CN107616979A - A kind of medicine of targeted therapy breast cancer and its application - Google Patents

Abstract

The invention discloses a kind of medicine of targeted therapy breast cancer and its application.Inventor is by studying for a long period of time, find that aluminum sulfate can differentiate identification human normal tissue and cancerous tissue first, change cancer cell physiological characteristic, suppress cancer cells secrete multiple proteins hydrolase, suppress the metabolic function of cancer cell mitochondria, so as to realize antitumor action, the medicine can be incorporated into the DNA of cancer cell, promote its cracking, quickly strong convergence it can condense cell surface glycoprotein, effectively change the protein structure of cell surface, disabling signal path, change Cell microstructure, and combined with succinate dehydrogenase in cell mitochondrial, effectively control cancer cell multiplication and transfer, 99.8~99.9% or even 100% are up to the inhibiting rate of cancer cell.Aluminum sulfate Small side effects, safe, treatment breast cancer has the advantages that dosage is micro-, rapid-action, short treating period, can reduce knurl body, apoptosis-induced, Inhibit proliferaton rapidly, final fragmentation comes off, and capturing breast cancer to the mankind has great importance.

Description

A kind of medicine of targeted therapy breast cancer and its application

Technical field

The present invention relates to a kind of new opplication of compound, the medicine of more particularly to a kind of targeted therapy breast cancer and its should With.

Background technology

It is well known that malignant tumour is the frequently-occurring disease and common disease of a kind of serious threat human health, with viral disease, The three challenges of geriatric disease and referred to as modern medicine, its pathogenesis not yet illustrate completely, and therapeutic effect is also unsatisfactory. According to incompletely statistics, the research and development of the average annual tumour in the whole world and medical expense are paid wages not low tens billion of, and to country, society and individual bring Massive losses.

Female mammary gland is made up of skin, fibr tissue, corpus mamma and fat, and breast cancer is occurred on mammary gland gland The malignant tumour of skin tissue.99% occurs only to account for 1% in women, male in breast cancer.

Mammary gland is not the vitals for maintaining human life activity, and breast cancer in situ is not fatal；But due to breast cancer The cell loss characteristic of normal cell, connects between cell loose, easily comes off.Cancer cell once comes off, and free cancer is thin Born of the same parents can send out whole body with blood or lymph, form transfer, threat to life.Breast cancer turns at present threatens women body and mind to be good for The kinds of tumor of health.

Global breast cancer incidence is in rising trend always since the late 1970s.8, U.S. women is in life 1 people is just had to suffer from breast cancer.China is not the country occurred frequently of breast cancer, but unsuitable optimistic, in recent years China's breast cancer incidence Growth rate is but higher by national 1~2 percentage point occurred frequently.According to National Cancer Center and prevention and control of diseases office of the Ministry of Public Health 2012 The pathogenesis of breast carcinoma data in 2009 announced are shown：National tumour registration area breast cancer incidence occupies female malignant 1st, female mammary gland cancer morbidity (rough and careless) whole nation adds up to 42.55/10 ten thousand, and city is 51.91/10 ten thousand, and rural area is 23.12/10 ten thousand.

One latest survey result shows that the city such as Shanghai, Beijing and Guangzhou breast cancer, which has become, to threaten most women Big malignant tumour.Amplification exceedes the incidence of disease between 17/,100,000 till now 52.98/10 ten thousand, 35 years of 1972 200%.

Estimate according to the World Health Organization, the whole world is annual to be there are about 1,200,000 women and be diagnosed to suffer from breast cancer.In China, breast cancer has been As " number one killer " of modern female.

Breast cancer has turned into the great public health problem of current social.From whole world Death Rate of Breast Cancer the 1990s Show downward trend；To find out its cause, one is the development of breast cancer screening, make the ratio of early-stage cases increase；Second, breast The development of gland cancer complex treatment, improves curative effect.Breast cancer has turned into one of optimal entity tumor of curative effect.

As what breast cancer biological behaviour was recognized deepens continuously, and the transformation and renewal for the treatment of concept, breast cancer Treatment enter complex treatment epoch, form the Therapeutic mode that breast cancer local treatment and whole body therapeutic are laid equal stress on.Doctor's meeting According to the health with patient by stages of tumour, as one sees fit using operation, radiotherapy, chemotherapy, endocrine therapy, Biological target therapy And the multiple means such as traditional Chinese medicine auxiliary treatment.Surgical operation breast cancer diagnosis, by stages with played in complex treatment it is important Effect.Radiotherapy is that growth, the breeding of cancer cell are destroyed using radioactive ray, has the function that to control and eliminate cancer cell.Perform the operation, put Treatment belongs to local treatment.Chemotherapy is a kind for the treatment of side for suppressing cancer cell division using cancer therapy drug, destroying cancer cell Method, abbreviation chemotherapy.Endocrine therapy is to adjust organism endocrine function using the method for medicine or removal endocrinal glands, is subtracted The secretory volume of few endocrine hormone, so as to reach the purpose for the treatment of breast cancer.Molecular targeted therapy is the most active in recent years One of research field, it is the new antitumoral curative with too many levels mechanism of action compared with chemotherapeutics.Chinese traditional treatment is swollen Knurl emphasizes regulation and the principle of balance, recovers and strengthen the resistance against diseases of internal body, so as to reach equilibrium between yin and yang treatment tumour Purpose.Chemotherapy, endocrine therapy, targeted therapy and Chinese medicine, belong to whole body therapeutic.Doctor can be simultaneous in therapeutic process The local treatment and whole body therapeutic of patient is cared for, morning, patient with breast cancer in mid-term are striven for curing, the life-span is extended to patients with terminal, carried High quality of life.

The surgical operation of breast cancer includes mammary gland and axillary gland two parts.Breast operation with a hook at the end operation on breast (protect Breast conserving surgery) and total mastectomy.Axillary gland operation has SLND and ALND.Sentinel Knot biopsy is that only excision sentinel lymph node, after testing sentinel lymph node transfer carry out ALND again, and also someone is referred to as To protect armpit operation.Breast-consering surgery has strict Operation indication, does not accomplish that all patient with breast cancers can be carried out also at present Breast-consering surgery.The patient with breast cancer for being not suitable for Breast-consering surgery is also needed to cut off breast, doctor can use shaping surgery technology Reconstructive breast.Breasst reconstruction can use autotissue rebuilding, can also use prosthese to rebuild.Can be while tumor resection be performed the operation Breasst reconstruction is carried out, also can after treatment is finished, every review result is rebuild when normal.Breasst reconstruction is carried out not interfere with The wholistic therapy of breast cancer.

Existing treatment method is with high costs, course for the treatment of length, less effective.Therefore, it is badly in need of a kind of breast cancer for the treatment of at present Medicine is to solve the matter of great urgency of cancer patient.

Aluminum sulfate is a kind of common sulfate, the precipitating reagent in paper industry as sizing materials such as gum rosin, wax emulsions, Make flocculant in water process, can also make the interior of foam annihilator and stay agent, the white raw material of manufacture alum, aluminium, oil decolourizes, deodorization Agent, the auxiliary material etc. of some drugses.The the first big purposes of aluminum sulfate for constituting about total output 50% is to be used for papermaking, and second largest purposes is Flocculant is made in drinking water, industrial water and Industrial Wastewater Treatment, constitutes about aluminum sulfate total output 40%.When into this kind of water After adding aluminum sulfate, can generate glue, can adsorb and be settled out bacterium, the aluminium hydroxide of colloid and other suspensions wadding Piece, the color and taste of water are can control in drinking water treatment.

In terms of medicine, aluminum sulfate also has a small amount of application, and such as all states are burned, and Wu Shurong, Chen Jinyun, wait compound aluminum sulfates Prescription research [J] liberation medical academy journals of solution, 1981 (2) disclose intratumoral injection tumor of bladder achieve compared with The effect of good, to the cure rate of early stage tumor of bladder up to 91.1%.Later stage is further investigations have shown that compound aluminum sulfate injection There is certain therapeutic action to tumor of bladder, but subsequent 30 years for many years, have no that aluminum sulfate can be used for treating other Tumour.

The content of the invention

It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of medicine of targeted therapy breast cancer and its answer With.

The technical solution used in the present invention is：

A kind of targeted drug for treating breast cancer, its active component include aluminum sulfate.

As the further improvement of said medicine, aluminum sulfate is aluminum sulfate more than injection stage, or pure with the following method Change obtains：

1) by aluminum sulfate and ultra-pure water mixed dissolution, aluminum sulfate solution is obtained；

2) refined filtration is carried out to aluminum sulfate solution, freeze-drying obtains aluminum sulfate.

As the further improvement of said medicine, medicine is ejection preparation.Further, ejection preparation is selected from injection powder pin Agent, parenteral solution.

As the further improvement of said medicine, the mass mixing ratio of aluminum sulfate and ultra-pure water is 1：(2~5), after dissolving The ultra-pure water purifying used.Further, the mass mixing ratio of aluminum sulfate and ultra-pure water is 1：3, the quality such as use after dissolving Ultra-pure water purifies.

As the further improvement of said medicine, refined filtration is carried out to aluminum sulfate solution using the filter membrane that aperture is 0.22 μm.

As the further improvement of said medicine, aluminum sulfate is selected from the hydrate of aluminum sulfate.

The present invention captures after the research of many decades, and finds " aluminum sulfate " first, can differentiate identification human normal tissue With cancerous tissue, its cancerous tissue is voluntarily come off from normal structure separation, while can differentiate and identify normal cell and cancer cell and select Selecting property dies of hunger the breakthrough first of cancer cell.It is that the most fast most definite and low toxicity of the treatment curative effect to malignant tumour is efficient so far, There is no inhibitory action substantially to normal cell, cancer cell can be killed rapidly and reduce knurl body generation primacy chemical drug, its curative effect is far excellent Traditional anti-cancer medicine at present, its birth can save ten hundreds of patient vitals, and the malignant tumour that is particularly suitable for use in solid carcinoma is controlled Treat.

Inventor is after many decades and puts into huge fund and is studied, and researches and develops world forefront primacy series anticancer of successfully having Special efficacy new drug, its curative effect are treatment of the century-old mankind so far for malignant tumour solid carcinoma, chemicotherapy, operation and modern any controlled What treatment means can not be realized, malignant tumour solid carcinoma can upon administration gradually voluntarily separated from human normal tissue and comes off.

Pharmacology：The critical path of Apoptosis is as follows：(1), cancer cell physiological characteristic can be changed.(2), cancer cells secrete is suppressed Multiple proteins hydrolase.(3), the metabolic function of cancer cell mitochondria is suppressed, so as to realize antitumor action.(4), the medicine energy It is attached in the DNA of cancer cell, promotes its cracking.(5), can quick strong convergence cohesion cell surface glycoprotein.(6), effectively control Cancer cell multiplication and transfer.(7), the medicine can effectively change the protein structure of cell surface.(8), disabling signal path.(9), change Become Cell microstructure.(10) and with succinate dehydrogenase in cell mitochondrial combined so as to change its biological effect.(11), because normal Otherwise cell sucks regular this irregular characteristic of cancer cell.(12), the compound can identify differentiate normal structure with it is improper Tissue, normal cell do not suppress normal cell with abnormal cell and selectively hungry to death and killing cancer cell so that malignant tumour The physianthropy important breakthrough that fragment comes off gradually is presented in entity cancerous tissue, 99.8~99.9% is up to tumor control rate, very To reaching 100%.

Toxicity：It is heavy dose of to have no obvious toxic-side effects through many decades research and test.

Drug effect：There are micro- dosage, rapid-action, short treating period, Small side effects, treating above-mentioned malignant tumour can contract rapidly Tubercle body, apoptosis-induced, Inhibit proliferaton.

Usage and dosage：Such as following table：

Sequence number	Gross tumor volume (cm3)	The sodium chloride injection of pharmaceutical quantities × 0.9%	Injecting method
				1	Solid carcinoma 2 × 2 × 2	1000mg×3mL	Local injection
2	Solid carcinoma 4 × 4 × 4	2000mg×6mL	Local injection
				3	Solid carcinoma 6 × 6 × 6	3000mg×9mL	Local injection
4	Solid carcinoma 8 × 8 × 8	4000mg×12mL	Local injection
				5	Solid carcinoma 10 × 10 × 10	5000mg×15mL	Local injection
6	Solid carcinoma 12 × 12 × 12	6000mg×18mL	Local injection

Note：The drug effect time is 3~5 minutes, and cancer cell starts tune occur to die, and prohibits after injection in 25~30 minutes Only walk about, avoid drug effect from losing.

Function is with curing mainly：Breast cancer.

Cell experiment as shown by data aluminum sulfate has obvious to human breast cancer cell (MCF-7 and MDA-MB-231) propagation Inhibitory action, its IC₅₀Respectively MCF-7 (14.598mg/ml), MDA-MB-231 (10.220mg/ml)；To 2 kinds of human breast carcinomas Cell has obvious apoptosis-promoting effect, and has concentration-effect relation；The cell cycle of 2 kinds of human breast cancer cells can be hindered It is stagnant in G₀/G₁Phase, and there is concentration-effect relation.

By injecting aluminum sulfate preparation for treating breast cancer, there are micro- dosage, rapid-action, short treating period, Small side effects, Knurl body, apoptosis-induced, Inhibit proliferaton can be reduced rapidly, if early detection its malignant tumour volume is in 2cm × 2cm × 2cm to 3cm × 3cm × 3cm, it can be achieved only enter a pin, 36h to 72h activity of tumor cells all disappears, and capturing breast cancer to the mankind has Important realistic meaning.

Brief description of the drawings

Fig. 1 is the HPLC chromatogram of aluminum sulfate after purification；

Fig. 2 is the pets of acute toxicity testing；

Fig. 3 is concentration-inhibiting rate curve of the aluminum sulfate to different breast cancer cells；A, B is respectively MCF-7, MDA-MB- The concentration of 231 cells-inhibiting rate curve；

Fig. 4 is influence of the aluminum sulfate to Cells Proliferation of Human Breast Cancer, and arrow represents non-viable non-apoptotic cell；

Fig. 5 is influence of the aluminum sulfate to apoptosis in breast cancer cell line MCF-7；In figure, A is vehicle control group, and B is aluminum sulfate 10mg/ml groups, C are aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Fig. 6 is aluminum sulfate in the influence figure of human breast cancer cell SGC-7901 apoptosis, A is vehicle control group, and B is sulfuric acid Aluminium 10mg/ml groups, C are aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Fig. 7 is influence of the aluminum sulfate to Apoptosis of Breast Cancer, and arrow represents nucleus shrinkage, prompts for apoptotic cell；

Fig. 8 is influence of the aluminum sulfate to the apoptosis in breast cancer cell line MCF-7 cycle；In figure, A is vehicle control group, and B is sulphur Sour aluminium 10mg/ml groups, C are aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Fig. 9 is influence of the aluminum sulfate to the human breast cancer cell MDA-MB-231 apoptosis cycles；In figure, A is vehicle control group, B is aluminum sulfate 10mg/ml groups, and C is aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups.

Embodiment

Confirm the testing data of chemical constitution

Experiment commission Hunan Province's Experimental Animal Center (Drug Safety Evaluation Center of Hunan Province's progress).

First, new drug title, molecular formula and molecular weight

Chinese name：Aluminum sulfate

Molecular formula：Al₂(SO₄)₃·18H₂O, molecular weight：666.4.

2nd, the method for confirming chemical constitution

1st, determination of moisture

1.1 test condition：

Instrument：V-30 cassette moisture tellers

Method：《Chinese Pharmacopoeia》The first method of aquametry of four general rules of version in 2015 0832.

1.2 measurement result

Moisture measurement result in table 1, aluminum sulfate

1.3rd, parse

Calculated according to sulfuric acid constructed of aluminium and moisture, the number containing the crystallization water is 18 in structure.

The measure of aluminium element

2.1st, test condition

Instrument：Agilent 240-DUO original absorption spectrometers

Method：Using graphite oven atomic absorption, aluminium element content in aluminum sulfate sample is determined.

2.2nd, test result

Aluminium element test result in table 2, aluminum sulfate sample

As a result show：The mean percent content of aluminium element is 8.15% in aluminum sulfate sample.

2.3rd, parse

According to above-mentioned determination of moisture, calculated by 18 crystallizations water, the ratio that aluminium element accounts for molecular weight is 8.11%, and above-mentioned The aluminium element content of atomic absorption detecting is consistent, further demonstrates that and contains aluminium element and 18 crystallizations water in sample.

The measure of sulfate ion

3.1st, test condition

Instrument：ICS900 ion chromatographs

Method：Using 25mM sodium hydroxides as leacheate, chromatographic column is Dionex IonpacTM As18 (4 × 250mm), Flow velocity is 1.0ml/min, sample size 25ul；Using anhydrous sodium sulfate as reference substance, calculated and contained with external standard method by peak area Amount.

3.2nd, test result

Sulfate radical content measurement result in table 3, aluminum sulfate

* it is actual concentrations.

After measured, it is 45.5% containing sulfate ion in sample.

3.3rd, parse

Sample chromatogram figure is consistent with reference substance chromatogram retention time, shows the sulfate radical containing high concentration in sample；Root According to moisture and aluminium element assay result, ion-chromatographic determination sulfate ion content is used as 43.4%, with molecule knot Sulfate ion molecular weight accounting is 43.2% basically identical in structure.

4th, conclusion

According to determination of moisture, aluminium element assay, sulfate radical content measurement result comprehensive analysis, this product molecular formula is Al₂(SO₄)₃·18H₂O。

, can be directly using the high-purity sulphuric acid aluminium (analysis of manufacturer production in order to thoroughly prevent three-waste free discharge to avoid purifying Pure or pharmaceutical grade), but have to carry out strict quality control from manufacturer source.

The purifying of aluminum sulfate

1) aluminum sulfate is pressed 1:3 mass ratio is dissolved in pure water；

2) with etc. the pure water of quality clean, refined filtration removal of impurities；

3) refined solution after refined filtration is freeze-dried to obtain the aluminum sulfate purified of fine white powder.

The HPLC chromatogram of aluminum sulfate is as shown in Figure 1 after purification, it is seen that the purity of aluminum sulfate after purification has before relatively purifying Larger raising.Being freeze-dried obtained aluminum sulfate has more preferable dissolubility.

With reference to experiment, technical scheme is further illustrated.

Safety experiment：

Experimental Animal Center experiment portion of safety experiment commission Zhongshan University is carried out, experimental design reference：

1. the tertiary cloud of Xu, the pharmacological experimental methodology third edition

2.2014 editions medicine safety pharmacology investigative technique guidelines.

Specific experiment method is as follows：

Experiment material

Aluminum sulfate, molecular formula：Al₂(SO₄)₃·18H₂O, molecular weight：666.4.

First, acute toxicity test

1. test objective：

Observe aluminum sulfate after single is given in certain time whether caused toxic reaction, for the preliminary poison for understanding medicine Property effect and its toxicity target organ, provide foundation for follow-up clinical test.

2nd, experimental animal and rearing conditions

SPF levels kunming mice 40,20 ± 2g, male and female half and half.Animal productiong supplying unit：In Zhongshan University experimental animal Heart production department, experimental animal production licence number are：SCXK (Guangdong) 2011-0029, animal quality verification of conformity： No.440085000 buys the date：On 08 15th, 2016；Animal identification method：With saturation picric acid by animal different parts Fur applies dye and represents different animals number, and different animal husbandry cages is made a distinction with animal feeding load card mark.Raising temperature 20~26 DEG C of degree；Humidity 40RH%~70RH%；Rate of ventilation：Receptacle is more than 15 times/hour；Stocking density：Group support, per cage Not more than 6.Use feed：The large and small mouse pellet of SPF levels, provided by Beijing Ke Ao Co., Ltds.Control group and administration group Pets are as shown in Figure 2.

3rd, experimental method：Mouse is randomly divided into control group and administration group, it is specific as follows：

Table 4, acute toxicity test packet and dosage

Group	Medicine	Dosage (mg/kg)	Capacity is administered	Size of animal
					Control group	Physiological saline	/	0.2ml/10g bw	10 female 10 heros
Administration group	Aluminum sulfate solution	2000	0.2ml/10g bw	10 female 10 heros

The compound method of aluminum sulfate solution is：Physiological saline 5ml, peace of the injection equipped with aluminum sulfate are accurately extracted with syringe In cuing open, mix 10~20min of standing and fully dissolve to obtain.

Method of administration：Gastric infusion.

Administration frequency and observing time：Gastric infusion 1 time, observation post administration 14 days.

Testing index：Clinical observation：The general symptom of observation animal daily；Measured body weight：In administration D0, D3, D7, D14 Weigh the weight of animals；Organ coefficient determines：In the 15th day put to death animal, anatomic observation main organs abnormal conditions, core, liver, 5 spleen, lung, kidney internal organs are weighed.

Result treatment and analysis：Handled with statistic software SPSS 24, calculate the average weight and organ coefficient of two groups of animals And make comparisons.

Experimental result：

During experiment, control group, administration group have no dead mouse and shown no obvious abnormalities.

Administration group the weight of animals is compared with control group, and there was no significant difference, refers to table 5.

The comparison of table 5, acute toxicity mouse weight

Organ coefficient statistical result refers to table 6.

The comparison of table 6, acute toxicity mice organs coefficient

* no significant difference (the P compared with control group<0.05) mouse all survives, and none is dead.

Conclusion：

Aluminum sulfate has preferable security, dosage 2000mg/kg, 0.2ml/10g bw0,2ml/10gbw physiology Salt solution, have no obvious toxic-side effects.

Breast cancer cell growth suppresses check experiment

Experiment commission Hunan Province's Experimental Animal Center (Drug Safety Evaluation Center of Hunan Province) is carried out.

1st, experiment material

1.1 tested material：Compound is prepared：Aluminum sulfate 9g is weighed, adds 0.9% sodium chloride injection 15ml, and vibrate extremely All dissolvings, produce 600mg/ml aluminum sulfate solutions, and this is maximum concentration working solution, will be standby after mother liquor progress bacteria removing. Mother liquor is configured to 200 successively with 0.9% sodium chloride injection, 60,20,6,2,0.6mg/ml working solution.

1.2 positive control drug：Cis-platinum (DDP), lot number：SJJMI-IE, Tokyo HuaCheng Industry Co., Ltd；5 FU 5 fluorouracil (5-FU), lot number：HFBM160120325008, Amresco company.Positive control drug is prepared：DDP or 5-FU2mg is weighed, with new Fresh complete medium is configured to 100mM mother liquor, then mother liquor is configured to fresh complete medium 200 successively, 60,20,6, 2nd, 0.6 μM of working solution.

1.3 main material：

1.4 key instrument：

2nd, test method

2.1 cell culture

MCF-7, MDA-MB-231 cell covered with is taken, using the DMEM in high glucose complete medium containing 10%FBS, in 37 DEG C, 5%CO₂Cultivated in incubator, according to cell growth status, 1~2d is passed on or changed liquid, standby to exponential phase.

2.2CCK-8 methods detect cell proliferation test

Take the logarithm the phase growth MCF-7, MDA-MB-231 cell with every hole 5 × 10³Individual cell is inoculated in 96 hole cell trainings Support in plate, after 12h cell attachments, vehicle control group, positive control drug (DDP) group, positive control drug (5-FU) group, sulphur are set Sour aluminium group (0.3-300mg/ml), every group of 5 multiple holes.Vehicle control group is with fresh DMEM culture medium incubated cells completely, sulfuric acid Aluminium group is respectively with the fresh DMEM incubated cells completely containing final concentration of 0.3-300mg/ml aluminum sulfate, DDP and 5-FU groups difference With the fresh DMEM incubated cells completely containing final concentration of 100,30,10,3,1,0.3 μM of compounds.Incubated by above-mentioned processing mode The μ l of CCK-8 10 are added after hatching cell 72h in every hole, continue to cultivate the suction for using ELIASA to measure each hole at 450nm after 1h Luminosity.Using vehicle control group OD values as 100% cell viability, the ratio of remaining each group OD values and vehicle control group OD values is relative Vigor.Toxicity of the compound to MCF-7, MDA-MB-231 cell is evaluated with cell proliferation inhibition rate, if having cell propagation During inhibiting rate ＞ 100%, the systematic error of instrument is judged to, based on 100%.

The detection of 2.3 Apoptosis

2.3.1 the double dye method detection Apoptosis of Annexin V-FITC and PI

MCF-7, MDA-MB-231 cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵/ hole Density is seeded in 6 orifice plates, after 12h cell attachments, sets vehicle control group, aluminum sulfate group (10,30,100mg/ml), often 5 multiple holes of group.Vehicle control group with fresh DMEM culture medium incubated cells completely, aluminum sulfate group respectively with containing final concentration of 10, 30th, the fresh DMEM incubated cells completely of 100mg/ml aluminum sulfate.After 6h, conventional digestion collects cell, adds 500 μ l Cell is resuspended in Binding Buffer buffer solutions, and cell is transferred in 1.5ml EP pipes, 5 μ l of addition Annexin V-FITC, 5 μ l PI, 15min is incubated under the conditions of room temperature lucifuge, with flow cytomery apoptosis situation.

2.3.2 fluorescence colour detects Apoptosis

Cell is handled according to 2.3.1 methods, after compound handles 6h, adds Hoechst 33342 per hole in 6 orifice plates Dyeing liquor 1ml, fully covers cell, is placed in 37 DEG C of culture 20-30min.Dyeing liquor is discarded, glimmering after being washed 2-3 times with PBS Fluoroscopic examination is carried out under light microscope.

Influence of the 2.4 Flow cytometry compounds to the breast cancer cell cycle

MCF-7, MDA-MB-231 cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵/ hole Density is seeded in 6 orifice plates, after 12h cell attachments, sets vehicle control group, aluminum sulfate group (100,30,10mg/ml), often 5 multiple holes of group.Vehicle control group with fresh DMEM culture medium incubated cells completely, aluminum sulfate group respectively with containing final concentration of 10, 30th, the fresh DMEM incubated cells completely of 100mg/ml aluminum sulfate.After 6h, conventional digestion collects cell, is consolidated with 70% cold ethanol It is fixed to stay overnight, add 5 μ l PI, room temperature lucifuge is incubated 30min, with the flow cytomery cell cycle.

2.5 statistical analysis

Data are handled using the statistical softwares of SPSS 16.0, measurement data withRepresent, the ratio of two sample averages Examined compared with using Student T-Test, the comparison of mean is using One-way ANOVA inspections, P between multisample group<0.05 represents It is statistically significant, P<0.01 represents that examined difference is very significant.

3rd, evaluation of result

Influence of 3.1 aluminum sulfate to Cells Proliferation of Human Breast Cancer

After the compound processing cell of micro- Microscopic observation various concentrations, there is cell proliferation rate and slow down, cell fragment Increase, space between cells increases, phenomena such as shape of sand vacuole occurs in cell.Incubation time has clear and definite correlation to cell state together, About after 12h is co-cultured, being rounded occurs in cell, the phenomenon of shrinkage；After 24h is co-cultured, there is part cell and swell, cell is saturating Photosensitiveness is deteriorated, intercellular gap increase；After 48h is co-cultured, there is shape of sand vacuole in cell, the situations such as cell rupture occurs； After 72h is co-cultured, shape of sand vacuole like cell substantially completely ruptures, without complete under 300,100,30mg/ml concentration conditions The cell of cellular morphology, only see and be condensed into stain shape on a small quantity.Cell co-cultures with test sample or positive control drug DDP and 5-FU After 72h, cell propagation is substantially suppressed, and has concentration-effect relation, with significant difference compared with vehicle control group (P<0.01).Cell inhibitory effect is the results detailed in Table 7.

The influence of table 7, different compounds to Cells Proliferation of Human Breast Cancer

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.IC₅₀Represent Suppress the concentration of 50% tumour cell, Emax represents the maximal percentage inhibition to tumour cell.

Aluminum sulfate is to concentration-inhibiting rate curve of breast cancer cell as shown in figure 3, A, B are respectively MCF-7, MDA-MB- 231 cells.

Influence of the aluminum sulfate to Cells Proliferation of Human Breast Cancer is as shown in figure 4, arrow represents non-viable non-apoptotic cell.It can understand from figure Find out that aluminum sulfate can promote breast cancer cell downright bad in ground.

Influence of 3.2 aluminum sulfate to Apoptosis of Breast Cancer

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes breast cancer cell 6h, collect cell, Annexin Apoptosis is detected after the double dyes of V-FITC/PI.Cell after double dyes can be divided into 4 groups by flow cytometer：Q1-UL represents machinery damage Hinder cell (Annexin V-/PI+)；Q1-UR non-viable apoptotic cells (Annexin V+/PI+)；Q1-LL survivaling cells (Annexin V-/PI-)；Q1-LR viable apoptotic cells (Annexin V+/PI-).Experimental result is as shown in table 8：

The influence of table 8, aluminum sulfate to cell apoptosis in human breast cancer

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.

As a result show：The MCF-7 cells late apoptic and non-viable non-apoptotic cell number handled through aluminum sulfate is apparently higher than Vehicle controls Group (P<0.05), and there is concentration-effect relation；The MCF-7 cells late apoptic and non-viable non-apoptotic cell number handled through aluminum sulfate is bright It is aobvious to be higher than vehicle control group (P<0.05), and there is concentration-effect relation；The MDA-MB-231 cell late periods handled through aluminum sulfate Apoptosis and non-viable non-apoptotic cell number are apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation.

Influence of the aluminum sulfate to apoptosis in breast cancer cell line MCF-7 is as shown in Figure 5；In figure, A is vehicle control group, and B is sulphur Sour aluminium 10mg/ml groups, C are aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Influence of the aluminum sulfate to human breast cancer cell MDA-MB-231 apoptosis is as shown in Figure 6；In figure, A is vehicle control group, B is aluminum sulfate 10mg/ml groups, and C is aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Influence of the aluminum sulfate to Apoptosis of Breast Cancer is as shown in fig. 7, arrow expression nucleus shrinkage, it is thin to prompt for apoptosis Born of the same parents.

Influence of 3.3 aluminum sulfate to the breast cancer cell cycle

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes breast cancer cell 6h, collect cell, it is cold with 70% After ethanol is fixed, after PI dyeing, the flow cytometry analysis cell cycle, experimental result is as shown in table 9.

The influence of table 9, aluminum sulfate to the human breast cancer cell cell cycle

Note：* represent compared with vehicle control group, P<0.05, * * expressions are compared with vehicle control group, P<0.01.

As a result show：After aluminum sulfate processing, G₀/G₁Cell proportion substantially increases, G₂/ M phase ratios significantly reduce, and show it G mainly is arrested in by breast cancer cell to Cells Proliferation of Human Breast Cancer inhibitory action₀/G₁Phase, it is prevented to enter the S phases.

Influence of the aluminum sulfate to the apoptosis in breast cancer cell line MCF-7 cycle is as shown in Figure 8；In figure, A is vehicle control group, B For aluminum sulfate 10mg/ml groups, C is aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups；

Influence of the aluminum sulfate to the human breast cancer cell MDA-MB-231 apoptosis cycles is as shown in Figure 9；In figure, A is solvent pair According to group, B is aluminum sulfate 10mg/ml groups, and C is aluminum sulfate 30mg/ml groups, and D is aluminum sulfate 100mg/ml groups.

In summary, under this experiment condition, aluminum sulfate can significantly inhibit 2 kinds of Cells Proliferation of Human Breast Cancer, and with dense Degree-effect relation, under a high concentration condition, with the extension of time, can kill cancer cell completely, it will can hinder the cell cycle It is stagnant in G₀/G₁Phase, inducing cell apoptosis.This compound performance suppression cancer cell concentration is higher, is mg/ml levels, may be peculiar with it Mechanism of action it is related.The compound may directly act on tumor cell surface, can change the physiological characteristic of cancer cell, thin It after cellular surface aggregation, can effectively change the protein structure of cell surface, cause the albumen precipitation of cell surface and cytoplasm, Cause cell permeability degradation, space between cells is shunk, and reduces the splitting ability of tumour cell, effectively controls the propagation of cell And transfer.After compound enters into the cell, cancer cells secrete multiple proteins hydrolase can be suppressed, cancer can be bonded directly to In the DNA of cell, cause DNA cracking, and effectively suppress the metabolic function of cancer cell mitochondria, with reference to the amber in mitochondria Amber acidohydrogenase, change its biological effect, cause Cell microstructure to change, disabling signal path, disturb tumour cell Growth metabolism, induced tumor Apoptosis, realize the effect for killing cancer cell.

It is recommended that：

Finished as zoopery mouse tumor transplanting maturation is injected into pin administration, answer embolism to enter needle passageway or closing inserting needle Passage, while nursing must be singly only isolated, there are certain smell and the small bulge of appearance because drug injection finishes more or less, mouse is such as The nursing of fruit colony, which occurs, mutually baits and causes liquid medicine outflow to influence drug effect.As human tumor inserting needle does not need embolism inserting needle to lead to Road is closed into needle passageway.

Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art For art personnel, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications Also within protection scope of the present invention.

Claims (10)

1. application of the aluminum sulfate in targeted therapy breast cancer medicines are prepared.

2. application according to claim 1, it is characterised in that：The formulation of medicine is ejection preparation.

3. application according to claim 2, it is characterised in that：Ejection preparation is selected from injection powder injection, parenteral solution.

4. application according to claim 1, it is characterised in that：Aluminum sulfate is selected from the hydrate of aluminum sulfate.

A kind of 5. targeted drug for treating breast cancer, it is characterised in that：Its active component includes aluminum sulfate.

6. targeted drug according to claim 5, it is characterised in that：Aluminum sulfate is aluminum sulfate more than injection stage, or is made Purifying obtains with the following method：

1) by aluminum sulfate and ultra-pure water mixed dissolution, aluminum sulfate solution is obtained；

2) refined filtration is carried out to aluminum sulfate solution, freeze-drying obtains aluminum sulfate freeze-dried powder.

7. targeted drug according to claim 5, it is characterised in that：Medicine is ejection preparation.

8. targeted drug according to claim 7, it is characterised in that：Ejection preparation is selected from injection powder injection, parenteral solution.

9. targeted drug according to claim 6, it is characterised in that：The mass mixing ratio of aluminum sulfate and ultra-pure water is 1：(2 ~5) the ultra-pure water purifying, used after dissolving.

10. the targeted drug according to claim 6 or 9, it is characterised in that：Using the filter membrane that aperture is 0.22 μm to sulfuric acid Aluminum solutions carry out refined filtration.