CN105534963B - Liver lures cancer agent and preparation method thereof - Google Patents
Liver lures cancer agent and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to biological technical field, is related to a kind of method that liver lures cancer agent, liver to lure the preparation method of cancer agent and lure cancer agent to establish primary liver cancer model based on liver.Wherein, it is DEN non-aqueous solution, e.g. DEN oil solutions or DEN sesame oil solutions that liver, which lures cancer agent,.The present invention by by DEN be dissolved in sesame oil oral administration add mouse take medicine compliance, overcome the individual difference between the water-soluble modeling mouse of DEN；At 20 weeks, water-soluble group of DEN and oral high dose group mouse naked eyes visual tumors tubercle, but 1 times/week of oral administration group mouse death rate is minimum, i.e., long-term low frequency administration can substantially reduce the death rate during modeling.The invention provides the method that a kind of strong, the time-consuming short and liver low fatal rate of stability lures cancer agent, liver to lure the preparation method of cancer agent and lure cancer agent to establish primary liver cancer model based on liver.
Description
Technical field
The invention belongs to biological technical field, is related to preparation method and base that a kind of liver lures cancer agent, liver to lure cancer agent
The method that cancer agent establishes primary liver cancer model is lured in liver.
Background technology
Primary carcinoma of liver is one of most common alimentary system malignant tumour, and in China, its morbidity and mortality is generation
Boundary is the first, is major disease (the The management of hepatocellular carcinoma in for seriously endangering health
Asia：A guideline combining quantitative and qualitative
Evaluation.BiosciTrends, 2010,6：283-287).Regrettably its mechanism not yet illustrates completely.Therefore,
The pathogenesis of liver cancer is furtherd investigate, the effective means of preventing and treating liver cancer is found, can preferably serve human health (A
decade’s studies on metastasis of hepatocellular carcinoma.J Cancer Res Clin
Oncol, 2004,130：187-196).
The mechanism of liver cancer is studied, first has to establish the animal model similar to human liver cancer naturally-occurring process.
Common liver cancer animal model has：Spontaneous hepatocellular carcinoma model, transplantation type liver cancer model, transgenosis liver cancer model and chemically induced
Property liver cancer model.Spontaneous hepatocellular carcinoma is uneven because a situation arises, it is difficult to a large amount of similar oncology materials are obtained in a short time,
And observing time is grown, experiment consuming is larger, therefore the less use in actual research work.Transplanted hepatoma model manipulation is numerous and diverse,
Success rate depends on the maturity of technology, therefore more difficult acquisition；Transgenic animals liver cancer model manufacturing technology requires high, price
Costliness, the country carry out still less；Chemically induced property liver cancer model is easy to operate, and cost is low, and can preferably simulate mankind liver
The natural pathologic process of cancer, preparation method is simple, is one of the most frequently used modeling method (progress of liver cancer animal model.
World Chinese digestion magazine, 2011,19：1275-1278).Chemically induced type liver cancer model most classical way is by diethyl
Nitrosamine (DEN) is soluble in water, gives mouse and freely drinks (Chemoprevention by Lipid-soluble Tea
Polyphenols in Diethylnitrosamine/Phenobarbital-induced Hepatic Pre-cancer
Lesion.Anticancer Res, 2014,34：683-693), the defects of such, is that dosage is difficult control, and mouse is suitable
Answering property is poor, and individual difference is big, and the death rate is high.
With the fast development of nanometer technology, nano material has targeting positioning, sustained-release and controlled release medicine, drugloading rate because of it
Height, improve the advantages that drug absorption rate and be used widely in fields such as drug delivery, treatment of cancer, gene therapies
(Codelivery of doxorubicin and curcumin with lipid nanoparticles results in
improved efficacy of chemotherapy in liver cancer.International Journal of
Nanomedicine, 2015,10：257-270), but utilize its negative biological effect, Disease model field application not yet
Appear in the newspapers.
The content of the invention
In order to solve the technical problem in the presence of background technology, the present invention and then to provide a kind of stability strong, time-consuming
Short and low fatal rate liver lures cancer agent, liver to lure the preparation method of cancer agent and lures cancer agent to establish Primary Hepatic based on liver
The method of cancer model.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of liver lures cancer agent, it is characterised in that：It is diethylnitrosamine (DEN) non-aqueous solution that the liver, which lures cancer agent,.
Preferably, it is DEN oil solutions that liver provided by the present invention, which lures cancer agent,.
Preferably, it is DEN sesame oil solutions that liver provided by the present invention, which lures cancer agent,.
Preferably, liver provided by the present invention lures the DEN oil solutions that cancer agent is nanosizing.
Preferably, the content of the DEN in the DEN oil solutions of nanosizing provided by the present invention is not less than 20.625mg
ml-1。
Preferably, the content of the DEN in the DEN oil solutions of nanosizing provided by the present invention is 20.625mgml-1-
41.25mg/ml-1。
It is a kind of to be used to prepare the method that liver as described above lures cancer agent, it is characterised in that：Methods described includes following step
Suddenly：
1) DEN and egg yolk lecithin are dissolved in sesame oil, oil phase is prepared；The final concentration of the DEN is 10.97%
V/V-21.94%V/V；The final concentration of the egg yolk lecithin is 6%W/V；
2) Tween 80 and glycerine are dissolved in ultra-pure water, aqueous phase is prepared；The final concentration of the Tween 80 is
1.25%V/V；The final concentration of the glycerine is 2.81%V/V；
3) under conditions of 60 DEG C of waters bath with thermostatic control, oil phase that step 1) is prepared is instilled into the water that step 2) is prepared
Xiang Zhong, 15min is stirred under conditions of 1200r/min；The volume ratio of the oil phase and aqueous phase is 1：4；
4) the high shear 2min under conditions of 10000rpm, the emulsified particles that particle diameter is 100nm-200nm is finally given；
5) under conditions of 1000bar high-pressure homogeneous 5 times i.e. obtain the DEN oil solutions of nanosizing.
A kind of method for establishing primary liver cancer model that cancer agent is lured based on liver as described above, it is characterised in that：Institute
The method of stating comprises the following steps：
1) 6-8 week old SPF level males Kunming KM mouse, body weight 18-22g are selected, raising adapts to environment 1 week；
2) cancer agent is lured by the foregoing liver of the body weight orally administration of male Kunming KM mouse weekly, continuous modeling 20 weeks, built
Vertical primary liver cancer model of mice；
3) model is identified.
Preferably, when it is DEN sesame oil solutions to use liver to lure cancer agent in step 2), male elder brother is pressed in the step 2)
It is 16.5mg/kg that the foregoing liver of body weight orally administration of bright KM mouse, which lures the dosage of cancer agent,；Administration frequency is every
Week 1 time, 3 times or 7 times.
Preferably, when it is the DEN oil solutions of nanosizing that the liver used in step 2), which lures cancer agent, in the step 2)
It is 8.25mg/kg to lure the dosage of cancer agent by the foregoing liver of body weight orally administration of male Kunming KM mouse；Administration
Frequency is 1 times a week.
Compared with prior art, the present invention has advantages below and effect：
Cancer agent, preparation method are lured the present invention relates to a kind of liver and establish rat primary liver based on what liver lured cancer agent
The method of cancer model, from 6-8 week old SPF level male KM mouse, lure cancer agent DEN, continuous modeling by body weight orally administration weekly
20 weeks, establish primary liver cancer model of mice.It is primary to be successfully established mouse by changing DEN formulations and administration frequency by the present invention
Property liver cancer model, the model has that modelling success rate is high, mortality of animals is low during modeling, high dose DEN sesame oil (1
Times/week) group mouse the death rate be 25%, survive 12 mouse in have 5 naked eyes visual tumors tubercles, liver cancer incidence is
31.25%；It has been observed that DEN is water-soluble and high dose DEN sesame oil group mouse under light microscopic, hence it is evident that the visible knot formed with envelope
Stove is saved, cell arrangement structure disturbance, size, is come in every shape, the obvious thickening of chromatin, kytoplasm enriches, it is seen that pathologic mitosis
Picture, break up visible macronucleus in poor liver cell, show that cell atypia is obvious；There is liver cell change in low dosage DEN sesame oil groups
Property necrosis, cell infiltration, a large amount of vacuole steatosises, and DEN nanometer micro emulsions group then it is clearly visible with envelope formed knot
Stove is saved, cell size, form differ, and karyomorphism is irregular, it is seen that tumor giant cell, nuclear fission picture are obvious.This method is simple
Easy, modeling is short experimental period, can establish preferable liver cancer animal model；After DEN nanosizings, DEN can be strengthened to liver
Damaging action, there is provided the theoretical foundation of primary carcinoma of liver animal model is established in a kind of shorter time.The present invention is by by DEN
The compliance that sesame oil oral administration adds mouse medication is dissolved in, overcomes the individual difference between the water-soluble modeling mouse of DEN；
At 20 weeks, water-soluble group of DEN and oral high dose group mouse naked eyes visual tumors tubercle, but 1 times/week of oral administration group mouse
The death rate is minimum, i.e., long-term low frequency administration can substantially reduce the death rate during modeling.Primary is established using DEN oil solutions
Liver cancer model has the advantage that：1. stability is strong；2. operation letter is convenient；3. animal compliance increases, the death rate reduces.It is based on
The development of nanometer technology at present, the present invention is using the long-term low frequency administrations of nanosizing DEN.Research is found, molten with relatively low-dose DEN oil
Liquid administration control, the damage that the principle for being targetted and being sustained using nanometer formulation makes the DEN of nanosizing to liver strengthen.
Brief description of the drawings
Figure 1A be modeling during at the 0th, 5,10,15,20 week each group mouse weight change schematic diagram；
Each group mouse weight change schematic diagram when Figure 1B is the 20th week during modeling；
Fig. 2 is to lure mouse death rate schematic diagram during cancer in embodiment 1-3；
Each group mouse liver naked eyes take piece into consideration when Fig. 3 is 20 weeks in embodiment 1-3；
Each group mouse liver histopathology takes piece into consideration when Fig. 4 is 20 weeks in embodiment 4.
Embodiment
Technical scheme is described in further detail below in conjunction with specific embodiment.It is it should be understood that following
Content should not be any limitation as to protection scope of the present invention in any way.
For the problems such as stability in above-mentioned conventional liver cancer animal model foundation is poor, time-consuming, the death rate is high, this hair
First bright purpose is the provision of a kind of optimization method for establishing primary liver cancer model of mice.
Second object of the present invention is the provision of the method for long-term low frequency induction primary liver cancer model, improves tradition
The problems such as death rate of chemical induction liver cancer model is high.
Third object of the present invention is the provision of nanosizing DEN (nano-DEN) and is used as a kind of more preferable Primary Hepatic
The theoretical foundation of cancer derivant.
The inventive concept of the present invention is as follows：
Chemical induction type liver cancer model is to study one of best model of liver cancer, and DEN is most widely used one kind so far
Chemical induction type liver cancer model preparation (Molecular pathology of early stage chemically induced
Hepatocaricinogenes.Pathol Int, 2009,59：605-622).And nitrosamine compound is prevalent in
Cereal, milk, cheese, tobacco and wine, bacon, barbecue, ocean fish, canned food and drinking-water in (in food N- nitroso compounds with
The progress China treatment and prevention of tumour magazine of relation between tumor, 2008,15：151-155).DEN is dissolved in water by traditional modeling method
In, give mouse and freely drink, but often experimental period is long, individual difference is big, and (chemically medicine lures death rate height
The progress medical research magazines of hair liver cancer model, 2009,38：12-14).In experimentation, due to the limit of a variety of conditions
System, especially in terms of practice, the restriction in terms of the death rate needs that optimization is more preferable, faster modeling method badly.
In order to which more preferable simulation people may be because of poisonous, harmful chemical in chronic environmental, and long-term use
Hepatotoxic medication causes the hepar damnification even situation of liver cancer, and this experiment set up low dosage DEN sesame oil group and nano-DEN groups.
At 20 weeks, low dosage DEN sesame oil group mouse livers visible cell is downright bad, cell infiltration and fatization, but finds no swollen
Knurl tubercle is formed.And have 4 naked eyes visual tumors tubercles in 16 mouse that nano-DEN groups are all survived, though there are 2 mouse
Naked eyes tumor nodule, but visual tumors tubercle under mirror are had no, liver cancer incidence is 37.5%.Show DEN after nanosizing to liver
It is dirty to produce even more serious damage.Nano-DEN is expected to as establishing the more preferable derivant of primary carcinoma of liver.
In order to realize first purpose, present invention employs following technical measures：
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 3 groups at random, every group of 16 mouse：Normal group,
Conventional free water；Water-soluble group of DEN, gives 16.5mg/kg (i.e. 2.5ml10g by body weight daily-1) the DEN aqueous solution, and often
It supplements deficiency with common filter water；Oral high dose group, presses body weight orally administration 16.5mg/kg (i.e. 4 μ l10g daily-1)
DEN sesame oil solutions (effect of sesame oil is attractant, and mouse is extremely sensitive to the taste of sesame oil, with olive oil, fish oil or other
Oil solution is compared, and mouse most eating sesame oil, the fragrance of sesame oil masks DEN penetrating odor).Research optimization rat primary
The method for building up of liver cancer model, improve the problem of traditional chemical induces the big individual difference of liver cancer model, poor compliance.
In order to realize second purpose, present invention employs following technical measures：
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 4 groups at random, every group of 16 mouse：Normal group,
Conventional free water；7 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN sesame oil it is molten
Liquid (7 times/week)；3 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN sesame oil solutions
(3 times/week)；1 group of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN sesame oil solutions (1
Times/week).The method for building up of research optimization primary liver cancer model of mice, improve the death rate of traditional chemical induction liver cancer model
The problem of high.
In order to realize the 3rd purpose, present invention employs following technical measures：
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 4 groups at random, every group of 16 mouse：Normal group,
Conventional free water；1 group of oral low dosage, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) DEN sesame oil it is molten
Liquid (1 times/week)；Nano-carrier group, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) nano-carrier (1 times/week)；
Nanosizing DEN groups, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) nanosizing DEN (1 times/week).Research optimization
The method for building up of primary liver cancer model of mice, improve the problem of traditional chemical induces the experimental period of liver cancer model long.
The present invention uses the widely used Kunming KM mouse in laboratory, using DEN to lure cancer agent, by oral administration, and 20 weeks
When be successfully established primary liver cancer model of mice, and the defects of overcome DEN water-soluble modelings.Because induction liver cancer is by small
Dosage stimulates liver canceration for a long time, and general experimental period needs 18-30 weeks, and by after DEN nanosizings, DEN pairs can be strengthened
The damaging action of liver, therefore nano-DEN is expected to that primary carcinoma of liver animal model can be established in shorter time.
The primary liver cancer model of mice method for building up of the orally administration DEN sesame oil solutions of embodiment 1 optimization
Animal：Tested SPF levels Kunming KM male mices 48 (being provided by Hubei Province Animal Experimental Study center), body weight
18-22g, free water diet, raising room temperature are maintained at 25 ± 1 DEG C, and the light and shade cycle is 12 hours.
Lure the preparation of cancer agent：
(1) the DEN aqueous solution：347.5 μ l (i.e. 330mg) DEN stostes are taken, are added in 5000ml sterile distilled waters, i.e. 66 μ
g·ml-1The DEN aqueous solution；
(2) high dose DEN sesame oil solutions：0.5ml (i.e. 470mg) DEN stostes are taken to add in 10.894ml sesame oil, i.e.,
41.25mg·ml-1DEN sesame oil solutions.
The DEN sesame oil solutions of different content can be prepared according to which.
1st, the foundation of primary liver cancer model
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 3 groups at random, every group of 16 mouse：Normal group,
Conventional free water；Water-soluble group of DEN, gives 16.5mg/kg (i.e. 2.5ml10g by body weight daily-1) the DEN aqueous solution, and often
It supplements deficiency with common filter water；Oral high dose group, presses body weight orally administration 16.5mg/kg (i.e. 4 μ l10g daily-1)
DEN sesame oil solutions.After continuous modeling 20 weeks, the dislocation of each group residue mouse is put to death, and is opened abdominal cavity, is taken liver.
2nd, mice behavior and liver are substantially seen
The observation mouse state of mind daily, diet, hair change, and weigh weekly, record every group of dead mouse number；
After modeling 20 weeks, the form, color, the macroscopic cancerous node number of quality and liver surface of each group residue mouse liver are observed；Meter
Calculate mouse survival rate and liver cancer incidence, number of mice/each group total mice × 100% of the survival of survival rate=every group；Liver cancer is sent out
Number of mice/each group total mice × 100% of the generation liver cancer of raw rate=every group.
3rd, experimental result
Normal group mouse is vivaciously active, reaction is flexible, fur gloss, and it is withered that different degrees of hair color occurs in remaining each group mouse
Dry, jaundice, tarnish, it is slow in action.Water-soluble group of DEN and high dose DEN sesame oil groups mouse weight are small significantly lower than normal group
Mouse.Each group changes of weight, see Figure 1A and Figure 1B, wherein, NG：Normal group；NVG：Nano-carrier group；DWG：Water-soluble group of DEN；
HDO7G：High dose DEN sesame oil group 7 times a week；HDO3G：High dose DEN sesame oil group 3 times a week；HDO1G：High dose DEN sesame oil
Group 1 times a week；LDO1G：Low dosage DEN sesame oil group 1 times a week；ND1G：Nanometer DEN groups 1 times a week.Compared with normal group, * * *
P<0.001.It can be seen that water-soluble group of DEN and high dose DEN sesame oil groups mouse weight are small significantly lower than normal group
Mouse.The death rate of mouse is detailed as shown in Figure 2, wherein, NG：Normal group；NVG：Nano-carrier group；DWG：Water-soluble group of DEN；HDO7G：
High dose DEN sesame oil group 7 times a week；HDO3G：High dose DEN sesame oil group 3 times a week；HDO1G：High dose DEN sesame oil weekly 1
Secondary group；LDO1G：Low dosage DEN sesame oil group 1 times a week；ND1G：Nanometer DEN groups 1 times a week.Compared with normal group, * * * P<
0.001.During modeling, normal group does not occur dead mouse, does not find the generation of primary carcinoma of liver, sees Fig. 3 (a).At 20 weeks,
The death rate of water-soluble group of mouse of DEN is 75%, and 4 mouse of survival have naked eyes visual tumors tubercle, sees Fig. 3 (c), and liver cancer occurs
Rate is 25%.(7 times/week) group mouse death rates of high dose DEN sesame oil are 75%, and 4 mouse of survival have naked eyes visual tumors
Tubercle, is shown in Fig. 3 (d), and liver cancer incidence is 25%.Each group death mouse gross examination is then shown in that its liver enlargement, color are partially yellow, portion
Divide visible edge to black, have no tumour.
The different frequency of embodiment 2 orally administration DEN sesame oil solutions induction primary liver cancer model of mice compare
Animal：Tested SPF levels Kunming KM male mices 64 (being provided by Hubei Province Animal Experimental Study center), body weight
18-22g, free water diet, raising room temperature are maintained at 25 ± 1 DEG C, and the light and shade cycle is 12 hours.
Lure the preparation of cancer agent：0.5ml (i.e. 470mg) DEN stostes are taken to add in 10.894ml sesame oil, i.e. 41.25mg
ml-1DEN sesame oil solutions.
1st, the foundation of primary liver cancer model
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 4 groups at random, every group of 16 mouse：Normal group,
Conventional free water；7 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN sesame oil it is molten
Liquid (7 times/week)；3 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN sesame oil solutions
(3 times/week)；1 group of oral high dose, presses body weight orally administration 16.5mg/kg (i.e. 4 μ l10g daily-1) DEN sesame oil it is molten
Liquid (1 times/week).After continuous modeling 20 weeks, the dislocation of each group residue mouse is put to death, and is opened abdominal cavity, is taken liver.
2nd, mice behavior and liver are substantially seen
The observation mouse state of mind daily, diet, hair change, and weigh weekly, record every group of dead mouse number；
After modeling 20 weeks, the form, color, the macroscopic cancerous node number of quality and liver surface of each group residue mouse liver are observed；Meter
Calculate mouse survival rate, number of mice/each group total mice × 100% of the survival of survival rate=every group；Liver cancer incidence=every group of hair
Number of mice/each group total mice × 100% of raw liver cancer.
3rd, experimental result
Normal group mouse is vivaciously active, reaction is flexible, fur gloss, and it is withered that different degrees of hair color occurs in remaining each group mouse
Dry, jaundice, tarnish, it is slow in action, and with the increase of administration frequency and dosage, mouse exacerbation of symptoms.High dose DEN
Sesame oil group mouse weight is significantly lower than normal group mouse.Each group changes of weight, is shown in Fig. 1.The death rate of mouse refers to Fig. 2 institutes
Show.During modeling, normal group does not occur dead mouse, does not find the generation of primary carcinoma of liver, sees Fig. 3 (a).It is high at 20 weeks
(7 times/week) group mouse death rates of dosage DEN sesame oil are 75%, and 4 mouse of survival have naked eyes visual tumors tubercle, see Fig. 3
(d), liver cancer incidence is 25%.The death rate of (3 times/week) group mouse of high dose DEN sesame oil is 37.5%, and survive 10 mouse
In have 5 naked eyes visual tumors tubercles, see Fig. 3 (e), though there is 1 mouse to have no naked eyes tumor nodule, visual tumors knot under mirror
Section, liver cancer incidence are 37.5%.The death rate of (1 times/week) group mouse of high dose DEN sesame oil is 25%, and survive 12 mouse
In have 5 naked eyes visual tumors tubercles, see Fig. 3 (f), liver cancer incidence is 31.25%.Each group death mouse gross examination is then shown in
Its liver enlargement, color are partially yellow, and partially visible edge blacks, and has no tumour.
The primary liver cancer model of mice method for building up of the orally administration nanosizing DEN sesame oil solutions of embodiment 3 optimization
Animal：Tested SPF levels Kunming KM male mices 64 (being provided by Hubei Province Animal Experimental Study center), body weight
18-22g, free water diet, raising room temperature are maintained at 25 ± 1 DEG C, and the light and shade cycle is 12 hours.
Lure the preparation of cancer agent：
(1) low dosage DEN sesame oil solutions：0.25ml (i.e. 235mg) DEN stostes are taken to add in 11.144ml sesame oil, i.e.,
20.625mg·ml-1DEN sesame oil solutions；
(2) Nano-DEN and nano-carrier：DEN and egg yolk lecithin are dissolved in sesame oil oil phase is prepared, DEN's
Final concentration is 10.97%V/V, and the final concentration of egg yolk lecithin is 6%W/V.Tween 80 and glycerine are dissolved in ultra-pure water
Aqueous phase is prepared, Tween 80 final concentration is 1.25%V/V, and the final concentration of glycerine is 2.81%V/V.In 60 DEG C of thermostatted waters
Under conditions of bath, oil phase is instilled in aqueous phase, stirs 15min under conditions of 1200r/min, the volume ratio of oil phase and aqueous phase is
1：4.The high shear 2min under conditions of 10000rpm, the emulsified particles that particle diameter is 100nm-200nm is finally given,
High-pressure homogeneous 5 times are to obtain the DEN oil solutions (nano-DEN) of nanosizing under conditions of 1000bar, DEN end in nano-DEN
Concentration is 20.625mgml-1.Nano-carrier oil phase is that egg yolk lecithin is dissolved in sesame oil, and the final concentration of egg yolk lecithin is
6%W/V；Remaining step is identical with preparing for nano-DEN.
1st, the foundation of primary liver cancer model
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 4 groups at random, every group of 16 mouse：Normal group,
Conventional free water；1 group of oral low dosage, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) DEN sesame oil it is molten
Liquid (1 times/week)；Nano-carrier group, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) nano-carrier solution (1 time/
Week)；Nano-DEN groups, by body weight orally administration 8.28mg/kg (i.e. 4 μ l10g-1) nano-DEN solution (1 times/week).Even
After continuous modeling 20 weeks, the dislocation of each group residue mouse is put to death, and is opened abdominal cavity, is taken liver.
2nd, mice behavior and liver are substantially seen
The observation mouse state of mind daily, diet, hair change, and weigh weekly, record every group of dead mouse number；
After modeling 20 weeks, the form, color, the macroscopic cancerous node number of quality and liver surface of each group residue mouse liver are observed；Meter
Calculate mouse survival rate and liver cancer incidence, number of mice/each group total mice × 100% of the survival of survival rate=every group；Liver cancer is sent out
Number of mice/each group total mice × 100% of the generation liver cancer of raw rate=every group.
3rd, experimental result
Normal group mouse is vivaciously active, reaction is flexible, fur gloss, and it is withered that different degrees of hair color occurs in remaining each group mouse
Dry, jaundice, tarnish, it is slow in action.Compared with normal group, remaining each group mouse weight has no significant change.Each group body weight becomes
Change, see Fig. 1.In detail as shown in Figure 2, it can be seen that during modeling, each group mouse small the death rate of mouse does not occur
Mouse is dead.At 20 weeks, in addition to nano-DEN groups, remaining each group mouse does not find that liver surface has the generation of obvious tubercle, sees
Fig. 3 (a, b, g)；There are 4 naked eyes visual tumors tubercles in 16 mouse that nano-DEN groups are all survived, though there are 2 mouse not
See naked eyes tumor nodule, but visual tumors tubercle under mirror, liver cancer incidence is 37.5%, sees Fig. 3 (h).
The 20th week hepatic tissue pathology is seen in the rat primary liver cancer that embodiment 4 is induced based on nanosizing DEN sesame oil solutions
Animal：Tested SPF levels Kunming KM male mices 108 (being provided by Hubei Province Animal Experimental Study center), body weight
18-22g, free water diet, raising room temperature are maintained at 25 ± 1 DEG C, and the light and shade cycle is 12 hours.
Lure the preparation of cancer agent：
(1) the DEN aqueous solution：347.5 μ l (i.e. 330mg) DEN stostes are taken, are added in 5000ml sterile distilled waters, i.e. 66 μ
g·ml-1The DEN aqueous solution；
(2) high dose DEN sesame oil solutions：0.5ml (i.e. 470mg) DEN stostes are taken to add in 10.894ml sesame oil, i.e.,
41.25mg·ml-1DEN sesame oil solutions.
(3) low dosage DEN sesame oil solutions：0.25ml (i.e. 235mg) DEN stostes are taken to add in 11.144ml sesame oil, i.e.,
20.625mg·ml-1DEN sesame oil solutions；
(4) Nano-DEN and nano-carrier：DEN and egg yolk lecithin are dissolved in sesame oil oil phase is prepared, DEN's
Final concentration is 10.97%V/V, and the final concentration of egg yolk lecithin is 6%W/V.Tween 80 and glycerine are dissolved in ultra-pure water
Aqueous phase is prepared, Tween 80 final concentration is 1.25%V/V, and the final concentration of glycerine is 2.81%V/V.In 60 DEG C of thermostatted waters
Under conditions of bath, oil phase is instilled in aqueous phase, stirs 15min under conditions of 1200r/min, the volume ratio of oil phase and aqueous phase is
1：4.The high shear 2min under conditions of 10000rpm, the emulsified particles that particle diameter is 100nm-200nm is finally given,
High-pressure homogeneous 5 times are to obtain the DEN oil solutions (nano-DEN) of nanosizing under conditions of 1000bar, DEN end in nano-DEN
Concentration is 20.625mgml-1.Nano-carrier oil phase is that egg yolk lecithin is dissolved in sesame oil, and the final concentration of egg yolk lecithin is
6%W/V；Remaining step is identical with preparing for nano-DEN.
1st, the foundation of primary liver cancer model
Raised in advance from Kunming KM mouse and adapt to environment after one week, be divided into 8 groups at random, every group of 16 mouse：Normal group,
Conventional free water；Water-soluble group of DEN, gives 16.5mg/kg (i.e. 2.5ml10g by body weight daily-1) the DEN aqueous solution, and often
It supplements deficiency with common filter water；7 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1)
DEN sesame oil solutions (7 times/week)；3 groups of oral high dose, by body weight orally administration 16.5mg/kg (i.e. 4 μ l10g-1) DEN
Sesame oil solution (3 times/week)；1 group of oral high dose, presses body weight orally administration 16.5mg/kg (i.e. 4 μ l10g daily-1)
DEN sesame oil solutions (1 times/week)；1 group of oral low dosage, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) DEN
Sesame oil solution (1 times/week)；Nano-carrier group, by body weight orally administration 8.25mg/kg (i.e. 4 μ l10g-1) nano-carrier it is molten
Liquid (1 times/week)；Nano-DEN groups, by body weight orally administration 8.28mg/kg (i.e. 4 μ l10g-1) nano-DEN solution (1 time/
Week).After continuous modeling 20 weeks, the dislocation of each group residue mouse is put to death, and is opened abdominal cavity, is taken liver.
2nd, mouse liver pathological study
The liver lobus dexter of all experimental mices (including early stage is dead) is cut, 10% paraformaldehyde solution fixes 24h, often
FFPE is advised, 4 μm of continuous paraffin sections are made in slicer；Synchronous roasting piece, then dewaxes, graded ethanol aquation, and haematine-she
Red colouring (hematoxylin and eosin, H＆E) dyes.Gradient alcohol dehydration, dimethylbenzene is transparent, resin mounting；Under light microscopic
Endochylema dye is red, and nuclei dyeing is blueness.
3rd, experimental result
In Fig. 4, a：Normal group；b：Nano-carrier group；c：Water-soluble group of DEN；d：High dose DEN sesame oil group 7 times a week；e：It is high
Dosage DEN sesame oil group 3 times a week；f：High dose DEN sesame oil group 1 times a week；g：Low dosage DEN sesame oil group 1 times a week；h：Nanometer
DEN groups 1 times a week.Scale is 100 μm, and the upper right corner is the enlarged drawing (400 times) of Local map (100 times) shown in black surround.Light microscopic
Lower observation, normal and nano-carrier group mouse liver cell marshalling, core is in the same size, and is evenly distributed, and hepatic cell cords surrounds
Central vein, radially distribute, sinus hepaticus is clear, sees Fig. 4 (a, b).And death early stage murine liver tissue microscopy is then visible acute
Large area swelling of liver cell, necrosis, there is not yet tumour is formed.DEN is water-soluble and high dose DEN sesame oil group mouse, hence it is evident that visible tool
The tubercle stove for having envelope to be formed, cell arrangement structure disturbance, size, comes in every shape, the obvious thickening of chromatin, kytoplasm enriches, can
See pathologic mitosis picture, break up visible macronucleus in poor liver cell, show that cell atypia is obvious, see Fig. 4 (c, d, e,
f).There is degeneration of liver cells necrosis, cell infiltration in low dosage DEN sesame oil groups；Liver rope is disorderly, sinus hepaticus distortion；Liver cell volume
There are circular or oval vacuolization, nucleus to be located at side, see Fig. 4 (g) in increase, kytoplasm.And nano-DEN group mouse, it is bright
The aobvious visible tubercle stove formed with envelope, cell size, form differ, and karyomorphism is irregular, it is seen that tumor giant cell, core
Mitotic figure is obvious, sees Fig. 4 (h).
Claims (1)
1. a kind of method for preparing liver and luring cancer agent, the liver lure the DEN sesame oil solutions that cancer agent is nanosizing, the nanosizing
DEN sesame oil solutions in DEN content be 20.625 mgml-1 -41.25 mg·ml-1；The DEN is diethyl Asia
Nitramine；
It is characterized in that：It the described method comprises the following steps：
1）DEN and egg yolk lecithin are dissolved in sesame oil, oil phase is prepared；The final concentration of the DEN be 10.97%V/V-
21.94%V/V；The final concentration of the egg yolk lecithin is 6%W/V；
2）Tween 80 and glycerine are dissolved in ultra-pure water, aqueous phase is prepared；The final concentration of the Tween 80 is 1.25%
V/V；The final concentration of the glycerine is 2.81%V/V；
3）60oUnder conditions of C waters bath with thermostatic control, by step 1）The oil phase being prepared instills step 2）In the aqueous phase being prepared,
15 min are stirred under conditions of 1200 r/min；The volume ratio of the oil phase and aqueous phase is 1：4；
4）The min of high shear 2 under conditions of 10000 rpm, finally give the emulsified particles that particle diameter is 100 nm-200 nm；
5）High-pressure homogeneous 5 times are to obtain the DEN sesame oil solutions of nanosizing under conditions of 1000 bar.
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| Mouse hepatic neoplasm formation induced by trace level and low frequency exposure to diethylnitrosamine through β-catenin signaling pathway;Yu-Sang Li等;《Toxicology Research》;20151023;第5卷(第1期);第210-223页，特别是摘要，第211页右栏第5-19行 * |
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