CN108403941A - A kind of Chinese medicine composition and its preparation method and application for treating cancer of pancreas - Google Patents
A kind of Chinese medicine composition and its preparation method and application for treating cancer of pancreas Download PDFInfo
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Abstract
The present invention discloses a kind of Chinese medicine composition and its preparation method and application for treating cancer of pancreas.The Chinese medicine composition is made of following raw material:20 25 parts by weight of Chinese yam, 48 parts by weight of rhizoma atractylodis, 15 20 parts by weight of jujube, 10 20 parts by weight of Semen Lablab Album, 58 parts by weight of pawpaw, 58 parts by weight of Morinda officinalis, 58 parts by weight of rhizoma arisaematis, 10 15 parts by weight of Snakegourd Fruit, 10 15 parts by weight of winter jasmine, 15 25 parts by weight of strophanthus divaricatus, 5 12 parts by weight of Akebia Fruit, 27 parts by weight of edible tulip, 10 15 parts by weight of champion bauhinia stem, 15 parts by weight of Himalayan mayapple fruit, 27 parts by weight of Rhizoma Acori Calami, 5 15 parts by weight of herba aristolochiae.The Chinese medicine composition has invigorating spleen to remove dampness, eliminating the phlegm heat-clearing, stagnation resolvation promoting blood circulation, warm stomach qi-regulating and other effects.Zoopery shows that high, middle dosage the Chinese medicine composition can obviously improve cancer of pancreas Nude Mouse Model mouse tumour inhibiting rate, spleen index, cell cycle, Apoptosis, block the expression of Hedgehog signal path key molecules, while without side-effects.The Chinese medicine composition will have broad application prospects in terms for the treatment of cancer of pancreas.
Description
Technical field
The present invention relates to a kind of Chinese medicine compositions and its preparation method and application, more particularly to a kind of to treat in cancer of pancreas
Drug composition and its preparation method and application.The invention belongs to pharmaceutical technology fields.
Background technology
Cancer of pancreas (Pancreatic Cancer) refers to the alimentary system malignant tumour that body external pancreatic secretion portion occurs.Closely
Ascendant trend is presented in whole world pancreas cancer morbidity, illness rate, the death rate within several years, wherein our national rural area pancreas
Cancer morbidity is higher than urban area more, meanwhile, our national cancer of pancreas year diagnosis and the death rate already exceed North America prosperity
Country (Lin QJ, Yang F, Jin C, et al.Current status and progress of pancreatic
Cancer in China [J] .World J Gastroenterol, 2015,21 (26):7988-8003.), these prompt us
Country needs to pay much attention to cancer of pancreas.
Cancer of pancreas onset be mostly it is hidden attack, rarer specific symptom and sign, this can cause cancer of pancreas early diagnosis, early stage
Treatment is more difficult, many patients existing transfer mostly when cancer of pancreas is made a definite diagnosis, and is easy to send out to liver, peritonaeum, lung and part leaching
It fawns on, the treatment of operative treatment compatibe drug is the preferred plan for the treatment of of pancreatic cancer, still, cancer of pancreas operative mortality rate, postoperative multiple
Hair rate is relatively high, and 5 annual survival rates are relatively low.Therefore, it is necessary to urgently propose the strategy of new treatment cancer of pancreas.
In recent years, much researches show that the generation of Hedgehog signal paths and cancer of pancreas and its development are closely related.
In embryonic development, Hedgehog signal paths are highly conserved, which can participate in a variety of groups of body of regulation and control
Knit allelotaxis.Hedgehog signal paths include ligand (Shh), membrane receptor (Ptch and Smo), nuclear factor (G1i family
Race), (Qin S, Sun D, Li H, the et al.The Effect of SHH-Gli Signaling Pathway such as regulatory factor
on the Synovial Fibroblast Proliferation in Rheumatoid Arthritis[J]
.Inflammation, 2016,39 (2):503-512.).In general, normal body usually can't detect Hedgehog signals
Access is expressed, it has already been proven that Several Kinds of Malignancy highly expressed extremely with Hedgehog signal paths it is closely related (Skoda AM,
Simovic D, Karin V, et al.The role of the Hedgehog signaling pathway in cancer:
A comprehensive review [J] .Bosn J Basic Med Sci, 2018,18 (1):8-20.), such as cancer of pancreas
(Gu J, Saiyin H, Fu D, et al.Stroma A Double Edged Sword in Pancreatic Cancer:A
Lesson From Targeting Stroma in Pancreatic Cancer With Hedgehog Signaling
Inhibitors [J] .Pancreas, 2018,47 (4):382-389.), gastric cancer, cervical carcinoma, prostate cancer, liver cancer, oophoroma,
Breast cancer, colorectal cancer, carcinoma of mouth, Skin Squamous Cell Carcinoma etc..It has confirmed, Hedgehog signal paths occur in most of Pancreas cancer patients
Activation, this will change cancer of pancreas biological property, can especially be detected in pancreas in Pancreas cancer patients early stage
Hedgehog signal paths Shh, Ptch, Smo, Gli high expression, it is seen then that Hedgehog signal paths rise important in cancer of pancreas
Effect.
There are three kinds of ligands Shh, Ihh and Dhh in Hedgehog signal paths, wherein ligand Shh researchs are more.Match
Body Shh is secreting type signal protein highly conserved in Hedgehog signal paths, is also activation Hedgehog signal paths
And the key protein of iuntercellular adjustment effect is played, ligand Shh is similar to cholesterol transfer enzyme effect, has self catalysis processing energy
Power can also release Ptch and inhibit to Smo and activate Smo (Du Z, Zhou F, Jia Z, et al.The hedgehog/Gli-
1 signaling pathways is involved in the inhibitoryeffect of resveratrol on
Human colorectal cancer HCT116 cells [J] .Iran J Basic Med Sci, 2016,19 (11):
1171-1176.).Ptch is membrane protein receptor, can be divided into Ptch1 and Ptch2, Ptch1 and Ptch2 all have in conjunction with Shh and
Inhibit Smo effects.Ptch is Hedgehog signal path key albumen, and Ptch up-regulated expressions are the activation of Hedgehog signal paths
Key factor, mutation Ptch loses inhibiting effect to Smo, and Smo activates downstream Gli, causes Hedgehog signal path target bases
Because of activation (Wang LW, Lin H, Lu Y, et al.Sonic hedgehog expression in a rat model of
Chronic pancreatitis [J] .World J Gastroenterol, 2014,20 (16):4712-4717.).Smo is original
Oncogene is Hedgehog signal path key members.When lacking Shh under normal circumstances, Smo activity can be pressed down by Ptch
System, and Smo plays control action to the downstream transcription factor Gli states of activation.Gli mainly include three kinds of molecule Gli1, Gli2,
Gli3 and Gli1, Gli2, Gli3 structure are particularly similar.Gli1, Gli2, Gli3 have 5, DNA structure domain zinc fingers, can be with
Hedgehog signal paths downstream target gene (GAC CAC CCA) particular sequence in conjunction with and play regulatory role.Wherein, Gli1 has
Potent activating transcription factor effect, Gli2 mainly exist in the form of activating transcription factor, and Gli3 mainly plays transcription inhibitory factor
Effect.
Data shows that cancer of pancreas Hedgehog signal paths can be over-expressed by abnormal activation with Gli1, and be combined
Downstream target gene (GAC CAC CCA) particular sequence in conjunction with and (Gan H, Liu H, Zhang H, the et al.SHh- that plays a role
Gli1 signaling pathway promotes cell survival by mediating baculoviral IAP
Repeat-containing 3 (BIRC3) gene in pancreatic cancercells [J] .Tumour Biol, 2016,
37(7):9943-9950.), reverse feedback regulation ring is formed, the Ptch transcriptions of Hedgehog signal paths is lured into, target gene is made to live
Change, causes Hedgehog signal paths out of control.It has confirmed, Gli target genes are related to growth of tumour cell, apoptosis, transfer, new
Angiogenic is formed and Epithelial and stromal conversion, for example, Gli can inducing cyclin D1 expression and promote pancreatic cancer cell to increase
It grows, directly activation can cause epithelial tissue ingredient to mesenchymal transformation factor transcription and enhance pancreatic cancer cell wellability, directly
The expression of induction Bcl-2 etc. anti-apoptosis factors while inhibiting Bax etc. and promote antiapoptotic factors expression, apoptosis of pancreatic cancer cell can be inhibited, most
The key protein Ptch of whole direct regulation and control Hedgehog signal paths is activated and is promoted Cell Proliferation of Pancreatic Cancer Cell.As it can be seen that effectively resistance
Disconnected Hedgehog signal paths can inhibit pancreatic cancer cell growth, invasion and transfer, this is the active strategy for the treatment of of pancreatic cancer.
Currently, all drug can treat cancer of pancreas both at home and abroad, such as 5 FU 5 fluorouracil (Wang WB, Yang Y, Zhao
YP, et al.Recent studies of 5-fluorouracil resistance in pancreatic cancer [J]
.World J Gastroenterol, 2014,20 (42):15682-15690.), antioxidant, anti-apoptotic drug, white lamb's-quarters
Reed alcohol (Xu Q, Zong L, Chen X, et al.Resveratrol in the treatment of pancreatic
Cancer [J] .Ann N Y Acad Sci, 2015,1348 (1):10-19.), cis-platinum etc., still, many drugs all have one
Determine the side effect of degree, harm can be brought to the body of Pancreas cancer patients, for this purpose, actively seeking Small side effects or making without pair
With, and very effective treatment pancreatic cancer drug is particularly important.
Theory of traditional Chinese medical science thinks that The spleen has the function to transport and transform nutrients, it will performance, which is transported, the basic functions such as conveys, digests and assimilates.The spleen has the function to transport and transform nutrients can
It is divided into two aspects of transporting and transforming nutrients from foodstuff and transformation of body flaud, spleen will promote the tissues such as internal organs, channels and collaterals to obtain fully by transportation capabilities
Nutrition.Spleen transportation capabilities are healthy and vigorous, prevent from the generation of the pathological products such as wet water, phlegm and retained fluid, dampness, blood stasis, heat evil.Once machine
Burnt dys-splenism will will appear spleen deficiency in body, and spleen deficiency is then wooden strongly fragrant, and spleen deficiency then gives birth to wet, spleen deficiency then dysfunction of the spleen in transportation and transformation, from outward appearance to inner essence strongly fragrant
And change heat, it forms spleen asthenia and dampness retention and induces cancer of pancreas, so cancer of pancreas needs invigorating spleen to remove dampness.In addition, body spleen deficiency then damages spleen, companion
There is inside and outside damp and hot heresy to be harmonious, stored in heat toxin, stay for a long time and do not go, gradually at lump, phlegm, heat, the stasis of blood, blood knot is caused to gather in spleen, it will and shape
At carcinoma, so cancer of pancreas needs eliminating the phlegm heat-clearing, resolving sputum promoting blood circulation.Further more, then transportation and transformation of water and food is not normal for body spleen deficiency, channels is obstructed,
Liver and gall disorder of qi, liver and gall mechanism of qi are obstructed, and suffocating of liver and gall is not normal, and with healthy tendency loss, diseases caused by external factors are damp and hot, diet is not normal, it is final and
At carcinoma, so cancer of pancreas needs regulating qi and dredging collateral.Pass through the disease pathogenic analysis on theory of traditional Chinese medical science basis as a result, as it can be seen that cancer of pancreas
Need invigorating spleen to remove dampness, eliminating the phlegm heat-clearing, resolving sputum promoting blood circulation, regulating qi and dredging collateral.
Chinese medicine is relatively strong with synergistic effect, side effect is smaller, drug target is more, drug is with integrally-regulated equivalance
Pole feature is delaying cancer of pancreas process, enhancing immune function, raising oxidation resistance, decrease inflammatory reaction, protection body normal
Cell etc. have unique positive advantage, this for clinical treatment cancer of pancreas bring hope (Cao Nida, Xu Jiaoya, it is old
It is refined, wait Chinese medicine compound prescriptions WCA-P and its side of tearing open to inoculate influence [J] of tumor to human pancreas cancer BxPC-3 cell suspension nude mouses
Western traditional Chinese medicine, 2017,30 (1):7-10.).The preferable and without side-effects Chinese medicine composition of curative effect is found as a result, carries out pancreas
The treatment work of cancer is just particularly important.
Invention content
The purpose of the present invention is to provide a kind of Chinese medicine compositions and preparation method thereof for treating cancer of pancreas.
In order to achieve the above object, present invention employs following technological means:
The present invention selects 16 kinds of Chinese medicines such as Chinese yam, rhizoma atractylodis, jujube, Semen Lablab Album, pawpaw, Morinda officinalis, rhizoma arisaematis, Snakegourd Fruit, meets
The Chinese traditional medicine composition of spring flower, strophanthus divaricatus, Akebia Fruit, edible tulip, champion bauhinia stem, Himalayan mayapple fruit, Rhizoma Acori Calami, herba aristolochiae composition treatment cancer of pancreas
Object.In order to prove that the effect of the traditional Chinese medicine composition for treating cancer of pancreas, the present invention have carried out following tests:
Germicidal efficacy growing state of the Chinese medicine composition to cancer of pancreas BxPC-3 cell strains.The results show that with Chinese medicine
The increase of composition concentration and its extension of Chinese medicine composition intervention time, Chinese medicine composition is to cancer of pancreas BxPC-3 cell strains
Inhibiting effect also gradually increases, and illustrates that there are docs-effects for pharmacological action of the Chinese medicine composition to cancer of pancreas BxPC-3 cell strains
Relationship.After Chinese medicine composition active intervention, Chinese medicine composition is high, middle dosage has cancer of pancreas BxPC-3 cell strain growths
Inhibiting effect;Meanwhile compared with 5 FU 5 fluorouracil group, Chinese medicine composition height, middle dose group the 3rd, 4,5,6, pancreas on 7d time points
Gland cancer BxPC-3 cell strain viable counts change unobvious (P > 0.05), and Chinese medicine composition low dose group the 3rd, 4,5,6,
Cancer of pancreas BxPC-3 cell strain viable counts obviously increase (P > 0.05) on 7d time points, and indication Chinese medicine composition is high, middle dose
Amount and 5 FU 5 fluorouracil are equivalent to the inhibiting effect of cancer of pancreas BxPC-3 cell strains, and Chinese medicine composition low dose therapy cancer of pancreas
The effect of BxPC-3 cell strains is poor.Synthesis display, Chinese medicine composition is high, middle dosage can inhibit cancer of pancreas BxPC-3 cell strains
Growth, and this inhibiting effect is identical as 5 FU 5 fluorouracil.
Measuring influence of the Chinese medicine composition to the locomitivity level of cancer of pancreas BxPC-3 cell strains.As a result table
It is bright, after Chinese medicine composition and 5 FU 5 fluorouracil treatment, Chinese medicine composition high dose, Chinese medicine composition middle dosage, 5- fluorine urine
Pyrimidine has very strong inhibiting effect, as dosage increases, Chinese medicine composition pair to cancer of pancreas BxPC-3 cell strain locomitivities
The locomitivity influence of cancer of pancreas BxPC-3 cell strains will be more obvious;Meanwhile Chinese medicine composition low dosage is to cancer of pancreas
The locomitivity of BxPC-3 cell strains influences poor.Synthesis display, Chinese medicine composition is high, middle dosage can inhibit cancer of pancreas
The locomitivity of BxPC-3 cell strains, this positive intervention effect are more apparent.
This research chooses cancer of pancreas BxPC-3 cell strains and builds cancer of pancreas BxPC-3 Nude Mouse Model mouse.With model
Group compares, and Chinese medicine composition high dose, Chinese medicine composition middle dosage, Chinese medicine composition low dosage and 5 FU 5 fluorouracil are to cancer of pancreas
Equal obvious inhibiting effect (the P of transplanted tumor in nude mice<0.05), wherein Chinese medicine composition high dose group tumor killing effect is fine.Each experimental group
Tumour inhibiting rate is all higher than 30%, according to the antitumor validity standard of Chinese herbal medicine and《Modern tumor therapeutic agent》Correlation standard
(tumour inhibiting rate>30%), it was initially believed that Chinese medicine composition high dose, Chinese medicine composition middle dosage, Chinese medicine composition low dosage are to pancreas
Gland cancer BxPC-3 transplantable tumors are inhibited.In general, spleen mainly generates B cell, body can be evaluated by measuring spleen
Immune function, this experiment using electronic balance detect mouse spleen index.Experimental result shows, Chinese medicine composition high dose group,
Chinese medicine composition middle dose group, Chinese medicine composition low dose group mouse spleen index are higher, illustrate Chinese medicine composition high dose group, in
It is more that drug composition middle dose group, Chinese medicine composition low dose group mice spleen mainly generate B cell, prompts the high agent of Chinese medicine composition
Amount group, Chinese medicine composition middle dose group, Chinese medicine composition low dose group mouse humoral immune level are stronger, prompt the immune of body
Function is stronger;5 FU 5 fluorouracil group mouse spleen index reduces, and illustrates the reduction of 5 FU 5 fluorouracil group mouse humoral immune level, prompts
The immune function of body is poor, and side effect is larger.Synthesis display, Chinese medicine composition high dose, Chinese medicine composition middle dosage, Chinese medicine
Composition low dosage has antitumor action, side effect smaller.
This research by flow cytometry analysis showed, Chinese medicine composition high dose group, Chinese medicine composition low dose group,
5 FU 5 fluorouracil group and model group relatively in, significant change has occurred in the cancer of pancreas BxPC-3 transplantable tumor cell cycles, wherein Chinese medicine
Composition high dose group, Chinese medicine composition middle dose group cancer of pancreas BxPC-3 transplantable tumor G1 phases obviously rise, and the G2 phases are decreased obviously
(P<0.05) cancer of pancreas BxPC-3 transplantable tumor cell cycles G2 shorten after, showing Chinese medicine composition height, middle dosage effect, and the G1 phases prolong
It is long, prompt pancreatic cancer cell retardance in the G1 phases.Meanwhile in Chinese medicine composition high dose group, Chinese medicine composition low dose group, 5- fluorine
During uracil group is compared with model group, cancer of pancreas BxPC-3 transplantable tumors apoptosis rate apparent increase (P<0.05).It is comprehensive
It has been shown that, Chinese medicine composition high dose, Chinese medicine composition middle dosage by by cancer of pancreas BxPC-3 transplantable tumors cell-cycle arrest in
It G1 phases and its reduces apoptosis rate and plays antitumor action.
Data shows that Hedgehog signal paths are highly conserved, and Hedgehog tables are usually can't detect in normal human
It reaches, and cancer of pancreas Hedgehog signal path height is expressed, cancer of pancreas and Hedgehog signal path Shh, Ptch1, Smo exception tables
Up to closely related (Dosch JS, Pasca di Magliano M, Simeone DM.Pancreatic cancer and
hedgehog pathway signaling:New insights [J] .Pancreatology, 2010,10 (3):151-
157.).It has confirmed, malignant cell can secrete Shh, as secretory signaling molecule to itself and peripheral cell production
Raw effect.In pancreas carcinogenesis early stage, Hedgehog signaling pathways are activated, and Pancreas ductal epithelium is caused to occur pernicious turn
Become, Ptch1 is used as membrane protein receptor on Hedgehog signal paths, and the recipient for being Hedgehog signal paths is to transmit again
Person releases Ptch1 to Smo inhibiting effect, so that Smo is transmitted signal intracellular, activation downstream transcription factor Gli families are adjusted
Related expression of target gene.After Gli1 activation, nucleus can be entered from cytoplasm, make Glil activation downstream gene expression up-regulations, with
This induces cancer of pancreas.This research is from internal angle, using immunohistochemistry, the sides blot Real-time PCR and Western
Method, inquires into whether the effect of Chinese medicine composition anti-pancreatic cancer is worked by this signal path.Immunohistochemistry results show,
Hedgehog signal paths Shh, Ptch1, Smo are positioned at cytoplasm and/or cell membrane, and Gli1 is positioned at cytoplasm and/or thin
Karyon.It detects that Hedgehog signal paths Shh, Ptch1, Smo high are expressed in cancer of pancreas BxPC-3 Transplanted tumor models, passes through
After Chinese medicine composition is intervened, compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Equal low expression (the P of pyridine group cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signal paths Shh, Ptch1, Smo<0.05) in,
Drug composition low dose group cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signal paths Shh, Ptch1, Smo high are expressed, and are said
Bright Chinese medicine composition high dose, Chinese medicine composition middle dosage can lower cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signals
Access Shh, Ptch1, Smo expression.
Using Real-time PCR and Western blot, more precisely quantitatively detection Hedgehog signals are logical for this research
Road activation levels.The results show that model group cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signal paths Shh, Ptch1,
Smo, Gli1 high are expressed.When Shh overexpressions, Shh is combined with Ptch1 releases inhibition to Smo, promote zinc finger turn it is green because
Son is detached with Sufu, so that Gli in cytoplasm is detached with Cos2 formation compounds and micro-pipe, Gli enters nucleus activation target gene
Transcription causes Cell Proliferation of Pancreatic Cancer Cell, illustrates that the Shh high expression of cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signal paths is
Activate one of the inducement of Hedgehog accesses.After Chinese medicine composition and 5 FU 5 fluorouracil treatment, Chinese medicine composition high dose
Cancer of pancreas BxPC-3 Transplanted tumor models Hedgehog is promoted to believe after group, Chinese medicine composition middle dose group, 5 FU 5 fluorouracil group intervention
Number access Shh, Ptch1, Smo, Gli1 are expressed in each group to be lowered, to inhibit Hedgehog signal paths to activate.But
Chinese medicine composition low dosage is dry to cancer of pancreas BxPC-3 Transplanted tumor model Hedgehog signal paths Shh, Ptch1, Smo, Gli1
Preact is weaker.Synthesis display, Chinese medicine composition high dose, Chinese medicine composition middle dosage can lower cancer of pancreas BxPC-3 transplanting
Tumor model Hedgehog signal paths Shh, Ptch1, Smo, Gli1 expression, it is seen then that Hedgehog signal paths Shh, Ptch,
Smo, Gli1 play an important role during cancer of pancreas occurrence and development.
For the side effect of effective evaluation Chinese medicine composition, it is small that this experiment also measured were cancer of pancreas BxPC-3 Transplanted tumor models
The change level of brain, the heart, liver, kidney and the Lung Exponent and mouse weight different time points of mouse.This experimental results showed that, compared to mould
Type group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese medicine composition low dose group, 5 FU 5 fluorouracil group pancreas
Brain, the heart, liver, kidney and the Lung Exponent of gland cancer BxPC-3 Transplanted tumor model mouse change unobvious (P > 0.05);Illustrate Chinese traditional medicine composition
The high, medium and low dosage of object is on cancer of pancreas BxPC-3 Transplanted tumor model mouse important organs without influence.It was administered simultaneously in each group mouse
Cheng Zhong, cancer of pancreas BxPC-3 Transplanted tumor model mouse weights are presented ascendant trend, illustrate the high, medium and low dosage pair of Chinese medicine composition
Cancer of pancreas BxPC-3 Transplanted tumor model mouse weights are without influence.Compared with model group, the high, medium and low dosage group pancreas of Chinese medicine composition
Gland cancer BxPC-3 transplantable tumor mouse 0d, 7d, 14d, 28d weight changes unobvious (P > 0.05);In illustrating also by statistical analysis
The high, medium and low dosage of drug composition is on cancer of pancreas BxPC-3 Transplanted tumor model mouse weights without influence.Final display, Chinese traditional medicine composition
It is important that object will not change cancer of pancreas BxPC-3 Transplanted tumor model mouse while treating cancer of pancreas BxPC-3 Transplanted tumor model mouse
Organ index and weight illustrate that Chinese medicine composition is without side-effects, you can, middle dosage development high with Chinese medicine composition safe to use is controlled
Treat cancer of pancreas BxPC-3 Transplanted tumor model mouse.
The above experiment shows that Chinese medicine composition height, middle dosage (43.48g/kg, 21.74g/kg) can obviously improve cancer of pancreas
Nude Mouse Model mouse tumour inhibiting rate, spleen index, cell cycle, Apoptosis, can also improve to cancer of pancreas nude mice model
Tumor model mice Hedgehog signal path key molecules Shh, Ptch1, Smo, Gli1 expression, more to cancer of pancreas transplanted tumor in nude mice
Model mice cerebral index, cardiac index, liver index, renal index, Lung Exponent and weight illustrate Chinese medicine composition without influence, final evaluation
It is effective to treat cancer of pancreas, and Chinese medicine composition is without side-effects, illustrates that the Chinese medicine composition will have in terms for the treatment of cancer of pancreas
Wide application prospect.
On the basis of the studies above, the present invention proposes a kind of Chinese medicine composition for treating cancer of pancreas, the Chinese medicine
Composition is made of each raw material of following parts by weight:Chinese yam 20-25 parts by weight, rhizoma atractylodis 4-8 parts by weight, jujube 15-20 parts by weight,
Semen Lablab Album 10-20 parts by weight, pawpaw 5-8 parts by weight, Morinda officinalis 5-8 parts by weight, rhizoma arisaematis 5-8 parts by weight, Snakegourd Fruit 10-15 weight
Part, winter jasmine 10-15 parts by weight, strophanthus divaricatus 15-25 parts by weight, Akebia Fruit 5-12 parts by weight, edible tulip 2-7 parts by weight, Kowloon
Rattan 10-15 parts by weight, Himalayan mayapple fruit 1-5 parts by weight, Rhizoma Acori Calami 2-7 parts by weight, herba aristolochiae 5-15 parts by weight.
Wherein, Chinese yam, mild-natured, sweet in flavor, converge to the spleen channel, stomach, kidney channel are promoted the production of body fluid with invigorating the spleen, nourishing the stomach benefit lung, the puckery essence of kidney tonifying
Effect;Rhizoma atractylodis, warm-natured, acrid flavour, hardship, converge to the spleen channel, stomach, Liver Channel, with effect that invigorating spleen and dispelling wind-evil, eliminating dampness cold dispelling, improving eyesight solution are strongly fragrant;
Jujube, warm-natured, sweet in flavor, converge to the spleen channel, stomach have effects that invigorating the spleen tonifying Qi, tranquilizing and blood nourishing, mitigate pharmacological property;Semen Lablab Album, it is mild-natured,
It is sweet in flavor, light, converge to the spleen channel, stomach, have effects that inducing diuresis and reducing edema, invigorating the spleen diuresis and in relieve summer heat;Pawpaw, it is warm-natured, it is sour, puckery, return
Liver Channel, the spleen channel, stomach have effects that dampness removing and stomach, Shugan Zhitong, inducing diuresis to remove edema;Morinda officinalis, slightly warm in nature is acrid flavour, sweet, returns
Liver Channel, kidney channel have effects that dampness removing kidney tonifying, wind-dispelling strengthening tendons;Rhizoma arisaematis, warm-natured, bitter, pungent, return liver warp, lung channel, the spleen channel, tool
Have eliminating phlegm and diminishing swelling, dispel pathogenic wind and remove dampness, dissipating bind of calming the frightened the effect of;Snakegourd Fruit, cold in nature, sweet in flavor, bitter, distributed in lung channel, stomach, large intestine channel have
The effect of eliminating the phlegm heat-clearing, the wide chest of dissipating bind, moisturize laxation, sharp gas detumescence;Winter jasmine, it is mild-natured, it is sweet in flavor, puckery, return kidney channel, bladder warp,
Have effects that clearing heat and detoxicating, inducing diuresis to remove edema, promoting blood circulation and stopping pain;Strophanthus divaricatus, mild-natured, sour, converge to the spleen channel, Liver Channel, stomach have clear
Thermalization is stagnant, help digestion dampness removing the effect of;Akebia Fruit, cold in nature, bitter return stomach, Liver Channel, gallbladder channel, bladder warp, soothing the liver with stagnation resolvation,
The effect of vital energy regualting and blood circulation-promoting, diuresis desinsection;Edible tulip, cold in nature, sweet in flavor, micro-pungent, return liver warp, the spleen channel have blood stasis dispersing and phlegm expelling, heat-clearing solution
Effect of poison;Champion bauhinia stem, mild-natured, bitter, pungent, converge to the spleen channel, Liver Channel, stomach have promoting blood circulation by removing wind, damp dispelling and pain relieving, regulating qi to disperse stagnation
The effect of;Himalayan mayapple fruit, warm-natured, bitter return stomach, Liver Channel, lung channel, have the work(of promoting blood circulation dehumidifying, dispelling wind stopping cough, removing toxic substances analgesic
Effect;Rhizoma Acori Calami, it is warm-natured, it is bitter, pungent, return stomach, the heart channel of Hang-Shaoyin, has effects that stomach warming pain relieving, yang-tonifying anti-inflammatory;Herba aristolochiae, warm-natured, taste
Hardship, converge to the spleen channel, Liver Channel have effects that regulating qi-flowing for relieving pain, activating blood and promoting diuresis.
Wherein, it is preferred that the Chinese medicine composition is made of each raw material of following parts by weight:22.5 parts by weight of Chinese yam,
6 parts by weight of rhizoma atractylodis, 17 parts by weight of jujube, 15 parts by weight of Semen Lablab Album, 6.75 parts by weight of pawpaw, 6.75 parts by weight of Morinda officinalis, Tian Nan
6.5 parts by weight of star, 12 parts by weight of Snakegourd Fruit, 12.5 parts by weight of winter jasmine, 19.5 parts by weight of strophanthus divaricatus, 9 parts by weight of Akebia Fruit, mountain are kind
4.5 parts by weight of mushroom, 12 parts by weight of champion bauhinia stem, 9 parts by weight of 3.75 parts by weight of Himalayan mayapple fruit, 4.5 parts by weight of Rhizoma Acori Calami and herba aristolochiae.
Further, the application the invention also provides Chinese medicine composition in the drug for preparing treatment cancer of pancreas.
Wherein, it is preferred that the auxiliary material needed for preparations shaping is added into Chinese medicine composition of the present invention, according to drug
Preparation conventional method makes clinically suitable various pharmaceutical preparations.
Wherein, it is preferred that the pharmaceutical preparation is water decoction, capsule, pill, granule, pulvis, tablet or oral
Liquid.
Further, the invention also provides a kind of method preparing the Chinese medicine composition, include the following steps:
(1) parts by weight as described above weigh each raw material;
(2) after each raw material for weighing step (1) mixes, the distilled water of 8-15 times of raw material gross weight is added, impregnates
1.5-3h decocts 1.5-3h later, and filtering obtains first time Chinese medicine composition filtrate and residue;
(3) distilled water with step (2) same volume is added in the Chinese medicine composition residue obtained to step (2), impregnates
Chinese medicine composition residue 1.5-3h, decocting herb composition residue 1.5-3h after filtering, obtain second of Chinese medicine composition filter
Liquid;
(4) first time Chinese medicine composition filtrate and second Chinese medicine composition filtrate are merged, mixing is to get in described
The water decoction of drug composition.
In the described method, it is preferred that further include that the water decoction that will be obtained is put in container for evaporation, and by container for evaporation
It is put in the electric heating constant-temperature blowing drying box of laboratory and 6d is dried for 72 DEG C, 8 times a day, per minor tick 3h, obtain Chinese traditional medicine composition
Object dry cream;Obtained Chinese medicine composition dry cream is crushed using conventional grinder, crushed material passes through 100 mesh, 304 stainless steel
Screen filtration 2 times, 400 mesh, 304 stainless steel mesh filter 2 times, finally obtain Chinese medicine composition dry powder, dispense, in -80 DEG C of refrigerators
It saves backup.
In the described method, it is preferred that 6 layers of white gauze, 17 layers of white yarn cloth are respectively adopted successively in filtering for the first time
Filtering, second of filtering are respectively adopted 8 layers of white gauze, 19 layers of white yarn cloth and filter successively.
Compared to the prior art, the beneficial effects of the invention are as follows:
Inventor is according to theory of traditional Chinese medical science basis, in conjunction with the disease interpretation of the cause, onset and process of an illness of cancer of pancreas, select Chinese yam, rhizoma atractylodis, jujube,
Semen Lablab Album, Morinda officinalis, rhizoma arisaematis, Snakegourd Fruit, winter jasmine, strophanthus divaricatus, Akebia Fruit, edible tulip, champion bauhinia stem, Himalayan mayapple fruit, hides Chang at pawpaw
Pu, herba aristolochiae composition treatment cancer of pancreas Chinese medicine composition, wherein Chinese yam, rhizoma atractylodis, jujube, Semen Lablab Album, pawpaw, Morinda officinalis can be with
The effect of the effect of playing invigorating spleen to remove dampness, rhizoma arisaematis, Snakegourd Fruit, winter jasmine, strophanthus divaricatus can play eliminating the phlegm heat-clearing, Akebia Fruit, mountain
Arrowhead, champion bauhinia stem, Himalayan mayapple fruit can play the effect of stagnation resolvation promoting blood circulation, select Rhizoma Acori Calami, herba aristolochiae that can play warm stomach qi-regulating
Effect.The synthesis display Chinese medicine composition has invigorating spleen to remove dampness, eliminating the phlegm heat-clearing, stagnation resolvation promoting blood circulation, warm stomach qi-regulating and other effects.The present invention
Proposition a kind of new, effective technological means is provided for the treatment of cancer of pancreas.
Description of the drawings
Influence of Fig. 1 Chinese medicine compositions to cancer of pancreas BxPC-3 cell strain growth situations;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Fig. 2 Chinese medicine compositions to cancer of pancreas BxPC-3 cell strain cut distances;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Fig. 3 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model mouse tumor quality;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Fig. 4 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model mouse spleen index;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Fig. 5 Chinese medicine compositions to the cancer of pancreas BxPC-3 Nude Mouse Model cell cycles;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Fig. 6 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model apoptosis rates;
Note:Compared with model group:*P<0.05;
Fig. 7 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 expression;
Note:Compared with model group:*P<0.05,#P > 0.05;
Fig. 8 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 albumen relative expression quantity;
Note:Compared with model group:*P<0.05,#P > 0.05;
Fig. 9 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 gene;
Note:Compared with model group:*P<0.05,#P > 0.05;
Influence of Figure 10 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model important organ indexes;
Note:Compared with model group:*P > 0.05;Compared with high dose group,#P > 0.05;
Influence of Figure 11 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model mouse weights.
Note:Compared with model group:*P > 0.05;Compared with high dose group,#P > 0.05.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will with description and more
To be clear, but the embodiment is merely to illustrate the present invention, does not constitute any restrictions to protection scope of the present invention.This field skill
Art personnel should be understood that without departing from the spirit and scope of the invention can be to the details and shape of technical solution of the present invention
Formula is modified or is replaced, but these modifications and replacement are each fallen in protection scope of the present invention.
The preparation of 1 Chinese medicine composition of embodiment
The Chinese medicine composition is made of each raw material of following parts by weight:22.5 parts by weight of Chinese yam, 6 parts by weight of rhizoma atractylodis,
17 parts by weight of jujube, 15 parts by weight of Semen Lablab Album, 6.75 parts by weight of pawpaw, 6.75 parts by weight of Morinda officinalis, 6.5 parts by weight of rhizoma arisaematis,
12 parts by weight of Snakegourd Fruit, 12.5 parts by weight of winter jasmine, 19.5 parts by weight of strophanthus divaricatus, 9 parts by weight of Akebia Fruit, 4.5 parts by weight of edible tulip,
12 parts by weight of champion bauhinia stem, 9 parts by weight of 3.75 parts by weight of Himalayan mayapple fruit, 4.5 parts by weight of Rhizoma Acori Calami and herba aristolochiae.
The preparation of 2 Chinese medicine composition of embodiment
The Chinese medicine composition is made of each raw material of following parts by weight:It is 20 parts by weight of Chinese yam, 4 parts by weight of rhizoma atractylodis, big
15 parts by weight of jujube, 12 parts by weight of Semen Lablab Album, 8 parts by weight of pawpaw, 5 parts by weight of Morinda officinalis, 6 parts by weight of rhizoma arisaematis, 14 weight of Snakegourd Fruit
Part, 10 parts by weight of winter jasmine, 25 parts by weight of strophanthus divaricatus, 10 parts by weight of Akebia Fruit, 2 parts by weight of edible tulip, 15 parts by weight of champion bauhinia stem,
5 parts by weight of Himalayan mayapple fruit, 2 parts by weight of Rhizoma Acori Calami, 10 parts by weight of herba aristolochiae.
The preparation of 3 Chinese medicine composition of embodiment
The Chinese medicine composition is made of each raw material of following parts by weight:It is 25 parts by weight of Chinese yam, 8 parts by weight of rhizoma atractylodis, big
20 parts by weight of jujube, 20 parts by weight of Semen Lablab Album, 5 parts by weight of pawpaw, 8 parts by weight of Morinda officinalis, 7 parts by weight of rhizoma arisaematis, 10 weight of Snakegourd Fruit
Part, 15 parts by weight of winter jasmine, 15 parts by weight of strophanthus divaricatus, 5 parts by weight of Akebia Fruit, 5 parts by weight of edible tulip, 10 parts by weight of champion bauhinia stem,
2 parts by weight of Himalayan mayapple fruit, 7 parts by weight of Rhizoma Acori Calami, 5 parts by weight of herba aristolochiae.
The preparation of 4 Chinese medicine composition of embodiment (dry powder)
(1) weight as described below weighs each raw material:
Chinese yam 22.5g, rhizoma atractylodis 6g, jujube 17g, Semen Lablab Album 15g, pawpaw 6.75g, Morinda officinalis 6.75g, rhizoma arisaematis 6.5g,
Snakegourd Fruit 12g, it winter jasmine 12.5g, strophanthus divaricatus 19.5g, Akebia Fruit 9g, edible tulip 4.5g, champion bauhinia stem 12g, Himalayan mayapple fruit 3.75g, hides
The crude drug weight of calamus 4.5g, herba aristolochiae 9g, totally 16 kinds of crude drugs, every part of Chinese medicine composition are 167.25g, prepare 4 times of amounts
Chinese medicine composition (crude drug 669g).
(2) 669g Chinese medicine compositions are put into 15000mL experiments in medium-sized round-bottomed flask, distilled water 7500mL are added,
Crude drug 669g Chinese medicine compositions are impregnated into 2.5h in distilled water 7500mL, use distilled water 7500mL decocting herbs to combine later
Object 2.5h is filtered, it will obtain Chinese medicine composition original respectively by 6 layers of white hospital gauze, 17 layers of white hospital gauze
The first time Chinese medicine composition filtered fluid and residue of medicinal material 669g.
(3) the Chinese medicine composition residue obtained after filtering the first time Chinese medicine composition of Chinese medicine composition crude drug 669g
15000mL experiments are put into in medium-sized round-bottomed flask, distilled water 7500mL is added, are obtained after impregnating the filtering of first time Chinese medicine composition
The Chinese medicine composition residue 2.5h obtained decocts 2.5h, is carried out respectively by 8 layers of white hospital gauze, 19 layers of white hospital gauze
After filtering, it will obtain second of Chinese medicine composition filtered fluid.
(4) merge first time Chinese medicine composition filtered fluid and second of Chinese medicine composition filtered fluid;
(5) after the two mixing, all filtered fluids are put in container for evaporation, and container for evaporation is put in laboratory electric heating perseverance
6d (temperature is 72 DEG C) is dried in warm air dry oven (Tianjin Stettlen Instrument Ltd.), 8 times a day (between every time
Every 3h) check liquid evaporation situation in drying box, the Chinese medicine for being equivalent to Chinese medicine composition crude drug 669g will eventually be obtained
Composition dry cream 201.32g, this is equivalent to Chinese medicine composition paste-forming rate=[Chinese medicine composition of Chinese medicine composition crude drug 669g
Dry cream (g)/Chinese medicine composition crude drug (g)] × 100%, according to formula by being calculated (201.32g/669g) × 100%
=30.09%, it is seen then that the Chinese medicine composition paste-forming rate for being equivalent to Chinese medicine composition crude drug 669g is 30.09%.
(6) the Chinese medicine composition dry cream that step (5) obtains thoroughly is crushed using conventional grinder, crushed material passes through
304 stainless steel mesh (100 mesh) filter 2 times, and 304 stainless steel mesh (400 mesh) filter 2 times, finally obtain and are equivalent to Chinese medicine group
The Chinese medicine composition dry powder 195.27g for closing object crude drug 669g, be equivalent to the Chinese medicine composition flour extraction of crude drug 669g=[in
Drug composition dry powder (g)/Chinese medicine composition crude drug (g)] × 100%, by being calculated (195.27g/669g) × 100%
=29.19%, it is seen then that the Chinese medicine composition flour extraction for being equivalent to Chinese medicine composition crude drug 669g is 29.19%.
Experimental formula Chinese medicine composition crude drug weight is 669g, finally obtains Chinese medicine composition dry cream 201.32g, Chinese medicine
Composition dry powder 195.27g, i.e. pulverizer are crushed and screen filtration loses 6.05g, Chinese medicine composition loss late=[Chinese traditional medicine composition
Object dry cream (g)-Chinese medicine composition dry powder (g)]/Chinese medicine composition dry powder (g) × 100%, by [(201.32g- is calculated
195.27g)/201.32g] × 100%=3.01%, it is seen then that Chinese medicine composition crude drug weight is the Chinese medicine composition of 669g
Loss late is 3.01%.
Finally, 4 times of Chinese medicine composition crude drug 669g can obtain Chinese medicine composition dry powder after distilled water decocts
195.27g, i.e. 1g Chinese medicine compositions dry powder are equivalent to Chinese medicine composition crude drug 3.43g.By Chinese medicine composition dry powder 195.27g
It is dispensed, 20g/ bags, last 1 bag of 15.27g, amounts to 10 bags, while marking every bag of Chinese medicine composition dry powder weight, -80 DEG C of ice
It is saved backup in case.
Application of 5 Chinese medicine composition of embodiment in treating cancer of pancreas
1 material
1.1 animal
Experiment male 70 4-6 weeks BALB/c nude mices, weight (18 ± 2) g, by Harbin Medical University's preclinical medicine
Teaching and research room professor Wu Shuliang purchase is dissected in Shanghai Si Laike zooperies Co., Ltd by institute, and animal for research credit number is
SCXK (Shanghai) 2012-0002, all animal for research are suitable at Heilongjiang University of Chinese Medicine's Drug safety assessment center
By (SPF grades) feedings under the conditions of the Li Baolong deputy director's normal sterile of Heilongjiang University of Chinese Medicine's safety evaluation center under temperature and humidity
It supports, gastric infusion.
1.2 drug
The dry powder that embodiment 4 is prepared.In addition, positive control medicine 5 FU 5 fluorouracil of the present invention is by Qiqihar medicine
Institute associate professor Zhang Yingbo buys in Sigma Co., USA.
1.3 reagent
RPMI-1640 culture mediums, trypsase are purchased from HyClone companies;Fixing powder (CW0062), highly sensitive chemistry
Luminescence detection kit (CW0049A), anti-GAPDH (CW0100A) are century bio tech ltd purchased from health;
BxPC-3 humanized's pancreas cancer cell strains are purchased from Shanghai Inst. of Life Science, CAS cell resource center;Medical X
Radiographic film (034402311) is bought in Xiamen Rui Ke medical equipments Co., Ltd;RNeasy Mini kits are purchased from
QIAGEN companies;Anti-Shh (2207), anti-Gli1 (3538) are purchased from INT companies of the U.S.;Anti-Ptch1
(ab39266), anti-Smo (ab72130) is purchased from abcam companies of the U.S.;SDS (L5750), Tween20 (P1379) are purchased from
In Sigma companies;PrimeScript RT kits are purchased from DaLian, China TaKaRa Biotechnology companies;Albumen is taken out
It puies forward reagent A (P0028-1) and is purchased from green skies company;Other domestic analyses are pure by Qiqihar Medical College associate professor Zhang Yingbo
It provides.
1.4 instrument
Electric heating constant-temperature blowing drying box is purchased from Tianjin Stettlen Instrument Ltd.;HMIAS high-definition colors are cured
It learns picture and text analysis system and is purchased from thousand screen image technology Co., Ltd of Wuhan;PLZOZ-S electronic balances are purchased from plum Teller-Tuo Li
Multiple instruments Co., Ltd;SIMF124 ice machines are purchased from SANYO companies of Japan;Tissue exhibition piece machine is public purchased from Germany Leica
Department;TGL-16G desk centrifuges are purchased from Anting Scientific Instrument Factory, Shanghai;Tissue processor is purchased from Leica companies of Germany;Stream
Formula cell instrument is purchased from U.S. company BD;Protein blot electrophoresis apparatus is purchased from 61 instrument plant of city of BeiJing, China;CO2Incubator is purchased
From in Thermo companies of the U.S.;Ultra-clean operation console is purchased from Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China;Experimental animal
Gas exchanges machine is purchased from Suzhou instrument and equipment factory;Real-time quantitative PCR detector is purchased from Bio Rad Laboratories etc..
2 methods
The setting of 2.1 Chinese medicine composition dosage
Chinese medicine composition includes Chinese yam 22.5g, rhizoma atractylodis 6g, jujube 17g, Semen Lablab Album 15g, pawpaw 6.75g, Morinda officinalis
6.75g, rhizoma arisaematis 6.5g, Snakegourd Fruit 12g, winter jasmine 12.5g, strophanthus divaricatus 19.5g, Akebia Fruit 9g, edible tulip 4.5g, champion bauhinia stem
12g, Himalayan mayapple fruit 3.75g, Rhizoma Acori Calami 4.5g, herba aristolochiae 9g, totally 16 kinds of crude drugs, the crude drug weight of every part of Chinese medicine composition are
167.25g/ adult's standard weights.167.25g/ adult standard weight be by Heilongjiang University of Chinese Medicine Zhou Zhongguang professor and it is black
Routine dose after the attached First Hospital division of endocrinology Li Ming associate chief physicians identification of Longjiang university of TCM.The 167.25g/ at
People's standard weight is according to mouse and body surface area's ratio (0.0026:1) it calculates, obtains Chinese medicine composition dosage=(167.25g
× 0.0026)/0.02kg=21.74g/kg, it is seen that Chinese medicine composition middle dosage is 21.74g/kg.According to Chinese medicine composition height
Dosage is 2 times of Chinese medicine composition middle dosage, and Chinese medicine composition low dosage is the 1/2 of Chinese medicine composition middle dosage, therefore, real
It tests and sets Chinese medicine composition high dose as (21.74g/kg) × 2=43.48g/kg, Chinese medicine composition low dosage is (21.74g/
Kg)/2=10.87g/kg.Finally, the present invention be set separately the high, medium and low dosage of Chinese medicine composition be respectively 43.48g/kg,
21.74g/kg、10.87g/kg。
The setting of 2.2 positive drug dosage
Positive drug 5 FU 5 fluorouracil routine dose is 150~300mg, and the body surface area ratio according to mouse and people is
(0.0026:1) calculated, it will obtain positive drug 5 FU 5 fluorouracil routine dose minimum value be (150 × 0.0026 ×
50) mg/kg=19.5mg/kg and maximum value are (300 × 0.0026 × 50) mg/kg=39mg/kg, and it is unit to be converted into molecule
After g, acquisition positive drug 5 FU 5 fluorouracil routine dose minimum value is 0.0195g/kg and maximum value is 0.039g/kg, is passed through
Heilongjiang University of Chinese Medicine Zhou Zhongguang is taught and the attached First Hospital division of endocrinology Li Ming deputy director doctor of Heilongjiang University of Chinese Medicine
Teacher evaluates, this experimental selection positive drug 5 FU 5 fluorouracil 0.03g/kg carries out experimental work as routine dose.
2.3 cell culture
This experiment, which is taken out, is equipped with cancer of pancreas BxPC-3 cell strain (Shanghai Inst. of Life Science, CAS cellular resources
Center provide) cryopreservation tube, be placed in 37 DEG C of water and melt rapidly, use TGL-16G desk centrifuges (Town in Shanghai pavilion scientific instrument
Factory) 2000rpm/10min centrifugations, frozen stock solution is discarded, 10 times of precooling RPMI1640 complete culture solutions are added and rinse 1 time, are inoculated in
Culture bottle is placed in 5%CO2, 37 DEG C, the CO of saturated humidity2Culture in incubator (Thermo companies of the U.S.), the next day change liquid.Culture
After cancer of pancreas BxPC-3 cell strains are in exponential phase at single layer and cancer of pancreas BxPC-3 cell strains, with 0.25% tryptose
Enzyme (containing EDTA) digestion, collects cancer of pancreas BxPC-3 cell strains at passage.
2.4 measure cell growth
Dead or non-viable non-apoptotic cell cell membrane can be penetrated according to trypan blue and combines DNA features, it can be by these not
It is distinguished by cell, that is, living cells of Trypan Blue.This experiment detects cancer of pancreas BxPC-3 cell strains using Trypan Blue
Growth, identify living cells and evaluate primary action effect of the drug to cancer of pancreas BxPC-3 cell strains.Experiment, which takes, to be in
The cancer of pancreas BxPC-3 cell strain single cell suspensions of exponential phase, trypsin digestion, phosphate buffer wash 3 times, system
Standby cancer of pancreas BxPC-3 cell strains, adjust a concentration of the 1 × 10 of cell4A cell/mL selects 300 μ L to be seeded in the culture of 24 holes
Different drugs is added, in 5%CO in plate2, 37 DEG C of sterile CO2Incubator (Thermo companies of the U.S.) culture 1,2,3,4,5,6,
7d.Single cell suspension is taken per d, is 9 according to the ratio of single cell suspension and trypan blue:1,4% trypan blue is added and mixes, in 3min
It counts, collects data statistic analysis, evaluate the growing state of cancer of pancreas BxPC-3 cell strains.
2.5 measure cell motility
This experiment logarithmic growth phase cancer of pancreas BxPC-3 cell strains, trypsin digestion, phosphate buffer washing 3
It is secondary, cancer of pancreas BxPC-3 cell strain single cell suspensions are prepared, a concentration of the 1 × 10 of cell is adjusted5A cell/mL.Black pen is 6
Well culture plate draws horizontal line behind, every 0.8cm, per hole at least across 3 lines.Cancer of pancreas BxPC-3 cell strains are added in every hole
About 1 × 105A cell/mL after cultivating 1d, behind perpendicular to 6 well culture plates, cut is drawn using horizontal line.Use phosphate buffer
3 times/hole of cell is washed, lower cancer of pancreas BxPC-3 cell strains are drawn in removal, in 5%CO2, 37 DEG C of sterile CO2Incubator (the U.S.
Thermo companies) culture.Normal complete medium is added in control group, and different drugs is added in other each groups, continues to cultivate 2d.It presses
0h, for 24 hours, 48h take cell sample, shoot at cut, measure distance between the cell cut of cancer of pancreas BxPC-3 cell strains, pancreas
The locomitivity of gland cancer BxPC-3 cell strains can be evaluated using the cell healing of cancer of pancreas BxPC-3 cell strains.
Calculate cell healing:Cell healing (value)=l- (48h cut distance/0h cuts distance).
2.6 establish model
Article that bibliography Heilongjiang University of Chinese Medicine professor Zhou Zhongguang delivers (Zhou ZG, Zhang CY, Fei HX,
et al.Phenolic alkaloids from Menispermum dauricum inhibits BxPC-3 pancreatic
Cancer cells by blocking of Hedgehog signaling pathway [J] .Pharmacogn Mag,
2015,11 (44):690-697.) establish cancer of pancreas BxPC-3 Transplanted tumor models.Cancer of pancreas BxPC-3 cell strains are collected in this experiment,
A concentration of 5 × 10 are adjusted with RPMI1640 complete mediums6A/mL cell suspensions are spare.Every mouse 0.2mL is (containing about cell number 5
×106It is a) to be inoculated in the right armpit of nude mice subcutaneous, establish cancer of pancreas BxPC-3 Transplanted tumor models.Meanwhile setting blank group (only
Injecting normal saline) it is subcutaneous in the right armpit of nude mice, further verify cancer of pancreas BxPC-3 Transplanted tumor models.As cancer of pancreas BxPC-3
Cell strain (5 × 106A/mL) the subcutaneous flat or Circular lump of the right armpit of inoculation 7~10d nude mices, and the right armpit of blank group nude mice is subcutaneous
Do not occur flat or Circular lump, illustrates that cancer of pancreas BxPC-3 Transplanted tumor models are successfully established.
2.7 animal packet
This experiment determines 50 cancer of pancreas BxPC-3 Transplanted tumor models, using table of random number at random by 50 cancers of pancreas
BxPC-3 Transplanted tumor model mouse are divided into five groups, 10/group, including model group, Chinese medicine composition high dose group (abbreviation high dose
Group), Chinese medicine composition middle dose group (abbreviation middle dose group), Chinese medicine composition low dose group (abbreviation low dose group), 5- fluorine urine
Pyrimidine group.In addition, this experiment need to select same background, with monthly age nude mice 10 be control group.
2.8 animals are administered
Model group:It is inoculated with cancer of pancreas BxPC-3 cell strains 5 × 106A/mL, through the isometric physiology salt of murine oral gavage
Water;Chinese medicine composition high dose group:It is inoculated with cancer of pancreas BxPC-3 cell strains 5 × 106A/mL, through murine oral gavage Chinese medicine group
Close object 43.48g/kg;Chinese medicine composition middle dose group:It is inoculated with cancer of pancreas BxPC-3 cell strains 5 × 106A/mL, through murine oral
Gavage Chinese medicine composition 21.74g/kg;Chinese medicine composition low dose group:It is inoculated with cancer of pancreas BxPC-3 cell strains 5 × 106A/mL,
Through murine oral gavage Chinese medicine composition 10.87g/kg;5 FU 5 fluorouracil group:It is inoculated with cancer of pancreas BxPC-3 cell strains 5 × 106
A/mL, through murine oral gavage positive drug 5 FU 5 fluorouracil 0.03g/kg.This experiment is through murine oral gavage successive administration
28d。
2.9 measure tumour inhibiting rate and spleen index
After each group cancer of pancreas BxPC-3 Transplanted tumor model mouse pharmaceutical interventions 28d, PLZOZ-S electronic balances (Mei Te is used
Le-support benefit Instrument Ltd.) next day weighs, and it takes off neck and puts to death cancer of pancreas BxPC-3 Transplanted tumor model mouse, alcohol disinfecting,
Ultra-clean operation console (Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China) sterile working completely strips cancer of pancreas BxPC-3 transplanting
The knurl and spleen of tumor model mice, normal saline flushing, filter paper blot, and weigh the swollen of cancer of pancreas BxPC-3 Transplanted tumor model mouse
Tumor and spleen weight.
Calculate tumour inhibiting rate:Tumour inhibiting rate (%)=(model group tumor quality-experimental group tumor quality)/model group average tumor
Quality × 100%;
Calculate spleen index:Spleen index (mg/g)=spleen quality (mg)/mouse weight (g).
2.10 measuring the cell cycle
After experiment, the tumor tissue block of each group cancer of pancreas BxPC-3 Transplanted tumor model mouse is cut, in ultra-clean operation console
(Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China) sterile working completely strips knurl, rejects tumor surface fibrous capsule,
It is placed on ice pan, shreds, grinds, be prepared into the knurl single cell suspension of cancer of pancreas BxPC-3 Transplanted tumor model mouse, be adjusted to thin
Born of the same parents a concentration of 2 × 106/mL.It is handled by cell inoculation in 6 orifice plates and with Chinese medicine composition, 5 FU 5 fluorouracil or physiological saline
24h.And it is fixed overnight with 70% ethyl alcohol at -20 DEG C, washing.1mL suspensions are taken to add RNase 1.6mL (1mg/mL), 37 DEG C of water
Bath is incubated 30min, and 1500rpm/min centrifuges 5min, precipitation plus 0.4mL phosphate buffers, shakes mixing.Use propidium iodide
(100μg/mL;Sigma-Aldrich 10min) is dyed, flow cytometer (U.S. company BD) is used in combination to be detected analysis pancreas
The tumour cell cycle of cancer BxPC-3 Transplanted tumor model mouse.
2.11 measuring Apoptosis
After experiment, cancer of pancreas is cut in ultra-clean operation console (Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China)
The tumor tissue block of BxPC-3 Transplanted tumor model mouse prepares the single cell suspension of cancer of pancreas BxPC-3 transplantable tumors, it is dense to be adjusted to cell
Degree is 2 × 106/ mL is handled for 24 hours with Chinese medicine composition, 5 FU 5 fluorouracil or physiological saline, is added by cell inoculation in 6 orifice plates
Enter fluorescence dye liquor, is incubated 30min, is protected from light and vibrates frequently.According to manufacturer scheme Annexin-V:The detection examination of FITC apoptosis
The apoptosis of tumor cells that agent box I measures cancer of pancreas BxPC-3 Transplanted tumor model mouse is horizontal.
2.12 immunohistochemistry detect protein expression
After experiment, in ultra-clean operation console (Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China) materials cancer of pancreas
The tumor tissues of BxPC-3 Transplanted tumor model mouse, 10% formalin fix 4h, and graded ethanol is dehydrated, and dimethylbenzene is transparent
30min, 45 DEG C of immersion 1h of soft wax, 55 DEG C of immersion 1h of hard wax, embedding device embedding, tissue is cut into 4 μ m thicks and is sliced by slicer, more
Poly-L-Lysine glass slide drags for piece, 60 DEG C of baking piece 12h, and dimethylbenzene dewaxes to water, 3%H2O2It is incubated at room temperature 10min, distilled water punching
It washes, microwave stove heat repairs 5min, room temperature cooling, and serum is incubated at room temperature 20min, discards serum, and one is added dropwise by reagent specification
Anti-, 37 DEG C are incubated 2h, and PBS rinses 5min, and secondary antibody 37 DEG C of incubations 30min, PBS are added and rinse 3min, DAB colour developing 10min, flowing water
It rinses, redyes 5min, by dehydration, transparent, mounting, the tumour of microscopic observation cancer of pancreas BxPC-3 Transplanted tumor model mouse
Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1 expressions, using HMIAS high-definition color medicine
Picture and text system (thousand screen image technology Co., Ltd of Wuhan) carry out analysis Hedgehog signal path key molecules Shh, Ptch1,
The OD values of Smo, Gli1.
2.13 Western blot detect protein expression
After experiment, the tumor tissues of cancer of pancreas BxPC-3 Transplanted tumor model mouse is taken to be homogenized on ice, 4 DEG C of centrifugations,
12000rpm centrifuges 10min, and -20 DEG C of refrigerators of albumen sample preserve, and carry out protein quantification using BCA methods, are counted on standard curve
Albumen concentration is calculated to obtain, 5min is denaturalized in boiling water, sds page, 75V electrophoresis 1h, electricity are prepared in 80 DEG C of preservations of packing ﹣
20min is balanced after swimming, 75V electricity transferring films 2h, 4 DEG C of closings are overnight.Primary antibody is added:GAPDH(1:3000)、Shh(1:800)、Ptch
(1:1000)、Smo(1:2000)、Gli1(1:2000), 37 DEG C of incubation 2h.Primary antibody is removed, is rinsed.Secondary antibody, 37 DEG C of incubations are added
1h.Secondary antibody is removed, is rinsed.It is rinsed by X-ray film exposure, development, fixing, clear water in darkroom, it is color using HMIAS fine definition
Color medicine picture and text system (thousand screen image technology Co., Ltd of Wuhan) carries out analysis cancer of pancreas BxPC-3 Transplanted tumor model mouse
The relative expression quantity of Hedgehog signal path key molecules Shh, Ptch1, Smo, Gli1.
2.14 RT-PCR detect gene expression
After experiment, take the tumor tissues of cancer of pancreas BxPC-3 Transplanted tumor model mouse according to Trizol kit explanations
Book extracts mRNA.Primer is by Shanghai Sheng Gong bio-engineering corporations compounding design, wherein GAPDH:5 '-AGA AGG CTG of upstream
GGG CTC ATT TG-3 ', 5 '-AGG GGC CAT CCA CAG TCT TC-3 ' of downstream;Shh:5 '-GCT CGG TGA of upstream
AAG CAG AGA ACT-3 ', 5 '-CCA GGA AAG TGA GGA AGT CG-3 ' of downstream;Smo:5 '-CTG GTG of upstream
TGG TTT GGT TTG TG-3 ', 5 '-CAG GTG GAA GTA GGA GGT CTT G-3 ' of downstream;Ptch1:Upstream 5 '-
CTC CTT TGC GGT GGA CAA-3 ', 5 '-CCT CAG CCT TAT TCA GCA TTT C-3 ' of downstream;Gli1:Upstream
5 '-ATC CTT ACC TCC CAA CCT CTG T-3 ', 5 '-CCA ACT TCT GGC TCT TCC TGT A-3 ' of downstream.
According to kit specification procedure operation, PCR reaction systems are prepared, are used real-time quantitative PCR detector (Bio Rad Laboratories)
It is measured, usesMethod data processing experimental data and the tumour for analyzing cancer of pancreas BxPC-3 Transplanted tumor model mouse
The mRNA relative expression quantities of Hedgehog signal path key molecules Shh, Ptch1, Smo, Gli1.
2.15 detection weight and organ index
After each group cancer of pancreas BxPC-3 Transplanted tumor model mouse detect, PLZOZ-S electronic balances (plum Teller-is used
Hold in the palm benefit Instrument Ltd.) cancer of pancreas BxPC-3 Transplanted tumor model mouse weights are weighed, count cancer of pancreas BxPC-3 transplantable tumors
Weight data changes model mice during the administration, and is analyzed.In addition, after experiment, mouse, sacrificed by decapitation are captured
Mouse dissects mouse, materials cancer of pancreas BxPC- on ultra-clean operation console (Dong Lianhaer instrument manufacturings Co., Ltd of BeiJing, China)
The internal organs such as brain, the heart, lung, kidney, the liver of 3 Transplanted tumor model mouse, washing internal organs, suck dry moisture, use PLZOZ-S electronic balances
(plum Teller-support benefit Instrument Ltd.) weighs Organ weight, starts to calculate cancer of pancreas according to organ index calculation formula
BxPC-3 Transplanted tumor model mice organs indexes.
Calculation formula:Organ index (mg/g)=Organ weight (mg)/mouse weight (g).
2.16 statistical analysis
Using SPSS19.0 software analysis datas, experimental data indicates that measurement data carries out test of normality using x ± s
Analysis, more comparison among groups use one-way analysis of variance data, P<0.05 indicates that difference has statistical significance, 0.05 tables of P >
Show that difference does not have statistical significance.
3 results
3.1 influence of the Chinese medicine composition to cancer of pancreas BxPC-3 cell strain growth situations
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group the 3rd, 4,5,6, cancer of pancreas BxPC-3 cell strain viable counts significantly reduce (P on 7d time points<0.05), Chinese medicine group
Close object low dose group the 3rd, 4,5,6,7d time points upper cancer of pancreas BxPC-3 cell strain viable counts change unobvious (P >
0.05).(being shown in Table 1, Fig. 1).
1 Chinese medicine composition of table to cancer of pancreas BxPC-3 cell strain growth situations influence (N=6,1 × 104)
Note:Compared with model group:*P<0.05,#P > 0.05
Influence of 3.2 Chinese medicine compositions to cancer of pancreas BxPC-3 cell strain locomitivities
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group cancer of pancreas BxPC-3 cell strain 48h cut distances obviously increase (P<0.05), cell healing is substantially reduced (P<
0.05), and Chinese medicine composition low dose group cancer of pancreas BxPC-3 cell strain 48h cuts distance and cell healing change it is unknown
Aobvious (P > 0.05).(being shown in Table 2, Fig. 2).
2 Chinese medicine composition of table to cancer of pancreas BxPC-3 cell strain locomitivities influence (N=9)
Note:Compared with model group:*P<0.05,#P > 0.05
Influence of 3.3 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model tumour inhibiting rates
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group, Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Model mouse tumor quality are substantially reduced (P<
0.05).Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese medicine composition low dose group, positive drug 5- fluorine urine
The tumour inhibiting rate of pyrimidine group be respectively (47.8372 ± 6.7827) %, (36.8352 ± 8.4470) %, (32.2523 ±
5.3657) %, (54.7148 ± 7.1300) %.As it can be seen that Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese medicine
Composition low dose group, 5 FU 5 fluorouracil group all have inhibiting effect to cancer of pancreas BxPC-3 Nude Mouse Model mouse, empty
Bai Zuwei generates cancer of pancreas BxPC-3 transplanted tumor in nude mice.Wherein, Chinese medicine composition high dose group and 5 FU 5 fluorouracil group cancer of pancreas
The tumor killing effect of BxPC-3 Nude Mouse Model mouse is fine.(being shown in Table 3, Fig. 3).
3 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model mouse tumor quality and tumour inhibiting rate influence (N=10)
Note:Compared with model group:*P<0.05
Influence of 3.4 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model spleen indexs
The result shows that compared with model group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Model mice spleens refer to
Number is substantially reduced (P<0.05), Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese medicine composition low dose group
Cancer of pancreas BxPC-3 Nude Mouse Model mouse spleen index no difference of science of statistics (P>0.05).(being shown in Table 4, Fig. 4).
4 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model mouse spleen index influence (N=8)
Note:Compared with model group:*P<0.05,#P > 0.05
3.5 Chinese medicine compositions are to cancer of pancreas BxPC-3 Nude Mouse Model Cell cycle influences
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
The pyridine group cancer of pancreas BxPC-3 Nude Mouse Model mouse G1 phases obviously increase (P < 0.05), and the G2 phases are substantially reduced (P <
0.05), and the Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Model mouse G1 phases change unobvious (P >
0.05);Compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5 FU 5 fluorouracil group cancer of pancreas
The BxPC-3 Nude Mouse Model mouse S phases are substantially reduced (P < 0.05), and Chinese medicine composition low dose group cancer of pancreas BxPC-3 is naked
The mouse Transplanted tumor model mouse S phases change unobvious (P > 0.05);Compared with model group, Chinese medicine composition high dose group, Chinese medicine group
Conjunction object middle dose group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Model mouse G2 phases are substantially reduced (P < 0.05),
And the Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Model mouse G2 phases change unobvious (P > 0.05).(see
Table 5, Fig. 5).
5 Chinese medicine composition of table to the cancer of pancreas BxPC-3 Nude Mouse Model cell cycles influence (N=6)
Note:Compared with model group:*P<0.05,#P > 0.05
Influence of 3.6 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model apoptosis rates
The result shows that model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, low dose of Chinese medicine composition
Amount group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Model mouse apoptosis rate be respectively (5.2933 ±
0.9249) %, (7.5717 ± 0.6930) %, (8.1983 ± 0.7523) %, (8.1667 ± 0.7392) %, (8.0067 ±
1.0206) %.Compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5 FU 5 fluorouracil group, in
Drug composition low dose group cancer of pancreas BxPC-3 Nude Mouse Model apoptosis rates are substantially reduced (P < 0.05).It (is shown in Table
6, Fig. 6).
Influence of 6 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model apoptosis rates
Note:Compared with model group:*P<0.05
3.7 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 protein expression
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1 albumen
Expression OD values are substantially reduced (P < 0.05), and Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Models
Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1 protein expression OD values change unobvious (P > 0.05).(see
Table 7, Fig. 7).
7 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
Ptch1, Smo, Gli1 expression influence (N=8, OD value)
Note:Compared with model group:*P<0.05,#P > 0.05
3.8 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 albumen relative expression quantity
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1 albumen
Relative expression quantity is substantially reduced (P < 0.05), and Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Models
Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1 albumen relative expression quantities change unobvious (P > 0.05).
(being shown in Table 8, Fig. 8).
8 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
Ptch1, Smo, Gli1 albumen relative expression quantity influence (N=7)
Note:Compared with model group:*P<0.05,#P > 0.05
3.9 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
The influence of Ptch1, Smo, Gli1 gene
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, 5- fluorine urine are phonetic
Pyridine group cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path keys molecules Shh, Ptch1, Smo, Gli1 mRNA
Expression quantity expression is substantially reduced (P < 0.05), and Chinese medicine composition low dose group cancer of pancreas BxPC-3 Nude Mouse Models
The expression of Hedgehog signal path key molecule Shh, Ptch1, Smo, Gli1mRNA expression quantity changes unobvious (P > 0.05).
(being shown in Table 9, Fig. 9).
9 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model Hedgehog signal path key molecules Shh,
Ptch1, Smo, Gli1 gene influence (N=7,2-△△Ct)
Note:Compared with model group:*P<0.05,#P > 0.05
Influence of 3.10 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model important organ indexes
The result shows that compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese traditional medicine composition
Object low dose group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Models brain, the heart, liver, kidney and Lung Exponent change unknown
Aobvious (P > 0.05), illustrates the high, medium and low dosage of Chinese medicine composition and 5 FU 5 fluorouracil to cancer of pancreas BxPC-3 transplanted tumor in nude mice
Model important organ is without influence;Compared with Chinese medicine composition high dose group, low dose of Chinese medicine composition middle dose group, Chinese medicine composition
Amount group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Models brain, the heart, liver, kidney and Lung Exponent change unobvious (P >
0.05), illustrate even if increase Chinese medicine composition dosage, Chinese medicine composition to cancer of pancreas BxPC-3 Nude Mouse Models brain, the heart,
Liver, kidney and Lung Exponent are also without influence.(being shown in Table 10, table 11, Figure 10).
10 Chinese medicine composition of table is to cancer of pancreas BxPC-3 Nude Mouse Model mouse cerebral indexes, cardiac index, liver index
Influence (N=8)
Note:Compared with model group:*P > 0.05;Compared with high dose group,#P > 0.05
11 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model mouse renal index, Lung Exponent influence (N=8)
Note:Compared with model group:*P > 0.05;Compared with high dose group,#P > 0.05
Influence of 3.11 Chinese medicine compositions to cancer of pancreas BxPC-3 Nude Mouse Model weight
The result shows that in each group cancer of pancreas BxPC-3 Nude Mouse Model mouse administration process, cancer of pancreas BxPC-3 is naked
Ascendant trend is presented in mouse Transplanted tumor model mouse weight, illustrates the high, medium and low dosage of Chinese medicine composition to cancer of pancreas BxPC-3 nude mices
Transplanted tumor model mouse weight is without influence.Compared with model group, Chinese medicine composition high dose group, Chinese medicine composition middle dose group,
Chinese medicine composition low dose group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Model mouse 0d, 7d, 14d, 28d bodies
Change unobvious (P > 0.05) again, further illustrates the high, medium and low dosage of Chinese medicine composition to cancer of pancreas BxPC-3 nude mice models
Tumor model mice weight illustrates that the high, medium and low dosage of Chinese medicine composition is naked to cancer of pancreas BxPC-3 with 5 FU 5 fluorouracil without influence
Mouse Transplanted tumor model mouse weight is without influence;Compared with Chinese medicine composition high dose group, Chinese medicine composition middle dose group, Chinese medicine group
Close object low dose group, 5 FU 5 fluorouracil group cancer of pancreas BxPC-3 Nude Mouse Model mouse weights change unobvious (P >
0.05), illustrate that Chinese medicine composition is to cancer of pancreas BxPC-3 Nude Mouse Model Mice Bodies even if increasing Chinese medicine composition dosage
Weight is also without influence.(being shown in Table 12, Figure 11).
12 Chinese medicine composition of table to cancer of pancreas BxPC-3 Nude Mouse Model mouse weights influence (N=8)
Note:Compared with model group:*P > 0.05;Compared with high dose group,#P > 0.05.
Claims (8)
1. a kind of Chinese medicine composition for treating cancer of pancreas, which is characterized in that the Chinese medicine composition is each by following parts by weight
Raw material forms:Chinese yam 20-25 parts by weight, rhizoma atractylodis 4-8 parts by weight, jujube 15-20 parts by weight, Semen Lablab Album 10-20 parts by weight, pawpaw
5-8 parts by weight, Morinda officinalis 5-8 parts by weight, rhizoma arisaematis 5-8 parts by weight, Snakegourd Fruit 10-15 parts by weight, winter jasmine 10-15 parts by weight,
Strophanthus divaricatus 15-25 parts by weight, Akebia Fruit 5-12 parts by weight, edible tulip 2-7 parts by weight, champion bauhinia stem 10-15 parts by weight, Himalayan mayapple fruit 1-
5 parts by weight, Rhizoma Acori Calami 2-7 parts by weight and herba aristolochiae 5-15 parts by weight.
2. Chinese medicine composition as described in claim 1, which is characterized in that the Chinese medicine composition is each by following parts by weight
Raw material forms:22.5 parts by weight of Chinese yam, 6 parts by weight of rhizoma atractylodis, 17 parts by weight of jujube, 15 parts by weight of Semen Lablab Album, 6.75 weight of pawpaw
Part, 6.75 parts by weight of Morinda officinalis, 6.5 parts by weight of rhizoma arisaematis, 12 parts by weight of Snakegourd Fruit, 12.5 parts by weight of winter jasmine, strophanthus divaricatus 19.5
Parts by weight, 9 parts by weight of Akebia Fruit, 4.5 parts by weight of edible tulip, 12 parts by weight of champion bauhinia stem, 3.75 parts by weight of Himalayan mayapple fruit, Rhizoma Acori Calami
9 parts by weight of 4.5 parts by weight and herba aristolochiae.
3. application of the Chinese medicine composition as claimed in claim 1 or 2 in the drug for preparing treatment cancer of pancreas.
4. application as claimed in claim 3, which is characterized in that system is added into Chinese medicine composition as claimed in claim 1 or 2
Auxiliary material needed for agent molding, clinically suitable various pharmaceutical preparations are made according to pharmaceutical preparation conventional method.
5. application as claimed in claim 4, which is characterized in that the pharmaceutical preparation be water decoction, capsule, pill,
Granula, pulvis, tablet or oral solution.
6. a kind of method preparing Chinese medicine composition as claimed in claim 1 or 2, which is characterized in that include the following steps:
(1) each raw material is weighed according to parts by weight as claimed in claim 1 or 2;
(2) after each raw material for weighing step (1) mixes, the distilled water of 8-15 times of raw material gross weight is added, impregnates 1.5-
3h decocts 1.5-3h later, and filtering obtains first time Chinese medicine composition filtrate and residue;
(3) distilled water with step (2) same volume is added in the Chinese medicine composition residue obtained to step (2), impregnates Chinese medicine
Composition residue 1.5-3h, decocting herb composition residue 1.5-3h after filtering, obtain second of Chinese medicine composition filtrate;
(4) first time Chinese medicine composition filtrate and second of Chinese medicine composition filtrate are merged, mixing is to get the Chinese medicine group
Close the water decoction of object.
7. method as claimed in claim 6, which is characterized in that further include that the water decoction that will be obtained is put in container for evaporation, and
Container for evaporation is put in the electric heating constant-temperature blowing drying box of laboratory and 6d is dried for 72 DEG C, 8 times a day, per minor tick 3h, is obtained
To Chinese medicine composition dry cream;Obtained Chinese medicine composition dry cream is crushed using conventional grinder, crushed material passes through 100 mesh
304 stainless steel mesh filter 2 times, and 400 mesh, 304 stainless steel mesh filters 2 times, finally obtains Chinese medicine composition dry powder, dispense ,-
It is saved backup in 80 DEG C of refrigerators.
8. method as claimed in claim 6, which is characterized in that 6 layers of white gauze, 17 layers of white are respectively adopted in filtering for the first time
Gauze filters successively, and second of filtering is respectively adopted 8 layers of white gauze, 19 layers of white yarn cloth and filters successively.
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