CN100341581C - Method of thalidomide for effective interventing oral cancer in animal model - Google Patents
Method of thalidomide for effective interventing oral cancer in animal model Download PDFInfo
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- CN100341581C CN100341581C CNB200510028311XA CN200510028311A CN100341581C CN 100341581 C CN100341581 C CN 100341581C CN B200510028311X A CNB200510028311X A CN B200510028311XA CN 200510028311 A CN200510028311 A CN 200510028311A CN 100341581 C CN100341581 C CN 100341581C
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Abstract
The present invention relates to a method for active interference of salvianolic acids B in an oral cancer animal model, which comprises the following procedures of oral cancer animal model arrangement, salvianolic acid B medicine interference, histological examination and pathology evaluation. The method for active interference of salvianolic acids B in an oral cancer animal model of the present invention has the advantages that a DMBA model for inducing the cheek pouch cancerization of golden hamsters is reasonably adopted; medicine interference is continuously carried out for 12 weeks from the seventh week, and the experimental time is moderate, which is convenient for the implementation of the present invention; selected dosage is 10 mg. kg<-1>. d<-1>, the medicine acts on animals in a drench mode, and the function of the salvianolic acids B is fully performed.
Description
Technical field
The present invention is about the method for activating blood herbs intervening oral carcinoma animal model, especially in regard to the method for salvianolic acid B intervening oral carcinoma animal model.
Background technology
Oral cancer is one of modal malignant tumor of incidence.Since its easy transfer, poor prognosis, and the prevention of oral cancer is particularly important.Cancer chemoprevention has become at present and has reduced one of most promising means of oral cancer incidence rate.In view of the prolonged application of cancer chemoprevention medicine, exploitation is the direction place of current cancer chemoprevention based on the medicine of natural plants or food.
Activating blood circulation to dissipate blood stasis is one of basic rule of treatment of Chinese traditional treatment tumor.Clinical research shows: promoting blood flow and remove blood stasis drug has microcirculation improvement, anticoagulation, anticancer growth and multiple effects such as transfer, human body immunity improving function.Secular clinical practice confirms that also the utilization promoting blood flow and remove blood stasis drug is treated various tumors and can be obtained result preferably.
In numerous blood-activating and stasis-removing, Radix Salviae Miltiorrhizae be clinical in promoting blood flow and remove blood stasis drug simply the most commonly used.Effect aspects such as vascular permeability, antioxidation, antiinflammatory and antitumor for Radix Salviae Miltiorrhizae protection heart microvascular endothelial cell, inhibition VEGF have deep research at present, and think that TANSHINONES is an antineoplastic component main in the Radix Salviae Miltiorrhizae.Salvianolic acid B is a kind of that content is the highest in the red sage root water soluble ingredient, function of promoting blood circulation to disperse blood clots is the strongest.Whether has antitumor action equally for salvianolic acid B, still planless at present research report.
Angiogenesis plays an important role to growth of tumor and transfer.Result of study shows that the generation of cancer-related blood vessel starts from the precancerous lesion stage, and the pernicious transformation that these new vesselses and precancerosis decrease is closely related, and thinks that precancerous lesion proceeds to the important step that the blood vessel phase may be the pernicious transformation of cell.Week was once observed discovery to the canceration overall process of golden hamster cheek pouch canceration model on an equal basis: white macula is followed increasing the weight of of its epithelial dysplasia degree as a kind of precancerous lesion, and angiogenesis is promptly arranged.Therefore, by the very possible pernicious transformation that suppresses some precancerous lesions such as oral leukoplakia of angiogenesis inhibitor treatment, thereby reach the purpose of preventing oral cancer.
Thalidomide is at first with a kind of calm antiemetic listing.Because it has serious teratogenesis, listing this medicine soon just is under an embargo and has used.In recent years, studies show that thalidomide has the effect of obvious inhibition angiogenesis, and have the generation that a large amount of interaction energies that studies confirm that this medicine angiogenesis inhibitor suppresses new vessels in the tumor at present, and then suppress growth of tumor.Yet relevant this medicine still lacks complete information to the effect of oral cancer.
The present invention uses the oral cancer animal model, tentatively inquires into salvianolic acid B and the thalidomide prophylactic-therapeutic effect to oral cancer, and the application in cancer chemoprevention and oncotherapy provides the scientific theory foundation for activating blood herbs and vasoinhibitor.
Summary of the invention
The object of the present invention is to provide a kind of method of thalidomide for effective interventing oral cancer in animal model.
The objective of the invention is to realize by following technical method: (1) sets up oral cancer animal model, (2) salvianolic acid B pharmaceutical intervention, (3) histological examination, (4) pathological evaluation.In the research of oral cancer and precancerous lesion, need provide a good reproducibility, stable that set up easily again, have an animal model to similar growth characteristics of human oral cancer and biological behaviour with precancerous lesion.The inductive golden hamster cheek pouch canceration model of DMBA is the ideal tools of further investigation oral precancerous lesion and oral cancer, its generation development and histological change procedure are very similar to the evolution of human mouth cancer, can reproduce a plurality of steps in the oral cancer generating process, it is normal epithelial, simple property epithelial proliferation, paraplasm, cancer in situ, and infiltrating carcinoma.
Result of the present invention shows: after giving the continuous 6 all DMBA stimulations of golden hamster cheek pouch mucosa, when the 18th week, the golden hamster cheek pouch mucosa shows the infringement of white macula sample, papilloma and three kinds of sick damages of cancer, and micromicro shows normally on it, multiple histopathology such as property hypertrophy, paraplasm and squamous cell carcinoma shows merely.The incidence rate of positive controls scale cancer is 47.1% among the present invention, and wherein about 70% scale cancer is to develop from papilloma.Results reported such as this result and Ning Li are close.Yet obviously the canceration rate of report was low more in the past for canceration rate of the present invention, and Bao Dao canceration rate was mostly more than 75% in the past.Analyzing its reason may and stop with the inductive time of carcinogen of the present invention inducing the time of back observation short relevant.Only there were 6 weeks the time that the present invention is coated with DMBA, stopped being coated with behind the carcinogen and observed for 12 weeks.And research in the past to be coated with the time of carcinogen be 12-16 week, stopping DMBA stimulating the back to continue to observe 8-14 week.The purpose that the present invention stimulates only for the DMBA in 6 weeks of golden hamster cheek pouch mucosa is to induce the formation oral cancer to lure cancer late stage model, have the precancerous lesions such as oral leukoplakia and the oral cancer high-risk group of contacted carcinogen once clinically with this modeling, if any smoking history or just the crowd of smoking.
Result of the present invention also shows: salvianolic acid B lures the canceration rate of cancer model in late period to be reduced to 5.6% from 47.1% the golden hamster cheek pouch mucosal carcinoma, as seen this medicine can prevent effectively that the golden hamster cheek pouch mucosa from developing to cancer from the cancer last stage, and the canceration procedural representation of golden hamster cheek pouch mucosa is gone out significant blocking effect.Although from pathological examination results, the salvianolic acid B group has the cheek pouch mucous epithelium of quite a few animal to show in various degree paraplasm, do not get rid of in these animals of prolongation in time part even the probability (this await prolonging in afterwards the experiment laboratory observation time confirm) of canceration is all arranged, but salvianolic acid B prevents the effectiveness of golden hamster cheek pouch mucosa canceration and remains conspicuous, at least it can significantly suppress the canceration of cheek pouch mucosa in early days, postpones the time that the cheek pouch mucosal carcinoma occurs.
Promoting blood circulation to restore menstrual flow, microcirculation improvement are one of main effects of salvianolic acid B.Known anoxia is a kind of enabling signal of angiogenesis in the body, it can pass through oxygen deficient induction factor 1 α (Hypoxia-inducible factor 1 α, HIF-1 α) induces the expression of a series of angiogenesis factors, as VEGF (VEGF), insulin like growth factor (insulin-likegrowth factor), inducibility nitricoxide synthase (inducible nitric oxidesynthase) etc.Infer thus: salvianolic acid B is by improving the blood circulation of whole body and local organization, and the blood that increases tissue supplies and nutrition, improves partial anoxic conditions, avoids the activation of HIF-1 α, and then suppresses the expression of angiogenesis factor, avoids the unlatching of angiogenic switch.Existing report: salvianolic acid B increases effect at the external inductive vascular permeability of VEGF (VEGF) that can significantly suppress.As for salvianolic acid B whether also have influence on the tumor-blood-vessel growth process in some other relevant somatomedin, and HIF-1 α whether participate in the canceration of salvianolic acid B prevention oral precancerous lesion with and concrete mechanism of action, we further study.
On the other hand, salvianolic acid B also has very strong lipotropism matter oxidation and free radical scavenging effect.Whether this two kinds of mechanism have also participated in the canceration process that salvianolic acid B prevents oral precancerous lesion, still require further study and are inquired into.
The present invention shows: thalidomide can significantly suppress the formation of golden hamster cheek pouch mucosa cancer, and cancer rate is reduced to 5.9% from 47.1%.The result of this result and Hoon Myoung etc. is quite different.Hoon etc. do not observe the inhibitory action of thalidomide to the oral cancer growth in the transplanted tumor model of oral cancer.We infer that this may be relevant with the difference of testing selected animal model and experimental design.What the transplanted tumor model formed is the dystopy tumor, this tumor aspects such as biological nature and angiogenesis with occur in intraoral tumor and compare and may have bigger difference.And the anti-angiogenic characteristic that existing at present scholar proposes anti-angiogenic medicaments also has organ specificity, and this specific character may also be to cause thalidomide can not suppress to transplant one of reason of the oral cancer growth under mouse skin.Yet a noticeable problem is among the present invention, and the thalidomide group has 3 animals to occur one-sided eyelid paralysis midway in experiment, can not open eyes homonymy upper limb congestion, stiff, the downright bad last lethal situation that infects.We infer that this may cause by one-sided limbs generation venous thrombosis.And this situation does not all appear in other several treated animals.This shows that though thalidomide has remarkable antitumor effect, its toxic and side effects is bigger, also is very important in a clinical use problem.
Await in research from now on, further to be confirmed that the cancer mechanism that presses down that it is concrete also awaits deep research as for salvianolic acid B and thalidomide inhibitory action to oral cancer.
Its advantage of the method for the disclosed a kind of thalidomide for effective interventing oral cancer in animal model of the present invention shows: (1) reasonably selects for use DMBA to induce the golden hamster cheek pouch canceration model, the purpose that the present invention stimulates only for the DMBA in 6 weeks of golden hamster cheek pouch mucosa is to induce the formation oral cancer to lure cancer late stage model, have the precancerous lesions such as oral leukoplakia and the oral cancer high-risk group of contacted carcinogen once clinically with this modeling, if any smoking history or just the crowd of smoking; (2) in continuous 12 weeks in pharmaceutical intervention time of the present invention since the 7th week, experimental period is moderate, is convenient to the invention process; (3) to select dose for use be 10mgkg in the present invention
-1D
-1, the approach of gavaging acts on animal, has given full play to the action effect of salvianolic acid B.
Description of drawings
Fig. 1 shows normal cheek pouch mucosa (HE dyeing, amplification 400)
Fig. 2 shows simple property hypertrophy (HE dyeing, amplification 400)
Fig. 3 display abnormality hypertrophy (HE dyeing, amplification 400)
Fig. 4 shows papilloma (HE dyeing, amplification 100)
Fig. 5 shows papilloma canceration (HE dyeing, amplification 100)
Fig. 6 shows scale cancer (HE dyeing, amplification 100)
The specific embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1
One. laboratory animal and medicine
1. age in the selection of laboratory animal and grouping: 6-8 week, 80 of the male Syria Golden Hamster of body weight 80-100 gram, the cleaning level provides (credit number: SCXK (Shanghai) 2002-2003) by Shanghai Slac Experimental Animal Co., Ltd..Sub-cage rearing, 5 in every cage is raised with the golden hamster special feed, and drinking water is a distilled water.Be divided into A, B, C, D and E5 group at random, each 20 of A, B and C groups, each 10 of D group and E groups.
2. chemical carcinogens: with dimethyl phenylpropyl alcohol anthracene (7,12-Dimethylbenz (a) anthracene, DMBA) (sigma) and analytical pure acetone soln are mixed with the DMBA solution of 5g/L.
3. medicine: the former medicine of salvianolic acid B is provided by crude drug teaching and research room of pharmaceutical college of The 2nd Army Medical College and identifies.The former medicine of thalidomide is provided by Changzhou pharmaceutical factory.
Two. experimental technique
1. the foundation of oral cancer animal model
Open golden hamster right side cheek pouch with big straight forceps, in the scope of cheek pouch center 1cm * 1cm, make 10 times circular motion with No. 1 oil painting brush that dips in the 0.5%DMBA acetone soln.Fasting was prohibited water 2 hours behind the coating.On every Mondays, three, five each coatings 1 time, be coated with for 6 weeks altogether.
2. pharmaceutical intervention
(1) A group:, induce oral cancer as stated above from the 1st thoughtful the 6th week.Gavage the 1ml normal saline since continuous 12 every days in week in the 7th week, as positive controls.
(2) B group: the first six week handles and organizes with A.Gavage salvianolic acid B solution since continuous 12 every days in week in the 7th week.Dosage is decided to be 10 times of adult's conventional amount used, i.e. 10mgkg
-1D
-1
(3) C group: the first six week handles and organizes with A.Gavage the thalidomide suspension since continuous 12 every days in week in the 7th week.Dosage is 100mgkg
-1D
-1
(4) D group: the first six week, on every Mondays, three, five at golden hamster right side cheek pouch applied with acetone solution.Since the 7th week gavaging normal saline 1ml every day, totally 12 weeks are as the solvent control group.
(5) E group: whole experimental session is normally raised, and is left intact, as the blank group.
3. histological examination and pathology judgment criteria
(1) histological examination: cut the about 1.0cm of golden hamster right side cheek pouch pathological tissues * 1.0cm size, 10% neutral formalin is fixed, and conventional dehydration, embedding, cuts into slices and does HE dyeing.
The histological observation result
During 6 weeks, as seen coarse, the granular performance of animal cheek pouch surface local that part DMBA handled, with hyperemia in various degree, minority has the infringement of white macula sample, but does not have tumor to produce in these animals.
During 18 weeks, the animal cheek pouch mucosa that DMBA handled shows as simple property hypertrophy (Fig. 2), paraplasm (Fig. 3), papilloma (Fig. 4) and scale cancer (Fig. 5 and Fig. 6) on the histology.In the scale cancer that forms, about 70% is from papilloma development (Fig. 5).The A group has 3 animal deads, and tumor takes place 8 animals, is squamous cell cancer, has 3 animals to show epithelium mile abnormality hypertrophy, and 4 show epithelium moderate paraplasm, and 1 shows the severe epithelial paraplasm.The B group has 2 animal deads, squamous cell cancer takes place in 1 animal, has the cheek pouch epithelium of 3 animals to show as simple property hypertrophy, and 11 animals show epithelium mile abnormality hypertrophy, 2 animals show epithelium moderate paraplasm, and 1 shows the severe epithelial paraplasm.The C group has 3 animal deads, squamous cell cancer takes place in 1 animal, has the cheek pouch epithelium of 4 animals to show as simple property hypertrophy, and 8 animals show epithelium mile abnormality hypertrophy, 2 animals show epithelium moderate paraplasm, and 2 show the severe epithelial paraplasm.The situation of one side upper limb congestion, stiff last necrosis all appearred in 3 dead midway animals before death, simultaneously also with homonymy eyelid paralysis, situation about can not open eyes.The D group has 3 animal cheek pouch mucosas to show as inflammation, and all the other 7 is normal.The pathological observation result of E treated animal is normal epithelial.Compare with the A group, the carcinogenesis rate of salvianolic acid B group and thalidomide group obviously reduces (being respectively 47.1%, 5.6% and 5.9%) (p＜0.005).
(2) pathological evaluation standard: the scope of involving epithelium according to 12 diagnostic criterias of WHO oral precancerous lesion cooperation center and pathological changes is carried out grading diagnosis, double checking.
The pathological observation result of each treated animal cheek pouch mucosa sees table 1 for details.
The distribution situation of each experimental group cheek pouch mucosa different pathological status of table 1.
| Group (total) | Normally | Inflammation | Simple property hypertrophy | Paraplasm | Scale cancer | ||
| Gently | In | Heavy | |||||
| A organizes (17) | 0 | 0 | 1 | 3 | 4 | 1 | 8 |
| B organizes (18) | 0 | 0 | 3 | 11 ● | 2 | 1 | 1 ● |
| C organizes (17) | 0 | 0 | 4 | 8 ● | 2 | 2 | 1 ● |
| D organizes (10) | 7 | 3 | 0 | 0 | 0 | 0 | 0 |
● the significant difference that identical pathological state incidence rate compares in representing the treatment group and A organizing (● represent P＜0.05; ● ● represent P＜0.005)
4. statistical analysis
The tumor incidence rate of each experimental group compares with X 2 test.When P value＜0.05, be designated as difference statistical significance is arranged.All statistical analysiss all adopt SPSS 10.0 softwares to carry out.
Claims (1)
1, the application of salvianolic acid B in preparation treatment oral precancerous lesion medicine.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB200510028311XA CN100341581C (en) | 2005-07-29 | 2005-07-29 | Method of thalidomide for effective interventing oral cancer in animal model |
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| CNB200510028311XA CN100341581C (en) | 2005-07-29 | 2005-07-29 | Method of thalidomide for effective interventing oral cancer in animal model |
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| CN100341581C true CN100341581C (en) | 2007-10-10 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102743436A (en) * | 2012-07-30 | 2012-10-24 | 邵明川 | Application of effective part of salvianolic acid in preparing preparation for preventing and treating mouth and throat diseases |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6043276A (en) * | 1998-06-25 | 2000-03-28 | Georgetown University School Of Medicine | Compounds obtained from salvia species having antiviral activity |
| US6149915A (en) * | 1999-09-29 | 2000-11-21 | Shiva Biomedical, Llc | Treatment of diabetic nephropathy and microalbuminuria |
| CN1408353A (en) * | 2002-09-13 | 2003-04-09 | 上海天甲生物医药有限公司 | Use of tanshin polyphenolic acid B in preparing tumor curing medicine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6043276A (en) * | 1998-06-25 | 2000-03-28 | Georgetown University School Of Medicine | Compounds obtained from salvia species having antiviral activity |
| US6149915A (en) * | 1999-09-29 | 2000-11-21 | Shiva Biomedical, Llc | Treatment of diabetic nephropathy and microalbuminuria |
| CN1408353A (en) * | 2002-09-13 | 2003-04-09 | 上海天甲生物医药有限公司 | Use of tanshin polyphenolic acid B in preparing tumor curing medicine |
Non-Patent Citations (3)
| Title |
|---|
| 丹参对口腔癌细胞粘附和增殖能力的抑制作用 李芃等，现代口腔医学杂志，第13卷第2期 1999 * |
| 丹参活性成分的现代中药药理研究进展 柳丽等，中国野生植物资源，第22卷第6期 2003 * |
| 丹参现代研究概况与进展（续一）杜冠华等，医药导报，第23卷第6期 2004 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102743436A (en) * | 2012-07-30 | 2012-10-24 | 邵明川 | Application of effective part of salvianolic acid in preparing preparation for preventing and treating mouth and throat diseases |
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