tank constant-temperature incubation 30min.400ulPI dye liquors (propidium iodide) are added in each sample cell, 4 DEG C of refrigerators are placed in after fully mixing Lucifuge is incubated 30min.

(4) machine testing on：Cell after dyeing crosses 400 mesh nylon mesh, flow cytomery red fluorescence, excitation wavelength 488nm.Data analysis is carried out using the analysis softwares of Flowjo 10.0.

1.2 promote the confirmatory experiment of apoptosis activity

1.2.1 HE is dyed

Detect change of the T7-R-T peptides to tumour cell form.

Take the logarithm the SGC-7901 cells in growth period, human liver cancer cell HepG-2, people's kidney blastocyte 293t are added 2mL PBSs rinse 3 times, remove the cell for swimming in nutrient solution upper strata, add trypsin solution 1mL and fully digest Pancreatin is suctioned out rapidly afterwards, add after nutrient solution with pipettor repeatedly pressure-vaccum cell to forming single cell suspension, cell counting count board Count and adjust cell ultimate density as 5 × 10⁴Individual/mL.

Cover glass is put into after disinfecting in alcohol in 6 orifice plates, and the cell suspension prepared is dropped in into cover glass surface, 1mL/ Hole.15min is stood to cell attachment, 6 well culture plates are put in 37 DEG C, 5%CO₂Incubator, continue to cultivate 24h.Upper strata is abandoned in suction Nutrient solution, each hole of experimental group add 1mL T7-R-T (the μ g/mL of final concentration 300) in distinguishing, and (5-Fluorouracil is dense for positive drug group Spend 10 μ g/mL), negative control group (isometric serum-free 1640 culture medium), by 6 well culture plates in 37 DEG C, 5%CO₂Incubator In continue cultivate 48h.

Original fluid is abandoned in suction, is added 2mL PBS solutions in every hole, is jiggled 6 orifice plates, rinses 3 times repeatedly, to remove The nutrient solution remained in 6 orifice plates.95% alcohol fixation 15min is added in per hole.Alcohol is abandoned in suction, then is rinsed with PBS Three times.1mL haematine dye liquors are added per hole, dye liquor is spread completely on the cover slip, static 5min.Distillation is used after suctioning out haematoxylin Water rinses 3 times, until washing lotion becomes blue.1mL eosin stains are instilled again per hole, are made dye liquor that cover glass be completely covered, static 3min, are steamed Distilled water is rinsed 3 times.Each 1mL of ethanol solution that every hole sequentially adds 30%, 50%, 70%, 80%, 90% is dehydrated step by step.Then The ethanol solution that 95% is added per hole rinses 1min, is repeated 2 times.100% ethanol solution is added per hole and rinses 1min, is repeated 3 times. By cover glass take out insert in dimethylbenzene it is transparent twice, each 5min.Finally face-down slowly it is placed on have a cell during drop has Property natural gum slide on.24h, the photograph of optical microphotograph Microscopic observation are dried in 37 DEG C of thermostatic drying chambers.

1.2.2 Apoptosis by Flow Cytometry rate (Annexin V/PI)

(1) cell is collected：Exponential phase SGC-7901 cells, human liver cancer cell HepG-2, people's kidney embryo is thin Born of the same parents 293t is counted after fully digesting, and adjustment cell concentration is about 5 × 106/mL, 1000rpm centrifugation 5min, abandons supernatant.Often Kind cell is divided into administration group (the μ g/mL of final concentration 300), positive drug group (the μ g/mL of 5-Fluorouracil concentration 10), negative control group (isometric serum-free 1640 culture medium).

(2) cell, 1000rpm centrifugations 5min are washed with incubation buffer.

(3) cell is resuspended in addition 100ul label solutions after abandoning supernatant, and lucifuge is incubated 15min at room temperature.

(4) 1000rpm centrifuges 5min again, abandons supernatant, is incubated buffering washing 1 time.

(5) fluorescent solutions, 4 DEG C of refrigerator lucifuge 20min are added, and are suitably vibrated.

(6) machine testing on, the analysis results of Flowjo 10.0.

(7) result judges：Bivariate (FITC/PI) scatter diagram is selected, left lower quadrant is [PI-/Annexin V- FITC-], represent living cells；Right upper quadrant is [PI+/Annexin V-FITC+], represents non-viable apoptotic cell and non-viable non-apoptotic cell； Right lower quadrant is [PI-/Annexin V-FITC+], represents viable apoptotic cell；And left upper quadrant represents mechanical damage cell.

1.3 statistical method

Using the statistical analysis softwares of SPSS 21.0, as a result represented with mean ± standard deviation (x ± s), data using completely with The multisample mean of machine packet design compares carry out statistical procedures, and * P ＜ 0.05 represent there is significant difference, * * P ＜ 0.01 Expression has significant differences, has statistical significance.

2. experimental result and analysis

2.1 MTT results

According to MTT results, with the rise of T7-R-T peptide concentrations, the inhibiting rate of SGC-7901 cells gradually rises, and gradient becomes Change substantially, there is obvious concentration dependent, growth of tumour cell is significantly inhibited more than 480 μ g/mL concentration.To HepG-2's Inhibitory action is taken second place, and effect has obvious concentration dependent.Growth to 293t cells also has certain inhibitory action, but nothing Obvious graded.For T7-R-T peptides to SGC-7901, HepG-2,293t IC50 is respectively 379,643,948 μ g/mL, right SGC-7901 inhibitory action is the most notable.

2.2 growth curve results

T7-R-T peptides (300 μ g/mL) are to SGC-7901, the growth curve chart of tri- kinds of cell lines of HepG-2,293t, with number of days For abscissa, cell concentration is ordinate, as shown in figure 1, each group cell propagation is affected after administration 48h, cell concentration Begin to decline.It is stronger to SGC-7901, HepG-2 cytosis, suppressed cel l proliferation most at the 4th day and the 5th day respectively By force, cell quantity is reduced more than 50 percentage points, reaches minimum cell concentration at the 5th day, weaker to 293t cytosiies, T7-R-T peptides have the function that stronger Inhibit proliferaton to two kinds of tumour cells, and have obvious time dependence, after administration It can continue to suppress growth of tumour cell.Growth curve experiment is consistent with MTT experiment result.

2.3 HE coloration results

As shown in Fig. 2 compared with control group, after T7-R-T acts on 48h, SGC-7901 and HepG2 cell inhibitory effects Effect is more obvious, and cell quantity is reduced, and cellular morphology changes, cell rounding, volume-diminished, cell nuclear hyperchromatism, kytoplasm aggegation, HepG2 is better than to SGC-7901 effects；And T7-R-T is weaker to 293t cytosiies, cell quantity is slightly reduced, morphologic change It is smaller.Illustrate that T7-R-T can influence SGC-7901 and HepG2 cellular morphologies and can suppress two kinds of tumor cell proliferations, and to 293t The form and proliferative effect of cell are less obvious.

2.4 flow cytometer detection Apoptosis

This experiment uses Annexin V-FITC/PI double-stainings, double using flow cytomery Apoptosis, selection Variable scatter diagram.As shown in Figure 3A, left lower quadrant is double-negative result [PI-/Annexin V-FITC-], represents normal cell； Left upper quadrant represents mechanically damaged cell [PI+/Annexin V-FITC-], in this experiment damaging cells be in compared with Low-ratio.Right upper quadrant is double positive findingses [PI+/AnnexinV-FITC+], represents apoptosis late cell or non-viable non-apoptotic cell, right Lower quadrant represents apoptosis early stage cell [PI-/Annexin V-FITC+].It is thin that apoptosis is represented with the quadrant ratio addition of two, right side Born of the same parents' ratio, according to result shown in Fig. 3 B, the apoptosis rate of SGC-7901 and HepG-2 cells is respectively 25.8% He after administration 22.6%, there is significant difference compared with control group, and early apoptosis and late apoptic ratio are suitable, it was demonstrated that T7-R-T peptides have Play the role of to promote apoptosis of tumor cells.The apoptosis rate of 293t cells is 9.9%, lower slightly compared with apoptosis of tumor cells rate, and early stage Apoptotic cell ratio is decreased obviously, the cause of death for illustrating 293t cells be probably due to foreign substance influence cell growth, and The non-apoptosis-promoting effect for having played polypeptide.

2.5 flow cytometer detection cell cycle results

This experiment is with the mono- dye method processing cells of PI, flow cytomery cell cycle.Such as Fig. 4 and Fig. 5, as a result show Showing, SGC-7901 cells are after the effect of T7-R-T peptides, and the cell cycle changes, and S phase percentage significantly raises (P ＜ 0.05), G2/M phase percentage significantly reduces (P ＜ 0.05), and cell is arrested in the S phases, it is cannot be introduced into the G2/M phases, so as to suppress 7901 cells are bred.After HepG-2 cell administrations, the phases of G0/G 1 and s phases cell percentage significantly raise (P ＜ 0.05), G2/M Phase percentage highly significant reduces (P ＜ 0.01), and cell is arrested in G0/G1 phases and S phases, cannot be introduced into the G2/M phases, so as to Suppress HepG-2 cells propagation.And for 293t cells, after medication the cell cycle there is no significant change.Illustrate T7-R-T to cell The influence in cycle has certain selectivity, and two kinds of tumour cells can be produced with cell-cycle arrest effect, it is swollen to reach suppression The effect of tumor cell proliferation, and normal cell-cycle is had little to no effect.

The present invention has carried out the antitumor Activity determination of T7-R-T peptides, detects Inhibit proliferaton of the polypeptide to tumour cell respectively Activity, the influence for promoting apoptosis activity and cell cycle.

Antineoplastic polypeptide T7-R-T has been synthesized first, and Proliferation Ability of the medicine to four kinds of tumour cells have detected by MTT Rate (SGC-7901 cells, human liver cancer cell HepG-2, human lung cancer cell A549, human breast cancer cell line Bcap-37), finally Two kinds of more sensitive cell line-human stomach cancer cell lines (SGC-7901), human hepatoma cell strains (HepG-2) are filtered out, and are chosen People's kidney blastocyte strain (293t) is used as normal cell controls.Mtt assay, growth curve method detection medicine press down to the propagation of tumour cell Make and use, respectively the dose dependent and time dependence of detection effect；The double dye method detection medicines of HE decoration methods, Anexin V-PI Promotion apoptotic effect of the thing to tumour cell, the influence of change and apoptosis rate of the medicine to tumour cell form is detected respectively.PI Influence of single dye method (flow cytometer) detection medicine to tumour cell cycle.

The screening of cell is carried out by MTT colorimetric methods, determines pharmaceutically-active sensitive cells-gastric carcinoma cells SGC- 7901st, human liver cancer cell HepG-2 shows as research object, people kidney blastocyte 293t as control cell, research, polypeptide pair The Proliferation Ability degree of SGC-7901 cells strengthens with the increase of concentration, has significant graded, and HepG-2 is acted on Take second place, 293t is acted on most weak.Growth curve method show that after polypeptide effect, SGC-7901 and HepG-2 cells are dense by comparing Degree is significantly inhibited with the extension of number of days, also has certain reduction to act on the concentration of 293t cells.Both the above experiment knot Fruit is basically identical, shows that T7-R-T peptides can suppress two kinds of growth of tumour cell, and have dosage and time dependence.It is thin to 293t The inhibitory action of born of the same parents is weaker.

In HE Coloration experiments, T7-R-T administration groups are obvious to SGC-7901, HepG-2 cell inhibitory effect, show as cell Density reduces, and form is rounded, volume-diminished, nuclear hyperchromatism, kytoplasm aggegation, darkened.For 293t cells, cell shape after administration State changes unobvious.In the double dye method detection apoptosis experiments of Anexin V-PI, SGC-7901, HepG-2 and 293t cell after administration Apoptosis rate be respectively 25.8%, 22.6% and 9.9%, there is significant difference compared with control group, wherein SGC-7901, HepG-2 early apoptosis of cells and late apoptic ratio are suitable, it was demonstrated that T7-R-T peptides have the function that to promote apoptosis of tumor cells. And 293t early apoptosis of cells cells ratios are decreased obviously, the cause of death for illustrating 293t cells is probably due to foreign substance shadow Cell growth is rung, rather than has played the promotion apoptosis of tumor cells effect of polypeptide.In summary two kinds of experimental results, T7-R-T energy Enough promote apoptosis of tumor cells, especially SGC-7901 is acted on substantially, it is weaker to 293t apoptosis-promoting effect, have to tumour cell There is certain selectivity.

In the flow cytometer detection cell cycle tests, as a result show, SGC-7901 and HepG-2 cells act on through T7-R-T peptides Afterwards, G0/G1 phases and the rise of S phases percentage, G2/M percentage have reduction, and cell is arrested in G0/G1 phases and S phases, makes its nothing Method enters the G2/M phases, so as to suppress tumor cell proliferation.And for 293t cells, after medication the cell cycle substantially do not become Change.Illustrating the influence of T7-R-T cell cycles has certain selectivity.

In summary, T7-R-T can suppress tumor cell proliferation, promote its apoptosis, and produce cell-cycle arrest work With.It is especially most strong to SGC-7901 cytosiies, HepG-2 is taken second place, and the effect to 293t cells is weaker, illustrates T7-R-T Killing to tumour cell has certain selectivity.

Influences of the T7-R-T of embodiment 2 to tumour cell energetic supersession

The propagation of cell needs substantial amounts of energy to supply, and tumour cell is due to the particularity of energetic supersession, even in having Under conditions of oxygen also based on glycolysis, and substantial amounts of lactic acid is produced so that tumour cell is in acidic micro-environment, enter one Step promotes propagation and the transfer of tumour cell.This experiment is studied tumour energetic supersession, using hexokinase, pyruvic acid Kinase reagent box is measured to several enzymes of glycolytic pathway in SGC-7901, HepG-2 and 293t cell；Select butanedioic acid Dehydrogenase reagent box determines the succinate dehydrogenase activity in several cellular oxidation Phosphorylation events；Detected by lactic acid and ATP Several cell glycolysis metabolism end-product lactic acid of kit measurement and energetic supersession end-product ATP contents, by three kinds of cells The intervention effect of glycolysis and oxidative phosphorylation energetic supersession discloses influence of the T7-R-T peptides to tumour cell energetic supersession.

1. experimental method

1.1 cell interventions are with collecting

By the SGC-7901 cells of exponential phase, human liver cancer cell HEPG-2, people kidney blastocyte 293t PBS Rinsing 3 times, the digestion of 1mL trypsin solutions is added, micro- Microscopic observation to cell rounding brightens, and adds 1640 culture medium repeatedly Cell suspension is made in piping and druming, counts up to cell concentration as 2 × 10⁵Individual/mL.2mL cell suspensions are taken to add blake bottle (25cm²) in, 37 DEG C are put, 5%CO₂24h is cultivated in incubator.Every kind of cell is divided into administration group (T-7-R peptides, the μ g/mL of final concentration 300), positive Control group (5-Fu, 10 μ g/mL), negative control group (equivalent serum-free 1640), continue culture 48 hours after administration, with 0.25% After pancreatin 1mL digestion, add 2mL 1640 culture mediums containing serum that single cell suspension is made.Cell suspension is transferred in 4mL EP pipes, 2000rpm centrifuges 5min and abandons supernatant.Continue to centrifuge after adding 2mL 0.9% physiological saline, repeat above washing operation 2 times. Cell suspension is made in addition 1mL physiological saline after abandoning supernatant, and -20 DEG C of refrigerator storages are standby.

The processing of 1.2 samples

The processing of general sample：Cell suspension is added in quartzy cell homogeniser, is homogenized 6-8 under the conditions of 0 DEG C manually It is secondary, 1min is homogenized every time, is spaced 30s, and Microscopic observation after bulk cell fragment or intact cell until without that can preserve, if any big Block cell fragment or intact cell continue homogenate 6-8 times.Sample is stored in -20 DEG C of refrigerators.

ATP content sample treatments：The cell suspension gathered (is no less than 10⁶It is individual), add in the 300 hot distilled waters of μ L. It is homogenized in boiling water bath after crushing and continues to heat 10min, suspension is taken out into continuation whirlpool mixes 1min, if bulk cell is broken Piece after centrifuging, it is necessary to take supernatant to determine, otherwise can directly determine.

1.3 cellular protein concentrations determine

BCA methods measure protein concentration has the advantages that simple, high stability, high sensitivity and good compatibility.In alkaline bar Under part, Cu²⁺Cu is reduced to by albumen⁺, Cu⁺Reacted with BCA reagents and produce violet complex, then can use spectrophotometric measurement It is fixed.

Standard protein (20mg BSA) is added into 0.8mL protein standards to prepare in liquid, after vibration dissolving, is configured to 25mg/mL protein standard solution.(it can be used immediately after preparation, -20 DEG C permanently effective.) take 25mg/mL protein standard solution 20 μ L, 980 μ L normal saline dilutions are added to final concentration of 0.5mg/mL protein standards solution.Separately 100 μ L BCA reagents B are added Enter into 5mL BCA reagent As to be configured to 5.1mL BCA working solutions (being imitated in 24h).0.5mg/mL protein standards solution is pressed 0,1, 2,4,8,12,16,20 μ L are added in cuvette, are supplied DEG C with standard dilutions less than 20 μ L.Add 200 μ L in each cuvette 60 DEG C of incubation 30min of BCA working solutions.Under biological spectrophotometer, distilled water is returned to zero, and absorbance is determined at 562nm, draws mark Directrix curve.

The μ L of cell homogenates liquid 20 are added in cuvette, separately plus 60 DEG C of 200 μ L BCA working solutions are incubated 30min.Biology Under spectrophotometer, distilled water is returned to zero, and absorbance is determined at 562nm.Protein content is calculated according to standard curve.

1.4 glycolytic pathway enzyme activity determinations

1.4.1 hexokinase (HK) vitality test

(1) preparation of reagents：The sample treatment by the processing of aforementioned sample (processing of 1.2 samples).The 1mL of reagent two is taken to add 9mL distilled waters dilute, and now match somebody with somebody before use；The powder-injection of reagent three is diluted with 2mL distilled waters, now matched somebody with somebody before use；Take No. four reagents Add 7mL distilled waters to dilute, now match somebody with somebody before use.Reagent one: reagent two: reagent three: reagent four: reagent five is pressed in the preparation of working solution: Distilled water=8: 4: 2: 8: 0.4: 16 ratio is prepared.In working solution 24h effectively.The coupling that this experiment passes through hexokinase Reaction, under the premise of enough substrates are provided, reflect the activity of hexokinase by detecting the value added of optical density.

(2) ultraviolet specrophotometer wavelength is adjusted to 340nm, from 0.5cm optical path quartz colorimetric utensils, distilled water zeroing. Working solution is in 37 DEG C of water-bath 10min.

(3) test tube after numbering is respectively added into the μ L of sample 50, it is rapid to be separately added into 37 DEG C of working solution 0.96mL in each test tube Mix simultaneously timing.

(4) first absorbance A1 is read during 30s.

(5) reaction solution is refunded into test tube, 37 DEG C are continued accurately to read A2 values during water-bath 2min, 150s.

(6) HK vigor is calculated as follows：

Note：MM extinction coefficient=6.22；Reaction time=2min；Colorimetric optical path=0.5cm；Sample in reaction system Extension rate=1.01/0.05.

1.4.2 pyruvate kinase (PK) vitality test

(1) preparation of reagents：The sample treatment by the processing of above-mentioned aforementioned sample (processing of 1.2 samples).One 4 DEG C of reagent Preserve；Reagent two plus distilled water 2.8mL, -20 DEG C preserve 3-7 days effectively and can not multigelation；34 DEG C of preservations of reagent；Reagent Four plus distilled water 1.28mL, -20 DEG C of preservations；Reagent slender acanthopanax distilled water 2.6mL, -20 DEG C of preservations.

(2) ultraviolet specrophotometer is placed at wavelength 340nm, and from 0.5cm optical path quartz colorimetric utensils, distilled water returns to zero, Preheat 30min.

(3) by adding each reagent as follows：The 1.0mL of reagent one；The 0.05mL of reagent two；The 0.05mL of reagent three；Reagent four 0.025mL；The 0.05mL of reagent five.After reagent is mixed, 37 DEG C of water-bath 10min.

(4) add sample to be tested 0.02mL to mix, A1 values are determined after timing 30S, rapidly pour into reaction solution 37 in test tube DEG C water-bath 15min.

(5) A2 values are determined in 15.5min.

(6) PK vigor is calculated as follows：

Note：MM extinction coefficient=6.22；Reaction time=15min；Colorimetric optical path=0.5cm；Reaction solution cumulative volume =1.195mL；Sampling amount=0.22mL.

1.5 oxidative phosphorylation enzymes-succinate dehydrogenase (SDH) vitality test

(1) preparation of reagent：The sample treatment by the processing of above-mentioned aforementioned sample (processing of 1.2 samples).Reagent- 120mL, 4 DEG C are kept in dark place；The 6mL of reagent two, 4 DEG C are kept in dark place；The 6mL of reagent three, 4 DEG C are kept in dark place；Reagent four 12mL, 4 DEG C It is kept in dark place；The 6mL of reagent five, -20 DEG C are kept in dark place；The 6mL of reagent six, 4 DEG C are kept in dark place, in mentioned reagent 3 months effectively；Work Make liquid: reagent one: reagent two: reagent three: reagent four: reagent five: reagent six=80: 4: 4: 8: 4: 4mL, lucifuge is protected after preparation Deposit, matching while using.

(2) ultraviolet specrophotometer is placed at 600nm, from 1cm optical path cuvettes, distilled water zeroing, preheating 30min.Working solution is preheated more than 5min by 37 DEG C of water-baths, notices that lucifuge operates.

(3) the μ L of sample 100 are added into numbered test tube, add 2.6mL working solutions, mix simultaneously timing, rapidly will be anti- Liquid is answered to pour into cuvette, this process notices that lucifuge operates.

(4) A1 values are read during 5s, reading A2 values during 65S.

(5) SDH vigor is calculated as follows：

Note：Reaction time=1min；Sampling amount=0.1mL.

1.6 glycometabolism product assays determine

1.6.1 atriphos (ATP) assay

(1) preparation of reagent：(processing of 1.2 samples) ATP content sample treatments in as stated above, substrate solution I are added 10mL boiling distilled water dissolvings, 37 DEG C of water-baths preserve；Substrate solution II 20mL, 4 DEG C of preservations；It is molten that accelerator adds 1.52mL dilutions Solution, -20 DEG C of preservations；Precipitating reagent 5.5mL, 4 DEG C of preservations, in mentioned reagent 3 months effectively；Developer, 28mL solution As add 24mL Second liquid mixes, matching while using；Terminate liquid 50mL, room temperature preservation, in 3 months effectively；ATP standard items add the dissolving of 10mL distilled waters To 1mmoL/L ATP standard items.

(2) ultraviolet specrophotometer is placed at 636nm, from 0.5cm optical path cuvettes, distilled water zeroing, preheating 30min。

(3) four test tubes are taken to be respectively labeled as blank tube, standard pipe, measure pipe and control tube.Each pipe is tried by following add Agent, blank tube：1mmoL/L ATP standard items 30 μ L, substrate solution I 100 μ L, substrate solution II 200 μ L, the μ L of distilled water 30；Standard Pipe：1mmoL/L ATP standard items 30 μ L, substrate solution I 100 μ L, substrate solution II 200 μ L, the μ L of accelerator 30；Measure pipe：It is to be measured Sample 30 μ L, substrate solution I 100 μ L, substrate solution II 200 μ L, the μ L of accelerator 30；Control tube：Sample to be tested 30 μ L, substrate solution I The μ L of 100 μ L, substrate solution II 200, the μ L of distilled water 30.37 DEG C of water-bath half an hour after each pipe fully mixes.

(4) each pipe is separately added into the μ L of precipitating reagent 50, fully mixes, and respectively takes the μ L of supernatant 300 to enter after 4000rpm centrifugations 5min Row measure.

(5) the μ L of nitrite ion 500 mixings are separately added into, the addition μ L of terminate liquid 500 are fully mixed after standing 2min at room temperature, room Pour into cuvette and determine after the lower standing 5min of temperature, notice that 0.5cm cuvettes will be scrubbed fully, in case phosphorus pollutes.

(6) ATP contents are calculated as follows：

Note：Standard concentration=1 × 10³μmol/L；Extension rate=2 before sample measures.

1.6.2 lactic acid content determines

Cell PBS in logarithmic phase is rinsed, 0.25% Trypsin Induced, nutrient solution is prepared into cell suspension.Carefully Born of the same parents' tally counts, and adjusts its concentration as 2 × 10⁵Individual/mL.4 25mL Tissue Culture Flasks, every bottle of 3mL, culture are inoculated in respectively 24h.Next day, administration, CO₂Incubator continues to cultivate 48h.Nutrient solution is collected in EP pipes, 2000rpm, centrifugation 5 minutes, carefully Supernatant is pipetted into new EP pipes, it is effective in one month for composition measurement in nutrient solution, -20 DEG C of preservations.Kit is pressed in experiment Specification step is operated.Returned to zero with distilled water, absorbance is measured at 530nm, and record.Principle：Using NAD+ as hydrogen acceptor, LDH catalysis lactic dehydrogenases produce pyruvic acid, are that NAD+ is converted into NADH.Purple material is ultimately generated, detects and inhales at wavelength 530nm Luminosity is linear with lactic acid content.

2. experimental result and analysis

2.1 protein concentrations and standard curve

It is Y=0.1016X+0.0289 (R to draw albumen mark song equation after testing²=0.9912)；Each group protein content is shown in Table 1.

The each group protein content of table 1

2.2 hexokinases (HK) vitality test result

Hexokinase (HK) is first key enzyme of glycolytic pathway, and its activity height determines speed and the side of glycolysis To.HK can be catalyzed the first step reaction of glycolysis, glucose phosphorylation is generated G-6-P.

From following experimental result (table 2 and Fig. 6), SGC-7901 cells, human liver cancer cell HepG-2, people Upon administration, HK vigor is decreased obviously kidney blastocyte 293t, compared with control group, has significant differences (P ＜ 0.01).Prompting T7-R-T peptides can suppress hexokinase vigor in several cells, and hexokinase is cell glycolysis key Enzyme, cellular energy metabolism can be influenceed.

The T7-R-T peptides of table 2 act on the influence of tri- kinds of cell HK vigor of SGC-7901, HepG-2,293t

2.3 pyruvate kinases (PK) vitality test result

Pyruvate kinase (PK) can catalytic phosphatase formula enol pyruvic acid generation pyruvic acid, adjust glycolysis speed and Direction, it is last rate-limiting enzyme in glycolytic cycle.

From experimental result (table 3 and Fig. 7), SGC-7901 cells, human liver cancer cell HepG-2, people's kidney embryo Upon administration, PK vigor declines cell 293t, and compared with control group, SGC-7901 cells and 293t cells show that conspicuousness is poor Different (P ＜ 0.05), HepG-2 cells show significant differences (P ＜ 0.01).T7-R-T peptides are prompted to HepG-2 cell shadows Sound is larger, also has to SGC-7901 and 293t and has a certain impact, can be by suppressing the pyruvate kinase of glycolysis in cell Vigor, influence cellular energy metabolism.

The T7-R-T peptides of table 3 act on the influence of tri- kinds of cell PK vigor of SGC-7901, HepG-2,293t

2.4 succinate dehydrogenases (SDH) vitality test result

Succinate dehydrogenase (SDH) is the marker enzyme in glycoxidative phosphorylation, is uniquely to be present in line in tricarboxylic acid cycle Enzyme on mitochondrial membrane.SDH can be catalyzed butanedioic acid dehydrogenation and become fumaric acid, and FAD is that electron acceptor generates FADH2, its activity It is usually implemented as evaluating the index of tricarboxylic acid cycle operation degree, and the direct reflection of mitochondrial function.

From experimental result (table 4 and Fig. 8), T7-R-T peptides can cause SGC-7901 cells and HepG-2 cells SDH vigor is remarkably decreased (P ＜ 0.05), and the mitochondrial function of two kinds of tumour cells may be directly inhibited due to T7-R-T peptides, Reduce SDH vigor；May also be because which inhibits the generation of pyruvic acid in glycolytic pathway so that SDH lacks substrate and suppressed System.And the SDH vigor of 293t cells has almost no change, T7-R-T peptides are prompted to follow the tricarboxylic acids in normal cell aerobic oxidation Ring step is not had an impact, and its mitochondrial function is also not corrupted.Illustrate energetic supersession of the T7-R-T peptides to tumour cell Tool is influenceed to have a certain impact.

The T7-R-T peptides of table 4 act on the influence of tri- kinds of cell SDH vigor of SGC-7901, HepG-2,293t

2.5 lactic acid (LD) assay result

For lactic acid as cell glycolysis final product, its content is to detect the horizontal important evidence of glycolysis.

(table 5 and Fig. 9) is understood by following result, compared with control group, SGC-7901 and HepG-2 cell administrations group breast Acid content declines (P ＜ 0.01) in highly significant, and 293t cell LD contents are remarkably decreased (P ＜ 0.05), prompts T7-R-T peptides pair The glycolysis of tumour cell has a great influence, and can substantially reduce SGC-7901 cells and HepG-2 cells LD synthesis, improves two kinds Tumour cell acidic micro-environment, the LD contents of 293t cells are also had a certain impact, but compared with tumour cell, act on compared with It is weak.

The T7-R-T peptides of table 5 act on the influence of tri- kinds of cell lactic acid contents of SGC-7901, HepG-2,293t

2.6 atriphos (ATP) assay result

ATP is the direct Energy supply material of vital movement, is the final product of glycolysis and sugared aerobic oxidation.ATP represents thin Born of the same parents' totality production capacity is horizontal, indicates the metabolic state of cell, affects propagation, growth or even the existence of organism.When thin When born of the same parents are in apoptosis, necrosis or less toxic environment, ATP level can be decreased obviously.

From following result (table 6 and Figure 10), compared with control group, SGC-7901 cells and HepG-2 cell administrations Group ATP contents decline (P ＜ 0.01) in highly significant, and 293t cellular ATP contents are remarkably decreased (P ＜ 0.05), illustrate T7-R-T Peptide has a great influence to SGC-7901 cells and the generation of HepG-2 cellular energies, compared with two kinds of tumour cells, T7-R-T peptides pair The generation of 293t cellular energies influences smaller.

The T7-R-T peptides of table 6 act on the influence of tri- kinds of cellular ATP contents of SGC-7901, HenG-2,293t

The generation of control tumour cell energy is to suppressing tumor cell proliferation, promoting its apoptosis significant.Tumour is thin There are two parts in energy source required for born of the same parents, most importantly by glycolysis, under conditions of aerobic, glycolysis production capacity Quick but production little energy, is only the 1/18 of aerobic glycolysis production capacity, so just needing the active calibration of tumour cell glycolytic ferment Normal cell wants high several times, while tumour cell is also required to aerobic oxidation and provides small part energy.

Key enzyme hexokinase (HK) and the production of pyruvate kinase (PK) and glycolysis of glycolysis are have chosen in experiment Thing lactic acid (LD) come study T7-R-T peptides effect tri- kinds of cell glycolysis of SGC-7901, HepG-2,293t influence.With control group Compare, T7-R-T peptides can cause SGC-7901 cells and HepG-2 cell HK vigor, PK vigor, LD contents to be decreased obviously, right Two kinds of tumour cell HK vigor, LD contents have significant differences (P ＜ 0.01), (P relatively strong to HepG-2 cell PK vigor ＜ 0.01), SGC-7901 cells are taken second place (P ＜ 0.05).For non-tumor cell 293t, T7-R-T peptides can cause cell HK Vigor, PK vigor, LD contents have declined, but significantly lower than the influence to two kinds of tumour cells, prompt T7-R-T peptides to cause SGC-7901 cells and HepG-2 cell glycolytic ferments vigor decline and glycolysis metabolism product lactic acid content is reduced, and suppress sugared ferment Production capacity is solved, improves the acidic micro-environment of two kinds of near tumor cells, the glycolysis inhibitory action of two kinds of tumour cells is better than non- Tumour cell.

In experiment choose tricarboxylic acid cycle in succinate dehydrogenase (SDH) come study T7-R-T peptides effect SGC-7901, Tri- kinds of cell sugar of HepG-2,293t have the influence of oxidative phosphorylation production capacity.Succinate dehydrogenase (SDH) is in glycoxidative phosphorylation Marker enzyme, it is the enzyme being uniquely present on mitochondrial inner membrane in tricarboxylic acid cycle.Its activity is usually implemented as evaluation tricarboxylic acids and followed The index of inscription of loop degree, and the direct reflection of mitochondrial function.Tumour cell mainly by aerobic glycolysis mode production capacity, At least partially through aerobic oxidation production capacity, under T7-R-T peptides can cause the SDH vigor of SGC-7901 cells and HepG-2 cells notable Drop (P ＜ 0.05), one side T7-R-T peptides directly inhibit the mitochondrial function of two kinds of tumour cells, so that tumour cell line SDH vigor on mitochondrial membrane substantially reduces；On the other hand which inhibits the glycolytic cycle of tumour cell, the generation of pyruvic acid Reduce so that SDH lacks substrate and suppressed.And the SDH vigor of 293t cells has almost no change, T7-R-T peptides are prompted to align Tricarboxylic acid cycle step in normal cell aerobic oxidation is not had an impact, and its mitochondrial function is also not corrupted.Illustrate T7- Oxidative phosphorylation production capacity tool of the R-T peptides to tumour cell has a certain impact.

ATP is the direct Energy supply material of vital movement, is the storage of energy and utilizes center, the existence of direct reacting cells State, glycolysis and the final purpose of sugared aerobic oxidation are exactly a large amount of ATP of production capacity, improve energy needed for the various physiological activities of cell Amount, thus ATP represent cell totality production capacity level, compared with control group, T7-R-T peptides effect after SGC-7901 cells and HepG-2 cellular ATP contents decline highly significant (P ＜ 0.01), and the ATP contents of 293t cells have declined, but swollen less than two kinds Oncocyte, illustrate T7-R-T peptides to SGC-7901 cells and HepG-2 cellular energies generation have a great influence, this with previously for enzyme The result of study of vigor and metabolite is consistent.

In summary, T7-R-T peptides are mainly by suppressing SGC-7901 cells and HepG-2 cell glycolysis key enzymes-HK With PK activity, suppress its glycolysis production capacity process, reducing the generation of lactic acid improves acidic micro-environment, so as to reach suppression tumour The effect of cell growth, also there is certain inhibitory action to oxidative phosphorylation production capacity.To the sugar of non-tumor cell 293t cells Diphosphate pathway and tricarboxylic acid cycle also have certain inhibitory action, but act on weaker.It can be said that T7-R-T peptides are thin to tumour The Energy suppression of born of the same parents has certain specificity.

The T7-R-T peptides of embodiment 3 influence the Mechanism Study of tumour cell energetic supersession

The energetic supersession of tumour cell affects the various aspects of its vital movement, 70% in the energy required for cell From in glycometabolism, therefore glycometabolism mainly influences the utilization of energy.The first step of glycometabolism then needs to absorb glucose, due to thin The lipid bilayer Rotating fields of cellular surface by the GLUT (GLUT) on cell membrane, it is necessary to can just be achieved. Tumour cell is due to fast breeding, in hypoxia, often promotes hypoxia-inducible factor-1 alpha (HIF-1 α) expression, and one For aspect by signal transduction tumour cell fast breeding, on the other hand, HIF-1 α can improve the activity of glycolytic ferment, promote Enter tumour cell and produce big energy, accelerate tumor proliferation speed.Mono carboxylic transporter (MCT-4) is the transhipment egg on cell membrane In vain, lactic acid caused by promotion glycolysis transports extracellular, causes cellular acid microenvironment, this acidic micro-environment can be further Promote tumor cell proliferation and improve tumor cell drug resistance.This experiment uses Western-Blot experimental methods, visits medicine pair The influence of tumour cell GLUT-1, HIF-1 α, MCT-4 relative expression, T7-R-T peptides are disclosed to several cells from molecular level The mechanism of action of energetic supersession.

1. experimental method

1.1 collecting protein

By the SGC-7901 cells of exponential phase, human liver cancer cell HepG-2, people's kidney blastocyte 293t are counted Plate is counted until cell concentration is 2 × 10⁵Individual/mL.2mL cell suspension inoculations are taken respectively in blake bottle (25cm²) in, it is placed in 37 DEG C, 5%CO₂24h is cultivated in incubator.Every kind of cell is divided into administration group (T-7-R peptides, the μ g/mL of final concentration 300), positive control Group (5-Fu, 10 μ g/mL), negative control group (equivalent serum-free 1640), administration continue culture 48 hours.Nutrient solution is outwelled, and Blake bottle is tipped upside down on nutrient solution is blotted on filter paper.(4 DEG C) washing cells of the cold PBS of 2mL are added in every bottle of cell, then abandon PBS. Repeated washing is three times to wash away the residual protein in nutrient solution.Blake bottle is placed on ice after PBS is abandoned only, 30min is cracked, is Cell is fully cracked blake bottle will shake.After every bottle of cell adds 150 μ L lysates to crack, with cell scraper by cell scrape in Scrape, collect into 1.5mL centrifuge tubes.

In the presence of medicine, some cell is unable to adherent growth, and also the cell in culture medium should be collected by centrifugation. Culture medium is moved in 4mL PE pipes, 2500rpm centrifuges 5min under the conditions of 4 DEG C.Supernatant is abandoned, addition 2mL PBS, which are fully blown and beaten, to be washed Wash, continue to centrifuge.PBS repeated washings are used after abandoning supernatant once.After supernatant is abandoned in suction, 30min is cracked on ice, in cracking process fully Vibration ensures that cell cracking is complete.Two parts cell pyrolysis liquid is mixed in 4 DEG C, 12000rpm centrifugation 5min, takes supernatant to dispense In 1.5mL EP pipes.

20 μ L protein extracts are taken out in 1.5mL centrifuge tubes, for protein quantification.Remaining protein sample adds its four points One of volume albumen sample-loading buffer, boil 5min.- 20 DEG C of refrigerators are stored in after 12000rpm centrifugations 5min.

1.2 protein quantification experimental procedures are with reference to embodiment 2.

1.3 protein electrophoresis

Protein sample is adjusted to isoconcentration, loading after fully mixing, applied sample amount is 20 μ L.Drive the gas under offset plate before loading away Bubble.The swimming lane loading on both sides is using isometric 1 × loading buffer, with 1 × loading buffer by Marker (3 μ L) adjust to isometric with sample.Loading in the following order, Marker, negative control group, administration group, positive drug group.During electrophoresis, Upper strata glue selects 80V voltages, when sample is run to separation gel, uses 110V voltages instead.General electrophoresis time is in 2.5h or so.Work as egg When white sample is moved to glass plate bottom, closes power supply and terminate electrophoresis.

1.4 transferring film

Glue is unloaded, according to target molecular weight of albumen cuts glue, HIF-1 alpha molecule amount 116KD, MCT-4 molecular weight 50KD, Actin Molecular weight 43KD, GLUT-1 molecular weight 55KD, NC films and filter paper are soaked into 5-30min in transferring film liquid before transferring film.This experiment is adopted With half-dried shifting method, connect electric turn trough and be adjusted to 15V, 5min.

1.5 antibody incubation

Take the film out, closed with milk powder confining liquid, 60r/min shaking table room temperatures 1h.An antibody is diluted from TBST, by HIF- 1 α (1: 500), MCT-4 (1: 500), GLUT-1 (1: 500), β-Actin (1: 200) dilution.By the antibody diluted and film one Rise and be incubated, 60r/min room temperatures 1h.It is put into sealer bag, sealing machine sealing, 4 DEG C overnight.Washed with TBST, 80r/min, 5min, Washing 3-4 times.The secondary antibody of dilution is added on film and is incubated together, 60r/min room temperatures 1h.

1.6 developments and scanning

Horseradish enzyme makes substrate react, and produces brown precipitate in reactive site.DAB developer solutions point drips to NC films, Room temperature lucifuge development 5-20min, observation colour developing change, upper machine scanning.

2. experimental result and analysis

Protein expression in 2.1 7901 cells

Western blotting results show：SGC-7901 cell β-Actin bands are neat and gray value difference ± Within 10, illustrating that each group protein content is identical has comparativity.From experimental result (Figure 11), compared with control group, SGC- 7901 cell administration group HIF-1 α, MCT-4 and GLUT-1 contents are in highly significant downward trend (P ＜ 0.01).

Protein expression in 2.2 HepG-2

Western blotting results show：HepG-2 cell β-Actin bands are neat and gray value difference is ± 10 Within, illustrating that each group protein content is identical has comparativity.From experimental result (Figure 12), compared with control group, HepG-2 is thin Born of the same parents' administration group HIF-1 alpha contents are remarkably decreased (P ＜ 0.05), and two kinds of protein contents of MCT-4 and GLUT-1 do not have significant change.

Protein expression in 2.3 293t

Western blotting results show：293t cell β-Actin bands are neat and gray value difference ± 10 with Interior, illustrating that each group protein content is identical has comparativity.From experimental result (Figure 13), compared with control group, 293t cells are given Medicine group HIF-1 α, MCT-4 and tri- kinds of protein contents of GLUT-1 do not have significant change.

The characteristics of tumor tissues are due to its fast-growth, tumor vasculature is abnormal in addition, causes its blood supply to reduce, therefore Anoxic is the state of tumour cell generally existing.Micro-environmental hypoxia can stimulate hypoxia inducible factor (HIF) transcription, and HIF Activation, can promote the expression of GLUT (GLUT) increases, and strengthens the vigor of glycolytic ferment, so as to promote tumour cell Glycolysis reaction, produce lactic acid and increase, and the effect of mono carboxylic transport protein (MCT) be then will be newborn caused by tumour cell Acid transporter forms the acidic micro-environment of tumour to extracellular, further promotes propagation, transfer and the angiogenesis of tumour cell. The present invention is after it have detected T7-R-T effects, three kinds of important albumen HIF-1 α, MCT-4 and GLUT-1 tables during tumor metabolic Up to change.From experimental result, HIF-1 α, MCT-4 and GLUT-1 expression is all compared with control group, in SGC-7901 cells Significantly reduce, only have HIF-1 alpha expressions to reduce in HepG-2 cells, and three kinds of protein expressions do not become significantly in 293t cells Change.In conjunction with the embodiments 2 and embodiment 1 experimental result, prompt T7-R-T peptides may be by suppressing tumour cell HIF-1 α, MCT-4 and GLUT-1 expression, the transport of the enzyme, lactic acid of intake, the glycolysis of tumour cell glucose is disturbed, so as to suppress sugar Glycolysis process production capacity process simultaneously improves the acidic micro-environment of tumour cell and reaches the inhibited proliferation to tumour cell, right Different tumour cells may have specificity.

The antineoplastic of market is mostly broad-spectrum action at present, lacks targeting, and hardly possible passes through cell membrane, uptake ratio and utilization Rate is low.Therefore we pointedly carry out multiple transformation by biological synthesis method to tumor chalone, obtain a kind of endogenous, The strong antineoplastic polypeptide of Small side effects, targeting：T7-R-T peptides.

The growth of any cell all be unable to do without energy supply.The energy of normal cell is mainly derived from glycometabolism, i.e. grape The oxidation Decomposition of sugar.The oxidation Decomposition approach of glucose includes sugared aerobic oxidation and glycolysis, under conditions of oxygen abundance, grape Glycoxidative decompose produces ATP；Under anoxic conditions, then ATP is mainly produced by glycolysis.Now, it is distributed on cell membrane Glucose transporter just played an important role in glycolytic pathway-by glucose by being transported in extracellular capillary into thin Intracellular.Intracellular glucose is entered by glycolytic ferment catabolism such as hexokinases (HK), then in pyruvate kinase (PK) in the presence of, pyruvic acid is ultimately generated.Under aerobic environment, pyruvic acid enters mitochondria by transport protein, and oxidation is de- Carboxylic generates acetyl coenzyme A, and CO is generated into tricarboxylic acid cycle exhaustive oxidation₂And H₂O.Under anaerobic environment, pyruvic acid takes off in lactic acid Lactic acid is converted into the presence of hydrogen enzyme.And the metabolism of tumour cell and normal cell are different, energy generation is mainly reflected in Thank with metabolism.Tumour cell can more actively absorb glucose and glutamine, carry out aerobic glycolysis (Warburg effects).This to seem uneconomic energy supply mode be necessary to tumour cell, and it had both been tumour cell Propagation energy is provided, be also provided for the raw material of biosynthesis.The feature of tumour cell glycometabolism is that sugar consumption is notable, sugar Glycolysis strengthen, this be probably tumor tissues fast-growth cause local oxygen supply deficiency and tumour cell metabolic characteristics change Event.

The present invention five kinds of tumour cells first by MTT preliminary experiment primary dcreening operations, SGC-7901 is filtered out according to experimental result Cell and HepG-2 cells choose 293t cells as normal cell controls as research object.5-FU quilts in the present invention Positive drug is elected to be, to evaluate the reliability of the effect of by reagent and experiment.5-Fluorouracil (5-FU) is in antineoplastic The antimetabolite of one synthesis, and the anti-miazines medicine that clinical practice is most wide, it is phonetic that effective fluorine urine is converted into the cell Pyridine deoxynucleotide, deoxyribouridine acid can be blocked to be converted into thymidylic acid, suppress tumour cell metabolism so as to reach Purpose.The positive drug selection preferably of the present invention is the energetic supersession inhibitor with antitumous effect, such as hexokinase Inhibitor -1,5-anhydroglucitol, 3-BrPA；PFK 1 inhibitor-levamisol；Lactic dehydrogenase enzyme level Agent-xanthosine.But several glycolytic pathway inhibitor are more effective in part Experiment in vitro above, clinical not true Curative effect is cut, therefore can not be used as positive drug.

In antitumor activity experiment, the Inhibit proliferaton activity of T7-R-T peptides is have detected respectively and promotes apoptosis activity.Adopt With mtt assay, growth curve method, HE decoration methods, flow cytometry (the double dye methods of Annexin V-PI), PI is mono- contaminates preliminary proof T7-R-T peptides have antitumor activity, can suppress two kinds of tumor cell proliferations, promote its apoptosis, but to the propagation of 293t cells Also there is certain influence, effect is weaker, but from the point of view of the double dye results of Annexin V-PI, mechanical damage cell is more, cell late period Apoptosis and necrosis rate are higher, it is possible to and it is due to that 293t cell itself vitality is weak compared with tumour cell, experimental implementation and various The application of reagent also grows on it to be caused necessarily to influence.

Influence of the T7-R-T peptides to tumour cell energetic supersession, including detection to enzyme activity and to metabolite content Detection.Cell glucose metabolism process key enzyme-HK and PK are have chosen, the two is respectively first of glycolytic cycle and last Key enzyme, influence of the polypeptide to glycolytic cycle can be reflected.The alternative choosing for selecting cellular oxidation Phosphorylation events enzyme-SDH, SDH Selecting can either reflect whether polypeptide has an impact to aerobic oxidation process, and and can reflects the functional status of mitochondria.Experiment is also to two Kind energetic supersession product LD and ATP content are detected, and wherein LD is the final product of cell glycolysis, and ATP is all thin The end-product of born of the same parents' energetic supersession, represent total energy supply situation.Experimental result shows that T7-R-T peptides are to three kinds of cell glycolysis Key enzyme --- HK and PK activity has inhibitory action, preliminary to illustrate that polypeptide suppress cell glycolytic cycle.To two kinds Tumour cell SDH has suppression, but influence is had no on 293t cells SDH, and SDH is unique existing metabolism on mitochondrial inner membrane Enzyme, its active stable tendency illustrates that T7-R-T peptides do not have an impact the tricarboxylic acid cycle and oxidation phosphoric acid of 293t cells after medication Change process, its mitochondrial function and activity are also not corrupted.And the SDH vigor of two kinds of tumour cells is remarkably decreased, due to The metabolism of tumour cell is not related to tricarboxylic acid cycle process, and guess is probably that T7-R-T peptides suppress tumour cell glycolytic cycle Afterwards, its energy supply has been blocked, has caused Cytostatic to tumor cell, and has accelerated its Apoptosis process, has made the items of tumour cell raw Life activity and organelle function are destroyed, then are probably that T7-R-T peptides exacerbate tumour one of the reason for SDH activity decreases Cell mitochondrial damages.Illustrate that influence of the T7-R-T peptides to cellular energy metabolism process has specificity, the pass of glycolytic cycle Key enzyme is probably the target spot of its effect.

Finally our expression to three kinds of important regulatory protein HIF-1 α, MCT-4 and GLUT-1 in energy metabolism Amount is detected, and tentatively probes into mechanism of the T7-R-T peptides to tumour cell energetic supersession.GLUT-1 acts on the initial rank of glycolysis Section, can assist in cellular uptake glucose.MCT-4 acts on glycolysis end stage, can transport lactic acid to extracellular, make tumour Microenvironment acidification residing for cell, promote HIF-1 alpha expressions in turn.From the point of view of experimental result, after medication in SGC-7901 cells HIF-1 α, MCT-4 and GLUT-1 expression all significantly reduce, and only have HIF-1 alpha expressions to reduce in HepG-2 cells, and 293t is thin Three kinds of protein expressions do not have significant changes in born of the same parents.With reference to experimental result before, it is inferred that T7-R-T peptides can suppress HIF-1 α expression in SGC-7901 cells and HepG-2 cells, so as to suppress HK and PK activity in glycolytic pathway, suppress thin Born of the same parents' glycolytic cycle, tumour cell energy supply is hindered, suppression tumor cell proliferation is finally reached, promotes the effect of its apoptosis. And the protein expression for 293t cells, energy supply and survival condition are not produced and significantly affected.

Tumour cell energetic supersession is influenceed by many factors such as the relevant enzyme of signal path, metabolism and metabolins, does Tumor metabolic process is disturbed, can targeted inhibition growth of tumour cell, propagation and transfer.Tune of the present invention from tumour cell energetic supersession The detection of section albumen, key enzyme is started with, the mechanism of the preliminary energetic supersession for disclosing tumour cell and with tumor cell proliferation and withering Relation between dying, it is intended to which finding T7-R-T peptides influences the major target class of tumour energetic supersession, and the zymetology for oncotherapy is special Different diagnosis, tumor prevention and treatment, the exploitation of antineoplastic all have important directive significance.At present, many antineoplastics Thing side effect is stronger, the research and development of antineoplastic polypeptide medicine, can not only reduce dosage, reduces toxic side effect, and synthesize Conveniently, drug cost is reduced, is expected to exploitation as a kind of anti-cancer agent safe and effective, that film ability is worn with targeting.It is generalized to Among antitumor clinical practice, it will have a tremendous social and economic benefits.

The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Sequence table

<110>Harbin University of Commerce

<120>It is a kind of to target cell-penetrating peptide, the medicine with it and application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 17

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Ala Val Pro Ile Arg Arg Lys Arg Arg Arg Cys Asn Tyr Tyr Ser Asn

1 5 10 15

Ser

Claims (10)

1. one kind targeting cell-penetrating peptide, it is characterised in that the targeting cell-penetrating peptide includes the amino acid sequence shown in SEQ ID NO.1, Amino acid sequence shown in SEQ ID NO.1 is AVPIRRKRRRCNYYSNS.

2. one kind targeting cell-penetrating peptide medicine, it is characterised in that including targetting cell-penetrating peptide described in claim 1.

3. a kind of injection, it is characterised in that including targetting cell-penetrating peptide described in claim 1.

4. application of the cell-penetrating peptide in antineoplastic is prepared is targetted described in claim 1.

5. target cell-penetrating peptide described in claim 1 preparing the medicine for suppressing proliferative activity o f tumor, promoting tumour cell to wither Application in the medicine of medicine, the medicine of influence tumour cell energetic supersession and/or the suppression tumour cell glycolysis died.

6. application of the cell-penetrating peptide in treatment and/or prevention gastric cancer medicament is prepared is targetted described in claim 1.

7. target cell-penetrating peptide described in claim 1 preparing the medicine for suppressing proliferation of human gastric cancer cell activity, promoting stomach cancer cell to wither Application in the medicine of medicine, the medicine of influence stomach cancer cell energetic supersession and/or the suppression stomach cancer cell glycolysis died.

8. application of the cell-penetrating peptide in treatment and/or the medicine that prevents liver cancer is prepared is targetted described in claim 1.

9. target cell-penetrating peptide described in claim 1 preparing the medicine for suppressing hepatoma cell proliferation activity, promoting liver cancer cells to wither Application in the medicine of medicine, the medicine of influence liver cancer cells energetic supersession and/or the suppression liver cancer cells glycolysis died.

10. a kind of encode the nucleotide sequence that cell-penetrating peptide is targetted described in claim 1.