CN107648260A - A kind of medicine of targeted therapy of liver cancer and its application - Google Patents

Abstract

The invention discloses a kind of medicine of targeted therapy of liver 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, oral medication liver 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 liver cancer to the mankind has important practical significance.

Description

A kind of medicine of targeted therapy of liver 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 of liver cancer and its application.

Background technology

Shown according to global cancer statistical report in 2012：Whole world liver cancer in 2012 newly sends out the people of liver cancer 74.8 ten thousand, all The 6th is ranked in cancer morbidity, positioned at lung cancer, breast cancer, colorectal cancer, stomach cancer, after prostate cancer：It is dead then 69.5 ten thousand people, after lung cancer, stomach cancer, occupy the 3rd.National tumour statistics shows liver cancer newly hair China in 2012 within 2012 Male's onset of liver cancer rate is 293,318 people, and women onset of liver cancer rate is 101,452 people, and men and women shares 394770 people morbidity, accounted for The 52.7% of global new hair liver cancer patient, the qualified whole world first.And the liver cancer patient of China is still increasing, it is contemplated that In 2015, Chinese male onset of liver cancer rate was 326,698, and women onset of liver cancer rate is 111,017, and both are added 437,715 People, equivalent to the population at a county town.The population of China only accounts for the 1/4 of total world population, and liver cancer patient has accounted for the whole world It is 1/2 more.

Liver cancer is liver malignancy, can be divided into primary and the major class of Secondary cases two.Characters of Primary Malignant Tumors of Liver originates from In the epithelium or mesenchymal tissue of liver, the former is referred to as primary carcinoma of liver, is that China is occurred frequently, very harmful malignant tumour；Afterwards Person is referred to as sarcoma, more rare compared with primary carcinoma of liver.Secondary cases or metastatic hepatic carcinoma mean that the multiple organs of whole body rise The malignant tumour in source is invaded to liver.Typically it is more common in the organs such as stomach, biliary tract, pancreas, Colon and rectum, ovary, uterus, lung, mammary gland The hepatic metastases of malignant tumour.

The cause of disease of primary carcinoma of liver and definite molecular mechanism are not fully understood, it is now recognized that its morbidity is multifactor, more The complex process of step, influenceed by environment and therefore double factor.Epidemiology and experimental study data show, hepatitis B Malicious (HBV) and HCV (HCV) infection, aflatoxins, contaminated drinking water, alcohol, hepatic sclerosis, sex hormone, nitrosamines Material, trace element etc. are all related to onset of liver cancer.Secondary carcinoma of liver (metastatic hepatic carcinoma) can be by different approaches, such as with blood Liquid, lymph transfer directly invade profit liver and form disease.

1. primary carcinoma of liver

(1) the normal symptom of symptom early liver cancer is without specificity, and the symptom of mid and late liver cancer is then more, and common clinical manifestation has Hepatalgia, abdominal distension, receive it is poor, weak, become thin, progressive hepatomegaly or upper abdomen enclosed mass etc.；Some patientss have low-heat, jaundice, abdomen Rush down, UGB；Occurs acute abdomen performance etc. after Rupture of Hepatocellular Carcinoma.Also there are symptom unobvious or be showed only as the disease of transfer stove Shape.

(2) sign：Early liver cancer is often without obvious positive sign or only similar to hepatic sclerosis sign.Mid and late liver cancer generally occurs within The signs such as liver enlargement, jaundice, ascites.In addition, merging hepatic sclerosis person often has liver palms, spider angioma, male mammary gland increase, lower limb water Swell.Each metastasis site corresponding sign is may occur in which during generation extrahepatic metastases.

(3) complication common are UGB, rupture of hepatocellular carcinoma, liver renal failure etc..

2. secondary carcinoma of liver

(1) clinical manifestation of primary tumo(u)r is mainly seen in the patient of no hepatopathy medical history, and hepatic metastasis still belongs to early stage, do not occurred Corresponding symptom, and primary tumo(u)r symptom substantially belongs to middle and advanced stages more.The more inspections in primary treatment of the secondary carcinoma of liver of such patient, Found in follow-up.

(2) the vexed swollen uncomfortable or secret anguish of the more main suit's upper abdomens of the clinical manifestation patient of secondary carcinoma of liver or hepatic region, as the state of an illness is sent out Exhibition, patient's appearance is weak, appetite is poor, becomes thin or generates heat.It can be laid one's hand on and the liver of enlargement in middle upper abdomen during physical examination, or quality is hard The hard hard tubercle for having tenderness, patients with terminal may occur in which anaemia, jaundice and ascites etc..The clinical manifestation of such patient is similar to primary Property liver cancer, but General development is relatively slow, and degree is also relatively light, doubted more when doing the various inspections of liver and transfer may, enter One step inspection finds primary tumo(u)r in operations research.Some patientss can not find primary carcinoma stove through a variety of inspections.

(3) existing primary tumo(u)r, the clinical manifestation for also having secondary carcinoma of liver are mainly seen in primary tumo(u)r and hepatic metastasis cancer Non- early stage, patient have clinical caused by primary tumo(u)r in addition to liver is similar to the symptom of primary carcinoma of liver, sign Performance, such as knot, the carcinoma of the rectum hepatic metastases when can and meanwhile with bowl evacuation habit, fecal character change and have blood in stool.

Taken the circumstances into consideration to carry out individual comprehensive therapy according to the different phase of liver cancer, be the key for improving curative effect；Treatment method bag The methods of including operation, hepatic artery ligation, hepatic arterial chemoembolization, radio frequency, freezing, laser, microwave and chemotherapy and radiotherapy. Biological therapy, traditional Chinese medical herbal treatment liver cancer also more application.

1. operative treatment

Operation is to treat the first choice of liver cancer, and most efficient method.Operation method has：Radical-ability hepatectomy, Palliative Hepatectomy etc..

Unresectable primary liver cancer using hepatic artery ligation, hepatic arterial chemoembolization in art, can be penetrated as the case may be Frequently, the treatment such as freezing, laser, microwave has the effect of certain.Primary carcinoma of liver is also one of indication of row orthotopic liver transplantation.

2. chemotherapy

Find that cancerous swelling can not be cut off through laparotomy exploration, or the successive treatment person as tumour Palliative surgery, it can be moved using liver Arteries and veins and (or) pump in portal vein (subcutaneously burying device for casting) make local chemotherapy embolism；To estimating unresectable person, also may be used Row Interventional radiology, it is intubated through electing property of femoral artery to arteria hepatica, injection suppository (conventional such as iodized oil) and anticarcinogen Therefore row TAE, some patientss can obtain the chance of surgery excision.

3. radiotherapy

Preferable to ordinary circumstance, liver function is fair, without hepatic sclerosis, no jaundice, ascites, without hypersplenia and oesophagus Varication, cancerous swelling relatively limit to, there is no DISTANT METASTASES IN and be unsuitable for surgery excision or recurrence after operation person, can use radiation for Main complex treatment.

4. biological therapy

Conventional has immune ribonucleic acid, interferon, interleukin 2, thymic peptide etc., can be with chemotherapy combined application.

5. traditional Chinese medical herbal treatment

Take diagnosis and treatment based on an overall analysis of the illness and the patient's condition, the method that tonification and purgation in combination, often with other therapy fit applications.To improve body's resistance to disease, change Kind overall health of patients and symptom, mitigate chemotherapy, radiotherapy adverse reaction.

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 medicine for treating liver cancer at present Thing 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 of liver cancer and its answer With.

The technical solution used in the present invention is：

A kind of targeted drug for treating liver 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：Liver cancer.

Cell experiment as shown by data aluminum sulfate has obvious to human liver cancer cell (SMMC-7721 and MHCC-97H) propagation Inhibitory action, its IC₅₀Respectively SMMC-7721 (28.860mg/ml), MHCC-97H (14.465mg/ml)；To 2 kinds of human liver cancers Cell has obvious apoptosis-promoting effect, and has concentration-effect relation；Can be by the cell-cycle arrest of 2 kinds of human liver cancer cells In G₀/G₁Phase, and there is concentration-effect relation.

By injecting aluminum sulfate preparation for treating liver cancer, there is micro- dosage, rapid-action, short treating period, Small side effects, energy It is rapid to reduce knurl body, apoptosis-induced, Inhibit proliferaton, as its malignant tumour volume of early detection 2cm × 2cm × 2cm to 3cm × 3cm × 3cm, can be achieved that only a pin need to be entered, 36h to 72h activity of tumor cells all disappears, the mankind are captured liver cancer have it is 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 liver cancer cells；A, B is respectively SMMC-7721, MHCC- The concentration of 97H cells-inhibiting rate curve；

Fig. 4 is influence of the aluminum sulfate to hepatoma cell proliferation, and arrow represents non-viable non-apoptotic cell；

Fig. 5 is influence of the aluminum sulfate to human liver cancer cells Hep G2 apoptosis；In figure, 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. 6 is aluminum sulfate in the influence figure of high-transfer human liver cancer cell SGC-7901 apoptosis, 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. 7 is influence of the aluminum sulfate to hepatoma cell apoptosis, and arrow represents nucleus shrinkage, prompts for apoptotic cell；

Fig. 8 is influence of the aluminum sulfate to the human liver cancer cells Hep G2 apoptosis cycle；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. 9 is influence of the aluminum sulfate to the high-transfer human liver cancer cell MHCC-97H apoptosis cycles；In figure, A is Vehicle controls Group, B are 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

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.

Liver 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- fluorine urine is phonetic Pyridine (5-FU), lot number：HFBM160120325008, Amresco company.Positive control drug is prepared：DDP or 5-FU2mg is weighed, is used Fresh complete medium is configured to 100mM mother liquor, then mother liquor is configured to fresh complete medium 200 successively, 60,20, 6th, 2,0.6 μM of working solution.

1.3 main material：

1.4 key instrument：

2nd, test method

2.1 cell culture

SMMC-7721, MHCC-97H 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.

2.2 CCK-8 methods detect cell proliferation test

Take the logarithm the phase growth SMMC-7721, MHCC-97H cell with every hole 5 × 10³Individual cell is inoculated in 96 hole cells In culture plate, after 12h cell attachments, set vehicle control group, positive control drug (DDP) group, positive control drug (5-FU) group, Aluminum sulfate group (0.3-300mg/ml), every group of 5 multiple holes.Vehicle control group is with fresh DMEM culture medium incubated cells completely, sulphur Sour 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 components Not with the fresh DMEM incubated cells completely containing final concentration of 100,30,10,3,1,0.3 μM of compounds.By above-mentioned processing mode The μ l of CCK-8 10 are added after incubated cell 72h in every hole, continue using ELIASA to measure each hole at 450nm after cultivating 1h Absorbance.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 phase To vigor.Toxicity of the compound to SMMC-7721, MHCC-97H cell is evaluated with cell proliferation inhibition rate, if having cell During proliferation inhibition 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

SMMC-7721, MHCC-97H cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵/ hole Density be seeded in 6 orifice plates, after 12h cell attachments, vehicle control group, aluminum sulfate group (10,30,100mg/ml) are set, Every group of 5 multiple holes.Vehicle control group is with fresh DMEM culture medium incubated cells completely, and aluminum sulfate group is respectively with containing final concentration of 10th, 30, 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 liver cancer cells cycle

SMMC-7721, MHCC-97H cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵/ hole Density be seeded in 6 orifice plates, after 12h cell attachments, vehicle control group, aluminum sulfate group (100,30,10mg/ml) are set, Every group of 5 multiple holes.Vehicle control group is with fresh DMEM culture medium incubated cells completely, and aluminum sulfate group is respectively with containing final concentration of 10th, 30, the fresh DMEM incubated cells completely of 100mg/ml aluminum sulfate.After 6h, conventional digestion collects cell, 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 hepatoma cell proliferation

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 hepatoma cell proliferation

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 liver cancer cells as shown in figure 3, A, B are respectively SMMC-7721, MHCC- 97H cells.

Influence of the aluminum sulfate to hepatoma cell proliferation is as shown in figure 4, arrow represents non-viable non-apoptotic cell.Can be clearly from figure Find out, aluminum sulfate can promote liver cancer cells downright bad.

Influence of 3.2 aluminum sulfate to hepatoma cell apoptosis

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes liver cancer cells 6h, collect cell, Annexin V- Apoptosis is detected after the double dyes of FITC/PI.Cell after double dyes can be divided into 4 groups by flow cytometer：Q1-UL represents mechanical damage Cell (Annexin V-/PI+)；Q1-UR non-viable apoptotic cells (Annexin V+/PI+)；Q1-LL survivaling cells (AnnexinV-/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 apoptosis of human hepatoma cell

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 SMMC-7721 cells late apoptic and non-viable non-apoptotic cell number handled through aluminum sulfate is apparently higher than solvent Control group (P<0.05), and there is concentration-effect relation；The SMMC-7721 cells late apoptic handled through aluminum sulfate and necrosis Cell number is apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation；The MHCC-97H handled through aluminum sulfate is thin Born of the same parents' late apoptic 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 human liver cancer cells Hep G2 apoptosis is as shown in Figure 5；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；

Influence of the aluminum sulfate to high-transfer human liver cancer cell MHCC-97H apoptosis is as shown in Figure 6；In figure, A is Vehicle controls Group, B are 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 hepatoma cell apoptosis 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 liver cancer cells cycle

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes liver cancer cells 6h, cell is collected, with 70% cold second After alcohol 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 liver 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 liver cancer cells to hepatoma cell proliferation inhibitory action₀/G₁Phase, it is prevented to enter the S phases.

Influence of the aluminum sulfate to the human liver cancer cells Hep G2 apoptosis cycle is as shown in Figure 8；In figure, A is Vehicle controls Group, B are 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 the high-transfer human liver cancer cell MHCC-97H apoptosis cycles is as shown in Figure 9；In figure, A is solvent Control group, B are 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 hepatoma cell proliferations, and with concentration- Effect relation, under a high concentration condition, with the extension of time, can kill cancer cell completely, it can be by cell-cycle arrest In G₀/G₁Phase, inducing cell apoptosis.This compound performance suppression cancer cell concentration is higher, is mg/ml levels, may be distinctive with it Mechanism of action is related.The compound may directly act on tumor cell surface, can change the physiological characteristic of cancer cell, in cell After surface aggregation, it can effectively change the protein structure of cell surface, cause the albumen precipitation of cell surface and cytoplasm, lead Cause cell permeability degradation, space between cells is shunk, and reduces the splitting ability of tumour cell, effectively control the propagation of cell with And transfer.After compound enters into the cell, cancer cells secrete multiple proteins hydrolase can be suppressed, it is thin cancer can be bonded directly to In the DNA of born of the same parents, cause DNA cracking, and effectively suppress the metabolic function of cancer cell mitochondria, with reference to the amber in mitochondria Acidohydrogenase, change its biological effect, cause Cell microstructure to change, disabling signal path, disturb the life of tumour cell Long metabolism, induced tumor Apoptosis, realizes 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 of liver cancer medicine is 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 liver 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.