CN108893518A - A kind of drug and detection method that can inhibit human cervical carcinoma cell proliferation - Google Patents
A kind of drug and detection method that can inhibit human cervical carcinoma cell proliferation Download PDFInfo
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- CN108893518A CN108893518A CN201810761026.6A CN201810761026A CN108893518A CN 108893518 A CN108893518 A CN 108893518A CN 201810761026 A CN201810761026 A CN 201810761026A CN 108893518 A CN108893518 A CN 108893518A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/58—Reptiles
- A61K35/583—Snakes; Lizards, e.g. chameleons
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Abstract
The invention belongs to technical field of organic chemistry, disclose a kind of drug test method that human cervical carcinoma cell can be inhibited to be proliferated, and HE dyeing is observed, the Hela cell of control group, nucleus dyes darkviolet by haematoxylin under the microscope；Hela cell after Agkistrodon acutus snake venom processing for 24 hours, as concentration increases, cell volume reduces, and cytoplasm is fine and close, forms fine and close mass, the fine and close deep dye of nuclear chromatin.As snake venom concentration increases, the inhibiting rate of Hela cell shows as the trend risen, is in concentration dependent, each experimental group and control group are than statistically significant.The present invention solves the problems, such as that fresh snake venom is not able to maintain in wine liquid and inhibits human cervical carcinoma cell activity by the compatibility of science using stabilizer.The snake venom emulsus wine prepared in the present invention has the function of inhibition human cervical carcinoma cell growth, and inhibiting rate reaches 20%.
Description
Technical field
The invention belongs to technical field of organic chemistry more particularly to it is a kind of can inhibit human cervical carcinoma cell be proliferated drug and
Detection method.
Background technique
Currently, the prior art commonly used in the trade is such：The western Hunan is long-noded pit viper source area.Snake venom is precious since ancient times,
Long-noded pit viper snake venom tops the list, and is known as the title of " soft gold ", but than gold preciousness.People from western Hunan rescues people with snake venom, when can be traced to time immemorial
Phase.People from western Hunan recruits snake art, is exactly the witness for using snake venom earliest.《Compendium of Materia Medica》It carries：" snake, spring go out winter hiding, are good at collection sun
Gas is cultivated the moral character and nourished the nature, and whole body is precious."《Sheng Nong's herbal classic》It carries：It is " interior to reach internal organs, outer thorough skin, promoting blood circulation by removing wind, hundred poison of removing, benefit
Vital essence, bowl spares gas ".China is to treat one of the country cured the disease using snake earliest.Class is used as medicine China's successive dynasties Traditional Chinese Medicine masterpiece all
It has a detailed record, and is widely used, such as the Western Han Dynastry《Legendary god of farming's book on Chinese herbal medicine》, beam《Bencao jizhu》, Song《Kai Baoben
Grass》, it is bright《Compendium of Materia Medica》,《Bencao Tujing》,《Bencao Shiyi》With《Chinese poisonous snake learns document》,《Guizhou medicinal animal medicine
Allusion quotation》,《Pharmacopoeia of People's Republic of China》,《Chinese medicine dictionary》,《Chinese animal drugs》Deng.Chinese Patent Application No.：
2016102149912, denomination of invention：A kind of top-quality wine emulsus wine and preparation method thereof, a kind of top-quality wine of disclosure of the invention
The weight ratio of emulsus wine, including modulation wine and edible glue, the modulation wine and edible glue is 99.1-99.7；The modulation wine packet
Include the raw material of following parts by weight：8-12 parts of flos caryophylli, 4-6 parts of sweet osmanthus, 788-1182 parts of glutinous rice, 39.4-118.2 parts of rock sugar.Its
Preparation method is that edible glue is added to closed stirring in hot water to melt completely to the edible glue, obtains edible sol solution；It will adjust
Alcoholic with edible sol solution is filling after mixing, refrigerates to obtain emulsus wine.Although foregoing invention discloses the production of emulsus wine, but
It is that but cannot achieve the preparation of snake venom emulsus wine, fresh snake venom stable suspersion cannot be made in white wine wine liquid and keep snake venom enzyme activity
Property.
In conclusion problem of the existing technology is：The production method of emulsus wine cannot achieve the preparation of snake venom emulsus wine,
It cannot make fresh snake venom stable suspersion in white wine wine liquid and keep snake venom enzymatic activity.If simple is mixed into snake venom stoste
In white wine wine liquid, the alcohol component in that white wine can make active constituent in reptilase be denaturalized and make snake venom shrink aggressiveness journey flocculence
State can not be really dissolved in wine liquid, and active enzyme component in this case just can not be really played in snake venom when using medicament to internal
The inhibiting effect of cancer rill is even likely to the case where user's ophidism occur.
Solve the difficulty and meaning of above-mentioned technical problem：Difficulty is that snake venom to be made uniformly and is adequately melted into white wine wine liquid
The active constituent of reptilase not variability is also kept while in the middle, must thus add other suitable ingredients to reach
It is made into the viper venom medicament that can effectively inhibit cancer cell.
Summary of the invention
In view of the problems of the existing technology, the present invention provides it is a kind of can inhibit human cervical carcinoma cell be proliferated drug and
Detection method.
The invention is realized in this way a kind of drug that human cervical carcinoma cell can be inhibited to be proliferated, described to inhibit people's uterine neck
The drug of cancer cell multiplication is Agkistrodon acutus snake venom.
Another object of the present invention is to provide a kind of detection method packets of drug that human cervical carcinoma cell can be inhibited to be proliferated
It includes：
Step 1, by white wine and stabiliser solution with volume ratio 19：1 mixing, stirring；
Step 2 fresh snake venom is added in the mixed liquor containing white wine and stabiliser solution of preparation, stirring；
Mixed liquor is detected the effect grown to human cervical carcinoma cell with mtt assay by step 3.
The micro enzyme reaction colorimetric method of mtt assay, that is, tetramethyl azo azoles salt.MTT participates in reacting as its substrate, forms blue
Formazan (Formazan) particle is deposited on intracellular or cell peripheral, is blue solution after hydrochloric acid-isopropanol dissolution, passes through
Enzyme-linked immunosorbent assay instrument measures the light absorption value in each hole at 490nm.Cell Proliferation in reaction system is calculated according to the size of OD value
Degree.
(1) adherent Hela cell is taken, cell is digested by the operating method of passage, cell suspension, cell is made
Concentration is 2.5 × 103~5 × 104A/ml (amount that the plate number and every hole for having calculated needs in advance add, to control cell suspension
Amount).
(2) 96 orifice plates are taken, in cell suspension inoculation, (1 control group and 9 medicine groups are dense by 10 group inoculations
Degree), every group of 8 multiple holes, every hole 200ul, which is put into, to be continued to cultivate in carbon dioxide incubator.
(3) after cell is adherent and is paved with board bottom, suspension, the drug-treated cell of concentrations above gradient, every hole are sucked
The complete culture solution of equivalent is added in 200ul, control group, is put into carbon dioxide incubator and continues to cultivate 72h.
(4) 96 orifice plates are taken out, supernatant is sucked, 100ulMTT liquid is added in every hole, continues to cultivate 4h, sucks supernatant, often
DMSO150ul is added in hole.
(5) 96 orifice plates are placed in microplate reader, shake 15min, wavelength is adjusted to 490nm and detects every hole OD value, then calculates
The inhibiting rate of each group.Inhibiting rate=(1- experimental group OD value/control group OD value) × 100%.
(6) for example above-mentioned method and step of HFL1 cell is done into MTT toxicity test, detected.
The detection method of drug that human cervical carcinoma cell can be inhibited to be proliferated further comprises：HE dyeing, in microscope
Lower observation, the Hela cell of control group, nucleus dye darkviolet by haematoxylin；After Agkistrodon acutus snake venom processing for 24 hours
Hela cell, as concentration increases, cell volume reduces, and cytoplasm is fine and close, forms fine and close mass, the fine and close deep dye of nuclear chromatin.
Further, it is described can inhibit human cervical carcinoma cell be proliferated drug detection method with snake venom concentration increase,
The inhibiting rate of Hela cell shows as the trend risen, is in concentration dependent, each experimental group and control group are than statistically significant.
Further, the Agkistrodon acutus snake venom of the detection method of the drug that human cervical carcinoma cell can be inhibited to be proliferated is to Hela
The growth of cell and HFL1 cell has depression effect, and Hela cell inhibits to be more obvious as snake venom concentration increases, and cell is gradually
Death illustrates with dose dependent, and Agkistrodon acutus snake venom compares HFL1 cell to the inhibitory effects on proliferation of Hela cell
Inhibitory effects on proliferation is eager to excel.
In conclusion advantages of the present invention and good effect are：The present invention utilizes stabilizer, solution by the compatibility of science
Fresh snake venom of having determined is not able to maintain the problem for inhibiting human cervical carcinoma cell activity in wine liquid.The snake venom emulsus prepared in the present invention
Wine has the function of inhibition human cervical carcinoma cell growth, and inhibiting rate reaches 20%.
Detailed description of the invention
Fig. 1 is the detection method flow chart of the drug provided in an embodiment of the present invention that human cervical carcinoma cell can be inhibited to be proliferated.
Fig. 2 is control group provided in an embodiment of the present invention and snake venom wine group schematic diagram；
In figure：(a) control group；(b) snake venom wine group.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
It is an object of the invention to overcome to make fresh snake venom that snake venom be kept to inhibit people palace in wine liquid in the prior art
The activity problems of neck growth of cancer cells provide a kind of snake venom method for preparing medicated wine, realize that fresh snake venom wine liquid is kept through the invention
Inhibit the active preparation method of growth of human cervical carcinoma Hela.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
The drug Agkistrodon acutus snake venom provided in an embodiment of the present invention that human cervical carcinoma cell can be inhibited to be proliferated.
As shown in Figure 1, the detection method provided in an embodiment of the present invention that human cervical carcinoma cell can be inhibited to be proliferated includes following
Step：
S101：By white wine and stabiliser solution with volume ratio 19：1 mixing, stirring；
S102：Fresh snake venom is added in the mixed liquor containing white wine and stabiliser solution of preparation, stirring；
S103：By mixed liquor, the effect grown to human cervical carcinoma cell is detected with mtt assay.
The micro enzyme reaction colorimetric method of mtt assay, that is, tetramethyl azo azoles salt.MTT participates in reacting as its substrate, forms blue
Formazan (Formazan) particle is deposited on intracellular or cell peripheral, is blue solution after hydrochloric acid-isopropanol dissolution, passes through
Enzyme-linked immunosorbent assay instrument measures the light absorption value in each hole at 490nm.Cell Proliferation in reaction system is calculated according to the size of OD value
Degree.
(1) adherent Hela cell is taken, cell is digested by the operating method of passage, cell suspension, cell is made
Concentration is 2.5 × 103~5 × 104A/ml (amount that the plate number and every hole for having calculated needs in advance add, to control cell suspension
Amount).
(2) 96 orifice plates are taken, in cell suspension inoculation, (1 control group and 9 medicine groups are dense by 10 group inoculations
Degree), every group of 8 multiple holes, every hole 200ul, which is put into, to be continued to cultivate in carbon dioxide incubator.
(3) after cell is adherent and is paved with board bottom, suspension, the drug-treated cell of concentrations above gradient, every hole are sucked
The complete culture solution of equivalent is added in 200ul, control group, is put into carbon dioxide incubator and continues to cultivate 72h.
(4) 96 orifice plates are taken out, supernatant is sucked, 100ulMTT liquid is added in every hole, continues to cultivate 4h, sucks supernatant, often
DMSO150ul is added in hole.
(5) 96 orifice plates are placed in microplate reader, shake 15min, wavelength is adjusted to 490nm and detects every hole OD value, then calculates
The inhibiting rate of each group.Inhibiting rate=(1- experimental group OD value/control group OD value) × 100%.
(6) for example above-mentioned method and step of HFL1 cell is done into MTT toxicity test, detected.
Application principle of the invention is further described with reference to the accompanying drawing.
Value-added inhibiting rate is grown to Hela cell for Agkistrodon acutus snake venom, increases with the increase of concentration, there is concentration
Dependence.Stabilizer is the suspending agent for being called CMC cellulose sodium salt bought from upper seamount Pu Chemical Co., Ltd., after addition
It can allow fresh snake venom venom stable suspersion in white wine wine liquid and keep snake venom enzymatic activity.
According to table 1 the results show that the inhibiting rate of Hela cell shows as the trend risen as snake venom concentration increases, it is in
Concentration dependent, each experimental group and control group are than statistically significant (p<0.05).
After HE dyeing, observe under the microscope, the Hela cell of control group, nucleus dyes darkviolet by haematoxylin, carefully
Born of the same parents are clear in structure, clear border.And the Hela cell after Agkistrodon acutus snake venom processing for 24 hours, as concentration increases, cell volume
It reduces, cytoplasm is fine and close, forms fine and close mass, the fine and close deep dye of nuclear chromatin.
Agkistrodon acutus snake venom has depression effect to the growth of Hela cell and HFL1 cell, and Hela cell is dense with snake venom
Degree, which increases, to be inhibited to be more obvious, and cell is gradually dead, illustrates with dose dependent, and increasing of the Agkistrodon acutus snake venom to Hela cell
The inhibitory effects on proliferation for growing depression effect comparison HFL1 cell is eager to excel.
1 snake venom wine of table is to human cervical carcinoma cell inhibited proliferation
Note：Each experimental group is compared with control group, *：P<0.05.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated, which is characterized in that described to inhibit people's uterine neck
The detection method of the drug of cancer cell multiplication includes：
Step 1, by white wine and stabiliser solution with volume ratio 19：1 mixing, stirring；
Step 2 fresh snake venom is added in the mixed liquor containing white wine and stabiliser solution of preparation, stirring；
Mixed liquor is detected the effect grown to human cervical carcinoma cell with mtt assay by step 3.
2. the detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated as described in claim 1, which is characterized in that described
The detection method HE dyeing for the drug that human cervical carcinoma cell can be inhibited to be proliferated, is observed, the Hela cell of control group under the microscope,
Nucleus dyes darkviolet by haematoxylin；Hela cell after Agkistrodon acutus snake venom processing for 24 hours, as concentration increases, cell
Volume-diminished, cytoplasm is fine and close, forms fine and close mass, the fine and close deep dye of nuclear chromatin.
3. the detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated as described in claim 1, which is characterized in that described
The detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated is increased with snake venom concentration, and the inhibiting rate of Hela cell is shown as
The trend of rising is in concentration dependent, and each experimental group and control group are than statistically significant.
4. the detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated as described in claim 1, which is characterized in that described
Life of the Agkistrodon acutus snake venom of the detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated to Hela cell and HFL1 cell
Long to have depression effect, Hela cell inhibits to be more obvious as snake venom concentration increases, and cell is gradually dead, illustrate with dosage according to
Lai Xing, and the inhibitory effects on proliferation that Agkistrodon acutus snake venom compares HFL1 cell to the inhibitory effects on proliferation of Hela cell is eager to excel.
5. the detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated as described in claim 1, which is characterized in that described
The detection method for the drug that human cervical carcinoma cell can be inhibited to be proliferated specifically includes：
(1) adherent Hela cell is taken, cell is digested by the operating method of passage, cell suspension, cell concentration is made
It is 2.5 × 103~5 × 104A/ml；
(2) 96 orifice plates are taken, in cell suspension inoculation, are inoculated with by 10 groups, every group of 8 multiple holes, every hole 200ul is put into dioxy
Change and continues to cultivate in carbon incubator；
(3) after cell is adherent and is paved with board bottom, suspension is sucked, the drug-treated cell of concentrations above gradient, every hole 200ul,
The complete culture solution of equivalent is added in control group, is put into carbon dioxide incubator and continues to cultivate 72h；
(4) 96 orifice plates are taken out, supernatant is sucked, 100ulMTT liquid is added in every hole, continues to cultivate 4h, sucks supernatant, every hole adds
Enter DMSO150ul；
(5) 96 orifice plates are placed in microplate reader, shake 15min, wavelength is adjusted to 490nm and detects every hole OD value, then calculates each group
Inhibiting rate.Inhibiting rate=(1- experimental group OD value/control group OD value) × 100%；
(6) for example above-mentioned method and step of HFL1 cell is done into MTT toxicity test, detected.
6. the detection method detection for the drug that one kind can inhibit human cervical carcinoma cell to be proliferated as described in claim 1 can inhibit people
The drug of cervical cancer cell proliferation, which is characterized in that the drug that human cervical carcinoma cell can be inhibited to be proliferated is agkistrodon acutus snake
Poison.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106309441A (en) * | 2016-10-12 | 2017-01-11 | 南京康凯生物科技有限公司 | Application of Fistulains A in preparation of drugs for treating cervical cancer |
| CN107022459A (en) * | 2017-06-07 | 2017-08-08 | 吉首大学 | A kind of snake venom emulsus wine and preparation method thereof |
| CN107574209A (en) * | 2016-07-05 | 2018-01-12 | 陈栋梁 | A kind of preparation method of homoarginine hybrid peptide and its application in treatment of human cervical cancer |
-
2018
- 2018-07-12 CN CN201810761026.6A patent/CN108893518A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574209A (en) * | 2016-07-05 | 2018-01-12 | 陈栋梁 | A kind of preparation method of homoarginine hybrid peptide and its application in treatment of human cervical cancer |
| CN106309441A (en) * | 2016-10-12 | 2017-01-11 | 南京康凯生物科技有限公司 | Application of Fistulains A in preparation of drugs for treating cervical cancer |
| CN107022459A (en) * | 2017-06-07 | 2017-08-08 | 吉首大学 | A kind of snake venom emulsus wine and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| EMANUELLY BERNARDES-OLIVEIRA等: "Bothrops jararaca and Bothrops erythromelas Snake Venoms Promote Cell Cycle Arrest and Induce Apoptosis via the Mitochondrial Depolarization of Cervical Cancer Cells", 《EVID BASED COMPLEMENT ALTERNAT MED》 * |
| 于源华主编: "《生物工程与技术专业基础实验教程》", 30 June 2016, 北京理工大学出版社 * |
| 李渭敏等: "蛇毒血凝酶用于宫颈癌根治术凝血的效果", 《广东医学》 * |
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