CN107616980A - A kind of medicine for treating stomach cancer and its application - Google Patents

Abstract

The invention discloses a kind of medicine for treating stomach 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 stomach 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 stomach cancer to the mankind has important practical significance.

Description

A kind of medicine for treating stomach cancer and its application

Technical field

The present invention relates to a kind of new opplication of compound, more particularly to a kind of medicine for treating stomach cancer and its application.

Background technology

The annual new hair stomach cancer 934,000 in the whole world at present, wherein having nearly 400,000 in inland of China；It is ill and dead Die twice that rate exceedes world average level.The data that Chinese Anti-Cancer Association director and stomach cancer Professional Committee disclose show flat Just there are a Chinese to die from stomach cancer within every three minutes.

Stomach cancer is derived from the malignant tumour of gastric epithelial cell, accounts for the 95% of stomach malignancy.Stomach cancer is fallen ill in China Rate is very high, and gastric cancer mortality accounts for first of malignant tumour, and the average gastric cancer mortality in the whole nation is up to 20/,100,000, and male is higher than female Property, man：Female about 3: 1.In all kinds of cancers, stomach cancer is one of most commonly seen cancer.The morning of curing gastric cancer and evening, effect is very much not Equally.Early carcinoma of stomach Post operation five year survival rate can reach 90%~95%.If stomach cancer development reaches an advanced stage, gastric cancer mortality It is just higher.

Ann doctors Chao of American Cancer Society's epidemic research and mechanism for monitoring point out that U.S.'s Men's and Women's are because inhaling Cigarette causes the assessment ratio respectively 28% and 14% of mortality of gastric carcinoma.Therefore, stomach cancer is one to grow tobacco associated tumors.Researcher By the perspective death rate research of country, have detected smoked a cigarette to men and women's property, stomach cancer that cigar, tobacco pipe or smokeless tobacco are related it is dead Die rate.As a result find, in the follow-up period of 1982~1996 years, 996 mortality of gastric carcinoma of generation in 467788 men, 58853 509 mortality of gastric carcinoma occur in example woman.Researcher points out that the man's for inhaling cigar (2.29) and cigarette (2.16) is more Variable regulation ratio (RR) highest.The RR of smoking phase is raised.The mortality of gastric carcinoma danger of woman of smoking a cigarette also dramatically increases.Just It is 1.49 in the RR of the woman to smoke a cigarette.Once the RR of smoking woman was 1.36.And the RR of the man once to smoke a cigarette is 1.55；Once inhaled The RR of cigarette man is 1.81.Once the RR for smoking the man of more than one tobacco products is 1.57.With chronic indigestion or stomach- The death rate of the man of duodenal ulcer disease history is higher.The RR of the man to smoke a cigarette is 3.45.Inhaling the man of cigar The RR of son is 8.93.

Metastasis symptom：The transfer probability of late gastric cancer is bigger, typically can direct spreading to neighbouring pancreas, liver, horizontal stroke Colon etc., also can be through Lymph Node Metastasis to stomach peripheral lymph node and distant place lymph node, and some accessible matter on left clavicle is not lived firmly Dynamic lymph node.Liver, lung, brain, bone, ovary etc. can be also transferred to by blood circulation, so as to ascites, jaundice, liver occur The symptoms such as dirty enlargement.The increase of cancerous swelling in itself can also cause the complication such as gastric perforation, bleeding, necrosis, obstruction.Before late gastric cancer is dead Symptom also have spitting blood, melena or stool blood positive etc..

Operation is still that can cut off the radical treatment means of stomach cancer, and recent meta analysis shows chemoradiation therapy can make Patient clinical is benefited, but NACT also lacks conclusive evidence new adjuvant chemotherapies to local advanced gastric as conventional therapy It is feasible by stages that cancer, which reduces, is further studied in progress.For late gastric cancer, median survival interval can be put forward by appeasing chemotherapy It is high further to improve chemotherapy effective percentage, the new target of chemotherapy combined to chemotherapy regimens of 7.5~12 months containing CPT-11, Japanese yew class Another approach is opened up into the treatment for stomach cancer to medicine.Existing curing gastric cancer high cost, side effect are big, sick after treatment The life quality of people is poor.Therefore, it is badly in need of a kind of medicine for treating stomach cancer at present 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 provide a kind of medicine for treating stomach cancer and its application.

The technical solution used in the present invention is：

A kind of medicine for treating stomach cancer, its active component include aluminum sulfate.

As the further improvement of said medicine, aluminum sulfate is aluminum sulfate more than food-grade, 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 oral formulations.Further, oral formulations are selected from soluble in the stomach oral Tablet, gastric-dissolved capsule agent, floating in stomach agent, granule, liquid preparation.

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, stomach cancer is selected from papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, mucus Gland cancer, signet ring cell cancer, adenosquamous carcinoma, squamous carcinoma, class cancer, undifferentiated carcinoma, Gastric-ulcer Cancer.

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：Oral maximum dosage：500mg/ grains, 4~6 every time, take day four times.One month course for the treatment of, 3 ~5 courses for the treatment of are until recovery from illness, long-term use have no apparent side effect.

Function is with curing mainly：Cure mainly stomach cancer, including but not limited to papillary adenocarcinoma, tubular adenocarcinoma, poorly differentiated adenocarcinoma, muccus gland Cancer, signet ring cell cancer, adenosquamous carcinoma, squamous carcinoma, class cancer, undifferentiated carcinoma, Gastric-ulcer Cancer.

Cell experiment as shown by data aluminum sulfate is bred equal to three-type-person's stomach cancer cell (MGC80-3, BGC823, SGC-7901) There is obvious inhibitory action, its IC₅₀Respectively MGC80-3 (6.927mg/ml), BGC823 (8.839mg/ml) and SGC-7901 (15.902mg/ml)；There is obvious apoptosis-promoting effect to three-type-person's stomach cancer cell, and there is concentration-effect relation；Can be by three The cell-cycle arrest of kind gastric carcinoma cells is in G₀/G₁Phase, and there is concentration-effect relation.

By oral aluminum sulfate preparation for treating stomach 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, stomach cancer is captured to the mankind and had important practical significance.

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 stomach cancer cells；1A, 1B, 1C be respectively MGC80-3, The concentration of BGC823, SGC-7901 cell-inhibiting rate curve；

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

Fig. 5 is influence of the aluminum sulfate to gastric carcinoma cells MGC80-3 apoptosis；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 influence of the aluminum sulfate to human gastric adenocarcinoma (low differentiation) BGC823 apoptosis；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；

Fig. 7 is aluminum sulfate in the influence figure of human gastric adenocarcinoma 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. 8 is influence of the aluminum sulfate to apoptosis in gastric cancer, and arrow represents nucleus shrinkage, prompts for apoptotic cell；

Fig. 9 is influence of the aluminum sulfate to the gastric carcinoma cells MGC80-3 apoptosis cycles；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；

Figure 10 is influence of the aluminum sulfate to human gastric adenocarcinoma (low differentiation) the BGC823 apoptosis cycles；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；

Figure 11 is aluminum sulfate in the influence figure in human gastric adenocarcinoma SGC-7901 apoptosis cycles, and 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.

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.

Growth of Gastric 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 by test product：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.

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

MGC80-3, BGC823, SGC-7901 cell covered with is taken, is cultivated completely using the DMEM in high glucose containing 10%FBS Base, 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 With.

2.2 CCK-8 methods detect cell proliferation test

Take the logarithm the phase growth MGC80-3, BGC823, SGC-7901 cell with every hole 5 × 10³Individual cell is inoculated in 96 holes In Tissue Culture Plate, after 12h cell attachments, vehicle control group, positive control drug (DDP) group, positive control drug (5- are set FU) group, aluminum sulfate (0.3-300mg/ml), every group of 5 multiple holes.Vehicle control group is incubated thin with fresh DMEM culture mediums completely Born of the same parents, aluminum sulfate group is respectively with the fresh DMEM incubated cells completely containing final concentration of 0.3-300mg/ml aluminum sulfate, DDP and 5-FU Group is respectively 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 The μ l of CCK-8 10 are added after mode incubated cell 72h in every hole, continue to measure at 450nm respectively using ELIASA after cultivating 1h The absorbance in hole.Using vehicle control group OD values as 100% cell viability, the ratio of remaining each group OD values and vehicle control group OD values For relative activity.Toxicity of the compound to MGC80-3, BGC823, SGC-7901 cell is evaluated with cell proliferation inhibition rate, if going out During existing cell 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

MGC80-3, BGC823, SGC-7901 cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵The density in/hole is seeded in 6 orifice plates, after 12h cell attachments, set vehicle control group, aluminum sulfate (10,30,100mg/ Ml), 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 For 10,30, 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 gastric cancer cell cycle

MGC80-3, BGC823, SGC-7901 cell in exponential phase are taken, conventional digestion collects cell, with 5 × 10⁵The density in/hole is seeded in 6 orifice plates, after 12h cell attachments, set vehicle control group, aluminum sulfate group (100,30, 10mg/ml), every group of 5 multiple holes.Vehicle control group with fresh DMEM culture medium incubated cells completely, aluminum sulfate group respectively with containing Final concentration of 10,30, the fresh DMEM incubated cells completely of 100mg/ml aluminum sulfate.After 6h, conventional digestion collects cell, uses 70% cold ethanol fixes the PI for overnight, adding 5 μ l, and 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 proliferation of human gastric cancer cell

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 proliferation of human gastric cancer cell

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 to concentration-inhibiting rate curve of stomach cancer cell as shown in figure 3,1A, 1B, 1C distinguish MGC80-3, BGC823, SGC-7901 cell.

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

Influence of 3.2 aluminum sulfate to apoptosis in gastric cancer

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes stomach cancer cell 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 (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 gastric carcinoma cells apoptosis

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 MGC80-3 cells late apoptic and non-viable non-apoptotic cell number handled through aluminum sulfate is apparently higher than solvent pair According to group (P<0.05), and there is concentration-effect relation；The BGC823 cells late apoptic and non-viable non-apoptotic cell number handled through aluminum sulfate Apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation；The SGC-7901 cells early stage handled through aluminum sulfate Apoptosis cell is apparently higher than vehicle control group (P<0.05), and there is concentration-effect relation.

Influence of the aluminum sulfate to gastric carcinoma cells MGC80-3 apoptosis 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 gastric adenocarcinoma (low differentiation) BGC823 apoptosis is as shown in Figure 6；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；

Influence of the aluminum sulfate to human gastric adenocarcinoma SGC-7901 apoptosis is as shown in Figure 7；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 apoptosis in gastric cancer is as shown in figure 8, arrow expression nucleus shrinkage, it is thin to prompt for apoptosis Born of the same parents.

Influence of 3.3 aluminum sulfate to gastric cancer cell cycle

Use concentration for 10,30, after 100mg/ml aluminum sulfate intervenes stomach cancer cell 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 gastric carcinoma cells MGC80-3 cell cycles

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 stomach cancer cell to Approach of gastric carcinoma cells proliferation inhibited₀/G₁Phase, it is prevented to enter the S phases.

Influence of the aluminum sulfate to the gastric carcinoma cells MGC80-3 apoptosis cycles is as shown in Figure 9；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 human gastric adenocarcinoma (low differentiation) the BGC823 apoptosis cycles is as shown in Figure 10；In figure, A is molten Matchmaker's 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；

Influence of the aluminum sulfate to the human gastric adenocarcinoma SGC-7901 apoptosis cycles is as shown in figure 11；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.

In summary, under this experiment condition, aluminum sulfate can significantly inhibit 3 kinds of proliferation of human gastric cancer cell, 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.

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. aluminum sulfate is preparing the application in treating gastric cancer medicament.

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

3. application according to claim 2, it is characterised in that：Oral formulations be selected from oral tablet soluble in the stomach, gastric-dissolved capsule agent, Floating in stomach agent, granule, liquid preparation.

4. the application according to Claims 2 or 3, it is characterised in that：Oral formulations are sustained release preparation.

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

6. application according to claim 1, it is characterised in that：Stomach cancer is selected from papillary adenocarcinoma, tubular adenocarcinoma, low differentiation gland Cancer, myxoadenocarcinoma, signet ring cell cancer, adenosquamous carcinoma, squamous carcinoma, class cancer, undifferentiated carcinoma, Gastric-ulcer Cancer.

A kind of 7. medicine for treating stomach cancer, it is characterised in that：Its active component includes aluminum sulfate.

8. medicine according to claim 7, it is characterised in that：Aluminum sulfate is aluminum sulfate more than food-grade, or using such as Lower method purifies to obtain：

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.

9. medicine according to claim 8, 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. medicine according to claim 8 or claim 9, it is characterised in that：It is molten to aluminum sulfate using the filter membrane that aperture is 0.22 μm Liquid carries out refined filtration.