CN110478377A - Phellinus is as the application for being used for tumor - Google Patents
Phellinus is as the application for being used for tumor Download PDFInfo
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Abstract
Phellinus is as the application for being used for tumor, it is related to field of pharmaceutical technology, and Phellinus strain and growth course include as follows: one, Phellinus strain: Boydii Phellinus pore fungi.Two, substituting stuff cultivation step: 1, substituting stuff cultivation is with the technology mode of the mixed raw materials such as sawdust, wheat bran cultivation Phellinus；2, generation material bacteria stick fabrication processing: stock → spice → pack → sterilizing → cooling → inoculation.Three, medium component: with ramulus mori sawdust, the sawdusts such as toothed oak tree (Mongolian oak), robur are major ingredient, and wheat bran, dregs of beans are auxiliary material.Phellinus of the present invention is as the application for being used for tumor; it can be improved Phellinus bacterium survival rate; it is able to demonstrate that Phellinus has preferable antitumor action; the tumours such as lung cancer, liver cancer, gastric cancer, colon cancer, cervical carcinoma, breast cancer, oophoroma, leukaemia, prostate cancer, melanoma, neuroblastoma tool is had a better effect, there is important economic benefit and social benefit.
Description
Technical field
The present invention relates to field of pharmaceutical technology, and in particular to Phellinus is as the application for being used for tumor.
Background technique
Phellinus kind, be accredited as rust leather hole Zoopagales Hymenochaetales, Hymenochaetaceae Hymenochaetaceae,
Phellinus hole Pseudomonas Sanghuangporus；Chinese name: Boydii Phellinus pore fungi, popular name: cloves Phellinus, Classification system:
Sanghuangporus baumii.Homonymus or synonymum: Inonotus baumii Boydii fibre pore fungi；Phellinus baumii Boydii
Phellinus.
Number of patent application: Phellinus kind involved in 201410788390.3 are as follows: Phellinus igniarius (L.e ×
Fr.) Quel (phelliuns igniarius of Polyporaceae), number of patent application: Phellinus involved in 201410788390.3 is " Duan Musang
It is yellow ", Phellinus bacterium is grown in " in the tree section of the trees such as robur ", and trees are directly cut into segment, and Phellinus bacterium grows in tree section
Come, inoculation survival rate is low.
Summary of the invention
It is survived in view of the defects and deficiencies of the prior art, the present invention intends to provide one kind can be improved Phellinus bacterium
Rate is able to demonstrate that Phellinus has the Phellinus of preferable antitumor action as the application for being used for tumor.
To achieve the above object, the technical solution adopted by the present invention is that: Phellinus as be used for tumor application,
Phellinus strain and growth course include as follows:
Phellinus strain:
Sanghuangporus baumii Boydii Phellinus pore fungi (homonymus or synonymum: Inonotus baumii Boydii fibre pore fungi；
Phellinus baumii Boydii Phellinus).
Substituting stuff cultivation step:
1, substituting stuff cultivation (Substitute cultivation) is with the technology of the mixed raw materials such as sawdust, wheat bran cultivation Phellinus
Mode；
2, generation material bacteria stick fabrication processing: stock → spice → pack → sterilizing → cooling → inoculation.
Medium component:
With ramulus mori sawdust, the sawdusts such as toothed oak tree (Mongolian oak), robur are major ingredient, and wheat bran, dregs of beans are auxiliary material.
The raw materials such as the sawdust, wheat bran, dregs of beans answer fresh, dry, color it is normal, without mildew, without agglomeration, free from extraneous odour, wood
It to be sieved before bits use, sieve diameter 8mm~12mm.
Culture medium compositing formula: the sawdusts 75%~80% such as ramulus mori sawdust or toothed oak tree (Mongolian oak), robur, wheat bran 13%~
18%, dregs of beans 0%~3%, sugar 0%~2.5%, quick lime 1.5%.It is stirred after adding water, water content control exists
62%~65%.
The present invention the beneficial effects are as follows: Phellinus of the present invention be used as tumor application, energy
Enough improve Phellinus bacterium survival rate, be able to demonstrate that Phellinus have preferable antitumor action, to lung cancer, liver cancer, gastric cancer, colon cancer,
The tumours such as cervical carcinoma, breast cancer, oophoroma, leukaemia, prostate cancer, melanoma, neuroblastoma have preferable treat
Effect has important economic benefit and social benefit.
One, Phellinus anti tumor activity in vitro is evaluated
(1) experimental material
1, cell strain
Lung cancer, liver cancer, gastric cancer, cancer of pancreas, bladder cancer, colon cancer, cervical carcinoma, breast cancer, oophoroma, leukaemia, forefront
43 kinds of cell strains such as gland cancer, melanoma, neuroblastoma；Liver cancer, gastric cancer, cancer of pancreas, bladder cancer, colon cancer, breast cancer,
Leukaemia, melanoma, human neuroblastoma cells are supplied by pharmaceutical college, Medical University Of Anhui；Lung cancer, cervical carcinoma, prostate
Cancer cell is bought in Wuhan Pu Nuosai Life Science Co., Ltd；Ovarian cancer cell: purchase receives biology in north.
2, drug and reagent
RPMI-1640 culture medium: purchase is in GE life science；
DMEM culture medium: purchase is in GE life science；
Ham ' S F12K culture medium is purchased from the limited publicity of Wuhan Pu Nuosai Life Science；
Fetal calf serum: it is purchased from Hangzhou Chinese holly biomaterial research institute.It is placed in -20 DEG C of refrigerators to save, 4 DEG C of refrigerator overnights are melted
Change, is used after 56 DEG C of water-bath 30min are inactivated；
Fluorouracil: it is purchased from Qilu Pharmaceutical Co., Ltd.；
Penicillin: it is purchased from HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory；
Streptomysin: it is purchased from Shenzhen south China south pharmaceutical Co. Ltd；
Dimethyl sulfoxide (DMSO): it is purchased from Sigma company；
Cell Proliferation and citotoxicity detection kit (CCK-8 kit): purchase is in the rich biology of shellfish；
50mm296 orifice plates: Tissue Culture Flask is purchased from Corning Incorporated
3, main solution is prepared
DMEM cell culture fluid: DMEM powder one wraps, Sodium Pyruvate 0.11g, glutamine 0.58g, sodium bicarbonate 2.0g,
Tri-distilled water is added and is settled to 1000ml, 0.22 μm of filtration sterilization is placed in -20 DEG C after packing and saves backup.37 DEG C of water-baths before use
Melt, l0 is added afterwards5IU/L penicillin, 100mg/L streptomysin, (10% fetal calf serum is for cultivating mammary gland for 10% calf serum
Cancer cell).
Phosphate buffer (PBS): 8.0g NaCl, 0.2g KCl, 2.9g Na2HP0412H20,0.2g KH2P04；Dissolution
In appropriate tri-distilled water, pH value is adjusted to 7.2, is settled to 1L, filtering dispenses, and after high pressure sterilization, -20 DEG C are saved backup.
4, instrument and equipment
4 DEG C, -20 DEG C of low temperature refrigerators: SANYO GS company；
- 80 DEG C of ultra low temperature freezers of MDF-U40865 type: SANYO GS company；
SB25-12DTD type supersonic wave cleaning machine: Ningbo Xin Zhi company；
DHG-9243BS-III type electric heating constant-temperature blowing drying box: Shanghai new talent；
Tri- hole electric heating constant temperature sink of DK-8D: Hefei Cologne company；
3-16K type refrigerated centrifuge: Sigma company；
TGL high speed freezing centrifuge: Changsha Xiang Zhi centrifugal apparatus Co., Ltd；
2306-Z type CO2 incubator: Shellab company, the U.S.；
Vertical pressure steam sterilizer: Shenan Medical Appliances Factory, Shanghai
SW-CJ-IF type clean work station: Suzhou purifies Products；
Rios83olPE ultrapure water machine: Millipore Corp., U.S. product；
MULI SKAN MK3 microplate reader: Thermo forma Instrument Ltd.；
Pipettor: French PIPETMA company；
× D-202 inverted microscope: Hefei Cologne；
(2) experimental method
2.1 cell culture
This experiment lung cancer used, liver cancer, gastric cancer, cancer of pancreas, bladder cancer, colon cancer, cervical carcinoma, breast cancer, oophoroma,
43 kinds of cell strains such as leukaemia, prostate cancer, melanoma, neuroblastoma are the cell strain of adherent growth, liver cancer, stomach
Cancer, cancer of pancreas, bladder cancer, colon cancer, cervical carcinoma, breast cancer, leukaemia, lymph cancer, human osteosarcoma, gastrointestinal stromal tumor, black
The fetal calf serum DMEM culture solution of plain tumor, human neuroblastoma cells containing 10%, lung cancer and ovarian cancer cell are used and contain 10%
Fetal calf serum 1640 culture medium, and blueness is added in fetal calf serum Ham ' S F12K culture solution of the prostate gland cancer cell containing 10%
The cultivating system culture of 100 μ g/mL of mycin l0IU/mL and streptomysin.Culture environment is 37 DEG C, 5% CO2Incubator, every 2 days
It is primary to replace culture solution, had digestive transfer culture when cell covers with culture bottle, (cell category is different, and passage ratio is different), take in pair
The cell in number growth period is tested.
2.2 CCK-8 methods detect cell proliferative conditions
Cell Counting Kit-8, abbreviation CCK-8 kit or CCK8 kit are a kind of extensive based on WST-8
The kit of quick, highly sensitive detection applied to cell Proliferation and cytotoxicity.WST-8 is a kind of change similar to MTT
Object is closed, in the presence of electronics coupled reagent, can be generated by Intramitochondrial some dehydrogenase reduction orange-yellow
formazan.WST-8 be MTT a kind of upgrading substitute products and MTT or other MTT similar products such as × TT, MTS compare
There is clear advantage.Firstly, the formazan that MTT is generated by Intramitochondrial some dehydrogenases reduction is not water-soluble, need
There is specific lysate to dissolve；And WST-8 and × TT, MTS generate formazan be all it is water-soluble, after can saving
Continuous dissolving step.Secondly, the formazan that formazan ratio × TT and MTS that WST-8 is generated are generated is more soluble.Again,
WST-8 ratio × TT and MTS are more stable, keep experimental result more stable.In addition, WST-8 and MTT, × TT etc. compare linear model
Enclose wider, sensitivity is higher.The more cell Proliferation the faster, then color is deeper；Cytotoxicity is bigger, then color is more shallow.For same
The cell of sample, the depth and cell number of color are in a linear relationship.Its absorbance value is measured at 450nm wavelength with microplate reader.
According to the absorbance value A measured, to judge living cells quantity, A value is bigger, and cell activity is stronger.
Cell is with 5 × 105The concentration of a/ml is inoculated in 96 well culture plates, and every hole inoculum concentration is 100 microlitres.To cell
After adherent, it is divided into experimental port (containing cell, culture medium, CCK8 solution and drug solution), control wells (containing cell, culture medium, CCK8
Solution and non-drug containing), blank well (containing culture medium, CCK8 solution without drug solution and cell), every group sets 5 multiple holes groups,
Every hole total volume is 100 microlitres, and after 48h, every hole is added 10 microlitres of CCK-8 solution, is continued after incubator is incubated for 4h, in abandoning
In on enzyme-linked immunosorbent assay instrument, with wavelength 450nm, reagent controls group zeroing is surveyed absorbance (A) value, is repeated 3 times, and takes it average
Value.It calculates according to the following formula:
Cell survival rate: [(As-Ab)/(AC-Ab)] × 100%
Cell inhibitory rate: [(AC-As)/(AC-Ab)] × 100%
As experimental port absorbance (contains cell, culture medium, CCK8 solution and drug solution)；
Ab blank well (containing culture medium, CCK8 solution without drug solution and cell)；
AC control wells (contain cell, culture medium, CCK8 solution and non-drug containing)
(3) experimental result
By to lung cancer, liver cancer, gastric cancer, cancer of pancreas, bladder cancer, colon cancer, cervical carcinoma, breast cancer, oophoroma, white blood
43 kinds of cells such as disease, prostate cancer, melanoma, neuroblastoma carry out high flux screening as the result is shown:
1, Phellinus is to hamster normal cell CHL, CHO without obvious inhibiting effect, and display: Phellinus is to the nontoxic pair of normal cell
Effect；
2, Phellinus is to lung cancer cell line A549, H460, liver cancer cell lines SMMC-7721, Bel-7402, gastric carcinoma cell lines
HGC-27, Colon cancer cell line HT-29, cervical cancer tumer line SiHa, breast cancer cell line MDA-MB-435, ovarian cancer cell line
SK-OV-3, A2780, Leukemia Cell Lines MOLT-4, CCRF-CEM, prostate cancer cell line PC3, K-1735
A375, LOCIMV, neuroblastoma cell SH-SY5Y, inhibitory effect is more obvious, in drug test concentration in 80 μ g/mL
When, inhibiting rate is all larger than 80% (being shown in Table 1), and shows preferable selectivity.
3, Phellinus inhibits to make to remaining tumor cell line without obvious.
Inhibiting effect of 1 Phellinus of table to 41 tumour cells
Two, antitumor activity in Phellinus body
(1) therapeutic effect of the Phellinus to nude mice xenograft prostatic carcinoma
1, experimental material
1.1 experimental animals and cell strain
Balb/c Nude nude mice, male, weight 11-14g, 3-4 week old, purchase have in Jiangsu treasury medicine health biotechnology
Limit company raises animal experimental center SPF grades of thousand Anhui Province animal house, first adaptive feeding 7 days；Laboratory temperature control exists
20-25 DEG C, humidity is in 50%-60%, free water and feed.
Lung cancer PC-3 cell strain is bought in Wuhan Pu Nuosai Life Science Co., Ltd, with the fetal calf serum containing 10%
DMEM culture medium, in 37 DEG C, the CO that volume fraction is 5%2Routine culture in incubator under fully saturated damp condition, takes pair
Number growth period cell is tested.
1.2 experimental drug
5 FU 5 fluorouracil is provided by Tianjin KingYork Amino Acid Co., Ltd.；
DMEM cultivates powder, dimethyl sulfoxide (DMSO), and tetramethyl azo is tut-tuted blue (MTT), and dimethyl diaminophenazine chloride is U.S. sigma public affairs
Take charge of product；
Cell pyrolysis liquid: acetic acid is 1:1 configuration with dehydrated alcohol by volume；
Interleukin-11 (IL-1), the purchase of cell necrosis factor α (TNF-α) ELISA kit are in RD company；
NO kit buys thousand Nanjing and builds up Bioengineering Research Institute.
Remaining reagent is domestic AR rank.
The preparation of 1.3 passage tumor tissues
Lung cancer H526 cell, microscopically observation cell, cell arrangement is close, adherent growth, and cell is polygon, core point
It splits mutually common, has a cell overlap, cell circle is bright, full, and Cell tracking is close.Cell grows to 90% or more, collects after digestion
Cell, placenta orchid dye survival rate) 95%, adjusting cell concentration with PBS is 1 × 107/ml；0.2ml/ is only injected in the nude mice right side
Forelimb dorsal sc, after cell tumor formation, nude mice, lung cancer knurl interior generation 3 times are put to death in dislocation.After 3 generations, tumor tissue conduct
Knurl source carries out subcutaneous transplantation.
1.4 groupings and administration
When diameter of tumor is 0.4-0.5cm, 90 tumor formation nude mices are randomly divided into 6 groups, every group 15, are divided into model group
(without any processing, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group
(50mg/kg/d), administration group middle dose group (l00mg/kg/d), administration group high dose group (200mg/kg/d)；Successive administration 10
It.After being discontinued for 24 hours, cervical dislocation puts to death nude mice, takes tissue, Testing index on the super-clean bench.Every 2 days record tumours during administration
Long and short diameter and weight.
1.5 transplanting knurl weighings are surveyed dizzy with volume
Gastric infusion 10 days, second day eyeball took blood after last dose, and nude mice is put to death in dislocation, and 75% ethyl alcohol impregnates
Afterwards, right fore skin of back is cut off, knurl is removed, removes the nonneoplastic tissue on knurl surface, claims knurl weight；Every 2 days during administration
The major diameter (a) of dizzy knurl, minor axis (b) are surveyed with vernier caliper.
The calculating of 1.6 Relative tumors inhibition appreciation rate
The general status of close observation growth of transplanted human situation and nude mice after nude inoculation tumour cell was surveyed every 1 day
It measures nude mice weight and moves the major diameter (a) for being afraid of tumor, minor axis (b) and calculate gross tumor volume (V), Relative tumor body according to following formula
Product (RTV) and Relative tumor appreciation rate (T/C)；
V=a × b2/2；RTV=V/V0(V0For pre-neoplastic volume is administered, V is to put to death pre-neoplastic volume)；
T/C=treatment group RTV/ model group RTV × 100%；
The measurement of 1.7 tumor-bearing mice index and spleen index
After cervical dislocation puts to death nude mice, spleen, weighing are removed.Formula is index and spleen index=spleen weight (mg)/nude mouse
Weight (g)；
The measurement of 1.8 ConA induction splenic lymphocytes
Cervical dislocation put to death nude mice after, remove spleen, be placed in sterilized petri dishes cross 4 layer of 200 mesh nylon mesh screen and with inject
The grinding of device needle core is rinsed with the PBS of pre-cooling, is collected PBS in centrifuge tube, is centrifuged 2000rpm, 10min, goes upper liquid later,
Erythrocyte cracked liquid is added, is mixed even, static 5min, is centrifuged again, 2000rpm, 10min remove supernatant liquid.Trypan Blue
Cell count, living cells) 95%, adjusting splenocyte concentration with RPMI-1640 culture solution is 1 × 107/ml, is inoculated in 96 holes
The hole Na cell suspension is 10 μ 1 in plate, adds the ConA (5mg/ml) of 100 μ 1,5 multiple holes are arranged, cultivate in incubator
36h discards supernatant after culture, 1 serum-free medium of MTT (5mg/ml) and 180 μ of 20 μ 1 is added in every hole, continues
Cultivate 4h in incubator, be centrifuged (2000rpm, 5min), abandon supernatant, the DMSO in 1/ hole 150 μ is added, in microplate reader in
570nm measures absorbance (A) value.
The detection of 1.9 tumor-bearing mice peritoneal macrophages phagocytosis dimethyl diaminophenazine chloride ability
After cervical dislocation puts to death mouse, it is dipped in 1min in 75% ethanol solution, in operating in super-clean bench.Abdominal cavity
The PBS solution 5ml for injecting pre-cooling, gently rubs abdomen with hand, and intraperitoneal liquid is sucked out with 5ml syringe, in 4 DEG C from
The heart (12000rpm, 10min) discards supernatant, is cleaned twice with PBS, with the fetal calf serum RPMI-1640 culture containing 10%
Cell, cell count is resuspended in liquid, and adjusting cell concentration is 1 × 107A/ml is inoculated in 96 orifice plates, and every hole is 100 μ 1, separately
Add complete medium RPMI-1640 for empty mortar control group, be placed in cell incubator and cultivate 4h, remove non-attached cell to get
To required macrophage concentration.
0.075% 100 μ 1 of dimethyl diaminophenazine chloride normal saline solution is added in the every hole of the macrophage of 96 orifice plates, continues to cultivate
30min discards supernatant, is cleaned 3 times with PBS, and cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) 150 μ 1, room is added in every hole
Temperature stands 3h, after cell dissolution, measures absorbance (A) value at 570nm in microplate reader.
1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-1, TNF-α content in serum
After nude mice eyeball takes blood, it is placed at room temperature for 1h, is centrifuged 2000rpm, 10min, sucts clear liquid, serum is sub-packed in EP pipe
In, 50 μ 1/ pipe.- 80 DEG C of preservations are measured according to the measurement method of ELISA kit.
The content of NO in 1.11 nitrate reductase method side face nude mouse serums
After nude mice eyeball takes blood, it is placed at room temperature for 1h, 2000rpm, 10min is centrifuged, sucts clear liquid, serum is sub-packed in EP,
50 μ 1/ pipe.- 80 DEG C of preservations.With the content of NO kit detection serum NO, experimental procedure is in strict accordance with kit specification side
Method carries out.
The active detection of 1.12NK effector cell
(1) effector cell: normal sterile takes nude mice splenic lymphocytes, with the RPMI-1640 culture solution of 10% fetal calf serum
It is 1 × 10 that cell concentration, which is made,7The cell suspension of a/ml.
(2) preparation of target cell: taking the YAC-l cell of secondary culture 24-48h, with the RPMI-1640 of 10% fetal calf serum
After culture solution washes twice, it washed once with 0.5%BSA-RPMI-1640 culture solution, Trypan Blue confirmation cell survival rate >
95%, adjustment cell concentration is 3 × 105A/ml.
(3) determination of activity: in 96 orifice plates, effector cell, each 100 μ 1 of target cell is added in experimental port；Target cell control wells add
Enter each 100 μ 1 of target cell, culture solution；Effector cell, each 100 μ 1 of culture solution is added in effector cell's control wells, and 3 multiple holes are arranged.
It is placed in incubator after cultivating 4h, the MTT culture solution of 10 μ 1 is added in experimental port, is continuing to cultivate 4h.After culture, discard
The DMSO of 150 μ 1 is respectively added in clear liquid, experimental port, and it is thousands of to set microplate reader by 20 μ 1 of 0.05mol (pH10.5) glycine buffer
OD value is measured at 570nm.
NK cell activity calculation formula is as follows:
NK cell activity (%)=[1- (experimental group OD value-effector cell organizes OD value)/target cell group OD value] × 100%；
1.13 observing survival time of mice
Nude mice for observing life cycle continues to give water and diet, puts to death when each group nude mice failure or natural
Death records the death time, calculates mean survival time (MST) and increase in life span, calculation formula are as follows:
Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group is averagely deposited
Live time × 100%
1.14 data processing
Experimental data is indicated according to mean standard deviation (χ soil S), carries out statistical data processing using SPSS17.0 software,
Group compares to be examined with One-wayANOVA, and P < 0.05 thinks that difference is statistically significant.
2, experimental result
The success of 2.1 transplantable lung cancer model foundations
Tumor tissue suspension is injected into armpit after a week, i.e., the kick of visible subcutaneous mung bean size, visible knurl after several weeks
Product is increasing.
The changes of weight of 2.2 tumor bearing nude mices and the inhibiting rate of transplantable tumor
In the entire experiment process without nude mice death, each group nude mice can be movable, and drinking-water feed is normal, and weight has increasing
Add, it is not statistically significant between the weight of each group (P > 0.05).The knurl weight of administration group significantly reduced compared with model group (P <
0.01, P < 0.05), Phellinus 200mg/kg group knurl compared with 5-Fu group is swooned again reduces (P < 0.01), experimental result such as 1 institute of table
Show.
Influence (χ soil S, n=6) of 1 Phellinus of table to the inhibiting rate of the transplantable tumor of tumor bearing nude mice
* * P < 0.001, * * P < 0.01, * P < 0.05vs. model group；#P < 0.05, vs.5-Fu group
Influence of 2.3 Phellinus to transplanted tumor in nude mice volume change
During the experiment, the drinking-water of each group nude mice and feed are normal, and no nude mice is dead.Nude mice passes through internal notch graft
After kind tumor tissue suspension after 10 days, subcutaneous visible tubercle, diameter of tumor is about 0.3-0.5cm, tumor formation rate 100% after 15 days.It is naked
The subcutaneous transplantable lung cancer of mouse is nodositas, clear border.Before being administered the volume change of each group transplantable tumor do not have otherness (P >
0.05), during administration, the tumour growth of each dosage group of Phellinus and positive drug group is opposite to be slowed down, and model group is then on the contrary, move
It plants tumor to grow comparatively fast, ulceration phenomenon occurs in the knurl of individual nude mices.Each dosage group of Phellinus and positive drug group after administration
RTV have apparent difference (P < 0.05, P < 0.01) compared with model group.Each dosage group of Phellinus can significantly reduce transplantable tumor
Volume, and there is dose dependent, Relative tumor appreciation rate is gradually reduced, and the results are shown in Table 2:
Influence (χ soil S, n=6) of 2 Phellinus of table to transplanted tumor in nude mice volume change
* P < 0.01, * P < 0.05vs. model group
Influence of 2.4 Phellinus to tumor bearing nude mice index and spleen index
Drug and 5-Fu class index spleen are not statistically significant compared with the index and spleen index of model group, drug (100,
The index and spleen index for 200mg/kg) organizing tumor bearing nude mice increases compared with model group, and statistically significant (P < 0.05, P <
0.01).Experimental result is as shown in table 3.
Influence (χ scholar S, n=6) of 3 Phellinus of table to the small nude mice index and spleen index of lotus knurl
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.5 Phellinus to tumor bearing nude mice peritoneal macrophage phagocytosis dimethyl diaminophenazine chloride ability
Phellinus (100,200mg/kg) organizes the phagocytic activity of the peritoneal macrophage of tumor bearing nude mice and the phagocytosis energy of model group
Power compares, and phagocytic activity is remarkably reinforced and statistically significant (P < 0.05, P < 0.01), remaining organizes other phagocytic activity with mould
The phagocytic activity of type group is compared to more not statistically significant.Shown in experimental result table 4.
Influence (χ soil S, n=6) of 4. Phellinus of table to tumor bearing nude mice macrophage phagocytosis dimethyl diaminophenazine chloride ability
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.6 Phellinus to tumor bearing nude mice serum NO content
The level of the tumor bearing nude mice serum NO of human lung cancer H526 cell strain is significantly increased than normal nude mice control group, Phellinus
The NO content in tumor bearing nude mice serum can be significantly reduced, and have certain dosage according to lazy sexual intercourse.Experimental result such as Fig. 1 institute
Show.
Influence of 2.7 Phellinus to human lung cancer A594 cell strain tumor bearing nude mice NK cell killing activity
After tumor bearing nude mice successive administration 10 days, Phellinus group, positive drug group tumor bearing nude mice NK cell killing activity and mould
Type group is relatively significantly increased (P < 0.05, P < 0.01), and experimental result is as shown in Figure 2.
Influence of 2.8 Phellinus to the ConA tumor bearing nude mice spleen leaching the sixth of the twelve Earthly Branches cell proliferative response induced
Compared with normal group, ConA induction model group spleen leaching the sixth of the twelve Earthly Branches cell proliferative response significantly lower than normal group (P <
0.01), after successive administration 10 days, Phellinus group and 5-Fu group are more statistically significant with model group, (P < 0.01, P < 0.05) mulberry
Tumor bearing nude mice spleen leaching the sixth of the twelve Earthly Branches cell proliferative response enhancing that yellow high dose group can be such that ConA induces, restores to normal level.Experiment
As a result as shown in Figure 3.
Influence of 2.9 Phellinus to IL-1, TNF-α content in tumor bearing nude mice serum
After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of Phellinus can make point of IL-1 and TNF-α in nude mouse serum
Bleeding is flat to be improved, and compared with model group and statistically significant (P < 0.05, P < 0.01), it is as shown in the table for experimental result, according to table
5, data know that the expression of nude mice immune factor can be improved in Phellinus in table 6, are improved effect to the immunity of nude mice body.
Influence (χ soil S, n=6) of 5. Phellinus of table to IL-1 is secreted in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence (χ soil S, n=6) of 6. Phellinus of table to TNF secretion-α in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.10 Phellinus to tumor bearing nude mice life cycle
Nude mice is raised to its natural death in SPF grades of animal houses, records the death time.Positive drug group and Phellinus high dose group
The life cycle of tumor bearing nude mice can be extended, and statistically significant compared with model group (P < 0.05, P < 0.01).Record result
As shown in table 7.
Influence (χ soil S, n=7) of the 7. Phellinus A of table to tumor bearing nude mice life cycle
* P < 0.05, * * P < 0.01vs. model group
(2) therapeutic effect of the Phellinus to nude mice mortar blood disease transplantable tumor
1, experimental material
1.1 experimental animals and cell strain
NOD/SCID mouse, female, 6-7 week old, weight 18-22g, thousand Beijing China Fukang biotechnology shares of purchase are limited
Company raises in animal experimental center SPF grades of Anhui Province animal house, first adaptive feeding 7 days；Laboratory temperature is controlled in 20-
25 DEG C, humidity is in 50%-60%, free water and feed.
Acute progranulocyte mortar blood disease cell line NB4 cell strain buys thousand Chinese Academy of Sciences's Shanghai cell banks, with containing 10%
Fetal calf serum DMEM culture medium, 37 DEG C, volume fraction be 5% CO2, routine culture under fully saturated damp condition,
Logarithmic growth phase cell is tested.
1.2 experimental drug
5 FU 5 fluorouracil is provided by Tianjin KingYork Amino Acid Co., Ltd.；
DMEM cultivates powder, and dimethyl sulfoxide (DMSO), tetramethyl azo is bright blue (MTT), and dimethyl diaminophenazine chloride is U.S. sigma public affairs
Take charge of product；
Cell pyrolysis liquid: acetic acid is 1:1 configuration with dehydrated alcohol by volume；
Interleukin-11 (IL-1), the purchase of cell necrosis factor α (TNF-α) ELISA kit are in RD company；
NO kit buys thousand Nanjing and builds up Bioengineering Research Institute.
Remaining reagent is domestic AR rank.
The preparation of 1.3 passage tumor tissues
Acute promyelocytic leukemia cell strain NB4 cell, microscopically observation is round, and volume is larger, core/slurry ratio
Greatly, slurry is less in light blue；Core is round, and kernel understands that chromatin is uneven, grows in irregular coarse grain or agglomeration shape, cell
90% or more, cell is collected after digestion, placenta orchid dyes survival rate > 95%, collects logarithmic growth phase cell, disposable with abdominal cavity
Injection 3 × 106A/only, and after cell tumor formation, dislocation execution nude mice, knurl interior generation 3 times.After 3 generations, tumor tissue conduct
Knurl source carries out subcutaneous transplantation.
1.4 groupings and administration
When diameter of tumor is 0.3-0.5cm, 90 tumor formation nude mices are randomly divided into 6 groups, every group 15, are divided into model group
(without any processing, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group
(50mg/kg/d), administration group middle dose group (100mg/kg/d), administration group high dose group (200mg/kg/d)；Successive administration 10
It.After being discontinued for 24 hours, cervical dislocation puts to death nude mice, takes tissue, Testing index on the super-clean bench.Every 2 days record tumours during administration
Long and short diameter and weight.
1.5 transplanting knurl weighings and cubing
Gastric infusion 10 days, second day eyeball took blood after last dose, and nude mice is put to death in dislocation, and 75% ethyl alcohol impregnates
Afterwards, skin at left lower extremity is cut off, knurl is removed, removes the nonneoplastic tissue on knurl surface, claims knurl weight；Every 2 days use during administration
Major diameter (a), the minor axis (b) of vernier caliper measurement knurl.
The calculating of 1.6 Relative tumors inhibition appreciation rate
The general status of close observation growth of transplanted human situation and nude mice after nude inoculation tumour cell was surveyed every 1 day
The major diameter (a) of amount nude mice weight and transplantable tumor, minor axis (b) ' according to following formula calculating gross tumor volume (V), Relative tumor
Volume (RTV) and Relative tumor appreciation rate (T/C):
V=a × b2/2；RTV=V/V0(V0For pre-neoplastic volume is administered, V is to put to death pre-neoplastic volume)；
T/C=treatment group RTV/ model group RTV × 100%；
The measurement of 1.7 tumor-bearing mice index and spleen index
After cervical dislocation puts to death nude mice, spleen, weighing are removed.Formula is index and spleen index=spleen weight (mg)/nude mouse
Weight (g)；
1.8ConA induces the measurement of splenic lymphocytes increment reaction
Cervical dislocation put to death nude mice after, remove spleen, be placed in sterilized petri dishes cross 4 layer of 200 mesh nylon mesh screen and with inject
The grinding of device needle core is rinsed with the PBS of pre-cooling, is collected PBS in centrifuge tube, is centrifuged 2000rpm, 10min, goes upper liquid later,
Erythrocyte cracked liquid is added, is mixed even, static 5min, is centrifuged again, 2000rpm, 10min remove supernatant liquid.Trypan Blue
Cell count, living cells > 95%, adjusting splenocyte concentration with RPMI-1640 culture solution is 1 × 107A/ml is inoculated in 96 holes
Every hole cell suspension is 10 μ 1 in plate, adds the ConA (5mg/ml) of 100 μ 1,5 multiple holes are arranged, cultivate in incubator
36h discards supernatant after culture, the MTT (5mg/ml) and 180 μ l serum-free mediums of 20 μ l is added in every hole, continues
Cultivate 4h in incubator, be centrifuged (2000rpm, 5min), abandon supernatant, the DMSO in 150 holes μ l/ is added, in microplate reader in
570nm measures absorbance (A) value.
The detection of 1.9 tumor-bearing mice peritoneal macrophages phagocytosis dimethyl diaminophenazine chloride ability
After cervical dislocation puts to death mouse, it is dipped in 1min in 75% ethanol solution, in operating in super-clean bench.Abdominal cavity
The PBS solution 5ml for injecting pre-cooling, gently rubs abdomen with hand, and intraperitoneal liquid is sucked out with 5ml syringe, in 4 DEG C from
The heart (12000rpm, 10min) discards supernatant, is cleaned twice with PSB, with the fetal calf serum RPIM-1640 culture containing 10%
Cell, cell count is resuspended in liquid, and adjusting cell concentration is 1 × 107A/ml is inoculated in 96 orifice plates, and every hole is 100 μ l, separately
Adding complete medium RPMI-1640 is blank control group, is placed in cell incubator and cultivates 4h, remove non-attached cell to get
To required macrophage concentration.
The 100 μ l of dimethyl diaminophenazine chloride normal saline solution that the every hole of the macrophage of 96 orifice plates is added 0.075% continues to cultivate
30min discards supernatant, is cleaned 3 times with PBS, and cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) is added in every hole
150 μ l, are stored at room temperature 3h, after cell dissolution, measure absorbance (A) value at 570nm in microplate reader.
1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-1, TNF-α content in serum
After nude mice eyeball takes blood, it is placed at room temperature for 1h, is centrifuged 2000rpm, 10min, sucts clear liquid, serum is sub-packed in EP pipe
In, 50 μ 1/ pipe.- 80 DEG C of preservations are measured according to the measurement method of ELISA kit.
1.11 nitrate reductase methods measure the content of NO in nude mouse serum
After nude mice eyeball takes blood, it is placed at room temperature for 1h, is centrifuged 2000rpm, 10min, sucts clear liquid, serum is sub-packed in EP pipe
In, 50 μ l/ pipe.- 80 DEG C of preservations.With the content of NO kit detection serum NO, experimental procedure illustrates in strict accordance with kit
Book method carries out.
The active detection of 1.12NK effector cell
(1) effector cell: normal sterile takes nude mice splenic lymphocytes, with the RPMI-1640 culture solution of 10% fetal calf serum
It is 1 × 10 that cell concentration, which is made,7The cell suspension of a/ml.
(2) preparation of target cell: taking the YAC-1 cell of secondary culture 24-48h, with the RPMI-1640 of 10% fetal calf serum
After culture solution washes twice, it washed once with 0.5%BSA-RPMI-1640 culture solution, Trypan Blue confirmation cell survival rate >
95%, adjustment cell concentration is 3 × 105A/ml.
(3) determination of activity: in 96 orifice plates, effector cell, each 100 μ l of target cell is added in experimental port；Target cell control wells add
Enter each 100 μ 1 of target cell, culture solution；Effector cell, each 100 μ 1 of culture solution is added in effector cell's control wells, and 3 multiple holes are arranged.
It is placed in incubator after cultivating 4h, the MTT culture solution of 10 μ 1 is added in experimental port, is continuing to cultivate 4h.After culture, discard
The DMSO of 150 μ 1,20 μ l of 0.05mol (pH10.5) glycine buffer is respectively added in clear liquid, experimental port, set in microplate reader in
OD value is measured at 570nm.
NK cell activity calculation formula is as follows:
NK cell activity (%)=[1- (experimental group OD value-effector cell organizes OD value)/target cell group OD value] × 100%；
1.13 observing survival time of mice
Nude mice for observing life cycle continues to give water and diet, puts to death when each group nude mice failure or natural
Death records the death time, calculates mean survival time (MST) and increase in life span, calculation formula are as follows:
Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group is averagely deposited
Live time × 100%
1.14 data processing
Experimental data is indicated according to mean standard deviation (χ soil S), carries out statistical data processing using SPSS17.0 software,
Group compares to be examined with One-wayANOVA, and P < 0.05 thinks that difference is statistically significant.
2, experimental result
2.1 leukaemia Transplanted tumor models are successfully established
Tumor tissue suspension is injected into abdominal cavity after a week, i.e., the kick of visible subcutaneous mung bean size, visible knurl after several weeks
Product is increasing.
The changes of weight of 2.2 tumor bearing nude mices and the inhibiting rate of transplantable tumor
In the entire experiment process without nude mice death, each group nude mice can be movable, and drinking-water feed is normal, and weight has increasing
Add, it is not statistically significant between the weight of each group (P > 0.05).The knurl weight of administration group significantly reduced compared with model group (P <
0.01), Phellinus 200mg/kg group tumor weight compared with 5-Fu group reduces (P < 0.05, P < 0.01), and the results are shown in Table 1.
Influence (χ soil S, n=6) of 1 Phellinus of table to tumor bearing nude mice inhibiting rate
* P < 0.01, * P < 0.05vs. model group；#P < 0.05, ##P < 0.01vs5-Fu group
Influence of 2.3 Phellinus to transplanted tumor in nude mice volume change
During the experiment, the drinking-water of each group nude mice and feed are normal, and no nude mice is dead.Nude mice passes through internal notch graft
10 days after kind tumor tissue suspension, subcutaneous visible tubercle, diameter of tumor is about 0.4-0.6cm, tumor formation rate 100% after 15 days.Nude mice
Subcutaneous transplanted human hepatocellular carcinoma is nodositas, clear border.The volume change of each group transplantable tumor does not have otherness (P > 0.05) before being administered,
During administration, the tumour growth of each dosage group of Phellinus and positive drug group is opposite to be slowed down, and model group is then on the contrary, transplantable tumor is equal
Growth is very fast, and ulceration phenomenon occurs in the knurl of individual nude mices.After administration the RTV of each dosage group of Phellinus and positive drug group with
Model group more has apparent difference (P < 0.05, P < 0.01).Each dosage group of Phellinus can significantly reduce the volume of transplantable tumor,
And there is dose dependent, Relative tumor appreciation rate is gradually reduced, and the results are shown in Table 2:
Influence (χ soil S, n=6) of 2 Phellinus of table to transplanted tumor in nude mice volume change
* P < 0.01, * P < 0.05vs. model group
Influence of 2.4 Phellinus to tumor bearing nude mice index and spleen index
Phellinus (50mg/kg) group and 5-Fu class index spleen are not statistically significant compared with the index and spleen index of model group, mulberry
The index and spleen index of yellow (100,200mg/kg) group tumor bearing nude mice increases compared with model group, and statistically significant (P < 0.05,
P<0.01).Experimental result is as shown in table 3.
Influence (χ soil S, n=6) of 3 Phellinus of table to the small nude mice index and spleen index of lotus knurl
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.5 Phellinus to tumor bearing nude mice peritoneal macrophage phagocytosis dimethyl diaminophenazine chloride ability
Phellinus (100,200mg/kg) organizes the phagocytic activity of the peritoneal macrophage of tumor bearing nude mice and the phagocytosis energy of model group
Power compares, and phagocytic activity is remarkably reinforced and statistically significant (P < 0.05, P < 0.01), remaining organizes other phagocytic activity with mould
The phagocytic activity of type group is compared to more not statistically significant.Shown in experimental result table 4.
Influence of 4 Phellinus of table to tumor bearing nude mice peritoneal macrophage phagocytosis dimethyl diaminophenazine chloride ability
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.6 Phellinus to tumor bearing nude mice serum NO content
The level of the tumor bearing nude mice serum NO of acute promyelocytic leukemia NB4 cell strain is than normal nude mice control group
Significant to increase, the NO content in tumor bearing nude mice serum can be significantly reduced in Phellinus, and has certain dosage according to lazy sexual intercourse.Experiment
As a result as shown in Figure 4.
Influence lotus of 2.7 Phellinus to acute promyelocytic leukemia NB4 cell strain tumor bearing nude mice NK cell killing activity
Tumor nude mice successive administration is after 10 days, the NK cell killing activity of medicine group tumor bearing nude mice be significantly increased compared with model group (P <
0.05, P < 0.01), experimental result is as shown in Figure 5.
Influence of 2.8 Phellinus to the ConA tumor bearing nude mice splenic lymphocytes induced
Compared with normal group, ConA induction model group splenic lymphocytes significantly lower than normal group (P <
0.01), after successive administration 10 days, medicine group and 5-Fu group are more statistically significant (P < 0.01, P < 0.05) with model group, medicine
The tumor bearing nude mice splenic lymphocytes enhancing that object high dose group can be such that ConA induces, restores to normal level.Experiment
As a result as shown in Figure 6.
Influence of 2.9 Phellinus to IL-1, TNF-α content in tumor bearing nude mice serum
After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of Phellinus can make point of IL-1 and TNF-α in nude mouse serum
Bleeding is flat to be improved, and compared with model group and statistically significant (P < 0.05, P < 0.01), it is as shown in the table for experimental result, according to table
5, data know that the expression of nude mice immune factor can be improved in Phellinus in table 6, are improved effect to the immunity of nude mice body.
Influence (χ soil S, n=6) of 5 Phellinus of table to IL-l is secreted in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence (χ soil S, n=6) of 6 Phellinus of table to TNF secretion-α in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.10 Phellinus to tumor bearing nude mice life cycle
Nude mice is raised to its natural death in SPF grades of animal houses, records the death time.Positive drug group and drug high dose group
The life cycle of tumor bearing nude mice can be extended, and statistically significant compared with model group (P < 0.05, P < 0.01).Record result
As shown in table 7.
Influence (χ soil S, n=7) of 7 Phellinus of table to tumor bearing nude mice life cycle
* P < 0.05, * * P0.01vs. model group
(3) therapeutic effect of the Phellinus to nude mice leaching the sixth of the twelve Earthly Branches cancer transplantable tumor
1, experimental material
1.1 experimental animal and cell strain
BALB/C (nu/nu) nude mice, male, weight 15-20g, 5-6 week old are bought in Beijing China Fukang biotechnology stock
Part Co., Ltd, raises in animal experimental center SPF grades of Anhui Province animal house, first adaptive feeding 7 days；Laboratory temperature control
At 20-25 DEG C, humidity is in 50%-60%, free water and feed.
Mouse lymph lymphoma TMD8 cell strain is bought in Chinese Academy of Sciences's Shanghai cell bank, with the DMEM of the fetal calf serum containing 10%
Culture medium, in 37 DEG C, the CO that volume fraction is 5%2, routine culture under fully saturated damp condition, logarithmic growth phase cell
It is tested.
1.2 experimental drug
5 FU 5 fluorouracil is provided by Tianjin KingYork Amino Acid Co., Ltd.；
DMEM cultivates powder, and dimethyl sulfoxide (DMSO), tetramethyl azo is bright blue (MTT), and dimethyl diaminophenazine chloride is U.S. sigma public affairs
Take charge of product；
Cell pyrolysis liquid: acetic acid is 1:1 configuration with dehydrated alcohol by volume；
Interleukin-11 (IL-1), the purchase of cell necrosis factor α (TNF-α) ELISA kit are in RD company；
NO kit buys thousand Nanjing and builds up Bioengineering Research Institute.
Remaining reagent is domestic AR rank.
The preparation of 1.3 passage tumor tissues
Mouse lymph lymphoma EL4 cell, microscopically observation cell is round, some visible cell cores, cell grow to 90% with
On, cell is collected after digestion, placenta orchid dyes survival rate > 95%, and adjusting cell concentration with PBS is 1 × 107/ml；0.2ml/ is only
It is injected in oxter blank space on the right side of nude mice, after cell tumor formation, dislocation execution nude mice, knurl interior generation 3 times.After 3 generations, tumor
Tissue carries out subcutaneous transplantation as knurl source.
1.4 groupings and administration
When diameter of tumor is 0.4-0.5cm, 90 tumor formation nude mices are randomly divided into 6 groups, every group 15, are divided into model group
(without any processing, allow its ad lib), positive drug group (fluorouracil, 5-Fu, 20mg/kg/d), administration group low dose group
(50mg/kg/d), administration group middle dose group (1OOmg/kg/d), administration group high dose group (200mg/kg/d)；Successive administration 10
It.After being discontinued for 24 hours, cervical dislocation puts to death nude mice, takes tissue, Testing index on the super-clean bench.Every 2 days record tumours during administration
Long and short diameter and weight.
1.5 transplanting knurl weighings and cubing
Gastric infusion 10 days, second day eyeball took blood after last dose, and nude mice is put to death in dislocation, and 75% ethyl alcohol impregnates
Afterwards, skin at armpit is cut off, knurl is removed, removes the nonneoplastic tissue on knurl surface, claims knurl weight；During administration every 2 days with trip
Mark major diameter (a), the minor axis (b) of calliper to measure knurl.
The calculating of 1.6 Relative tumors inhibition appreciation rate
The general status of close observation growth of transplanted human situation and nude mice after nude inoculation tumour cell was surveyed every 1 day
The major diameter (a) of amount nude mice weight and transplantable tumor, minor axis (b) ' according to following formula calculating gross tumor volume (V), Relative tumor
Volume (RTV) and Relative tumor appreciation rate (T/C):
V=a × b2/2；RTV=V/V.(V0For pre-neoplastic volume is administered, V is to put to death pre-neoplastic volume)；
T/C=treatment group RTV/ model group RTV × 100%；
The measurement of 1.7 tumor-bearing mice index and spleen index
After cervical dislocation puts to death nude mice, spleen, weighing are removed.Formula is index and spleen index=spleen weight (mg)/nude mouse
Weight (g)；
1.8ConA induces the measurement of splenic lymphocytes increment reaction
Cervical dislocation put to death nude mice after, remove spleen, be placed in sterilized petri dishes cross 4 layer of 200 mesh nylon mesh screen and with inject
The grinding of device needle core is rinsed with the PBS of pre-cooling, is collected in thousand centrifuge tube of PBS, is centrifuged 2000rpm, 10min, go upper liquid later,
Erythrocyte cracked liquid is added, is mixed even, static 5min, is centrifuged again, 2000rpm, 10min remove supernatant liquid.Trypan blue dye
Color, cell count, living cells > 95%, adjusting splenocyte concentration with RPMI-1640 culture solution is 1 × 107A/ml, is inoculated in 96
Every hole cell suspension is 10 μ 1 in orifice plate, adds the ConA (5mg/ml) of 100 μ 1,5 multiple holes are arranged, cultivate in incubator
36h discards supernatant after culture, 1 serum-free medium of MTT (5mg/ml) and 180 μ of 20 μ 1 is added in every hole, continues
4h is cultivated in incubator, is centrifuged (2000rpm, 5min), supernatant is abandoned, and the DMSO in 1/ hole 150 μ is added, it is thousands of in microplate reader
570nm measures absorbance (A) value.
The detection of 1.9 tumor-bearing mice peritoneal macrophages phagocytosis dimethyl diaminophenazine chloride ability
After cervical dislocation puts to death mouse, it is dipped in 1min in 75% ethanol solution, in operating in super-clean bench.Intraperitoneal injection is pre-
Cold PBS solution 5ml, gently rubs abdomen with hand, and intraperitoneal liquid is sucked out with 5ml syringe, be centrifuged in 4 DEG C (12000rpm,
Supernatant 10min) is discarded, is cleaned twice with PBS, cell is resuspended with the fetal calf serum RPMI-1640 culture solution containing 10%, carefully
Born of the same parents count, and adjusting cell concentration is 1 × 107A/ml is inoculated in 1,000 orifice plates, and every hole is 100 μ 1, separately plus complete medium
RPMI-1640 is blank control group, is placed in cell incubator and cultivates 4h, and it is required huge to get arriving to remove non-attached cell
Phagocyte concentration.
0.075% 100 μ 1 of dimethyl diaminophenazine chloride normal saline solution is added in the every hole of the macrophage of 96 orifice plates, continues to cultivate
30min discards supernatant, is cleaned 3 times with PBS, and cell pyrolysis liquid (acetic acid: dehydrated alcohol=1:1) 150 μ 1, room is added in every hole
Temperature stands 3h, after cell dissolution, measures absorbance (A) value at 570nm in microplate reader.
1.10 enzyme linked immunosorbent assays (ELISA method) detect IL-l, TNF-α content in serum
After nude mice eyeball takes blood, it is placed at room temperature for 1h, is centrifuged 2000rpm, 10min, sucts clear liquid, serum is sub-packed in EP pipe
In, 50 μ 1/ pipe.- 80 DEG C of preservations are surveyed dizzy according to the measurement method of ELISA kit.
The content of NO in 1.11 nitrate reductase method side face nude mouse serums
After nude mice eyeball takes blood, it is placed at room temperature for 1h, is centrifuged 2000rpm, 10min, sucts clear liquid, serum is sub-packed in EP pipe
In, 50 μ 1/ pipe.- 80 DEG C of preservations.With the content of NO kit detection serum NO, experimental procedure illustrates in strict accordance with kit
Book method carries out.
The active detection of 1.12NK effector cell
(1) effector cell: normal sterile takes nude mice splenic lymphocytes, with the RPMI-1640 culture solution of 10% fetal calf serum
It is 1 × 10 that cell concentration, which is made,7The cell suspension of a/ml.
(2) preparation of target cell: taking the YAC-1 cell of secondary culture 24-48h, with the RPMI-1640 of 10% fetal calf serum
After culture solution washes twice, it washed once with 0.5%BSA-RPMI-1640 culture solution, Trypan Blue confirmation cell survival rate >
95%, adjustment cell concentration is 3 × 105A/ml.
(3) determination of activity: in 96 orifice plates, effector cell, each 100 μ 1 of target cell is added in experimental port；Target cell control wells add
Enter each 100 μ 1 of target cell, culture solution；Effector cell, each 100 μ 1 of culture solution is added in effector cell's control wells, and 3 multiple holes are arranged.
It is placed in incubator after cultivating 4h, the MTT culture solution of 10 μ 1 is added in experimental port, is continuing to cultivate 4h.After culture, discard
The DMSO of 150 μ 1,20 μ 1 of 0.05mol (pH10.5) glycine buffer is respectively added in clear liquid, experimental port, set in microplate reader in
OD value is measured at 570nm.
NK cell activity calculation formula is as follows:
NK cell activity (%)=[1- (experimental group OD value-effector cell organizes OD value)/target cell group OD value] × 100%；
1.13 observing survival time of mice
Nude mice for observing life cycle continues to give water and diet, puts to death when each group nude mice failure or natural
Death records the death time, calculates mean survival time (MST) and increase in life span, calculation formula are as follows:
Increase in life span (%)=(medication group mean survival time-model group mean survival time)/model group is averagely deposited
Live time × 100%
1.14 data processing
Experimental data is indicated according to mean standard deviation (χ soil S), carries out statistical data processing using SPSS17.0 software,
Group compares to be examined with One-wayANOVA, and P < 0.05 thinks that difference is statistically significant.
2, experimental result
2.1 lymph cancer Transplanted tumor models are successfully established
Tumor tissue suspension is injected into armpit after a week, i.e., the kick of visible subcutaneous mung bean size, visible knurl after several weeks
Product is increasing.
The changes of weight of 2.2 tumor bearing nude mices and the inhibiting rate of transplantable tumor
In the entire experiment process without nude mice death, each group nude mice can be movable, and drinking-water feed is normal, and weight has increasing
Add, it is not statistically significant between the weight of each group (P > 0.05).The knurl weight of administration group significantly reduced compared with model group (P <
0.01), Phellinus 100mg/kg, 200mg/kg group tumor weight compared with 5-Fu group reduces (P < 0.05, P < 0.01), as a result such as
Shown in table 1.
Influence (χ soil S, n=6) of 1 Phellinus of table to tumor bearing nude mice inhibiting rate
* P < 0.01, * P < 0.05vs. model group；#P < 0.05, ##P < 0.01vs5-Fu group
Influence of 2.3 Phellinus to transplanted tumor in nude mice volume change
During the experiment, the drinking-water of each group nude mice and feed are normal, and no nude mice is dead.Nude mice passes through internal notch graft
In kind tumor tissue suspension latter week, subcutaneous visible tubercle, diameter of tumor is about 0.3-0.5cm, tumor formation rate 100% after 15 days.Nude mice
Subcutaneous transplanted human hepatocellular carcinoma is nodositas, clear border.The volume change of each group transplantable tumor does not have otherness (P > 0.05) before being administered,
During administration, the tumour growth of each dosage group of Phellinus and positive drug group is opposite to be slowed down, and model group then on the contrary,
Transplantable tumor is grown comparatively fast, and ulceration phenomenon occurs in the knurl of individual nude mices.Each dosage group of Phellinus and positive drug after administration
The RTV of group has apparent difference (P < 0.05, P < 0.01) compared with model group.Each dosage group of Phellinus can significantly reduce transplanting
The volume of tumor, and there is dose dependent, Relative tumor appreciation rate is gradually reduced, and the results are shown in Table 2:
Influence (χ soil S, n=6) of 2 Phellinus of table to transplanted tumor in nude mice volume change
* P < 0.01, * P < 0.05vs. model group
Influence of 2.4 Phellinus to tumor bearing nude mice index and spleen index
Phellinus (50mg/kg) group and 5-Fu class index spleen are not statistically significant compared with the index and spleen index of model group, mulberry
The index and spleen index of yellow (100,200mg/kg) group tumor bearing nude mice increases compared with model group, and statistically significant (P < 0.05,
P<0.01).Experimental result is as shown in table 3.
Influence (χ soil S, n=6) of 3 Phellinus of table to the small nude mice index and spleen index of lotus knurl
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.5 Phellinus to tumor bearing nude mice peritoneal macrophage phagocytosis dimethyl diaminophenazine chloride ability
Phellinus (100,200mg/kg) organizes the phagocytic activity of the peritoneal macrophage of tumor bearing nude mice and the phagocytosis energy of model group
Power compares, and phagocytic activity is remarkably reinforced and statistically significant (P < 0.05, P < 0.01), remaining organizes other phagocytic activity with mould
The phagocytic activity of type group is compared to more not statistically significant.Shown in experimental result table 4.
Influence of 4 Phellinus of table to tumor bearing nude mice peritoneal macrophage phagocytosis dimethyl diaminophenazine chloride ability
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.6 Phellinus to tumor bearing nude mice serum NO content
The level of the tumor bearing nude mice serum NO of mouse lymph lymphoma EL4 cell strain is significantly increased than normal nude mice control group,
The NO content in tumor bearing nude mice serum can be significantly reduced in Phellinus, and has certain dosage according to lazy sexual intercourse.Experimental result such as Fig. 7
It is shown.
Influence of 2.7 Phellinus to mouse lymph lymphoma EL4 cell strain tumor bearing nude mice NK cell killing activity
After tumor bearing nude mice successive administration 10 days, the NK cell killing activity of medicine group tumor bearing nude mice has aobvious compared with model group
It writes and improves (P < 0.05, P < 0.01), experimental results are shown in figure 8.
Influence of 2.8 Phellinus to the ConA tumor bearing nude mice splenic lymphocytes induced
Compared with normal group, ConA induction model group splenic lymphocytes significantly lower than normal group (P <
0.01), after successive administration 10 days, medicine group and 5-Fu group are more statistically significant (P < 0.01, P < 0.05) with model group, medicine
The tumor bearing nude mice splenic lymphocytes enhancing that object high dose group can be such that ConA induces, restores to normal level.Experiment
As a result as shown in Figure 9.
Influence of 2.9 Phellinus to IL-1, TNF-α content in tumor bearing nude mice serum
After tumor bearing nude mice successive administration 10 days, the middle and high dosage group of Phellinus can make point of IL-1 and TNF-α in nude mouse serum
Bleeding is flat to be improved, and compared with model group and statistically significant (P < 0.05, P < 0.01), it is as shown in the table for experimental result, according to table
5, data know that the expression of nude mice immune factor can be improved in Phellinus in table 6, are improved effect to the immunity of nude mice body.
Influence (χ soil S, n=6) of 5 Phellinus of table to IL-1 is secreted in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence (χ soil S, n=6) of 6 Phellinus of table to TNF secretion-α in tumor bearing nude mice serum
* P < 0.05, * * P < 0.01vs. model group
Influence of 2.10 Phellinus to tumor bearing nude mice life cycle
Nude mice is raised to its natural death in SPF grades of animal houses, records the death time.Positive drug group and drug high dose group
The life cycle of tumor bearing nude mice can be extended, and statistically significant compared with model group (P < 1.05, P < 1.01).Record result
As shown in table 7.
Influence (χ soil S, n=7) of 7 Phellinus of table to tumor bearing nude mice life cycle
* P < 0.05, * * P < 0.01vs. model group
Detailed description of the invention
Fig. 1 is impact analysis figure of the Phellinus to lung cancer tumor bearing nude mice serum NO content；Wherein, * P < 0.05, * * P <
0.01vs. model group；Normal group of #P < 0.05, ##P < 0.01vs..
Fig. 2 is impact analysis figure of the Phellinus to lung cancer tumor bearing nude mice NK cell killing activity；Wherein, * P < 0.05, * * P <
0.01vs. model group.
Fig. 3 is the influence that Phellinus induces lung cancer tumor bearing nude mice ConA spleen leaching the sixth of the twelve Earthly Branches cell proliferative response；Wherein, * P <
0.05, * * P < 0.01vs. model group；Normal group of ##P < 0.01vs..
Fig. 4 is impact analysis figure of the Phellinus to leukaemia tumor bearing nude mice serum NO content；Wherein, * P < 0.05, * * P <
0.01vs. model group；Normal group of #P < 0.05, ##P < 0.01vs..
Fig. 5 is impact analysis figure of the Phellinus to leukaemia tumor bearing nude mice NK cell killing activity；Wherein, * P < 0.05, * * P <
0.01vs. model group.
Fig. 6 is the influence that Phellinus induces leukaemia tumor bearing nude mice ConA splenic lymphocytes；Wherein, * P <
0.05, * * P < 0.01vs. model group；Normal group of ##P < 0.01vs..
Fig. 7 is impact analysis figure of the Phellinus to lymph cancer tumor bearing nude mice serum NO content；Wherein, * P < 0.05, * * P <
0.01vs. model group；Normal group of #P < 0.05, ##P < 0.01vs..
Fig. 8 is impact analysis figure of the Phellinus to lymph cancer tumor bearing nude mice NK cell killing activity；Wherein, * P < 0.05, * * P <
0.01vs. model group.
Fig. 9 is the influence that Phellinus induces lymph cancer tumor bearing nude mice ConA splenic lymphocytes；Wherein, * P <
0.05, * * P < 0.01vs. model group；Normal group of ##P < 0.01vs..
Specific embodiment
Embodiment 1: Phellinus granule preparation
Phellinus 2000g is weighed, is prepared as follows:
(a) add 15 times of water amounts for the first time, decoct 2 hours, for the second time plus 12 times of water are measured, and are decocted 2 hours, are merged medical fluid, medicine
Liquid filtration, takes filtrate, and when being concentrated into relative density 1.10-1.15 (60 DEG C), 95% ethyl alcohol, which is added, makes alcohol content reach 80%, sinks
It forms sediment, stands 24 hours, filtering obtains sediment, the recovered ethyl alcohol of filtrate obtains Aqueous extracts；
(b) Aqueous extracts are concentrated, and dry, pulverize, get dry extract powder；
(c) drying precipitate crushes, obtains precipitated powder；
(d) gained dried cream powder and precipitated powder are mixed well with appropriate dextrin, binder granulation is made with ethanol, it is dry,
Granule is made.
Embodiment 2: Phellinus granule preparation
Phellinus 3000g is weighed, is prepared as follows:
(a) add 95% ethyl alcohol, 15 times of amount refluxing extractions twice, 3 hours for the first time, second 2 hours.Merge medical fluid, medical fluid
Filtration recycles ethyl alcohol, obtains alcohol extract；
(b) dregs of a decoction after alcohol extracting add water to cook twice jointly, and for the first time plus 15 times of water are measured, and decoct 2 hours, second plus water
12 times amount, decoct 2 hours, merge medical fluid, medical fluid filtration, take filtrate, be concentrated into relative density be 1.10-1.15 (60 DEG C) when,
95% ethyl alcohol, which is added, makes alcohol content reach 80%, and precipitating stands 24 hours, and Aspirate supernatant recycles ethyl alcohol, obtains Aqueous extracts；
(c) gained alcohol extract and Aqueous extracts merge, and concentration dry, pulverize, get dry extract powder；
(d) gained dried cream powder is mixed well with appropriate amount of starch, binder granulation is made with ethanol, it is dry, particle is made
Agent.
Embodiment 3: Phellinus tablet preparation
Phellinus 25000g is weighed, is prepared as follows:
(a) add 15 times of water amounts for the first time, decoct 2 hours, for the second time plus 12 times of water are measured, and are decocted 2 hours, are merged medical fluid, medicine
Liquid filtration, takes filtrate, and when being concentrated into relative density 1.10-1.15 (60 DEG C), 95% ethyl alcohol, which is added, makes alcohol content reach 70%, sinks
It forms sediment, stands 24 hours, Aspirate supernatant recycles ethyl alcohol, obtains Aqueous extracts；
(b) it is concentrated, dry, pulverize, get dry extract powder；
(c) drying precipitate crushes, obtains precipitated powder；
(d) gained dried cream powder and precipitated powder are mixed well with suitable multitudinous sugar of swooning, binder granulation is made with ethanol, it is dry,
Appropriate magnesium stearate is added to mix, tablet is made.
Embodiment 4: Phellinus capsule preparation
Phellinus 15000g is weighed, is prepared as follows:
Above-mentioned raw materials medicine is prepared as follows:
(a) add 80% ethyl alcohol, 15 times of amount refluxing extractions twice, 3 hours for the first time, second 2 hours.Merge medical fluid, medical fluid
Filtration recycles ethyl alcohol, obtains alcohol extract；
(b) dregs of a decoction after alcohol extracting add water to cook twice jointly, and for the first time plus 15 times of water are measured, and decoct 2 hours, second plus water
12 times amount, decoct 2 hours, merge medical fluid, medical fluid filtration, take filtrate, be concentrated into relative density be 1.10-1.15 (60 DEG C) when,
95% ethyl alcohol, which is added, makes alcohol content reach 80%, and precipitating stands 24 hours, and Aspirate supernatant recycles ethyl alcohol, obtains Aqueous extracts；
(c) gained alcohol extract and Aqueous extracts merge, and concentration dry, pulverize, get dry extract powder；
(d) gained dried cream powder and appropriate differential silica gel are mixed well, binder granulation is made with ethanol, it is dry, it is added
Appropriate magnesium stearate mixes, and is packed into snap fit capsule.
Embodiment 5: Phellinus soft capsule preparation
(a) auxiliary material including soybean oil, beeswax, sorbic anhydride list olein and glycine is taken to dissolve by heating, with above-mentioned reality
Example 1 or embodiment 2 or embodiment 3 or the mixing of 4 gained dried cream powder of embodiment are applied, stirs evenly, is ground with colloid mill, obtain soft capsule
Content.
(b) by soft capsule content together with the capsule skin made of gelatin, glycerol, water, disintegrating agent and colorant, using pressure
Preparation method is suppressed, is formed, washed ball, drying, picked, polishing, soft capsule is made.
Embodiment 6: Phellinus powder preparation
Phellinus 6000g is weighed, is prepared as follows:
Phellinus is crushed, 80 meshes is crossed, is distributed into every bag of 2g.
Phellinus strain used by present embodiment is Boydii Phellinus pore fungi, and the strain is at present in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center receives preservation, deposit number are as follows: CGMCC No.14799.
Basic principles and main features and advantages of the present invention of the invention have been shown and described above.The skill of the industry
Art personnel it should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe
The principle of the present invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these
Changes and improvements all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and
Its equivalent thereof.
Claims (4)
1. Phellinus is as the application for being used for tumor, it is characterised in that Phellinus strain and growth course are comprising as follows:
Phellinus strain:
Sanghuangporus baumii Boydii Phellinus pore fungi (homonymus or synonymum: Inonotus baumii Boydii fibre pore fungi；
Phellinus baumii Boydii Phellinus).
Substituting stuff cultivation step:
(1), substituting stuff cultivation (Substitute cultivation) is with the technology mould of the mixed raw materials such as sawdust, wheat bran cultivation Phellinus
Formula；
(2), generation material bacteria stick fabrication processing: stock → spice → pack → sterilizing → cooling → inoculation.
Medium component:
With ramulus mori sawdust, the sawdusts such as toothed oak tree (Mongolian oak), robur are major ingredient, and wheat bran, dregs of beans are auxiliary material.
2. Phellinus according to claim 1 is as the application for being used for tumor, it is characterised in that: the sawdust,
The raw materials such as wheat bran, dregs of beans answer fresh, dry, color it is normal, without mildew, without agglomeration, free from extraneous odour, to be sieved, sieve before sawdust use
Bore dia 8mm~12mm.
3. Phellinus according to claim 1 is as the application for being used for tumor, it is characterised in that: culture medium composition
Formula: the sawdusts 75%~80% such as ramulus mori sawdust or toothed oak tree (Mongolian oak), robur, wheat bran 13%~18%, dregs of beans 0%~3%,
Sugar 0%~2.5%, quick lime 1.5%.It is stirred after adding water, water content is controlled 62%~65%.
4. Phellinus according to claim 1 is as the application for being used for tumor, it is characterised in that: tumour includes lung
Cancer, liver cancer, gastric cancer, cancer of pancreas, bladder cancer, colon cancer, cervical carcinoma, breast cancer, oophoroma, leukaemia, prostate cancer, melanin
Tumor, neuroblastoma.
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| CN201910715342.4A CN110478377A (en) | 2019-08-05 | 2019-08-05 | Phellinus is as the application for being used for tumor |
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| CN201910715342.4A CN110478377A (en) | 2019-08-05 | 2019-08-05 | Phellinus is as the application for being used for tumor |
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Application publication date: 20191122 |