CN106265629A - The application in esophageal carcinoma chemoprophylaxis of the different rhamnol - Google Patents

The application in esophageal carcinoma chemoprophylaxis of the different rhamnol Download PDF

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CN106265629A
CN106265629A CN201610662904.XA CN201610662904A CN106265629A CN 106265629 A CN106265629 A CN 106265629A CN 201610662904 A CN201610662904 A CN 201610662904A CN 106265629 A CN106265629 A CN 106265629A
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rhamnol
different
esophageal carcinoma
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cell
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董子钢
常俊标
董子明
赵四敏
刘康栋
李洪林
江亚南
赵继敏
王艳红
别荣海
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Zhengzhou University
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Abstract

The application belongs to treatment of cancer and prevention technique field, is specifically related to the application in the esophageal carcinoma is prevented of the different rhamnol.Different rhamnol is for the Carcinogenesis of the chemoprophylaxis esophageal carcinoma.The general design idea of the application is: in the rat esophagus Carcinogenesis of NMBA induction, by affecting the incidence rate of precancerous lesion, multiformity, and judge that different rhamnol is judged by the expression of Ki67, c Jun index for the elemental abundances of the esophageal carcinoma by ImmunohistochemistryMethods Methods.Different rhamnol, by building the rat esophagus carcinogenesis model process of NMBA induction, is intervened the esophageal carcinoma and is occurred the effect of development to do concrete evaluation by the application.Result shows, the different rhamnol of debita spissitudo has preferably effect for the prevention of esophageal carcinoma pathological process, provides the probability of new thinking and reference for the chemoprophylaxis of the esophageal carcinoma.

Description

The application in esophageal carcinoma chemoprophylaxis of the different rhamnol
Technical field
The application belongs to treatment of cancer and prevention technique field, is specifically related to different rhamnol in esophageal carcinoma chemoprophylaxis Application.
Background technology
The esophageal carcinoma is modal malignant tumor in the world, in causing the modal tumor of cancer mortality, occupies Six, prognosis is excessively poor, and five year survival rate is only 8 ~ 12%.Esophageal squamous cell carcinoma (esophageal squamous cell Carcinoma, ESCC) and adenocarcinoma of esophagus (esophageal adenocarcinoma, EAC) be two main group of the esophageal carcinoma Knit type.ESCC is the modal hypotype of developing country, but is mainly EAC in the U.S. and other western countries' esophageal carcinoma. In recent years, although esophageal carcinoma early diagnosis, early discovery technology have had great progress, the chemotherapy and radiation of standard is the most clinically Apply decades, but the overall survival of most of clinical middle and advanced stage patient is still not improved, and has pointed out and has only emphasized tumor Early discovery and treatment and ignore tumor prevention, result produces little effect.
Whether cancer can prevent？As far back as 20th century the mid-80, the World Health Organization (WHO) carries the most clearly Having gone out the viewpoint of 3 1/3, the cancer of i.e. 1/3 can be prevented, and the cancer patient of 1/3 can cure, patient's active treatment of 1/3 After can improve the quality of living, extend life cycle.Existing research is thought, esophageal canceration is a multistage Progressive symmetric erythrokeratodermia evolution (normal-basal cell hyperplasia-atypical hyperplasia-cancer in situ-esophageal squamous cell carcinoma), and establish the general of precancerous lesion Read.Esophageal precancerous lesion presents the unstable characteristic of clearly bi-directional development, i.e. these pathological changes can be developed to the direction of cancer, it is possible to Return to more slight pathological changes, or maintain same state the most constant.If this feature prompting of precancerous lesion of cancer of esophagus exists Getting involved in early days and intervene, precancerous lesion may be reversed or evolution is blocked, thus suppresses generation and the development of the esophageal carcinoma.More Gene and/or the precancerous lesion mechanism of understanding well precancerous lesion of cancer of esophagus can be to determining that the key that precancerous lesion develops is divided Son plays a decisive role, accordingly, it would be desirable to exploitation is for the safe and effective targeted drug of these key genes, and then reverses, presses down Make, or stopping precancerous lesion progressing to cancer further.Therefore the animal mould that can simulate carcinogenesis of human esophageal carcinoma is built Type can be that esophageal carcinoma chemoprophylaxis provides a good thinking.
Esophageal carcinoma generation development is the result of environmental factors and interaction of genes.By to Henan area Esophageal Cancer The epidemiological investigation of district crowd, finds Methyl jasmonate (N-nitrosomethylbenzylamine, NMBA) With the esophageal carcinoma, there is close positive correlation.The rat esophagus squamous cell canceration model of NMBA induction at present, extensively Ground is for assessing the pathophysiological processes of the esophageal carcinoma, to confirm chemoprophylactic drug application prospect in human studies.Greatly Mus is developed into squamous cell carcinoma from normal epithelium of esophagus and experienced by hypertrophy, leukoplakia, atypical hyperplasia, papilloma, this Plant strain responses very much like with human pathological's process, can fully simulate the process of people's epithelium of esophagus canceration, use this animal Model can inquire into the molecular mechanism of canceration course of esophageal further, thus for finding molecular target, and be that screening is dry further Cancer chemoprevention medicine that is pre-or that delay esophageal carcinoma generation to develop provides a platform.
In esophageal carcinoma chemoprophylaxis research, in addition to vitamin and various trace elements, multiple compounds is also used to taste Examination carries out the chemoprophylaxis research of the esophageal carcinoma, such as tea polyphenols, freeze-dried strawberry, gallic acid, antioxidation class and nonsteroidal anti-inflammatory Medicines etc., these compounds prevent generation and the development of the esophageal carcinoma by different targeted moleculars, but its mechanism of action also needs to Study deeper into ground.But preventing target spot according to the predetermined esophageal carcinoma, screening safety is good, the compound of excellent in efficiency, low toxicity It it is the basic engineering target carrying out esophageal carcinoma prevention.
Different rhamnol is a kind of quercetin derivative in multiple medicinal plants.Quercetin is a kind of natural flavonoid Compound, is widely present in plurality of Chinese, and research shows, Quercetin is by regulation oncogene c-fos and c-jun gene table Reach, and Suppressor p53, p21, p27, the expression of oncogene c-myc, c-src etc. plays suppression tumor cell proliferation, Promote the effect of apoptosis, and the signal transduction pathway such as MEK-ERK, STAT3 are all had regulating and controlling effect.Same point of different rhamnol The research in tumor of the isomer isorhamnetin is the most, isorhamnetin can by reduce apoptosis suppressor bcl-2 express, Increase pro apoptotic protein Bax quantity and activate caspase activity inducement apoptosis of tumor cells, and to MEK-ERK, The signal transduction pathway such as PKC, JNK all have regulating and controlling effect.And research about different rhamnol antitumor action is considerably less, different Mus Lee's alcohol only research report mistake in skin carcinoma and breast carcinoma, in skin carcinoma, different rhamnol stops skin by its target spot Erk The conversion process of cancerous cell, it can also suppress the propagation of breast cancer cell simultaneously.And about the pass of different rhamnol Yu the esophageal carcinoma System then lacks corresponding research.
Summary of the invention
The present invention is by building the animal model of the rat esophagus canceration of NMBA induction, for different rhamnol and esophageal carcinoma cancer Change relation has carried out preliminary study, it was demonstrated that different rhamnol may be used for preventing the canceration of the esophageal carcinoma, have the most potential should By value.
The technical solution used in the present invention is as described below.
The application in esophageal carcinoma chemoprophylaxis of the different rhamnol, for preventing the Carcinogenesis of epithelium of esophagus.
Specifically, different rhamnol is when epithelium of esophagus immortalized cells (SHEE cell), time below 10 μMs of concentration pair Less in cytotoxicity, and epithelium of esophagus immortalized cells can be suppressed to convert；
During for preventing esophageal carcinoma pathological changes, by 70 ~ 350mg/m2Dose application；Furthermore, when rat, 70 ~ 350mg/m2Dosage, is equivalent to dose,equivalent 10 ~ 50 mg/kg, and rat esophagus carninomatosis can be prevented to become；When mice, according to 70~350mg/m2Dosage, is equivalent to dose,equivalent 15.4 ~ 78.54mg/kg, can prevent mice esophageal carcinoma pathological changes, for people Time, be equivalent to dose,equivalent 1.5 ~ 7mg/kg.
The general design idea of the application is: in the rat esophagus Carcinogenesis of NMBA induction, by affecting precancerous lesion Incidence rate, multiformity, and judge that the expression of ki67, c-jun index comes different rhamnol by ImmunohistochemistryMethods Methods Elemental abundances with the esophageal carcinoma is judged.
In experimentation, applicant passes through experiment in vitro, and screening obtains the optimal different rhamnol for SHEE cell Concentration, and confirm suppression SHEE cell that different rhamnol can the be strong cell proliferation in Carcinogenesis and grappling is non-depends on Rely type growth.
Further, by building the rat esophagus carcinogenesis model of NMBA induction, different rhamnol is intervened the esophageal carcinoma The effect that development occurs has done concrete evaluation.Result shows, the different rhamnol of debita spissitudo is for the pre-armour of esophageal carcinoma pathological changes There is preferably effect, provide the probability of new thinking and reference for the chemoprophylaxis of the esophageal carcinoma.
Accompanying drawing explanation
What Fig. 1 was different rhamnol on SHEE cell proliferation and conversion capability affects result, and wherein A is by CCK8 test kit Detecting different rhamnol experimental result Cytotoxic to SHEE, in figure, the survival rate of 10 μMs of different rhamnol cells is about 80%；B Showing SHEE cell growth inhibition result, its result for different rhamnol, when 0 ~ 10 μM, different rhamnol is to SHEE cell Inhibitory action there is time and dose dependent；C is that different rhamnol affects result to SHEE cell transformation capacity, its knot Fruit shows, along with the rising of different rhamnol concentration, cell transformation capacity and clone's quantity decline, different rhamnol can suppress EGF The conversion of induction epithelium of esophagus immortalized cells SHEE；
Fig. 2 is experiment packet situation, and wherein A is solvent control group, subcutaneous injection sterilized water, 3 times a week, and totally 5 weeks；0.5% carboxylic first Base sodium cellulosate gavage (10ml/kg)；B is NMBA group, subcutaneous injection NMBA(0.5mg/kg), 3 times a week, totally 5 weeks (0 week to 5 Week)；C is zengshengping tablet group, starts gavage zengshengping tablet (4g/kg), at 0 week to 5 week subcutaneous injection NMBA(0.5mg/kg from-1 week), 3 times a week；D is different rhamnol low dose group, starts the different rhamnol of gavage (10mg/kg) from-1 week, at 0 week to 5 perithelium bets Penetrate NMBA(0.5mg/kg), 3 times a week；E is different rhamnol high dose group, starts the different rhamnol of gavage (50mg/kg) from-1 week, At 0 week to 5 week subcutaneous injection NMBA(0.5mg/kg), 3 times a week；Note: ↑ represent NMBA injection time, at 0 ~ 5 week, note weekly Penetrate 3 times；
When Fig. 3 is 35 weeks, each experimental group rat esophagus mucosa squamous epithelial cancer ki67 expression (× 400), wherein A is solvent pair According to group sample drawing, B is Methyl jasmonate group sample drawing, and C is zengshengping tablet group sample drawing, and D is different rhamnol low dose group sample Product figure, E is different rhamnol high dose group sample drawing, and F is that each experimental group ki67 positive cell percentage is (compared with NMBA group, poor Different statistically significant, * represents that P < 0.05, * * represents that P < 0.01, * * * represents P < 0.001)；
When Fig. 4 is 35w, each experimental group rat esophagus mucosa squamous epithelial cancer c-jun expression (× 400), wherein A is solvent pair According to group sample drawing, B is Methyl jasmonate group sample drawing, and C is zengshengping tablet group sample drawing, and D is different rhamnol low dose group sample Product figure, E is different rhamnol high dose group sample drawing, and F is that each experimental group c-jun positive cell percentage is (compared with NMBA group, poor Different statistically significant, * represents that P < 0.05, * * represents that P < 0.01, * * * represents P < 0.001)；
During Fig. 5 is for experiment, rat general physiology statistical indicator situation, wherein A is each group of rat body weight situation, compares for each group No significant difference；B is that each group of rat chow takes in situation, compares no significant difference for each group, and C is each group of rat Drinking-water situation, compares no significant difference for each group；
Fig. 6 is rat lesion type statistical conditions, respectively group rat pathological changes (hypertrophy, leukoplakia, atypical hyperplasia) all with NMBA group is compared, and concrete condition is:
Hypertrophy, zengshengping tablet group no significant difference, different rhamnol low dose group difference statistically significant (P< 0.05), Different rhamnol high dose group difference statistically significant (P<0.0001)；
Leukoplakia, zengshengping tablet group, different rhamnol low dose group and different rhamnol high dose group difference the most statistically significant (P <0.000)；
Atypical hyperplasia, zengshengping tablet group difference statistically significant (P< 0.05), different rhamnol low dosage difference has statistics to anticipate Justice (P< 0.01), different rhamnol high dose group difference the most statistically significant (P< 0.000) (* representsP< 0.05, * * representsP < 0.01, * * * representsP< 0.001)；
Fig. 7 is rat esophagus mucosa HE dyeing pathological grading canonical reference figure, and wherein A is NMBA group rat esophagus mid-stream sample Morphologic observation figure；In B, a is normal esophageal mucosa, and b is Esophageal Mucosa proliferative lesion, and c is that Esophageal Mucosa white macula sexually transmitted disease (STD) becomes, d For Esophageal Mucosa atypical hyperplasia (× 400)；
Fig. 8 is 15 weeks, 25 weeks, 35 weeks time rat esophagus mucosa infection statistical result.
Hypertrophic change, compared with matched group, NMBA group 15 weeks, 25 weeks, 35 weeks time preneoplastic lesions showed increased, difference The most statistically significant (P < 0.05)；
Leukoplakia, 35 weeks NMBA group rat esophagus pathological changes and 15 weeks, within 25 weeks, NMBA group is compared, and leukoplakia increases, and difference has Statistical significance (P < 0.05)；
Atypical hyperplasia, 35 weeks NMBA groups and 15 weeks, within 25 weeks, NMBA group compares lesion degree seriously, and difference is statistically significant (P<0.05)。
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is described in detail, before introducing embodiment, the most right In the present invention, part Experiment material used and animal model build and are briefly discussed below.
Cell strain: SHEE cell strain (Shantou human embryonic esophageal epithelial cell Line, people's embryo esophageal epithelial cell Shantou strain, also referred to as Immortalized Esophageal Epithelial Cells strain, by Medical College of Shantou University cancer pathology Research department professor Li Enmin give), cell strain uses 10% FBS/DMEM culture medium, 37 DEG C of constant temperature, 5% CO in incubator2Bar Under part cultivate, standby.
Different rhamnol: provided by department of chemistry of Zhengzhou University synthetic, by the processes such as rutin etherificate, hydrolysis are manually closed One-tenth is prepared from, and main preparation process is:
First, take rutin (20g, 33 mmol) in 500 mL round-bottomed flasks, add DMF solvent 160 mL, be stirred at room temperature to reed Fourth is completely dissolved；
Add K2CO3(22.7 g, 165 mmol) are heated to 50 DEG C in oil bath；
Being slow added into benzyl bromide a-bromotoluene 4.5eq.(to note: the addition TLC to be seen of benzyl bromide a-bromotoluene, reaction produces two main point, wherein The principal point of top to be richer than following principal point, if not being richer than following point, then adds the benzyl bromide a-bromotoluene of 1eq., extends the response time, Until the point of top than following point dense just can stopped reaction, otherwise subsequent reactions yield can be by considerable influence), reaction stops After, it being slowly added to aqueous acetic acid (volume ratio, acetic acid: water=1:1), regulation PH is neutral；
Adding substantial amounts of water, ethyl acetate extracts, and TLC detects whether that extraction is completely；
Being washed with water twice after organic facies being merged, organic facies is evaporated to obtain bronzing dope；
Second, the above-mentioned bronzing dope being evaporated is added 30mL concentrated hydrochloric acid, 100mL ethanol, is heated to reflux TLC detection after 4h Whether react complete；
The bottle of cooling is put in refrigerator 1h, sucking filtration, and frozen water washes twice, and yellow solid 60 DEG C is dried；
Under normal circumstances, the yield in above-mentioned first, second two steps is about 80 ~ 90%；
3rd, take yellow solid in second step (20g, 35mmol) in 250mL round-bottomed flask, add 80mL dry DMF, then Add K2CO3(8.7g, 63 mmol), are added dropwise to CH3I(2.6ml, 42 mmol), 30 DEG C of stirrings (if reaction carries out not exclusively, Suitably add CH3I)；
After reaction completely, shrend is gone out reaction, and ethyl acetate extraction three times, water washes twice, and anhydrous sodium sulfate is dried, and is evaporated post Obtain faint yellow solid, PE/EA/DCM=80/15/15；
Product once obtains faint yellow solid (yield about about 50%) with absolute ethanol washing；
4th, take gained faint yellow solid 15g in third step and throw in hydrogenation reaction kettle, be initially charged oxolane 110mL and stir Mix to dissolving, add methanol 110mL, Pd/C 1.5g；
Gas displacement three times, reaction 15h, TLC detect whether that reaction is completely；
Take out reactant liquor after reaction completely, filter Pd/C with kieselguhr, cross post, polarity: oxolane/dichloromethane=8/1, obtain Yellow solid (yield about about 90%)；
Product synthetic route is as follows:
。
The structure of esophageal canceration animal model:
In animal model building process, rat used is: male, the F344 rat (purchased from Beijing dimension tonneau China) of 4 ~ 5 week old；Mould In type building process, rat is raised by 5/cage standard conditions, constant temperature (22～25 DEG C), constant humidity (40～60%), every 12 hours Light dark cycle once, freely eats for animal through autoclaved cleaning grade animal feed and water；Within two weeks, change a mouse cage, And regular clean animal room；
Animal model building process is with reference to Stoner GD be NMBA induced rat esophageal canceration model construction process (Stoner GD et.al., Etiology and chemoprevention of esophageal squamous cell carcinoma, Carcinogenesis, 2001,22 (11): 1737 ~ 1746), particularly as follows:
By the amount of subcutaneous injection NMBA 0.5mg/kg, (it is revised as also dependent on needs: for 3 times/all, continuous 5 weeks for 1 time/week, continuously 15 weeks) mode builds rat esophagus canceration animal model, and puts to death a collection of rat respectively 15 weeks, 25 weeks and 35 weeks, thus Stage by stage rat esophagus precancerous lesion process classified and quantify (refinement), being beneficial to find what each stage changed Gene, by analyzing the molecular mechanism of precancerous lesion, thus selects chemoprophylaxis target spot to provide theoretical foundation for different phase；
In the most following embodiment, when carrying out compound prevention esophageal canceration experiment, give NMBA subcutaneous injection the last week to Give medicine gavage, carry out the chemoprophylaxis of medicine.
It is to be understood that if carried out the compound-treated experiments of the esophageal carcinoma, can after NMBA subcutaneous injection completes, then Carry out compound gavage, carry out pharmaceutical intervention altogether 35 weeks.
Embodiment 1
Owing to different rhamnol is when carrying out biologic applications, certain toxicity be there may be for cell, thus first need different Fructus rhamni (Rhamnus davurica Pall.) Alcohol carries out preliminary assessment for toxicity and the growth effect of cell, and related experiment process is briefly discussed below.
Cytotoxicity experiment
Experimentation is: SHEE cell is with 1 × 104Individual/every hole be inoculated in 96 orifice plates carry out cultivating (10% FBS/DMEM, 37 DEG C, 5% CO2), after 12 hours, change fresh culture and add variable concentrations different rhamnol (0 μM, 1 μM, 2.5 μMs, 5 μMs, 10 μMs, 20 μMs), after cultivating 24 and 48 hours respectively, every hole adds 10 μ L CCK-8(cell counting kit-8, Japan Colleague), continue to cultivate 2 hours, finally in microplate reader, 450nm carries out absorbance detection.
Experimental result is as shown in Figure 1A.It can be seen that pass through the detection of CCK-8 test kit from Figure 1A, it appeared that 0 ~ The different rhamnol of 10 μMs is the least on the impact of cell, but the different rhamnol of 20 μMs shows certain cytotoxicity.Thus 10 μMs are selected as experiment maximum dose level in subsequent experimental.
Cell proliferation experiment
Experimentation is: SHEE cell is with 5 × 103Individual/every hole be inoculated in 96 orifice plates carry out cultivating (10% FBS/DMEM, 37 DEG C, 5% CO2), after 12 hours, change fresh culture and add variable concentrations different rhamnol (0 μM, 1 μM, 2.5 μMs, 5 μMs, 10 μMs), respectively after cultivating 0,24,48,72 and 96 hours, every hole adds the CCK-8 of 10 μ L, continues cultivation 2 hours, finally Microplate reader carries out under 450nm wavelength condition absorbance detection.
Experimental result is as shown in Figure 1B.
It can be seen that the different rhamnol effect SHEE cell of 1 μM, 2.5 μMs concentration 24,48,72 and 96 hours from Figure 1B After, SHEE cell is not had inhibitory action, the different rhamnol of 5 μMs, 10 μMs concentration is inhibited to SHEE cell, and in Now there is time and dose dependent.
Anchorage Independent growth experiment
Experimentation is: on 6 orifice plates, every hole spreads into 3 mL BME culture medium (containing 10% FBS and 0.5% agar), to be solidified after Spread and have 8 × 10 into suspendible3The upper strata glue (1 mL BME culture medium, containing 10% FBS and 0.33% agar) of individual SHEE cell, Incubator (37 DEG C, 5% CO2) cultivate after 7 days, treat Clone formation, utilize microscope to carry out colony count.
Experimental result is as shown in Figure 1 C.
From Fig. 1 C it can be seen that with EGF(epithelical cell growth factor) group compares, DMSO(dimethyl sulfoxide) solvent pair SHEE cell according to group does not has clonality；After SHEE cell is induced by EGF, cell forms clone, but adds different Mus Lee's alcohol can suppress the SHEE cell clonal formation that EGF induces, and along with the rising of different rhamnol concentration, clone's quantity is gradually Declining, illustrate in the case of low toxic concentration, different rhamnol can suppress the SHEE cell clonal formation energy that EGF induces Power.
In above-mentioned cell research, with people's epithelium of esophagus SHEE cell as object, select 0 μM, 1 μM, 2.5 μMs, 5 μMs, 10 μM, the different rhamnol of 20 μMs of concentration carry out toxicity test, filter out safe drugs concentration, concentration be defined as 0 μM, 1 μM, 2.5 μM、 5μM、10μM.By further proliferation experiment, it was observed that SHEE cell is had by the different rhamnol of 5 μMs, 10 μMs concentration Inhibited proliferation.The SHEE cell of normal SHEE cell and EGF induction is detected finally by grappling non-dependent growth experiment Conversion capability, and the inhibitory action of the SHEE cell transformation that EGF is induced by different rhamnol can be detected, it was observed that different rhamnol exists There is under the conditions of hypotoxic the suppression normal cell effect to tumour cell transformation.
The to sum up conclusion such as cytotoxicity, cell proliferation, grappling experiment, it can be assumed that, the different rhamnol of less than 10 μMs concentration To the toxicity of SHEE cell at tolerance interval, and in this concentration range, different rhamnol has increasing to SHEE cell Grow inhibitory action；And the SHEE transformation induced EGF due to different rhamnol is inhibited, thus can tentatively recognize Fixed, different rhamnol has the suppression normal cell effect to tumour cell transformation under low consistency conditions, in other words, in safety On different rhamnol drug level scope (when less than 10 μMs), can effectively suppress the propagation of SHEE cell and pernicious turn Change.
Embodiment 2
The present invention is configured to basis with the synchronization of esophageal canceration animal model, by synchronizing in NMBA induction canceration course of esophageal Add different rhamnol, by different rhamnol for the impact of model construction process, thus to the change to esophageal canceration of the different rhamnol Learn preventive effect and make preliminary assessment.
In experimentation, for evaluating the impact for esophageal canceration of the different rhamnol, at esophageal carcinoma animal model building process In, carry out preliminary assessment by arranging different experiments group with the elemental abundances to different rhamnol.Specific experiment group sets Put as in figure 2 it is shown, specific as follows (newly purchased rat adapted to environment after one week, then by following group grouping experiment):
First group: solvent control group (rat quantity n=20), conventional raising, subcutaneous injection sterilized water, three-times-weekly, totally five weeks； Press the carboxymethyl cellulose of the dosage gavage 0.5% of rat body weight 1mL/100g, once a day, totally 35 weeks simultaneously；
Second group: NMBA model group (rat quantity n=33), conventional raising, subcutaneous injection NMBA, three-times-weekly, totally five weeks；
3rd group: zengshengping tablet group (rat quantity n=8), on the basis of NMBA injects, simultaneously with zengshengping tablet to rat oral gavage (4g/ Kg), once a day, totally 35 weeks；
4th group: different rhamnol low dose group (rat quantity n=10), on the basis of NMBA injects, by 10mg/kg dosage by different Rhamnol is suspended in 0.5% carboxymethyl cellulose and carries out gavage, once a day, carries out altogether 35 weeks；
5th group: different rhamnol high dose group (rat quantity n=10), on the basis of NMBA injects, by 50mg/kg dosage by different Rhamnol is suspended in 0.5% carboxymethyl cellulose and carries out gavage, once a day, carries out altogether 35 weeks.
In experimentation, the free diet of rat.When 15 weeks and 25 weeks, from first group and second group, take 5 respectively Only with 10 rats, dissect rat after anesthesia and (analyze for ease of other detections, blood can be separated at when dissected abdominal aortic blood The preservation after starching that purifies the blood is standby to-80 DEG C), esophagus is taken out and stringer is cut open, be divided into upper, middle and lower three sections, the preservation of half frozen section In liquid nitrogen, in case the extraction and analysis of follow-up DNA, RNA, albumen, second half fixes 24 hours in the neutral formalin of 10%, And carry out paraffin embedding and HE dyeing, in case follow-up histopathology evaluation and analysis.When 35 weeks at the end of experiment, put to death each Organize all rats, and retain esophagus to carry out concrete immunohistochemical analysis and otherwise assay.
Histological evaluation, in all Esophageal Mucosa samples of each rat after mainly dyeing paraffin embedding HE Normal mucosa, hypertrophy, leukoplakia, the type of atypical hyperplasia carry out judging, count and adding up；
Immunohistochemical analysis, after mainly esophagus sample being carried out immunohistochemical staining, then enters properties of samples Row judges, immunohistochemical staining process is: after paraffin-embedded sample routine dewaxing aquation, with the citric acid of 0.01mol Salt repair liquid microwave 10min repairs antigen, drips H2O2Close endogenous peroxydase (room temperature 10 min), first antibody Ki67(1:50), c-jun (1:50) (4 DEG C overnight), dropping two anti-(incubated at room 30 min), DAB develops the color, and distillation washing is eventually Only colour developing, haematoxylin redyes, wash, break up after fully washing return indigo plant, conventional dehydration is transparent, neutral gum mounting, simultaneously with PBS One is replaced anti-to make negative control.
At the end of 35w, the expression of each experimental group rat esophagus mucosa squamous epithelial cancer ki67 and epithelium c-jun such as figure 3, shown in Fig. 4, it can be seen that compared to NMBA group, each experimental group is respectively provided with significant difference, and compared to zengshengping tablet Group, along with different rhamnol consumption is different, its action effect also has and to a certain degree optimizes.
And the morphological indexes such as the body weight of rat, amount of drinking water, quantity of food is as it is shown in figure 5, can from figure during testing Going out, each group rat does not has notable difference in terms of diet, body weight.
The epithelial tissue of rat esophagus is carried out morphological observation, and carries out Esophageal Mucosa HE dyeing pathological grading and system Meter.During morphological observation, by two pathologists under double blinding state, light Microscopic observation esophagus Histopathologic changes is also remembered Record (× 100) different pathological classification percentage under each visual field.The precancerous lesion pathological grading of esophagus is as it is shown in fig. 7, have Body grade scale is: the change of Esophageal Mucosa epithelium is divided into normal, hypertrophy, mucous membrane white spot, atypical hyperplasia 4 type；
Normally, normal esophageal squamous epithelial cancer, unchanged；
Hypertrophy, Accretive Type pathological changes is divided into basal cell hyperplasia, epithelial cell (spine cell) hypertrophy；
Mucous membrane white spot, basal cell and spine cell significantly increase, and the basal cell of hypertrophy forms more than 3-6 layer, can account for holostrome 1/2；
Atypical hyperplasia, basal layer cell active proliferation, level is disorderly and more, and basad portion highlights, and can exceed epithelium holostrome 2/3；Cell increases, and size and form are inconsistent, arrange the most whole.
Rat esophagus canceration situation statistical result is as shown in Fig. 6 and following table.
Different rhamnol is on F344 rat esophagus canceration degree and the impact of quantity:
Note:aRepresent compared with NMBA group, difference statistically significant (P< 0.05),bRepresenting compared with NMBA group, difference has system Meter meaning (P< 0.01),cRepresent compared with NMBA group, difference statistically significant (P< 0.001).
From Fig. 6 and upper table statistical result it can be seen that control rats only has preneoplastic lesions, and Methyl jasmonate group Rat occurs that hypertrophy, leukoplakia, atypical hyperplasia precancerous lesion, different rhamnol high dose group and low dose group there is also increasing Life, leukoplakia, the precancerous lesion of atypical hyperplasia, but compare with Methyl jasmonate group, pathological changes substantially alleviates, and difference has Statistical significance, does not finds the heart simultaneously, liver, kidney, and spleen etc. is abnormal the most further, to F344 rat esophagus epithelium in NMBA model group In canceration evolution process, the number of each pathological changes (hypertrophy, leukoplakia, atypical hyperplasia) carries out adding up (mean ± standard deviation), tool Body result is as shown in Fig. 8 and following table:
15w, 25w, 35w rat esophagus mucosa infection statistical result:
Note: a represents compared with matched group, difference statistically significant (P < 0.05)；B represents compared with 35 weeks NMBA groups, difference Statistically significant (P < 0.05).
NMBA group 15 weeks, 25 weeks, 35 weeks time hypertrophy showed increased, compare with matched group difference statistically significant (P < 0.05).Matched group does not finds leukoplakia and atypical hyperplasia, and NMBA group was at 15 weeks, 25 weeks, within 35 weeks, all there is leukoplakia And atypical hyperplasia, and as time went on, pathological changes gradually increases.
Leukoplakia, 35 weeks NMBA group rat esophagus pathological changes and 15 weeks, within 25 weeks, NMBA group is compared, and leukoplakia increases, poor Different statistically significant (P < 0.05)；
Atypical hyperplasia, 35 weeks NMBA groups and 15 weeks, within 25 weeks, NMBA group compares lesion degree seriously, the statistically significant (P of difference <0.05)。
The observed result of summary optical microscope and statistical result, it can be clearly seen that, NMBA induce 15 weeks, 25 When week, 35 weeks, in epithelium of esophagus, the pathological changes showed increased such as hypertrophy, leukoplakia and atypical hyperplasia, the result shows NMBA success is induction of the generation of rat esophagus carcinogenesis process.

Claims (3)

  1. The most different rhamnol application in esophageal carcinoma chemoprophylaxis, it is characterised in that for preventing the Carcinogenesis of epithelium of esophagus.
  2. The different rhamnol the most as claimed in claim 1 application in esophageal carcinoma chemoprophylaxis, it is characterised in that by 70 ~ 350mg/m2 Dosage, is used for preventing esophageal carcinoma pathological changes.
  3. The different rhamnol the most as claimed in claim 1 application in esophageal carcinoma chemoprophylaxis, it is characterised in that for SHEE cell Time, different rhamnol consumption is: not less than 0, but less than 10 μMs.
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