CN102657674A - Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities - Google Patents
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Abstract
The invention relates to an application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities. The Trichoderma pseudokoningii extracellular polysaccharides having the anticancer activities can reduce the vitality of K562 cells in vitro when they are applied to tumor growth inhibition (auxiliary) medicines, so intracellular active oxygen and free calcium ion concentrations are improved, the expression abundance of mRNA of p53 and Bax genes is improved, the expression of the Bc1-2 transcription level is reduced, the ectropion of cell membrane phosphatidylserine of the human chronic granulocyte leukemia cells K562 is induced, and the nuclear polycondensation and the fragmentation of K562 are induced to generate apoptotic bodies and apoptosis; and in-vivo administration can increase weights of tumor bearing mice and alleviate growth speeds and weights of tumors of the tumor bearing mice, thereby tumor cell killing and tumor growth inhibiting purposes are reached.
Description
Technical field
The present invention relates to a kind of application, belong to medical technical field with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity.
Background technology
According to World Health Organization's statistical data, by 2009, the whole world average expected life-span arrived 68 years old.But the heavy metal pollution of the electromagnetic pollution of modern society, soil and water body, air pollution or the like have caused increasing harm to human health.Along with the sustainable growth of global aged tendency of population aggravation and world's total population, and the trouble cancer very dangerous behavior that increases year by year (particularly remarkable with smoking in developing country or the area), make global cancer patient's quantity sharply increase.The statistical data of GLOBOCAN in 2008 shows, about 56% cases of cancer and 64% cancer mortality person wherein 24% occur in China from developing country or area in about 1,270 ten thousand cases of cancers and 7,600,000 cancer mortality persons.China cancer patient's existence patient and healing patient are merely 13%, have every year 1800000 people therefore sick dead approximately, and cancer has become the No.1 killer of serious threat China people ' s health.Along with continuing to increase of pathogenesis of cancer and death toll, the financial burden that brings because of cancer and the harmful effect of socio-economic development more and more significantly displayed.
Operation, radiotherapy, chemotherapy are three great tradition methods of treatment tumor, but these methods are also brought serious adverse and complication to the patient.Show that according to recent statistics the tumor mortality case is many to be owing to the complication that causes in operation, radiotherapy, the chemotherapy process causes.And above-mentioned Therapeutic Method expensive, prognosis are relatively poor, seek evident in efficacy, side effect is little, the antitumor drug of cost and Therapeutic Method are significant.
Polysaccharide (polysaccharide) is polyhydroxylated polymer that contains aldehyde radical or ketone group and the derivant thereof that is connected to form through glycosidic bond by the monosaccharide more than 10.Polysaccharide has biological function widely, not only can be used as the solvent of intravital energy supply material and cell, also participates in processes such as cell recognition, body's immunity adjusting, intercellular substance transportation, transformation, apoptosis.Polysaccharide has good biological action at aspects such as antitumor, antiinflammatory, antiviral, blood sugar lowering, defying age, anticoagulations; Advantages such as and side effect is less, and wherein fungus polysaccharide is strong with its anti-tumor activity, toxicity is little cause the extensive attention of Chinese and overseas scholars.Fungus polysaccharide is isolated a kind of active polysaccharide from fungus sporophore, mycelium, fermentation liquid.Lentinan, krestin, grifolan etc. have got into clinical practice as antitumour auxiliary drug.Over nearly 20 years, along with the develop rapidly of subjects such as biochemistry, molecular biology, people have had more and more deep understanding to the active function of polysaccharide and complex thereof.The anti-tumor activity of fungus polysaccharide mainly plays a role both ways: the one, directly act on tumor cell, through changing the tumor cell membrane biochemical characteristic, regulating cell proliferation and expression of apoptosis-related genes inhibition growth of tumour cell; The 2nd, immune cell activated, human body immunity improving power.Can obviously raise p53 expression of gene in the Lewis lung cancer cell like the Dihuang polysaccharide treated in vitro; Phellin polysaccharides can suppress growth, the invasion and attack of SW480 cell, and regulates Wnt/ β-catenin path.
Culture presevation has very strong growth potential number for the trichoderma pseudokiningii of CGMCC No.1443 (Trichoderma pseudokoningii) separates acquisition from corn straw, a large amount of extracellular polysaccharide of secretion in the liquid fermentation and culture process.The trichoderma pseudokiningii crude extracellular polysaccharide comprises various ingredients, adopts different isolation and purification methods can obtain multiple homogeneous polysaccharide, because the difference that its structure and monosaccharide are formed has different physiologically actives.Research shows that trichoderma pseudokoningii exocellular polysaccharide has excellent immunocompetence, can promote the secretion of spleen lymphocyte proliferation and IL-2, strengthens the phagocytic activity and the activity of acid phosphatase of peritoneal macrophage.IL-2 and TNF-alpha content significantly improve in normal mouse that trichoderma pseudokoningii exocellular polysaccharide filling stomach is handled and the immunocompromised model mice serum, strengthen the immunoreation of mice delayed, improve thymus index and spleen index.A kind of trichoderma pseudokoningii exocellular polysaccharide is disclosed like Chinese patent document CN101220101A (application number is 200810014047.8).This polysaccharide is characterised in that (1) is detected through the efficient gel filtration chromatography, and it has single symmetrical peak, and molecular weight is 18325; (2) sulfuric acid-phynol method and the phenylphenol method through improvement detects, and its neutral polyoses content is 65.2%, and glucuronic acid content is 32.6%; (3) GC through alditol acetate analyzes, and its monosaccharide consists of rhamnose, glucose and galactose, and mol ratio is RHA: GLC: GAL=5.6: 2.7: 1.0.After this polysaccharide reduced fully, the mol ratio of rhamnose, glucose and galactose was RHA: GLC: GAL=14.5: 9.3: 1.0, and its to contain molar content be 26.6% glucuronic acid.The method for preparing of this trichoderma pseudokoningii exocellular polysaccharide comprises the preparation of crude polysaccharides and the purification of polysaccharide.This trichoderma pseudokoningii exocellular polysaccharide has purposes widely in preparing raising mammalian immune active medicine or functional food and in the medicine for preparing treatment or adjuvant therapy of tumors, functional food.
At present, the functional study of trichoderma pseudokoningii exocellular polysaccharide has become the focus of trichoderma pseudokiningii bacterium research, but trichoderma pseudokoningii exocellular polysaccharide does not also appear in the newspapers to the research of the influence of human chronic myeloid leukemia cell K562 growth.
Summary of the invention
The present invention is directed to the deficiency of prior art, a kind of trichoderma pseudokoningii exocellular polysaccharide and application thereof that suppresses the K562 cells growth activity that have is provided.
The term explanation
The expression abundance of mRNA: refer to the average mark subnumber that mRNA expresses in certain cell.
Seveage reagent: a kind of protein denaturant can make the protein distortion from solution, separate out.
Technical scheme of the present invention is following:
Have the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity and suppress the application in tumor growth medicine or the ancillary drug in preparation.Said medicine can be at external reduction K562 cell viability; Improve its intracellular reactive oxygen species generation and free calcium ion concentration; Improve the expression abundance of p53, Bax gene mRNA, the expression of downward modulation Bcl-2 transcriptional level causes that human chronic myeloid leukemia cell K562 cell membrane phospholipid acyl serine turns up; Induce K562 nucleus bunching, cracked generation apoptotic body, apoptosis takes place; Vivo medicine-feeding can increase the tumor-bearing mice body weight, slows down tumor-bearing mice growth of tumor speed and tumor and weighs, and kills tumor cell thereby reach, and suppresses the purpose of tumor growth.
Above-mentioned application; Said molecular weight with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity is 31.9KDa, and the mol ratio that monosaccharide is formed is a rhamnose: xylose: fucose: mannose: glucose: galactose=16.2: 14.4: 1: 25.8: 23.6: 48.1.
Above-mentioned application, said trichoderma pseudokoningii exocellular polysaccharide with inhibition K562 cells growth activity prepares through following steps:
(1) with culture presevation number for the trichoderma pseudokiningii of CGMCC No.1443 (Trichoderma pseudokoningii) is inoculated in activation in the PDA culture medium, make the activation mycelia;
(2) the activation mycelia that step (1) is made is transferred in the PDB fermentation medium, is 25 ℃～30 ℃ in cultivation temperature, and the 120rpm shaking table was cultivated 9～11 days, made liquid fermentation liquid;
(3) liquid fermentation liquid that step (2) is made is through removing by filter mycelia, and filtrating is evaporated to 20%～30% of original volume through 60 ℃, concentrated solution; The alcoholic solution that in the concentrated solution that is cooled to room temperature, adds triploid long-pending 95% (volumetric concentration) then; 4 ℃ are spent the night, centrifugal, collecting precipitation;
The precipitate with deionized water of (4) step (3) being collected is dissolved, and adds the seveage reagent vibration deproteinising of lysate volume 1/3 then, makes raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours through weak-base anion-exchange resin D-301R; Adopt DEAE-Fast Flow anion exchange gel and Sephadex G-75 gel separation purification, make trichoderma pseudokoningii exocellular polysaccharide (EPS-1) solution of purification;
The trichoderma pseudokoningii exocellular polysaccharide solution of the purification that (6) step (5) is made makes trichoderma pseudokoningii exocellular polysaccharide after vacuum lyophilization;
The seveage reagent of said step (4) is by chloroform and 4: 1 by volume mixed of n-butyl alcohol.
The activatory time is 20～26h in the said step (1), and activation temperature is 25 ℃～30 ℃.
Cultivation temperature in the said step (2) is 28 ℃.
Centrifugal condition in the said step (3) is: the centrifugal 5min of 10000rpm.
Has a trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity by what above-mentioned method for preparing made; Its molecular weight is 31.9KDa; Monosaccharide consists of rhamnose, xylose, fucose, mannose, glucose, galactose, and mol ratio is 16.2: 14.4: 1: 25.8: 23.6: 48.1.
Beneficial effect
Trichoderma pseudokoningii exocellular polysaccharide of the present invention is applied to suppress in tumor growth medicine or the ancillary drug; Can improve its intracellular reactive oxygen species generation and free calcium ion concentration at external reduction K562 cell viability, improve the expression abundance of p53, Bax gene mRNA; The expression of downward modulation Bcl-2 transcriptional level; Cause that human chronic myeloid leukemia cell K562 cell membrane phospholipid acyl serine turns up, induce K562 nucleus bunching, cracked generation apoptotic body, apoptosis takes place; Vivo medicine-feeding can increase the tumor-bearing mice body weight, slows down tumor-bearing mice growth of tumor speed and tumor and weighs, and kills tumor cell thereby reach, and suppresses the purpose of tumor growth.
Description of drawings
Fig. 1 trichoderma pseudokoningii exocellular polysaccharide efficient gel permeation chromatography collection of illustrative plates;
Fig. 2 trichoderma pseudokoningii exocellular polysaccharide monosaccharide composition analysis;
Fig. 3 trichoderma pseudokoningii exocellular polysaccharide treated in vitro is to the active influence of human chronic myeloid leukemia cell K562 in-vitro multiplication;
Wherein:
*After the expression t check, compare with matched group and to be significant difference (P＜0.05)
*After the expression t check, compare with matched group and to be utmost point significant difference (P＜0.01)
Fig. 4 trichoderma pseudokoningii exocellular polysaccharide treated in vitro is to the influence of human chronic myeloid leukemia cell K562 form;
Wherein: A is a matched group; B, C, D are respectively trichoderma pseudokoningii exocellular polysaccharide 0.2mg/ml concentration, 0.6mg/ml concentration, 1mg/ml concentration processed group.
Fig. 5 trichoderma pseudokoningii exocellular polysaccharide treated in vitro induces human chronic myeloid leukemia cell K562 that apoptosis takes place;
Wherein: A is a matched group; B, C, D are respectively trichoderma pseudokoningii exocellular polysaccharide 0.2mg/ml concentration, 0.6mg/ml concentration, 1mg/ml concentration processed group.
The influence that Fig. 6 trichoderma pseudokoningii exocellular polysaccharide treated in vitro is expressed human chronic myeloid leukemia cell K562p53, Bcl-2, BaxmRNA;
Fig. 7 trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding is to the influence of human chronic myeloid leukemia cell K562 tumor-bearing mice tumor growth;
The specific embodiment
Below in conjunction with embodiment the present invention is done further elaboration, but institute of the present invention protection domain is not limited thereto.
Among the embodiment, trichoderma pseudokiningii (Trichoderma pseudokoningii) is available from Chinese common micro-organisms culture presevation administrative center, and culture presevation number is CGMCC No.1443; DEAE-Fast Flow anion exchange gel, Sephadex G-75 gel are available from GE company; Weak-base anion-exchange resin D-301R is available from Tianjin Nanjing University resin company limited; SRB is available from Sigma-Aldrich company; Hoechst 33258 dyeing liquors, Annexin V-FITC apoptosis test regent box are available from green skies Bioisystech Co., Ltd; Human chronic myeloid leukemia cell K562 is available from Shanghai Inst. of Life Science, CAS cell resource center; Trypan blue is available from the biological company limited of Shanghai, Shanghai space, and TRIZOL reagent is available from Invitrogen company; The reverse transcription test kit is available from Takara company; Other reagent are commercial reagent commonly used, this area, analytical pure.
A kind of method for preparing with active anticancer trichoderma pseudokoningii exocellular polysaccharide comprises the steps:
(1) with culture presevation number for the trichoderma pseudokiningii of CGMCC No.1443 (Trichoderma pseudokoningii) is inoculated in the PDA culture medium, activation 24h under 28 ℃ of conditions makes the activation mycelia;
Said PDA culture medium, every liter of component is following: Rhizoma Solani tuber osi 200g, sucrose 20g, agar 2g, water is settled to 1000mL, natural pH;
(2) the activation mycelia that step (1) is made is transferred in the PDB fermentation medium, 28 ℃ of cultivation temperature, and the 120rpm shaking table was cultivated 10 days, made liquid fermentation liquid;
Said PDB fermentation medium, every liter of component is following: Rhizoma Solani tuber osi 200g, sucrose 20g, water is settled to 1000mL, natural pH;
(3) liquid fermentation liquid that step (2) is made is removed mycelia through 4 layers of filtered through gauze; Filtrating is evaporated to 25% of original volume through 60 ℃; Get concentrated solution, in the concentrated solution that is cooled to room temperature, add the alcoholic solution of triploid long-pending 95% (volumetric concentration) then, 4 ℃ are spent the night; The centrifugal 5min of 10000rpm, collecting precipitation;
The precipitate with deionized water dissolving of (4) step (3) being collected; Get lysate; Add the seveage reagent thermal agitation deproteinising of lysate volume 1/3 then, seveage reagent is made by chloroform and 4: 1 by volume mixed of n-butyl alcohol, makes raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours through weak-base anion-exchange resin D-301R, adopt DEAE-Fast Flow anion exchange gel and Sephadex G-75 gel separation purification, make the trichoderma pseudokoningii exocellular polysaccharide solution of purification;
The trichoderma pseudokoningii exocellular polysaccharide solution of the purification that (6) step (5) is made makes and has the trichoderma pseudokoningii exocellular polysaccharide (EPS-1) that suppresses the K562 cells growth activity after vacuum lyophilization.
The EPS-1 that makes through said method is a white solid state, is cotton-shaped;
Detect through infrared spectrum, find that its characteristic peak that polysaccharide is arranged absorbs, and proves that EPS-1 is a polysaccharide;
That IKI reaction, biuret reaction, uv absorption testing result show is not starch-containing among the EPS-1, protein, nucleic acid impurity;
Measuring the EPS-1 molecular weight through efficient permeation chromatography is 31.9KDa (as shown in Figure 1);
Utilize gas chromatography to record its monosaccharide and consist of rhamnose, xylose, fucose, mannose, Fructus Vitis viniferae, galactose, mol ratio is 16.214.4: 1: 25.8: 23.6: 48.1 (as shown in Figure 2) show that EPS-1 is a kind of heteropolysaccharide.
The EPS-1 treated in vitro is to the influence of human chronic myeloid leukemia cell K562 proliferation activity
SRB is a kind of protein bound dyestuff; Can combine to appear a kind of bright pink with the basic amino acid of cell protein after trichloroacetic acid is fixing; And its change in color is directly proportional with albumen in the living cells, measures absorbance with enzyme-linked immunosorbent assay instrument, can obtain the numerical value of cell protein content accurately; Calculate cell number with this, the proliferation activity of reflection cell.
Experimental technique
Get the human chronic myeloid leukemia cell K562 that is in exponential phase, processing density is 1 * 10
4Individual/ml cell suspension, be inoculated in 96 orifice plates 180 μ L/ holes.After cultivating 24h; Every hole adds the trichoderma pseudokoningii exocellular polysaccharide solution 20 μ L (final concentration be 0.2,0.4,0.6,0.8,1mg/ml) of variable concentrations, and the total liquid measure in every hole is 200 μ L, and each drug level is established 6 multiple holes; And establish blank and (do not have cell; Only add the RPMI-1640 culture fluid) and normal control hole (do not add medicine, add equivalent RPMI-1640 culture fluid), 37 ℃, 5%CO
2, saturated humidity (following cell culture condition herewith) cultivated 24,48,72 hours.After cultivating end, every hole adds trichloroacetic acid (TCA) solution of 50 μ L4 ℃ 50wt%, and making the final concentration of TCA is 10wt%, puts into fixedly 1h of 4 ℃ of refrigerators.Deionized water wash 5 times dries the SRB that every hole, back adds 100 μ L 0.4wt%, and room temperature is placed 30min; Discard each hole liquid; With 1wt% acetic acid washing 5 times, every hole adding 150uL pH is 10.5 10mmol/L Tris base (Tris) solution, vibration 5min; All dissolve until dyestuff, use enzyme-linked immunosorbent assay instrument 490nm place to measure OD value and record.
Suppression ratio=(matched group OD value-processed group OD value)/control wells OD value * 100%
The trichoderma pseudokoningii exocellular polysaccharide that embodiment 1 makes has inhibitory action at external proliferation activity to human chronic myeloid leukemia cell K562, and experimental result is seen Fig. 3.Trichoderma pseudokoningii exocellular polysaccharide has significantly suppressed the in-vitro multiplication of human chronic myeloid leukemia cell K562, and has time and dose dependent.The variable concentrations trichoderma pseudokoningii exocellular polysaccharide is handled 24h, 48h and 72h, and the growth of human chronic myeloid leukemia cell K562 has all been produced inhibitory action, and suppression ratio is up to 41%.
Embodiment 3
The trichoderma pseudokoningii exocellular polysaccharide treated in vitro is to the influence of human chronic myeloid leukemia cell K562 chromatin form
Permeability of cell membrane changes during cell generation apoptosis; The chromatin bunching also produces apoptotic body; Use can be observed nuclear metamorphosis after having membrane permeability and can specificity combining the Hoechst33258 dyeing liquor dyeing of DNA intuitively under inverted fluorescence microscope.
Experimental technique
The sterility cover slide is placed in six orifice plates, plant human chronic myeloid leukemia cell K562 and hatch 24h.The trichoderma pseudokoningii exocellular polysaccharide solution irritation cell that adds variable concentrations next day removed culture fluid after 48 hours, added the 0.5ml fixative, fixing 10 minutes.Fixative is removed in suction, and PBS cleans the back and adds 0.5ml Hoechst 33258 dyeing liquors, dyes PBS flush away dyeing liquor 5 minutes.Observe under the fluorescence microscope and take pictures, excitation wavelength is about 350nm, about emission wavelength 460nm.
The trichoderma pseudokoningii exocellular polysaccharide treated in vitro that embodiment 1 makes causes the change of human chronic myeloid leukemia cell K562 nuclei dyeing chromaticness form, result such as Fig. 4.Hoechst 33238 dyeing liquor coloration results show; Matched group (A) cell size evenly, form is regular, nucleus dyeing is more shallow, trichoderma pseudokoningii exocellular polysaccharide processed group (B, C, D) cell membrane shrinkage, nuclei dyeing chromaticness bunching engrain; Character is irregular, apoptotic body occurs.The generation of apoptotic body is the distinctive marks of cell generation apoptosis, explains that trichoderma pseudokoningii exocellular polysaccharide induces human chronic myeloid leukemia cell K562 that apoptosis takes place.
Embodiment 4
The EPS-1 treated in vitro induces human chronic myeloid leukemia cell K562 that apoptosis takes place
To turn up be one of important symbol of early apoptosis of cells to Phosphatidylserine on the cell membrane; Annexin-V a kind ofly has the calcium ion dependency phospholipids incorporate albumen of high-affinity to Phosphatidylserine, can be used as the specific probe of the Phosphatidylserine that turn to the cell membrane outer surface.
Experimental technique
The human chronic myeloid leukemia cell K562 of the trophophase of taking the logarithm, adjustment concentration is 5 * 10
5Individual/ml, plant in six orifice plates, hatch 24h.Add variable concentrations trichoderma pseudokoningii exocellular polysaccharide solution next day, harvesting behind the processing 48h.Get 5-10 ten thousand cells, add 195 μ L Annexin V-FITC and combine liquid, add 5 μ L Annexin V-FITC then, room temperature (20-25 ℃) lucifuge was hatched 10 minutes; Centrifugal 5 minutes of 1000g abandons supernatant, adds 190 μ LAnnexin V-FITC and combines liquid re-suspended cell gently; Add 10 μ L propidium iodide dyeing liquors, mixing gently, the ice bath lucifuge is placed; Go up immediately machine testing, Annexin V-FITC exciting light is a green fluorescence, and the propidium iodide exciting light is a red fluorescence.
The trichoderma pseudokoningii exocellular polysaccharide treated in vitro that embodiment 1 makes induces human chronic myeloid leukemia cell K562 that apoptosis takes place, and the result sees Fig. 5.Human chronic myeloid leukemia cell K562 is after trichoderma pseudokoningii exocellular polysaccharide is handled 48h; With respect to matched group; Viable apoptotic cell (right lower quadrant) ratio of the human chronic myeloid leukemia cell K562 of processed group rises to 20.5% from 0.1%; Late period apoptosis and non-viable non-apoptotic cell (right upper quadrant) bring up to 18.75% from 1.7%, improve with concentration, and the apoptotic cell number progressively increases; Be dose dependent, explain that trichoderma pseudokoningii exocellular polysaccharide can induce human chronic myeloid leukemia cell K562 that apoptosis takes place.
Embodiment 5 trichoderma pseudokoningii exocellular polysaccharide treated in vitro are to the influence of the mRNA expression of human chronic myeloid leukemia cell K562p53, Bcl-2, Bax
P53, Bcl-2, Bax are the marker gene in the apoptosis of tumor cells process, and wherein p53 can induce and regulate apoptosis process, and the Bax/Bcl-2 ratio is that cell is to judge whether cell the important indicator of apoptosis takes place.
Experimental technique
To be in the secular human chronic myeloid leukemia cell K562 of logarithm and be inoculated in six orifice plates, after 24 hours, add the trichoderma pseudokoningii exocellular polysaccharide mother solution, make that its final concentration is 0.2,0.6,1mg/ml, and establish matched group.Harvesting behind the stimulation 24h uses TRIZOL reagent to extract total RNA, and reverse transcription is to carry out the polymerase chain reaction according to each target gene sequences design primer behind the cDNA, and primer sequence is seen table 1.
The trichoderma pseudokoningii exocellular polysaccharide treated in vitro that embodiment 1 makes raises the transcriptional level of K562 cell p53 and Bax, reduces the mRNA abundance of Bcl-2, result such as Fig. 6.With the raising of trichoderma pseudokoningii exocellular polysaccharide concentration, the mRNA expression of p53 and Bax improves, the down-regulated expression of the mRNA of Bcl-2, and the Bax/Bcl-2 ratio improves, and the K562 cell generation apoptosis that trichoderma pseudokoningii exocellular polysaccharide is handled is described.
Table 1 polymerase chain reaction the primer sequence
Embodiment has the trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding influence heavy to tumor-bearing mice body weight, gross tumor volume and tumor that suppresses human chronic myeloid leukemia cell K562 growth activity for 6 one kinds
The take the logarithm human chronic myeloid leukemia cell K562 of trophophase, trypan blue chromoscopy living cells content is more than 95%, and (every liter contains: potassium dihydrogen phosphate 0.27g with the PBS buffer; Sodium hydrogen phosphate 1.42g; Sodium chloride 8g, potassium chloride 0.2g, pH 7.4) to regulate cell density be 1 * 10
8Individual/ml, 50 of the nude mices of the subcutaneous vaccination BALB/C-nu/nu of right side omoplate portion strain, 0.2ml/ is only.Be selected to the tumor mice after one week; Be divided into 4 groups at random; Matched group, trichoderma pseudokoningii exocellular polysaccharide 25mg/kg.day group, 50mg/kg.day group, 100mg/kg.day group, cyclophosphamide 20mg/kg.day group, 10 every group, average weight difference is no more than 1 gram between group.The trichoderma pseudokoningii exocellular polysaccharide normal saline solution of variable concentrations is in set time every day gastric infusion, and matched group gives the equivalent normal saline, successive administration 20 days.Observe the growing state of respectively organizing mice, per 5 days weighing mice body weight are measured gross tumor volume.Last administration 24 back sacrificed by exsanguination mices strip subcutaneous solid tumor tumor piece, remove fat and connective tissue, weigh.
The trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding that embodiment 1 makes can suppress the heteroplastic transplantation growth of tumor, promotes the increase of tumor-bearing mice body weight, and the result sees table 2, table 3, Fig. 7.The gastric infusion of trichoderma pseudokoningii exocellular polysaccharide shown in the table 2 is to the influence of tumor-bearing mice body weight; Matched group tumor-bearing mice weight increase is slow; The tumor-bearing mice weight increase that trichoderma pseudokoningii exocellular polysaccharide filling stomach is handled is obviously accelerated, and the mice average weight was higher by 16.06% than matched group after wherein 100mg/kg.day organized 20 days.The influence that the gastric infusion of trichoderma pseudokoningii exocellular polysaccharide shown in the table 3 is heavy to tumor-bearing mice tumor tumor, with the increase of dosage, the tumor-bearing mice tumor heavily descends, and tumour inhibiting rate improves, and has dose-dependent effect, and the tumour inhibiting rate of 100mg/kg.day group is up to 46.62%.Shown in Figure 7 with the variation of administration process tumor-bearing mice gross tumor volume, processed group mouse tumor volume gathers way and is slower than matched group.The above results explanation trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding can increase the tumor-bearing mice body weight, suppresses the tumor-bearing mice tumor growth.
Table 2 trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding is to the influence (n=10,
) of K562 tumor-bearing mice body weight (gram)
| Handle | 0d (before the administration) | | 10d | 15d | 20d | ||
| 0 (matched group) | 19.2±1.1 | 20.8±1.2 | 23.9±1.4 | 24.1±1.1 | 24.9±1.2 | ||
| 25mg/kg.day | 18.5±0.9 | 21.6±1.1 | 24.4±1.5 | 25.1±1.3 | 26.5±1.4 | ||
| 50mg/kg.day | 19.1±1.3 | 21.9±1.3 | 25.7±1.2 | 27.9±1.5 | 28.6±1.6 | ||
| 100mg/kg.day | 18.8±1.2 | 21.5±1.1 | 25.1±1.3 | 26.2±1.3 | 28.9±1.4 |
The influence that table 3 trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding is heavy to K562 tumor-bearing mice tumor tumor
| Handle | Tumor heavy (gram) | Tumour inhibiting rate (%) |
| Matched group | 1.63 | |
| 25mg/kg.day | 1.35 | 17.18 |
| 50mg/kg.day | 1.13 | 30.67 |
| 100mg/kg.day | 0.87 | 46.62 |
Claims (6)
1. have the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity and suppress the application in tumor growth medicine or the ancillary drug in preparation.
2. application as claimed in claim 1; It is characterized in that; Said molecular weight with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity is 31.9KDa, and the mol ratio that monosaccharide is formed is a rhamnose: xylose: fucose: mannose: glucose: galactose=16.2: 14.4: 1: 25.8: 23.6: 48.1.
3. application as claimed in claim 1 is characterized in that, described trichoderma pseudokoningii exocellular polysaccharide prepares through following steps:
(1) with culture presevation number for the trichoderma pseudokiningii of CGMCC No.1443 (Trichoderma pseudokoningii) is inoculated in activation in the PDA culture medium, make the activation mycelia;
(2) the activation mycelia that step (1) is made is transferred in the PDB fermentation medium, is 25 ℃～30 ℃ in cultivation temperature, and the 120rpm shaking table was cultivated 9～11 days, made liquid fermentation liquid;
(3) liquid fermentation liquid that step (2) is made is through removing by filter mycelia, and filtrating is evaporated to 20%～30% of original volume through 60 ℃, concentrated solution; The alcoholic solution that in the concentrated solution that is cooled to room temperature, adds triploid long-pending 95% (volumetric concentration) then; 4 ℃ are spent the night, centrifugal, collecting precipitation;
The precipitate with deionized water dissolving of (4) step (3) being collected, the seveage reagent that adds lysate volume 1/3 then shakes deproteinising, makes raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours through weak-base anion-exchange resin D-301R, adopt DEAE-Fast Flow anion exchange gel and Sephadex G-75 gel separation purification, make the trichoderma pseudokoningii exocellular polysaccharide solution of purification;
The trichoderma pseudokoningii exocellular polysaccharide solution of the purification that (6) step (5) is made makes trichoderma pseudokoningii exocellular polysaccharide after vacuum lyophilization.
4. application as claimed in claim 3 is characterized in that, the activatory time is 20～26h in the said step (1), and activation temperature is 25 ℃～30 ℃.
5. application as claimed in claim 3 is characterized in that, the cultivation temperature in the said step (2) is 28 ℃.
6. application as claimed in claim 3 is characterized in that, the centrifugal condition in the said step (3) is: the centrifugal 5min of 10000rpm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210105395 CN102657674B (en) | 2012-04-11 | 2012-04-11 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
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| CN 201210105395 CN102657674B (en) | 2012-04-11 | 2012-04-11 | Application of Trichoderma pseudokoningii extracellular polysaccharides having anticancer activities |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103027924A (en) * | 2012-11-26 | 2013-04-10 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103816370A (en) * | 2014-03-17 | 2014-05-28 | 鞠芳 | Chinese medicament group for assisting chemotherapy |
| CN104726515A (en) * | 2015-03-20 | 2015-06-24 | 江南大学 | Method for extracting bacterial exopolysaccharide rich in fucose |
| CN105685766A (en) * | 2014-11-27 | 2016-06-22 | 丰益(上海)生物技术研发中心有限公司 | Microalgae broth exopolysaccharide and its preparation method and use |
| CN109929765A (en) * | 2019-03-20 | 2019-06-25 | 武汉大学 | One plant of Cryptococcus and its exocellular polysaccharide and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220101A (en) * | 2008-01-23 | 2008-07-16 | 山东大学 | Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof |
-
2012
- 2012-04-11 CN CN 201210105395 patent/CN102657674B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220101A (en) * | 2008-01-23 | 2008-07-16 | 山东大学 | Trichoderma pseudokoningii exocellular polysaccharide, preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 石磊: "拟康氏木霉产胞外多糖的分离纯化及组成初步研究", 《曲阜师范大学学报(自然科学版)》 * |
| 陈靠山: "功能多糖的研究进展", 《皖南医学院学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103027924A (en) * | 2012-11-26 | 2013-04-10 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103027924B (en) * | 2012-11-26 | 2015-02-25 | 皖南医学院 | Application of trichoderma pseudokoningii exopolysaccharide as medicine for treating gastric cancer |
| CN103816370A (en) * | 2014-03-17 | 2014-05-28 | 鞠芳 | Chinese medicament group for assisting chemotherapy |
| CN105685766A (en) * | 2014-11-27 | 2016-06-22 | 丰益(上海)生物技术研发中心有限公司 | Microalgae broth exopolysaccharide and its preparation method and use |
| CN104726515A (en) * | 2015-03-20 | 2015-06-24 | 江南大学 | Method for extracting bacterial exopolysaccharide rich in fucose |
| CN109929765A (en) * | 2019-03-20 | 2019-06-25 | 武汉大学 | One plant of Cryptococcus and its exocellular polysaccharide and application |
| CN109929765B (en) * | 2019-03-20 | 2020-10-13 | 武汉大学 | Cryptococcus lactis and exopolysaccharide and application thereof |
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| CN102657674B (en) | 2013-10-30 |
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