CN105169402B - Target drug-carried nanometer and its application of immunization therapy cancer of pancreas - Google Patents

Abstract

The invention provides a kind of targeting vector for being used to treat cancer of pancreas, it includes：CD44v6 single-chain antibodies and nano-carrier IONP PEI for targets identification pancreatic cancer cell；Wherein CD44v6 single-chain antibodies are coupled with nano-carrier IONP PEI.On the other hand, the invention provides a kind of nanoparticle for being used to treat cancer of pancreas, it includes foregoing targeting vector, silence RNA and gemcitabine chemotherapeutics, wherein, silence RNA and gemcitabine are compound on foregoing targeting vector.The present invention constructs non-viral gene vector that is efficient, safe, having target function.Silence RNA and gemcitabine are coupled by nano-carrier, and are coupled with anti-CD44v6 single-chain antibodies, by gene therapy, chemotherapy and Biological target therapy use in conjunction, new approaches and scientific basis are provided for the research and development of anti-pancreatic cancer newtype drug.

Description

Target drug-carried nanometer and its application of immunization therapy cancer of pancreas

Technical field

The present invention relates to the targeting vector for treating cancer of pancreas, nanoparticle and its application

Background technology

Cancer of pancreas is one of common malignant tumor of digestive tract, has incidence of occult, the easily low spy of transfer, long-term survival rate Point.Radical surgery excision is to treat the prefered method of cancer of pancreas, but has belonged to late period when most pancreatic adenocarcinoma patients make a definite diagnosis, and is performed the operation Resection rate<20%, chemotherapy is essential therapeutic arsenals, but due to the presence of chemotherapy of tumors resistance, chemotherapeutics effect is not satisfactory, New medicine is developed more effectively to treat the problem of cancer of pancreas is clinical in the urgent need to address.From 1997, gemcitabine It is the first-line drug of clinical anti-pancreatic cancer treatment, but is not made a breakthrough in terms of late result and prognosis, its reason May be relevant to gemcitabine resistance with tumour cell.(the B lymphoma of B lymthoma moloney murine leukemia virus insert district 1 Moloney murine leukemia virus insertion region 1, Bmi-1) albumen is in mankind's Several Kinds of Malignancy Middle high expression, as head and neck neoplasm, breast cancer, cancer of pancreas, its expression are relevant with the occurrence and development of tumour.Bmi-1 participates in cell Cycle, stem cells hyperplasia, the regulation and control of differentiation and aging, can also inducing cell early stage conversion.Research shows to suppress Bmi-1's Expression can suppress the propagation of pancreatic cancer cell, it might even be possible to reverse malignant biological characteristics, the increase pancreatic cancer cell of pancreatic cancer cell Drug susceptibility.The RNA interference of siRNAs (small interference RNA) inductions is specific silencing of target genes Tool, but be a lack of effective carrier and limit its vivo applications.At present polyethyleneimine (polyethyleneimine, PEI) be most widely used nano-carrier, it has positive charge, can be combined with the cell membrane with negative charge, obtain compared with High transfection efficiency, played a role into the cell so as to which siRNA be brought into.Gemcitabine and biological targeting Drug combination are carried High curative effect and reduction reaction toxicity are one of Recent study fields the most active.SiRNAs and chemotherapeutics is used in combination, Chemotherapy and gene therapy use in conjunction are obtained into more powerful, more longlasting synergistic antitumor to act on.Using nano material by chemotherapy Drug encapsulation, and contain siRNAs nano-carrier while be delivered to intracellular, the acute toxicity of medicine can be lowered and improve curative effect. In addition, nano-carrier with coupled antibody, can identify the characteristic of antigen using antibody target, by the gene contained or drug targeting Tumour cell is transported to, lowers the non-specific toxicities of normal tissue cell while Antineoplastic effect is strengthened.IONP (magnetic iron oxide nanoparticles) is that one kind to cell delivery medicine and can have magnetic resonance The carrier of (magnetic resonance imaging, MRI) imaging function.Pancreatic cancer cell overexpression protection adhesion factor CD44v6, can be as the target of identification cancer of pancreas.Anti- CD44v6 single-chain antibodies (the anti-CD44v6single chain Variable fragment, scFvCD44v6) there is high affinity and targeting, identify pancreatic cancer cell with can targetting. Inventor's early-stage Study shows, carries medicine (gene) nanoparticle and single-chain antibody carries out crosslinking and Immunological nanometer particles are made, can carry High medicine reaches the purpose for improving curative effect, reducing toxic side effect to the selectivity of tumour cell.

The content of the invention

The invention provides a kind of targeting vector for being used to treat cancer of pancreas, it includes：It is thin for targets identification cancer of pancreas The CD44v6 single-chain antibodies and nano-carrier IONP-PEI of born of the same parents；Wherein CD44v6 single-chain antibodies and nano-carrier IONP-PEI Coupling.CD44v6 single-chain antibodies identify that cancer of pancreas is thin with can targetting in vivo and in vitro after being coupled with nano-carrier IONP-PEI Born of the same parents.

On the other hand, be used to treat the nanoparticle of cancer of pancreas the invention provides a kind of, it include foregoing targeting vector, And silence RNA, wherein, silence RNA is equipped on foregoing targeting vector.

Specifically, silence RNA is the silence RNAs, i.e. siBmi-1 of Bmi-1 genes.This is equipped with siBmi-1 nanometer Particulate can silence pancreatic cancer cell Bmi-1 gene expressions, cause Panc-1 Cell clonalities weaken, the cell cycle resistance It is stagnant, propagation it is suppressed, migration and invasion and attack reduce.

Further, nanoparticle includes gemcitabine chemotherapeutics (hereinafter referred to as gemcitabine), gemcitabine with SiBmi-1 carries (compound) on cancer of pancreas targeting vector altogether.This has carried gemcitabine altogether and siBmi-1 nanoparticle can be effective Ground silence Bmi-1 gene expressions, inducing cell apoptosis, have cooperative effect in terms of tumor suppression.

The present invention constructs non-viral gene vector that is efficient, safe, having target function.By siRNAs and gemcitabine It is coupled by nano-carrier, and is coupled with anti-CD44v6 single-chain antibodies, gene therapy, chemotherapy and Biological target therapy is combined Using, for anti-pancreatic cancer newtype drug research and development provide new approaches and scientific basis.

Brief description of the drawings

Fig. 1 is IONP-PEI (non-targeted group) and scFv-IONP-PEI (targeting group) when N/P ratios are 20 in embodiment 1 Fluorescence microscopy Microscopic observation after transfection Panc-1 cells；

Fig. 2 is the cell endocytic of scFv-IP/siRNAs nanoparticles and distribution in the cell in embodiment 1, FITC- Mouse anti human 6 × His tag the antibody specificities and scFv of mark_CD44v6With reference to, therefore endocytosis scFv-IP/siRNAs Panc-1 cells are in green fluorescence under fluorescence microscope；

Fig. 3 is IONP-PEI (non-targeted group) and scFv-IONP-PEI (targeting group) when N/P ratios are 20 in embodiment 1 Transfection efficiency, (* vs lipo, #vs scFv-IONP-PEI, P<0.05)；

Fig. 4 is in embodiment 1, and IP/siBmi-1 and scFv-IP/siBmi-1 transfect cell proliferation after Panc-1 cells Influence；

Fig. 5 be embodiment 1 in, IP/siBmi-1 and scFv-IP/siBmi-1 transfection Panc-1 cells after to cell clone The influence of Forming ability；

Fig. 6 be embodiment 1 in, IP/siBmi-1 and scFv-IP/siBmi-1 transfection Panc-1 cells after cell migration, The influence of invasive ability；

Fig. 7 is the living body fluorescent imaging in embodiment 1：IP/siRNAs and scFv-IP/siRNAs different times in vivo (different time points fluorescent material is in vivo after mouse tail vein injection IP/siRNAs and scFv-IP/siRNAs for the distribution of section Distribution)；

Fig. 8 is the in vitro fluorescence imaging in embodiment 1：Mouse tail vein injects IP/siRNAs and scFv-IP/ respectively After siRNAs, mouse is euthanized after 48 hours, each histoorgan is taken out and carries out in vitro tissue imaging；

Fig. 9 is the transmission electron microscope picture of scFv-Gem-IONP compounds in embodiment 2；

Figure 10 is gemcitabine release profiles in scFv-Gem-IONP compounds in embodiment 2；

Figure 11 is Panc-1 cells cytotoxicity (Gem, Gem-IONP, scFv- after different disposal group in embodiment 2 Gem-IONP, scFv-Gem-IONP/siBmi-1 are after various concentrations act on Panc-1 cells to the lethal effect of cell)；

Figure 12 be embodiment 2 in, different disposal group transplanted tumor in nude mice histological examination, mouse tail vein injection PBS (control), after IP-SCR, IP-siBmi-1 and scFv-IP-siBmi-1, experimental endpoints carry out each group transplanting tumor tissue Bmi-1 dyeing detects silence Bmi-1 expressions in each tissue Bmi-1 protein expressions situation detection each group body；

Figure 13 be embodiment 2 in, different disposal group transplanted tumor in nude mice histological examination, mouse tail vein injection PBS (control), Gem, Gem-IONP, scFv-Gem-IONP, scFv-Gem-IONP/siBmi-1, experimental endpoints transplant each group Tumor tissue carries out Bmi-1, Bcl-2, Bax dyeing detection each group Bmi-1 Gene silencing efficacies and apoptosis-related protein expression.

Embodiment

Embodiment 1

The targeting vector of the present embodiment includes CD44v6 single-chain antibodies and nano-carrier IONP-PEI, and it is equipped with siBmi-1.The content of the present embodiment includes experimental method and Analysis of conclusion two parts.

First, experimental method

1st, cell culture

Human pancreas cancer cell strain Panc-1 is in the DMEM in high glucose culture medium containing 10% hyclone, 37 DEG C of constant temperature, 5%CO₂ Cultivated in incubator.

2nd, targeting vector is built

ScFvCD44v6 (CD44v6 single-chain antibodies) is draped over one's shoulders according to Chinese patent 2011102402903,2011102402871 The method synthesis of dew.ScFvCD44v6 and PEI in mass ratio 1：1 coupling, process are summarized as follows：

1. proportionally preparing IONP-PEI compounds, mal-PEG-NHS is dissolved in sterile deionized water, with scFv according to Mol ratio is 1:10 add in compound, mix, and are incubated at room temperature 30min；

2. according to mass ratio it is 1 by the scFvCD44v6 prepared and PEI:1 adds in above-mentioned compound, and 4 DEG C overnight；

3. removing free antibody using preceding 5KD MILLIPORE ultra-filtration centrifuge tubes 3000r/min centrifugations 10min, obtain The targeting vector scFv-IONP-PEI (scFv-IP) of purifying.

3rd, the preparation of scFv-IP/siRNAs compounds (targeting vector for carrying siBmi-1)

It is that 5 μ g, IONP and PEI mass ratioes are 0.75 by IONP mass, prepares IONP-PEI compounds；According to different N/ P ratios, a certain amount of siRNAs solution and IP solution are gently mixed to (medium size sample loading gun gently pressure-vaccum 5 in deionized water It is secondary), incubation 20 minutes is stored at room temperature, that is, obtains the IP/siRNAs compounds of different N/P values.

4th, the distribution of fluorescence microscope scFv-IP/siRNAs nanoparticles in the cell

(1) Panc-1 cells are transfected with IP/siRNAs, scFv-IP/siRNAs respectively, detailed process is as follows：

A, Panc-1 cells are seeded in 24 orifice plates, and 5 × 10⁴/ hole, adherent growth 24 is small in antibiotic-free complete medium When.

B, siRNAs concentration is 100nmol/L.IP/FAM-siRNAs compounds are prepared by N/P ratios=20, mix rear chamber Temperature stands 20 minutes, then adds in 100 μ l transfection special culture medias Opti-MEM and mixes.

C, old DMEM culture mediums in 24 orifice plates are discarded, the fresh plasma-free DMEM mediums of 400ul are added per hole.Then will Above-mentioned 100 μ l mixtures are added dropwise in orifice plate, are gently shaken up.37 DEG C, 5%CO₂Cultivated in incubator.

D, it is to observe IP/FAM-siRNAs compounds in distribution situation intracellular Panc-1, after transfecting 6 hours, removal Old culture medium, after PBS gently washes cell one time, add 1ml PBS.Then in fluorescence microscopy Microscopic observation cell fluorescent images. Lucifuge operates.

(2) after transfecting 6 hours, old culture medium is discarded, is washed three times with PBS, 500 μ l paraformaldehydes (4%) rooms are added per hole Temperature fixes 5 minutes.

(3) PBS gently washes cell 2 times.

(4) 200 μ l 1%BSA/PBS are added per hole, at room temperature low speed shaking table closing 30min.

(5) confining liquid is removed, PBS gently washes cell 2 times.

(6) it is incubated secondary antibody.Add FITC mark mouse and resist 6 × His tag monoclonal antibodies (10 μ g/ml), it is low at room temperature Fast shaking table, lucifuge are incubated 30min.

(7) secondary antibody is removed, PBS is added and gently washes cell 2 times.

(8) core is contaminated.The working solutions of Hoechest 33342 dye core 5min.

(9) PBS gently washes cell 2 times.Add a small amount of PBS, then in fluorescence microscopy Microscopic observation, take pictures.

5th, transfection efficiency is analyzed

By N/P ratios 20 formed scFv-IP/FAM-siRNAs compounds, and using IP/FAM-siRNAs compounds as pair According to transfection Panc-1 cells.SiRNAs concentration is 100nmol/L, flow cytomery each group transfection efficiency.Transfection process is specific It is as follows：

(1) transfection efficiency for measure IP/FAM-siRNAs to Panc-1 cells, by the IP/FAM- of different NP ratios SiRNAs compounds abandon supernatant after transfecting 6 hours, are washed 3 times with PBS, with 0.25% pancreatin-Na₂EDTA is cell from 24 holes Digested on plate, 900rpm is centrifuged 4 minutes, abandons supernatant.

(2) cell is resuspended in PBS, and 900rpm is centrifuged 4 minutes, removes supernatant.

(3) cell, flow cytometer detection is resuspended with 0.5ml PBS.It is lucifuge operation above.

6th, siBmi-1 transfects Panc-1 cells

ScFv-IP/siBmi-1 or IP/si Bmi-1 compounds are formed by N/P ratios 20, transfect Panc-1 cells, with IP/SCR is as control.SiRNAs concentration is 100nmol/L, chooses N/P=20 by IP to Panc-1 cell transfectings siBmi- 1.Panc-1 cell transfectings the previous day plating density is 2 × 10⁵/ hole (6 orifice plate).SiBmi-1 concentration is 100nmol/L.By N/ P ratios=20 prepare IP/FAM-siRNAs compounds, are stored at room temperature after mixing 20 minutes, then add 100 μ l and transfect special training Support and mixed in base Opti-MEM.Old DMEM culture mediums in 24 orifice plates are discarded, the fresh serum-free DMEM cultures of 400ul are added per hole Base.Then above-mentioned 100 μ l mixtures are added dropwise in orifice plate, gently shaken up.37 DEG C, 5%CO₂Cultivated in incubator.Transfection 6 Supernatant is abandoned after hour, fresh complete DMEM culture mediums is added and continues culture 48 hours or 72 hours, carry out subsequent experimental respectively.

7th, qRT-PCR is tested

(1) Trizol methods extracting RNA

1. after transfection 48 hours, PBS treated DEPC gently washs cell 3 times, 6 orifice plates add 1ml Trizol per hole, Be stored at room temperature 3 minutes, blow and beat cell repeatedly, and be transferred to 1.5mlEP pipes, with 1ml sample loading guns repeatedly pressure-vaccum to can be with without naked eyes Precipitation, is then stored at room temperature 5 minutes.

2. each EP pipes add 1/5 volume (0.2ml) chloroform, after covering tightly lid, firmly rock 15 seconds, quiet 5 minutes of room temperature.

3. 4 DEG C, 12,000 × g is centrifuged 15 minutes.

4. carefully drawing upper strata aqueous phase (about 400ul) into another new 1.5mlEP pipes, inside contain cell total rna.

5. adding isometric isopropanol (about 400ul), turn upside down 8 times, be stored at room temperature 10 minutes.

6. 4 DEG C, 12,000 × g is centrifuged 10 minutes.

7. removing supernatant, each EP pipes add 75% ethanol (being matched somebody with somebody with DEPC water) of 1ml precoolings on ice, clean tube wall, as far as possible White flock precipitate thing is not touched (for RNA).

8. 4 DEG C, 12,000 × g is centrifuged 5 minutes.

9. supernatant is removed, drying at room temperature 10 minutes (for salt content in control RNA, as far as possible cleared ethanol).

10. often pipe adds 20-30ul DEPC water, dried RNA is dissolved.

(2) RNA agarose electrophoresis

1. electrophoresis tank is handled：Detergent wash clean；Water rinses；Ethanol is dried；3% hydrogen peroxide solution is filled, is stood 10min；0.1%DEPC is rinsed.

2. electrophoretic buffer (5 × TBE)：Used time is diluted to 0.5 × TBE working solutions.

3. prepare 2% Ago-Gel：0.80g agarose 0.5 × tbe buffer liquids of+40ml are melted in micro-wave oven.

4. agarose to be melted is cooled to 60 DEG C or so, adds after 3ul collaurums mix and be poured slowly into electrophoresis tank, note Meaning does not cause bubble, plugs comb, is stored at room temperature 60 minutes.

5. taking out comb, it is carefully added into after 10ulRNA solution and 6 × sample-loading buffers of 2ul are mixed in well.

6. electrophoresis 30 minutes or so under 5v/cm voltage, now bromophenol blue band go to electrophoresis tank center, in gel imaging Instrument irradiates lower observation band with UV, to determine RNA purity.

(3) trace dna protein assay measure RNA OD₂₆₀And OD₂₈₀Value

Trace dna analyzer measure extraction RNA OD values, to assess its purity and calculate its concentration, wherein OD₂₃₀It is worth generation Table salt content, OD₂₆₀Represent nucleic acid content, OD₂₈₀Value represents protein content, OD₃₂₀Value represents the absorbance of solvent.OD₂₆₀/ OD₂₈₀Ratio between 1.8-2.0, show RNA without obvious protein contamination.RNA original liquid concentrations=(OD₂₆₀-OD₃₂₀) × dilute Release multiple × 40 (ng/ μ l).

(4) cDNA synthesis

1. following reactant mixture (operating on ice) is added in PCR pipe

2. gently mix, the micro centrifuge centrifugation 3-5 seconds.

3. reverse transcription reaction condition is as follows

37℃ 15min

85℃ 5sec

4. the cDNA synthesized is put -20 DEG C and saved backup.

(5) Real Time PCR react

1. following PCR reaction solutions (operating on ice) are added per hole in 96 hole PCR plates

2. 4 DEG C, 3,000 × g is centrifuged 3 minutes.

3. carry out Real Time PCR reactions by following reaction condition

8th, Western blot are tested

(1) total protein of cell extracts

Transfection extracts total protein of cell after 72 hours, and process is as follows：

1. discarding old culture medium, cell is gently washed with PBS one time.

2. 100 μ l RIPA and 1 μ l PMSF (final concentration of 1mM) are added per hole.

3. cell scrape it is several under, lysate is fully contacted with cell, on ice crack 30 minutes.

4. 4 DEG C, 12,000 × g is centrifuged 30 minutes.

5. carefully drawing supernatant, inside contain total protein of cell.

(2) determination of protein concentration

BCA-100 protein quantification kit measurements protein concentration (96 well plate method), detailed process is as follows：

By A：B=50：1 prepares A+B mixed liquors, i.e., each reaction uses 200 μ l Solution A+4 μ l Solution B, used after mixing, i.e., with i.e. use.Each sample will do 3 parallel holes.

1. the preparation of BSA standard items and sample：Sample is prepared with PBS, is diluted 5 times (the μ lPBS of 5 μ l albumen stoste+20), is made Concentration to be determined is located at the linear segment of standard curve.Each reaction prepares 3 parallel determinations.Standard curve typically takes 5-6 Individual point, the specific concentration of each point is determined according to the concentration of sample.BSA is also diluted with PBS.The order of according to the form below adds BSA Standard items or sample and Solution A+B mixed liquors, mix.

3. close the lid mixing 30 seconds, 37 DEG C are incubated 30 minutes.

4. it is cooled to room temperature.

5. 490nm absorbance (OD values) is determined on ELIASA.

6. with Excel software analysis and standard curve is drawn, R²The curves of ＞ 0.99 can use.According to the absorbance of unknown sample Value, uses formula：Y=0.2729x+0.0793 (y=absorbances, x=protein concentrations, R²=0.9994) unknown sample can be drawn The concentration of product, actual concentrations need to be multiplied by the extension rate of sample.

(3) glue.It is sequentially prepared 12% separation gel and 5% concentration glue.

(4) preparation of albumen sample：It is identical (25 μ g) per hole loading specimen amount, it is separately added into 0.2mlEP pipes：Sample Albumen (25 μ g)+4 μ 5 × loading of l buffer+RIPA (containing 1%PMSF), the μ l of cumulative volume 20.

(5) albuminous degeneration.Protein sample and 5 × albumen sample-loading buffer mix, and are placed in 100 DEG C of water-baths 8 minutes, instantaneously from The heart, is cooled to room temperature, and direct loading or -80 DEG C save backup.

(6) loading.Per the μ l of hole 20, blank well adds 15 μ l RIPA+5 μ l 5 × albumen sample-loading buffers, pre- on edge hole Contaminate albumen marker.

(7) electrophoresis.Glue 60V constant pressure electrophoresis is concentrated, when bromophenol blue to separation gel, 120V constant pressure electrophoresis is used instead, to bromine phenol Indigo plant stops electrophoresis to separation gel bottom.

(8) transferring film.Pvdf membrane cuts off the upper right corner with label orientation, and then the pvdf membrane marked is placed in methanol and activated It is put into after 10min standby in transfering buffering liquid.Gel is removed from glass plate, in a certain order by sponge, filter paper, solidifying Glue, pvdf membrane are placed in electricity and turned on folder, are put into electric turn trough, 150mA Constant Electric Currents turn 60min.

(9) close.After transferring film, pvdf membrane is taken out, TBST washes 5min × 3 time, is placed in room temperature in Block buffer and closes 2h.

(10) it is incubated primary antibody.With antibody diluent dilution primary antibody (Tubulin mAb 1:1000, Bmi-1mAb 1： 2000).Pvdf membrane is cut according to pre-dyed albumen marker sizes, is incubated primary antibody, 4 DEG C of incubator overnights respectively.TBST washes film 5min × 3 It is secondary.

(11) it is incubated secondary antibody.Secondary antibody (1 is diluted with antibody diluent：1000), it is incubated at room temperature secondary antibody 2 hours.TBST washes film 5min × 3 time.

(12) expose.Pvdf membrane is placed in exposure box, is uniformly coated with chemical luminescence for liquid, film is covered, is exposed in darkroom 0.5-5min.Film is placed in developer solution and developed, is fixed in fixing solution.Observe band.With Image J software analysis images.

9th, cell proliferation test

(1) Panc-1 cells are seeded in 96 orifice plates, and 1 × 10⁴/ hole, adherent growth 24 is small in antibiotic-free complete medium When.

(2) old culture medium is removed, is separately added into IP/SCR and IP/siBmi-1 (N/P=20) compounds and fresh culture Totally 100 μ l are transfected, and siRNAs amounts are set as 100nM contained by each compound.

(3) 37 DEG C, 5%CO₂Fresh antibiotic-free complete medium is replaced by after being cultivated 6 hours in incubator, per hole 100ul, continue culture 7 days.One block of plate is taken out at random within every 24 hours, cell absorbance is determined with MTS methods.

10th, cell cycle analysis

(1) Panc-1 cells continue culture 48 hours after IP/SCR or IP/siBmi-1 transfections, abandon supernatant, precooling PBS Wash twice, in 0.25% Trypsin Induced collection cell to centrifuge tube, 900rpm is centrifuged 3 minutes, sedimentation cell.It is careful to absorb Supernatant, the nutrient solution of about 50 microlitres or so of residual, to avoid siphoning away cell.The PBS of about 1 milliliter of ice bath precooling is added, is resuspended thin Born of the same parents, 900rpm are centrifuged 3 minutes, sedimentation cell, carefully absorb supernatant, and the PBS of about 50 microlitres or so of residual is thin to avoid siphoning away Born of the same parents.Gently attack centrifuge tube bottom avoids cell agglomerating with appropriate cell dispersion.

(2) cell is fixed：Add in 1 milliliter of ethanol of ice bath precooling 70%, gently piping and druming mixes, and 4 DEG C of fixations are overnight. 900rpm is centrifuged 3 minutes, sedimentation cell.It is careful to absorb supernatant, the PBS of about 1 milliliter of ice bath precooling is added, cell is resuspended, washes altogether Wash twice.Gently attack centrifuge tube bottom avoids cell agglomerating with appropriate cell dispersion.

(3) preparation of propidium iodide stain liquid：Appropriate propidium iodide stain is prepared according to the quantity of detected sample Liquid, i.e., with i.e. use：

(4) dye：0.5 milliliter of propidium iodide stain liquid is added in per solencyte sample, cell is resuspended slowly and fully and sinks Form sediment, 37 DEG C of lucifuge warm bath 30 minutes.Immediately flow cytometer is detected.

(5) flow cytometer detection and analysis：Red fluorescence is detected at excitation wavelength 488nm wavelength with flow cytometer, simultaneously Detect light scattering situation.Gating, light-scattering analysis and cell DNA content analysis are carried out using Cellquest and ModFit softwares.

11st, Clone forming Test

(1) Panc-1 cells are seeded in 24 orifice plates, and 5 × 10⁴/ hole, adherent growth 24 is small in antibiotic-free complete medium When.

(2) old culture medium is removed, is separately added into IP/SCR and IP/siBmi-1 (N/P=20) compounds and fresh culture Totally 500 μ l are transfected, and siRNAs amounts are set as 100nM contained by each compound.

(3) 37 DEG C, 5%CO₂Fresh antibiotic-free complete medium is replaced by after being cultivated 6 hours in incubator, per hole 500ul, continue culture 48 hours.

(4) old culture medium is discarded, PBS washes cell three times, and cell is collected with 0.25% Trypsin Induced, prepares single thin Born of the same parents' suspension, cell is suspended in standby in the DMEM complete mediums of 10% hyclone.

(5) plate clone forms experiment：

(a) take 2000 cells to be inoculated in 6 orifice plates, add complete medium 2ml per hole, and gently rotate, make cell point Dissipate uniform.37 DEG C are put, 5%CO₂Quiescent culture 14 days in incubator, there is more macroscopic clone.The hole of repeated inoculation three.

(b) abandoning supernatant, carefully embathed with PBS 2 times.Pure methanol 2mL is added, fixes 15 minutes.Then fixer is removed, Add appropriate Wright-Giemsa applications dyeing liquor to dye 10~30 minutes, then slowly wash away dyeing liquor with flowing water, air is done It is dry, directly visually observe.

(6) soft-agar cloning forms experiment：

(a) by 1.2% low melting-point agarose and 2 × DMEM complete mediums (containing 2 × antibiotic and 20%FBS) with 1: 1 volume ratio is mixed with 0.6% bottom-layer agar, and each hole 1.4ml greenhouses solidify in 6 orifice plates.

(b) by 1.2% low melting-point agarose and 2 × DMEM complete mediums (containing 2 × antibiotic and 20%FBS) with 1: 1 volume ratio in sterile test tube mutually mix after, then into pipe add 0.2mL cell suspension (totally 5000 cells), fully Mix, injection is covered with 0.6% bottom-layer agar culture plate, by the double agar layers of formation.It is stored at room temperature about 1 hour, treats upper strata fine jade After fat solidification, insert in 37 DEG C of 5%CO2 incubators and cultivate 14 days.Every three days complete DMEM culture medium 1mls of the supplement containing 10%FBS.

(c) counted under low-powered microscope containing clones more than 50 cells, calculate cell colony formation rate, SpotII collections Image.

12nd, migrate and attack experiment

(1) before invasion and attack experiment, the upper chamber in Transwell cells spreads 250 μ g/ml Matrigel glue, is placed in 37 DEG C 4 small When, it is standby after gelling is solid.Migration test need not spread glue.Migration is identical with invasion and attack experiment following steps.

(2) Panc-1 cells digest, and use nothing after IP/SCR or IP/siBmi-1 is transfected 48 hours from 24 orifice plates Serum DMEM in high glucose culture medium is resuspended, by 1 × 10⁴/ hole is seeded in the upper chamber of Transwell cells, and adds 0.1%BAS and put down Weigh osmotic pressure.DMEM in high glucose culture mediums of the 500 μ l containing 10% hyclone is added in lower room and induces its migration, through 8 μm of apertures Polycarbonate membrane.

(3) 37 DEG C, 5%CO₂Cultivated under the conditions of saturated humidity 20 hours (migration tests) or 40 hours (invasion and attack experiment).

(4) cell is taken out, upper chamber face cell is gently wiped with cotton swab, room temperature is fully dried.

(5) pure methanol fixes lower room face cell 15min at room temperature.

(6) PBS is washed 2 times, and cotton swab dries upper chamber face, air-dries 5min at room temperature.

(7) Wright-Giemsa applications dyeing liquor dyes 30 minutes, is washed 3 times with distilled water, and room temperature fully air-dries.

(8) micro- Microscopic observation is placed in, 6 100 × visuals field are taken per cell, counting wears theca cell number, tries to achieve single regard Theca cell number is worn in wild being averaged.

13rd, internal cancer of pancreas targeting Journal of Sex Research

(1) Xenografts in nude mice is formed

Female BAl BIc/C (nu/nu) nude mice that body weight is 14g ± 1g is chosen, by 1 × 10⁷In exponential phase Cell suspension is made with 300 μ lPBS in Panc-1 cells, is subcutaneously injected in nude mice dorsal scapular area, tumor formation rate ＞ 90%.

(2) living body fluorescent is imaged

ScFv-IP is mixed with the 20 μ g siRNAs that nir dye Cy5.5 is marked by N/P=20, forms scFv-IP/ Cy5.5-siRNAs compounds, through nude mice tail vein injection after being diluted with PBS.The forward fluorescence imaging scanning of injection.Injection after Row fluorescence imaging scans again by 12h, 24h, 48h.The fluorescence signal for observing Panc-1 subcutaneous transplantation knurls changes.

(3) in vitro fluorescence imaging

After injecting 24h, hyperfluorescence is still presented in targeting group tumor locus, and non-targeted group of tumor region fluorescence signal is obvious Weaken, still have strong fluorescence to 48 hours targeting group tumor regions, and non-targeted group of fluorescence intensity weakens clearly.By nude mice Excessively anesthesia is put to death, and is peeled off the vital tissue organ such as tumour and the heart, liver,spleen,kidney, is carried out fluorescence imaging again.

14th, statistical method

Compare between two groups of means and examined using t, compared between multigroup mean using one-way analysis of variance.Using SPSS 17.0for windows statistical softwares carry out statistical procedures.Significant difference level is p ＜ 0.05.

2nd, Analysis of conclusion

1st, the cell endocytic of scFv-IP/siRNAs nanoparticles and distribution in the cell

As shown in figure 1, siRNAs is distributed in cytoplasm after scFv-IP/Cy3-siRNAs transfection Panc-1 cells, and target Non-targeted group is considerably higher than to the fluorescence intensity after group transfection.The mouse of FITC marks resists 6 × Histag monoclonal antibodies glimmering Green fluorescence is shown under light microscope, it specifically can be combined with scFvCD44v6, therefore only endocytosis scFv-IP/ SiRNAs cell just shows green fluorescence.As shown in Figure 2：All cells for having Cy3-siRNAs red fluorescences to occur have Green fluorescence occurs, and shows that scFvCD44v6 is successfully connected on IP and by cell endocytic, siRNAs is brought into cytoplasm.

2nd, transfection efficiency is analyzed

The cell of Successful transfection is green fluorescence in flow cytomery, and green cells represent than row brief introduction The transfection efficiency of transfectional cell.As shown in Figure 3：The transfection efficiency of IP/FAM-siRNAs groups is (59.87% ± 5.53%)；scFv- The transfection efficiency of IP/FAM-siRNAs groups is (89.75% ± 2.21%).As a result show, when siRNAs transfection concentrations are identical, target It is higher than non-targeted group to the transfection efficiency of group, illustrates scFv_CD44v6More siRNAs can be promoted to be transported into the cell.

3rd, the checking of siBmi-1 silences Bmi-1 gene expressions

After IP/siBmi-1 and scFv-IP/siBmi-1 processing Panc-1 cells, Bmi-1 genes are in mRNA and the table of albumen It is down to (60.00% ± 7.07%) vs (37.00% ± 2.83%) (p ＜ 0.05) of control respectively up to amount, (40.80% ± 4.92%) vs (36.85% ± 7.93%) (p ＜ 0.05).Prompting scFv-IP targeting groups have more preferable gene silencing function.

4th, cell proliferation experiment

In order to verify after targeting group gene silencing with non-targeted group in terms of the biological function of Panc-1 cells is influenceed it is right Than having carried out subsequent experimental.As shown in figure 4, after processing, in the 7 day time of cell growth, the cell number of targeting group is low always In non-targeted group, in experimental endpoints, two groups of cell number is respectively (0.98 ± 0.04) × 10⁴(targeting group) vs (1.23 ± 0.06)×10⁴(non-targeted group) (p ＜ 0.05).

5th, cell cycle analysis

After targeting group and non-targeted group of processing cell, the cell number in the G0/G1 phases is respectively (69.27% ± 2.46%) Vs (57.25% ± 1.33%) (p ＜ 0.05), the cell numbers of S phases be respectively (12.47% ± 2.46%) vs (26.39% ± 1.34%) (p ＜ 0.05).As a result show, targeting group is by more cell blocks in the G0/G1 phases.

6th, Clone forming Test

As shown in figure 5, after targeting group processing cell, the cell colony of formation is less, smaller, and clonality is weaker.

7th, migrate and attack experiment

As shown in fig. 6, Panc-1 cell migrations and invasion and attack are equal after targeting group and non-targeted group of difference silence Bmi-1 gene Significantly reduced compared with control group, and targeting group is respectively less than non-targeted group：(89 ± 8.02) vs (158 ± 7.78) (p ＜ 0.05) (89.3 ± 8.02) vs (137 ± 9.61) (p ＜ 0.05).

8th, internal cancer of pancreas targeting

Xenografts in nude mice model is established, targetings of the observation scFv-IP in nude mouse to cancer of pancreas combines.Tail vein ScFv-IP/Cy5.5-siRNAs compounds are injected, Cy5.5-siRNAs dosages are 20 μ g/ animals.NIRF imagings can be observed Aggregations of the Cy5.5-siRNAs NPs in tumor tissues.As shown in Figure 7：12 hours after injection, targeting group and non-targeted group it is equal Show more fluorescent material and be gathered in tumor tissues, reach most strong.Continue to observe 24 hours and 48 hours, targeting group tumour Position and non-targeted group of tumor locus fluorescence all gradually weaken.But targeting group tumor locus fluorescence intensity is consistently higher than non-targeted Group.By animal euthanasia after 48 hours, separate the vitals such as tumor tissues and the heart, liver,spleen,kidney and carry out fluorescence again and be in Picture, as shown in figure 8, the tumor tissues of targeting group show higher fluorescence intensity, prompt scFv-IP/Cy5.5-siRNAs NPs Mainly absorbed by tumor tissues, the fluorescence intensity of non-targeted group of tumor tissues is significantly lower than targeting group, prompts by tumor tissues institute The Cy5.5-siRNAs amounts of intake are less than targeting group.

Embodiment 2

The targeting vector of the present embodiment includes CD44v6 single-chain antibodies and nano-carrier IONP-PEI, and it has carried Ji altogether His shore of west and siBmi-1.The content of the present embodiment includes experimental method and Analysis of conclusion two parts.

First, experimental method

1st, cell culture

Human pancreas cancer cell strain Panc-1 is in the DMEM in high glucose culture medium containing 10% hyclone, 37 DEG C of constant temperature, 5%CO₂ Cultivated in incubator.

2nd, the Gem-IONP carrier IONP of gemcitabine (carry) and svFv-Gem-IONP (carry gemcitabine and SiBmi-1 targeting vector) preparation and sign

(1) in ice bath, by triethylamine (Triethylamine) and acid-sensitive tetrapeptide (9-fluorenylm Ethoxycarbonyl (Fmoc)-Gly-Phe-Leu-Gly (GFLG)-nitrophenyl ester peptide) according to 7.5umol：6.8 μm of ol are added in 1.0mol acetonitrile solution.

(2) by gemcitabine and triethylamine (Triethylamine) according to 13.6umol：13.6umol be dissolved in 1ml go from In sub- water.

(3) solution of above-mentioned (2) is added in (1), after rotation mixes 5min, mixed liquor is placed in ice salt bath and reacted 100min。

(4) gradient pressure method removes the acetonitrile and water in mixed liquor, adds water to be settled to 1ml.With 6M HCIs to PH 2.0, the 4 DEG C of preservations of mixture that will be obtained.

(5) obtained solution is washed repeatedly with 1M hydrochloric acid, and last solution is removed with gradient pressure method.Finally obtain Solid is thoroughly drained to obtain solid powder (Fmoc)-GFLG-Gem in vacuum desiccator.

(6) solid dissolving that will be obtained with 0.4ml dimethyl imide (N, N-dimethylmethanamide), to 100ul piperidines (piperidine) is added in solution, room temperature concussion 5min is mixed, placed reaction liquid into ice salt bath, add immediately Acetic acid.After dichloromethane (dichloromethane) extractive reaction liquid, vacuum desiccator freezes compound to GFLG-Gem Thing.

(7) 108.6 μ g diimine (EDAC) and 196.9 μ g N- hydroxy ambers are added into 1mg IONPs solution Amber acid imide (sulfo-NHS), react at room temperature 15min.Obtained amidated IONPs and 420.4ug GFLG-Gem is compound 4 DEG C of amidation process of thing obtain Gem-IONP compounds.

(8) 1.33mg PEI are added to room temperature reaction 30min in IONP-Gem solution and obtain Gem-IONP-PEI.

(9) 47.1ug maleimides-polyethylene glycol-succinimidyl succinate (mal-PEG-NHS) is added to 30min is reacted at room temperature in above-mentioned solution.By 750ug scFv_CD44v6Above-mentioned solution is added to, 4 DEG C obtain scFv-Gem- overnight IONP (i.e. scFv-Gem-IONP-PEI, can be with compound siBmi-1).(the GFLG)-Gem-IONP and scFv- finally obtained (GFLG)-Gem-IONP compounds are purified with magnetic separtor, filter filtration sterilization.

(10) by Gem-IONP and svFv-Gem-IONP its surface characteristics of transmission electron microscope observing and particle size.

3rd, gemcitabine drug release test

(1) 10ul scFv-Gem-IONP are dissolved in 90ul PBSs to (cathepsin B final concentration is 1uM), each time point prepares two pipes, and pH value is adjusted into 5.5 and 7.4 respectively.

(2) 37 DEG C of incubations, at 0,6,12,24,36,48,72,96 hour, each pH value took out a pipe, ultrafiltration respectively Pipe is filtered off except IONP, collects filtered fluid, high-efficient liquid phase analysis method (acetonitrile：Water (20:80, v/v), containing 0.01% trifluoro second Acid, flow velocity 0.3mL/min, wavelength 270nm), gemcitabine concentration in upper machine testing solution.

(3) the gemcitabine medication amount of each time point release, mapping are calculated.

4th, cell toxicity test

Combine cytotoxicity of the gemcitabine immune nano compound to Panc-1 to assess siBmi-1, it is real to carry out MTS Test.Every group sets 3 multiple holes, and the experiment is independently repeated 3 times, and gained numerical value is expressed as mean ± standard error.

(1) Panc-1 cells are seeded in 96 orifice plates, and 1 × 10⁴/ hole, adherent growth 24 is small in antibiotic-free complete medium When.Different disposal is added, experiment packet is：Gem, Gem-IONP, svFv-Gem-IONP, and scFv-Gem-IONP/siBmi- 1, siRNAs amount is per hole 10pmol.Gem concentration is 1-20uM, depending on PEI-IONP is according to various concentrations Gem.Untreated cell As blank control.37 DEG C, 5%CO₂Cultivated 72 hours in incubator.

(2) culture medium is removed, 100 μ l are added per hole and contain the serum free medium of 20ul MTS solution, 37 DEG C, 5%CO₂ Continue culture 4 hours in incubator.

(3) returned to zero with blank group, ELIASA detection 490nm absorbances.

Cell survival rate (%)=(A_{Experimental group}/A_{Control group}) × 100%.

5th, Apoptosis assay

(1) Panc-1 cells are seeded in 6 orifice plates, and 2 × 10⁵/ hole, adherent growth 24 is small in antibiotic-free complete medium When.Different disposal is added, 1. Control 2. Gem 3. Gem-IONP 4. scFv-Gem-IONP 5. scFv-Gem-IONP/ siBmi-1.SiRNAs amounts are per hole 10pmol, and Gem concentration is 5uM, 37 DEG C, 5%CO₂Cultivated 72 hours in incubator.

(2) specific transfection method is as follows：

A, siRNAs concentration is 100nmol/L.IP/FAM-siRNAs compounds are prepared by N/P ratios=20, mix rear chamber Temperature stands 20 minutes, then adds in 100 μ l transfection special culture medias Opti-MEM and mixes.

B, old DMEM culture mediums in 24 orifice plates are discarded, the fresh plasma-free DMEM mediums of 400ul are added per hole.Then will Above-mentioned 100 μ l mixtures are added dropwise in orifice plate, are gently shaken up.37 DEG C, 5%CO₂Cultivated in incubator.

(3) Apoptosis is detected：FITC-ANNEXIN V/PI staining for flow cytometry detection methods

Principle：Early stage apoptosis, the phosphatidyl serine (PS) on the inside of cell membrane outer can be translated into thin On the outside of after birth.The Annexin V of FITC marks are selectively combined with PS, in green fluorescence.Non-viable non-apoptotic cell or apoptosis late cell, Cell membrane integrity is lost, and Annexin V-FITC and propidium iodide (PI) can make its dyeing.Therefore, with Annexin V- After FITC and PI dyeing, normal living cells is not colored；The cell FITC of apoptosis early stage is positive, PI is negative；Non-viable non-apoptotic cell and The cell in apoptosis late period can be dyed by Annexin V-FITC and PI simultaneously.

Colouring method：

1. abandoning old culture medium, rinsed twice with PBS, in 0.25% Trypsin Induced collection cell to centrifuge tube, 900rpm is centrifuged 3 minutes, sedimentation cell.It is careful to absorb supernatant, avoid siphoning away cell.About 1 milliliter of PBS is added, cell is resuspended, 900rpm is centrifuged 3 minutes, sedimentation cell, carefully absorbs supernatant.Gently attack centrifuge tube bottom avoids cell with appropriate cell dispersion It is agglomerating.

2. often pipe adds 500 μ l Binding Buffer, gently piping and druming prepares single cell suspension.

3. often pipe adds FITC/ANNEXIN V 5ul, room temperature lucifuge is incubated 15 minutes after mixing, and then often pipe adds PI Dye liquor 5ul, flow cytometer detects immediately after being incubated at room temperature 5 minutes.

6th, Western blot are tested

Apoptosis-related protein Bcl-2, Bax detection Western blot methods, it is specific as follows：

(1) total protein of cell extracts

Transfection extracts total protein of cell after 72 hours, and process is as follows：

A, old culture medium is discarded, cell is gently washed with PBS one time.

B, 100 μ l RIPA and 1 μ l PMSF (final concentration of 1mM) are added per hole.

C, cell scrape it is several under, lysate is fully contacted with cell, on ice crack 30 minutes.

D, 4 DEG C, 12,000 × g is centrifuged 30 minutes.

E, supernatant is carefully drawn, inside contains total protein of cell.

(2) determination of protein concentration

BCA-100 protein quantification kit measurements protein concentration (96 well plate method), detailed process is as follows：

A, by A：B=50：1 prepares A+B mixed liquors, i.e., each reaction uses 200 μ l Solution A+4 μ l Solution B, used after mixing, i.e., with i.e. use.Each sample will do 3 parallel holes.

B, the preparation of BSA standard items and sample：Sample is prepared with PBS, is diluted 5 times (the μ lPBS of 5 μ l albumen stoste+20), is made Concentration to be determined is located at the linear segment of standard curve.Each reaction prepares 3 parallel determinations.Standard curve typically takes 5-6 Individual point, the specific concentration of each point is determined according to the concentration of sample.BSA is also diluted with PBS.The order of according to the form below adds BSA Standard items or sample and Solution A+B mixed liquors, mix.

C, close the lid mixing 30 seconds, and 37 DEG C are incubated 30 minutes.

D, it is cooled to room temperature.

E, 490nm absorbance (OD values) is determined on ELIASA.

F, with Excel software analysis and standard curve is drawn, R²The curves of ＞ 0.99 can use.According to the suction of unknown sample

Shading value, use formula：Y=0.2729x+0.0793 (y=absorbances, x=protein concentrations, R²=0.9994) can be with The concentration of unknown sample is drawn, actual concentrations need to be multiplied by the extension rate of sample.

(3) glue.It is sequentially prepared 12% separation gel and 5% concentration glue.

(4) preparation of albumen sample：It is identical (25 μ g) per hole loading specimen amount, it is separately added into 0.2mlEP pipes：Sample Albumen (25 μ g)+4 μ 5 × loading of l buffer+RIPA (containing 1%PMSF), the μ l of cumulative volume 20.

(5) albuminous degeneration.Protein sample and 5 × albumen sample-loading buffer mix, and are placed in 100 DEG C of water-baths 8 minutes, instantaneously from The heart, is cooled to room temperature, and direct loading or -80 DEG C save backup.

(6) loading.Per the μ l of hole 20, blank well adds 15 μ l RIPA+5 μ l 5 × albumen sample-loading buffers, pre- on edge hole Contaminate albumen marker.

(7) electrophoresis.Glue 60V constant pressure electrophoresis is concentrated, when bromophenol blue to separation gel, 120V constant pressure electrophoresis is used instead, to bromine phenol Indigo plant stops electrophoresis to separation gel bottom.

(8) transferring film.Pvdf membrane cuts off the upper right corner with label orientation, and then the pvdf membrane marked is placed in methanol and activated It is put into after 10min standby in transfering buffering liquid.Gel is removed from glass plate, in a certain order by sponge, filter paper, solidifying Glue, pvdf membrane are placed in electricity and turned on folder, are put into electric turn trough, 150mA Constant Electric Currents turn 60min.

(9) close.After transferring film, pvdf membrane is taken out, TBST washes 5min × 3 time, is placed in room temperature in Block buffer and closes 2h。

(10) it is incubated primary antibody.With antibody diluent dilution primary antibody (Tubulin mAb 1:1000, Bmi-1mAb 1： 2000).Pvdf membrane is cut according to pre-dyed albumen marker sizes, is incubated primary antibody, 4 DEG C of incubator overnights respectively.TBST washes film 5min × 3 It is secondary.

(11) it is incubated secondary antibody.Secondary antibody (1 is diluted with antibody diluent：1000), it is incubated at room temperature secondary antibody 2 hours.TBST washes film 5min × 3 time.

(12) expose.Pvdf membrane is placed in exposure box, is uniformly coated with chemical luminescence for liquid, film is covered, is exposed in darkroom 0.5-5min.Film is placed in developer solution and developed, is fixed in fixing solution.Observe band.With Image J software analysis images.

7th, cancer of pancreas research is suppressed in vivo

When gross tumor volume reaches~100mm³, tumor-bearing mice is randomly divided into 8 groups (n=6), receives the single dose of different pharmaceutical respectively Amount processing：(i) PBS (control), (ii) IP/SCR, (iii) IP/siBmi-1, (iv) scFv-IP/siBmi-1, (v) Gem, (vi) Gem-IONP, (vii) scFv-Gem-IONP, (viii) scFv-Gem-IONP/siBmi-1 (20 μ g/ animals).Every three days Observe animal ordinary circumstance and tumour growth situation.With vernier caliper measurement tumor size, gross tumor volume=(L × W is calculated²)/ 2, wherein L are tumour most major diameters, and W is vertical with L.Experimental endpoints are arranged to 21 days, by animal euthanasia when reaching experimental endpoints.Take Go out tumor tissues and carry out histopathological analysis.The tumour of stripping is fixed with 4% paraformaldehyde at once, FFPE, prepares 6 μm Section, H＆E dyeing, immunohistochemical staining analysis are carried out respectively.

2nd, Analysis of conclusion

1st, the preparation of Gem-IONP and scFv-Gem-IONP nanoparticles and measure

Gemcitabine forms GFLG-Gem by chemical reaction and acid-sensitive tetrapeptide, is adding EDAC and sulfo-NHS reactions Under the conditions of, by sucking action between amidation process and positive and negative charge, scFv, Gem and IONP coupling are compounded to form ScFv-Gem-IONP, and ultimately form and can both be coupled gemcitabine, siRNAs compound scFv-Gem- can be loaded again IONP-PEI.As shown in figure 9, formation and the pattern of polymer are further verified by transmission electron microscope.The compound of formation is rule Then spherical, is evenly distributed, centered on 3-5 IONP small particles, around around single-chain antibody, gemcitabine, PEI and siRNA.The diameter of these particulates is distributed in 60nm or so, and particle diameter is small, beneficial to the endocytosis of cell.The electric charge of particle surface institute band draws Play coloring agent excessively to adsorb, so as to cause particle color deeper.

2nd, gemcitabine drug release test

10ul scFv-Gem-IONP are dissolved in the 90ul containing 1uM (final concentration) cathepsin B (cathepsin B) PBS, the link between IONP and Gem be by acid-sensitive tetrapeptide (9-fluorenylmethoxycarbonyl (Fmoc)- Gly-Phe-Leu-Gly (GFLG)-nitrophenyl ester peptide) between chemical reaction be coupled together, organize Cathepsin B can specifically hydrolyze this polypeptide, and so as to which the chemical bond of link be disconnected, gemcitabine is discharged.Tissue Cathepsin B is primarily present in lysosome in the cell, this environmental simulation intracellular environment.Nanoparticle enters intracellular, By the histone enzyme hydrolysis in lysosome, gemcitabine is discharged, plays tumor-inhibiting action.As shown in Figure 10：In PH5.5, Gemcitabine rate of release and amount are compared with PH7.4 height.Tumour cell metabolism is vigorous so that tumor tissue sections are in faintly acid, root Understood according to experimental result, Nano medication tumor tissues can faster a greater amount of releases, discharged in normal structure it is less, so as to both may be used To reduce the lethal effect to normal cell, tumor killing effect can be improved again.

3rd, cell toxicity test

As shown in 11 figures：In different gemcitabine concentration, Gem, Gem-IONP, scFv-Gem-IONP, scFv-Gem- IONP/siBmi-1 each group cytotoxicities gradually strengthen：The Gem joint siBmi-1 nanometer medications groups of targeting are higher than alone targeting Gem nanometers, the cytotoxic effect of targeting group are better than non-targeted group again, and this three groups cytotoxic effect is above alone Ji Xi His shore medicine group.It is 1 μM in gemcitabine concentration, Gem, Gem-IONP, scFv-Gem-IONP, scFv-Gem-IONP/ SiBmi-1 cell viabilities be respectively (98.33% ± 0.80%), (94.73% ± 1.46%), (89.79% ± 1.94%), (89.36% ± 2.81%) (p ＜ 0.05)；At 5 μM be respectively (79.80% ± 1.71%), (72.91% ± 1.82%), (62.13% ± 2.87%), (56.33% ± 5.13%) (p ＜ 0.05)；Cell viability difference (60.67% when concentration is 20 μM ± 4.04%), (51.67% ± 3.51%), (44.00% ± 2.65%), (39.00% ± 1.73%) (p ＜ 0.05).Experiment As a result show, gemcitabine nanoparticle and siBmi-1 have collaboration tumor-inhibiting action.

4th, Apoptosis assay

Each group Apoptosis ratio：Control 0%, Gem 8.46%, Gem-IONP 9.54%, scFv-Gem-IONP 13.59%, scFv-Gem-IONP/siBmi-117.53%.Flow cytometer detection prompts gemcitabine nanoparticle compared with gemcitabine medicine Thing group has stronger cytotoxicity, and siBmi-1 and gemcitabine have synergy, the experimental result and cytotoxicity experiment result Unanimously.

5th, apoptosis-related protein expression

Apoptosis-related protein Bcl2 is in Gem, Gem-IONP, scFv-Gem-IONP, scFv-Gem-IONP/siBmi-1 tetra- Expression quantity gradually reduces in group, and relative expression quantity is 100 respectively, (58.56 ± 1.99), (52.93 ± 1.99), (10.77 ± 1.97).And increasing trend then occurs in Bax, relative expression quantity is 100, (188.65 ± 11.50), (216.74 ± 2.96), (668.42±16.92).As a result the ability of natural death of cerebral cells is promoted gradually to strengthen after showing this four groups of processing cells.scFv- Gem-IONP/siBmi-1 groups have stronger rush apoptosis capacity compared with scFv-Gem-IONP, then can further illustrate gemcitabine Collaboration tumor-inhibiting action between siBmi-1 be present.

6th, tumor inhibition and histotomy ImmunohistochemistryResults Results

In being tested more than, we, which have proven to silence Bmi-1 genes, to suppress pancreatic cancer cell in cytologic experiment aspect Growth, and living body fluorescent imaging experiment display targeting nano-carrier can by siRNA target imported into tumour cell.And And in vitro experimental verification each medicine group Gem, Gem-IONP, scFv-Gem-IONP, scFv-Gem-IONP/siBmi-1 to pancreas The influence of the toxic action of adenocarcinoma cell.Nude mice is grouped (i) PBS (control), (ii) IP/SCR, (iii) on this basis IP/siBmi-1, (iv) scFv-IP/siBmi-1, (v) Gem, (vi) Gem-IONP, (vii) scFv-Gem-IONP, (viii) ScFv-Gem-IONP/siBmi-1 (20 μ g/ animals).Medicine is entered in nude mouse through tail vein injection, observation suppresses cancer of pancreas Cell growth.All treatment group tumours compared with control group (PBS) group tumour it is small, non-targeted gene therapy group IP/siBmi-1 with SCR groups are compared, gross tumor volume and weight reduction unobvious, without statistical significance.But target gene group scFv-IP/ SiBmi-1 has obvious reduction compared with SCR control groups and non-targeted group, and volume is smaller, and weight is lighter.In gemcitabine medicine group In, also more non-targeted medicine group Gem-IONP and simple Gem groups are smaller, lighter by targeted drug group scFv-Gem-IONP；And altogether It is most light to carry Gem and siBmi-1 groups body weight in all packets, volume is minimum.To PBS (control), IP/SCR, IP/siBmi- 1 and scFv-IP/siBmi-1 groups tumor tissue section carries out expression of the Immunohistochemical study Bmi-1 genes in each group.Knot Fruit is as shown in figure 12：PBS groups and SCR groups Bmi-1 expression quantity are higher, IP/siBmi-1 and scFv-IP/siBmi-1 expression quantity Obvious to reduce, targeting group reduces more；Prompting targeting group and non-targeted group siBmi-1 can be brought into tumour cell, and And targeting group has reached more preferable interference effect.To PBS (control), Gem, Gem-IONP, scFv-Gem-IONP, scFv- Gem-IONP/siBmi-1 carries out Bmi-1, Bcl-2, Bax dyeing.As a result it is as shown in figure 13：Compared with other each groups, scFv- Gem-IONP/siBmi-1 group Bmi-1 expression quantity reduces.Bcl-2 expression quantity combines siBmi-1 in trend, Gem is gradually reduced The expression quantity of group is minimum；Bax expression then shows opposite trend, gradually rises, in scFv-Gem-IONP/siBmi-1 groups Expression quantity highest.It is consistent with Western blot results.Result above is prompted, scFv_CD44v6The carrier of modification can obtain height Tumour-specific, increase the siRNAs concentration of tumor by local, strengthen siBmi-1 GVT；Gemcitabine and siBmi-1 Use in conjunction has stronger synergistic antitumor effect.

Claims (1)

1. a kind of nanoparticle for being used to treat cancer of pancreas, it includes targeting vector and silence RNA, wherein, the silence RNA is equipped on the targeting vector；

The targeting vector includes the CD44v6 single-chain antibodies and nano-carrier IONP- for targets identification pancreatic cancer cell PEI, wherein the CD44v6 single-chain antibodies are coupled with the nano-carrier IONP-PEI；

The silence RNA is the silence RNAs, i.e. siBmi-1 of Bmi-1 genes；

The nanoparticle further comprises gemcitabine chemotherapeutics, the gemcitabine chemotherapeutics and the siBmi-1 It is loaded in altogether on the cancer of pancreas targeting vector.