CN109876079A

CN109876079A - The application of Haizao Yuhu Tang and its active constituent fucoxanthine in the drug of preparation treatment liver cancer

Info

Publication number: CN109876079A
Application number: CN201910335460.2A
Authority: CN
Inventors: 朱全刚; 张树辉; 张欢欢; 陈中建; 朱聪聪; 潘会君; 柴蓉蓉
Original assignee: SHANGHAI DERMATOLOGY HOSPITAL
Current assignee: SHANGHAI DERMATOLOGY HOSPITAL
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2019-06-14

Abstract

The invention discloses the application of Haizao Yuhu Tang and its active constituent fucoxanthine in the drug of preparation treatment primary carcinoma of liver to investigate the antitumaous effect of Haizao Yuhu Tang and its active constituent fucoxanthine by establishing human liver cancer transplantable tumor nude mice model；Effect of the fucoxanthine to liver cancer cells is investigated from cell viability, Clone formation, cell cycle, Apoptosis etc.；Using proteomics methodology binding molecule biology techniques, cell key protein expression level is investigated.Its advantage is shown: (1) present invention obtains Haizao Yuhu Tang for the first time can be used for treating the conclusion of liver cancer, and curative effect is good, has no toxic side effect, it the life cycle that patient can effectively be extended, prevents from recurring, improves patients ' life quality, obtains long-term health existence.(2) the albumen network group that fucoxanthine plays adjustment effect is disclosed for the first time, and new approach is provided to the research of liver cancer treatment.

Description

Haizao Yuhu Tang and its active constituent fucoxanthine are in the drug of preparation treatment liver cancer Application

Technical field

The present invention relates to pharmaceutical technology fields, specifically, being that Haizao Yuhu Tang and its active constituent fucoxanthine are being made Application in the drug of standby treatment liver cancer.

Background technique

Primary carcinoma of liver is a kind of common digestive system tumor, and there is onset concealment, the course of disease to change, and fast, grade malignancy is high Feature, life cycle is short, poor prognosis, and has the aggressive and very high recurrence rate of height.Based on counting, global cancer is popular The estimation for the database GLOBOCAN that disease is learned, 2018 global 840,000 people of liver cancer neopathy, ranking the 7th, and PLC mortality number is only Inferior to lung cancer and gastric cancer, it is up to 780,000 people.In China about 31.9 ten thousand people of PLC mortality in 2014, arranged according to tumor invasion number of cases Position is only second to lung cancer, gastric cancer, colon cancer, ranks the 4th, and is only second to lung cancer according to the ranking of tumor mortality number of cases, is second place, Thus one of the emphasis tumour of always China's research.

Primary carcinoma of liver diagnosis and treatment specification (version in 2011) points out that the treatment means of primary carcinoma of liver have surgical operation to control at present Treatment, local ablation, arteria hepatica intervention, radiotherapy and systematic treating (systemic therapy), and systematic treating includes molecular targeted Drug therapy, systemic chemotherapy, traditional Chinese medicine treatment.But advanced stage has often been reached when HCC makes a definite diagnosis, and has both made to perform the operation, Postoperative recurrent rate Also higher, long-term survival rate is low, therefore, it is quite necessary to find curative effect is obvious, toxicity is small, elongated strap tumor life span and The liver cancer treatment method improved the quality of living.

Liver cancer belongs in traditional Chinese medicine " tympanites ", " liver product ", " accumulation ", " fat gas ", " spleen product ", " mass at the right hypochondrium ", " mass located in the upper or lower abdomen ", " cruelly The disease categories such as disease ", " Disorder lump in the abdomen ", " hypochondriac pain ", " jaundice ", " bulging ", " consumptive disease ", etiology and pathogenesis is considerably complicated, and modern Chinese medicine is more Think that tumour is the impairing the spleen and stomach or feelings will is strongly fragrant long or overstrain internal injury by internal organs deficiency of qi and blood, weakness of the spleen and the stomach or feeding desorder, Or the invasion of six external factors which cause diseases cult poison, cause to accumulate in heat toxin, qi depression to blood stasis, even liver kidney is impaired；The pathological products such as poison, stasis of blood form cause again simultaneously Cause of disease element, and interact, reciprocal causation, so as to cause body healthy tendency further weak, the malicious stasis of blood is mutually tied into cancer.Face On bed, firmly such as stone, right flank swells hard sharp ache the liver cancer mostly above abdominal mass matter, radiates to shoulder back, the increase of lump progressive, Tenderness and syntexis, loss of appetite, out of strength, abdominal distension etc. are primary symptom, are with fever, diarrhea, bleeding etc. and disease, advanced stage or with Huang The performance such as subcutaneous ulcer, ascites, stupor, adjusting and runing yang qi and blood should be taken into account with clearing heat and detoxicating, dissolving stasis resolving sputum, resolving hard lump by treating.

Traditional Chinese medicine has unique in terms of the generation that prevents liver cancer, raising life quality, reduction recurrence, extension Curative effect, reducing phlegm and resolving masses are one of common therapies of liver cancer.It is primary that Sun Guizhi etc. observes qi and activate blood circulation softening hard masses disintoxication treatment middle and advanced stage Property liver cancer clinical effectiveness discovery, this method have improve liver cancer patient clinical symptoms effect, liver can be significantly improved in conjunction with chemotherapy Cancer patient cancer stove coefficient of stabilization and life quality coefficient of stabilization, and liver cancer short term effect can be improved.Peng Haiyan is using Chinese medicines groups such as seaweed At tonifying liver softening hard masses side, select 100 primary carcinoma of liver patients diagnosed, treated with 2 months for 1 course for the treatment of.After treatment, portion Divide and alleviate 8, stablize 75, deteriorates 17；Recent remission rate is 8%, knurl coefficient of stabilization 83%.It can be seen that tonifying liver softening hard masses side is treated Primary carcinoma of liver has certain curative effect.

There are about more than 10,000 Chinese medicines and more than 50,000 Chinese medicine compound prescriptions, half a century comes to nearly 5000 kinds of Chinese medicine and close in China 500 compounds carry out tumor suppression screening and clinical verification, have developed the novel formulation of a collection of Chinese patent drug, crude drug, reliable curative effect has been A large amount of clinical practices are confirmed.It is the Haizao Yuhu Tang of main ingredient by seaweed, kelp, thallus laminariae, fritillaria, half using seaweed, kelp, thallus laminariae Summer, Fructus Forsythiae, dried orange peel, green peel, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Angelicae Pubescentis, Radix Glycyrrhizae composition, have the function of resolving phlegm and softening hard masses, eliminating goiter and stasis." surgery is just Ancestor " in point out " to control goiter from the beginning of swollen or hard or red, but not broken person.Two clock of water decocts eight points, measures disease up and down, food front and back Take it ".It is clinical often to use it to treatment simplex goiter, thyroid adenoma, the proliferation of mammary gland, hyperplasia of prostate, non-alcoholic Fatty liver disease, gallbladder polyps, chronic lymphocytic leukemia, brain tumor.

Kelp, thallus laminariae are used as thallus laminariae in tcm clinical practice medication, are algae Laminariaceae plant sea-tangle or wing The thallus of algae section plant thallus laminariae, thallus laminariae, pharmacological property is cold, salty, has effects that resolving hard lump, dissolving phlegm benefit water, for treating goitre Tumor, scrofula, testiclar gall, the illnesss such as phlegm retention oedema.Its medical value is known very early, and beam generation " Mingyi Bielu " is recorded " soft Hard dissipating bind, dissolving phlegm, Li Shui " effect cure mainly goiter, scrofula, testiclar gall, phlegm retention oedema.Thallus Laminariae (Thallus Eckloniae) extract fucoxanthine (fucoxanthin, FX), also known as fucoxanthin, seaweed Huang matter, are a kind of natural Beta-carotenes, are had antitumor, anti-inflammatory, anti- The multiple pharmacological effects such as oxidation, weight-reducing, current study show that, fucoxanthine is to cutaneum carcinoma, colon cancer, hematological system tumor, preceding The Several Kinds of Malignancy such as column gland cancer and liver cancer have obvious effect, have not to the growth of a variety of human tumor cells such as A549, HepG2 With the inhibiting effect of degree.

Proteomics is studied in protein group integral level, and main includes studying intracellular dynamic change Protein constituent；Expression and posttranslational modification state；Interaction and connection between protein.It applies each hatching egg White matter omics technology studies interaction of certain protein in the function and protein population in complicated cellular environment.Egg White matter group is divided into Different Proteomics and expression proteomics again.Wherein, Different Proteomics are to divide by comparing The difference that protein is expressed in cell, tissue or body fluid under different raw pathological conditions is analysed, the key of target point protein matter is filtered out Molecule obtains the information to interact between the function and differential protein of corresponding gene, the comprehensive sheet for illustrating disease of dynamic Matter.So proteomics research is being disclosed on solving biology significant problem with its distinctive method of thinking and technological means The rule of the vital movements such as growth and development, apoptosis, differentiation, signal transduction and metabolic regulation has new breakthrough, to inquire into major disease Mechanism, disease diagnosis prevention and treatment and new drug development provide important theoretical basis.

Chinese patent application: CN103191162B discloses a kind of Akebia Fruit seed extract in treatment Primary Hepatic cancer drug In application, the application refer to be used to prepare using Akebia Fruit seed extract as active constituent treatment primary carcinoma of liver drug Preparation.But whether confront firmly such as about the resolving phlegm and softening hard masses of this patent but Haizao Yuhu Tang and its active constituent fucoxanthine effect Does the liver cancer abdominal mass of stone have active treatment effect? it is had not been reported at present about these.

For the present invention based on traditional Chinese medicine basic theory, China's Liver Cancer status of combining closely establishes human liver cancer transplantable tumor Mouse investigates the antitumor action of Haizao Yuhu Tang and its active constituent fucoxanthine, and rock algae Huang is investigated in terms of cell viability The effect to liver cancer cells of matter；And then in terms of cell-cycle arrest, apoptosis-induced, the effect of fucoxanthine is inquired into；Using The quantitative proteomics method of the iTRAQ technology combination high performance liquid chromatography-tandem mass (LC-MS/MS) of isotope labelling, Analysis and identification fucoxanthine antihepatocarcinoma effect GAP-associated protein GAP, are illustrated based on bioinformatic analysis binding molecule biological experiment The biological function of fucoxanthine modulin, discloses its key effect albumen, explores its mechanism of action from system level.

Summary of the invention

The first purpose of this invention is the middle deficiency for the prior art, provides a kind of new application of Haizao Yuhu Tang.

Second object of the present invention is in view of the deficiencies of the prior art, to provide a kind of new application of fucoxanthine.

To realize above-mentioned first purpose, the technical solution adopted by the present invention is that:

Application of the Haizao Yuhu Tang in the drug of preparation treatment liver cancer, the liver cancer is primary carcinoma of liver.

Application of the Haizao Yuhu Tang in the drug that preparation inhibits hepatoma cell proliferation, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

Application of the Haizao Yuhu Tang in the drug for preparing liver cancer apoptosis reducing, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell, the Haizao Yuhu Tang are by up-regulation Bax expression come liver cancer apoptosis reducing.

Application of the Haizao Yuhu Tang in the drug that preparation inhibits the liver cancer cells period, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell, the Haizao Yuhu Tang are to inhibit the liver cancer cells period by inhibiting the expression of CyclinD1.

To realize above-mentioned second purpose, the technical solution adopted by the present invention is that:

Application of the fucoxanthine in the drug of preparation treatment liver cancer, the liver cancer is primary carcinoma of liver.

Application of the fucoxanthine in the drug that preparation inhibits hepatoma cell proliferation, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

Application of the fucoxanthine in the drug that preparation inhibits liver cancer cells Clone formation, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

Application of the fucoxanthine in the drug for preparing liver cancer apoptosis reducing, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

Application of the fucoxanthine in the drug that G1 phase cell-cycle arrest occurs for preparation induction liver cancer, the liver cancer cells It is Hep3B cells, MHCC97h cell.

Fucoxanthine reduces RPS6KA1, NOP2, MIF, CDK12, STAT3, MDH2, ATP5I egg in liver cancer cells in preparation It is white, improve application in liver cancer cells in the drug of SQSTM1 albumen, the liver cancer cells are Hep3B cells, MHCC97h thin Born of the same parents.

The invention has the advantages that:

1, the present invention obtains Haizao Yuhu Tang for the first time can be used for treating the conclusion of liver cancer, and therapeutic effect is good, nontoxic secondary makees With, can effectively extend the life cycle of patient, prevent from recurring, improve patients ' life quality, make patient obtain long-term health.For the first time The albumen network group that fucoxanthine plays adjustment effect is disclosed, new approach is provided to the research of liver cancer treatment.

2, the present invention investigates Haizao Yuhu Tang and its active constituent rock algae is yellow by establishing human liver cancer transplantable tumor nude mice model Matter antitumaous effect；Fucoxanthine is investigated to liver cancer cells from cell viability, Clone formation, cell cycle, Apoptosis etc. Effect；Using proteomics methodology binding molecule biology techniques, cell key protein expression level is investigated, it is yellow to explore rock algae The antihepatocarcinoma effect approach of matter develops Haizao Yuhu Tang and its active constituent fucoxanthine new application, and suffers from for liver cancer The treatment of person provides new therapeutic scheme, can be applied to well clinically.

Detailed description of the invention

Attached drawing 1 is influence of the Haizao Yuhu Tang to Hep3B cell transplantation tumor nude mice, note: A: the influence to knurl weight；B: to tumor The influence of volume；C: nude mice changes of weight；D: nude mice tumor volume change；E: transplanted tumor in nude mice picture.Compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.

Attached drawing 2 is influence of the HT to Hep3B cell transplantation tumor nude mice ALT, AST, note: compared with Control group, * P < 0.05。

Attached drawing 3 is each group nude mouse tumor tissue pathologies change (HE dyeing).

Attached drawing 4 is each group nude mouse tumor tissue Bax protein expression (400 ×).

Attached drawing 5 is each group nude mouse tumor tissue CyclinD1 protein expression (400 ×).

Attached drawing 6 is influence of the FX to Hep3B cell transplantation tumor nude mice, note: A: the influence to knurl weight；B: to the shadow of knurl product It rings；C: nude mice changes of weight；D: nude mice tumor volume change；E: transplanted tumor in nude mice.Compared with Control group, * P < 0.05；With Control group compares, * * P < 0.01.

Attached drawing 7 is influence of the FX to Hep3B cell transplantation tumor nude mice ALT, AST, note: compared with Control group, * P < 0.05。

Attached drawing 8 is each group nude mouse tumor tissue pathological slice result (HE dyeing).

Attached drawing 9 is each group nude mouse tumor tissue Bax protein expression (400 ×).

Attached drawing 10 is each group nude mouse tumor tissue CyclinD1 protein expression (400 ×).

Attached drawing 11 is cell growth curve, note: A:Hep3B cell；B:MHCC97h cell.

Attached drawing 12 is that FX acts on 24,48, influence of the 72h to Hep3B cell viability.Note: compared with Control group, * P < 0.05；Compared with Control group, * * P < 0.01.A:FX is acted on for 24 hours；B:FX acts on 48h；C:FX acts on 72h.

Attached drawing 13 is that FX acts on 24,48, influence of the 72h to MHCC97h cell viability, note: compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.A:FX is acted on for 24 hours；B:FX acts on 48h；C:FX acts on 72h.

Attached drawing 14 is influence of the FX to Hep3B, MHCC97h cell clonal formation, note: compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.A: group of cells Clone formation；B:Hep3B cell clone vigor；C: MHCC97h cell clone vigor.

Attached drawing 15 be various concentration FX processing after Hep3B cell cycle testing result, note: compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.A:FX10 μM, 14 μM of processing Hep3B cells 24,48h cell cycles detect As a result；B:24h cell cycle distribution；C:48h cell cycle distribution.

Attached drawing 16 is the cell cycle testing result of MHCC97h after various concentration FX processing, note: with Control group ratio Compared with * P < 0.05；Compared with control group, * * P < 0.01.A:FX10 μM, 14 μM of processing MHCC97h cells for 24 hours, 48h cell Cycle detection result；B:24h cell cycle distribution；C:48h cell cycle distribution.

Attached drawing 17 be various concentration FX processing after Hep3B Apoptosis testing result, note: compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.A:FX10 μM, 14 μM of processing Hep3B cells for 24 hours, 48h Apoptosis inspection Survey result；B:24h Apoptosis；C:48h Apoptosis.

Attached drawing 18 is the Apoptosis testing result of MHCC97h after various concentration FX processing, note: with Control group ratio Compared with * P < 0.05；Compared with control group, * * P < 0.01.A:FX10,14 μM of processing MHCC97h cells 24,48h cells wither Die testing result；B:24h Apoptosis；C:48h Apoptosis.

Attached drawing 19 is SDS-PAGE electrophoresis.

Attached drawing 20 is assessment and the statistical analysis for identifying map, note: A: peptide fragment error substep statistical chart；B: peptide fragment is identified Electric charge number distribution statistics figure.

Attached drawing 21 is assessment and the statistical analysis for identifying peptide fragment, note: A: peptide section sequence distribution of lengths statistical chart；B: peptide fragment PSM number divides statistical chart；C: peptide fragment marking Data-Statistics figure；D: the leakage enzyme site number statistical number of peptide fragment.

Attached drawing 22 is assessment and the statistical analysis for identifying albumen, note: A: the peptide fragment number statistical figure that protein matches arrive；B: egg The matched PSM number distribution statistical chart of white matter；C: molecular weight of albumen distribution statistics figure；D: the peptide fragment coverage rate distribution of albumen is identified Statistical chart；E: the isoelectric point distribution statistics figure of albumen is identified.

Attached drawing 23 is global clustering analysis chart.

Attached drawing 24 is that differential protein PANTHERGO-Slim biological processes is analyzed for 24 hours for FX10 μM of processing, note: data mark in figure Label are followed successively by GOterm (GONO.), percentage.

Attached drawing 25 is FX10 μM of processing 48h differential protein PANTHERGO-Slim biological processes analysis, note: data mark in figure Label are followed successively by GOterm (GONO.), percentage.

Attached drawing 26 is FX processing differential protein PANTHERGO-Slim Ceil Wall Composition of Alkalophliic for 24 hours, note: in figure data label according to Secondary is GOterm (GONO.), percentage.

Attached drawing 27 be FX processing 48h differential protein PANTHERGO-Slim Ceil Wall Composition of Alkalophliic, note: in figure data label according to Secondary is GOterm (GONO.), percentage.

Attached drawing 28 be FX processing for 24 hours differential protein PANTHERGO-Slim molecular function analyze, note: in figure data label according to Secondary is GOterm (GONO.), percentage.

Attached drawing 29 be FX processing 48h differential protein PANTHERGO-Slim molecular function analysis, note: in figure data label according to Secondary is GOterm (GONO.), percentage.

Attached drawing 30 is FX effect Hep3B cell interactions between protein network for 24 hours.

Attached drawing 31 is FX effect Hep3B cell 48h interactions between protein network.

Attached drawing 32 is the influence of FX10,14 μM of effect Hep3B cell 48h to key difference protein expression level.

Specific embodiment

The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after having read the content of the invention recorded, art technology Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

The effect experiment of 1 Haizao Yuhu Tang of embodiment

Antitumor effect in Haizao Yuhu Tang body is inquired into this experiment by establishing human liver cancer cell Hep3B Transplanted tumor model Fruit.

1.1 experimental animal

Experimental animal: health male BALB/c nude mice 30, male 4 weeks, 15~20g of weight, are purchased from Shanghai Si Laike reality Company of Animals Ltd. is tested, quality certification number: 2015000549528, experimental animal credit number: SYXK (Shanghai) 2013-0109.

1.2 cell strain

Hep3B cells cell is received in Changhai Academy of Traditional Chinese Medicine, hospital.

1.3 experimental drug

Hydrochloride for injection Doxorubicin (DoxorubicinHydrochlorideforinjection, DOX), Shenzhen ten thousand are happy Pharmaceutcal corporation, Ltd's production, batch number 1703E3.

Haizao Yuhu Tang (HaizaoYuhuTang, HT): seaweed 30g, thallus laminariae 15g, kelp 5g, fritillaria 15g, prepared RHIZOMA PINELLIZE without adju-vant L0g, green peel 6g, dried orange peel l0g, Radix Angelicae Sinensis 15g, Rhizoma Chuanxiong l0g, Fructus Forsythiae l0g, Radix Glycyrrhizae 6g.Single medicinal material is formulated granula, and three nine-day periods after the winter solstice medicine is public Department's production.

1.4 main agents

1.5 laboratory apparatus

2 experimental methods

2.1 cell culture

Human liver cancer cell Hep3B, which is used, contains 10% fetal calf serum and 1 × 105UL^{- 1}Streptomysin, 1 × 105UL^{- 1}Penicillin DMEM culture solution, in 37 DEG C, 5%CO₂It is cultivated in incubator.It changes liquid within the 2nd day after passage, reaches 70~80% to cell density Shi Jinhang passage, the good cell of logarithmic growth phase upgrowth situation is for testing.

The raising of 2.2 nude mices

30 BALB/c male mice sub-cage rearings are in the attached Yueyang Clinical Medical Institute zoopery of Shanghai Univ. of Traditional Chinese Medicine In the SPF environment of center, free diet, SPF grades of animal pellet feed raisings, environment temperature control is between 20~25 DEG C, humidity Control is 60%.

2.3 plant tumor

Nude mice adaptable fed 7 days.The Hep3B cell of logarithmic growth phase is collected, serum-free DMEM is resuspended and counts, and adjusts Cell number is to 2 × 10⁷/ ml, aseptic cotton carrier dip chelated iodine disinfection nude mice injection site (right side oxter) skin, subcutaneous injection 0.2ml(4×10⁶A cell) cell suspension, aseptic cotton carrier pressing injection pin hole 30 seconds to prevent cell suspension spilling.

2.4 medicine preparation

Haizao Yuhu Tang particle be dissolved in respectively 96.2ml (for high dose group), 192.4ml (for middle dose group), In 384.8ml (being used for low dose group) water, heating water bath dissolution；Hydrochloride for injection Doxorubicin (DOX) is molten with 0.9% sodium chloride Liquid is diluted to 1mg/ml.

2.5 groupings and processing

Selection tumor size is nude mice 30 of 5mm or so after planting tumor, according to knurl product size layering grouping, is divided into 5 Group, i.e. blank control group (Control group), doxorubicin hydrochloride group (DOX group), Haizao Yuhu Tang high dose group (HTH), seaweed Beautiful pot soup middle dose group (HTM), Haizao Yuhu Tang low dose group (HTL), every group 6.Each nude mice is re-measured after the completion of grouping The size of transplantable tumor counts indifference between determining group, starts to treat.

The physiological saline stomach-filling one time a day of Control group；DOX group is injected intraperitoneally DOX1mg/kg on the right side of the nude mice, and daily 1 It is secondary, until treatment end；For HTH, HTM, HTL group according to " experimental pharmacology " method, it is opposite that every kg body weight occupies body surface area Ratio is to calculate, and the K value of every 20g mouse Meeh-Rubner formula is 9.1, HTH, HTM, HTL dosage is respectively 660,330, 165mg/kg, daily stomach-filling 0.5ml/20g.Situations such as diet, the activity, the state of mind of nude mice are observed in experimentation, excrement Character and situations such as to extraneous stimulate the reaction.Weighing in every 3 days, measurement tumor size 1 time.

It after medication 21 days, weighs in next day, plucks eyeball method and take blood, cervical dislocation puts to death animal, and operating scissors are used in fixation of facing upward Skin is opened, exposure tumour visually observes tumour growth situation.Tumor mass is stripped completely with eye scissors sterile working, when putting to death rat By conventional visual inspection Xenografts in nude mice and each vitals such as lung, liver, stomach and intestine, kidney and adrenal gland, abdominal aorta whether there is or not Transplantable tumor DISTANT METASTASES IN, takes tumor mass, and after weighing, taking pictures, tumor tissues are put into fixer containing general organization by normal saline flushing It is fixed in the test tube of (neutrality), is used for pathological examination.

2.6 pathological examination

2.6.1 organization embedding is sliced

(1) fixed: tissue being taken out from the general organization fixer (neutrality) fixed more than for 24 hours, is used in draught cupboard Scalpel is whole by tumor tissues equating, will repair the tissue cut and corresponding label is put in dewatering box.

(2) it is dehydrated: putting dewatering box into hanging basket in successively graded ethanol is dehydrated in dewaterer.75% alcohol 4h- 85% alcohol 2h-90% alcohol 2h-95% alcohol 1h- dehydrated alcohol I30min- dehydrated alcohol II30min- alcohol benzene 5-10min- Dimethylbenzene I5-10min- dimethylbenzene II5-10min- wax I1h- wax II1h- wax III1h.

(3) it embeds: being organized in embedding machine for wax will have been soaked and embedded.The wax of thawing is first put into embedding frame, to wax Tissue is taken out out of dewatering box before solidification and is put into embedding frame according to the requirement in embedding face and sticks corresponding label.In -20 It is cooling DEG C to freeze platform, wax stone is taken out from embedding frame after wax solidification and modifies wax stone.

(4) it is sliced: the wax stone trimmed being placed on paraffin slicing machine and is sliced, piece is 4 μm thick.Slice floats on booth piece machine 40 On DEG C warm water will tissue flattening, picked up with glass slide, and put into and bake piece in 60 DEG C of baking ovens.It is taken out after water dries wax roastingization, often Temperature saves backup.

2.6.2HE dyeing

Experimental procedure:

(1) paraffin section de-waxing is to water: slice being successively put into I 20min- dimethylbenzene of dimethylbenzene, II 20min- dehydrated alcohol I 10min- dehydrated alcohol, II 10min-95% alcohol 5min-90% alcohol 5min-80% alcohol 5min-70% alcohol 5min- steams Distilled water is washed.

(2) bush uniformly dyeing nucleus: being sliced into Harris bush uniformly dyeing 3-8min, originally wash, 1% hydrochloride alcohol point Change the several seconds, tap water rinses, and 0.6% ammonium hydroxide returns indigo plant, and flowing water rinses.

(3) Yihong contaminates cytoplasm: being sliced in eosin stain and dyes 1-3min.

(4) it is dehydrated mounting: slice is sequentially placed into 95% alcohol I5min-95% alcohol II5min- dehydrated alcohol, I 5min- It is dehydrated transparent in II 5min- dimethylbenzene of dehydrated alcohol, I 5min- dimethylbenzene, II 5min, slice is taken out from dimethylbenzene and slightly dried, Neutral gum mounting.

(5) sediments microscope inspection, Image Acquisition analysis.Nuclear targeting is blue, and cytoplasm is red.

2.6.3 immunohistochemistry

Operating procedure:

(1) paraffin section de-waxing is to water: slice being successively put into I 15min- dimethylbenzene of dimethylbenzene, II 15min- dimethylbenzene I 5min- dehydrated alcohol of III15min- dehydrated alcohol, II 5min-85% alcohol 5min-75% alcohol 5min- distillation washing.

(2) antigen retrieval

EDTA antigen retrieval: histotomy is placed in Yu Wei in the reparation box for filling with EDTA antigen retrieval buffer (PH9.0) Carry out antigen retrieval in wave furnace, moderate heat 8min is to boiling, truce 8min heat preservation, in low fire 7min, guard against dry plate.Natural cooling, will Slide is placed in PBS (PH7.4) to be washed 3 times in decolorization swinging table, each 5min.

Citric acid antigen reparation: histotomy is placed in the pressure cooker for filling citric acid antigen reparation buffer (PH6.0). It is boiled that liquid will be repaired, slice will be submerged in reparation liquid, lid pot cover, slide is placed in by timing 3min after jet, natural cooling Washing 3 times, each 5min are shaken in decolorization swinging table in PBS (PH7.4).

(3) block endogenous peroxydase: slice is put into 3% hydrogenperoxide steam generator (hydrogen peroxide: pure water=1:9), room Temperature, which is protected from light, is incubated for 25min, slide is placed in PBS (PH7.4), decolorization swinging table shakes washing 3 times, each 5min.

(4) serum is closed: 3%BSA uniform fold tissue is added dropwise in group change circle, room temperature closes 30min.Primary antibody is goat Source is closed with rabbit anteserum, other sources are closed with BSA.

(5) add primary antibody: gently getting rid of confining liquid, the primary antibody that PBS is prepared by a certain percentage is added dropwise on slice, slice is laid flat In 4 DEG C of overnight incubations in wet box.(in wet box plus a small amount of water prevents antibody from evaporating)

(6) add secondary antibody: slide, which is placed in PBS (PH7.4) on decolorization swinging table, shakes washing 3 times, each 5min.Slice is slightly The secondary antibody (HRP label) that kind corresponding to primary antibody is added dropwise after drying in circle covers tissue, is incubated at room temperature 50min.

(7) DAB develops the color: slide, which is placed in PBS (PH7.4) on decolorization swinging table, shakes washing 3 times, each 5min.Slice The DAB developing solution of Fresh is added dropwise after slightly drying in circle, developing time is controlled under microscope, the positive is brown color, originally Water rinses slice color development stopping.

(8) redye nucleus: Harris haematoxylin redyes 3min or so, originally washes, and 1% hydrochloride alcohol breaks up the several seconds, Tap water rinses, and ammonium hydroxide returns indigo plant, and flowing water rinses.

(9) be dehydrated mounting: by slice be sequentially placed into 75% alcohol 5min-85% alcohol 5min- dehydrated alcohol, I 5min- without It is dehydrated transparent in II 5min- dimethylbenzene of water-ethanol, I 5min, slice is taken out from dimethylbenzene and slightly dried, neutral gum mounting.

2.7ALT, AST detection

After nude mice to be plucked to the blood standing 1h that eyeball takes blood to acquire, 3500rmp, 12min centrifugation take supernatant, automatic biochemical Machine testing on analyzer.

2.8 data processing

Statistical analysis is carried out to experimental data using GraphPadPrism7.0 software, as a result with means standard deviation (x ± s) it indicates.Group difference is examined using independent sample bilateral T-, and Multiple range test is examined using ANOVA and Bonferroni, P value It is considered as significant difference less than 0.05.

3 results

3.1 nude mice general states

30 nude mices of this experiment construct Hep3B cell transplantation tumor model, and inoculation is locally shown in skin mound when cell is subcutaneously injected, the Three days accessible subcutaneous nodules of inoculation position, such as grain of rice is big within the 5th day, and such as mung bean is big within the 7th day, and tubercle protuberance is in skin, and 12 days It is all modeled successfully at 5mm tumour is greater than afterwards.After Transplanted tumor model is built up, each group nude mice diet, action and the equal table of the state of mind Now normal, inoculating cell area skin color is normal, and tumor mass is in irregular shape or ellipse, sharpness of border, and subcutaneous mobility is good It is good, no ulceration, without infection.

Influence of 3.2 Haizao Yuhu Tangs to Hep3B transplantable tumor mouse weight and growth of transplanted human

This research has been successfully established human liver cancer cell Hep3B Transplanted tumor model, to investigate Haizao Yuhu Tang in integral level Anti-tumor activity, every 3 days weighing nude mice weight over the course for the treatment of, as a result no significant difference, tentatively illustrate HT to nude mice without Obvious toxic-side effects, as shown in Fig. 1 and table 1, compared with the control group, HT high, middle dosage significantly inhibit tumour growth (P < 0.05)。

1 HT of table to human liver cancer cell Hep3B transplantable tumor tumor weight influence (N=6)

Note: compared with Control group,^*P < 0.05；Compared with control group,^**P < 0.01.

Compared with the control group, HT high, middle dosage significantly inhibit tumour growth (P < 0.05), inhibit according to knurl product gained Rate is respectively 52.4%, 48%；Meanwhile in investigating influence of the HT to Hep3B cells cell transplantation tumor knurl weight, the knot that obtains Fruit and knurl product slightly have difference, but almost the same, and compared with the control group, the high, medium and low dosage group of HT significantly inhibits tumour growth (P < 0.05).As shown in table 2.

2 HT of table to human liver cancer cell Hep3B transplantable tumor knurl product influence (N=6)

3.3 Haizao Yuhu Tangs are to Hep3B cells cell transplantation tumor nude mice liver function influence

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) are liver function important indicators.Liver Cancer patient ALT and AST increase.Shown in table 3, table 4 and Fig. 2, including control group is generally high in interior each group nude mouse serum AST, ALT value In normal value, thus it is speculated that may be related with liver cancer xenograft models, and ALT is substantially reduced HTH group compared with the control group, there is conspicuousness Difference (P < 0.05).It can be seen that HT can protect liver to a certain extent.FX high dose group also has the tendency that reducing transaminase.Together When, DOX is in relatively low-dose to this then without remarkable effect.

3 HT of table to Hep3B cell transplantation tumor nude mice ALT influence (N=6)

Note: compared with Control group,^*P < 0.05.

4 HT of table to Hep3B cell transplantation tumor nude mice AST influence (N=6)

3.4HE dyeing

For observation, tumor tissue pathology changes, and experiment has carried out HE dyeing to each group tumor tissues.As shown in figure 3, light Microscopically observation is learned as it can be seen that each group liver cancer cells differ in size, atypia is obvious, and the growth of control group tumor tissue cell is good Good, tumour cell arrangement is close, and interstitial is few, and core is big, and deep to contaminate, kernel is obvious, and blood is abundant, it is seen that pathology Mitotic index, core Shape is different；DOX group and HT each group nuclear pyknosis, pathology nuclear fission picture are reduced, and are increased necrosis region with dosage and increased, and have amount Effect relationship.

3.5 immunohistochemistry

(1) influence of the Haizao Yuhu Tang to each group tumor tissues Bax protein expression

The therapeutic effect of tumour and the apoptosis of tumour cell are closely related.Bcl-2 family is closely related with Apoptosis A genoid, wherein bax is that promote gene and the bcl-2 expression in the cell of apoptosis be to determine apoptosis weight Want factor.

As shown in figure 4, compared with the control group, HT each group Bax expression enhancing illustrates that HT can raise Bax expression, infers HT energy Enough induce Hep3B tumor tissue cell apoptosis.

(2) influence of the Haizao Yuhu Tang to tumor tissues CyclinD1 protein expression

G1-S cell cycle, point time limit determine cell continue be proliferated or enter the G0 phase, or permanently leave the cell cycle into Enter terminal differentiation or death.Positivity regulatory factor of the CyclinD1 as the starting point G1-S phase of cell division cycle, accumulation can Cause the cell cycle out of control, accelerate cell division, be that is be proved have the oncogene of direct relation with tumour, turns in the G1-S phase It plays a crucial role in changing, also there is Clinical pathological study to show the tumour of cell CyclinD1 overexpression and series of human cancer It shifts related with prognosis mala].Fig. 5 shows the CyclinD1 protein positive expression cell number and control group of HT high, middle dose group Compare positive cell number to decline in dose dependent, it is seen that HT can be by inhibiting the expression of CyclinD1 to inhibit liver cancer cells all Phase.

4 brief summaries

This experiment has investigated influence of the HT to tumor growth in vivo by establishing hepatocellular carcinoma xenografts in nude mice cell Hep3B transplantable tumor, Brief summary is as follows:

(1) Haizao Yuhu Tang (660,330mg/kg) can dose dependent significantly inhibit nude mice Hep3B growth of transplanted human, according to Calculating inhibiting rate according to knurl product is 52.4%, 48%, and low dosage is without obvious inhibiting effect；

(2) Haizao Yuhu Tang improves liver function to a certain extent, and wherein Haizao Yuhu Tang high dose group has statistics poor It is different.

(3) HE staining analysis is found, the visible different degrees of tumour cell of DOX group, HTH group, HTM group, HTL group tumor tissues Focal necrosis.

(4) it is found by immunohistochemical analysis, HT various dose can inhibit CyclinD1 protein expression in various degree, simultaneously Enhance Bax protein expression.Its antitumor action may be related with inducing cell cycle arrest, apoptosis, inhibition cell Proliferation.

The effect experiment of 2 fucoxanthine of embodiment

This experiment establishes human liver cancer cell Hep3B Nude Mouse Model, investigates in FX body antitumous effect and external Influence to liver cancer cells vigor, cell cycle, apoptosis.

One, resisting liver cancer activity is studied in fucoxanthine body

1 material

1.1 experimental animal

1.2 cell strain

1.3 experimental drug

Fucoxanthine reference substance (Fucoxanthin, FX, purity: 98%), solarbio production, article No.: SF8180- 10mg。

1.4 main agents

1.5 laboratory apparatus

2 experimental methods

2.1 cell culture

The raising of 2.2 nude mices

2.3 plant tumor

2.4 medicine preparation

Hydrochloride for injection Doxorubicin is diluted to 1mg/ml with 0.9% sodium chloride solution；More than 0.5% sodium carboxymethylcellulose pyce is mixed Outstanding fucoxanthine is made into the fucoxanthine suspension that concentration is 2mg/ml.

2.5 groupings and processing

Selection tumor size is nude mice 30 of 5mm or so after planting tumor, according to knurl product size layering grouping, is divided into 5 Group, i.e. blank control group (Control group), doxorubicin hydrochloride group (DOX group), fucoxanthine high dose group (be labeled as FXH), Fucoxanthine middle dose group (being labeled as FXM), fucoxanthine low dose group (being labeled as FXL), every group 6.Weight after the completion of grouping The size of each transplanted tumor in nude mice is newly measured, indifference between determining group is counted, starts to treat.

Blank control group physiological saline stomach-filling one time a day；Doxorubicin hydrochloride 1mg/ is injected intraperitoneally in DOX group on the right side of nude mice Kg, one time a day, until treatment end；The administration of FXH, FXM, FXL group reference literature, gives 50,25,12.5mg/kg, often respectively Day stomach-filling.Situations such as diet, the activity, the state of mind of nude mice are observed in experimentation, fecal character and to extraneous stimulate the reaction Situations such as.Weighing in every 3 days, measurement tumor size 1 time.

It after medication 21 days, weighs in next day, plucks eyeball method and take blood, cervical dislocation puts to death animal, and operating scissors are used in fixation of facing upward Skin is opened, exposure tumour visually observes tumour growth situation.Tumor mass is stripped completely with eye scissors sterile working, when putting to death rat By conventional visual inspection Xenografts in nude mice and each vitals such as lung, liver, stomach and intestine, kidney and adrenal gland, abdominal aorta whether there is or not Transplantable tumor DISTANT METASTASES IN, takes tumor mass, and after weighing, taking pictures, tumor tissues are put into and consolidate containing 4% paraformaldehyde by normal saline flushing Determine to be fixed in the test tube of liquid, is used for pathological examination.

Calculate tumor control rate.

Tumor control rate (%)=(1- administration group average knurl weight/control group average knurl weight) × 100%.

2.6 pathological examination

With the 2.6 of embodiment 1.

2.7ALT, AST detection

With the 2.7 of embodiment 1.

2.8 data processing

3 results

The influence that 3.1 fucoxanthines grow Hep3B cells cell transplantation tumor

This research has been successfully established human liver cancer cell Hep3B transplantable tumor mouse model, to investigate fucoxanthine in whole water Flat anti-tumor activity, as shown in table 5, table 6 and Fig. 6, compared with the control group, it is raw that FX group is high, middle dosage also significantly inhibits tumour It is long, it is 52.5%, 44.8% according to knurl product gained inhibiting rate；Meanwhile FX is being investigated to Hep3B cells cell transplantation tumor tumor In the influence of weight, result and the knurl product obtained slightly has difference, but almost the same, and compared with the control group, FXH group also significantly inhibits Tumour growth (P < 0.05), though FXM, FXL group inhibit tumour growth, there was no significant difference.In addition, over the course for the treatment of every 3 Day weighs nude mice weight, and as a result no significant difference, tentatively illustrates FX to nude mice without obvious toxic-side effects.

5 FX of table to Hep3B cell transplantation tumor nude mouse tumor knurl weight influence (N=6)

6 FX of table to Hep3B cell transplantation tumor nude mouse tumor knurl product influence (N=6)

3.2 pairs of Hep3B cells cell transplantation tumor nude mice liver function influences

As shown in table 7, table 8 and Fig. 7, including control group is generally higher than normal value in interior each group nude mouse serum AST, ALT value, It may be related with liver cancer xenograft models.FX high dose group also has the tendency that reduction transaminase, but not statistically significant.Meanwhile DOX is in relatively low-dose to this then without remarkable effect.

7 FX of table to Hep3B cell transplantation tumor nude mice ALT influence (N=6)

3.3HE dyeing

As shown in figure 8, the arrangement of control group tumour cell is close, karyon is larger, and karyomorphism is different, and visible Mitotic index；DOX The visible a few place's tumour cell focal necrosis of group tumor tissues, necrotic tissue are in pink colour, necrotic tumor cells nuclear pyknosis dye deeply or Fragmentation is dissolved, visible a small amount of inflammatory cell infiltration in the connective tissue wrapped up outside tumour；The visible many places tumour of FXH group tumor tissues Cell focal necrosis, necrosis region are in pink colour, and necrotic tumor cells nuclear pyknosis dye or fragmentation deeply dissolves, and visible a small amount of calcification； FXM group and FXL group tumor tissues also have different degrees of necrosis.

3.4 immunohistochemistry

(1) to the influence of each group tumor tissues Bax protein expression

As shown in Figure 9, compared with the control group, FX each group Bax expression enhancing illustrates that FX can raise Bax expression, infers FX energy Enough induce Hep3B tumor tissue cell apoptosis.

(2) to the influence of tumor tissues CyclinD1 protein expression

Figure 10 shows the CyclinD1 protein positive expression cell number positive cell compared with the control group of FX high, middle dose group Number declines in dose dependent, it is seen that FX can be by inhibiting the expression of CyclinD1 to inhibit the liver cancer cells period.

Two, the external antihepatocarcinoma effect research of fucoxanthine

1 material

1.1 experimental cell

Hep3B cells cell is received in Changhai Academy of Traditional Chinese Medicine, hospital, and human liver cancer MHCC97h cell is received big in Fudan University Learn attached hepatopathy research institute, Zhong Shan hospital.

1.2 experimental drug

1.3 experiment reagent

1.4 laboratory apparatus

2 experimental methods

2.1 drugs composition and preparation

Hydrochloride for injection Doxorubicin (DOX) is dissolved into 2mg/ml mother liquor with 0.9% sodium chloride solution；

2ml DMSO dissolves 10mg fucoxanthine (FX), dispenses after being made into the fucoxanthine mother liquor that concentration is 7.6mM, -20 It DEG C saves backup.

2.2 draw growth curve

Single cell suspension is made in Hep3B, MHCC97h cell of logarithmic growth phase, cell concentration is adjusted, with 2000 The density of cells/well is inoculated in 96 orifice plates, and cell is completely adherent after for 24 hours, be divided into adriamycin positive control group (i.e. DOX group, 1.25 μ g/ml), FX group (20,10 μM), blank control group (i.e. Control group, give complete medium).Culture plate is being trained Feeding case is incubated for 24 respectively, 48,72, after 96h, abandon medical fluid, DMEM solution of the 100 μ l containing 10%CCK8 be added to every hole, is placed in Continue to take out after being incubated for 1h in incubator, microplate reader measures the absorbance at 450nm.Experiment is in triplicate.

2.3 influence using Cellcountingkit-8 (CCK-8 method) detection FX to cell viability

Hep3B, MHCC97h cell of logarithmic growth phase, are inoculated in 96 orifice plates with the density of 4000 cells/wells, When cell adherent growth is in good condition, be divided into adriamycin positive control group (i.e. DOX group, 1.25 μ g/ml), FX group (concentration according to Secondary is 25,20,15,10,5 μM), blank control group (i.e. Control group, give complete medium).By culture plate in incubator Be incubated for 24,48, after 72h, medical fluid is sucked out, DMEM solution of the 100 μ l containing 10%CCK-8 is added to every hole, is placed in incubator Continue to take out after being incubated for 1h, microplate reader measures the absorbance at 450nm.Experiment is in triplicate.

Cell survival rate %=[(A0-Ab)]/[(Ac-Ab)] × 100%

A0: experimental port (contains cell culture fluid, CCK-8, drug)

Ac: control wells (contain cell culture fluid, CCK-8)

Ab: blank well (culture solution, CCK-8 without cell and drug)

2.4 colony formation

(1) cell of logarithmic growth phase with 0.25% trypsin digestion and is blown and beaten into individual cells respectively, and thin Born of the same parents are suspended in spare in the DMEM culture solution of 10% fetal calf serum.

(2) cell suspension is made into the dilution of gradient multiple, every group of cell is connect with the density of every 1000 cells of ware respectively respectively In six orifice plates of kind 37 DEG C of pre-temperature culture solutions containing 2mL, it is placed in 37 DEG C, 5%CO₂And in the cell incubator of saturated humidity, patch (10,14 μM) of FX cultures are given after wall respectively, until the 7th day changes the liquid once.

(3) when observe there is macroscopic clone in culture dish when, terminate culture.Supernatant is abandoned, is carefully embathed with PBS 2 times.The fixed cell 1mL room temperature of 4% paraformaldehyde is added to fix 30 minutes.Fixer is removed, 1ml Giemsa dye liquor is added to disseminate 30 points Clock, ddH₂O washes away dyeing liquor, is stored at room temperature drying.

(4) it takes pictures, counts.

The experiment of 2.5 cell cycles

By Hep3B, MHCC97h cell with 1 × 10⁶The density of a cell/ware is inoculated in 6cm culture dish, FX (10,14 μM) After effect 24,48h, flow cytometry detects intracellular DNA content.

Operating method:

(1) PBS washs cell, and 1000rpm 5min is collected by centrifugation, and adjustment cell concentration is 1 × 10⁶/ ml takes 1ml slender Born of the same parents' suspension；

(2) after the single cell suspension centrifugation prepared, supernatant is removed, 150 μ l pre-cooling PBS is added in cell, side is gently vortexed Dehydrated alcohol 350 the μ l, 4 DEG C of fixed 2h of pre-cooling is added dropwise to overnight in side.

(3) PBS washes away fixer.

(4) 100 μ l RNaseA solution are added in cell precipitation, cell, 37 DEG C of water-bath 30min is resuspended.

(5) 400 μ lPI dyeing liquors are added to mix, 4 DEG C are protected from light incubation 30min.

(6) machine testing on records red fluorescence at excitation wavelength 488nm.

Cell cycle each phase is divided into G0/G1 phase, S phase and G2/M phase, the streaming histogram of acquisition corresponds to each cell Period, software calculate the cell percentage of each phase.

Experiment is repeated 3 times.

2.6 cell apoptosis assay

By Hep3B, MHCC97h cell with 1 × 10⁶The density of a cell/ware is inoculated in 6cm culture dish, FX (10,14 μM) After effect 24,48h, the bis- dye methods of fluidic cell AnnexinV-FITC/PI carry out Apoptosis detection.

Operating procedure:

(1) 10 × BindingBuffer is diluted to 1 × Binding Buffer with PBS；

(2) cell is collected: using collecting after the trypsin digestion cell without EDTA, being centrifuged 5min in room temperature 1000rpm, is received Collect cell；

(3) cell washs: cell is resuspended with the PBS of pre-cooling, removes supernatant after 1000rpm centrifugation 5min；

(4) (cell concentration is adjusted to 1 × 10 to 1 × BindingBuffer suspension cell of 100 μ l of addition⁶)；

(5) AnnexinV-FITC and PI label: each group is separately added into the PI of the AnnexinV-FITC and 5 μ l of 5 μ l (PI is not added in AnnexinV-FITC group, and AnnexinV-FITC is not added in PI group, and AnnexinV-FITC and PI label is not added in blank group) It after mixing, is protected from light, is incubated at room temperature 15min.

(6) each group adds 1 × Binding Buffer of 400 μ l before upper machine.

Detection is completed in 1h, experiment is repeated 3 times.

2.7 data processing

Statistical analysis is carried out to experimental data with GraphPad.Prism7.0 software, as a result with mean value ± standard error Poor (Means ± SEM) is indicated.Group difference uses independent sample bilateral T- inspection statistics method, and ANOVA is used when Multiple range test It is examined with Bonferroni, P value is considered as significant difference less than 0.05.

3 results

3.1 draw growth curve

Cell growth curve is one of the basic parameter of cultivated cytobiology characteristic, and determines the important of cell viability Index.The Hep3B cell and MHCC97H cell kind of logarithmic growth phase enter a large amount of divisions in 96 orifice plates after incubation period Exponential phase of growth.In control group, with the extension of cultivated days, the OD value measured is in rising trend, indicates thin in its hole Born of the same parents' number is being increasing, and cell is grown in lasting progress.After the FX of various concentration processing, OD value is compared with control group in now Drop trend, Hep3B cell and the growth of MHCC97H cell receive obvious inhibition, and action time is longer, and the cell number of growth is got over It is few, prompt FX to significantly suppress the proliferation function of Hep3B cells, MHCC97H cell in vitro, as shown in figure 11.

3.2CCk-8 method detects influence of the FX to Hep3B, MHCC97h cell viability

Further to detect effect of the FX to tumour cell, we give Hep3B, MHCC97h cell FX group (concentration respectively Be followed successively by 25,20,15,10,5 μM) and clinical commonly used drug DOX1.25 μ g/ml, with CCk-8 method detect respectively FX effect 24, 48, the cell viability after 72h.

As shown in table 9 and Figure 12, FX acts on the influence generated after Hep3B cell 24,48,72h to cell viability.FX15μM Act on Hep3B cell for 24 hours i.e. statistically significant (P < 0.05).FX acts on Hep3B cell 72hIC35, IC50 value 10、14μM。

9 FX of table effect 24,48,72h to Hep3B cell viability influence (N=3)

< 0.05；Compared with control group,^**P < 0.01.

20 μM of effect MHCC97h cells of FX are statistically significant for 24 hours (P < 0.05), extend at any time and dosage increases work With significantly, as shown in table 10 and Figure 13, FX acts on MHCC97h cell 72h IC35, IC50 value is respectively 16.9,20.5 μM. Subsequent experimental takes FX effect Hep3B cell 72h IC35, IC50 value to carry out, that is, choosing 10,14 μM is experimental concentration.

10 FX of table effect 24,48,72h to MHCC97h cell viability influence (N=3)

Note: compared with Control group, * P < 0.05；Compared with control group, * * P < 0.01.

Influence of 3.3 fucoxanthines to human liver cancer cell Hep3B, MHCC97h Clone formation

Cell clonal formation experiment can reflect that the adherent survival rate situation of cell and reflection proliferation form clone's colony shape At situation.This experimental result is it can be found that fucoxanthine group decreased significantly compared with control group clone's size and colony counts. It is 90.13%, 10 μM of groups 22.63% of FX, 14 μM of FX that Hep3B each group cloning efficiency, which is respectively as follows: control group cloning efficiency, Group 11.87% has conspicuousness (P < 0.01) compared with control group difference.MHCC97h each group cloning efficiency is respectively as follows: control group 57.87%, 14 μM of 10 μM of groups 26.33% of FX, FX groups 16.03%, there is different differences compared with control group, and high dose group has aobvious It writes sex differernce (P < 0.01).As shown in table 11 and Figure 14.

11 various concentration FX of table to Hep3B, MHCC97h cell clonal formation influence (N=3)

The influence of 3.4 cell cycles

It is acted on whether caused by inducing cell cycle arrest to investigate the cell inhibitory effect of FX, experiment uses fluidic cell The double dye method detection FX of art induce Hep3B, MHCC97h cell cycle distribution situation.As shown in 12,13 and Figure 15 of table, FX 10,14 μ After M is respectively acting on Hep3B cell for 24 hours, G1 phase cell content rises to 43.64%, 50.15% from 36.49% respectively, has aobvious It writes sex differernce (P < 0.01), while the S phase gradually decreases；After acting on 48h, increases with dosage and see in phase cell cycle G1 Cell dramatically increases, and cell content rises to 56.34%, 64.27% from 44.2%, has significant difference (P < 0.01), together When, S phase cell distribution significantly reduces, and G2 phase cell distribution is also reduced.

FX induces MHCC97h cell cycle distribution situation as shown in 14,15 and Figure 16 of table, after 10,14 μM of FX effects for 24 hours G1 phase cell content rises to 46.15%, 51.98% from 38.99% respectively, wherein 14 μM of groups of FX have statistical difference (P < 0.05), the S phase gradually decreases；And after 10,14 μM of effect 48h of FX, G1 phase cell content rises to 55.25% from 40.44%, 59.32%, there is significant difference (P < 0.01), the S phase significantly reduces.

12 various concentration FX of table processing for 24 hours after Hep3B cell cycle testing result (N=3)

13 various concentration FX of table handle 48h after Hep3B cell cycle testing result (N=3)

14 various concentration FX of table processing for 24 hours after MHCC97h cell cycle testing result (N=3)

15 various concentration FX of table handle 48h after MHCC97h cell cycle testing result (N=3)

The influence of 3.5 pairs of Apoptosis

According to cell viability, cell cycle stage, project has investigated FX (10,14 μM) and has acted on Hep3B and MHCC97h respectively To the influence of Apoptosis after cell 24,48h, it is shown in Table 16,17 and Figure 17,18.10 μM of effect Hep3B cells of FX for 24 hours after apoptosis Rate is 7.32%, statistically significant (P < 0.05)；14 μM of FX effects are afterwards for 24 hours 11.37% to apoptosis rate, are had significant Sex differernce (P < 0.01), after FX10,14 μM of effect 48h, apoptosis rate 13.66%, 17.48%, compared with the control group There is significant difference (P < 0.01).

16 various concentration FX of table processing Hep3B apoptosis testing result (N=3)

By table 17 and Figure 18 as it can be seen that FX10 μM of effect MHCC97h cell apoptosis rate is 9.34% for 24 hours, promote apoptosis without obvious It acts on (P > 0.05), effect 48h apoptosis rate is 12.26%, there is significant difference (P < 0.01)；FX14 μM effect for 24 hours, 48h Apoptosis rate is respectively 15.42%, 16.45% afterwards, has apoptosis-promoting effect to cell, there is significant difference (P < compared with the control group 0.01)。

17 various concentration FX of table processing MHCC97h apoptosis testing result (N=3)

4 brief summaries

(1) FX (50,25mg/kg) can dose dependent significantly inhibit nude mice Hep3B growth of transplanted human, according to tumor stereometer Calculating inhibiting rate is 52.5%, 44.8%, and low dosage is without obvious inhibiting effect；

(2) FX can significantly inhibit Hep3B cells, MHCC97h cell viability, and FX15 μM of effect Hep3B cell has for 24 hours Statistical significance (P < 0.05), FX20 μM of effect MHCC97h cell is statistically significant for 24 hours (P < 0.05), prolongs at any time Long and concentration increasing action is more significant.It is respectively 10,14 μM that FX, which acts on Hep3B cell 72hIC35, IC50 value, FX effect MHCC97h cell 72hIC35, IC50 value is respectively 16.9,20.5 μM.

(3) after FX acts on Hep3B cell and MHCC97h, cloning size and colony counts compared with control group be decreased significantly. Hep3B each group cloning efficiency is respectively as follows: control group 90.13%, FX10 μM of group 22.63%, FX14 μM of group 11.87% and relatively compares Group difference has conspicuousness (P < 0.01)；MHCC97h each group cloning efficiency is respectively as follows: control group 57.87%, FX10 μM of group 26.33%, 14 μM of groups 16.03% of FX, there is different differences compared with control group, and high dose group has significant difference (P < 0.01).

(4) FX10,14 μM be respectively acting on Hep3B cell for 24 hours after, G1 phase cell content rises to respectively from 36.49% 43.64%, 50.15%, there is significant difference (P < 0.01)；After acting on 48h, increases with dosage and see in cell cycle G1 Phase cell dramatically increases, and cell content rises to 56.34%, 64.27% from 44.2%, there is significant difference (P < 0.01).

FX10,14 μM of effect MHCC97h cells for 24 hours after G1 phase cell content rise to 46.15% respectively from 38.99%, 51.98%, wherein FX14 μM of group has statistical difference (P < 0.05)；After FX10,14 μM of effect 48h, G1 phase cell content from 40.44% rises to 55.25%, 59.32%, there is significant difference (P < 0.01).

(5) FX10,14 μM act on Hep3B cell for 24 hours after apoptosis, apoptosis rate is respectively 7.32%, 11.37%, it is statistically significant (P < 0.05)；Apoptosis rate is respectively 13.66%, 17.48% after acting on 48h, and is compareed Group more has significant difference (P < 0.01).

Apoptosis rate is 9.34% to FX10 μM of effect MHCC97h cell for 24 hours, without obvious apoptosis-promoting effect (P > 0.05), effect 48h apoptosis rate is 12.26%, there is significant difference (P < 0.01)；Apoptosis rate is respectively after FX14 μM of effect 24,48h 15.42%, 16.45%, there is apoptosis-promoting effect to cell, there is significant difference (P < 0.01) compared with the control group.

3 proteome analysis of embodiment

The method that this experiment uses proteomics investigates FX modulate tumor Cell differentials protein expression water from system level It is flat, and it is verified in conjunction with Westernblot method, to explore FX antitumor action approach.

One, influence of the fucoxanthine to human liver cancer cell Hep3B difference between the effects protein science

1 material

1.1 reagent

1.1.1 main agents

1.1.2 reagent preparation

(1) the reversed phase chromatography separation experiment under the conditions of high pH:

Mobile phase A: 98%ddH₂O, 2% acetonitrile (pH 10)；Mobile phase B: 98% acetonitrile, 2%ddH₂O(pH 10)； ddH₂O ammonium hydroxide tune pH value to 10.

(2) nanoliter level reverse-phase chromatography-Q Exactive protein analysis:

Mobile phase A: 100% ultrapure water, 0.1% formic acid；Mobile phase B: 100% acetonitrile, 0.1% formic acid

1.2 laboratory apparatus

2 research methods

After acting on Hep3B cells cell 24,48h respectively with the FX that concentration is 10 μM, total protein of cell is extracted, using same The quantitative proteomics method of the iTRAQ technology combination high performance liquid chromatography-tandem mass (LC-MS/MS) of position element label, point The protein of analysis identification differential expression, with the protein function of bioinformatic analysis differential expression.

2.1 protein extraction

(1) it is added in sample appropriate lysisbuffer (7M urea, 2M thiocarbamide), is vortexed and mixes.

(2) ultrasound 60s, 0.2son, 2soff, amplitude 22%.

(3) room temperature extracts 30min.

(4) 4 DEG C, 15000g centrifugation 20min, collect supernatant, packing freezes in -80 DEG C.

2.2Bradford method protein quantification

Protein concentration is measured using Bradford method.BSA is dissolved into the standard egg of series of concentrations with lysisbuffer White, sample, which carries out certain multiple dilution with lysisbuffer, falls in its final concentration in the bent range of mark, the sample and mark that have diluted Quasi- product respectively take 10 μ l to be protected from light 20min with 300 μ l protein quantification dyestuffs respectively, measure standard items with microplate reader and sample exists Light absorption value under 595nm draws standard curve.Each sample protein concentration is calculated according to curve equation.

2.3 proteolysis (FilterAidedSamplePreparation, FASP)

Operating procedure:

(1) 100 μ g protein solutions is taken to be placed in centrifuge tube after protein quantification.

(2) 4 μ l Reducing Reagent, 60 DEG C of reaction 1h are added.

(3) 2 μ l Cysteine-Blocking Reagent, room temperature 10min are added.

(4) protein solution after reductive alkylation is added in the super filter tube of 10K, 12000rpm is centrifuged 20min.

(5) DissolutionBuffer100 μ l is added, 12000rpm is centrifuged 20min.

(6) collecting pipe is replaced, trypsase is added in super filter tube, 37 DEG C of reactions are overnight.

(7) the peptide fragment solution 12000rpm centrifugation 20min after enzymolysis, digestion is collected in bottom of the tube.

(8) 50 μ l Dissolution Buffer5,12000rpm centrifugation 20min are added in super filter tube, merge with upper step, Collect the sample after 100 μ l enzymatic hydrolysis is obtained.

2.4iTRAQ label

113 mark control group (CON-24h) for 24 hours, and 114 labels 48h control group (CON-48h), rock algae is yellow for 24 hours for 117 labels Matter group (FX-24h), 118 labels 48h fucoxanthine group (FX-48h).

Operating procedure:

(1) iTRAQ reagent equilibrates to room temperature, centrifugation to tube bottom.

(2) 150 μ l isopropanols, vortex oscillation, centrifugation to tube bottom are separately added into.

(3) 50 μ l samples (100 μ g enzymolysis product) is taken, iTRAQ reagent is dosed in sample, vortex oscillation, centrifugation is extremely Tube bottom reacts at room temperature 2h.

(4) 100 μ l water are added and terminate reaction.

(5) sample after mixed mark, vortex oscillation, centrifugation.

(6) freezen protective is stand-by after vacuum refrigeration centrifugal drying.

Reversed phase chromatography separation under the conditions of 2.5 high pH

Operating procedure:

(1) sample after mixed mark is dissolved with 100 μ l mobile phase As, and 14000g is centrifuged 20min, takes supernatant.

(2) (45 DEG C of column temperature, Detection wavelength 214nm), detection system situation are separated using the BSA that 400 μ g are digested.

(3) 100 μ l sample loadings are taken.

(4) flow velocity 0.7ml/min, separation gradient are shown in Table 18.

Reversed phase chromatography separation gradient under the conditions of table 18 is pH high

2.6 nanoliter level reverse-phase chromatography-Q Exactive carry out protein analysis

2.6.1 operating procedure

(1) component for obtaining high pH reverse phase separation 20 μ l, 2% methanol, 0.1% formic acid redissolve.

(2) 12000rpm is centrifuged 10min, draws supernatant loading.

(3) 10 μ l of sandwich method loading.

(4) Loading Pump flow velocity 350nl/min, 15min.

(5) flow velocity 350nl/min is separated, separation gradient is shown in Table 19.

19 nanoscale reverse-phase chromatography protein analysis of table separates gradient

2.6.2 mass spectrometry parameters are arranged

Source parameters:

Spray voltage:2.1kv；Capillary Temperature:250 DEG C；Ion Source:EASY-Spray source；DP:100；

Full MS:

Resolution:70000FWHM；Full ScanAGC target:1e6；Full Scan Max.IT:60ms； Scan range:350-1800m/z；

Dd-MS2:

Resolution:17500FWHM；AGC target:5e6；Maximum IT:70ms；Intensity Threshold:5.00E+03；Fragmentation Methods:HCD；NCE:29%；Top N:20；

The processing of 2.7 mass spectrometric datas

Experiment uses Uniprot Human database.

The mass spectral analysis of iTRAQ is completed by Thermo Q-Exactive type mass spectrum, and the mass spectrum original document of generation uses The mating business software Proteome Discoverer2.1 of Thermo company is handled.

The setting of 20 retrieval parameter of table

3 results

3.1SDS-PAGE electrophoresis

Every sample takes 15 μ g to carry out SDS-PAGE electrophoresis, and coomassie brilliant blue staining 30min decolourizes up to clear background, Relative quantification accurately clear sample strip band is obtained, as shown in figure 19.

3.2 protein qualitative results statistics

Mass spectrometric data after processing, obtains protein qualitative results, is shown in Table 21.High confidence level protein sum is identified altogether 5092.

21 protein identification result of table statistics

3.3 quantification of protein results statistics

Mass spectrometric data after processing, obtains quantification of protein as a result, setting fold differences 2 times or more as significant difference, sees Table 22.10 μM of FX effects for 24 hours, obtain up-regulation differential expression protein 97, lower differential expression protein 87, and totally 184 It is a；10 μM of effect 48h of FX obtain up-regulation differential expression protein 30, lower differential expression protein 43, totally 184.

22 quantification of protein result of table statistics

The assessment of 3.4 qualification results and statistical analysis

The qualification process of protein group is to carry out peptide fragment identification with mass spectrum, then derive first by protein digestion at peptide fragment Possible protein.Therefore, in order to assess the overall condition of proteome data obtained, we are from map, peptide fragment and egg These three white levels are assessed from each physicochemical property etc..

3.4.1 assessment and the statistical analysis of map are identified

(1) identify the error distribution of map: i.e. the molecular weight error of peptide fragment depends on mass spectrographic precision and levels of collimation, Theoretical upward peak is better closer to 0, and whole distribution is most of all in the error range being arranged when searching library.

By protein digestion at peptide fragment, identified with the molecular weight error that mass spectrum carries out peptide fragment, peptide fragment peak value closer to 0, mass spectrographic precision and levels of collimation are better.

(2) the charge number distribution of peptide fragment ion identification: in QE/Fusion instrument, the peptide fragment of general divalent and trivalent is easy It is accredited, high charge is difficult to be accredited.

By protein digestion at peptide fragment, carry out peptide fragment identification with mass spectrum, divalent, trivalent, 4 valences peptide fragment qualification result as schemed 20。

3.4.2 assessment and the statistical analysis of peptide fragment are identified

It identifies that the physicochemical property of peptide fragment reacts mass spectrographic appraisal, therefore tends to assess its technological layer, Ke Yicong The following aspects carries out analysis and assessment, as a result sees Figure 21:

(1) peptide segment length: the amino acid length range for the peptide fragment that the Mass Spectrometric Identification of calorific power arrives, the too short peptide of sequence were reacted Section (less than 7 amino acid) due to form it is simple most of be filtered, too long peptide fragment (generally greater than 40 amino acid) by It is too high-leveled and difficult to be arrived by Mass Spectrometric Identification in molecular weight；

(2) the PSM number distribution that each peptide fragment identifies: the case where reaction peptide fragment is captured by mass spectrum, average each peptide fragment The PSM number of capture is fewer, illustrates that mass spectrum can more effectively be avoided repeating to sample and obtaining more as a result, and average PSM quantity It is more, then illustrate that the reliability of single peptide fragment identification is higher；

(3) distribution of peptide fragment ion marking: being the score value distribution of the map comparison of theoretical peptide fragment map and measurement, the score value It is higher to illustrate that matching is more reliable.

(4) peptide fragment leakage enzyme site number distribution: reacting the thorough degree of digestion, and the peptide fragment that it is 0 that leakage, which is cut and counted, is more, illustrates digestion It is more thorough, it is more advantageous to identifying.

3.4.3 assessment and the statistical analysis of albumen are identified

The physicochemical property of identification protein has reacted the identification of process and protein group sample that albumen is derived in peptide fragment Situation, therefore the assessment of existing technological layer, also there is the assessment of biology level, and assessment point can be carried out from the following aspects Analysis:

(1) the matched peptide number of segment distribution of protein

The protein identification efficiency of the matched peptide number of segment distribution reaction peptide fragment of protein and the repeatability of protein identification.It is flat The peptide fragment of each protein matches is fewer, illustrates that the protein identification efficiency of peptide fragment is higher；Conversely, average each protein matching Peptide fragment it is more, illustrate that the abundance of these protein is more balanced, average each certified number of repetition of protein is more.

(2) the matched PSM number distribution of protein

Since PSM number can be used for quantifying for map counting, this index general description different proteins abundance The distributed number of horizontal protein.

(3) molecular weight distribution of protein is identified

React the distribution situation of the molecular weight of the albumen integrally identified.

(4) identification albumen is by the distribution of peptide fragment coverage rate

Since protein group takes shotgun to identify, often an albumen only has the identification of a few peptide fragment, namely only The sequence of partially protein is covered by peptide fragment.The index shows the certified situation of protein, and coverage rate is higher, for explaining egg The isomers of white matter etc. is more advantageous.

(5) the isoelectric point distribution of albumen is identified

The distribution situation of the isoelectric point of identified protein is as shown in figure 22.

The aggregate analysis of 3.5 qualification results and differential protein screening

3.5.1 global analysis

The quantitative values of the different label of (FDR < 0.01) obtain after removal has 0 result in result after extracting PD search To file, Figure 23 is obtained after clustering processing, abscissa respectively represents FX effect 24,48h and control group 24,48h, Mei Yilie For a sample, the similitude based on gene expression between sample is clustered, gene expression is closer between sample, and that leans on is closer.It is vertical to sit Mark represents gene clusters, and a gene of behavior one clusters the similitude expressed in the sample based on gene, and expression is closer, leans on It is closer.Color range represents gene expression abundance, and red to indicate that up-regulation is more obvious more again, green is represented to lower more again and is more obvious.By As it can be seen that after FX processing, Hep3B cell protein expression levels differ greatly figure.

3.5.2 differential protein screens

Variance analysis is carried out using the method for the SignificancesA of Maxquant, reconciles and lowers in FX effect 24,48h Differential protein is shown in Table 23,24,25,26 (analysis result takes pvalue < 0.05,2 times of Foldchange >).

23 FX of table processing Hep3B cell expresses the albumen of up-regulation afterwards for 24 hours

24 FX of table processing Hep3B cell expresses the albumen of downward afterwards for 24 hours

The albumen of up-regulation is expressed after 25 FX of table processing Hep3B cell 48h

The albumen of downward is expressed after 26 FX of table processing Hep3B cell 48h

3.6 functional annotations and analysis

3.6.1GO analysis

Gene ontology (Gene Ontology, GO) is function, positioning and the movable standard vocabulary for describing gene Table has tree structure, is the most widely used ontology of molecular biology field.Gene and gene product vocabulary cover biology Three aspects.Bioprocess (Biological Process, BP) is generated by one or more molecular function sequential combinations Chain of events, a process is made of multiple and different steps；Cellular component (CellularComponent, CC) description Each part of cell and extracellular environment；Molecular function (Molecular Function, MF) can be described as molecular level Activity (activity), such as be catalyzed (catalytic) or combine (binding) activity.The annotation of GO is very many and diverse and careful, The PANTHER (Protein analysis through evolutionary is used for statistical analysis macroscopically Relationships) Classification System Version 13.1 (http://www.pantherdb.org) is infused Release completion.

(1) BP is annotated

BP is analyzed as shown in Figure 24,25, after FX10 μM acts on 24,48h respectively, with control group differential protein bioprocess master Metabolism process, biological regulation, cell component tissue, cellular processes etc. are concentrated on, effect is for 24 hours with effect 48h without obvious Difference.

(2) CC is annotated

It is found by CC analysis, after 10 μM of FX acts on 24,48h respectively, differential protein cellular component master compared with the control group It is distributed in cell component, organelle, cell membrane, contains protein complex.In addition, differential protein is thin after FX10 μM of effect for 24 hours Born of the same parents' component is also distributed in cell connection, and cellular component is variant on extracellular matrix after acting on 48h.As shown in Figure 26,27.

(3) MF is annotated

Molecular function annotation analysis is as shown in Figure 28,29, after FX10 μM of effect 24,48h, with control group differential protein molecule The function of activity, transcripting regulating activity is mainly adjusted with catalytic activity, combination, molecular function regulator, transport activity, structure Energy.

3.6.2 signal path is analyzed

KEGG is the database of an integrator gene group, chemistry and system function information, which can be counted using one kind The knowledge that the form of calculation captures and histological examination obtains forms system function knowledge base, and the computer mould as biosystem is planned The system of the cell of gene catalogue and higher level, species and ecosystem-level obtained in the genome being completely sequenced Function association gets up.With DAVID 6.8 to FX difference between the effects albumen carry out KEGG analysis (https: // David.ncifcrf.gov/), it is enriched to hsa05200:Pathways in cancer；Hsa05202: Transcriptional misregulation in cancer；The signals such as hsa04010:MAPK signalingpathway Access, GAP-associated protein GAP have WNT2B, BCL2 etc..

Difference signal access after 27 FX of table effect

3.6.3 protein interaction analysis

From the interactive network between albumen group can system disclose protein group function, and to illustrate growth, development, point The vital movements rules such as change, apoptosis and bioelectric detecting mechanism are most important, can be discussion major disease mechanism, treatment and new drug Exploitation provides important theoretical basis.

This research application String (https: //string-db.org, Version 11), analyze differentially expressed protein between Interaction network, to find correlation and the potential function group of differential protein.

Differential protein after interactions between protein net analysis FX effect for 24 hours, finds in addition to the relatively independent albumen of a few functions, greatly Most albumen have interaction in functioning, and prompting FX effect, there may be multiple protein collective effects.Confidence level is set It is 0.4, forms network after hiding the node disconnected in a network, as shown in figure 30.Make with String net analysis FX As seen in figure 31 with differential protein after 48h.

To in the analysis of network key node selection, this project comprehensive Degree, Closeness, Betweenness into Row.It is known as Connected degree (Degree) in network with the number of node K other nodes connecting, the higher node of Connected degree, in net Stronger with the relevance of other nodes in network, Connected degree is greater than all twice of node connectivity median of nodes of network and is known as Hub node.

Tightness (Closeness) is to be measured by the transmitting distance between node to the different degree of node, node K The distance of each node and most short is reached, prompts its tightness higher, corresponding node K is at the central area of network, weight Spending also can be higher, compared with calculating degree, calculates in node all in network is considered in by tightness, effectively utilizes The network topology of node.

Jie's degree (Betweenness) of node K is equal to always most short by the shortest path Zhan of this node in whole network figure The ratio in path carries out pitch point importance evaluation, the bigger expression section of Jie's degree by calculating Jie's degree of all nodes in network The frequency that point K occurs in shortest path is higher, while also closer with the relationship of other nodes, then, different degree is also got over It is high.Jie's degree is able to reflect the frequency of node institute's " flowing through " in complex network relationship, is the behavioral characteristics of node in a network.

This project is based on interactions between protein network analysis as a result, according to Degree, Closeness, Betweenness ranking, Determine that albumen RPS6KA1, NOP2, MIF, CDK12, SQSTM1, STAT3, MDH2, ATP5I may play the part of in FX plays a role Key player.

Two, key difference albumen is verified

Front portion studies have shown that FX transplants tumor growth in vivo, inducing cell cycle arrest, and apoptosis occurs.Albumen Group is learned research shows that FX controllable tumour cell multiple protein expression variation, interactions between protein analysis shows that albumen RPS6KA1, NOP2, MIF, CDK12, SQSTM1, STAT3, MDH2, ATP5I may play an important role in FX plays a role.This section uses Westernblot method verifies the above albumen, to explore FX action pathway.

1 experimental material

1.1 experiment reagent

1.2 laboratory apparatus

2 methods

2.1 reagent preparation

(1) 10%SDS

(2) 10%APS

(3) 1.5mol/LTrisHCl (pH=8.8)

(4) 0.5mol/LTrisHCl (pH=6.8)

(5) 1mol/LTrisHCl (pH=7.5)

(6) 30%Acr/Bic (30%T, 2.67%C)

(7) 10 × electrophoretic buffers

(8) 10 × transferring film buffers

(9) 10 × TBS buffers

(10) TBS-T buffer

(11) confining liquid

(12) antibody diluent

The extraction of 2.2 total protein of cell

(1) test consumptive material and instrument to prepare: liquid transfer gun head needed for experimentation, that centrifuge tube shifts to an earlier date sterilizing, drying is spare；4 DEG C pre-cooling low-temperature and high-speed centrifuge；Prepare ice chest.

(2) enter to add 10 μ l PMSF and 10 μ l phosphatase inhibitor cocktails in every 1ml lysate.

(3) collect supernatant: culture dish is set on ice chest, and transfer supernatant to EP is managed, and centrifugation takes supernatant, is marked, -20 DEG C of preservations.

(4) it washes ware: the PBS of 4 DEG C of pre-coolings is added, crossing method is removed PBS after gently shaking 1min, is repeated 2 times.

(5) it collects cell: using cell scraper concentrated cell, PBS is added to be transferred to cell in marked EP pipe, be centrifuged 2min, PBS are washed twice.

(6) cell cracking: 200-800 μ l is added and cracks liquid mixture, is placed on shaking table, cracks 20min on ice.

(7) it is centrifuged: in low-temperature and high-speed centrifuge, 4 DEG C, 16000rpm centrifugation 10min.

(8) it collects protein sample: the supernatant in centrifuge tube is transferred in new centrifuge tube, mark.

(9) take 5 μ l for determination of protein concentration, after the mixing of 1 × sample-loading buffer is added in remaining sample, boiling water bath 10min, So that every 100 μ l of pipe packing, -80 DEG C of refrigerators freeze after albumen is sufficiently denaturalized.

2.3 determining the protein quantity

Standard protein and sample protein solution are prepared according to BCA determination of protein concentration kit specification.Operating procedure is such as Under:

(1) in BCA reagent A: the ratio of reagent B=50:1 prepares BCA working solution, mixes well.BCA working solution is current existing Match, is placed at room temperature for and keeps stablizing in for 24 hours；

(2) 25 μ l standard items samples are separately added into 96 orifice plates；

(3) it takes 12.5 μ l of protein sample that PBS12.5 μ l is added, dilutes 2 times.2-3 multiple holes of each sample；

(4) all holes respectively add 200 μ l BCA working solutions, mix well；

(5) 37 DEG C of incubation 30min；

(6) OD value is measured at microplate reader 562nm, draws standard curve, calculates sample protein concentration.

2.4 western blot test

(1) vertical electrophoresis glass plate is cleaned, dried, fixed；6%-14% is prepared according to the molecular mass of destination protein The separation gel of concentration.

(2) separation gel is fed into glass plate interlayer, isopropanol sealing liquid prepares concentration glue after glue polymerization completely to be separated.

(3) upper layer water sealing liquid is removed, concentration glue, insertion loading wells comb are poured into, concentration is fixed.

(4) electrophoresis tank is injected into electrophoretic buffer, takes out comb, lateral opening plus 2 μ l pre-dyed albumen Marker, remaining intermediate hole Add albumen after being denaturalized.

(5) power on, starting voltage 80V, after sample after concentration glue-line is pressed into a flat line, adjustment voltage is extremely 120V。

(6) stop electrophoresis when bromophenol blue indicator reaches offset plate bottom, take out gel, and after impregnating 20s with methanol Pvdf membrane (washing away methanol), filter paper immerse transferring film liquid 10min together.

(7) it is successively stacked from top to bottom on transferring film instrument " filter paper-gel-pvdf membrane-filter paper ", when superposition pays attention to excluding gas Bubble.Power on, total current number is adjusted according to the membrane area of 5.5mA/cm2, is arranged according to the relative molecular mass of destination protein Transferring film time, generally 1.5h.

(8) after transferring film, pvdf membrane is taken out, immerses in the beginning of spring red colouring liquid and dyes 1min, whether observation transferring film is complete.

(9)ddH₂O washes away the beginning of spring red colouring liquid, closes 2h with 10% skimmed milk power-PBST confining liquid shaken at room temperature.

(10) primary antibody diluted antibody, 4 DEG C of overnight incubations.

(11) l × TBST is washed film 3 times, room temperature horizontal oscillations, each 10min.

(12) 5%BSA-PBST prepares secondary antibody, is incubated at room temperature 2h.

(13) after secondary antibody is incubated for, l × PBST is washed film 3 times, room temperature horizontal oscillations, each 10min.

(14) it develops the color.By the A liquid and B liquid mixed in equal amounts reaction 1min in ECL kit, it is added dropwise on pvdf membrane, is placed in On ChemiDocTMMP gel imaging system scanning board, the every 100s-200s run-down of software is set, according to band fluorescence intensity Selection exposes preferable band picture and carries out gray value analysis.

3 results

This part content is analyzed according to interactions between protein as a result, having investigated FX to key node egg according in last point of content The influence of white RPS6KA1, NOP2, MIF, CDK12, SQSTM1, STAT3, MDH2, ATP5I.

If Figure 32 shows, FX10,14 μM act on Hep3B cell after, key node albumen RPS6KA1, NOP2, MIF, CDK12, STAT3, MDH2, ATP5I increase protein content with FX concentration and lower, and SQSTM1 up-regulation increases protein content with FX concentration Up-regulation.

RPS6KA1 (Ribosomal protein S6kinase alpha-1), is Ribosomal protein α -1, is led to Peroxophosphoric acid CDK inhibitor C DKN1B participates in Cycle Regulation, which can promote CDKN1B and 14-3-3 protein knot It closes, it is prevented to be transferred to nucleus, and then inhibit cell cycle G1 progress.It can inhibit gastric cancer in vitro after being overexpressed NFIX Proliferation, migration and the invasion of SGC7901 cell line, and it may be NFIX that immunoprecipitation, which combines mass spectrographic method discovery RPS6KA1, Interaction protein, phosphorylation NFIX can enhance its ability of regulation and control to downstream gene.

NOP2 (Probable 28S rRNA (cytosine (4447)-C (5))-methyltransferase) i.e. kernel Antigen 2, phytochemicals SFN, UA and BA are the kernel stress stimuli in breast cancer cell, can induce oxidative stress and egg White matter carbonylation causes uneven B1/ layers of adhesion A/C ratio of layer adhesion, stratum nucleare change in organization and nuclear morphology abnormal.Phytochemistry The kernel stress reaction of mediation is related to nucleoid and synthesizes inhibition to RRN3 transposition and to rRNA, leads to phosphoric acid-S6 signal and kernel NOP2 and WDR12 protein level reduces, to reduce translation efficiency, and then inhibits Cells Proliferation of Human Breast Cancer.

Macrophage migration inhibition factor MIF (Macrophage migration inhibitory factor) is a kind of The critical mediator of unique cell factor and host defense plays a role in tumour, chronic inflammation and autoimmune disease. Some researches show that the expression for lowering MIF can effectively inhibit the proliferation of lung carcinoma cell, induce its apoptosis, and effect may be by The expression for regulating and controlling the downstream target gene that NF- κ B signal access mediates is realized.MIF antibody can obviously inhibit rat colorectal mucosa cancer Become, may be related with rat large intestine exception crypts lesion quantity and MIF expression is inhibited, MIF antibody is expected to become large intestine cancerous precaution With the novel targets for the treatment of.

CDK12, that is, cell cycle protein dependent kinase 12 is a kind of transcription associated kinase, participates in including that DNA damage is anti- It answers, develop and the various cell processes such as cell differentiation and montage and premessenger RNA processing.Overexpression table of the CDK12 in tumour Bright CDK12 has a possibility that carcinogenic nature, similar to other transcription associated kinases.

SQSTM1 (Sequestosome-1), i.e. SQSTM1/p62, and abbreviation p62, for needed for the big autophagy of selectivity from Bite receptor, may participate in cell differentiation, apoptosis, immune response and the channel K+ adjusting, by the way that vesicle is retained in core Zhou Yunzhong Body tissue attracts specific vesicle associated adapter after RNF26 ubiquitination in participating in, and forms a molecular bridge, inhibits core week The homologous vesicle in region, and organize the endosomal of effective cargo transport.Adjust and/or degrade thereon reduce with the formation of tumour, The promotion of cancer and related to the resistance for the treatment of, plays key effect in autophagy and apoptosis.

Signal transduction and (the Signal transducer and activator of transcriptional activators 3 Oftranscription 3, STAT3) mediated cell is to interleukins class, KITLG/SCF, LEP and other growth factors The signal transducer and transcription activation factor of reaction.STAT3 is once activated, just by the synergistic activation factor (such as NCOA1 or MED1) Recruit the promoter region of target gene.Cycle Regulation is participated in by inducing to express from the G1 phase to the key gene of S phase, Such as CCND1.Apoptotic effect can be played and transcribing BIRC5 expression under LEP is activated.Cytoplasm STAT3 passes through inhibition The big autophagy of EIF2AK2/PKR activity suppression.IL-22 promotes human osteosarcoma cell proliferation and invasion, can by IL-22 antibody or STAT3siRNA is reversed.RhoGTPase activator protein 24 (ARHGAP24) adjusts Sorafenib to cream by STAT3 signal path The anticancer activity of gland cancer MDA-MB-231 cell.Natural killer cells (NK) is damaged the toxicity of liver cancer (HCC) cell in HCC The reason of may be anti tumor immune response failure.MiRNAs is considered as the important regulatory factor of NK cell development and function, MiR-506 enhances NK cell to the toxicity of liver cancer cells by targeting STAT3.

Malate dehydrogenase M DH2 (Malatedehydrogenase2) is used as a kind of metabolizing enzyme, in kinds of tumors It is overexpressed.Studies have found that being over-expressed in its endometrial tissues, and related with the grade of cancer.Westernblot, Real-time PCR and immunofluorescence dyeing show that MDH2 inhibits the expression of PTEN, and co-locate with PTEN in carcinoma of endometrium In cytoplasm, meanwhile, proliferation, perforation and apoptosis experiment show MDH2 by inhibit PTEN promote endometrial carcinoma cell proliferation, Migration and invasion, while inhibiting the apoptosis of Endometrial carcinoma cell line.In addition, E2 inhibits the expression of PTEN by GPR30, but Enhance the expression of MDH2.MDH2 gene over-expresses in clinical prostate cancer sample.It is new auxiliary that MDH2 is overexpressed patient's receiving The recurrence-free survival phase (RFS) after chemotherapy is helped to be obviously shortened.Compared with benign prostate epithelial cell, in prostate cancer cell line The expression of MDH2 increases.Cell Proliferation can be reduced by the stable knockout MDH2 of shRNA in prostate cancer cell line and increased more Western paclitaxel-sensitive.In addition, MDH2shRNA enhances the metabolism effect of the alcohol-induced JNK signal activation of Taxotere and induction Rate is low.

ATP5I (ATPsynthasesubunite, mitochondrial) is mitochondrial ATP synthase subunit e, there is research table Bright, the human ATP synthase subunit E (hAS-e) of antisense can be by reducing the suppression of mitogenesis original activated protein kinase (MAP) approach Cell Proliferation processed.The growth of the Antisense Suppression human liver cancer cell of atp synthase subunit E.It tests excessive in cancerous tissue using one The cDNA segment of expression, the normal difference expression gene between liver cancer tissue of comparison.Homology analysis shows the sequence and hAS-e Sequence it is identical.In addition, Northernblot analysis shows that, in 11 (91%) liver cancer samples, the hAS-e of 10 (91%) with Corresponding normal tissue is compared to overexpression.The hAS-e of antisense is introduced in BEL-7404 human liver cancer cell, discovery antisense turns Dye can reduce the activation of serum map kinase, and the downward of hAS-e causes cell growth to be suppressed.

To sum up, these key proteins have regulation cycle progression, macrophage it is mobile inhibit, mediated signal transduction and Transcription activating promotes the effects of autophagy, may play with FX and inhibit Hep3B cell Proliferation, reduces cell viability and cell colony It is formed and inducing cell cycle arrest, cells apoptosis is related.

4 brief summaries

(1) experiment uses iTRAQ quantitative proteomics, identifies map 77064 that peptide fragment is matched to altogether, peptide fragment 33820, unique peptide fragment is 27883, and the protein sum of high confidence level is 5092.

(2) after mass spectrometric data processing, through quantification of protein, fold differences 2 times or more is set as significant difference, at FX10 μM Reason handles comparison in difference result for 24 hours with control group for 24 hours as it can be seen that up-regulation differential protein totally 97, lowers differential protein totally 87；Place Manage 48h compared with the control group result difference as it can be seen that up-regulation differential protein 30, lower 43.

(3) GO analysis is carried out to differential protein, studies the function of differentially expressed protein.BP analysis is found, poor after FX effect M-band bioprocess is concentrated mainly on metabolism process, biological regulation, cell component tissue, cellular processes etc., and effect is for 24 hours With effect 48h no significant difference.CC analysis finds that after FX10 μM acts on 24,48h respectively, differential protein is thin compared with the control group Born of the same parents' component is mainly distributed on cell component, organelle, cell membrane, contains protein complex.In addition, after F X10 μM effect for 24 hours, Differential protein cellular component is also distributed in cell connection, and cellular component is variant on extracellular matrix after acting on 48h.MF points Analysis prompt, after FX effect, differential protein molecule mainly has catalytic activity, combination, molecular function regulator, transport activity, knot Structure adjusts activity, the function of transcripting regulating activity.KEGG analysis finds that differential protein related pathways mainly have hsa05200: Pathwaysincancer；Hsa05202:Transcriptionalmisregulationin cancer；Hsa04010: MAPKsignalingpathway。

(4) difference egg after being acted on Degree, Closeness, Betweenness FX by String network synthesis It is white to be analyzed, it is discovery RPS6KA1, NOP2, MIF, CDK12, SQSTM1, STAT3, MDH2, ATP5I etc. albumen Connected degree, tight Density and node Jie's degree are higher, by Westernblot method, study the above protein expression level variation.It is verified, RPS6KA1, NOP2, MIF, CDK12, STAT3, MDH2, ATP5I protein level under FX effect are lowered, SQSTM1 protein level Up-regulation, it is consistent with protein science qualification result.These key proteins may be played with FX to be inhibited Hep3B cell Proliferation, reduces carefully Born of the same parents' vigor and cell colony are formed and inducing cell cycle arrest, cells apoptosis are related.

This experiment finds that Haizao Yuhu Tang and FX can inhibit CyclinD1 albumen table in various degree by immunohistochemical analysis It reaches, while enhancing Bax protein expression.Its antitumor action may be with inducing cell cycle arrest and apoptosis, inhibition cell Proliferation It is related.

According to In vivo study as a result, project further grinds Haizao Yuhu Tang active constituent FX extracorporeal anti-tumor function Study carefully.Influence of the CCk-8 experimental study FX to Hep3B cells, MHCC97h cell Proliferation, it is seen then that change with administration time, it is empty White cellular control unit quantity persistently increases, FX short time low dosage to cell without obvious effect, it is FX15 μM smaller to impact cell. As dose time changes, FX20,25 μM of obvious inhibition cell Proliferations, effect is significant after 24h.It is living that FX can significantly inhibit cell Power, FX15 μM of effect Hep3B cell is i.e. statistically significant for 24 hours, extends at any time and dosage increasing action is more significant.FX effect Hep3B cell 72hIC35, IC50 value is respectively 10,14 μM, effect MHCC97h cell 72hIC35, IC50 value is respectively 16.9, 20.5 μM, inhibit growth of tumour cell significant effect.

Liver cancer cells maintain the feature of its malignant proliferation by regulating and controlling the bioprocess such as itself period, apoptosis.With normal hepatocytes The cell cycle of cell compares, and liver cancer cells have the following characteristics that the starting in 1. internal liver cancer cells period independent of the external world Proliferation signal；2. the division cycle of liver cancer cells shortens, phase critical period G1 especially in proliferation process obviously contracts It is short；3. the ratio disorder of cell cycle each phase, G1 phase, S phase ratio are reduced, and the ratio of G2 phase and M phase increase；4. in division Being immortalized property of cell in the process, Apoptosis ability decline, is in unconfined vegetative state.The evil in liver cancer cells period Property feature mainly regulated and controled by its complicated cycle regulating network.

The network system of various regulatory factor complicated composition, most importantly cyclin in cell cycle (cyclins)-cell cycle protein dependent kinase (cyclindependentkinase, CDKs)-cyclin relies on Property kinase inhibition albumen (CDKinhibitor, CKI) system.CDK is the core protein of cell cycle regulating network, and expression is lived The change of property directly influences the length of cell cycle, decides the process of cell, growth, differentiation, movement with body cell, The generation of apoptosis and tumour, development, transfer relationship are close.And CKI is negative regulatory factor.CyclinD1 is as a kind of cell week Phase regulatory factor is that is had been found have the oncogene of direct relation with tumour.CyclinD1 can lead to CDK4 in conjunction with CDK4 Activation, the CDK4 of activation can make the protein product phosphorylation of retinoblastoma tumor susceptibility gene (retinoblastoma, Rb) (pRb), the effect for inhibiting transcription factor E2F is lost, starts the transcription of S phase related gene, so that cell be made to enter S by the G1 phase Phase, G1 → S time limit point decide that cell is that the synthesis of beginning DNA continues to be proliferated or enters the G0 phase, or permanently leaves cell Period enters terminal differentiation or death.It can be seen that CyclinD1-CDK4-pRb is cell week in the control of cell cycle Phase important regulatory protein plays a crucial role in the G1-S phase converts.In this experiment, FX10,14 μM be respectively acting on Hep3B cell After for 24 hours, G1 phase cell content rises, and has significant difference (P < 0.01), while the S phase gradually decreases；After acting on 48h, with agent Amount increases and sees and dramatically increase in G1 cell cycle, cell phase have significant difference (P < 0.01), meanwhile, S phase cell distribution It significantly reduces, G2 phase cell distribution is also reduced.FX induces MHCC97h cell cycle distribution situation and Hep3B almost the same, G1 phase cell content rises to 46.15%, 51.98% from 38.99% respectively after FX10,14 μM of effects for 24 hours, wherein FX14 μM Group has statistical difference (P < 0.05), and the S phase gradually decreases；And after FX10,14 μM of effect 48h, G1 phase cell content from 40.44% rises to 55.25%, 59.32%, has significant difference (P < 0.01), the S phase significantly reduces.

As it can be seen that G1 phase cell-cycle arrest occurs after (10,14 μM) of FX effects.And in vivo experiment, use immunohistochemistry Method confirms that in FX mechanism, CyclinD1 protein expression is increased with dosage and reduced, and thus speculates in this experiment and occurs The G1 phase blocks may be related with CyclinD1 protein expression is lowered.Fucoxanthine also can induce kinds of tumor cells Cycle Arrest, Such as by up-regulation p21WAF1/CIP1 albumen, activation NF- κ b, AP1, Akt, JAK/STAT access inducing cell cycle arrest in G0/G1, M phase etc., however in Hep3B cells, MHCC97h cell-cycle arrest, if being worked by the above access has To further study.

The generation of tumour is not only related with the abnormality proliferation of cell and differentiation, also related with the exception of Apoptosis.Induction Apoptosis of tumor cells is the key that oncotherapy point of penetration.Apoptosis is dead different from the special cell of one kind of meronecrosis Die form.

Fucoxanthine can induce various kinds of cell and apoptosis occur, such as human neuroblastoma cell system GOTO, prostate cancer Cell PC3, DU145, LNCaP, human promyelocytic leukemia HL-60, mainly by changing, mitochondrial membrane is penetrating to swash The apoptosis-related proteins signal paths such as caspase living play a role.Hepatoma cell apoptosis is adjusted by number of ways, is adjusting line grain During body membrane permeability, Bcl-2 family protein plays key effect.In this experiment, FX (10,14 μM) acts on Hep3B Apoptosis after cell 24,48h, apoptosis rate are respectively 7.32%, 11.37% and 13.66%, 17.48%.Effect Apoptosis rate is respectively 9.34%, 15.42% and 12.26%, 16.45% after MHCC97h cell 24,48h, in conjunction with immune group It is related that change related experiment result infers that FX induction Hep3B cells apoptosis may raise Bax protein expression with it.

ITRAQ (isobaric tagsfor relative and absolute quantitation) technology is a kind of Based on the opposite and absolute quantitation technology of external equal heavy labels, which utilizes isotope reagent labeling polypeptide end ammonia Base or lysine side chain amino groups repeat the high sensitivity of low-abundance protein detection through high-resolution mass spectrometer Tandem analysis Property it is good, the protein expression quantity between various samples can be compared simultaneously, separate and identify hundreds and thousands of protein, can be maximum simultaneously " the full group information " of the acquisition protein of change, is the common High Throughput Screening Assay of quantitative proteomics in recent years.With this side Whether method can screen the protein of fucoxanthine antitumaous effect, and whether the antitumaous effect of fucoxanthine is by multiple proteins tune Control expression is to realize that there is not been reported.

After 10 μM of FX of this experiment selection acts on Hep3B cell 24,48h, cell is collected, extracts total protein, mass spectrometric data After processing, sum 5092, high confidence level protein are identified altogether, set fold differences 2 times or more as significant difference, it is seen then that Effect raises differential expression protein 97 afterwards for 24 hours, lowers differential expression protein 87, totally 184；It is raised after effect 48h Differential expression protein 30, lower differential expression protein 43, totally 184.

Protein group is identified, first by protein digestion at peptide fragment, carries out peptide fragment identification with mass spectrum, then deriving can The protein of energy.Test to assess the overall condition of proteome data obtained, from map, peptide fragment and albumen these three Level is assessed from each physicochemical property etc..The error distribution for identifying map is the molecular weight error of peptide fragment, depends on matter The precision and levels of collimation of spectrum, this empirical theory upward peak is close to 0, and whole distribution is most of all in being arranged when searching library Error range.By protein digestion at peptide fragment, peptide fragment identification, divalent, trivalent, the peptide fragment qualification result affirmative of 4 valences are carried out with mass spectrum. It identifies the assessment of peptide fragment and statisticallys analyze the PSM number distribution identified from peptide segment length, each peptide fragment, peptide fragment ion is given a mark Distribution, the distribution of peptide fragment ion marking, peptide fragment leakage enzyme site number distribution are started with, it is seen then that the matching of peptide fragment ion is reliable, and digestion is thorough Bottom.The assessment for identifying albumen and statistical analysis are as it can be seen that the matched peptide number of segment distribution of protein, the matched PSM number of protein point Cloth, identification albumen are distributed by the isoelectric point of the distribution of peptide fragment coverage rate, identification albumen.

Gene ontology (Gene Ontology, GO) is function, positioning and the movable standard vocabulary for describing gene Table has tree structure, is the most widely used ontology of molecular biology field.Gene and gene product vocabulary cover biology Three aspects.Cellular component (Cellular Component, CC) describes each part and the extracellular environment of cell；Molecule function Energy (Molecular Function, MF) can be described as the activity (activity) of molecular level, such as be catalyzed (catalytic) Or combine (binding) activity；Bioprocess (Biological Process, BP) is orderly by one or more molecular functions The chain of events of combination and generation, a process are made of multiple and different steps.

This experimental analysis discovery mainly has after FX10 μM acts on 24,48h respectively with control group differential protein cellular component Cell component, organelle, extracellular region, cell membrane, macromolecular complex.In addition, FX10 μM acts on rear visible cell for 24 hours respectively and connects It connects, act on visible cell epimatrix after 48h.In terms of molecular function, after FX10 μM of effect 24,48h, with control group differential protein master Concentrate on catalytic activity, combination, transport activity, structure adjusting activity aspect.In terms of bioprocess, after effect 24,48h, with Control group comparing difference albumen is concentrated mainly on the side such as molecular processes, metabolism process, biological regulation, stimulate the reaction, bio-adhesive Face.

By the network String integrated use Degree, Closeness, Betweenness to differential protein after FX effect It is analyzed, it is discovery RPS6KA1, NOP2, MIF, CDK12, SQSTM1, STAT3, MDH2, ATP5I etc. albumen Connected degree, close Degree and node Jie degree are higher, by Westernblot method, study the above protein expression level variation.It is verified, RPS6KA1, NOP2, MIF, CDK12, STAT3, MDH2, ATP5I FX effect under compared with the control group protein level lower, SQSTM1 with it is right Compare protein level up-regulation according to group, it is consistent with protein science qualification result.

Cell cycle protein dependent kinase (CDK) is the essential mediator of various cell processes.They are divided into two Asias Family: cell cycle relevant CDK (CDK1,2,4,6), they directly adjust the progress by the individual cells phase of the cycles, with And the relevant CDKs (CDK7,8,9,11,12,13) of transcription, adjust genetic transcription.The C-terminal knot of these tyrosine phosphorylations Rbp1 Structure domain, and Rbp1 is the maximum subunit and various transcription regulaton factors of rna plymerase ii (RNApolII).CDK in tumour It often lacks of proper care in cell, can be used for the therapeutic targets of the wide spectrum of tumour.

Eukaryotic transcription is complicated and regulation is stringent, and cell processes include differentiation and the reaction to extracellular stimulus, depends on turning Record horizontal adjusting.In addition, transcription and other events, accurate such as mRNA processing, montage, chromatin remodeling and histone modification Coordinate most important for normal stechiology.Therefore, when the imbalance of these processes, generation and the hair of cancer can be driven Exhibition.Transcription factor is often mutated in cancer cell and represents typical oncogene and tumor suppressor.These mutation lead to base Because expressing the change of program, and there may be the dependences to certain transcription regulaton factors, keep cancer cell addicted to its activity, this Phenomenon is referred to as " transcription habituation ", provides chance for the novel therapeutic intervention of cancer.

CDK12 is a kind of transcription relevant CDK, CTD of phosphorylatable RNApolII, it reacts DNA damage, montage and Break up most important.It has reported CDK12 mutation in various malignant tumours and has been overexpressed.

RPS6KA1 (RibosomalproteinS6kinasealpha-1), is Ribosomal protein α -1, and length is 90kDa acts on ERK (MAPK1/ERk2 and MAPK3/ERK1) downstream signal transduction, and the mediate transcription factor CREB1, ETV1/ The mitosis of ER81 and NR4A1/NUR77 and stress-induced activation, are translated, and lead to by RPS6 and EIF4B phosphorylated regulation It overregulates mTOR signal and the apoptotic function of BAD and DAPK1 is inhibited to come mediated cell proliferation, survival and differentiation.Pass through phosphoric acid Change CDK inhibitor C DKN1B and participate in Cycle Regulation, which can promote CDKN1B in conjunction with 14-3-3 protein, prevent It is transferred to nucleus, and then inhibits cell cycle G1 progress.Gastric cancer SGC7901 can be inhibited in vitro thin after being overexpressed NFIX Proliferation, migration and the invasion of born of the same parents system, and it may be the interaction of NFIX that immunoprecipitation, which combines mass spectrographic method discovery RPS6KA1, Albumen, phosphorylatable NFIX enhance its ability of regulation and control to downstream gene.

Kernel be stress sensor, activity impaired is considered as a kind of anticancer strategy.In addition in ribosomes biosynthesis Main function outside, kernel also participate in cell cycle progress and stress signal adjusting.NOP2(Probable28SrRNA (cytosine (4447)-C (5))-methyltransferase) i.e. nucleolar antigen 2, participate in the assembling of ribosomes large subunit.S- Adenosyl-L-methione dependence transmethylase, the position C5 of cytimidine 4447 in possible specific methylation 28SrRNA.It can It can work adjusting the cell cycle and increasing in kernel activity relevant to cell Proliferation.Phytochemicals SFN, UA and BA For the kernel stress stimuli in breast cancer cell, oxidative stress and protein carbonylation can induce, cause B1/ layers of layer adhesion to glue Even A/C ratio imbalance, stratum nucleare change in organization and nuclear morphology are abnormal.The kernel stress reaction that Phytochemistry mediates is related to nucleoid Inhibition is synthesized to RRN3 transposition and to rRNA, phosphoric acid-S6 signal and kernel NOP2 and WDR12 protein level is caused to reduce, thus Translation efficiency is reduced, and then inhibits Cells Proliferation of Human Breast Cancer.

Macrophage migration inhibition factor MIF (Macrophage migration inhibitory factor) is a kind of The critical mediator of unique cell factor and host defense plays a role in tumour, chronic inflammation and autoimmune disease. Whole body inhibits MIF that can significantly increase antitumor cell toxic T lymphocyte (CTL) Th1 response during mice tumors grew；With table Neuroblastoma cell up to MIF is compared, the induced strong antitumor CTL response in transplantable tumor mouse after silencing MIF.It grinds Study carefully and show to lower the expression of MIF and can effectively inhibit the proliferation of lung carcinoma cell, induces its apoptosis, effect may be by The expression for the downstream target gene that NF- κ B signal access mediates is realized.MIF antibody can obviously inhibit rat colorectal mucosa canceration, can Can be related with rat large intestine exception crypts lesion quantity and MIF expression is inhibited, MIF antibody is expected to become colorectal cancer prevention and treatment Novel targets.

Cell cycle protein dependent kinase (CDK) is the key regulator of cell cycle progression and transcription, the mistake of CDK Tune is the tumorigenic frequent event of driving.CDK12, that is, cell cycle protein dependent kinase 12 is a kind of transcription associated kinase, Participate in various cell processes, including DNA damage reaction, development and cell differentiation and montage and premessenger RNA processing.CDK12 is prominent Become and expand in different types of malignant tumour, including the function loss mutation in high-level serous ovarian cancer, and leads It causes to assume that CDK12 is tumor suppressor.On the contrary, overexpression of the CDK12 in other tumours shows that CDK12 has carcinogenic spy Property a possibility that, to other transcription associated kinases it is similar.

SQSTM1 (Sequestosome-1) is autophagy receptor needed for selectively big autophagy, serves as more ubiquitin cargos and oneself The bridge between body is bitten, is directly interacted with cargo to be degraded and the autophagy dressing agent of MAP1LC3 family, it can be with WDFY3 mono- Act the formation for participating in cytoplasm ubiquitin inclusion body (P62 body, ALIS/ aggregation sample inducement structure) and autophagy degradation.It may participate in thin Born of the same parents' differentiation, apoptosis, immune response and the channel K+ adjusting, by by vesicle be retained in core Zhou Yunzhong participate in body tissue, After RNF26 ubiquitination, attract specific vesicle associated adapter, form a molecular bridge, inhibits the homologous vesicle in core week region, And organize the endosomal of effective cargo transport.SQSTM1/p62, and abbreviation p62, adjusting and/or degrade thereon reduces and tumour It is formed, the promotion of cancer and related to the resistance for the treatment of, plays key effect in autophagy and apoptosis.

Malate dehydrogenase M DH2 (Malatedehydrogenase2) is used as a kind of metabolizing enzyme, Endometrial Carcinomas It is over-expressed in tissue, and related with the grade of cancer.Westernblot, real-time PCR and immunofluorescence dyeing show MDH2 suppression The expression of PTEN has been made, and has been co-located in the cytoplasm of carcinoma of endometrium with PTEN, meanwhile, proliferation, perforation and apoptosis are real It tests and shows that MDH2 by inhibiting PTEN to promote endometrial carcinoma cell proliferation, migration and invasion, inhibits Endometrial carcinoma cell line Apoptosis.In addition, E2 inhibits the expression of PTEN by GPR30, but enhance the expression [41] of MDH2.MDH2 gene is in clinic It is over-expressed in prostate cancer sample.MDH2 is overexpressed patient and receives recurrence-free survival phase (RFS) obviously contracting after new adjuvant chemotherapy It is short.Compared with benign prostate epithelial cell, the expression of MDH2 is increased in prostate cancer cell line.In prostate cancer cell line Cell Proliferation and increase Docetaxel sensibility can be reduced by stablizing knockout MDH2 by shRNA.In addition, MDH2shRNA enhances Taxotere alcohol-induced JNK signal activation and the metabolic efficiency of induction are low.

Therefore, these key proteins may play with FX and inhibit Hep3B cell Proliferation, reduce cell viability and cell colony It is formed and inducing cell cycle arrest, cells apoptosis is related.

The present invention confirms the antitumor of Haizao Yuhu Tang and its active constituent fucoxanthine by inside and outside experimental study Effect, provides basis for tcm clinical anti-cancer applications.Through iTRAQ quantitative protein group, interactions between protein analysis and binding molecule Biological experiment discloses fucoxanthine and regulates and controls key protein group, inquired into fucoxanthine antihepatocarcinoma effect approach.

Obtained conclusion: (1) Haizao Yuhu Tang and its active constituent fucoxanthine can dose dependent significantly inhibit nude mice Hep3B growth of transplanted human；Haizao Yuhu Tang improves tumor bearing nude mice liver function to a certain extent；

(2) fucoxanthine obviously inhibits Hep3B cells cell Proliferation and cell viability in vitro, cell clone size and Form number decline, it may be possible to G1 phase cell-cycle arrest be occurred by induction Hep3B cells cell, MHCC97h cell, promoted Apoptosis plays antitumaous effect.

(3) researches show that the controllable multiple proteins of fucoxanthine for protein science: cellular portions, organelle, film, macromolecular are multiple Close object, these albumen have the function of catalytic activity, transcriptional activity, combination, molecular function regulator etc. mostly, participate in cell into Journey, metabolism process, biological regulation；Related pathways mainly have Pathwaysincancer, Transcriptionalmisregula tionincancer；MAPKsignalingpathway etc..Interactions between protein analyzes Binding experiment verifying display: interactions between protein network Key protein has cell cycle regulation process, inhibits mobile macrophage, mediated signal transduction and transcription activating, promotes autophagy The effects of；FX may be by adjusting the protein exhibits inducing cell cycle arrest, apoptotic effect, so that Hep3B cell be inhibited to increase It grows, vigor, Colony forming.

Haizao Yuhu Tang antihepatocarcinoma effect in nude mouse is clear, energy liver function protecting, and without obvious toxic-side effects, soft Hard dissipating bind effect is significant to clinical application is instructed, and proteomics discloses the albumen that fucoxanthine plays adjustment effect Network group.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, without departing from the principle of the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as Protection scope of the present invention.

Claims

1. application of the Haizao Yuhu Tang in the drug of preparation treatment liver cancer, the liver cancer is primary carcinoma of liver.

2. application of the Haizao Yuhu Tang in the drug that preparation inhibits hepatoma cell proliferation, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

3. application of the Haizao Yuhu Tang in the drug for preparing liver cancer apoptosis reducing, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell, the Haizao Yuhu Tang are by up-regulation Bax expression come liver cancer apoptosis reducing.

4. application of the Haizao Yuhu Tang in the drug that preparation inhibits the liver cancer cells period, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell, the Haizao Yuhu Tang are to inhibit the liver cancer cells period by inhibiting the expression of CyclinD1.

5. application of the fucoxanthine in the drug of preparation treatment liver cancer, the liver cancer is primary carcinoma of liver.

6. fucoxanthine preparation inhibit hepatoma cell proliferation drug in application, the liver cancer cells be Hep3B cells, MHCC97h cell.

7. application of the fucoxanthine in the drug that preparation inhibits liver cancer cells Clone formation, the liver cancer cells are human liver cancers Hep3B, MHCC97h cell.

8. application of the fucoxanthine in the drug for preparing liver cancer apoptosis reducing, the liver cancer cells be Hep3B cells, MHCC97h cell.

9. application of the fucoxanthine in the drug that G1 phase cell-cycle arrest occurs for preparation induction liver cancer, the liver cancer cells are Hep3B cells, MHCC97h cell.

10. fucoxanthine reduces RPS6KA1, NOP2, MIF, CDK12, STAT3, MDH2, ATP5I egg in liver cancer cells in preparation It is white, improve application in liver cancer cells in the drug of SQSTM1 albumen, the liver cancer cells are Hep3B cells, MHCC97h thin Born of the same parents.