CN102125696A - Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof - Google Patents
Oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof Download PDFInfo
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Abstract
The invention discloses an oligomeric ribonucleic acid composite for inhibiting tumor growth and angiogenesis and application thereof. In the oligomeric ribonucleic acid composite, a main ingredient is double-stranded ribonucleic acid (RNA), wherein a nucleotide sequence in a first strand of the double-stranded RNA is shown by a sequence 1 in a sequence table, and a nucleotide sequence in a second strand of the double-stranded RNA and a reverse complementary strand of the first strand have homology of over 63.6 percent. In the oligomeric ribonucleic acid composite serving as a novel anti-tumor medicament of small interference RNA, all oligomeric ribonucleic acid ingredients in the composite are chemically-synthesized and modified oligomeric ribonucleic acid, so the composite has the characteristics that: the chemically-synthesized and modified oligomeric ribonucleic acid is difficult to degrade, has the long effect half-life period, and can be used for extracorporal experiments and intracorporal treatment. Compared with the medicaments with a single ingredient, the anti-tumor effect achieved by the combination of part of oligomeric ribonucleic acids is better.
Description
Technical field
The present invention relates to suppress tumor growth and angiogenesis nucleic acid oligomer compositions and application thereof.
Background technology
Small nucleic acid (miRNA) is the non-coding small nucleic acids of a class, and wide expression is regulated body important physical and pathological process in vivo.The generation of small nucleic acid and tumor, development and prognosis are closely related.Discovering in recent years, miRNA tumor tissues with normal structure between expression obviously different.MiRNA both can be used as proto-oncogene and had promoted developing of tumor, can suppress developing of tumor as antioncogene again, regulate the different mechanism of tumor development according to miRNA, they are formed different prescriptions or they are formed different prescriptions with other antineoplastic siRNA (siRNA), will more can treat neoplastic disease effectively than single miRNA.
Summary of the invention
The object of the present invention is to provide a kind of nucleic acid oligomer.
Nucleic acid oligomer provided by the invention is a kind of double-stranded RNA, and wherein: the nucleotide sequence of article one chain of described double-stranded RNA is shown in sequence in the sequence table 1; The reverse complemental chain of the nucleotide sequence of second chain and described article one chain has the homology more than 63.6%.Sequence 1 direction from left to right is 5 '-3 '.
The nucleotide sequence of above-mentioned second chain can be shown in sequence in the sequence table 2 or 3. Sequence 2 and 3 direction from left to right is 3 '-5 '.
Above-mentioned nucleic acid oligomer through as 1) or 2) modify the nucleic acid oligomer that obtains:
1) phosphodiester bond of the connection nucleotide of described nucleic acid oligomer is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described nucleic acid oligomer.
The DNA that gives expression to above-mentioned nucleic acid oligomer also belongs within protection scope of the present invention.
Another object of the present invention is to provide a kind of pharmaceutical composition that suppresses tumor growth.
The active ingredient in pharmaceutical of inhibition tumor growth provided by the invention is the nucleic acid oligomer compositions, can be following a) or b) or c):
A) modify body by VEGF-siRNA or its and above-mentioned nucleic acid oligomer is formed;
B) modify body by PTN-siRNA or its and above-mentioned nucleic acid oligomer is formed;
C) modify by VEGF-siRNA or its that body, PTN-siRNA or its are modified body and above-mentioned nucleic acid oligomer is formed;
Described VEGF-siRNA is a double chain nucleotide, and the nucleotide sequence of article one chain of described VEGF-siRNA is shown in sequence in the sequence table 4, and the nucleotide sequence of the second chain of described VEGF-siRNA is shown in sequence in the sequence table 5; Sequence 4 direction from left to right is 5 '-3 ', and sequence 5 direction from left to right is 3 '-5 '.
Described PTN-siRNA is a double chain nucleotide, and the nucleotide sequence of article one chain of described PTN-siRNA is shown in sequence in the sequence table 6, and the nucleotide sequence of the second chain of described PTN-siRNA is shown in sequence in the sequence table 7; Sequence 6 direction from left to right is 5 '-3 ', and sequence 7 direction from left to right is 3 '-5 '.
The modification body of described VEGF-siRNA is through following 1) or 2) modify the material that obtains:
1) phosphodiester bond of the connection nucleotide of described VEGF-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described VEGF-siRNA;
The modification body of described PTN-siRNA is through following 1) or 2) modify the material that obtains:
1) phosphodiester bond of the connection nucleotide of described PTN-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described PTN-siRNA.
Wherein, active component a) in, to modify the mass ratio of body be (0.5-2) for arbitrary described nucleic acid oligomer and described VEGF-siRNA or its among the claim 1-3: (0.5-2), preferably 1: 1;
Active component b) in, arbitrary described nucleic acid oligomer and described PTN-siRNA or its mass ratio of modifying body are (0.5-2) among the claim 1-3: (0.5-2), and preferably 1: 1;
Active component c) in, arbitrary described nucleic acid oligomer and described VEGF-s iRNA or its mass ratio of modifying body, PTN-siRNA or its modification body are (0.5-2) among the claim 1-3: (0.5-2): (0.5-2), and preferably 1: 1: 1.
Aforementioned pharmaceutical compositions comprises at least a and at least a pharmaceutically acceptable carrier in the above-mentioned nucleic acid oligomer molecule." pharmaceutically acceptable carrier " used herein should be compatible with the double stranded rna molecule in the pharmaceutical composition of the present invention.In a preferred embodiment, described " pharmaceutically acceptable carrier " is meant transfection reagent in the body, as polymine (PEI), and jetPEI (linear polyethylene imines), liposome, transferrins, folic acid etc.
Above-mentioned nucleic acid oligomer also belongs within protection scope of the present invention in reagent or the application in the reagent that preparation inhibition tumour cell cycle protein D 1 is expressed that preparation suppresses tumor growth.
Further, mentioned reagent can be the pharmaceutical composition of above-mentioned inhibition tumor growth.
Specifically, above-mentioned tumor is intestinal cancer, nasopharyngeal carcinoma, cervical cancer, hepatocarcinoma, breast carcinoma or pulmonary carcinoma.
Described nucleic acid oligomer is chemosynthesis, can carry out 2 ' fluoro (2 '-F), 2 ' methoxyl group (2 '-OMe), sulfo-(PS) and 2 ' deoxidation (2 '-deoxy) chemical modification.
Experimental result with chemosynthesis and the above-mentioned nucleic acid oligomer molecule transfection human nasopharyngeal epithelioma 1 CNE that modifies, colon-cancer cell strain HCT116, breast carcinoma cell strain MCF7, tumor cells such as cervical cancer cell strain Hela, hepatoma cell strain HepG2 shows that described nucleic acid oligomer miR-210 can effectively suppress the propagation of above-mentioned tumor cell.SiPTN also can suppress the propagation of above-mentioned tumor cell.SiVEGF and siPTN all can suppress tumor-blood-vessel growth.
Described nucleic acid oligomer miR-210 suppresses the expression of CCND1; SiVEGF suppresses the expression of VEGF, and siPTN suppresses the expression of PTN.By the combination of above-mentioned three kinds of different nucleic acid oligomer molecules, realize suppressing the effect of growth of tumor and angiogenesis.
Tumor cell inoculation in nude mice, is formed the described nucleic acid oligomer combination that experimental results show that for the treatment of with described nucleic acid oligomer after the tumor and can effectively suppress growth of tumor in vivo.
As the antitumor drug of novel small nut acids, chemosynthesis and the described nucleic acid oligomer of modifying are difficult for being degraded, and the long effect half-life is arranged, and can be used for experiment in vitro, more can be used for interior therapeutic.
Description of drawings
Figure 1A for mtt assay detect 2 '-OMe-miR-210 suppresses the bar diagram of nasopharyngeal carcinoma CNE cell proliferation, Figure 1B for mtt assay detect 2 '-bar diagram of OMe-siPTN inhibition Hela cell proliferation.
Fig. 2 for RT-PCR detect 2 '-OMe-miR-210** suppresses the expression of tumor cell CCND1.
Fig. 3 for RT-PCR detect 2 '-OMe-siVEGF suppresses the expression of tumor cell VEGF.
Fig. 4 for RT-PCR detect 2 '-PTN that OMe-siPTN suppresses tumor cell expresses, swimming lane 1-4 is respectively the cell of untransfected, the negative control of transfection random sequence (NC), transfection 2 '-OMe-siPTN24 hour, transfection 2 '-OMe-siPTN 48 hours.
Fig. 5 is 2 '-5 later tumor volume broken line graphs of OMe nucleic acid oligomer combined therapy nasopharyngeal carcinoma transplanted tumor.
Fig. 6 is 2 '-combination of OMe nucleic acid oligomer adds 6 heavily bar diagrams of later tumor of treatment nasopharyngeal carcinoma transplanted tumor.
Fig. 7 is 2 '-OMe nucleic acid oligomer combined therapy nasopharyngeal carcinoma transplanted tumor 6 times after tumor effect figure.
The specific embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be conventional method.
The preparation of embodiment 1, nucleic acid oligomer
The sequence of nucleic acid oligomer described in the present invention positive-sense strand is: 5 '-CUGUGCGUGUGACAGCGGCUGA-3 ' (SEQID No.1); Antisense strand is 3 '-UUGACACGCACACUGUCGCCGA-5 ' sequence (SEQ ID No.2) or 3 '-CACACGCCACCCGUCCCCGACG-5 ' (SEQ ID No.3).The VEGF-siRNA positive-sense strand is 5 '-GGAGUACCCUGAUGAGAUCTT-3 ' (SEQ ID No.4) among the present invention; Antisense strand is 3 '-TTCCUCAUGGGACUACUCUAG-5 ' (SEQ ID No.5); The PTN-siRNA positive-sense strand is 5 '-GCCCAAACCUCAAGCAGAATT-3 ' (SEQ ID No.6); Antisense strand is 3 '-TTCGGGUUUGGAGUUCGUCUU-5 ' (SEQ ID No.7); Random sequence described in the present invention is as negative control (NC), and the positive-sense strand sequence of NC is: 5 '-UUCUCCGAACGUGUCACGUTT-3 ' (SEQ ID No.8), the antisense strand sequence is: 3 '-TTAAGAGGCUUGCACAGUGCA-5 ' (SEQ ID No.9).A, G, C, U in described nucleic acid oligomer and the NC sequence represents adenine ribonucleotide, guanosint ribotide, cytosine ribonucleotide and uracil ribonucleotide, and T represents thymidylic acid.
Described nucleic acid oligomer and NC entrust Shanghai lucky agate (GenePharma) Pharmaceutical Technology Inc. synthetic and carry out following any one chemical modification: 2 ' methoxyl group (2 '-OMe), 2 ' fluoro (2 '-F), sulfo-and 2 '-deoxidation (2 '-deoxy), handle through lyophilizing then and obtain the nucleic acid oligomer powder, be used for the experiment of embodiment 2-4.
5 kinds of nucleic acid oligomers among the embodiment 1 are respectively: the nucleic acid oligomer negative control (called after NC) that nucleic acid oligomer (called after PTN-siRNA), sequence 8 and the sequence 9 that nucleic acid oligomer (called after VEGF-siRNA), sequence 6 and the sequence 7 that nucleic acid oligomer (called after miR-210**), sequence 4 and the sequence 5 that the nucleic acid oligomer that sequence 1 and sequence 2 are paired into (called after miR-210), sequence 1 and sequence 3 are paired into is paired into is paired into is paired into.
Wherein: miR-210 after 2 ' methoxyl group is modified called after 2 '-OMe-miR-210; MiR-210** after above-mentioned 2 ' methoxyl group is modified called after 2 '-OMe-miR-210**; VEGF-siRNA through 2 ' methoxyl group modify back called after 2 '-OMe-siVEGF, PTN-siRNA after the modification of 2 ' methoxyl group called after 2 '-OMe-siPTN.NC after 2 ' methoxyl group is modified called after 2 '-OMe-NC.
Above-mentioned synthetic method be derived from one piece nineteen ninety-five disclosed document: Wincott F, DiRenzo A, Shaffer C, GrimmS, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S and Usman N.Synthesis, deprotection, analysis and purification of RNA and ribozymes.Nucleic Acids Res.1995,23:2677-84.Whole chemosynthesis can roughly be divided into four process (1) oligomerization ribonucleic acid synthetic; (2) deprotection; (3) purifies and separates; (4) the desalination aseptic sterilization of annealing.
(1) the oligomerization ribonucleotide is synthetic
In that automated DNA/the RNA synthesizer (for example, Applied Biosystems EXPEDITE8909) goes up the RNA that sets synthetic 1 mM, setting each circulation coupling time simultaneously is 10-15 minute, starting material is that 5 '-O-of connecting of solid phase is to dimethoxy-thymidine holder, first circulates in and connects a base on the solid support, then in the n time (19 〉=n 〉=2) circulation, on the base that the n-1 time circulation connected, connect a base, repeat this circulation until finishing the synthetic of whole nucleotide sequences.
(2) deprotection
The solid support that is connected with RNA is joined in the test tube, and in this test tube, add ethanol/ethamine (volume ratio is 1: 3) of 1 milliliter, sealing then, place 55-70 ℃ of incubator, hatched 2-30 hour, taking-up is connected with the solid support of siRNA and with distilled water drip washing 2 times (each 1 milliliter), collects eluent, and at room temperature dry 30 minutes.Then, add the tetrahydrofuran solution (1M) of 1 milliliter of tetrabutyl ammonium fluoride, room temperature was placed 4-12 hour, added 2 milliliters of ethanol again, and collecting precipitation promptly obtains the crude product of little RNA.
(3) purifies and separates
It is in 1 mole/milliliter the ammonium acetate solution that the crude product of the RNA that obtains is dissolved in 2 ml concns, separates by the C18 high pressure liquid chromatography then, obtains the purified RNA product.
(4) desalination annealing
With concentration is ethanol water washing purified RNA product 2-4 time (each 2 milliliters) of 75 weight %, removing salt, and drying under the room temperature.Then with oligomerization ribonucleic acid mixed dissolution (10mM Tris in the buffer of 1-2 milliliter of positive-sense strand and antisense strand, pH=7.5-8.0,50mM NaCl), this solution is heated to 95 ℃, slowly this solution is cooled to room temperature then, and kept room temperature 16-22 hour, obtain containing the solution of double-stranded Microrna.
Experimental procedure is: with embodiment 1 obtain 2 '-OMe-miR-210 (being called for short miR-210 among the figure) and 2 '-OMe-siPTN (being called for short siPTN among the figure) and negative control 2 '-OMe-NC (being called for short NC among the figure) powder is dissolved in respectively in the sterilized water of no RNA enzyme, the nucleic acid oligomer solution that to be made into 3 kinds of final concentrations be 20pmol/L.Collect logarithmic (log) phase nasopharyngeal carcinoma CNE cell (available from the Shanghai cell bank, for 2 '-OMe-miR-210 and 2 '-OMe-NC transfection) or the Hela cell (available from the Shanghai cell bank, confession 2 '-OMe-siPTN and 2 '-OMe-NC transfection), adjust concentration of cell suspension, divide and plant in 96 orifice plates, every hole 150 μ l are totally 5000 cells; Place 37 ℃, contain 5%CO
2Incubator in make cell attachment, cultivated 6-24 hour.During transfection, respectively with 0.40 μ l nucleic acid oligomer solution (above-mentioned 2 '-F nucleic acid oligomer or 2 '-F nucleic acid oligomer negative control) and the lipofectamine 2000 (Invitrogen) of 0.20 μ l be diluted in respectively in the 20 μ l serum-free culture culture fluid (Opti-MEM), and at room temperature hatched 5 minutes, then above-mentioned nucleic acid oligomer solution is mixed with lipofectamine 2000 solution, complex is after room temperature leaves standstill 20 minutes, the 10%FBS-DMEM of 60ul is added in nucleic acid oligomer-liposome complex of 40 μ l, be added in the Tissue Culture Plate (cell growth fusion rate is 50-70%) again transfection 6-8 hour.For making cell synchronous, after transfection,, continue to cultivate 72 hours with being changed to the 2.5%FBS-DMEM culture medium more afterwards with cell serum starvation 12 hours; The careful suction removed supernatant 120ul, makes that the fertility culture fluid is 80 μ l in the hole, adds 20 μ lMTT (5mg/ml) again, and promptly 0.5% MTT continues to cultivate 4 hours; Suction goes supernatant to add the dimethyl sulfoxide DMSO of 100 μ l, measures the light absorption value in each hole at enzyme-linked immunosorbent assay instrument 492nm place; Be provided with simultaneously and transfer 0 hole (culture medium, MTT, dimethyl sulfoxide), every group is provided with 3 multiple holes; The result as shown in Figure 1, Figure 1A show with transfection 2 '-OMe-NC relatively, transfection 2 '-the cell OD value of OMe-miR-210 (Figure 1A) or 2 '-OMe-siPTN (Figure 1B) obviously reduces, illustrate 2 '-OMe-miR-210 or 2 '-OMe-siPTN can obviously suppress the propagation of tumor cell.
In the present embodiment, miR-210 through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify the back to the influence of tumor cell proliferation with through 2 ' methoxyl group (2 '-OMe) the effect there was no significant difference after the modification.
The RT-PCR that embodiment 3, nucleic acid oligomer suppress its expression of target gene detects
Use the l ipofectamine of Invi trogen
TM2000 liposomees carry out transfection, the rule of operation that all operations all provides by Invitrogen.With CNE or Hela cell inoculation (5X10 in 6 orifice plates
5Individual cells/well), supports liquid (available from GIBCO) cultivation after 16-24 hour, cell culture fluid is changed into the DMEM culture fluid that contains antibiotic-free with the DMEM that contains 10% hyclone.With the nucleic acid oligomer powder (2 '-OMe-miR-210**, 2 '-OMe-siPTN, 2 '-OMe-siVEGF and 2 '-OMe-NC) be dissolved in respectively in the sterilized water of no RNA enzyme, being made into final concentration is the nucleic acid oligomer solution of 20pmol/L.
Wherein, 2 '-OMe-miR-210** and 2 '-OMe-NC transfection CNE cell; 2 '-OMe-siPTN and 2 '-OMe-NC transfection Hela cell; 2 '-OMe-siVEGF and 2 '-OMe-NC transfection CNE cell.
During transfection, getting the above-mentioned oligonucleotide solution of 10 μ l and the lipofectamine 2000 of 5 μ l respectively is diluted in respectively in the 250 μ l serum-free culture culture fluid (Opti-MEM), and at room temperature hatched 5 minutes, then above-mentioned nucleic acid oligomer solution is mixed with lipofectamine 2000 solution, complex is after room temperature leaves standstill 20 minutes, nucleic acid oligomer-liposome complex of 500 μ l with after 1.5ml serum-free DMEM culture fluid mixes, be added in the Tissue Culture Plate, be changed to the 10%FBS-DMEM culture medium after 6 hours, respectively at 24,48 collecting cells carry out RT-PCR and detect.
The step that RT-PCR detects is: use the CNE or the Hela cell of 1ml TRIzol (invitrogen) described nucleic acid oligomer of cracking transfection or nucleic acid oligomer negative control, and extract total RNA by the rule of operation of the TRIzol description of product.Because the RNA effects of jamming may be by potential a spot of genomic DNA influence, experiment adopts DNase I (RNase-free) (TaKaRa) further to digest residual DNA among total RNA that degrades.With 37 ℃ of digestion of DNase I after 30 minutes, according to purify again total RNA and RNA carried out quantitatively and Quality Identification of the DNase I description of product.Reverse transcription reaction is to use the M-MLV reverse transcriptase system of TaKaRa company, and total RNA of 1ug is mixed with the Oligo dT of 0.5ug, heats 5 minutes, is cooled to 0 ℃ rapidly.Press the description of product and add buffer, hatched 1 hour for 42 ℃.CDNA behind the reverse transcription reaction, template detection target gene as the PCR reaction, transfection 2 '-expression of the pattern detection CCND1 of OMe-miR-210**, forward primer is 5 '-GCTGGAGCCCGTGAAAAAGA-3 ', downstream primer is 5 '-CTCCGCCTCTGGCATTTTG-3 '.Transfection 2 '-the pattern detection vegf expression forward primer of OMe-siVEGF is 5 '-GAGGGCAGAATCATCACGAA-3 '; Downstream primer is 5 '-GGGAACGCTCCAGGACTTAT-3 '.Transfection 2 '-the pattern detection PTN of OMe-siPTN expresses, and forward primer is 5 '-CACGGGAGGGCACTCGGACT-3 ', and downstream primer is 5 '-GTCTTCTGGCATTCGGCATTG-3 '.The PCR reaction system is 94 degree 5 minutes, 94 degree 1 minute, 54 degree 30 seconds, 72 degree 20 seconds, totally 28 circulations.The PCR product is carried out agarose gel electrophoresis.
The result is shown in Fig. 2-4, with the negative control group of transfection nucleic acid oligomer (2 '-OMe-NC, be called for short NC among the figure) relatively, transfection 2 '-CCND1 that OMe-miR-210** (being called for short miR-210** among the figure) obviously suppresses tumor cell expresses (Fig. 2), transfection 2 '-OMe-siVEGF (being called for short siVEGF among the figure) obviously suppresses the expression (Fig. 3) of tumor cell VEGF, and transfection 2 '-PTN that OMe-siPTN obviously suppresses tumor cell expresses (Fig. 4).Among Fig. 2 and Fig. 3, last figure is an electrophoretogram, and figure below is the rectangular histogram that the gel images analysis software changes into electrophoresis band relative expression quantity.
In the present embodiment, miR-210** through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify the back to the influence of the half-life test result of tumor cells expression CCND1 with through 2 ' methoxyl group (2 '-OMe) influence there was no significant difference after the modification.
Cultivate nasopharyngeal carcinoma CNE cell strain with the DMEM culture fluid that contains 10% hyclone, the cell of the trophophase of taking the logarithm is made single cell suspension, regulates cell concentration 5 * 10
7Individual cell/ml.At the dorsal part alar part subcutaneous injection 0.1ml tumor cell suspension of male BALB/C nude mice in 5 ages in week, make it form transplanted tumor.Nude mice is divided into eight groups, be respectively 2 '-OMe-NC (being called for short the NC group), 2 '-OMe-miR-210 (being called for short the miR-210 group), 2 '-OMe-miR-210 adds 2 '-OMe-siVEGFA (being called for short the miR-210+siVEGF group), 2 '-OMe-siVEGF (being called for short the siVEGF group), 2 '-OMe-siVEGF+2 '-OMe-siPTN (being called for short the siVEGF+siPTN group), 2 '-OMe-miR-210+2 '-OMe-siPTR (being called for short the miR-210+siPTN group), 2 '-OMe-miR-210+2 '-OMe-siVEGF+2 '-OMe-siPTN (being called for short the miR-210+siVEGF+siPTN group), 5 fluoro-uracil (being called for short 5 ' FU group).Inoculated tumour cell later first day and the 3rd day carries out tail vein injection respectively 1 time.Reach 50mm to gross tumor volume
3After change intratumor injection into.
The nucleic acid oligomer preparing preparation that the tail vein injection of each group is used is: single nucleic acid oligomer group (NC group, miR-210 group and siVEGF group) is to dissolve every group of corresponding nucleic acid oligomer of 100 μ g respectively with the sterilized water 165 μ l that do not contain the RNA enzyme; Duoupoly polynucleic acid group (miR-210+siVEGF group, siVEGF+siPTN group and miR-210+siPTN group) is mixing (total measurement (volume) 165ul) behind two kind of 50 μ g nucleic acid oligomer in dissolving every group respectively with two parts of sterilized water 87.5 μ l that do not contain the RNA enzyme; Three nucleic acid oligomer groups (miR-210+siVEGF+siPTN group) are to mix (total measurement (volume) 165ul) after dissolving the nucleic acid oligomer of three kind of 33.3333 μ g respectively with three parts of sterilized water 55 μ l that do not contain the RNA enzyme.
Each group adds transfection agents jetPEI 10 μ l (available from Polyplus company) more then, and incubated at room mixed with equal-volume 10% glucose solution after 15 minutes, and cumulative volume 350ul is in incubated at room laggard end of line intravenous injection in 15 minutes.The oligonucleotide consumption of a shot is 100 μ g/.5 fluoro-uracil usages are: 25ng/kg/ time, and lumbar injection.
The nucleic acid oligomer preparing preparation that intratumor injection is used is to dissolve 2 of 20 μ g '-OMe-nucleic acid oligomer with the sterilized water 11.3 μ l that do not contain the RNA enzyme.Single nucleic acid oligomer group is got the nucleic acid oligomer that sterilized water that 11.3 μ l do not contain the RNA enzyme dissolves 20 μ g, mix (total measurement (volume) 11.3ul) behind two kind of 10 μ g nucleic acid oligomer in duoupoly polynucleic acid group is dissolved every group respectively with two parts of sterilized water 5.65 μ l that do not contain the RNA enzyme, three nucleic acid oligomer groups are dissolved mixing (total measurement (volume) 11.3 μ l) behind three kind of 6.667 μ g nucleic acid oligomer respectively with three parts of sterilized water 3.77 μ l that do not contain the RNA enzyme.The consumption of transfection reagent is 1.2 μ l, adds equal-volume 10% glucose solution and mixes cumulative volume 25ul intratumor injection.The oligonucleotide consumption of a shot is 20 μ g/ points.Every injection in 2 days once, intratumor injection totally 5 times.5 fluoro-uracil usages are: 25ng/kg/ time, and lumbar injection.Inoculate back 3 days and use every three days major diameter (L) of vernier caliper measurement tumor and transverse diameter (S), calculate the approximate volumes of tumor.Computing formula is: V (mm
3)=0.5 * L * S
2Finish experiment behind the intratumor injection 5 times, put to death nude mice, take out tumor and weigh.
Figure 5 shows that the treatment group of the different nucleic acid oligomer combinations in treatment back and the correlated broken line graph of gross tumor volume of matched group, the increase of treatment group gross tumor volume is obviously little than matched group, and the matched group gross tumor volume is obvious ascendant trend.The tumor killing effect of nucleic acid oligomer combination miR-210+s iVEGF is best.
Fig. 6 shows after the treatment that on nasopharynx transplantation tumor nude mice model, the heavy obviously comparison of the tumor of treatment group nude mice is little according to the NC group, the inhibitory rate to 62.41% of nucleic acid oligomer combination miR-210+siVEGF.
Fig. 7 is after the oncotherapy, the oncotherapy design sketch of processed group and matched group.
In the present embodiment, miR-210 through 2 ' fluoro (2 '-F), sulfo-(PS) and 2 ' deoxidation modify the back to the inhibitory action of tumor growth with through 2 ' methoxyl group (2 '-OMe) influence there was no significant difference after the modification.
Mice serum AST detects data to table 1 for experiment finishes later on.Detect nude mice aspartate amino transferase (AST) according to aspartate amino transferase test kit (available from the middle north control Biological Co., Ltd. of giving birth to) description operation.10ml/ bottle R2 (liquid) in the test kit is joined in R1 (powder) bottle, be working solution after the dissolving, place room temperature to leave standstill.Getting fresh nude mice does not have haemolysis serum 20ul/ hole, joins in 96 orifice plates, and the working solution 200ul/ hole for preparing is also joined in 96 orifice plates mix homogeneously.Set 4 blank holes simultaneously.Reaction temperature insulation 1 minute, under the 340nm wavelength, read initial absorbance, pick up counting simultaneously, accurate 1, in the time of 2,3 minutes, read absorbance respectively, determine that the per minute mean light absorbency changes, the formula that provides to specifications calculates, and obtains the numerical value of AST.Result's demonstration does not have the significance rising with the ALT numerical value that nonuser PBS organizes relatively treatment group, and the no tangible hepatotoxicity of described nucleic acid oligomer treatment is described.
Table 1 detects tables of data to clear AST
| Group | Example number (N) | AST(U/L) | Standard error | The P value |
| PBS | 6 | 38.5 | 3.2 | ＞0.05 |
| NC | 6 | 32.6 | 7.2 | ＞0.05 |
| 5′Fu | 6 | 38.2 | 5.5 | ＞0.05 |
| miR-210+siPTN+siVEGF | 6 | 32.7 | 4.7 | ＞0.05 |
| si?PTN+siVEGF | 6 | 39.3 | 2.7 | ＞0.05 |
| miR-210+siPTN | 6 | 38.9 | 9.3 | ＞0.05 |
| siVEGF | 6 | 34.2 | 4.9 | ＞0.05 |
| miR-210 | 6 | 31.0 | 7.3 | ＞0.05 |
| miR-210+siVEGF | 6 | 42.4 | 4.6 | ＞0.05 |
Claims (10)
1. compositions that suppresses tumor growth, its active component be following a) or b) or c):
A) form by arbitrary described nucleic acid oligomer among VEGF-siRNA or its modification body and the claim 3-5;
B) form by arbitrary described nucleic acid oligomer among PTN-siRNA or its modification body and the claim 3-5;
C) form by arbitrary described nucleic acid oligomer among VEGF-siRNA or its modification body, PTN-siRNA or its modification body and the claim 7-9;
Described VEGF-siRNA is a double chain nucleotide, and the nucleotide sequence of its article one chain is shown in sequence in the sequence table 4, and the nucleotide sequence of second chain is shown in sequence in the sequence table 5;
Described PTN-siRNA is a double chain nucleotide, and the nucleotide sequence of its article one chain is shown in sequence in the sequence table 6, and the nucleotide sequence of second chain is shown in sequence in the sequence table 7;
The modification body of described VEGF-siRNA is through following 1) or 2) modify the material that obtains:
1) phosphodiester bond of the connection nucleotide of described VEGF-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described VEGF-siRNA;
The modification body of described PTN-siRNA is through following 1) or 2) modify the material that obtains:
1) phosphodiester bond of the connection nucleotide of described PTN-siRNA is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described PTN-siRNA.
2. compositions as claimed in claim 1 is characterized in that:
Active component a) in, to modify the mass ratio of body be (0.5-2) for arbitrary described nucleic acid oligomer and described VEGF-siRNA or its among the claim 3-5: (0.5-2), preferably 1: 1;
Active component b) in, arbitrary described nucleic acid oligomer and described PTN-siRNA or its mass ratio of modifying body are (0.5-2) among the claim 3-5: (0.5-2), and preferably 1: 1;
Active component c) in, arbitrary described nucleic acid oligomer and described VEGF-siRNA or its mass ratio of modifying body, PTN-siRNA or its modification body are (0.5-2) among the claim 3-5: (0.5-2): (0.5-2), and preferably 1: 1: 1.
3. a nucleic acid oligomer is a double-stranded RNA, it is characterized in that: the nucleotide sequence of article one chain of described double-stranded RNA is shown in sequence in the sequence table 1; The reverse complemental chain of the nucleotide sequence of second chain and described article one chain has the homology more than 63.6%.
4. nucleic acid oligomer as claimed in claim 3 is characterized in that: the nucleotide sequence of described second chain is shown in sequence in the sequence table 2 or 3.
5. as claim 3 or 4 described nucleic acid oligomers, it is characterized in that: described nucleic acid oligomer is through following 1) or 2) modify the nucleic acid oligomer that obtains:
1) phosphodiester bond of the connection nucleotide of described nucleic acid oligomer is modified, preferably the oxygen with described phosphodiester bond replaces with sulfur;
2), preferably described 2 '-OH is replaced with methoxyl group or fluorine or described 2 '-OH is carried out deoxidation modify to the modification of 2 '-OH of the ribose of described nucleic acid oligomer.
6. give expression to the DNA of arbitrary described nucleic acid oligomer among the claim 3-5.
7. arbitrary described nucleic acid oligomer is preparing reagent or the application in the reagent that preparation inhibition tumour cell cycle protein D 1 is expressed that suppresses tumor growth among the claim 3-5.
8. application as claimed in claim 7 is characterized in that: described reagent is arbitrary described pharmaceutical composition among the claim 3-5.
9. compositions as claimed in claim 1 or 2 is characterized in that: described tumor is intestinal cancer, nasopharyngeal carcinoma, cervical cancer, hepatocarcinoma, breast carcinoma or pulmonary carcinoma.
10. as claim 7 or 8 described application, it is characterized in that: described tumor is intestinal cancer, nasopharyngeal carcinoma, cervical cancer, hepatocarcinoma, breast carcinoma or pulmonary carcinoma.
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| CN102895672A (en) * | 2012-02-24 | 2013-01-30 | 中国科学院北京基因组研究所 | Composition for treating cancers and use thereof |
| CN102895672B (en) * | 2012-02-24 | 2013-11-20 | 中国科学院北京基因组研究所 | Composition for treating cancers and use thereof |
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