CN102657674B - 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 with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity, belong to medical technical field.
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, air pollution etc. have caused increasing harm to human health.Along with the sustainable growth of population in the world aging aggravation and world's total population, and the trouble cancer very dangerous behavior of increasing year by year (particularly remarkable with smoking in developing country or the area), so that global cancer patient's quantity sharply increases.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 only are 13%, have approximately every year 1800000 people therefore sick dead, 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 for the treatment of tumor, but these methods are also brought serious side effect 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 somewhat expensive, prognosis are relatively poor, and it is significant to seek antitumor drug and Therapeutic Method evident in efficacy, that side effect is little, expense is cheap.
Polysaccharide (polysaccharide) is the polyhydroxylated polymer that contains aldehyde radical or ketone group and the derivant thereof that is connected to form by glycosidic bond by the monosaccharide more than 10.Polysaccharide has widely biological function, not only can be used as the interior energy supply material of body and the solvent of cell, also participates in the 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, the 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 entered clinical practice as antitumour auxiliary drug.Over nearly 20 years, along with the develop rapidly of the 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, by changing the expression inhibiting growth of tumour cell of tumor cell membrane biochemical characteristic, adjusting cell proliferation and apoptosis-related genes; The 2nd, immune cell activated improves immunity of organisms.Can obviously raise the expression of p53 gene in the Lewis lung cancer cell such as 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 form has different physiologically actives.Studies show that trichoderma pseudokoningii exocellular polysaccharide has excellent immunocompetence, can promote the secretion of spleen lymphocyte proliferation and IL-2, strengthen phagocytic activity and the activity of acid phosphatase of peritoneal macrophage.IL-2 and TNF-alpha content significantly improve in the normal mouse that the trichoderma pseudokoningii exocellular polysaccharide gavage is processed and the immunodeficiency models mice serum, strengthen the immunoreation of mice delayed, improve thymus index and spleen index.A kind of trichoderma pseudokoningii exocellular polysaccharide is disclosed such as Chinese patent literature CN101220101A (application number is 200810014047.8).This polysaccharide is characterised in that (1) is detected by the efficient gel filtration chromatography, and it has single symmetrical peak, and molecular weight is 18325; (2) sulfuric acid-phynol method and the phenyl phynol method by improvement detects, and its neutral polyoses content is 65.2%, and glucuronic acid content is 32.6%; (3) GC by 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 preparation method of this trichoderma pseudokoningii exocellular polysaccharide comprises the preparation of crude polysaccharides and the purification of polysaccharide.This trichoderma pseudokoningii exocellular polysaccharide has widely purposes 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 yet there are no report to the research of human chronic myeloid leukemia cell K562 affects on the growth.
Summary of the invention
The present invention is directed to the deficiencies in the 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 gene expression abundance of mRNA: refer to that mRNA is at the average mark subnumber of certain cells.
Seveage reagent: a kind of protein denaturant can make the protein distortion separate out from solution.
Technical scheme of the present invention is as follows:
Have the application of trichoderma pseudokoningii exocellular polysaccharide in preparation inhibition tumor growth medicine or ancillary drug that suppresses the K562 cells growth activity.Said medicine can be at external reduction K562 cell viability, improve its intracellular reactive oxygen species generation and free calcium ion concentration, improve the gene 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; Vivo medicine-feeding can increase the tumor-bearing mice body weight, and the speed of growth and the tumor of slowing down the tumor-bearing mice tumor are heavy, thereby reach killing off tumor cells, suppress the purpose of tumor growth.
Above-mentioned application, described molecular weight with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity is 31.9KDa, and the mol ratio that monosaccharide forms is rhamnose: xylose: fucose: mannose: glucose: galactose=16.2: 14.4: 1: 25.8: 23.6: 48.1.
Above-mentioned application, described trichoderma pseudokoningii exocellular polysaccharide with inhibition K562 cells growth activity prepares as follows:
(1) culture presevation number is activated in the PDA culture medium for the trichoderma pseudokiningii of CGMCC No.1443 (Trichoderma pseudokoningii) is inoculated in, 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 removed mycelia after filtration, filtrate is evaporated to 20%～30% of original volume through 60 ℃, get concentrated solution, then the alcoholic solution that adds triploid long-pending 95% (volumetric concentration) in the concentrated solution that is cooled to room temperature, 4 ℃ are spent the night, centrifugal, collecting precipitation;
The precipitate with deionized water of (4) step (3) being collected is dissolved, and then adds the seveage reagent vibration deproteinising of lysate volume 1/3, makes raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours by weak-base anion-exchange resin D-301R, adopt DEAE-Fast Flow anion exchange gel and the separation and purification of Sephadex G-75 gel, 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 described step (4) is mixed by 4: 1 by volume ratio of chloroform and n-butyl alcohol.
The time of activation is 20～26h in the described step (1), and activation temperature is 25 ℃～30 ℃.
Cultivation temperature in the described step (2) is 28 ℃.
Centrifugal condition in the described 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 preparation method 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 be at external reduction K562 cell viability, improve its intracellular reactive oxygen species generation and free calcium ion concentration, improve the gene 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, and induces K562 nucleus bunching, cracked generation apoptotic body, apoptosis; Vivo medicine-feeding can increase the tumor-bearing mice body weight, and the speed of growth and the tumor of slowing down the tumor-bearing mice tumor are heavy, thereby reach killing off tumor cells, suppress 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 on the impact of human chronic myeloid leukemia cell K562 in-vitro multiplication activity;
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 on the impact of human chronic myeloid leukemia cell K562 form;
Wherein: A is 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 is induced human chronic myeloid leukemia cell K562 apoptosis;
Wherein: A is 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 impact 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 on the impact of human chronic myeloid leukemia cell K562 tumor-bearing mice tumor growth;
The specific embodiment
Below in conjunction with embodiment the present invention is further elaborated, but institute of the present invention protection domain is not limited to this.
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 the large resin company limited in south, Tianjin; SRB is available from Sigma-Aldrich company; Hoechst 33258 dyeing liquors, Annexin V-FITC cell apoptosis detection kit 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 preparation method 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, under 28 ℃ of conditions, activate 24h, make the activation mycelia;
Described PDA culture medium, every liter of component is as follows: 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;
Described PDB fermentation medium, every liter of component is as follows: 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, filtrate is evaporated to 25% of original volume through 60 ℃, get concentrated solution, then the alcoholic solution that adds triploid long-pending 95% (volumetric concentration) in the concentrated solution that is cooled to room temperature, 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, then the seveage reagent thermal agitation deproteinising that adds lysate volume 1/3, seveage reagent are mixed by 4: 1 by volume ratio of chloroform and n-butyl alcohol and make, and make raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours by weak-base anion-exchange resin D-301R, adopt DEAE-Fast Flow anion exchange gel and the separation and purification of Sephadex G-75 gel, 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 the trichoderma pseudokoningii exocellular polysaccharide (EPS-1) with inhibition K562 cells growth activity after vacuum lyophilization.
The EPS-1 that makes through said method is 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 polysaccharide;
That IKI reaction, biuret reaction, uv absorption testing result show is not starch-containing among the EPS-1, protein, nucleic acid impurity;
It is 31.9KDa (as shown in Figure 1) by high-effective penetrating chromatography determination EPS-1 molecular weight;
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 on the impact of human chronic myeloid leukemia cell K562 proliferation activity
SRB is a kind of protein bound dyestuff, can be combined with the basic amino acid of cell protein after trichloroacetic acid is fixing and present a kind of bright pink, and the variation of its color is directly proportional with albumen in the living cells, measure absorbance with enzyme-linked immunosorbent assay instrument, can obtain the accurately numerical value of cell protein content, 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, making 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, the total liquid measure in every hole is 200 μ L, 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, dry the SRB that rear every hole adds 100 μ L 0.4wt%, 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, until dyestuff all dissolves, 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 processed 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 on the impact of human chronic myeloid leukemia cell K562 chromatin form
Permeability of cell membrane changes during the cell apoptosis, the chromatin bunching also produces apoptotic body, use can be observed nuclear metamorphosis after having membrane permeability and Hoechst33258 dyeing liquor dyeing that can specific binding 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.Add the trichoderma pseudokoningii exocellular polysaccharide solution irritation cell of variable concentrations next day after 48 hours, remove culture fluid, add the 0.5ml fixative, fix 10 minutes.Suck fixative, add 0.5ml Hoechst 33258 dyeing liquors after PBS cleans, dyeed PBS flush away dyeing liquor 5 minutes.The fluorescence microscopy Microscopic observation is also taken pictures, and 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 is even, 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 apoptosis, illustrates that trichoderma pseudokoningii exocellular polysaccharide induces human chronic myeloid leukemia cell K562 apoptosis.
The EPS-1 treated in vitro is induced human chronic myeloid leukemia cell K562 apoptosis
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, adjusting 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 in conjunction with liquid, then add 5 μ L Annexin V-FITC, room temperature (20-25 ℃) lucifuge was hatched 10 minutes, centrifugal 5 minutes of 1000g abandons supernatant, adds 190 μ LAnnexin V-FITC in conjunction with liquid re-suspended cell gently, add 10 μ L propidium iodide dyeing liquors, mixing gently, the ice bath lucifuge is placed, and goes up machine testing immediately, Annexin V-FITC exciting light is green fluorescence, and the propidium iodide exciting light is red fluorescence.
The trichoderma pseudokoningii exocellular polysaccharide treated in vitro that embodiment 1 makes is induced human chronic myeloid leukemia cell K562 apoptosis, the results are shown in Figure 5.Human chronic myeloid leukemia cell K562 is after trichoderma pseudokoningii exocellular polysaccharide is processed 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%, necrosis and apoptosis cell in late period (right upper quadrant) brings up to 18.75% from 1.7%, improve with concentration, the apoptotic cell number progressively increases, be dose dependent, illustrate that trichoderma pseudokoningii exocellular polysaccharide can induce human chronic myeloid leukemia cell K562 apoptosis.
Embodiment 5 trichoderma pseudokoningii exocellular polysaccharide treated in vitro are on the impact 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 the whether important indicator of apoptosis of cell.
Experimental technique
To be in the long-term 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 genes of interest primers behind the cDNA, and primer sequence sees 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 amount 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 apoptosis that trichoderma pseudokoningii exocellular polysaccharide is processed is described.
Table 1 polymerase chain reaction the primer sequence
6 one kinds of trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding impacts heavy on tumor-bearing mice body weight, gross tumor volume and tumor with the human chronic myeloid leukemia cell K562 growth activity of inhibition of embodiment
The human chronic myeloid leukemia cell K562 of trophophase takes the logarithm, Trypan Blue checks that living cells content is more than 95%, (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 right side scapular region subcutaneous vaccination BALB/C-nu/nu strain, 0.2ml/ is only.Be selected to the tumor mice after one week, be divided at random 4 groups, 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 Mouse Weights are measured gross tumor volume.Last administration 24 rear 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 growth of heteroplastic transplantation tumor, promotes the increase of tumor-bearing mice body weight, the results are shown in Table 2, table 3, Fig. 7.The gastric infusion of trichoderma pseudokoningii exocellular polysaccharide shown in the table 2 is on the impact of tumor-bearing mice body weight, matched group tumor-bearing mice body weight increases slowly, the tumor-bearing mice body weight that the trichoderma pseudokoningii exocellular polysaccharide gavage is processed increases obviously and accelerates, and mice average weight contrast group was high by 16.06% after wherein 100mg/kg.day organized 20 days.The impact that the gastric infusion of trichoderma pseudokoningii exocellular polysaccharide shown in the table 3 is heavy on tumor-bearing mice tumor tumor, with the increase of dosage, tumor weight 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 on the impact of K562 tumor-bearing mice body weight (gram) (n=10,
)
| Process | 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 impact that table 3 trichoderma pseudokoningii exocellular polysaccharide vivo medicine-feeding is heavy on K562 tumor-bearing mice tumor tumor
| Process | 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 (2)
1. have the application of trichoderma pseudokoningii exocellular polysaccharide in preparation inhibition tumor growth medicine or ancillary drug that suppresses the K562 cells growth activity;
Described molecular weight with the trichoderma pseudokoningii exocellular polysaccharide that suppresses the K562 cells growth activity is 31.9KDa, and the mol ratio that monosaccharide forms is rhamnose: xylose: fucose: mannose: glucose: galactose=16.2:14.4:1:25.8:23.6:48.1.
2. application as claimed in claim 1 is characterized in that, described trichoderma pseudokoningii exocellular polysaccharide prepares as follows:
(1) with culture presevation number be CGMCC No.1443 trichoderma pseudokiningii (
Trichoderma pseudokoningii) be inoculated in the PDA culture medium and activate, 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 removed mycelia after filtration, filtrate is evaporated to 20%～30% of original volume through 60 ℃, get concentrated solution, then add the long-pending 95%(volumetric concentration of triploid in the concentrated solution that is cooled to room temperature) alcoholic solution, 4 ℃ are spent the night, centrifugal, collecting precipitation;
The precipitate with deionized water dissolving of (4) step (3) being collected, the seveage reagent that then adds lysate volume 1/3 shakes deproteinising, makes raw sugar solution;
(5) after the raw sugar solution that step (4) is made decolours by weak-base anion-exchange resin D-301R, adopt DEAE-Fast Flow anion exchange gel and the separation and purification of Sephadex G-75 gel, 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.
3
.Application as claimed in claim 2 is characterized in that, the time of activation is 20～26h in the described step (1), and activation temperature is 25 ℃～30 ℃.
4
.Application as claimed in claim 2 is characterized in that, the cultivation temperature in the described step (2) is 28 ℃.
5
.Application as claimed in claim 2 is characterized in that, the centrifugal condition in the described step (3) is: the centrifugal 5min of 10000rpm.
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