CN105535016A

CN105535016A - New use of nitrate in prevention and treatment of malignant tumors

Info

Publication number: CN105535016A
Application number: CN201510634364.XA
Authority: CN
Inventors: 王松灵; 庞宝兴; 曲兴民
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-09-30
Filing date: 2015-09-30
Publication date: 2016-05-04
Also published as: WO2017054336A1

Abstract

In the invention, a human oral squamous cell carcinoma cell line CAL27 is selected as a research object, an oral carcinoma nude mouse transplanted tumor model is established, sodium nitrate drinking-water (0.5mmol/Kg b.w.) is given, an animal is killed 1 month later, and a result shows that nitrate diet obviously alleviates rapid increase of the volume of oral carcinoma transplanted tumors, and application of the nitrate makes the body weight of the nude mouse have no obvious change. Additionally, sodium nitrate is added to oral carcinoma CAL27 cells in vitro, and a result shows that high concentrate nitrate can obviously inhibit proliferation of tumor cells, promotes apoptosis and obviously inhibits mitigation of the tumor cells. The inhibition effect of nitrate on the oral carcinoma, found for the first time, shows the importance of the nitrate in the prevention and treatment of the oral carcinoma and even whole body tumors.

Description

Nitrate prevents and treats the novelty teabag of malignant tumor

Technical field

The present invention relates to nitrate and the novelty teabag of related preparations in prevention and therapy malignant tumor thereof, belong to pharmaceutical sanitary field.

Background technology

Nitrate is a kind of inorganic matter that nature extensively exists, and plant can absorb and utilize nitrate in soil with nitrogen element required in supplying.Nitrate major part in food derives from green vegetables.Traditional viewpoint is thought, NO3-N and NO2-N class in diet has potential carcinogenic tendency, and people recognize subsequently, and nitrite only has with amine substance combination formation inferior ammonium nitrate compounds, and when reaching certain concentration, just can show carcinogenesis.At present, nitric oxide (the NitricOxide that nitrate and downstream passages thereof produce, NO) reducing blood pressure, regulate antiotasis, the definite effect bearing the aspects such as nerve signal transmission of signal molecule and immunomodulating has reached common recognition, this loading by exogenous nitrate, increases the expression of nitrate and downstream passages NO etc. thereof in body, for the control of some disease opens up a new way.

Oral squamous cell carcinoma is the modal malignant tumor of incidence, sickness rate has and increases trend in recent years, clinically because tumor growth is fast, wellability strong, normal early stage Cervical Lymph Node Metastasis occurs, often take the general treatment measures of operation, radiotherapy, chemotherapy and targeted therapy etc., prognosis is poor.

At present, research or the application of patent report nitrate diet in prevention and therapy tumor is not yet had.

Summary of the invention

The present inventor find, nitrate diet obviously slows down the quick growth of oral cancer transplanted tumor in nude mice, and the application of nitrate to nude mice body weight without significant change.

We find, adopt the diet being rich in nitrate transplantation tumor to increase fast, the nitrate concentration of its every daily ingestion is 0.5mmol/Kg.

We find, nitrate obviously can suppress propagation and the migration of cancer cell of oral cavity, promote the apoptosis of cancer cell of oral cavity.

Discovery of the present invention can be applicable to prevention, treatment oral cancer, also has reference to the treatment of general tumour.

Accompanying drawing explanation

Fig. 1 is the impact of nitrate diet on oral cancer CAL27 cell transplanted tumor in nude mice volume.Result shows that nitrate diet obviously slows down the quick growth of oral cancer transplanted tumor volume.

Fig. 2 is that nitrate diet transplants the impact of nude mice body weight to oral cancer CAL27.Result shows the application of nitrate to nude mice body weight without significant change.

Fig. 3 is the impact of nitrate on oral cancer CAL27 cellular morphology.Under result shows inverted microscope, cellular control unit adherent growth, cell is fusiformis or polygon, and endochylema enriches, and grows vigorous, and flanking cell growth is merged in flakes.After adding the Chile saltpeter of 100mM, CAL27 cell starts to diminish, become circle, and refractivity strengthens, and cell attachment reduces, and presents typical apoptosis form, the morphological change such as the coagulation of visible stain matter, karyopycnosis, karyorrhexis under mirror.

Fig. 4 is the impact of nitrate on oral cancer CAL27 cell proliferation.Result shows the propagation of the obvious inhibition tumor cell of high concentration nitrate (more than 30mM concentration).

Fig. 5 is the impact of nitrate on oral cancer CAL27 apoptosis.Result shows that the nitrate of 100mM obviously promotes apoptosis of tumor cells.

Fig. 6 is the impact of nitrate on oral cancer CAL27 cell migration.Result shows that 50mM obviously suppresses the migration of cancer cell of oral cavity.

Detailed description of the invention

1, the foundation of oral cancer Nude Mouse Model

Select 4 week age Female nude mice to be laboratory animal, cellar culture human mouth squamous cell carcinoma system CAL27, by 2.5 × 10 ⁷/ ml concentration, only injects into nude mice right hind groin subcutaneous with 0.2ml/.Each group of process factor is given when tumor body enters the quick rise period.

2, nitrate drinking water administration and transplanted tumor in nude mice are observed

Experiment is divided into matched group and Nitrate group, often organizes 10.Nitrate group gives Chile saltpeter drinking-water, and concentration is 2.5mmol/L, and the nitrate content of every every daily ingestion of rat is about 0.5mmol/Kgb.w., and continuous nitrate is drunk water 1 month, matched group normal water.Periodic logging transplanted tumor tumor volume and nude mice body weight.

3, the impact of nitrate oral cancer cell propagation, apoptosis and migration

(1) CCK8 detects propagation

100 μ l cell suspension are inoculated in 96 orifice plates, by culture plate in incubator preculture 24 hours (at 37 DEG C, under the condition of 5%CO2), subsequently culture plate is hatched 72 hours at incubator, the culture medium 100 μ l that every hole renews, and 10 μ lCCK-8 solution are added to every hole.Be determined at the absorbance at 450nm place by microplate reader after 2 hours.

(2) Flow cytometry apoptosis

1) cell inoculates 6 orifice plates, after drug treating, with the collected by trypsinisation not containing EDTA;

2) PBS washed cell 2 times are used, centrifugal, collecting cell;

3) the BindingBuffer suspension cell of 500 μ l is added;

4), after adding 5 μ lAnnexinV-FITC mixings, 5 μ lPropidiumIodide are added, mixing;

5) room temperature, lucifuge, reaction 5-15min;

6) in 1h, flow cytometer detects apoptosis situation.

(3) scarification detects cell migration

Cell is cultivated respectively in 6 orifice plates, Growth of Cells inhales sample rifle head by cell cut to converging rear use 200 μ l sterilizing completely, 1 × PBS washes 1 time, continue to cultivate 6h by the RPMI1640 culture medium containing 10% new-born calf serum, the RPMI1640 culture medium that cellular-restoring is used instead containing 0.5% hyclone again continues to cultivate, observe under inverted microscope machinery scratch after (0h) each cut position taking a picture at once, after 24h, again under inverted microscope, observe mechanical mark and take a picture.

4, statistical method

Adopt SPSS17.0 statistics software to carry out statistical analysis, many Sets of Measurement Datas compare employing ANOVA and analyze, and P < 0.05 has statistical significance.

Claims

1. nitrate is used for the prevention and therapy of oral cancer and general tumour.

2. application according to claim 1, is characterized in that, described nitrate is selected from Chile saltpeter, potassium nitrate, lime nitrate and combination thereof, comprises the food and extract that are rich in nitrate.

3. the application according to claim 1-2, is characterized in that, the dosage form of the medicine of described anti-curing oncoma is tablet, capsule, pill, injection, syrup, oral liquid, granule or patch.