CN104337803B - Isodon xerophilus B prime xerophilusin B application in preparation suppression tumor growth product - Google Patents
Isodon xerophilus B prime xerophilusin B application in preparation suppression tumor growth product Download PDFInfo
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- CN104337803B CN104337803B CN201410509939.0A CN201410509939A CN104337803B CN 104337803 B CN104337803 B CN 104337803B CN 201410509939 A CN201410509939 A CN 201410509939A CN 104337803 B CN104337803 B CN 104337803B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses Isodon xerophilus B prime Xerophilusin B application in preparation suppression tumor growth product, the invention provides and provide Xerophilusin B and have following 1 in preparation) and/or 2) application in the product of function: 1) prevent or treat tumour;2) suppression tumor cell proliferation;The chemical structural formula of described xerophilusin B is such as following formula I:
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of Isodon xerophilus B prime xerophilusin B in preparation
Application in suppression tumor growth product.
Background technology
The cancer of the esophagus is that worldwide is occurred frequently and one of the malignant tumour of serious threat human health and life, at present, and food
The incidence of disease of pipe malignant tumour occupy the 8th of whole malignant tumour.Adenocarcinoma of esophagus is suffered from different, China's cancer of the esophagus from American-European countries
Always based on dermoid cancer, and the incidence of disease of adenocarcinoma of esophagus has no significantly raised, more than 90% cancer of the esophagus case class
Type is esophageal squamous cell carcinoma, and therefore esophageal squamous cell carcinoma is a malignant neoplastic disease kind with Chinese characteristics.And in China, the cancer of the esophagus has
Having obvious geographical distribution clustering phenomena, high incidence is regional quite to be concentrated with high case fatality rate area.Province occurred frequently is Henan, river
North, Chongqing and Fujian Province, be secondly Xinjiang, Gansu, Shanxi, Anhui and Jiangsu Province.Country's anti-cancer does third time cancer screening
Data shows, from 20 century 70s at the beginning of 21 century, though its case fatality rate of respective regions has declined, but China's Incidence of Esophageal Cancer
The present situation that rate remains high still continues.
In recent years, find the natural plants effectively with notable antitumor activity and become research with Chinese herbal medicine extract
One of focus, puts down the natural plants as representative with vinca, taxanes and the cancer that disappears and Chinese herbal medicine extract is the most extensive
For clinical and receive good curative effect.Rabdosia (Isodon) is Labiatae (Labiatae) plant, for perennial herb
Shrub or fruticuli, be distributed mainly on east Asia and Africa is western, and worldwide there are about more than 150 kinds, and China is that this genus is planted
The country that goods and materials source is the abundantest, reserves are huge, there are about more than 90 kinds, 25 mutation, and all parts of the country are equal in addition to Qinghai with the Inner Mongol
Plant is had to be distributed, wherein most with southwestern all provinces and regions especially Yunnan Province's topological classes.For Rabdosia plant medical value
Utilize long-standing, wherein there are about more than 30 and plant former plant by among the people as medicinal materials, have clearing heat and detoxicating, promoting blood circulation and breaking stagnation,
The effects such as anti-inflammation, exploitation and research to this platymiscium the most both at home and abroad is vigorously developed.
Xerophilusin B be mainly distributed in Rabdosia plant (as Isodon xerophilus Isodon xerophilus,
Rabdosia coesta I.coetsa, Rabdosia adenoloma I.adenolomus, leaf fringe Rabdosia amethystoides I.phyllostachys etc.).The earliest by
Dong Sun Han etc. are isolated (Han-Dong Sun, et al., Journal of Natural from Isodon xerophilus
Products,2000,63(5),599-601.).Cytotoxic activity screening shows: xerophilusin B is thin to three-type-person's tumour
Born of the same parents system K562, HL-60 and MKN-28 all show remarkable activity, IC50It is [positive that value is respectively 0.73,0.29 and 0.17 μ g/mL
The IC of comparison medicine mitoxantrone (mitoxantrone)50Value is respectively 0.0334,0.29 and 0.02 μ g/mL] (Han-Dong
Sun,et al.,Journal of Natural Products,2000,63(5),599-601.);To three-type-person's tumour cell
It is that K562, MKN45 and HepG2 also show preferable activity, IC50Value is respectively 4.1,0.4 and 3.9 μM, and [positive control drug is suitable
The IC of platinum (cis-platin)50Value is respectively 2.7,3.5 and 3.3 μMs] (Han-Dong Sun, et al., Journal of
Natural Products,2007,70(8),1295-1301.);To three-type-person tumor cell line HT-29, BEL-7402 and SK-
OV-3 also shows certain activity, IC50Value is respectively 2.52,3.06 and 2.14 μ g/mL [positive control drug adriamycins
(ADR) IC50Value is respectively 0.08,0.12 and 0.47 μM] (Han-Dong Sun, et al., Archives of
Pharmacal Research,2011,34(12),2007-2014.);To five kinds of human tumor cell line HL-60, SMMC-7721,
A-549, MCF-7 and SW480 all show remarkable activity, IC50Value is respectively 0.8,1.3,2.0,1.1 and 0.9 μMs, and [positive is right
IC according to medicine cis-platinum (cis-platin)50Value is respectively 1.5,14.5,14.1,15.0 and 16.9 μMs] (Han-Dong Sun, et
al.,Journal of Natural Products,2011,74(5),1213-1220.);Xerophilusin B can suppress
The NO that in mouse macrophage RAW264.7, lipopolysaccharides (LPS) is induced generates (IC50=0.23 μM), mRNA can be suppressed raw simultaneously
Become (Jos é Luis R í os, et al., Journal of Natural Products, 2009,72 (9), 1269-1272.).
Up to now, the document report of ent-kaurane diterpenoid xerophilusin B anti esophageal cancer activity is had no.
Summary of the invention
It is an object of the present invention to provide the new application of Xerophilusin B.
The invention provides Xerophilusin B and have following 1 in preparation) and/or 2) application in the product of function:
1) prevent or treat tumour;
2) suppression tumor cell proliferation;
The chemical structural formula of described Xerophilusin B is such as following formula I:
Formulas I.
In above-mentioned application, described suppression tumor cell proliferation is by making cell block in G2/ M phase and/or promotion tumour are thin
Born of the same parents' apoptosis embodies.
In above-mentioned application, described promotion apoptosis of tumor cells is by reducing apoptosis pathway protein B CL-2/Bax in cell
Expressing quantity ratio embodies.
In above-mentioned application, described tumour is esophageal squamous cell carcinoma;Described tumour cell be esophageal squamous cell carcinoma cell KYSE-140,
KYSE-150, KYSE-450 or KYSE-510, described tumour cell is specially KYSE-150 or KYSE-450.
Another object of the present invention is to provide the other purposes of Xerophilusin B.
The invention provides Xerophilusin B and have 1 in preparation)-3) at least one function product in should
With:
1) make cell block in G2/ M the phase;
2) apoptosis of tumor cells is promoted;
3) the expressing quantity ratio of BCL-2/Bax is reduced.
In the said goods, described tumour is esophageal squamous cell carcinoma;Described tumour cell be esophageal squamous cell carcinoma cell KYSE-140,
KYSE-150, KYSE-450 or KYSE-510, described tumour cell is specially KYSE-150 or KYSE-450.
The 3rd purpose of the present invention is to provide a kind of antineoplastic.
The antineoplastic that the present invention provides, its active component is described compounds X erophilusin B.
Said medicine is made up of described compounds X erophilusin B and pharmaceutical carrier, and said medicine carrier can be medicine
The all of pharmaceutical carrier accepted in reason.
In said medicine, described tumour is esophageal squamous cell carcinoma.
Said medicine can be such that
Medicine 1:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, injects routinely and uses water, and essence filter, parenteral solution is made in embedding sterilizing.
Medicine 2:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, is dissolved in sterile water for injection, and stirring makes molten, filters with aseptic suction funnel, more aseptic essence filter, frozen drying
Rear aseptic sealing by fusing obtains powder-injection.
Medicine 3:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 9:1, makes pulvis.
Medicine 4:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient, pelletizing press sheet with excipient weight than the ratio for 1:5-1:10 in it.
Medicine 5:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, oral liquid preparation method makes oral liquid routinely.
Medicine 6:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 5:1 in it, makes capsule or granule or electuary.
Medicine 7:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 3:1 in it, makes capsule or granule or electuary.
The experiment proves that, from Rabdosia plant Isodon xerophilus (Isodon xerophilus), screen energy
Enough diterpene-kind compound Xerophilusin B effectively suppressing esophageal squamous cell carcinoma cell proliferation, are studied with experiment in vitro by internal
Verify that it can cause cell death by mechanism of action such as inducing cell apoptosis and Cycle Arrest, and by control compounds with
Effect in knurl body and normal organ tissue is distinguished by classic chemotherapy medicine, utilizes nude mice hepatic and renal function to detect, and investigates its poison
Property reaction, for be further used as antineoplastic lead compound provide evidence.Therefore, in view of in Rabdosia plant all kinds of newly
The continuous discovery of compound, and the remarkable result found in oncotherapy with research, along with Isodon xerophilus plant extract
Gradually deeply, the diterpene-kind compound with xerophilusin B as representative may be as oncotherapy in the research of chemical composition
Bring new approach.
Accompanying drawing explanation
Fig. 1 is Xerophilusin B1H-NMR spectral data (400MHz, CDCl3)。
Fig. 2 is Xerophilusin B13C-NMR spectral data (100MHz, CDCl3)。
Fig. 3 is Xerophilusin B ESI spectral data.
Fig. 4 is Xerophilusin B HREI spectral data.
Fig. 5 is compound xerophilusin B structure formula.
Fig. 6 is the Xerophilusin B impact on esophageal squamous cell carcinoma cell proliferation.
Fig. 7 is that Xerophilusin B is on esophageal squamous cell carcinoma cell and the impact of normal cell proliferation
Fig. 8 is Xerophilusin B IC50 value in esophageal squamous cell carcinoma cell and normal cell
Fig. 9 is the observation that KYSE-150 Yu KYSE-450 cytomorphology is affected by Xerophilusin B
Figure 10 is that Xerophilusin B is to KYSE-150 Yu KYSE-450 transplanted tumor in nude mice depression effect
Figure 11 is that Xerophilusin B is to KYSE-150 transplanted tumor in nude mice knurl volume depression effect
Figure 12 is that Xerophilusin B is to KYSE-450 transplanted tumor in nude mice knurl volume depression effect
Figure 13 is that Xerophilusin B is to KYSE-150 Yu KYSE-450 transplanted tumor in nude mice knurl weight depression effect
Figure 14 is Xerophilusin B learns impact to KYSE-150 Yu KYSE-450 transplanted tumor in nude mice knurl soma
Figure 15 is the Xerophilusin B impact on KYSE-150 Yu KYSE-450 cell cycle
Figure 16 is the Xerophilusin B impact on KYSE-150 Yu KYSE-450 apoptosis
Figure 17 is that Xerophilusin B is on the impact of β-actin protein content in KYSE-150 Yu KYSE-450 cell
Figure 18 is the impact that apoptosis-related protein in KYSE-150 Yu KYSE-450 cell is expressed by Xerophilusin B
Figure 19 is that Xerophilusin B is to the shadow of Bcl-2 Yu Bax protein expression in KYSE-150 Yu KYSE-450 cell
Ring
Figure 20 is the Xerophilusin B impact on NIH-3T3 Yu HEK-293 cell proliferation
Figure 21 is the Xerophilusin B shadow to KYSE-150 Yu KYSE-450 transplantable tumor nude mouse serum liver kidney merit index
Ring
Figure 22 is the Xerophilusin B impact on KYSE-150 Yu KYSE-450 transplantable tumor nude mice hepatic tissue
Figure 23 is the Xerophilusin B impact on KYSE-150 Yu KYSE-450 transplantable tumor nude mice nephridial tissue
Figure 24 is that Xerophilusin B is on the KYSE-150 transplantable tumor nude mice heart, lung, the impact of spleen tissue
Figure 25 is that Xerophilusin B is on the KYSE-450 transplantable tumor nude mice heart, lung, the impact of spleen tissue
Figure 26 is the Xerophilusin B impact on KYSE-150 Yu KYSE-450 transplantable tumor nude mice body weight
In above-mentioned accompanying drawing, xerophilusin B is abbreviated as spxl-3.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain, and remaining is such as
Under:
1, the required reagent of experiment
Table 1 is the required reagent of experiment
2, key instrument equipment
Table 2 is key instrument equipment
3, the preparation of solution:
1) compound xerophilusin B solution: xerophilusin B is dissolved in DMSO, the solution obtained;External
In experiment compound used therefor solution weigh 3.46mg xerophilusin B compound (molecular weight=346) crystalline powder with
10ul DMSO solvent mixes, and mixing to being i.e. configured to concentration after being completely dissolved is that 1mol/L compound stores stoste.Draw 1uL
I.e. being made into 1mmol/L compound in stoste addition 999ul culture medium and store liquid, dilution is configured to desired concn the most successively
Liquid, finally complements to the amount identical with maximum concentration experimental group by the DMSO in each concentration compound solution, to ensure each group
In compound solution, the concentration of DMSO is homogeneous, and in this research cell experiment, DMSO concentration is all 0.1% (v/v).Same method is joined
The xerophilusin B solution used in experiment in vivo processed, but it is used for the solvent diluting stoste after dissolving crystallized powder for emulsification
Agent 1%Pluronic F68.
2) ethylenediamine tetra-acetic acid (EDTA) solution: 37.2gEDTA sodium salt is joined in the distilled water of 160mL, and use magnetic
Power agitator carries out being sufficiently stirred for shaking up under the conditions of room temperature environment.Connect electronics acidometer, under conditions of real time readouts to
Being slowly added to NaOH powder in solution, until the pH value of solution is adjusted to 8.0, the amount of required NaOH is about 4g, with distillation
After water is settled to 200mL, process through high pressure steam sterilization, preserve under room temperature condition.The ultimate density of EDTA solution is
0.5mol/L。
3) TAE (Tris-Acetate-EDTA) electrophoretic buffer (50 ×): accurately weigh Tris alkali 121g, adds 400mL
In distilled water, it is sufficiently stirred for concussion with magnetic stirring apparatus, mixes and backward solution adds the glacial acetic acid of 28.55mL and 50mL is dense
Degree is the EDTA solution of 0.5mol/L, is eventually adding distilled water and is settled to 500mL, preserves under room temperature condition.
4) ethidium bromide (EB) solution: precise 0.2g ethidium bromide, is dissolved in 20mL water, abundant with magnetic stirring apparatus
After stir about 2 hours, solution is transferred in brown reagent bottle, or transfers the solution in the reagent bottle of tinfoil parcel,
Keep in Dark Place under room temperature condition.
5) dual anti-(penicillin and Streptomycin Solution) solution: penicillin and each 1,000,000 units of streptomysin be dissolved in 100mL without
In bacterium distilled water, it is configured to the dual anti-solution of 100 × concentration, wherein each with streptomysin containing penicillin in the dual anti-solution of every mL
10000 units, are then distributed in new Ep pipe, preserve under the conditions of-20 DEG C.It is diluted to finally with aseptic double-distilled water during use
Concentration is 100 units/mL.
6) cells frozen storing liquid: main component is the RPMI1640 serum free medium containing 80%, the dimethyl of 10% is sub-
Sulfone (DMSO) and the hyclone of 10%.
7) phosphate buffer (PBS): precise NaCl8.5g, KCl0.2g, Na2HPO4·2H2O powder 2.85g,
KH2PO4Powder 0.27g, is dissolved in aseptic double-distilled water, is settled to 1L, after standby after autoclave sterilization.
8) 10 × Tris-HCl buffer salt solution (TBS): first the Tris alkali of 80g NaCl, 24.2g is dissolved in 800mL
In aseptic double-distilled water, finally use HCl regulation pH value to 7.6.
9) the hematoxylic preparation of Harris: first with distilled water heating for dissolving alum (KAl (SO4)2), use absolute ethyl alcohol
Dissolve haematine and pour the KAl (SO dissolved again into4)2In distilled water, after boiling 1min, to be cooled a little, be slowly added to red oxygen
Change mercury (HgO) 0.5g, continue to be heated to dye liquor when becoming aubergine, cover bottleneck with gauze, with in 100mL every after Filter paper filtering
Dropping glacial acetic acid 5mL.
10) preparation of Yihong (eosin): first Yihong is dissolved in alcohol, after glass bar stirring and dissolving, every 100mL
1, middle dropping glacial acetic acid.
4, the cell used by experiment and cultural method
The esophageal squamous cell carcinoma cell used has following 4 strains: KYSE-140 (middle differentiated squamous-cell carcinomas), KYSE-150 (low differentiation squama
Cancer), (above-mentioned cell line is all documented in such as Publication about Document KYSE-450 (differentiated squama cancer) and KYSE-510 (differentiated squama cancer)
In: Shimada Y1, Imamura M, Wagata T, Yamaguchi N, Tobe T.Characterization
of21newly established esophageal cancer cell lines.Cancer.1992Jan15;69(2):
277-84.;The public can be from Cancer Hospital of Chinese Academy of Medical Sciences;Kunming Inst. of Botany, Chinese Academy of Sciences obtains).Above cell
System all identifies through STR.
For impact and the toxic reaction of research compound normal tissue cell, apply following 2 strain normal structure sources
Clone, is respectively as follows: MEC system NIH-3T3 (ATCC, CRL-1658), human embryonic kidney cell line HEK-293
(human embryonic kidney-293 (ATCC, CRL-1573), is purchased from ATCC cell bank.
The cultivation of esophageal squamous cell carcinoma cell uses RPMI1640 culture medium, the training of MEC system NIH-3T3
Supporting and use DMEM (Hyclone, USA) culture medium, the cultivation of human embryonic kidney cell line HEK-293 uses MEM (Hyclone, USA),
Wherein need the hyclone (Gibco) adding 10% and penicillin and the streptomysin dual anti-(Invitrogen) that concentration is 0.2%.
It is 37 DEG C of constant temperature that cell cultivates incubator condition, moisture constant, CO2Concentration maintain 5%.
Embodiment 1, the acquisition of compounds X erophilusin B (being abbreviated as spxl-3)
1) extraction and purification
A) as plant material (7.5 thousand after the Isodon xerophilus plant leaves part process ground, dry that will gather
Gram), during pulverization process is soaked in the acetone soln that volume is 12L in room temperature environment, within every 3 days, change the freshest acetone molten
Liquid.
B) plant material after soaking at room temperature filters, and collects filtered fluid.Collected filtered fluid uses and subtracts
Pressure distillating method obtains CE to remove solvent.
C) CE in b) is dissolved in the water (4L), with petroleum ether (totally 3 times, each 3L) and ethyl acetate (totally 3
Secondary, each 4L) extract extraction successively, collect concentrate and i.e. obtain ethyl acetate extraction medicinal extract (504 grams).
D) ethyl acetate extract extract (504 grams) use MCl resin is carried out desolventing technology, utilize 90%MeOH-H2O
After eluting, produce flaxen medicinal extract (423 grams).
E) the faint yellow medicinal extract (423 grams) in d) is taked silica gel column chromatography, use CHCl3-Me2CO (1:0 → 0:1) ladder
Degree wash-out processes and obtains A-G, 7 parts.
F) by the thick product B (33.4 grams) of gained in d) through silica gel column chromatography, oil Ether-Acetone (6:1 → 2:1) is used
Gradient system wash-out flow process elutes, final generation compound xerophilusin B (103.5mg).
2) Structural Identification
A) high performance liquid chromatography (High Performance Liquid Chromatography, HPLC) is utilized to measure
Xerophilusin B purity, to ensure that its purity reaches at least 98%.
B) use electronic micro balance to weigh appropriate compound powder, load in Ep pipe, draw DMSO solution and add EP pipe
In, repeatedly aspirate after mixing makes compound be completely dissolved with pipettor, be placed in-20 DEG C long-term kept dry.
Wherein NMR, ESI (Fig. 3) and the HREI spectral data (Fig. 4) of compound xerophilusin B are as follows:
Compound xerophilusin B:1H NMR(400MHz,pyridine-d5,δin ppm,J in Hz)δ1.68
(H-1a,d,13.4);1.00(H-1b,dd,13.4,4.5),1.48(H-2,m),1.35(H-3a,br d,13.2),1.10(H-
3b,dd,13.2,3.6),1.52(H-5,br s),4.21(H-6,br s),2.75(H-9,d,7.1),1.61(H-11a,dd,
15.3,6.7),1.50(H-11b,m),2.24(H-12a,m),1.42(H-12b,m),3.10(H-13,m),4.95(H-14,d,
6.5),6.17(H-17a,br s)5.27(H-17b,br s),1.08(H3-18,s),0.91(H3-19,s),5.47(H-20,
s).13C NMR(100MHz,pyridine-d5,δin ppm)δ27.7(C-1,t),19.0(C-2,t),41.3(C-3,t),
33.8(C-4,s),63.3(C-5,d),73.0(C-6,d),101.6(C-7,s),57.6(C-8,s),45.4(C-9,d),43.0
(C-10,s),17.3(C-11,t),26.2(C-12,t),40.3(C-13,d),70.1(C-14,d),199.8(C-15,s),
149.0(C-16,s),118.3(C-17,t),31.6(C-18,q),23.3(C-19,q),98.0(C-20,d).HRMS(m/z):
[M]+calcd.for HRMS(m/z):[M+Na]+calcd.for C20H26O5Na,369.1678;found,369.1681.
From1H-NMR and13C-NMR collection of illustrative plates (Fig. 1-Fig. 2) can show that the molecular formula of compound sxp-3 is C20H26O5, molecule
Quality is 346.41744, data above and NCBI (National Center for Biotechnology Information)
In database, in PubChem search library, No. 44445767 compound data information of CID are coincide, and raw with the drought of document report
The data consistent of plectranthin Xerophilusin B, its structural formula of compound is as shown in Figure 5.In sum, database is passed through
Retrieval also compares with data in literature, determines that this compound is Isodon xerophilus element xerophilusin B.
Embodiment 2, the Xerophilusin B application in suppression esophageal squamous cell carcinoma growth
1, the Xerophilusin B growth in vitro inhibitory action to esophageal squamous cell carcinoma clone
Measure inhibitory rate of cell growth experiment reagent and use Cell Counting Kit-8 (CCK-8) of Dojindo company
Kit, concrete grammar is as follows:
1) by through cultivation amplification after KYSE-140, KYSE-150, KYSE-450, KYSE-510 and NIH-3T3 with
HEK-293 cell, cultivates and reaches 70~80% to cell degrees of fusion, uses 0.25% trypsinization to process cell, then uses
Homogeneous cell suspension is made in the piping and druming of modified form RPMI-1640, DMEM or MEM complete medium, is placed in 15ml or 50ml and is centrifuged
Guan Zhong.
2) add complete medium to be diluted by resuspended cell suspension, make ultimate density reach 1.5 × 104Individual/mL,
Fully draw 200 μ L after mixing and join (every hole is containing about 2000 cells) in 96 orifice plates, be placed in the CO of 37 DEG C and 5%2Carefully
Born of the same parents' incubator hatches cultivation.When dripping cell suspension hand-hole, application 1mL rifle head is to reduce too small the caused cell of rifle head
Damage and death;Often add 3~behind 5 holes, for avoiding cell suspension sedimentation to cause cell concentration uneven, with pipettor or suction pipe
Again mix cell suspension;For quickening bed board speed to reduce difference between hole, the cell suspension in centrifuge tube is distributed into 1.5mL
Or 2mL Ep pipe.
3) 96 orifice plates are lateral opening not as experiment use, for avoiding hatching and in operating process near plank at subsequent cell incubator
The culture medium evaporation at edge, causes volume difference density unevenness between hole to affect reading, by lateral opening for surrounding and be not used in detection
Hole same volume culture medium or sterilized water in addition.
4) after bed board, lid is built, and on lid and orifice plate, mark experimentai batches, experimenter's name, concentration
Gradient and time, jiggle orifice plate even in order to cell paving.Hatch period Microscopic observation cell attachment and growing way situation in good time.
5), after cell attachment, after cell degrees of fusion reaches 50~60%, inhale and abandon Central Plains, each hole culture medium, the most often
Hole adds fresh culture and 10uL each concentration compounds X erophilusin B solution (solvent is DMSO), makes cell in every hole
The total amount of culture medium reaches 100 μ L, using the same day adding compound as the starting point (the 0th hour) of cell proliferation.Experiment point
Group arranges Xerophilusin B process group (cell+10uL Xerophilusin B solution+culture medium), DMSO control group
(cell+unitized dose amounts DMSO+ culture medium), blank group (cell+culture medium) and Background control group (culture medium).
6) compounds X erophilusin B concentration arranges 0 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs, 10 μMs and 250 Μ
M (dilutes with culture medium), totally 8 concentration gradients.
7) the 0th hour, the 6th hour, the 12nd hour, the 24th hour, the 48th hour and the 72nd after dosing is little respectively
Time in each hole of 96 orifice plates, add the CCK-8 of 10 μ l respectively detect liquid, add and fashionable uniformly drip along hole wall, in order to avoid in hole
Generate aeration OD and be worth reading, then at 37 DEG C and the CO of 5%2Cell culture incubator is hatched 0.5~1.5 hours.This
Detection time interval after place adds CCK-8 needs through different time points duplicate detection, in order to find out the optimum detection time,
It is excessive that overlong time then detects initial OD values, easily beyond its detection range after cell continued growth, reduces accuracy, and if
Interval time is too short, then may react not exclusively, cause false negative.0.5~1 hour it is generally for the esophageal squamous cell carcinoma time, just
Often general 0.5 hour of clone.
8) building orifice plate top cover, being placed on incubator nexine affects lucifuge to reduce incubator switch.If carrying out multiple batches of
Test and temporarily do not measure OD value, HCl solution or 1% (w/v) SDS solution of 10 μ l0.1M can be added in every hole, and
Hide culture plate to keep in Dark Place at ambient temperature.Measuring in 24 hours, absorbance will not change.For avoiding to be detectedization
Compound produces redox reaction with CCK-8 and causes experiment false positive, therefore before being added dropwise to CCK-8, and will with pipettor
In hole, culture medium is inhaled and is abandoned, and adds 200uL fresh culture.
9) sucking-off 100 μ l nutrient solution from the culture hole adding CCK-8 detection liquid, joins a new enzyme according to sequence number
In mark instrument color board, set ELIASA, throw off 96 well culture plate top covers, put into ELIASA bracket, after shaking 5~10 seconds, read training
Nutrient solution light absorption value at 450nm, every kind of cell experiment group arranges 6 multiple holes.
10) detection OD value result, the Background control wells OD value that each group all deducts in respective sets, with the time after cell dosing be
Transverse axis, starting point is the 0th hour, and cell culture fluid light absorption value (OD value) at 450nm, as the longitudinal axis, connects and forms propagation song
Line, and use Bonferroni inspection to compare the statistical significance of repeated measurement data on each time point.
11) each group OD value recorded is substituted into inhibiting rate computing formula: inhibiting rate (%)=(1 ODProcess group/ODDMSO group)×
In 100, calculate compound inhibiting rate.
Respectively after dosing behind 0 hour, 6 hours, 12 hours, 24 hours, 48 hours and 72 hours, apply Cell
Counting Kit-8 method detection xerophilusin B compound its concentration be 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and
The i.e. OD value of the light absorption value at 450nm when 10 μMs, draws increment curve and calculates growth inhibition ratio.
Result as shown in Figure 6, is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs for adding concentration gradient
Xerophilusin B compound 0 hour, 6 hours, 12 hours, 24 hours, 48 hours and after 72 hours, KYSE-140,
The growth curve situation of KYSE-150 cell, KYSE-450 cell and KYSE-510 cell, abscissa represents and adds compound
After 6 time detecting points, ordinate show using the light absorption value at 450nm is standardized process and as cell number
The measurement index (standardized cell index Normalized Cell Index) of amount.Can clearly find out for KYSE-150 thin
Born of the same parents, the passage in time of blank group cell demonstrates that cell number gradually increases, the linear trend that absorbance is gradually increased;With
Xerophilusin B compound concentration to be stepped up, the cell proliferation rate of its correspondence gradually slows down, growth curve trend
Decline, and when xerophilusin B compound concentration is 0.5 μM after 72 hours, compared with cellular control unit absorbance i.e.
Having notable growth inhibition effect (P < 0.01), equally under 1 μM, 2 μMs, 5 μMs and 10 μMs of concentration, cell proliferation is all significantly pressed down
System (P value all < 0.001), when compound reaches the highest 10 μMs, cell is at 6 little the most major part death, absorbance convergences
In 0 (Fig. 6 B).Similarly, for KYSE-450 cell, the passage in time of blank group cell demonstrate cell number also by
Cumulative many, that absorbance is gradually increased linear trends;Along with xerophilusin B compound concentration is stepped up, its correspondence
Cell proliferation rate gradually slows down, and growth curve trend declines, and xerophilusin B compound concentration after 72 hours
When being 1 μM, i.e. have notable growth inhibition effect (P < 0.01) compared with cellular control unit absorbance, equally 2 μMs, 5 μMs and
Under 10 μMs of concentration, cell proliferation is all suppressed significantly (P value all < 0.001), and when compound reaches the highest 10 μMs, cell is little 6
The most dead, absorbance levels off to 0.Equally, in KYSE-140 clone with KYSE-510 clone, all observe
There is phase same-action trend to xerophilusin B compound, but its effect is not thin with KYSE-450 at KYSE-150 cell
In born of the same parents significantly.Diterpene-kind compound in studying due to this is all dissolved as compound stock solution with DMSO solution or stores liquid, and it is right
Unknown in the cytotoxicity of esophageal squamous cell carcinoma cell, therefore the anti-GIA of compound for detecting in eliminating experiment is
Caused by DMSO solvent, above cell proliferation experiment is provided with DMSO control group, the most only adds the DMSO of same dose
Solvent, i.e. volume fraction are all 0.1% (v/v).Testing result shows, with the blank comprising only tumour cell and nutrient solution
Group is compared, and DMSO solvent cell proliferation ability is without impact, and cell itself is not had by the DMSO concentration dose used in experiment
Toxicity.
According to cell proliferation experiment data result, for more clearly comparative compound to Cytostatic to tumor cell effect
Effect, be converted and become cell proliferation inhibition rate.
As it is shown in fig. 7, for adding xerophilusin B compound effects inhibitory rate of cell growth situation after 72 hours,
Abscissa represents 6 concentration gradients of xerophilusin B compound, ordinate display inhibitory rate of cell growth, is adding difference
After the xerophilusin B compound of concentration, the inhibitory rate of cell growth of 4 kinds of esophageal squamous cell carcinoma cells is all with xerophilusin
The effect that the rising of B compound concentration and presenting progressively is incremented by.In KYSE-140 clone, after acting on 72 hours
Xerophilusin B compound is when its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, and growth inhibition ratio divides
It is not 0.61%, 6.50%, 12.66%, 29.74%, 78.36% and 98.01%;In KYSE-150 clone, effect
After 72 hours, xerophilusin B compound is when its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, growth
Inhibiting rate is respectively 0,8.61%, 25.60%, 76.46%, 99.05% and 99.46%;In KYSE-450 clone, make
With xerophilusin B compound after 72 hours when its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, raw
Long inhibiting rate is respectively 0,0,13.12%, 64.97%, 95.30% and 99.08%;In KYSE-510 clone, effect
After 72 hours, xerophilusin B compound is when its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, growth
Inhibiting rate is respectively 0,0,0,27.90%, 90.21% and 99.11%.
In HEK293 cell, after acting on 72 hours xerophilusin B compound its concentration be 0.1 μM, 0.5 μM,
When 1 μM, 2 μMs, 5 μMs and 10 μMs, growth inhibition ratio be respectively 0,0.36%, 3.44%, 35.77%, 83.32% and
98.97%.
In NIH-3T3 cell, after acting on 72 hours, xerophilusin B compound is 0.1 μM, 0.5 μ in its concentration
When M, 1 μM, 2 μMs, 5 μMs and 10 μMs, growth inhibition ratio is respectively 4.04%, 3.59%, 11.44%, 29.78%, 65.30%
And 94.30%.
Xerophilusin B compound inhibiting rate in two strain normal cells, is substantially less than in esophageal squamous cell carcinoma clone
KYSE-150 cell and the inhibiting rate in KYSE-450 cell.Further, compound effects 72 is calculated according to above inhibiting rate little
IC time after50Value, found that the IC in NIH-3T3 cell with HEK-293 cell50Value is respectively 2.61 μMs and 3.24 μMs,
Far above the IC at esophageal squamous cell carcinoma clone KYSE-150 cell (1.15 μMs) Yu KYSE-450 cell (1.70 μMs)50Value.
It is apparent that xerophilusin B compound to the growth inhibition effect of each esophageal squamous cell carcinoma clone all with
Concentration gradient raises and shows the trend gradually strengthened, and when xerophilusin B compound concentration reaches 10 μMs, it is respectively
Growth inhibition ratio in clone all reaches more than 90%, and in KYSE-150 cell and KYSE-450 cell two strain clone
In, when xerophilusin B compound concentration is only 1 μM of extremely low concentration with 2 μMs, it is possible to significantly inhibit tumour cell and increase
Growing, under 2 μMs of concentration, its inhibiting rate has all exceeded 50%, it can be seen that, xerophilusin in this two strains esophageal squamous cell carcinoma cell
B compound has higher lethal effect.
It addition, according to above xerophilusin B compound inhibiting rate in esophageal squamous cell carcinoma cell, calculate chemical combination
IC after thing effect 72 hours50Value.As shown in Figure 8, abscissa represents each clone title to result, and ordinate represents IC50Value (μ
M), the IC in KYSE-140 clone, KYSE-150 clone, KYSE-450 clone and KYSE-510 clone50Value
It is respectively 2.79 μMs, 1.15 μMs, 1.70 μMs and 2.62 μMs.From above numerical value it is apparent that xerophilusin Bization
Compound IC in esophageal squamous cell carcinoma cell system's KYSE-150 cell and KYSE-450 cell50It is thin that value is substantially less than KYSE-140
Born of the same parents and KYSE-510 cell, illustrate xerophilusin B compound to esophageal squamous cell carcinoma cell system's KYSE-150 cell and
The growth inhibition effect of KYSE-450 cell is relatively strong, therefore selects this two strains clone and proceeds cell function experiment.
2, the morphological observation after Xerophilusin B effect esophageal squamous cell carcinoma cell
Cells survival state and vigor can be reflected by observation by light microscope cellular morphology, utilize the high power lens naked eyes can
Observing directly the grown form of cell, cell membrane, cytoplasm (slurry) and nucleus, concrete grammar is as follows:
1) experiment packet: compounds X erophilusin B concentration sets 0 μM, 2 μMs and 5 μMs of three concentration gradients, Mei Genong
Degree respectively sets 3 multiple holes, before dosing, inhales with suction pipe and abandons original culture medium in hole, be added dropwise to fresh complete medium 2mL, six orifice plates
Middle experimental group adds the xerophilusin B solution 20uL/ hole that concentration is 200 μMs or 500 μMs, after therefore adding in system
Liquor strength is 2 μMs and 5 μMs.Control group adds equivalent DMSO solvent, often organizes and sets 3 parallel multiple holes.
2) application absolute ethyl alcohol or 95% cotton ball soaked in alcohol are by microscope carrier, eyepiece, object lens, adjusting knob and micro-
Mirror peripheral region strict sterilization.
3) by the Tissue Culture Flask after Secondary Culture or addition compound treatment or culture plate, screw bottle cap or compress top cover
After carefully take out from incubator.
4) open inverted microscope switch, regulate lamplight brightness, Tissue Culture Flask or culture plate are gently placed on objective table.
5) field of view from eyepiece, regulation object lens and the distance of blake bottle bottom surface, regulate and stop to time the most clear simultaneously.
6) move blake bottle or culture plate to select optimal viewing region, or regulation object lens multiple is so that apparent observation is thin
Micro-structural.
7) repeatedly adjust object lens and reached the clear visual field with objective table spacing, utilize camera shooting record cell to draw
Face.
8) suitably rock blake bottle to have seen whether floccule or granular substance, carefully discern whether as polluting so that and
Time process, in order to avoid causing experiment false positive.
Result is as it is shown in figure 9, for KYSE-150 cell, in blank group, cell arrangement is uniform, and Cell tracking is tight
Close, cellular morphology is normal, and in polygon, nucleus is oval, normal with cytoplasmic ratios, without cavity in cell cytoplasm, and training
Foster base color normal hyaline, without muddiness, waits impurity without cotton-shaped in culture medium, and acellular secretory granules, cell proliferation is vigorous.When adding
After entering the xerophilusin B compound of 2 μMs of concentration, within 24 hours, observe that cellular morphology changes, its membranolysis, cell
Propagation weakens, and cell number significantly reduces compared with Normal group, and cell shape is become round by polygon, and cell volume increases
Greatly, intracellular there is cavity, and nuclear shapes changes in irregular, and in nucleus, chromosome form changes, cell karyoplasmic ratio
Example changes, and cell surface highlights film bubble shape vesicle, and Cell tracking major part disappears, and part cell detachment culture plate diapire also wafts
Entering in cell culture medium, be suspended with particulate cellular secretion in culture medium, culture medium color is dimer, but and finds no muddy
Turbid or contamination phenomenon.When after the xerophilusin B compound adding 5 μMs of concentration, within 24 hours, most cells be can be observed complete
Entirely losing normal cell form, intracellular structure is disorderly, cellular membrane disruption, and cellular content spills, and cell surface and surrounding are the most attached
Having a large amount of film balloon-shaped structure, nucleus fragmentation, Cell tracking disappears, and cell adhesion ability is substantially reduced, slight oscillating and shaking
Culture plate can make attached cell depart from culture plate diapire, and culture medium color is dark and gloomy, suspension mortality cell and cell fragment,
But do not observe the sign of microorganism or pathogen contamination.
Equally, in the esophageal squamous cell carcinoma KYSE-450 cell after adding xerophilusin B compound, also been observed
Similar phenomenon.In blank group, cell arrangement is uniform, and Cell tracking is tight, and cellular morphology is normal, in polygonal, carefully
Karyon is oval or circle, and N/C is normal, and without cavity in cell cytoplasm, culture medium color normal hyaline, without muddiness, is cultivated
Waiting impurity without cotton-shaped in base, acellular secretory granules, cell proliferation is vigorous.When the xerophilusin Bization adding 2 μMs of concentration
After compound, within 24 hours, observing that cellular morphology changes, its membranolysis, cell proliferation weakens, cell number and Normal group
Comparing and significantly reduce, cell shape is become oval or circle from polygonal, and cell volume increases, and intracellular cavity occur, carefully
Karyon alteration of form is irregular, and in nucleus, chromosome form changes, and cytoplasm core ratio changes, and cell surface highlights film
Blister vesicle, Cell tracking major part disappears, and part cell detachment culture plate diapire also wafts in cell culture medium, culture medium
In be suspended with particulate cellular secretion, culture medium color is dimer, but and finds no muddiness or contamination phenomenon.When adding 5 μ
After the xerophilusin B compound of M concentration, overwhelming majority cell within 24 hours, be can be observed and loses normal cell form completely,
Intracellular structure is disorderly, cellular membrane disruption, and cellular content spills, and cell surface and surrounding are with a large amount of film balloon-shaped structures, carefully
Karyon fragmentation, Cell tracking disappears, and cell adhesion ability significantly reduces, and slight oscillating and shaking culture plate can make attached cell
Departing from culture plate diapire, culture medium color is dark and gloomy, suspension mortality cell and cell fragment, but has not also observed microorganism
Or the sign of pathogen contamination.
Thus, by morphologic observation, it appeared that and confirm that xerophilusin B compound is thin to esophageal squamous cell carcinoma
Intracellular growth has the most lethal, can significantly change cellular morphology and growth activity.
3, Xerophilusin B becomes the impact of knurl effect to nude mice in esophageal squamous cell carcinoma cell body
Experiment is purchased from Fukang bio tech ltd of Beijing China with BALB/cA-nu nude mice, left through 1 week nude mice
After right adaptation cage, animal used as test carrying out ear nail numbered packets at random, often group 5, is divided into 8 groups.Randomly select therein 4
Group, injects 2 × 10 respectively for every nude mice in oxter7Individual KYSE-150 cell, 3 groups additionally are noted in the oxter of every nude mice
Penetrate 2 × 107Individual KYSE-450 cell, animal feeding indoor raising 7 days, and the 3rd day and the 7th day measurement skin after inoculation
The size of lower one-tenth knurl.After hypodermic injection becomes knurl the 7th day, inoculation KYSE-150 cell is real with 8 groups of KYSE-450 cell
Test animal all to survive and all at subcutaneous formation transplantable tumor.
From the beginning of the 7th day after inoculating cell, compound experimental group gives every nude mice abdominal cavity injection 7.5mg/kg every day
The xerophilusin B compound solution 200uL of concentration or 15mg/kg concentration, cis-platinum group 1 2mg/kg concentration of injection in every 2 days
Cis-platinum dilution, blank Normal group is injected Isodose Pluronic F68 solution 200uL, be specifically grouped situation every day
As shown in Figure 10.Each group nude mice of inoculation KYSE-150 cell is injected 19 days after intraperitoneal injection first altogether, compound group
Accepting xerophilusin B compound lumbar injection altogether 19 times, cis-platinum group accepts cisplatin injections intraperitoneal administration 11 times altogether, connects
The each group nude mice planting KYSE-450 cell is injected 20 days after intraperitoneal injection first altogether, and compound group accepts altogether
Xerophilusin B compound lumbar injection 20 times, cis-platinum group accepts cisplatin injections intraperitoneal administration 11 times altogether.Below every group
In every nude mice measure the volume of transplantable tumor before administration, weigh nude mice body weight, and the animation of observation experiment nude mice.
In experimentation, the nude mice body weight of injection Pluronic F68 and compound xerophilusin B increases naturally
Adding, Quick off the mark, appetite is normal, and fur is intensive glossy;And the nude mice of injecting cis-platinum is One's spirits are drooping, tired sleeping few dynamic, fur is done
Puckery, it is off one's feed.Measurement data according to tumor size calculates gross tumor volume and draws tumor growth curve.KYSE-150 is thin in inoculation
Each group nude mice of born of the same parents and each group nude mice of inoculation KYSE-450 cell after inoculation the 26th day with the 27th day, use method of craning one
Putting to death experiment nude mice, carefully strip tumor tissues, record is taken pictures and is weighed afterwards.
In the animal used as test group of inoculation KYSE-150 group (Figure 11, A are curve, and B is phenotype), inject Pluronic F68
The blank group of solution has 1 nude mice owing to knurl body rapid development was exhaustion death in the 17th day, by the time accepting same treatment
Mend after nude mice enters group and participate in final statistical analysis.Low concentration compound by injection 7.5mg/kg concentration xerophilusin B
Group and the blank group of Pluronic F68 solution compare, found that tumour be grown in the 1st~4 day significantly
Difference, but the beginning in the 5th day after inoculating cell is until putting to death the 19th day of animal used as test, injects 7.5mg/kg concentration
It is molten that the volume of the low concentration compound group animal used as test transplantable tumor of xerophilusin B is significantly less than injection Pluronic F68
The blank group animal used as test of liquid, uses Bonferroni post t inspection to find that compound group is transplanted with blank group
Difference between the growth curve of knurl has statistical significance (p < 0.001).Equally, will injection 15mg/kg concentration
The high concentrations of compounds group of xerophilusin B compares with the blank group of Pluronic F68 solution, found that
Being grown in the 1st~3 day of tumour is not significantly different from, but the beginning in the 4th day after inoculating cell is until putting to death animal used as test
The 19th day, the volume of high concentrations of compounds group animal used as test transplantable tumor of injection 15mg/kg concentration xerophilusin B is bright
The aobvious blank group animal used as test less than injection Pluronic F68 solution, uses Bonferroni post t inspection to find
Difference between the growth curve of compound group and blank group transplantable tumor has statistical significance (p < 0.001).
Beginning in the 3rd day after inoculating cell, until putting to death the 19th day of animal used as test, injects the cis-platinum of cisplatin injections
The volume of group animal used as test transplantable tumor is significantly less than the blank group animal used as test of injection Pluronic F68 solution, uses
Bonferroni post t inspection find two groups of transplantable tumors growth curve between difference have statistical significance (p <
0.001).Result illustrates low concentration compound and the injection of lumbar injection 7.5mg/kg concentration xerophilusin B in vivo
The high concentrations of compounds of 15mg/kg concentration xerophilusin B can significantly inhibit the growth of KYSE-150 transplantable tumor.
Equally, in the animal used as test group of inoculation KYSE-450 group (Figure 12, A are curve, and B is phenotype), injection cis-platinum note
The cis-platinum group animal used as test penetrating liquid has 1 nude mice to cause gradually exhaustion and exhaustion in the 19th day due to cisplatin medicine toxic side effects
Death, the candidate nude mice accepting same treatment participate in final statistical analysis after entering group.Will injection 7.5mg/kg concentration
The low concentration compound group of xerophilusin B compares with the blank group of Pluronic F68 solution, result such as figure
Shown in 15A, find tumour be grown in the 1st~7 day be not significantly different from, but beginning in after inoculating cell the 8th day until
Put to death animal used as test the 20th day, the low concentration compound group animal used as test of injection 7.5mg/kg concentration xerophilusin B moves
The volume planting knurl is significantly less than the blank group animal used as test of injection Pluronic F68 solution, uses Bonferroni
Post t inspection find the difference between compound group and the growth curve of blank group transplantable tumor have statistical significance (p <
0.001).Equally, by high concentrations of compounds group and the Pluronic F68 solution of injection 15mg/kg concentration xerophilusin B
Blank group compare, found that tumour be grown in the 1st~6 day be not significantly different from, but from inoculating cell
After beginning in the 7th day until putting to death the 20th day of animal used as test, the high concentration of injection 15mg/kg concentration xerophilusin B
The volume of compound group animal used as test transplantable tumor is significantly less than the blank group animal used as test of injection Pluronic F68 solution, changes
Difference between the growth curve of compound group and blank group transplantable tumor has statistical significance (p < 0.001).
Beginning in the 3rd day after inoculating cell, until putting to death the 20th day of animal used as test, injects the cis-platinum of cisplatin injections
The volume of group animal used as test transplantable tumor is significantly less than the blank group animal used as test of injection Pluronic F68 solution, uses
Bonferroni post t inspection finds that the difference between the growth curve of two groups of transplantable tumors has statistical significance (p <
0.001).Result illustrates low concentration compound and the injection of lumbar injection 7.5mg/kg concentration xerophilusin B in vivo
The high concentrations of compounds of 15mg/kg concentration xerophilusin B can significantly inhibit the growth of KYSE-450 transplantable tumor.
For KYSE-150 transplantable tumor nude mice, within the 19th day after lumbar injection, put to death animal, carefully dissect and peel off transplantable tumor
Weigh afterwards, the low concentration compound animal used as test transplantable tumor of internal lumbar injection 7.5mg/kg concentration xerophilusin B
Weight is significantly less than the blank group animal used as test of lumbar injection Pluronic F68 solution, p < 0.01, two groups of transplantable tumor weights
Difference between amount has statistical significance;Equally, the high concentration of internal lumbar injection 15mg/kg concentration xerophilusin B
The blank group experiment that the weight of compound animal used as test transplantable tumor is significantly less than lumbar injection Pluronic F68 solution is dynamic
Thing, p < 0.001, the difference between two groups of transplantable tumor weight has statistical significance.Similarly, the cis-platinum of cisplatin injections is injected
Group animal used as test transplantable tumor weight be significantly less than injection Pluronic F68 solution blank group animal used as test, p <
0.001, the difference between two groups of transplantable tumor weight has statistical significance.Result explanation 7.5mg/kg concentration xerophilusin
The low concentration compound of B all can become at KYSE-150 cell with the high concentrations of compounds of 15mg/kg concentration xerophilusin B
Knurl experiment can suppress the growth (Figure 13 A) of tumour cell really.
Equally, within the 20th day after lumbar injection, putting to death animal for KYSE-450 transplantable tumor nude mice, knurl body finds after weighing,
The weight of the low concentration compound animal used as test transplantable tumor of internal lumbar injection 7.5mg/kg concentration xerophilusin B is obvious
Less than the blank group animal used as test of lumbar injection Pluronic F68 solution, p < 0.01, between two groups of transplantable tumor weight
Difference has statistical significance;The high concentrations of compounds experiment of internal lumbar injection 15mg/kg concentration xerophilusin B is dynamic
The weight of thing transplantable tumor is significantly less than the blank group animal used as test of lumbar injection Pluronic F68 solution, p < 0.001, and two
Difference between group transplantable tumor weight has statistical significance.The weight of the cis-platinum group animal used as test transplantable tumor of injection cisplatin injections
Amount is significantly less than the blank group animal used as test of injection Pluronic F68 solution, and p < 0.001, between two groups of transplantable tumor weight
Difference there is statistical significance.The low concentration compound of result explanation 7.5mg/kg concentration xerophilusin B and 15mg/
The high concentrations of compounds of kg concentration xerophilusin B all can effectively suppress swollen in KYSE-450 cell becomes knurl experiment
The growth (Figure 13 B) of oncocyte.
By KYSE-150 cell nude mice and KYSE-450 cell nude mice respectively subcutaneous vaccination esophageal squamous cell carcinoma cell 22 days with
Put to death experiment nude mice after 23 days, carefully separate subcutaneous transplantation knurl, after recording and weighing, nude mice model tumor tissue is cut into two
Point, a portion fixes 24 hours with neutral formalin solution at once, then forms wax stone with paraffin-embedded tissue and goes forward side by side
Row histopathologic slide, uses hematoxylin-eosin (Hematoxylin-Eosin, HE) to carry out immunohistochemical staining.Additionally
A part apply the most at once neutral formalin solution fixing give over to standby.When the subcutaneous transplantable tumor of anatomical isolation, permissible
Find low concentration compound animal used as test and 15mg/kg concentration xerophilusin of 7.5mg/kg concentration xerophilusin B
The knurl body of the high concentrations of compounds animal used as test of B, its knurl physique ground relatively consolidation, knurl external surface is wrapped up by layer of transparent tunicle,
Knurl body, without adhesion, can the most promptly be taken off during separation by knurl body itself and skin and surrounding subcutaneous tissue, observes after taking off
To the smooth ovalize in knurl surface, without obvious vascular distribution.Dissecting the sky cutting lumbar injection Pluronic F68 solution
During white control group experiment nude mice by subcutaneous knurl body, finding that knurl body is the most very large, knurl body region pachylosis, subcutaneous have extravasated blood, Ge Biepi
The ulcer that formed after knurl body ulceration seen from skin surface and oozing out, knurl surface hollow, knurl shape is irregular, during anatomical isolation with
Skin adhesion is tight, and knurl body tunicle ulceration also has cauliflower-shaped tubercle bulging, wet periphery (perimeter) musculature, seeks connections with in scapular region and upper
Limb surrounding bone, isolated knurl physique ground is more uniform, and knurl surface can be observed there is a large amount of vascular distribution, after gauze wiping
Hemorrhage substantially, quality relatively consolidation during incision, knurl body structure is finer and close.It addition, cut lumbar injection cisplatin injections dissecting
During cis-platinum group experiment nude mice by subcutaneous knurl body, find that knurl body scope is limited to, but knurl body region pachylosis, knurl surface is concavo-convex not
Flat, during anatomical isolation, knurl surface has tunicle to cover, less with skin adhesion, and during incision, knurl physique ground is harder, and knurl body structure causes
Close (Figure 11 B, 12B).
The transplantable tumor experiment of nude mouse group being vaccinated with esophageal squamous cell carcinoma clone KYSE-150 separated, through neutral first
Aldehyde fixer fixes, is dehydrated, after transparent, waxdip and FFPE, section, the section of each nude mice source tumor specimen is chosen
10 carry out SABC HE dyeing.
Result is as shown in figure 14.In HE stained slice under 100 times of visuals field amplified, in injection 7.5mg/kg concentration
The low concentration compound transplanted tumor in nude mice section of xerophilusin B observes the little necrosis region of patch shape spread all over, through 400
There is the phenomenon that the obvious pyknosis of nucleus, dyeing are deepened in the esophageal squamous cell carcinoma cell that local lesion be can be observed after times magnification field.
The high concentrations of compounds KYSE-150 nude mice model of 15mg/kg concentration xerophilusin B has been injected in anatomical isolation cutting
During knurl knurl body, finding that obvious large area cheese shape necrosis region or liquefaction stove occurs in its central part, content is formed after getting rid of
Bigger downright bad chamber, cuts into slices its HE under 100 times of fields of microscope amplified, can be observed at transplantable tumor knurl body central part
There is a cavity that large area liquefaction and necrosis causes, it can be seen that the cell tissue fragment of liquefaction in cavity.And in HE stained slice 400
Observe under the visual field amplified again, find that the esophageal squamous cell carcinoma cell of cavity periphery focus occurs that karyopyknosis, intensification of dyeing etc. are thin
The sign of born of the same parents' apoptosis.When Microscopic observation has injected the KYSE-150 blank group transplanted tumor in nude mice knurl of Pluronic F68 solution
During body, homogeneous without downright bad in finding its knurl body under 100 times of visuals field amplified in HE stained slice, tumour cell arrangement is tight
Close, in HE stained slice under 400 times of visuals field amplified, cell nucleus split coil method be can be observed obvious, chromosome is thick, cell
Core ratio is high, and cell proliferation capacity is vigorous.Microscopic observation has injected the cis-platinum group KYSE-150 experiment nude mice model of cisplatin injections
During knurl knurl body, in finding its knurl body under 100 times of visuals field amplified in HE stained slice, quality is tight, and tumour cell arrangement is tight
Close, connective tissue it is distributed between tumour cell, focal can be observed with the presence of stigma sheet meronecrosis stove, in HE stained slice
Under upper 400 times of visuals field amplified, cell nucleus split coil method be can be observed obvious, chromosome is thick, and nuclear ratio is high, and cell increases
Grow power vigorous.
Equally, under the same conditions the experiment of nude mouse group being vaccinated with esophageal squamous cell carcinoma clone KYSE-450 is separated
Transplantable tumor.Result as shown in figure 14, in HE stained slice under 100 times of visuals field amplified, is injecting 7.5mg/kg concentration
The large area cavity shape that i.e. can be observed to spread all in the low concentration compound transplanted tumor in nude mice section of xerophilusin B is downright bad
Stove and liquefaction stove, the esophageal squamous cell carcinoma cell that local lesion be can be observed after 400 times of magnification fields occurs that nucleus is obvious equally
Pyknosis, dyeing are deepened.In the high concentrations of compounds group transplantable tumor knurl body of 15mg/kg concentration xerophilusin B, find it
There is obvious large area cheese shape necrosis region or liquefaction stove in central part, and content forms bigger downright bad chamber after getting rid of, 100
The cavity that at transplantable tumor knurl body central part have large area liquefaction and necrosis cause be can be observed, it can be seen that liquid in cavity under times visual field
The cell tissue fragment changed.And under 400 times of visuals field, find that the esophageal squamous cell carcinoma cell of cavity periphery focus occurs that cell shape changes
The apoptotic signs such as change, karyopycnosis, dyeing intensification.Observe the blank group knurl body of injection Pluronic F68 solution
Time, spreading all over thick vessel lumen in finding its knurl body under 100 times of visuals field, the vessel lumen of expansion is by the tumour knurl of rapid development
Body compressing deformation is in irregular, and knurl body is homogeneous without downright bad, and tumour cell arranges closely, observable under 400 times of visuals field
Obvious to cell nucleus split coil method, chromosome is thick, and nuclear ratio is high, and cell proliferation capacity is vigorous.Microscopic observation injection cis-platinum
During the cis-platinum group transplantable tumor knurl body of parenteral solution, in finding its knurl body under 100 times of visuals field, quality is tight, and tumour cell arrangement is tight
Close, connective tissue it is distributed between tumour cell, focal can be observed with the presence of stigma sheet meronecrosis stove, in 400 times of visuals field
Under that cell nucleus split coil method be can be observed is obvious, chromosome is thick, and nuclear ratio is high, and cell proliferation capacity is vigorous.
The result of HE stained slice shows, compound xerophilusin B can effectively suppress interior tumor cell to increase
Grow, promote death of neoplastic cells, from Microscopic observation to a series of phenomenon speculate that it causes the reason machine of death of neoplastic cells
System may be for realize by apoptosis, and its concrete mechanism needs progressively to be verified by following experiment further.
4, the Xerophilusin B impact on esophageal squamous cell carcinoma cell cycle distribution
Cause esophageal squamous cell carcinoma cell dead with experiment in vitro in vivo for research compound xerophilusin B further
The reason die, suppressed knurl body to increase and mechanism, after agent-feeding treatment, collect cell and use ethanol to fix, passing through
PI dyeing uses Flow Cytometry detection analysis of compounds xerophilusin B (2 μMs and 5 μMs) for the cell cycle
Impact.It addition, use cycle stain detection technique to verify that tumour cell cycle is divided by compound xerophilusin B further
The impact of cloth.
1), after being got off with 0.25% trypsinization by KYSE-150 Yu the KYSE-450 cell being in logarithmic phase growth, phase is used
The complete medium piping and druming answered is resuspended, in cell counter counts and is inoculated in six orifice plates, and every porocyte number about 2 × 103Individual/
Hole.When carrying out cell inoculation, six orifice plates should be rocked up and down, to avoid cell in left and right the most gently after in cell suspension inlet hole
At cluster agglomeration and, cause superposition growth local overfill, affect subsequent treatment.
2) at 37 DEG C and the CO of 5%2Cell culture incubator is cultivated 12 hours, after treating that cell is the most adherent, observes with really
Determine cell degrees of fusion and reach 60~more than 70%.
3), before dosing, inhale with suction pipe and abandon original culture medium in hole, be added dropwise to fresh complete medium 2mL, real in six orifice plates
Test and group adds the xerophilusin B solution 20ul/ hole that concentration is 200 μMs or 500 μMs, system herb liquid after therefore adding
Concentration is 2 μMs and 5 μMs.Control group adds equivalent DMSO solvent, often organizes and sets 3 parallel multiple holes.
4), after dosing 24 hours, serum-free RPMI1640/DMEM/MEM culture medium is changed, at 37 DEG C and the CO of 5%2Carefully
Born of the same parents' incubator cultivated 12 hours or overnight cell is carried out synchronization process, then changing containing 10% hyclone
RPMI1640/DMEM/MEM complete medium continues in incubator and cultivates 24 hours.
5) original complete medium is abandoned in suction, rinses cell 3 times with PBS phosphate buffer, then uses 0.25% pancreatin
Vitellophag, uses containing the RPMI1640/DMEM/MEM complete medium suspension cell of 10% hyclone, through of short duration from
Use PBS phosphate buffer to rinse cell after the heart, of short duration centrifugal after supernatant discarded.
6) use after 1 × Buffer A buffer solution cell, of short duration centrifugal and discard under conditions of 1500 revs/min
Clear liquid, repeats above step 3 times, adds appropriate 1 × Buffer A buffer solution with re-suspended cell, collects and utilize cell counter
Carry out cell count, make the ultimate density of every porocyte suspension reach 1~2 × 106Individual/mL.
7) in cell suspending liquid, add 70% ethanol solution of 9 times of volumes, blow and beat mixing gently, solid under the conditions of 4 DEG C
Fixed at least 24 hours (4 DEG C of lucifuges are fixing overnight, or-20 DEG C of lucifuges are fixed for a long time).Should be noted during operation and can not acutely shake dress
The Ep having fixing cell manages, in order to avoid damaging cells.
8) before detection, being taken out by fixing cell, under conditions of 1500 revs/min, of short duration collected after centrifugation cell, makes afterwards
With 1 × Buffer A buffer solution cell to remove ethanol, fixing cell is resuspended in 1 × Buffer A buffering of 400 μ L
In liquid.
9) adding 100ul concentration in every hole is that the RNaseA solution of 1% is to avoid RNA to disturb so that it is final concentration reaches
0.25mg/mL, is placed in 37 DEG C of water baths and hatches 30 minutes.Water-bath it is placed in again equipped with after the Ep pipe application sealed membrane sealing of cell
In case, in order to avoid steam enters affects experimental result.
10) Ep pipe is taken out from water bath, wiping surface water smoke, it can be observed that in pipe, cell settlement is at the bottom of pipe, cuts
The most acutely shake or mix, rupturing in order to avoid damaging cells causes it.In every hole, add the PI solution of 5 μ L, keep away at ambient temperature
Light hatches at least 30 minutes.Then sucking in streaming pipe by suspension rapidly, application flow cytometer detects.Hand is used before detection
Refer to that bullet strikes streaming pipe gently, promotes that it mixes, to avoid blocking flow cytometer detection probe.
11) application ModFit LT3.2 (Verity Software House Co., USA) analysis software calculates and analyzes
G0/G1Phase, S phase and G2The percentage of each phase cell of/M phase.
For esophageal squamous cell carcinoma clone KYSE-150, as shown in figure 15, in Normal group, leftmost blue peak generation
The ratio shared by cell of apoptosis occurs in table all detections cell, and on the right side of it, the red peak of high point represents G1Phase cell institute accounting
Example, on the right side of this peak, slightly lower short red peak represents G2/ M phase cell proportion, the light gray short peak generation in the middle of two red peaks
Ratio shared by table S phase cell.After when adding 2 μMs of concentration xerophilusin B compound solutions 24 and being little, flow cytometry
Testing result shows its G2Average proportions shared by/M phase cell is 31.82%, higher than Normal group cell G2Shared by/M phase cell
Average proportions (22.27%), difference statistically significant (p < 0.001);Also detect that its G1Phase cell proportion is
32.46%, less than cellular control unit G1Average proportions (45.45%) shared by phase cell, difference statistically significant (p <
0.001);Additionally, its S phase cell proportion is 35.72%, higher than average proportions shared by Normal group cell S phase cell
(32.27%), although its difference is not statistically significant (p > 0.05), but its ratio is it is also seen that for rising trend.Working as
When the concentration of compound xerophilusin B reaches 5 μMs, the display of Flow cytometry result occurs the cell proportion of apoptosis to reach
39.67%, it is significantly higher than Normal group Apoptosis proportion (1.34%) and 2 μMs of xerophilusin B are real
Testing group Apoptosis proportion (2.84%), its difference all has significant statistical significance (p < 0.001).
Similarly, for esophageal squamous cell carcinoma clone KYSE-450, as shown in figure 15, when adding 2 μMs of concentration
Xerophilusin B compound solution is after 24 hours, and Flow cytometry result shows its G2Average specific shared by/M phase cell
Example is 31.78%, higher than Normal group cell G2Average proportions (19.65%) shared by/M phase cell, difference has statistics to anticipate
Justice (p < 0.001);Also detect that its G1Phase cell proportion is 33.91%, less than cellular control unit G1Shared by phase cell averagely
Ratio (51.89%), difference statistically significant (p < 0.001);Additionally, its S phase cell proportion is 34.31%, it is higher than
Average proportions (28.46%) shared by Normal group cell S phase cell, its difference statistically significant (p < 0.01).Work as chemical combination
When the concentration of thing xerophilusin B reaches 5 μMs, Flow cytometry result shows that it occurs the cell proportion of apoptosis high
Reach 62.05%, be significantly higher than Normal group Apoptosis proportion (1.56%) and 2 μMs of xerophilusin B
Experimental group Apoptosis proportion (1.75%), its difference all has significant statistical significance (p < 0.001).
Above-mentioned test result indicate that, in esophageal squamous cell carcinoma clone KYSE-150 cell and KYSE-450 cell, work as addition
After concentration is the compound xerophilusin B of 2 μMs, it is possible to the cycle of tumour cell is made a significant impact, its G2/ M the phase is thin
Born of the same parents' ratio significantly raises, G1Phase cell proportion is remarkably decreased, and difference all has statistical significance, and S phase cell has rising trend, by
This it can be said that under this low concentration xerophilusin B mainly make cell block in G by affecting the cell cycle2/ M the phase.And work as
When compound concentration is increased to high concentration 5 μMs, death of neoplastic cells cell concentration significantly increases, therefore, and high concentration
Xerophilusin B is mainly by promoting Apoptosis suppression cell proliferation.
5, Xerophilusin B impact apoptotic on esophageal squamous cell carcinoma
In order to verify that the cell caused about compound xerophilusin B in cytomorphology and cycle detection withers
The testing result died, further with Flow Cytometry, uses FITC-AnnexinV and PI to dye respectively and detection compound
The xerophilusin B impact on esophageal squamous cell carcinoma clone KYSE-150 cell Yu KYSE-450 Level of Apoptosis.
1), after being got off with 0.25% trypsinization by KYSE-150 Yu the KYSE-450 cell being in logarithmic phase growth, phase is used
The complete medium piping and druming answered is resuspended, in cell counter counts and is inoculated in six orifice plates, and every porocyte number about 2 × 103Individual/
Hole.When carrying out cell inoculation, six orifice plates should be rocked up and down, to avoid cell in left and right the most gently after in cell suspension inlet hole
At cluster agglomeration and, cause superposition growth local overfill, affect subsequent treatment.
2), before dosing, inhale with suction pipe and abandon original culture medium in hole, be added dropwise to fresh complete medium 2mL.
3) at 37 DEG C and the CO of 5%2In cell culture incubator cultivate 12 hours adherent after, make cell degrees of fusion reach 60~
More than 70%, six orifice plates add in experimental group the xerophilusin B solution 20uL/ hole that concentration is 200 μMs or 500 μMs, because of
After this adds, system herb liquid concentration is 2 μMs and 5 μMs.Control group adds equivalent DMSO solution.Often organize and set 3 parallel multiple holes.
4) 24h and 48 hours after dosing, inhale with suction pipe and abandon former culture medium, rinse with the PBS phosphate buffer of cooling
Cell 3 times, uses 0.25% trypsin digestion and cell, then with complete medium, cell is blown and beaten from six orifice plate walls flushing
Get off, use containing the RPMI1640/DMEM/MEM complete medium suspension cell of 10% hyclone, transfer to 15ml from
In heart pipe, after of short duration being centrifuged, clean cell 2 times with the PBS phosphate buffer of cooling.Note pancreatin can not contain
Ethylenediamine tetra-acetic acid (EDTA), cell dissociation time and PBS phosphate buffer scavenging period are unsuitable long, otherwise easily cause
The false positive of result.
5) 10 × AnnexinV in kit is combined buffer solution (0.1M Hepes/NaOH (pH 7.4), 1.4M's
The CaCl of NaCl, 25mM2) be diluted according to the ratio aseptic double-distilled water of 1:10, it is configured to streaming and combines buffer solution.
6) cell suspension is added in centrifuge tube, carry out of short duration centrifugal 5 minutes according to the speed conditions of 800r/min, use
The AnnexinV newly prepared combines buffer solution working solution, and cell is resuspended, then uses cell counter to carry out cell count,
The ultimate density making cell suspension reaches at least 1~2 × 106Individual/mL.
7) from cell suspension sucking-off 100 μ L (containing 1~2 × 105Individual cell) transfer to, in new aseptic Ep pipe, carry out
Operation it is put on ice for after mark.
8) adding the FITC-AnnexinV stain of 5 μ L in Ep pipe, in super-clean bench, lucifuge stands 15 at ambient temperature
~20 minutes.
9) adding the PI dye solution of 5 μ L in Ep pipe, lucifuge stands about 5 minutes at ambient temperature, notes for often
A kind of cell KYSE-140, KYSE-150, KYSE-450, KYSE-510, NIH-3T3 or HEK-293 to be measured, it is necessary to prepare single dye
With blank cell in order to set fluidic cell design parameter, i.e. reserve 3 pipe 100 μ L (containing 2 × 105Individual cell) cell hang
Liquid, wherein 1 pipe is not added with any reagent, and 1 pipe only adds the FITC-AnnexinV dye solution of 5 μ L, and 1 pipe only adds the PI dye of 5 μ L
Material solution.
10), on flow cytometer before machine, the AnnexinV adding 400 μ L in each Ep pipe combines buffer solution work
Liquid is hatched, within 15 minutes on machine testing.Should be noted during operation and can not acutely shake the pipe equipped with fixing cell, in order to avoid
Damaging cells.
11) being sucked by suspension rapidly in streaming pipe during detection, application flow cytometer detects.Detect front finger light
Flick and strike streaming pipe, promote that it mixes, to avoid blocking flow cytometer detection probe.
12) in the testing result of lower machine, the abscissa shown in streaming apoptosis figure is AnnexinV FITC, ordinate
Being PI, in upper left, upper right, lower-left and four, bottom right quadrant, right upper quadrant represents the cell of dead, and left lower quadrant is surviving
Cell, what right lower quadrant represented is the cell of apoptosis.
Testing result is as shown in Figure 16 A, C, for esophageal squamous cell carcinoma clone KYSE-150 cell, when adding 2 μMs of concentration
Xerophilusin B compound solution is after 24 hours, it can be seen that represent in the right lower quadrant of viable apoptotic cell in streaming figure
Cell proportion and represent the cell proportion of non-viable apoptotic cell, with the cell proportion phase of all quadrants in blank Normal group
Compare showed increased.Flow cytometry result shows the cell proportion i.e. early apoptosis of its FITC-AnnexinV stained positive
Cell is 21.67% with the summation ratio of non-viable apoptotic cell, higher than AnnexinV dyeing sun in blank Normal group cell
Property ratio (5.13%), difference tool be statistically significant (p < 0.001).When adding 2 μMs of concentration xerophilusin B
After compound solution 48 hours, in streaming figure, it can be seen that represent cell proportion in the right lower quadrant of viable apoptotic cell with
And represent the cell proportion of non-viable apoptotic cell, significantly increase compared with the cell proportion of all quadrants in blank Normal group.
Flow cytometry result shows that the cell proportion i.e. viable apoptotic cell of its FITC-AnnexinV stained positive withered with late period
The summation ratio of cell of dying is 35.17%, higher than the ratio of AnnexinV stained positive in blank Normal group cell
(6.07%), difference tool is statistically significant (p < 0.001).When adding, 5 μMs of concentration xerophilusin B compounds are molten
After liquid 24 hours, it will also be seen that the cell proportion represented in the right lower quadrant of viable apoptotic cell and generation in streaming figure
The cell proportion of table non-viable apoptotic cell, showed increased compared with the cell proportion of all quadrants in blank Normal group, wherein
Viable apoptotic cell ratio increase is the most notable.Flow cytometry result shows its FITC-AnnexinV stained positive
Cell proportion i.e. viable apoptotic cell is 88.47% with the summation ratio of non-viable apoptotic cell, and rising becomes apparent from, with blank just
Often in cellular control unit, the ratio of AnnexinV stained positive is compared, and difference tool is statistically significant (p < 0.001).When adding
Enter 5 μMs of concentration xerophilusin B compound solutions after 48 hours, it can be seen that represent viable apoptotic cell in streaming figure
Cell proportion in right lower quadrant and represent the cell proportion of non-viable apoptotic cell, with all quadrants in blank Normal group
Cell proportion is compared and is significantly increased.Flow cytometry result shows the cell proportion of its FITC-AnnexinV stained positive
I.e. viable apoptotic cell is 94% with the summation ratio of non-viable apoptotic cell, higher than AnnexinV in blank Normal group cell
The ratio of stained positive, difference tool is statistically significant (p < 0.001).
For esophageal squamous cell carcinoma clone KYSE-450 cell as shown in Figure 16 B, C, when adding 2 μMs of concentration xerophilusin
After B compound solution 24 hours, in streaming figure it can be seen that the cell proportion represented in the right lower quadrant of viable apoptotic cell with
And represent the cell proportion of non-viable apoptotic cell, showed increased compared with the cell proportion of all quadrants in blank Normal group.
Flow cytometry result shows that the cell proportion i.e. viable apoptotic cell of its FITC-AnnexinV stained positive withered with late period
The summation ratio of cell of dying is 21.67%, higher than the ratio of AnnexinV stained positive in blank Normal group cell
(4.07%), difference tool is statistically significant (p < 0.001).When adding, 2 μMs of concentration xerophilusin B compounds are molten
After liquid 48 hours, it can be seen that the cell proportion represented in the right lower quadrant of viable apoptotic cell and represent late period in streaming figure
The cell proportion of apoptotic cell, significantly increases compared with the cell proportion of all quadrants in blank Normal group.Flow cytometry
Testing result shows the cell proportion i.e. viable apoptotic cell of its FITC-AnnexinV stained positive and the total of non-viable apoptotic cell
Being 39.87% with ratio, higher than the ratio (4.53%) of AnnexinV stained positive in blank Normal group cell, difference has
It is statistically significant (p < 0.001).After when adding 5 μMs of concentration xerophilusin B compound solutions 24 and being little, at stream
It will also be seen that the cell proportion represented in the right lower quadrant of viable apoptotic cell and represent non-viable apoptotic cell in formula figure
Cell proportion, showed increased, wherein viable apoptotic cell ratio compared with the cell proportion of all quadrants in blank Normal group
Example increase is the most notable.Flow cytometry result shows the cell proportion of its FITC-AnnexinV stained positive the most in early days
The summation ratio of apoptotic cell and non-viable apoptotic cell is 90.97%, and rising becomes apparent from, and in blank Normal group cell
The ratio of AnnexinV stained positive is compared, and difference tool is statistically significant (p < 0.001).When adding 5 μMs of concentration
Xerophilusin B compound solution is after 48 hours, it can be seen that represent in the right lower quadrant of viable apoptotic cell in streaming figure
Cell proportion and represent the cell proportion of non-viable apoptotic cell, with the cell proportion phase of all quadrants in blank Normal group
Ratio significantly increases.Flow cytometry result shows the cell proportion i.e. early apoptosis of its FITC-AnnexinV stained positive
Cell is 96.57% with the summation ratio of non-viable apoptotic cell, far above AnnexinV dyeing in blank Normal group cell
Positive ratio, difference tool is statistically significant (p < 0.001).
The above results shows, compound xerophilusin B can promote esophageal squamous cell carcinoma clone KYSE-150 cell and
Apoptosis occurring in KYSE-450 cell, makes the cell proportion being in the apoptosis stage raise, difference tool is statistically significant.
6, Xerophilusin B is on the impact of apoptosis pathway protein expression in esophageal squamous cell carcinoma cell
Can draw from above experimental studies results, compound xerophilusin B can pass through inducing cell apoptosis
Suppress the esophageal squamous cell carcinoma cell proliferation of in vitro culture and suppress the growth of Inoculation transplantable tumor, for further further investigated chemical combination
Thing xerophilusin B causes apoptotic molecular mechanism, is extracted variable concentrations compound xerophilusin B effect
The albumen of esophageal squamous cell carcinoma cell front and back, and use Western blot Western blot detection technique quantitative detecting analysis to wither with cell
Die closely-related endogenous signaling pathway protein, i.e. caspase-9 Yu caspase-3 of dependent cells pigment c activity cascades
Activation signal path.
This research is used for detect cytochrome c, pro-caspase-9cleaved-caspase-9, pro-
The antibody of caspase-3, cleaved-caspase-3, pro-caspase-7, PARP, Bax and Bcl-2 protein expression level
Purchased from CST (Cell Signaling Technology, Danvers, MA, USA) company, the anti-human list of rabbit of above each albumen
Clonal antibody i.e. Rabbit anti-cytochrome c, pro-caspase-9cleaved-caspase-9, pro-
Caspase-3, cleaved-caspase-3, pro-caspase-7, PARP, Bax and Bcl-2monoclone
Antibody, purchased from CST company, the β-actin antibody as internal reference is purchased from Albgent (San Diego, USA) company.
Anti-and the above each antibody of β-actin two two is anti-is purchased from ProteinTech company (Chicago, USA).
Extract esophageal squamous cell carcinoma clone KYSE-150 and KYSE-450 total protein of cell, use BCA Protein Assay
Kit (Themo, Rockford, USA) kit preparation standard protein concentration gradient solution also detects sample to be tested protein concentration,
Carrying out total protein, one resists for rabbit anti-human monoclonal antibodies, and two resist for goat-anti rabbit.
It is 0.5 μM in the intracellular addition concentration cultivated, 1 μM, the xerophilusin B compound of 2 μMs and 5 μMs, add
Medicine extracts the antibody adding internal reference albumen β-actin after total protein of cell after 24 hours, find after development, under the same terms for
KYSE-150 cell, the cell after the xerophilusin B compound treatment of 0.5 μM, 1 μM and 2 μMs concentration, it is intracellular
The content of β-actin albumen and the content no significant difference of Normal group intracellular β-actin albumen, and work as compound concentration
When rising reaches 5 μMs, detect that intracellular β-actin protein content substantially reduces.Similarly, for KYSE-450 cell, 0.5
μM, cell after the xerophilusin B compound treatment of 1 μM and 2 μMs concentration, the content of its intracellular β-actin albumen
Though having lowered but without significant difference with the content of Normal group intracellular β-actin albumen, and when compound concentration raises
When reaching 5 μMs, detect that intracellular β-actin protein content is reduced to distinguish.Therefore, by 0.5 μM, 1 μM, 2 μMs of concentration
The concentration range (Figure 17) that detects as follow-up esophageal squamous cell carcinoma intracellular apoptotic signal pathway protein molecule content of compound.
As shown in figure 18, when adding 1 μM of denier in esophageal squamous cell carcinoma cell system KYSE-150 cell
After xerophilusin B compound, the expressing quantity making cytochrome c as Apoptosis promotive factor is significantly raised,
After compound concentration is increased to 2 μMs, cytochrome c protein expression significantly raises.The cytochrome c that expression raises swashs
The downstream cystine protein kinase caspase-9 that lives makes it be degraded into active by inactive pepsinogen form (47kDa)
Enzyme form (37kDa), it can be observed that along with the concentration of compound xerophilusin B gradually rises, Pro-caspase-
9 protein levels are substantially lowered, and simultaneous its shear pattern Cleaved caspase-9 albumen table level goes up step by step
Rise.When the key protein factor cytochrome c protein finding inducing apoptosis raises with Pro-caspase-9 protein content level
After, continue deeper into detection downstream cascade activating reaction chain on key factor caspase-3, testing result show along with
The concentration of compound xerophilusin B gradually rises, and Pro-caspase-3 zymogen protein level is substantially lowered, and simultaneously
It is gradually increasing along with its shear pattern Cleaved caspase-3 albumen is flat, and also caspase-3 kinases downstream detected
Pro-caspase-7 zymogen protein level with compound concentration level raise and reduce.As the receptor of DNA damage,
PRAP albumen can be degraded as cutting substrate by caspase family kinase, thus it is malfunctioning, finally to cause DNA damage to be repaired
Apoptosis.Research detects that PRAP protein level is gradually along with compound xerophilusin B concentration gradually rises
Declining, when concentration reaches 2 μMs, protein level is significantly lowered, and this has also confirmed examines in cytomorphology and fluidic cell apoptosis
The result obtained in survey.Equally, similar result be have also discovered for esophageal squamous cell carcinoma cell system KYSE-450 cell, along with
The expressing quantity that the concentration of compound xerophilusin B gradually rises cytochrome c is significantly raised, works as compound concentration
After being increased to 2 μMs, cytochrome c protein expression significantly raises.Pro-caspase-9 protein level is substantially lowered, and
Simultaneous its shear pattern Cleaved caspase-9 albumen table level steps up.Pro-caspase-3 zymogen protein
Level is substantially lowered, and simultaneous its shear pattern Cleaved caspase-3 albumen is put down and is gradually increasing, downstream
Pro-caspase-7 zymogen protein level raises with compound concentration level and reduces.Additionally also detect that along with compound
Xerophilusin B concentration gradually rises, and causes DNA to repair enzyme PARP (poly ADP-ribose polymerase) albumen
Level is gradually reduced.
In apoptosis regulation mechanism, the increase of mitochondrial membrane permeability can make the apoptosis activity factor of intraor extracellular enter
Mitochondria, or make Intramitochondrial apoptosis promotive factor enter in cell cytosol, thus cause apoptotic program to start, at this
During, the Bcl-2 albumen of the Bcl-2 protein family molecule being positioned on mitochondrial membrane, as promoting cells survival (i.e. anti-apoptotic)
The factor, plays the regulation effect of key, and another member's Bax albumen of this family is for promoting antiapoptotic factors simultaneously, it is possible to Bcl-2 egg
The turnover of the factor inside and outside Bai Gongtong regulation mitochondria.Therefore, for inquiring into whether compound xerophilusin B is by impact
The expression of Bcl-2 family protein promotes the apoptosis inducing factor in mitochondria to be released in cell, have detected addition compound
Bcl-2 albumen and Bax protein expression level in cell before and after xerophilusin B.β-actin is as internal reference albumen in application.
As shown in Figure 19 A, when adding xerophilusin Bization in esophageal squamous cell carcinoma cell system KYSE-150 cell
After compound, it can be observed that along with the concentration of compound xerophilusin B gradually rises, Bax protein level substantially raises,
There is downward trend in Bcl-2 protein expression level simultaneously.Similarly, when in esophageal squamous cell carcinoma cell system KYSE-450 cell
After adding xerophilusin B compound, also observe along with the concentration of compound xerophilusin B gradually rises, Bax
Protein level substantially raises, and downward trend occurs in Bcl-2 protein expression level simultaneously.Further, through to Bcl-2 albumen with
Bax expressing quantity is the analysis of ratio after β-actin calibrates, and finds along with compound xerophilusin B concentration gradually
Raising, Bcl-2/Bax ratio is gradually lowered, for KYSE-150 cell, when compound concentration is 0.5 μM, 1 μM and 2 μMs, its
Bcl-2/Bax ratio is respectively 0.72,0.2 and 0.18.For KYSE-450 cell, when compound concentration is 0.5 μM, 1 μM and 2
μM time, its Bcl-2/Bax ratio respectively 0.51,0.43 and 0.14, the most gradually successively decrease (Figure 19 B).
Thus, the above results shows, compound xerophilusin B is promoted by regulation BCL-2 with Bax protein level
Membrane passage increases, so that the entrance of ectogenic cytochrome c is intracellular or in mitochondria, causes cystine subsequently
Protease casepase-9, caspase-3 and caspase-7 albumen is changed into activity shape by inactive protein zymogen forms
Formula, and then there is a series of cascade activating reaction, finally resulting in PARP degraded, DNA damage cannot be repaired and active cell apoptosis
Program, cell is the most dead.
7, the safety evaluatio of Xerophilusin B anti-esophageal squamous cell carcinoma cell
1), the Xerophilusin B impact on normal clone NIH-3T3 with HEK-293 growth
For the safety evaluatio of compound be research compound whether can specific killing tumour cell thus become anti-
The important step of tumour medicine precursor.In order to whether research xerophilusin B can be to esophageal squamous cell carcinoma cell further
Specific killing action plays in system, have detected again compound xerophilusin B to the two i.e. mice embryonics of strain normal cell system
The toxic action of fibroblast NIH-3T3 and human embryonic kidney cell line HEK293, respectively after dosing 0 hour, 6 hours, 12
Hour, 24 hours, 48 hours and after 72 hours, application Cell Counting Kit-8 method detection xerophilusin Bization
Compound light absorption value i.e. OD value at the 450nm that its concentration is when 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, draws and increases
Value curve also calculates growth inhibition ratio.
As shown in figure 20, for add concentration gradient be 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs
Xerophilusin B compound 0 hour, 6 hours, 12 hours, 24 hours, 48 hours and after 72 hours, NIH-3T3 cell
Growth curve situation with HEK-293 cell.Can clearly find out that blank group is thin for NIH-3T3 Yu HEK-293 cell
Born of the same parents' passage in time demonstrates that cell number gradually increases, the linear trend that absorbance is gradually increased;Along with xerophilusin
B compound concentration is stepped up, and the cell proliferation rate of its correspondence gradually slows down, and growth curve trend declines.
According to cell proliferation experiment data result, for more clearly comparative compound to Cytostatic to tumor cell effect
Effect, be converted and become cell proliferation inhibition rate.As it is shown in fig. 7, for adding xerophilusin B compound effects 72
Inhibitory rate of cell growth situation after hour.In NIH-3T3 clone, xerophilusin B compound after acting on 72 hours
When its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, growth inhibition ratio is respectively 4.04%, 3.59%,
11.44%, 29.78%, 65.30% and 94.30%;In HEK-293 clone, xerophilusin after acting on 72 hours
B compound when its concentration is 0.1 μM, 0.5 μM, 1 μM, 2 μMs, 5 μMs and 10 μMs, growth inhibition ratio is respectively 0,0.36%,
3.44%, 35.77%, 83.32% and 98.97%.It addition, according to above xerophilusin B compound at esophageal squamous cell carcinoma
Inhibiting rate in cell, calculates compound effects IC after 72 hours50Value.As shown in Figure 8, at NIH-3T3 cell and HEK-
IC in 293 cells50Value is respectively 2.61 μMs and 3.24 μMs, far above at esophageal squamous cell carcinoma clone KYSE-150 cell (1.15 μ
M) with the IC of KYSE-450 cell (1.70 μMs)50Value.
Although it is apparent that xerophilusin B compound is to NIH-3T3 Yu HEK-293 two strain normal cell system
Growth inhibition effect all raise with concentration gradient and also show the trend gradually strengthened, but the most each concentration
Xerophilusin B compound is below xerophilusin B compound pair to the growth inhibition ratio of two strain normal cell systems
Esophageal squamous cell carcinoma clone KYSE-150 cell and the growth inhibition ratio of KYSE-450 cell, compound xerophilusin B has
Certain esophageal squamous cell carcinoma cell growth inhibition is specific.
2), the Xerophilusin B impact on nude mice liver kidney merit
By In vitro cell experiment detect compound xerophilusin B kill while esophageal squamous cell carcinoma cell right
There is relatively low toxicity in normal cell cording, come for inquiring into its toxic action introducing internal Xenografts in nude mice experiment further
The security of Comprehensive Evaluation compound xerophilusin B.Xenografts in nude mice experiment and intraperitoneal injection chemical combination in vivo
At the end of thing experiment, after using eyeball rear vein beard blood taking method to gather nude mice peripheral blood, carry out liver function and examine with kidney merit serum biochemistry
Survey.It is as shown in the table for testing result, detects project totally 16, and detection data result is as shown in table 3.
Table 3 is that Xerophilusin B is to KYSE-150 Yu KYSE-450 transplantable tumor nude mouse serum liver kidney merit biochemical indicator
Impact
Unit: ALT (U/L), AST (U/L), TP (g/L), ALB (g/L), GLOB (g/L), TBIL (μm ol/L), ALP (U/
L),GGT(U/L),GLU(mmol/L),UN(mmol/L),CREA(μmol/L),UA(μmol/L),CHO(mmol/L),TG
(mmol/L),LDH(U/L).(*p<0.05,**p<0.01,and***p<0.001).
Wherein ALT, AST, CREA and UN are the reflection the most representational biochemical indicators of hepatic and renal function.As illustrated in fig. 21,
For KYSE-150 cell transplantation knurl nude mice, the xerophilusin B compound that lumbar injection concentration is 7.5mg/kg 19 days
After, detect that serum alt level is 36.6U/L, with the ALT level (38.2U/L) of the control group of injection Pluronic F68
Compare, no significant difference (p > 0.05), at the xerophilusin B compound that lumbar injection concentration is 15mg/kg
After, serum alt level is 56.8U/L, compared with control group, and no significant difference (p > 0.05), and at lumbar injection
Concentration is in the nude mouse serum of every 2 days cisplatin injections of 2mg/kg/, detects that ALT level is up to 95.4U/L, far above other
Each group, difference is statistically significant (p < 0.001) compared with the control.Equally, also detected that injection concentration is 7.5mg/
Kg and concentration be 15mg/kg xerophilusin B compound after in serum AST level be respectively 145U/L and 160.2U/L,
Compared with control group, difference all not statistically significants (p value is equal > 0.05), and in the nude mouse serum of injection cis-platinum, detect
AST level is up to 361U/L, and far above other each group, difference is statistically significant (p < 0.001) compared with the control.Explanation
Compound xerophilusin B is on KYSE-150 nude mice hepatic functional index without impact, and liver function is created significantly by cis-platinum
Toxic reaction.Similarly, it is 7.5mg/kg's to KYSE-450 cell transplantation knurl nude mice in lumbar injection concentration
Xerophilusin B compound is after 20 days, and serum alt level is 43.4U/L, and injection concentration is 15mg/kg's
In the nude mouse serum of xerophilusin B compound, ALT level is 53.2U/L, and compared with control group (50.4U/L), difference is equal
Not statistically significant (p > 0.05).Equally, at the xerophilusin B compound that injection concentration is 7.5mg/kg, in serum
AST level is 143U/L, injection concentration be the xerophilusin B compound of 15mg/kg nude mouse serum in ALT level be
302U/L, compared with control group (158U/L), the equal not statistically significant of difference (p > 0.05) (Figure 21 B).
To KYSE-150 cell transplantation knurl nude mice at the xerophilusin B chemical combination that lumbar injection concentration is 7.5mg/kg
After thing 19 days, in serum, CREA level is 11.11 μm ol/L, and injection concentration is the xerophilusin B compound of 15mg/kg
Nude mouse serum in CREA level be 9.44 μm ol/L, compared with control group (11.74 μm ol/L), the equal not statistically significant of difference
(p>0.05).Equally, at the xerophilusin B compound that injection concentration is 7.5mg/kg, in serum, UN level is
8.31mmol/L, injection concentration be the xerophilusin B compound of 15mg/kg nude mouse serum in UN level be
7.49mmol/L, compared with control group (10.35mmol/L), the equal not statistically significant of difference (p > 0.05) (Figure 21 C, D).Similar
Ground, to KYSE-450 cell transplantation knurl nude mice the xerophilusin B compound that lumbar injection concentration is 7.5mg/kg 19 days
After, in serum, CREA level is 11.98 μm ol/L, and injection concentration is the nude mice of the xerophilusin B compound of 15mg/kg
In serum, CREA level is 9.92 μm ol/L, compared with control group (10.74 μm ol/L), the equal not statistically significant of difference (p >
0.05).At the xerophilusin B compound that injection concentration is 7.5mg/kg, in serum, UN level is 13.48mmol/L, note
UN level in the nude mouse serum of the xerophilusin B compound that concentration is 15mg/kg of penetrating is 10.98mmol/L, with control group
(12.49mmol/L) comparing, the equal not statistically significant of difference (p > 0.05) illustrates that compound xerophilusin B is to KYSE-
150 is non-toxic (Figure 21 C, D) with KYSE-450 nude mice renal function index.
3), the Xerophilusin B impact on nude mice organ-tissue
After nude mice by subcutaneous becomes knurl to terminate with lumbar injection experiment, visually observe the heart, liver, spleen, lung and kidney etc. after dissection main
Want internal organs, after observing its color, shape and size, carry out further histopathological examination.
Result is as shown in figure 22: sliced dye with SABC HE after, it was observed that 7.5mg/kg with 15mg/kg is dense in injection
The nude mice of the xerophilusin B compound of degree, its liver tunicle is complete, and lobuli hepatis structure is normal, and liver rope understands, arranges whole
Together, liver cell is complete, form rule, and liver cell nuclear is clear, big and justify, and endochylema enriches light dye, without sex change, necrosis, without fiber group
Knit hyperplasia.With Normal group indistinction.And inject the nude mice hepatic cell cords arrangement disorder of cis-platinum, in liver, fibrous septum is increased,
Broadening, leaflet is cut, sinus hepaticus is smudgy, and liver cell endochylema sky is bright, becomes and stove shape granular degeneration in diffusivity balloon sample, and
Visible focal necrosis, portal area blood vessel dilatation is congested.
Further, it was observed that nude mice kidney tunicle is complete in each group, glomerulus, renal tubule structure is normal, renal interstitial is without exception.
Spleen tunicle is complete, and spleen trabeculae structure is normal, red, white pulp ratio normal, without inflammatory cell infiltration and pigmentation (Figure 23).And
And, it was further observed that H&L section is without pathological change (Figure 24,25).Above pathological study to result also confirm
The cis-platinum that blood biochemistry detects is for the result of hepatotoxicity.
4), the Xerophilusin B impact on nude mice body weight
Detection for nude mice body weight can intuitively reflect the compound toxic action to body, thin at inoculation esophageal squamous cell carcinoma
After born of the same parents intraperitoneal injection, detect nude mice changes of weight every day, find through analysis of experimental data, as shown in fig. 26, for
KYSE-150 cell transplantation knurl nude mice, each group nude mouse rebalancing method after intraperitoneal injection, but elapse over time, injection
Its body weight of nude mice of 7.5mg/kg concentration xerophilusin B compound is all stable to be risen, and the average weight at the end of experiment is
18.12g, compared with the nude mice body weight (19.66g) of the nude mice of control group of injection Pluronic F68, no significant difference.
The nude mice average weight of injection 15mg/kg concentration xerophilusin B compound is 16.26g, although compares with control group and has
Statistical significance, but its reason should be nude mice of control group knurl bulk-growth rapidly, and knurl body is excessive, and therefore body weight increases substantially.And note
The nude mice body weight penetrating cis-platinum is gradually reduced, at the end of experiment average weight is called 15.01g, far below control group average weight,
Difference has conspicuousness (p < 0.001).Equally, for KYSE-450 cell transplantation knurl nude mice, after intraperitoneal injection, each group is naked
Mouse body weight equalizes, but elapses over time, injection 7.5mg/kg concentration and 15mg/kg xerophilusin B concentration compound
Its body weight of nude mice all stable rise, the average weight at the end of experiment is respectively 17.68g and 19.13g, with injection
The nude mice body weight (18.77g) of the nude mice of control group of Pluronic F68 is compared, no significant difference.And inject cis-platinum
Nude mice body weight is gradually reduced, at the end of experiment average weight is called 17.64g, far below control group average weight, difference has
Conspicuousness (p < 0.05) (Figure 26 B).Also cis-platinum that pathology and blood biochemistry detect has been confirmed for nude mice machine above in result
The result of body toxicity.
Comprehensive above experimental data and result are it was confirmed Xerophilusin B can effectively suppress Human esophageal squamous cell cancer cell
The propagation of KYSE-140, KYSE-150, KYSE-450 or KYSE-510, especially to KYSE-150, KYSE-450 Proliferation Ability
Better, and cause cell-cycle arrest and apoptosis-induced, it is applied to clinical treatment tumour for Chinese herbal medicine and composition thereof and establishes
Certain basis.
Prepared by embodiment 3, anti-esophageal squamous cell carcinoma medicine
According to above-described embodiment 2, it can be seen that Xerophilusin B can be as the work treating anti-esophageal squamous cell carcinoma medicine
Property composition, prepares anti-esophageal squamous cell carcinoma medicine according to traditional drug formulations, specific as follows:
Medicine 1:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, injects routinely and uses water, and essence filter, parenteral solution is made in embedding sterilizing.
Medicine 2:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, is dissolved in sterile water for injection, and stirring makes molten, filters with aseptic suction funnel, more aseptic essence filter, frozen drying
Rear aseptic sealing by fusing obtains powder-injection.
Medicine 3:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 9:1, makes pulvis.
Medicine 4:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient, pelletizing press sheet with excipient weight than the ratio for 1:5-1:10 in it.
Medicine 5:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, oral liquid preparation method makes oral liquid routinely.
Medicine 6:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 5:1 in it, makes capsule or granule or electuary.
Medicine 7:Xerophilusin B, and utilize mixed solvent, nano medicine crystal technology and cyclodextrin to make system
Agent, adds excipient with excipient weight than the ratio for 3:1 in it, makes capsule or granule or electuary.
Claims (8)
1.Xerophilusin B has following 1 in preparation) and/or 2) application in the product of function:
1) prevent or treat tumour;
2) suppression tumor cell proliferation;
The chemical structural formula of described Xerophilusin B is such as following formula I:
Formulas I;
Described tumour is esophageal squamous cell carcinoma.
Application the most according to claim 1, it is characterised in that:
Described suppression tumor cell proliferation is by making cell block in G2/ M phase and/or promotion apoptosis of tumor cells embody.
Application the most according to claim 2, it is characterised in that:
Described promotion apoptosis of tumor cells is by reducing the expressing quantity ratio body of apoptosis pathway protein B CL-2/Bax in cell
Existing.
4. according to described application arbitrary in claim 1-3, it is characterised in that:
Described tumour cell is esophageal squamous cell carcinoma cell KYSE-140, KYSE-150, KYSE-450 or KYSE-510.
Application the most according to claim 4, it is characterised in that:
Described tumour cell is KYSE-150 or KYSE-450.
6.Xerophilusin B has 1 in preparation)-3) at least one function product in application:
1) tumour cell is made to block in G2/ M the phase;
2) apoptosis of tumor cells is promoted;
3) the expressing quantity ratio of BCL-2/Bax in tumour cell is reduced;Described tumour is esophageal squamous cell carcinoma;
The chemical structural formula of described Xerophilusin B is such as following formula I:
Formulas I.
Application the most according to claim 6, it is characterised in that: described tumour cell be esophageal squamous cell carcinoma cell KYSE-140,
KYSE-150, KYSE-450 or KYSE-510.
Application the most according to claim 7, it is characterised in that: described tumour cell is KYSE-150 or KYSE-450.
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