CN103893190B - A kind of medical usage of buxus alkaloids compound - Google Patents
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Abstract
This division application discloses that a kind of buxus alkaloids compound is preparing the application in antitumor drug, experimental result shows, under the concentration nontoxic to wild type normal mouse embryo fibroblast, this compound optionally can kill the mouse tumor cell with tomour specific mutant gene p53, Ras, consistently therewith be, this compounds also has very strong killing activity to human colon cancer cells's strain of carrying p53 sudden change, and not strong to human colon cancer cells's strain killing activity of p53 wild type; The compound of this Buxus sinica (Rehd.et Wils.) class plant origin has the application potential of preparation for the personalized treatment medicine of tomour specific mutant gene p53, Ras.
Description
To be application number be the application 201310104292.9, March 28 2013 applying date, denomination of invention " medical usages of four kinds of buxus alkaloids compounds " divisional application.
Technical field
The invention belongs to field of medicaments, be specifically related to a kind of buxus alkaloids compound and preparing the application in antitumor drug.
Background technology
Tumor is the fatal disease of a class serious threat human health, and its pathogenesis is illustrated not yet completely, and therapeutic effect is also unsatisfactory.Current routine is used for most of medicine of oncotherapy, is all to design for the regulation and control of DNA replication dna in proliferation process or cytoskeleton.These medicines are while killing and wounding the tumor cell of fast breeding, required normal proliferating cells (as bone marrow stem cell etc.) is maintained to bodily fuctions and also has very large toxicity, cause connecing subject patient's body normal function to be badly damaged, resistivity for tumor declines on the contrary, is difficult to get good therapeutic effect.Therefore, utilize molecular targeted triage techniques, for tumor cell specific gene mutation product, screening can suppress the micromolecular compound of these tomour specific mutating molecules activity or expression, thus exploitation forms low toxicity, efficiently targeting personalized treatment medicine, has become the recent tendency of anti-tumor medicine research and development.At present, the most of targeted drug entering clinical practice is all that the research and development of external drugmaker are formed, and these target therapeutic agents are expensive, cause heavy financial burden to patient.Exploitation has low toxicity, efficiently the targeting personalized treatment medicine of independent intellectual property right, also becomes the important directions of domestic tumor transformation medical investigator.
Buxus sinica (Rehd.et Wils.) belongs to Buxaceae (Buxaceae) Buxus (Buxus) plant, evergreen shrubs, has another name called (blue or green bright short, short, yellow head Chinese mugwort in thousand, Rohdea japonica Roth, Cladrastis kentukea Rudd, Buxus sempervirens/common box), is mainly distributed in south China each province, district and Hubei; Be distributed in the stone ditch in streams or small stream bank more; Also cultivation is had.Buxus sinica (Rehd.et Wils.) is recorded in Compendium of Material Medica already as medicinal plants, says it: " leaf hardship is flat nontoxic ", " cures mainly married woman's difficult labour, enters use in DASHENG SAN, again main summer-heat day life furuncle; Mash painting ".The functions such as this medicine mainly contains promoting flow of QI and blood, treatment of arthritis and promoting collateral and channels, heat-clearing and toxic substances removing, dispels the wind, hemostasis.Among the people in order to treat malaria, syphilis, rheumatism, dermatitis and rabies.The active drug of Buxus sinica (Rehd.et Wils.) wood powder folklore treatment " cardiopathia ".This platymiscium total alkaloids generally has hypotensive effect; Buxus alkaloids acts on vagus nerve and heart, produces heartbeat slowly, ischemia, the conduction block of hole room and myocardial damage; Some plant total alkaloid also has inhibitory action to choline vinegar enzyme, have not been reported about content buxus alkaloids being used for the treatment of tumor.
Summary of the invention
The object of the present invention is to provide a kind of novel medical use of buxus alkaloids compound, namely this buxus alkaloids compound is preparing the application in antitumor drug, namely be applied in and prepare targeted therapy and carry in the cancer therapy drug of p53 and/or Ras mutated tumor, this buxus alkaloids compound is made medicine as the main active of antitumor drug, carrys out Therapeutic cancer as anti-tumor drugs targeting.
The compound of buxus alkaloids described in the present invention refers to one of compound shown in following formula I, formula II, formula III, formula IV:
Formula I:9,19-yclo-5
α, 9
β-pregnan-1-en-3-one, 20
α-(dimethylamino)-16
α– hydroxy-4,4,14-trimethyl-(7CI, 8CI); Cyclomicrobuxinine (KBA01), Chinese: 9,19-ring-5
α, 9
β-pregnane-1 (2)-alkene-3-ketone, 20
α-dimethylamino-16
α-hydroxyl-4,4,14-trimethyl-(7CI, 8CI);
Formula II:9,19 – Cyclo-5 α, 9
β– pregnan-20-one, 3
β-(dimethylamino)-16
α-hydroxy-14-methyl-4-methylene
-(7CI, 8CI); Buxippine K (KBA02), Chinese: 9,19-ring-5
α, 9
β-pregnane-20-ketone, 3
β-dimethylamino-16
α-hydroxyl-14-methyl-4-methylene-(7CI, 8CI);
Formula III: 9,19-Cyclo-5
α, 9
β-pregnan-17 (20)-en-16-one, 14-methyl-3
β-(methylamino)-4-methylene-(7CI, 8CI); Buxmicrophylline B (KBA03), Chinese: 9,19-ring-5
α, 9
β-pregnane-17 (20)-alkene-16-ketone, 3
β-dimethylamino-14-methyl-4-methylene-(7CI, 8CI);
Formula IV:9,19-Cyclo-5
α, 9
β-pregnane-4
α-hydroxymethyl, 3
β, 20
α-bis (dimethylamino)-16
α-hydroxy-4,14-dimethyl-(7CI, 8CI); Dihydrocyclomicrophylline (KBR18), Chinese: 9,19-ring-5
α, 9
β-pregnane-4
α-methylol, 3
β, 20
α-two (dimethylaminos)-16
α-hydroxyl-4,14-dimethyl-(7CI, 8CI);
One or more pharmaceutically acceptable adjuvants can also be added in application of the present invention, described adjuvant comprises the filler of pharmaceutical field routine, diluent, binding agent, excipient, absorption enhancer, filler, surfactant and stabilizing agent etc., also can add flavouring agent, pigment and sweeting agent etc. if desired.
Application of the present invention, except making capsule, can also make the various ways such as pill, powder, tablet, granule, oral liquid and injection.
In the present invention, targeting screen body system is based on transgenic mouse cell, builds targets neoplastic cells screening system, with the experimental technique in-vitro screening targeting anti-tumor reactive compound of MTT with modal tumour-specific mutant gene p53 and Ras.
The advantage of this targets neoplastic cells system is that genetic background is clear, there is different Tumor Suppressor Gene Mutations state and oncogene mutation state, targeting screening is carried out with these recombinant tumor cells, we can filter out the reactive compound for the specific mutagenesis carried in reconstitution cell more effectively, thus realize the object of targeting screening.
Mouse embryo fibroblasts (the mouse embryonic fibrobLast of wild type, MEF) cell in contrast, the application of this normal control cells in drug screening system can get rid of the cell killing effect caused because of cytotoxic effect, screening kills and wounds tumor cell is selective, and does not have virose natural active matter to normal cell.
Promoter and the reporter gene green fluorescent protein GFP of tumor suppressor gene p16 merge, construct the report carrier of p16 target molecule, these report carriers are transfected in the tumor cell of different genetic background, its reporter gene GFP can be expressed under active medicine induction, display fluorescence, thus filter out for the large flux of plant compound, fast targeting the antitumoral compounds acting on p16 promoter, there is the compound activating luciferase expression also to increase tumor cell more simultaneously p16 protein expression further by the method validation compound of protein immunoblot simultaneously.
Experimental result shows, under the concentration nontoxic to wild type normal mouse embryo fibroblast, KBA01, KBA02, KBA03 and KBR18 compound optionally can kill the mouse tumor cell with tomour specific mutant gene p53 and/or Ras, and less to the normal mouse embryo fibroblast toxicity of wild type; Consistently therewith be, this compounds also has very strong killing activity to human colon cancer cells's strain of carrying p53 sudden change, and it is not strong to human colon cancer cells's strain killing activity of p53 wild type, in addition, the GFP report carrier screening system display of gene promoter, KBA02 and 03 compound can activate the promoter of tumor suppressor gene p16, and these characteristics show that the compound of these four kinds of Buxus sinica (Rehd.et Wils.) class plant origins has the application potential of preparation for the personalized treatment medicine of tomour specific mutant gene p53, Ras and tumor suppressor gene p16.
Accompanying drawing explanation
Fig. 1 is that buxus alkaloids compound K BR18 of the present invention acts on p53-/-+V+Ras and p53-/-+S+Ras cell strain and reduces the testing result schematic diagram that molecular chaperones HSP70 expresses, in figure: DMSO36h is the DMSO process 36 hours of dissolved substance; DMSO12h is the DMSO process 12 hours of dissolved substance; KBR18 12h and 36h is the KBR18 process p53-/-+V+Ras cell of 5 μMs and p53-/-+S+Ras cell 12 hours and 36 hours;
Fig. 2 is the testing result schematic diagram that buxus alkaloids compound K BR18 and KBA01 of the present invention acts on that p53-/-+V+Ras and p53-/-+S+Ras cell strain reduces HSP90 expression;
Fig. 3 is the schematic diagram that p53-/-+V+Ras, the p53-/-+S+Ras cell of the compounds of this invention KBA02 process causes caspase-3 to cut; In figure, DMSO24h refers to DMSO process 24h; KBA02 8h and KBA02 24h refers to that compound K BA02 acts on p53-/-+V+Ras and p53-/-+S+Ras cell strain 8h and 24h;
Fig. 4 is the expression figure that the compounds of this invention KBA01, KBA02 and KBA03 compound reduces p53mut, HSP90, HSP70 albumen in human colon cancer cell HT29, KBA01, KBA02 and KBA03 process HT29 cell 24h and 48h.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail; but protection scope of the present invention is not limited to described content; in embodiment, method all adopts conventional method if no special instructions, uses examination if no special instructions, the reagent being conventional commercial reagent or adopting conventional method to configure.
embodiment 1:the separation and Extraction of four kinds of compounds and qualification
From botanical garden, Kunming gather little leaf boxwood (
buxus.microphylla) aerial parts 14Kg, plant species name is identified by Kunming plant institute Qiu Minghua researcher, and crude drug specimen deposits in Chinese Academy of Sciences's Kunming plant institute's phytochemistry and western plant resources sustainable utilization National Key Laboratory.
Little leaf boxwood aerial parts 14Kg, extracts 3 times, each 2 days with 70% acetone room temperature diafiltration after pulverizing, merge extractive liquid, is evaporated to and steams without acetone, obtains extractum 800 g, adds sour water 3000ml and dilutes tune PH=2, use 3000ml extraction into ethyl acetate respectively 3 times, obtain non-alkaloid part, acid liquid alkalization, to PH=10, obtains alkaloid moiety 135g 3 times with 3000ml chloroform extraction, (135 g) mix silica gel 180 g at normal pressure silica gel column (silica gel 400 g) Column chromatography to chloroform extraction part, chloroform-methanol (100:0,50:1,20:1,10:1,2:1) gradient elution, detects with TLC, obtains 5 parts (Fr.1-Fr.5), the Fr.2 part of chloroform-methanol 50:1 (18 g) column chromatographies on a silica gel column, with petroleum ether-acetone (20:1, 5:1) eluting obtains Fr.2.1 and Fr.2.2 two components, (4 g) continue upper silicagel column petroleum ether-acetone (40:1 to Fr.2.1, 20:1, 10:1) gradient elution, obtain petroleum ether-acetone (20:1) eluent, separation and purification after concentrated, and be further purified with Sephadex LH-20 and obtain 45 components, wherein component 8-15 is Buxippine K(KBA02) sterling (38 mg), component 21-28 is Buxmicrophylline B(KBA03) sterling (29 mg).
Through qualification
buxippine K: C
25h
39nO
2, white powder;
1h NMR (ppm, CDCl
3, 500 MHz):
δ h2.70 (m, H-3), 4.78,4.58 (s, H-30), 0.04,0.21 (d,
j=4.0, H-19), 1.10 (s, 18-CH
3), 0.80 (s, 32-CH
3), 2.24 (s, 3-N (CH
3)
2);
13c NMR (ppm, CDCl
3, 125 MHz):
δ c31.2 (t, C-1), 27.5 (t, C-2), 68.5 (d, C-3), 151.3 (s, C-4), 44.4 (d, C-5), 23.6 (t, C-6), 27.0 (t, C-7), 47.1 (d, C-8), 22.4 (s, C-9), 32.0 (s, C-10), 25.3 (t, C-11), 26.6 (t, C-12), 48.3 (s, C-13), 48.9 (s, C-14), 45.6 (t C-15), 71.2 (d, C-16), 70.0 (d, C-17), 20.7 (q, 18-CH
3), 20.4 (t, C-19), 210.7 (s, C-20), 31.2 (q, 21-CH
3), 103.4 (t, C-30), 20.3 (q, 32-CH
3), 42.4 (q, 3-N (CH
3)
2); E1-MS:m/z 385 [M]
+;
Buxmicrophylline B：C
24H
35NO, colorless needle;
1H NMR (ppm, CDCl
3, 500 MHz):
d H2.86 (dd,
J = 4.0, 11.6, H-3), 0.10, 0.35 (d,
J = 4.4, H-19), 0.94 (s, 18-CH
3), 1.29 (s, 32-CH
3), 2.46 (s, 3-NCH
3), 1.80 (d,
J = 7.6, 21-CH
3), 6.53 (q,
J = 7.6, H-20), 4.57 (s, H
a-30), 4.80 (s, H
b-30);
13C NMR (ppm, CDCl
3, 125 MHz):
d C31.7 (t, C-1), 23.4 (t, C-2), 63.5 (d, C-3), 153.6 (s, C-4), 44.1 (d, C-5)，23.4 (t, C-6), 26.4 (t, C-7), 45.4 (d, C-8), 22.7 (s, C-9), 32.4 (s, C-10), 25.5 (t, C-11), 34.4 (t, C-12), 42.3 (s, C-13), 46.7 (s, C-14), 49.3 (t, C-15), 206.5 (s, C-16), 146.5 (d, C-17), 13.2 (q, 18-CH
3), 29.3 (t, C-19), 130.2 (d, C-20), 20.8 (q, 21-CH
3), 101.0 (t, C-30), 24.1 (q, 32-CH
3), 34.5 (q, 3-NCH
3); EI-MS
m/z: 353 (M
+)。
From high mountain Buxus sinica (Rehd.et Wils.) (Buxus.rugulosa) the aerial parts 75Kg that Lijiang, yunnan gathers, plant species name is identified by Kunming plant institute Li Xiwen researcher, and crude drug specimen deposits in Chinese Academy of Sciences's Kunming plant institute's phytochemistry and western plant resources sustainable utilization National Key Laboratory.
High mountain Buxus sinica (Rehd.et Wils.) aerial parts 75 Kg, pulverizes rear 90% methanol heating and refluxing extraction 3 times, each 8 hours, merge extractive liquid, is evaporated to and steams without methanol, obtains extractum 1800 g, adds sour water dilution and adjusts PH=3, be extracted with ethyl acetate 2-3 time respectively, obtain non-alkaloid part, acid liquid alkalization is to PH=9, alkaloid moiety 180 g is obtained for 3 times with chloroform extraction, (180 g) mix silica gel 200 g at normal pressure silica gel column (silica gel 600 g) Column chromatography to chloroform extraction part, chloroform-methanol (100:0, 50:1, 20:1, 10:1, 2:1) gradient elution, detect with TLC, obtain 5 parts (Fr.1-Fr.5), (45 g) carry out silica gel column chromatography to the Fr.2 of chloroform-methanol 50:1, with petroleum ether-acetone (20:1, 5:1) eluting obtains Fr.2.1, Fr.2.2 two components, (14 g) continue upper silicagel column petroleum ether-acetone (40:1 to Fr.2.1 part, 20:1, 10:1) gradient elution, obtain petroleum ether-acetone (20:1) eluent part, separation and purification after concentrated, and be further purified with Sephadex LH-20 obtain cyclomicrobuxinine(KBA01) sterling (68 mg),
Elution fraction Fr.4 (12 g) after alkylamino silica gel column chromatography, is further purified obtains dihydrocyclomicrophylline(KBR18 with the Sephadex LH-20) sterling (35 mg) of chloroform-methanol 10:1.
Through qualification
cyclomicrobuxinine: C
24h
37nO
2, colorless needle;
1h NMR (ppm, CDCl
3, 500 MHz):
δ h4.56,4.30 (s, H-30), 0.12,0.31 (d,
j=5.0, H-19), 1.16 (s, 18-CH
3), 0.86 (s, 32-CH
3), 2.72 (s, 3-NCH
3);
13c NMR (ppm, CDCl
3, 125 MHz):
δ c30.4 (t, C-1), 31.6 (t, C-2), 62.4 (d, C-3), 147.9 (s, C-4), 47.8 (d, C-5), 23.8 (t, C-6), 25.6 (t, C-7), 44.6 (d, C-8), 23.7 (s, C-9), 32.0 (s, C-10), 27.2 (t, C-11), 31.2 (t, C-12), 48.3 (s, C-13), 48.4 (s, C-14), 46.2 (t, C-15), 71.7 (d, C-16), 70.4 (d, C-17), 20.7 (q, 18-CH
3), 27.8 (t, C-19), 211.4 (s, C-20), 31.3 (q, 21-CH
3), 103.9 (t, C-30) 20.9 (q, 32-CH
3), 31.7 (q, 3-NCH
3); ES1-MS:
m/z372 [M+H]
+;
Dihydrocyclomicrophylline A(
11): C
28H
50N
2O
2, colorless needle;
1H NMR (ppm, CDCl
3, 500 MHz):
δ H2.41 (dd,
J = 3.0, 12.5, H-3), 0.28, 0.54 (d,
J = 4.0, H-19), 0.86 (s, 18-CH
3), 1.02 (s, 32-CH
3), 2.17 (s, 20-N(CH
3)
2), 0.98 (d,
J = 6.4, 21-CH
3);
13C NMR (ppm, CDCl
3, 125 MHz):
δ C32.8 (t, C-1), 25.1 (t, C-2), 54.6 (d, C-3), 40.3 (s, C-4), 45.7 (d, C-5), 21.6 (t, C-6), 27.0 (t, C-7), 43.7 (d, C-8), 19.2 (s, C-9), 24.9 (s, C-10), 18.5 (t, C-11), 33.2 (t, C-12), 44.5 (s, C-13), 47.1 (s, C-14), 28.3 (t C-15), 72.4 (d, C-16), 52.3 (d, C-17), 11.1 (q, 18-CH
3), 30.2 (t, C-19), 57.6 (d, C-20), 18.8 (q, 21-CH
3), 71.2 (t, C-30), 9.6 (q, C-31), 20.9 (q, C-32), 39.2 (q, 20-N(CH
3)
2); EI-MS
m/z446 M
+。
embodiment 2: the anti-tumor activity experiment of the compounds of this invention
1, screen the structure of cell: take wild-type mice as carrier, utilize nearly source, the mouse embryo fibroblasts (p53 of molecular genetic background clearly p53 gene delection
-/-cell), build and carry p53S(common in human tumor and be abbreviated as S) and/or the mouse tumor cell (p53 of Ras gene mutation
-/-+ S+Ras, p53
-/-+ V+Ras), concrete steps are as follows:
(1) structure of the carrier of Ras mutant gene and the carrier of expression p53S mutant gene
The construction method of carrier is shown in method in patent ZL200910095184.3, namely conventional gene engineering method is adopted to be connected in PQCXIP carrier by p53S or Ras mutant gene cDNA, verified p53S or the Ras sequence be built in carrier by order-checking, the carrier built is at p53
-/-p53S or Ras mutant gene is introduced in cell.
(2) transfection of p53S or Ras mutant gene carrier
A () prepares phoenix cell
At day before transfection, phoenix cell (HEKC, for packing the virion with rotaring redyeing gene) is gone down to posterity (DMEM high glucose medium, 10%FBS serum are cultivated in 37 DEG C of CO2 gas incubator), for subsequent use;
B () extracts transfected plasmids
Extract p53S-PQCXIP plasmid with except endotoxic plasmid extraction kit, and measure plasmid concentration (operate and undertaken by test kit description);
(c) configuration rotaring redyeing system (10cm culture dish)
Mix1: add the Eagle culture medium that 24 μ g plasmid DNA improve in the DMEM(dulbecco's modified eagle medium of serum-free, Dulbecco) in, be settled to 1.5mL, mixing;
Mix2: add 60 μ L transfection reagents Lipofectamine2000 (Invitrogen) in the DMEM of serum-free, be settled to 1.5mL, after incubated at room 5min, mixes mix1 and mix2, mixes gently, incubated at room 20min; Supernatant in the phoenix Cell sap gone down to posterity in gettering step (a), then mix1 and mix2 mixed system is added in phoenix cell, and add DMEM and supply 8mL, adding serum to concentration after cultivating 5h in 37 DEG C of CO2 gas incubator is 10%, 37 DEG C of cultivations;
(d) difference 24h after transfection, two periods of 48h, adopt the phoenix Cell sap that 0.45 μm of aperture membrane filtration is cultivated, collect supernatant (namely containing the culture fluid of transfected virus), and in supernatant, add polybrene (cohesion amine, for improving the efficiency of virus infected cell), make polybrene final concentration be 10 μ g/mL, supernatant is joined recipient cell p53
-/-in cell, 37 DEG C of cultivations;
E () infects recipient cell 48h after, add puromycin (puromycin) (final concentration 2 μ g/mL) and screen, lasting screening about month, sets up the cell strain of stably express; By above method, p53S and Ras gene is proceeded to p53 jointly
-/-just p53 is obtained in cell
-/-+ S+Ras, proceeds to p53 jointly by empty carrier (vector, V) and Ras gene
-/-just p53 is obtained in cell
-/-two cells are added DMEM (10%FBS) and cultivate in 37 DEG C of CO2 gas incubator, for the screening of following compound by the stable cell line of+V+Ras respectively.
2, cell proliferation experiment: collect logarithmic (log) phase cell, adjustment concentration of cell suspension, p53-/-+V+Ras, p53-/-+S+Ras cell is inoculated on 96 orifice plates, 2500/hole, mouse embryo fibroblasts (wt cell) inoculates 5000/hole, human colon cancer cell strain HCT116(p53 wild type) and HT29(p53 saltant type) cell inoculates 5000/ hole, every hole solution final volume 200 μ L(edge hole culture medium is filled), HCT116 and HT29 cell culture in 10% FBS+1640 culture medium, 5%CO
2, 37 DEG C of cultivations, add the compounds of this invention of Concentraton gradient after 12h, arrange 5 gradients, are respectively 0.1 μM, 1 μM, 10 μMs, 30 μMs, 50 μMs, if 3 parallel holes; 5%CO
2, cultivate after 72 hours for 37 DEG C, every hole adds 20 μ L MTT solution (5mg/mL, i.e. 0.5%MTT), continues to cultivate 4h, stops cultivating, carefully sucks culture fluid in hole; Every hole adds 150 μ L dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, crystal is fully dissolved; Measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD490nm place, zeroing hole (culture medium, MTT, dimethyl sulfoxide) is set simultaneously.
Table 1: the anti-tumor activity of compound K BA01-03 and KBR18
Compound K BA01 as shown in Table 1, KBA02, KBA03 and KBR18 optionally act on p53
-/-+ V+Ras and p53
-/-the IC50 of+S+Ras, KBA01-03 and KBR18 compound is all less than 5 μMs and less to the toxicity of MEF cell, and its IC50 expresses target gene 5-10 doubly.
Table 2: compound K BA01, KBA02, KBA03 are at the Activity Results of human colon carcinoma
Table 2 shows KBA01, KBA02, KBA03 optionally acts on the human colon cancer cell strain HT29 of p53 sudden change, and it is relatively weak to the HCT116 colon cancer cell line activity of p53 wild type, further demonstrate that the action target spot of this compounds is mutant p53, KBR18 does not have anti-tumor activity at these two cell strains.
embodiment 4: immunoblot experiment
One, the pretreatment of protein sample and concentration determination
(1) by the p53 of exponential phase
-/-+ V+Ras, p53
-/-+ S+Ras cell KBR18, KBA01, KBA02 of 5 μMs and contrast DMSO process 8h, after 16h and 24h, cell is scraped gently from plate, 15mL centrifuge tube is put into together with culture medium, 4 DEG C, 4000 rpm (3082 ' g) centrifugal 5min, abandon supernatant, cell precipitation 1mL 1 × PBS is suspended gently, again cell suspension is proceeded in 1.5mL centrifuge tube, 13000 rpm (12470 ' g) centrifugal 1min, exhausted by supernatant, this cell precipitation is preserved in-80 DEG C, for subsequent use;
(2) by the cell pyrolysis liquid cracking of cell precipitation 0.2mL, (lysate volume visual cell precipitation is how many and determine, and be generally doubly good with the 3-5 of cell volume, then cell pyrolysis liquid concentration is excessively rare too much, loading volume increase; Then in the step of ultrasonic degradation, easily get bubble very little, cause albuminous degeneration), lysate formula is: 10mLM-PER(Mammalian Protein Extraction Reagent, and Thermo mammalian proteins extracts reagent), 50 μ L 200 mM Na
3vO
4, 50 μ L 200 mM PMSF(Phenylmethanesulfonyl fluorides), a slice Protease inhibitor cocktail Tablets(protease inhibitor complex, Roche); Be placed on shaking table by the cell suspension that with the addition of lysate, at 4 DEG C, maximal rate shakes 1h; Be placed on ice, carry out fragmentation with ultrasound probe, note first with pure water cleaning probe, ultrasonic 10s, pause 5s, 10 times repeatedly (probe can not encounter tube wall, and remain at below liquid level, and try not to produce bubble), last suspension retightens on decolorization swinging table, 4 DEG C, maximal rate shakes 1 h, and (10625 ' is g) centrifugal for 12000 rpm, 4 DEG C, 20 min, suct clearly in another 1.5mL centrifuge tube, this cell pyrolysis liquid is kept at-80 DEG C, for subsequent use;
(3) protein concentration (Protein Assay Kit is measured with Coomassie brilliant blue, Bio-rad): first use bovine serum albumin (BSA) titer to prepare the BSA solution of 1mg/mL, being diluted to Concentraton gradient is again 100 μ g/mL, 200 μ g/mL, 400 μ g/mL, 600 μ g/mL, the titer of 800 μ g/mL.The titer prepared respectively is got 20 μ L to join in 2mL Coomassie brilliant blue reagent, room temperature places 5 more than min, visible wavelength 595nm place's light-metering absorption value, calculating and plotting standard curve (R
2need 0.99 be greater than);
(4) the protein sample lysate getting 2 μ L amounts joins in 2mL Coomassie brilliant blue reagent, room temperature places 5 more than min, 595nm light-metering absorption value is (during OD value >0.8, need the protein lysate volume reducing reaction), tried to achieve the concentration of agnoprotein by standard curve, calculate loading volume.
Two, SDS-PAGE electrophoresis, concrete operations are as follows:
(1) install offset plate, whether inspection leaks, and suck dry moisture, first prepares separation gel, take concentration as the glue (glue that 1.5mm is thick) of 12%, is sequentially added into successively: 1.87mL H
2o, 3.375mL 1M Tris-HCl (pH 8.8), 3.6mL Acr:Bis(29:1), 90 μ L 10%SDS, 60 μ L 10%APS, 4.5 μ L TEMED(tetramethyl diethylamine), mixing, the gel solution of mixing to be added in offset plate with measuring pipette and (adherently to add, avoid producing bubble), then add 1mL water or n-butyl alcohol seals liquid level, after gelling is solid, the liquid of upper strata sealing is gone from the predominate of glue groove, blots excessive moisture with filter paper bar;
Etc. (2) be separated gelling solid after, adding each preparation of reagents by a certain percentage concentrates glue, take concentration as the glue (glue that 1.5mm is thick) of 4.5%, is sequentially added into successively: 2.17mL H
2o, 0.388mL 1M Tris-HCl (pH 6.8), 0.45mL Acr:Bis(29:1), 27 μ L 10%SDS, 15 μ L 10%APS, 2 μ L TEMED, after mixing, slowly add in offset plate, carefully plug comb;
(3) prepare boiling water bath, meanwhile, get 20 μ g albumen, calculate sample concentration and loading volume (no more than 50 μ L) according to standard curve and sample OD value, supply identical loading volume with 1 × PBS; 5 × loading buffer(adds 5% beta-mercaptoethanol before using, and the amount added is 1/5 of cumulative volume), mixing, clamps the centrifugal mouth of pipe of 1.5mL with Small clamp, processes 8min in boiling water bath;
(4) comb on offset plate is taken off, half-open being opened for bottom offset plate, electrophoresis liquid is poured in electrophoresis tank, no offset plate, by well-done albumen sample syringe or thin rifle head loading in gel glue hole, action wants slow, must guard against the glue hole overflowing into side, upper groove connects positive pole, lower groove connects negative pole, 30mA, about 1-1.5h, when bromophenol blue forward position arrives the bottom of glue, stop electrophoresis.
Three, transferring film
Prepare 1L transferring film buffer, before using, be first placed in 4 DEG C of pre-coolings, pour appropriate transferring film buffer in a reservoir into and soak filter paper 4,2, sponge, cellulose membrane or pvdf membrane 1, after electrophoresis terminates, take off offset plate, unload low glue frame, pry open short glass plate, gel is cut a little angle as loaded with marker; The concentrated glue excision of blob of viscose abandoned, separation gel takes out and is immersed in transferring film buffer, makes transferring film sandwich: negative pole-one deck sponge-two layers of filter paper-glue-cellulose membrane-two layers of filter paper-one deck sponge-positive pole.Notice that whole operating process is avoided producing bubble; " sandwich " is put into electrophoresis tank, fills transferring film buffer, note the both positive and negative polarity of electrophoresis tank; Electrophoresis tank puts into ice basin, and in 4 DEG C of chromatography cabinets, transferring film 3 hours (voltage is transferred to maximum by 180mA), also can transferring film spend the night.
Four, immuning hybridization and colour developing
(1) taken out by film, PBST washes film 1 time, and film is put into Block buffer (10% defatted milk), decolorization swinging table shakes 1 hour, room temperature;
(2) film is washed 3 times, each 5min with PBST; Enclosed by film in bag, add a kind of primary antibodie diluted by a certain percentage, primary antibodie can be selected: anti-p53Ab1 (clonePAb240) (1:250, Neomarker, CA), anti-PARP (1:500, Cell Signaling, MA), anti-γ-tubulin (1:5000, Upstate, NY), anti-HSP70, anti-HSP90(1:1000 Cell Signaling, MA); Drive bubble away, sealing, shakes and spends the night or 3h under room temperature by 4 DEG C;
(3) taken out by film, primary antibodie reclaims, and film PBST washes 3 times, 5min/ time; Again enclosed in bag by film, add two and resist, the two anti-sources according to primary antibodie are selected (as Mus source, rabbit source etc.), drive bubble away, sealing, shake 1 hour, room temperature;
(4) film is washed, 10min/ time, 3 times; Take out ECL(enhanced chemiluminescence, enhanced chemiluminescence) reagent, open developing machine, film is placed on clean disposable glove, face up, every ml of EC L reagent adds the hydrogen peroxide of 3 μ L 4%, is added in (the film 1mL of a 2 × 6cm) on film and reacts 1min after mixing; Film drop is fallen ECL liquid, moves on in magazine and (face up), be covered with transparent thin film, take darkroom, in darkroom, take out X-ray film, be pressed onto on film, the reaction 1-2min(concrete time is depending on fluorescence intensity), send developing machine to and develop a film;
Experimental result shows: 5 μMs of KBR18 reduce p53 at 36h
-/-+ V+Ras, p53
-/-the expressing quantity (see figure 1) of HSP70 in+S+Ras cell, 5 μMs of KBA01 and KBR18 reduce p53 simultaneously
-/-+ V+Ras, p53
-/-the expressing quantity (see figure 2) of HSP90 in+S+Ras cell, the p53-/-+V+Ras of 5 μMs of KBA02 process, p53
-/-+ S+Ras cell, there occurs the cutting of caspase-3 at 24h, the generation (see figure 3) of showed cell apoptosis, and these results suggest that the increase along with time and dosage, KBA01, KBR18 compound can cause p53
-/-+ V+Ras and p53
-/-the reduction of intracellular HSP70, the HSP90 expressing quantity of+S+Ras, simultaneously compound K BA01, KBA02 and KBA03 compound reduces the expression (Fig. 4) of Mutation p53, HSP90 and HSP70 albumen in human tumor cell line HT29 cell.
Claims (2)
1. structural formula is preparing the application in antitumor drug for the buxus alkaloids compound shown in formula IV,
Formula IV:9,19-ring-5
α, 9
β-pregnane-4
α-methylol, 3
β, 20
α-two (dimethylaminos)-16
α-hydroxyl-4,14-dimethyl-(7CI, 8CI).
2. the application of buxus alkaloids compound according to claim 1, is characterized in that: carry for the preparation of targeted therapy in the antitumor drug of p53 and/or Ras mutated tumor.
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| BUKUS ALKALOIDS. PART I. THE STRUCTURES OF THREE NEW ALKALOIDS, CYCLOMICROPHYLLINE-A, B, AND C, FROM B. MICROPHYLLA SIEB. ET ZUCC.;T.NAKANO等;《Tetrahedron Letters》;19641231;第5卷(第18期);全文 * |
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| CN103142621A (en) | 2013-06-12 |
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