CN108815147A - Niclosamidum is treating or preventing the application in melanoma - Google Patents

Abstract

The invention discloses niclosamidums to treat or prevent the application in melanoma, is related to melanoma and treats or prevents drug field.Research discovery niclosamidum of the invention has apparent tumor-inhibiting action to Humanmachine tumour, and niclosamidum can effectively inhibit the proliferation of melanoma；Niclosamidum can inhibit the migration and invasion of melanoma；Niclosamidum can promote the apoptosis of melanoma, it has further been found that the secretion that niclosamidum agent can improve the activity of the TYR enzyme in melanoma cells, promote melanin, prompts niclosamidum agent that can promote the terminal differentiation of melanoma cells.Research prompt niclosamidum of the invention has wide application value in treating or preventing the fields such as melanoma, has the prospect as anti-melanin tumor medicine.

Description

Niclosamidum is treating or preventing the application in melanoma

Technical field

The present invention relates to melanomas to treat or prevent drug field, in particular to niclosamidum in treatment or in advance Application in anti-melanin tumor.

Background technique

Melanoma grade malignancy is high, and transfer is early, and especially lethality is very high, and 80% has been accounted in cutaneum carcinoma.It is extremely early The melanoma of phase can be cured by operation.Since melanoma early symptom is unobvious, when most of Finding case It is insensitive to radiotherapy in middle and advanced stage, chemotherapy is relied primarily on, and it is poor to the sensibility of chemotherapeutics, easy drug resistance is black at present The bottleneck of melanoma treatment.Therefore to the treatment of melanoma need to by developing new drug tumor suppression or to resistance mechanism Research approach come It carries out.But the problems such as new drug research period is long, and risk is big, and biological safety is unknown, limits its development, resistance mechanism study into It postpones slow.

In recent years, drug repositions, i.e., old medicine is newly used, developed the new application of existing drug, receives more and more attention. Drug repositioning refers to the drug for known bio-safety, having grasped pharmacokinetics effect, seeks new pharmacodynamics effect, And drug indication is expanded into Other diseases.Since the existing drug used has been put into bio-safety, medicine for power Learn etc. has accumulated mass data, and obtains the verifying of practical application, and drug repositioning certainly will greatly cut down drug development Expense shortens the development time.

Dacarbazine is that U.S. FDA in 1975 is ratified to have been reported that for the drug for treating Advanced Malignant melanoma earliest Claim its objective efficient only 7%-13%, both just such Dacarbazine was always one of main chemotherapeutics of medical treatment. There are also Temozolomide, dacarbazine, cis-platinum, vincristine etc. for clinically used chemotherapeutics, but clinical effective rate is still bad. Since two thousand and ten, the whole world has listed multiple melanoma therapeutic agents, mainly there is Wei Luofeini, dabrafenib, Trimetinib Deng, wherein Wei Luofeini for BRAF V600E mutation melanoma suffer from clinical effective rate be up to 80%.Both just in this way, prestige Luo Feini can generate serious drug resistance after treating a period of time, this disadvantage limits Wei Luofeini in melanoma therapy field Extensive use.

Therefore for melanoma chemotherapeutic treatment, there are still following challenges：First is that more effectively chemotherapeutics is urgently developed, Second is that the problems such as new drug research period is long, and risk is big, and biological safety is unknown limits its development, resistance mechanism progress is slow Slowly.

In consideration of it, the present invention is specifically proposed.

Summary of the invention

The purpose of the present invention includes but is not limited to provide niclosamidum to treat or prevent answering in the fields such as melanoma With.Using Humanmachine tumour A375 cell strain as cell model, discovery niclosamidum has Humanmachine tumour bright for research of the invention Aobvious tumor-inhibiting action, niclosamidum can effectively inhibit the proliferation of melanoma；Niclosamidum can inhibit the migration of melanoma with Invasion；Niclosamidum can promote the apoptosis of melanoma, it has further been found that niclosamidum agent can improve in melanoma cells TYR enzyme activity, promote the secretion of melanin, prompt niclosamidum agent that can promote the terminal differentiations of melanoma cells.This The research prompt niclosamidum of invention has wide application value in treating or preventing the fields such as melanoma, has as anti- The prospect of melanoma drug.

The invention is realized in this way：

On the one hand, the present invention provides niclosamidums to prepare for treating or preventing the application in tumour medicine, described Tumour is melanoma.

Further, in some embodiments of the invention, effective usage amount of niclosamidum is 4mg/kg or more.

Further, in some embodiments of the invention, effective usage amount of niclosamidum is 4-16mg/kg.

Further, in some embodiments of the invention, effective usage amount of niclosamidum is 8-16mg/kg.

Further, in some embodiments of the invention, effective usage amount of niclosamidum is 16mg/kg.

Results of Animal of the invention shows that the niclosamidum for applying 4mg/kg or more dosage can obviously inhibit The volume of melanoma increases.

As niclosamidum applies the increase of dosage, the effect for inhibiting melanoma to increase is more obvious, and is reaching 16mg/ Under the conditions of the applied amount of kg, melanoma hardly increases.Result of study prompt applies 4mg/kg or more dosage to animal body Niclosamidum be able to suppress the growth of melanoma, it is preferable that the niclosamidum for applying 8mg/kg can significantly more inhibit The growth of melanoma, it is highly preferred that the niclosamidum for applying 16mg/kg can almost inhibit the increasing of melanoma It is long.

The applied amount of drug relative to existing melanoma, using used in niclosamidum treatment melanoma Effective applied amount is smaller, has stronger biological safety.

On the other hand, the present invention provides niclosamidums to prepare for inhibiting tumor cell proliferation, migration or invasion Application in inhibitor, the tumour cell are melanoma cells.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 1 μm of ol/L or more.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 1-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum be 1.82 μm of ol/L with On.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 2-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 4-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 8-16 μm of ol/L.

Cell experiment result of study of the invention shows that the niclosamidum for applying 1 μm of ol/L concentrations above can obviously press down Melanoma cells proliferation, transfer ability and invasive ability processed.As niclosamidum applies the increase of concentration, when concentration reaches 1.82 μm of ol/L are able to suppress the proliferation (IC of half melanoma cells₅₀=1.82 μM), with its inhibition of the increase of concentration Effect is more obvious.

On the other hand, the present invention provides niclosamidums to prepare answering in the promotor for promoting apoptosis of tumor With the tumour cell is melanoma cells.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 1 μm of ol/L or more.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 1-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 2-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 4-16 μm of ol/L.

Further, in some embodiments of the invention, effective application concentration of niclosamidum is 8-16 μm of ol/L.

Cell experiment result of study of the invention shows that the niclosamidum for applying 1 μm of ol/L concentrations above can obviously promote Into the apoptosis of melanoma cells.As niclosamidum applies the increase of concentration, the effect for promoting melanoma cells tune to die It is more obvious.

On the other hand, the present invention provides niclosamidums in preparing the promotor for promoting tumour cell terminal differentiation Application, the tumour cell be melanoma cells.

On the other hand, the present invention provides niclosamidums to prepare for improving the active tune of TYR enzyme in tumour cell The application in agent is saved, the tumour cell is melanoma cells.

On the other hand, the present invention provides niclosamidums to prepare the tune for promoting melanoma cells secretion melanin Save the application in agent.

On the other hand, the present invention provides a kind of active methods of TYR enzyme in raising melanoma cells comprising： Niclosamidum is contacted with melanoma cells.

On the other hand, the present invention provides a kind of methods of promotion melanoma cells secretion melanin comprising：By chlorine Nitre willow amine is contacted with melanoma cells.

During detecting inhibiting effect of the niclosamidum to melanoma, inventor be have been surprisingly found that：Chlorine nitre Willow amine can also promote the melanin production of melanoma, and through the processed cell of niclosamidum, drug concentration is bigger, melanin point Secrete it is more, by the detection to TYR enzyme, experimental result show TYR enzyme activity have the tendency that increasing.This result of study It prompts that the TYR enzyme activity in melanoma cells can be improved to melanoma cells application niclosamidum, promotes melanin point It secretes, and promotes the terminal differentiation of melanoma cells, cellular proliferative potential weakens with the enhancing of differentiation.

On the other hand, the present invention provides a kind of for treating or preventing the drug of melanoma, contains as activity The niclosamidum of ingredient and pharmaceutically acceptable auxiliary material.

It is in short, present invention research is using human melanoma cell strain as cell model, horizontal in vitro, it was demonstrated that niclosamidum Under physiological dose, there is apparent tumor-inhibiting action to Humanmachine tumour, there are various aspects to human melanoma cell biological behaviour Influence.It is tested by Trypan Blue cell count, CCK-8 method, violet staining etc., discovery niclosamidum can effectively inhibit The proliferation of melanoma；By cell scratch experiment and Transwell method, show that niclosamidum can inhibit moving for melanoma It moves and invades；Using Hoechst dyeing and flow cytometry etc., discovery niclosamidum can promote the apoptosis of melanoma.It is detecting Discovery melanin production increases with niclosamidum dosage and is increased in the process, then carries out TYR enzyme Activity determination, as a result mentions Show that it may promote the terminal differentiation of human melanoma cell, while cellular proliferative potential weakens with the enhancing of differentiation.

These results of study show that niclosamidum has wide application valence in treating or preventing the fields such as melanoma Value, for example, can be used for preparation treat or prevent melanoma drug, such as be used to prepare inhibit tumor cell proliferation, The inhibitor of migration or invasion, is used to prepare promotion tumour cell terminal at the promotor for being used to prepare promotion apoptosis of tumor The promotor of differentiation is used to prepare and improves the active regulator of TYR enzyme in tumour cell, is used to prepare promotion melanoma Cell is secreted in the fields such as the regulator of melanin.

Detailed description of the invention

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is the optical microphotograph sem observation image of A375 cell under niclosamidum effect in the embodiment of the present invention；

Fig. 2 is that the trypan blue living cells count in the embodiment of the present invention counts the survival rate that niclosamidum acts on lower A375 (* indicates the statistical difference between control group, * P<0.05, * * P<0.01, * * * P<0.001)；

Fig. 3 be the embodiment of the present invention in CCK-8 detect niclosamidum to the proliferation of A375 influence (* indicate with Statistical difference between control group, * P<0.05, * * P<0.01, * * * P<0.001)；

Fig. 4 is that the crystal violet staining assay in the embodiment of the present invention tests influence that niclosamidum is proliferated A375 (in figure：A： Violet staining；B：OD value, * indicate the statistical difference between control group, * P<0.05, * * P<0.01, * * * P< 0.001)；

Fig. 5 is that the cell scratch experiment in the embodiment of the present invention detects influence that niclosamidum migrates A375 (in figure：A: Optical microphotograph sem observation image；B:Scratch width accounts for the percentage of control group, * P<0.05)；

Fig. 6 shows for the Transwell Faxian in the embodiment of the present invention wears theca cell (in figure by violet staining：A： Optical microphotograph is schemed under the microscope, B：Theca cell number is worn, * indicates the statistical difference between control group, * P<0.05)；

Fig. 7 be the embodiment of the present invention in Transwell method after acetic acid elutes crystal violet OD value (* indicate with Statistical difference between control group, * P<0.05, * * P<0.01)；

Fig. 8 is A375 fluorescent image after the niclosamidum processing in the embodiment of the present invention (in figure：A:Fluorescence microscope Figure, B：Special-shaped core ratio, * indicate the statistical difference between control group, * P<0.05, * * P<0.01, * * * P<0.001)；

Fig. 9 is the Apoptosis assay result using Flow Cytometry in the embodiment of the present invention；

Figure 10 is the cell cycle analysis result using Flow Cytometry in the embodiment of the present invention；

Figure 11 is the influence result (A that the niclosamidum in the embodiment of the present invention generates A375 cell melanin:Melanin It compares, B:TYR enzyme activity OD value, * indicate the statistical difference between control group, * P<0.05, * * P<0.01, * * * P< 0.001)；

Figure 12 is that the niclosamidum in the embodiment of the present invention inhibits melanoma cells Xenografts in nude mice one-tenth knurl ability (in figure：A：Different time points small animal living body image B：Small animal living body imaging should be read, and * is indicated and control group Between statistical difference, * P<0.05, * * P<0.01, * * * P<0.001)；

Figure 13 is that the niclosamidum in the embodiment of the present invention inhibits melanoma cells nude mice by subcutaneous to transplant one-tenth knurl ability Tumor size difference.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Feature and performance of the invention are described in further detail with reference to embodiments.

Embodiment 1

Niclosamidum has cytotoxicity to melanoma cells A375, can inhibit its proliferation

1.1 Trypan Blue living cells counts

To by various concentration niclosamidum (0,1,2,4,8,16 μm of ol/L) handle A375 cell strain respectively 24/ 48/72 hour three time point carried out optical microphotograph discovery increasing with concentration under the microscope, the cell number of various time points It is reduced significantly, and in 16 μM of concentration groups of 72h, cell mortality (see Fig. 1).By Trypan Blue and live thin Born of the same parents' counting method detects, consistent with the result of microscopic observation, and A375 cell is compared with control group conspicuousness after finding drug-treated It reduces, and drug concentration is higher, the remaining fewer (P of viable count<0.05, see Fig. 2).

1.2CCK-8 method

Using CCK-8 method, the A375 handled the niclosamidum (0,1,2,4,8,16 μm of ol/L) by various concentration is thin Born of the same parents' strain was detected at 24/48/72 hour, the results show that compared with the control group, A375 cell strain CCK-8 is read after drug-treated Number significantly reduces, and drug concentration is higher, and CCK-8 reads lower (P<0.05, see Fig. 3).Pass through software CompuSyn points of analysis CCK-8 data are analysed, inhibiting rate curve is fitted, calculate the IC that niclosamidum inhibits A375 cell₅₀=1.82 μM.

1.3 crystal violet staining assay

To the A375 cell strain of the niclosamidum (0,1,2,4,8,16 μm of ol/L) of various concentration processing in 24/48/72h tri- A time point carries out violet staining, and the cell after dyeing is used spectrophotometer after the elution of 10% acetic acid again after scan image Reading.The results show that compared with the control group, the dyeing of drug-treated group obviously shoals, spectrophotometer measures the significant decrease of OD value (P<0.05, see Fig. 4).

Embodiment 2

Niclosamidum can inhibit the transfer ability of melanoma cells A375

2.1 cell scratch experiments

Using cell scratch experiment, the transfer ability of A375 cell strain is detected, is carried out using ImageJ 1.48v Image analysis.The results show that the scratch closing speed of niclosamidum processing group slows down (P compared with control group conspicuousness<0.01), especially After drug-treated 36 hours, control group scratch is almost closed completely, and drug-treated group is still with the presence of obvious scratch (see figure 5)。

2.2 Transwell methods

Using Transwell method, the invasive ability of A375 cell strain is detected, after violet staining, is used ImageJ 1.48v carries out image analysis.The results show that the A375 of various concentration niclosamidum (0,2,4,8 μm of ol/L) processing is thin After the 4/8/12h of born of the same parents, the film ability of wearing of the cell of drug-treated group significantly reduces (P than control group<0.01), drug concentration is bigger Cell-penetrating quantity reduce (see Fig. 6).

Cell after violet staining is read with spectrophotometer again after the elution of 10% acetic acid.The results show that with right It is compared according to group, the dyeing of drug-treated group obviously shoals, and spectrophotometer, which measures OD value, significantly reduces (P<0.01) it, can accurately reflect Microscopic observation result (see Fig. 7).

Embodiment 3

3.1 Hoechst dyeing

It is dyed using Hoechst, detects influence of the niclosamidum to A375 cell strain apoptosis.A375 cell is different dense The niclosamidum (0,1,2,4,8,16 μm of ol/L) of degree is handled 4/8/12 hour, is collected cell respectively and is carried out Hoechst dyeing, claps According to progress image analysis.

The results show that compared with the control group, what niclosamidum was observed under high power field of view after handling cell 8 hours withers It dies cell number conspicuousness to increase, statistically significant (P<0.05, see Fig. 8), and drug concentration is higher, the apoptotic cell observed It is more.

3.2 Flow Cytometry Apoptosis assays

Apoptosis assay is carried out using flow cytometer, niclosamidum is demonstrated again and promotes A375 cell strain apoptosis Ability.The niclosamidum (0,2,4,8 μm of ol/L) of A375 cell various concentration is handled 4/8/12 hour, cell is collected, presses Kit step is dyed with Annexin V-FITC/PI, carries out the detection of flow cytometer Apoptosis.As a result it is contaminated with Hoechst Color is consistent, and after niclosamidum is handled 8 hours, the early apoptosis of cells of 8 μM of concentration groups has conspicuousness increase compared with the control group (P<0.05, see Fig. 9).

3.3 flow cytometry cell cycle analysis

Cell cycle analysis, shadow of the detection niclosamidum to the A375 cell strain cell cycle are carried out using flow cytometer It rings.It uses the niclosamidum (0,2,4,8 μm of ol/L) of various concentration to handle respectively 4/8/12 hour in A375 cell, collects cell, Flow cytometer carries out cell cycle analysis after PI dyeing.The results show that compared with the control group, it is small through niclosamidum processing 8 Shi Hou, proliferation period cell (S phase, G2 phase and M phase) ratio compared with the control group without marked difference, show that niclosamidum may be to black The melanoma cell cycle is without influence (see Figure 10).

Embodiment 4

Niclosamidum can promote melanoma cells A375 cell melanin to generate

During detecting inhibiting effect of the niclosamidum to melanoma, discovery niclosamidum can also promote melanin The melanin production of tumor, through the processed cell of niclosamidum, drug concentration is bigger, and melanin secretion is more, by network ammonia The detection of sour enzyme, experimental result show that TYR enzyme activity has the tendency that increasing, demonstrate this experimental phenomena (P<0.05, see Figure 11).

Embodiment 5

Niclosamidum can inhibit Xenografts in nude mice one-tenth knurl ability

Using 44 weeks big Female nude mices, it is divided into drug-treated group (each 41 of 4mg/Kg, 8mg/Kg, 16mg/Kg) Do not add drug control group (1), the A375-Fluc cell marked through luciferase is inoculated with respectively at nude mice dorsal sc 2x10⁶It is a.Each group nude mice ordinary circumstance is good after inoculation, and freely, weight is slowly increased diet activity, during which carries out chlorine nitre daily Willow amine intraperitoneal administration is primary, every 2-3 days progress small animal living body imaging (IMAGING) it is primary.It is tagged on A375-Fluc cell Luciferase Catalysis experiments when inject the intracorporal luciferase substrate of nude mice, generate fluorescence, and fluorescence intensity and A375-Fluc The quantity of cell is directly proportional.Small animal living body imaging reflects Xenografts in nude mice tumor formation feelings by fluorescence intensity in real time Condition.All previous living imaging result, which summarizes, sees Figure 12.

It can be seen that：

1. detecting that fluorescence intensity gradually increases as nude mice feeding time extends after inoculation, 5 interior increase are delayed after inoculation Slowly, it then increases sharply after 9 days, fluorescence intensity increase is consistent with Xenografts in nude mice knurl growth pattern, shows animal model It constructs successfully；

2. the fluorescence intensity conspicuousness of niclosamidum processing group is lower than without drug-treated group, there is statistical difference (see figure 12-B, P<0.05), illustrate that niclosamidum can inhibit melanoma cells in Xenografts in nude mice one-tenth knurl ability；

3. visible significant difference (* * P between 3 kinds of various concentration niclosamidum processing groups<0.01, * * * P<0.001, Day13), then the concentration for prompting experiment in vivo niclosamidum is useful effect concentration；

4. whole nude mices survive to experiment and terminate, inoculating cell puts to death nude mice after 2 weeks, collects subcutaneous transplantation tumor, and measurement is each The size of knurl calculates volume.It can be seen that the subcutaneous transplantation tumor knurl conspicuousness of niclosamidum processing group is less than control group, Statistically significant (P<0.05, see Figure 13).

The above experiment is horizontal in vitro, it was demonstrated that niclosamidum is in life using Humanmachine tumour A375 cell strain as cell model It manages under dosage, has apparent tumor-inhibiting action to Humanmachine tumour, have various influences to the cell behaviors of A375.It is logical The experiment such as Trypan Blue cell count, CCK-8 method, violet staining is crossed, discovery niclosamidum can effectively inhibit melanoma Proliferation；By cell scratch experiment and Transwell method, show that niclosamidum can inhibit the migration of melanoma and invade It attacks；Using Hoechst dyeing and flow cytometry etc., discovery niclosamidum can promote the apoptosis of melanoma.In the detection process It was found that melanin production increases with niclosamidum dosage and increased, TYR enzyme Activity determination is then carried out, as a result prompts it can It can promote the terminal differentiation of A375, while cellular proliferative potential weakens with the enhancing of differentiation.

After carrying out cytologic experiment, prison in real time is then then imaged using the small animal living body using bioluminescence technique Survey the influence of the growing state and niclosamidum of tumour cell to it.Firefly luciferase will successfully imported 375 cell of (Firefly luciferase) Gene A, and it is transplanted to nude mice by subcutaneous.It can be seen from living imaging for several times naked Mouse subcutaneous transplantation tumor fluorescence intensity and knurl are grown simultaneously, and the fluorescence intensity conspicuousness of niclosamidum processing group is lower than without drug Processing group；Put to death nude mice make a collection of specimens it has also been found that niclosamidum processing group subcutaneous transplantation tumor knurl conspicuousness be less than control group, This all shows that niclosamidum can inhibit melanoma cells in Xenografts in nude mice one-tenth knurl ability.

Related experimental methods involved in above embodiments are with reference to as follows：

1.1 cell recovery

1. taking out cell strain from -196 DEG C of liquid nitrogen pipes, it is immediately placed in heat in 37 DEG C of water-baths being ready for and thaws, Slight concussion accelerates to thaw, 1min.

2. being transferred in centrifuge tube after cell is blown and beaten, 1000rpm centrifugation 3-5mi after suitable culture medium is added.

3. abandoning supernatant, appropriate culture medium (2ml) is added, after sufficiently piping and druming mixes, is inoculated in two culture dishes, slightly shakes Shaking culture dish makes it be evenly distributed.

4. after microscopic observation, it is put into cell incubator and cultivates, 37 DEG C, 5%CO₂。

1.2 cell culture

1. the good cell suspension of growth conditions is distributed into culture dish, and in culture dish subscript clear-cells title, training The information such as nutrient solution, treatment conditions, experimenter, experiment date.

2. culture dish is put into 37 DEG C, 5%CO₂Culture in constant incubator.

3. culture changes the time of liquid depending on situations such as cell density, health status.Generally replaced after 24-36 hours Culture medium continues to cultivate.

The passage of 1.3 cells

1. microscopic observation cell state, attached cell accounts for about 80% of the visual field or more, and dead cell is slightly more.

2. outwelling old culture medium in culture dish, remaining culture medium is cleaned with 2mlPBS, abandons it.

3. adding 1ml trypsin digestion and cell, culture dish is gently shaken, digests 1-3min, ground-glass-like to appear changes, under mirror Observation, it is seen that cell pseudopodium is withdrawn.

4. appropriate culture medium (2ml), which is added, terminates digestion, attached cell is blown and beaten repeatedly, make its completely de- wall and is dispersed, and is blown The process of beating avoids the occurrence of bubble and damaging cells.

5. the cell point that piping and druming is finished passes in the culture dish for having added good culture medium, mixing is rocked, is placed in incubator Culture.

1.4 cell cryopreservation

1. exponential phase of growth, the good cell of health status will be in, by the operation that cell passes on, vitellophag, preparation Cell suspension is placed in centrifuge tube.

2. a small amount of cell suspension (about 100 μ l) is taken to count cell concentration.

3. 1000g is centrifuged 3-5min by centrifuge tube trim.Supernatant is removed, appropriate cells frozen storing liquid is added, keeps cell dense Degree is 5 × 10⁶/ ml is sufficiently blown and beaten, and is uniformly mixed, and be sub-packed in freezen protective pipe, about 1.5ml/ pipe.Freezing tube wall And pipe covers clear marking freeze-stored cell title, culture solution, experimenter, freezes the information such as date.

4. cryopreservation methods：

1. conventional method：Cryopreservation tube is placed in 4 DEG C 30min → -20 DEG C 1h → -80 DEG C of 16~18h (or staying overnight) → liquid nitrogen container Stored for extended periods.Wherein -20 DEG C of no more than 1h, prevent ice crystal excessive, lead to cell mortality.

2. program cools down：The speed for being -1~-3 DEG C/min by the program setting of constant speed cooler, cell cryopreservation tube is put Enter wherein, -120 DEG C (- 80 DEG C or less) are dropped to by room temperature, are then placed on liquid nitrogen container stored for extended periods.

1.5 plating cells

1. taking the good culture dish of growth conditions, 80% or more coverage rate.

2. outwelling old culture medium, 2mlPBS buffer solution for cleaning remaining medium is added, abandons it.

3. 1ml trypsin digestion and cell is added, sufficiently after digestion, 2ml culture medium is added and terminates digestion, and blows and beats mixed It is even.

4. in centrifuge tube, constant volume mixes i.e. 1ml in 25ml, piping and druming for example, 24 orifice plates of paving, take 1/3 cell suspension.

5. liquid-transfering gun takes 900 holes μ l/ (24 orifice plate), 1800 holes μ l/ (12 orifice plate), cross rocks orifice plate, causes cell point Cloth is uniform, is placed in incubator cultivates under the microscope.

1.6 gradient medicine ordinances

1. niclosamidum storing liquid configures：Niclosamidum tablet is ground in mortar, is caused so that it becomes powder, takes 32.68mg (32.7mg) is dissolved in 20ml DMSO, is made into the storing liquid of 5mM, saves in -4 DEG C of refrigerators.

2. the equal specific concentrations administration that cell in the orifice plate (about 4-6h) after adherent 80%, carries out niclosamidum：0,1μM,2μ M、4μM、8μM、16μM。

For example, 24 orifice plates set 2 multiple holes, the storing liquid of 12.8 μ l is taken, 400 μ l are settled in EP pipe, 100 μ are taken after mixing The hole l/ (2 multiple holes) is left 200 μ l and the dilution mixing of 200 μ l culture mediums is added, takes 100 holes μ l/ (2 multiple holes), so analogize.Finally Obtain gradient concentration.

1.7 trypan blue viable counts and violet staining

1. observation for 24 hours, 48h and 72h administration after cell state, computer software record.

2.1-A Trypan Blue：

1. collecting each hole supernatant (about 1000 μ l) in EP pipe；

2. using PBS buffer solution (200 μ l) cleaning orifice, collect as in corresponding EP pipe；

3. digesting attached cell using trypsase (300 μ l), 1min is collected as in corresponding EP pipe；

4. being centrifuged 1000rpm, 1min, supernatant is abandoned；

5. 250 μ l cell diluents are added, piping and druming, which is resuspended, to be mixed；

6. taking 50 μ l cell suspensions, 50 μ l trypan blue dye liquors are added, piping and druming mixes, and dyes 3-5min, and cell counting board is read Number.

3.1-B violet staining：

1. drawing orifice plate supernatant, it is abandoned；

2. PBS buffer solution (200 μ l) is added to clean 2 times；

3. 4% paraformaldehyde is added, fixed cell 10min draws paraformaldehyde, abandons it；

4. PBS buffer solution (200 μ l) is added to clean 2 times；

5. crystal violet dye liquor (200 μ l), which is added, dyes 15-20min, draws, abandon it；

6. PBS buffer solution (200 μ l) is added to clean 2 times；

7. acetic acid (400 μ l), which is added, dissolves crystal violet, take in 100 μ l and 96 orifice plates, microplate reader reading.

1.8CCK-8 method

1. taking the good culture dish of growth conditions, 80% or more coverage rate abandons old culture medium, and PBS cleans remaining medium, Trypsase (1ml) digestion, adds culture medium (2ml) to terminate digestion, and piping and druming mixes.

2. taking 1/3, i.e. 1ml suspension, constant volume is learnt in 10ml, cell count plate technique：Cell density is 1.25x10^5 A/ml.

3. cell dilutes：9.6ml cell suspension is taken, constant volume is diluted to 10^5/ml in 12ml.

4. cell 96 orifice plates of paving, 100 holes μ l/, i.e. 10^4/hole.The outmost turns of 96 orifice plates add 100 μ l PBS to keep wet Profit.

5. (4-6h) dosing after cell is adherent completely, 10 holes μ l/.

6. control wells are arranged：100 μ l culture medium+20 μ lCCK-8 ,+20 μ l CCK- of+10 μ l niclosamidum of 90 μ l culture medium 8。

7.CCK-8 dyeing, CCK-8,20 holes μ l/ is added in 23h, 47h and 71h after dosing, i.e., 1h in advance adds CCK-8, trains Microplate reader is read after supporting case culture 1h.

1.9 cell scratch experiments

1. taking a dish covered with.Coverage rate is in 80-90%, conventional digestion piping and druming.

2. go 2/3, i.e. 2ml, be settled to 12ml with culture medium, piping and druming mixes, guarantee cell density 1-5x10^5/ ml。

In 3.12 orifice plates, every hole adds 900 μ l cell suspensions, if 2 multiple holes, are placed in incubator and cultivate.

4. after cultivating 4-6h, carrying out scratch after under the microscope, determining that cell is adherent.

5. scratch:It is marked on orifice plate, guarantees to streak when scratch in hole center, be compared directly using liquid transfer gun head Ruler, perpendicular to orifice plate, at the uniform velocity scratch.It is washed cell 2 times using PBS, removes the cell under being drawn, 900 μ l culture mediums are added, with ladder Spend the drug (0,1,2,4,8,16uM) of concentration.

6. setting time point observation (after administration immediately, 12h, for 24 hours, 36h), photograph to record under mirror.

1.10 Transwell method

1), migration experiment

1. bed board：A culture dish covered with is taken, coverage rate 90% or so abandons old culture medium, and PBS cleans remaining medium, Pancreatin (1ml) digestion completely, adds the culture medium (3ml) of serum-free to terminate digestion, and piping and druming mixes.

2. taking 3/4, i.e. 3ml suspension, guarantee that cell suspension cell density in 2.5x10^6/ml, is added on cell compartments Room, 200 holes μ l/, i.e. 5x10^5/hole.

3. room adds the culture medium containing 10%FBS, 600 holes μ l/ under cell compartments.

4. dosing：The storing liquid for taking 3.84 μ l is settled to 120 μ l in EP pipe, and 20 holes μ l/ (3 multiple holes) are taken after mixing, remains Lower 60 μ l are added the dilution of 60 μ l culture mediums and mix, and take 20 holes μ l/ (3 multiple holes), so analogize.

5. being cultivated in incubator after mixing.

6. observing the cell of 4/8/12h after dosing：

1. removing cell indoor culture solution up and down, washed 3 times, 2min/ times in PBS；

2. fixing 20min in 4% paraformaldehyde (lower 600 μ l of room)；

3. being washed 3 times with PBS, 2min/ times, adds 600 hole μ l/ of violet staining liquid, contaminate 20min；

4. being washed 2 times with PBS, and the indoor cell being colored, PBS on cell are wiped with cotton swab and is washed 2 times, 2min/ times；

5. under the microscope, random 100 times of 3 visuals field carry out cell count or 200 times of 5 visual field cell counts；

6. cell is placed in 24 orifice plates, 200 μ l acetic acid of lower interior addition is placed on shaking table and shakes 3min, causes to be colored Part is all dissolved in acetic acid, in microplate reader observed reading.

2), the cell Transwell reuses

1, for carrying out PBS cleaning by the cell compartments that acetic acid dissolves away violet staining, washes 3 times, 2min/ times, upper chamber adds 400 μ l PBS, lower room add 600 μ l PBS.

2 removal PBS, by cell compartments film bubble in trypsase, i.e., upper chamber adds 400 μ l, and lower room adds 600 μ l, digestion 30min-2h。

3 removal trypsase, are washed 3 times, 2min/ times with PBS.

4 impregnate in PBS, are placed in sterilization under ultraviolet lamp, time >=2h.

3), Transwell Matrigel

1 prepares Mareigel glue, thaws (dispensed, 160 μ l/ pipe) overnight, by the culture medium of glue and serum-free with 1：8 Dilution proportion mix, i.e. 160 μ l glue be added 1280 μ l serum free mediums, ice bath operation.

2 are put in the cell Transwell in 24 orifice plates, and 100 μ l glue are added in each cell compartments upper chamber, in 37 DEG C, 5% 4h is incubated in CO2 incubator.

3 inoculating cells：A culture dish covered with is taken, coverage rate 90% or so abandons old culture medium, PBS cleaning residual culture Base, pancreatin (1ml) digestion completely, add the culture medium (3ml) of serum-free to terminate digestion, and piping and druming mixes.3/4, i.e. 3ml suspension are taken, Guarantee cell suspension cell density in 2.5x10^6/ml or so, is added cell compartments upper chamber, 200 holes μ l/, i.e. 5x10^5/ Hole.

4. room adds the culture medium containing 10%FBS, 600 holes μ l/ under cell compartments

5 dosings：The storing liquid for taking 3.84 μ l is settled to 120 μ l in EP pipe, and 20 holes μ l/ (3 multiple holes) are taken after mixing, remains Lower 60 μ l are added the dilution of 60 μ l culture mediums and mix, and take 20 holes μ l/ (3 multiple holes), so analogize.

It is cultivated in incubator after 6 mixings.

The cell of 4/8/12h, carries out violet staining after 7 observation dosings.

1.11 Hoechst is dyed

1. cell climbing sheet is put into 12 orifice plates, 1/hole, conventional digestion cell, every 900 μ l cell suspension of hole, guarantee dense Degree reaches 1-5x10^5/ml.

2. cultivating the adherent rear cell dosing (0,1,2,4,8,16uM) of 4-6h.

3. sampling observation (4,6,8,10h)：

1. removing culture medium, PBS is washed 2 times, 2min/ times；

2. 4% paraformaldehyde fixes cell, 15min；

3. removing paraformaldehyde, PBS is washed 3 times, 2min/ times；

4. Hoechst is dyed, every hole adds 500 μ l dyeing liquors, 30min；

5. removing dyeing liquor, PBS is washed 3 times, 2min/ times；

7. carrying out cell climbing sheet patch using anti-fluorescence quenching, it is placed in wet box, is saved in 4 DEG C；

6. fluorescence microscopy, photographs to record under the microscope.

1.12 cell cycles and apoptosis

1. spreading 36 orifice plates, conventional digestion cell, every hole 2ml cell suspension guarantees that concentration reaches 5x10^5/ml.

2. cultivating the adherent rear cell dosing (0,1,2,4,8,16uM) of 4-6h.

3. sampling (4/8/12h)：

1. collecting cell：Supernatant cell (1ml), attached cell is digested after being washed with 1ml PBS with 500 μ l pancreatin, then is used After 500u lPBS is washed, all it is collected into centrifuge tube；

2. being centrifuged, 1000g, 3min, supernatant is abandoned；

3. 1ml PBS is added to be resuspended；

4. being centrifuged, 1000g, 3mi, supernatant is abandoned；

5. 70% ethyl alcohol of 1ml is added to fix, >=2h (12-24h is more preferably)；

6. being centrifuged, 1000g, 3min, supernatant is abandoned；

7. 1ml PBS, which is added, is resuspended cell；

8. being centrifuged, 1000g, 3min, supernatant is abandoned；

9. plus 500 μ l/ of dyeing liquor pipe, 37 DEG C of warm bath 30min can be saved in 4 DEG C, be observed in in-flow for 24 hours, streaming observation Fluorescence 488nm, red fluorescence.

The preparation of dyeing liquor：Buffer 6ml：300 μ l of propidium iodide：RNaseA120 μ l, final 6.42ml be (12 samples It prepares).

1.13 AnnexinV-FITC/PI cell apoptosis assay

1. spreading 26 orifice plates, conventional digestion cell, every hole 2ml cell suspension guarantees that concentration reaches 5x10^5/ml.

2. cultivating the adherent rear cell dosing (0,2,4,8uM) of 4-6h.

3. sampling (4/8/12h)

1. collecting cell：Supernatant cell (1ml), attached cell are digested after being washed with 1ml PBS with 500 μ l pancreatin, then with 500 After μ l PBS is washed, collection is dispensed into 4 centrifuge tubes；

2. 2000rpm, centrifugation, 5min；

3. abandoning supernatant, it is resuspended with 1ml PBS, centrifugation, 2000rpm, 5min；

4. abandoning supernatant, it is resuspended with 1ml PBS, centrifugation, 2000rpm, 5min；

5. abandoning supernatant, add the BindBuffer of 650 μ l that cell is resuspended；

6. respectively taking 150 μ l cell suspensions in 4 centrifuge tubes, it is dispensed into 3 EP pipes after mixing, makees blank control (blank, the mono- dye of PI, the mono- dye of AnnerxinV)；

7. after 5 μ l AnnexinV-FITC mixing are added in 500 μ l of residue, 5 μ lPI being added and mix；

8. room temperature is protected from light 5min；

9. carrying out the detection (AnnexinV-FITC/FL1, PI/FL2) of flow cytometer in 1h.

1.14 L-DOPA measure TYR enzyme

1. spreading 3 12 orifice plates, conventional digestion cell, every hole 2ml cell suspension guarantees that concentration reaches 5x10^5/ml.

2. cultivating the adherent rear cell dosing (0,1,2,4,8,16uM) of 4-6h.

3. sampling (24/48/72h)：

1. collecting cell：Supernatant cell (1ml), attached cell are digested after being washed with 1mlPBS with 200 μ l pancreatin, then with 300 μ After l PBS is washed, collection is dispensed into 6 EP；

2. being centrifuged, 1000g, 3min；

3. abandoning supernatant；Add 200 μ L of 1%TritonX-100 solution, sets 30min in -80 DEG C of refrigerators rapidly；Melt at room temperature It is completely severed cell；

4. 10000g is centrifuged 10min；Take 50 μ L supernatants into a 96 new orifice plates；

5. every hole adds 2mg/mLL-DOPA solution 10 μ L, 37 DEG C of reaction 2h；Naked eyes visual color change；

6. 490nm wavelength measures each hole absorbance value on enzyme-linked immunosorbent assay instrument.

1.15 living imagings study niclosamidum to the effect in body melanoma

(1) raising of nude mice

Using 16 4 weeks big female athymic nude mices, it is divided into four groups of groups：Control group (0mg/Kg), low-level chlorinated nitre Willow amine group (4mg/Kg), middle concentration niclosamidum group (8mg/Kg), high concentration niclosamidum group (16mg/Kg), weight 14- 16g is provided by Beijing Vital River Experimental Animals Technology Co., Ltd..Rearing conditions are specific pathogen free environment (SPF grades of rings Border) in：Room temperature is kept for 23~29 DEG C of constant temperature；Constant relative humidity 40~70%；Shroud, drink water used in raising nude mice, Feed etc. is handled by high pressure sterilization；20~50Pa of pressure difference in space air.

(2) cell is inoculated

In SPF grades of environment, using routine culture cell transplantation methods, collect good in logarithmic growth phase, health status A375-Fluc cell, inoculate to every nude mice two sides dorsal sc, every 100 μ l (4 × 106 of place's inoculating cell suspension It is a).Situations such as spirit of routine observation nude mice, diet, defecation, activity, and record the time of transplantable tumor appearance, growing state.

(3) drug is prepared

Niclosamidum is dissolved in 100% ethyl alcohol, the drug that compound concentration is 10mg/ml saves liquid, and working solution is with physiology salt Water dilution, while as a control group with physiological saline, the filter with 0.22 μm is distinguished filtration sterilization.Each dosage root It is scaled according to nude mice weight：Low concentration niclosamidum group 4mg/Kg, middle concentration niclosamidum group 8mg/Kg high concentration niclosamidum Group 16mg/Kg, control group 0mg/Kg (physiological saline).200 μ l/ mouse should be noted that piping and druming mixes medicine because it is also easy to produce precipitating Object.

(4) gastric infusion

From inoculating second day, stomach-filling is carried out according to dosage (0mg/Kg, 4mg/Kg, 8mg/Kg, 16mg/Kg) daily Niclosamidum is injected, is administered ten times altogether.

(5) small animal living body is imaged

According to nude mice health status and subcutaneous nodule growing state, per every about progress small animal living body imaging inspection in 2-3 days It surveys.D fluorescein (D-Luciferin) 10mg is first taken, is dissolved in the sterile PBS liquid of 100 μ l, oscillation mixes.By nude mice with different fluorine After alkane gas anesthesia, intraperitoneal injection D fluorescein.Nude mice activity 10 minutes is stimulated, to promote the absorption of D fluorescein.10 minutes Afterwards, by nude mice gas anesthesia again, it is put into small animal living body imaging system XenogenIVIS200 detection.It is carried using system soft Part carries out image analysis.

(6) nude mice is put to death, sample is collected

The 15th day after inoculation, nude mice is put to death using disconnected cervical approach, transplantable tumor is won and takes pictures, and measure gross tumor volume, more than 4% Polyformaldehyde is fixed and Liquid nitrogen storage is in case subsequent detection.

1.16 statistical analysis

Statistical analysis is carried out using SPSSv19.0 statistics software.It is examined using t and carries out continuous data analysis,

The statistical check of all data is all made of two-sided test, with P<0.05 indicates that difference has statistical significance.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1. niclosamidum is in preparation for treating or preventing the application in tumour medicine, which is characterized in that the tumour is black Plain tumor.

2. application according to claim 1, which is characterized in that effective usage amount of niclosamidum is 4mg/kg or more.

3. niclosamidum is in preparation for inhibiting the application in tumor cell proliferation, migration or the inhibitor of invasion, feature exists In the tumour cell is melanoma cells.

4. niclosamidum is preparing the application in the promotor for promoting apoptosis of tumor, which is characterized in that the tumour Cell is melanoma cells.

5. niclosamidum is preparing the application in the promotor for promoting tumour cell terminal differentiation, which is characterized in that described Tumour cell is melanoma cells.

6. niclosamidum is in preparation for improving the application in the active regulator of TYR enzyme in tumour cell, feature exists In the tumour cell is melanoma cells.

7. niclosamidum is in preparation for promoting melanoma cells to secrete the application in the regulator of melanin.

8. a kind of active method of TYR enzyme in raising melanoma cells, which is characterized in that it includes：By niclosamidum with Melanoma cells contact.

9. a kind of method for promoting melanoma cells secretion melanin, which is characterized in that it includes：By niclosamidum and black Plain oncocyte contact.

10. a kind of for treating or preventing the drug of melanoma, which is characterized in that it contains the chlorine nitre willow as active constituent Amine and pharmaceutically acceptable auxiliary material.