CN113975281B - Application of tertiary amine monomer in preparation of drug for inhibiting helicobacter pylori in oral cavity and stomach - Google Patents
Application of tertiary amine monomer in preparation of drug for inhibiting helicobacter pylori in oral cavity and stomach Download PDFInfo
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- CN113975281B CN113975281B CN202111470258.4A CN202111470258A CN113975281B CN 113975281 B CN113975281 B CN 113975281B CN 202111470258 A CN202111470258 A CN 202111470258A CN 113975281 B CN113975281 B CN 113975281B
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/5014—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 toxicity
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of anti-helicobacter pylori treatment, and discloses application of a tertiary amine monomer in preparation of medicines for inhibiting oral cavity and stomach helicobacter pylori. Determining the minimum inhibitory concentration and the minimum bactericidal concentration of the medicine through the culture of a flat plate and a bacterial suspension; determination of bacterial amount under the action of a drug and determination of the effect of the drug on the level of bacterial virulence: and (5) detecting the cell safety. Aiming at the situation that a local medicine for effectively treating helicobacter pylori infection is lacking under the existing condition and the drug resistance rate of helicobacter pylori antibiotics rises year by year, the invention combines the pH sensitive characteristic of DMAEM for the first time, acts the DMAEM on helicobacter pylori, has obvious bactericidal effect on helicobacter pylori, and can effectively prevent and treat helicobacter pylori infection. Meanwhile, DMAEM detection has good biological safety; has strong anti-helicobacter pylori growth and proliferation effects, thereby reducing the bad use of antibiotics, and further reducing the problems of antibiotic resistance and host flora disorder caused by the antibiotic resistance.
Description
Technical Field
The invention belongs to the technical field of anti-helicobacter pylori treatment, and particularly relates to application of a tertiary amine monomer in preparation of medicines for inhibiting oral cavity and stomach helicobacter pylori.
Background
At present, helicobacter pylori is a common infectious germ for human beings, the infection of which has become a global health problem, more than half of people worldwide infect helicobacter pylori, and the infection rate in China is 40-60%. Helicobacter pylori is a risk factor for a variety of diseases such as chronic gastritis, gastric ulcers, gastric adenocarcinoma, and even mucosa-associated lymphoid tissue lymphoma. Helicobacter pylori was listed by the world health organization International cancer research Institute (IARC) as a human class I carcinogen in 1994. 67% -80% of gastric ulcers and 95% of duodenal ulcers are caused by helicobacter pylori, and about 39% of gastric cancer is closely related to helicobacter pylori infection. Worldwide, gastric cancer affects over 100 tens of thousands of people each year and causes over 70 tens of thousands of people to die.
Oral delivery is the main transmission mode of helicobacter pylori, and a great deal of research shows that bad oral habits, such as lack of tooth brushing habits, can increase the risk of helicobacter pylori infection. Meanwhile, epidemiological researches show that the correlation exists between helicobacter pylori infection and oral diseases such as periodontal disease, oral leukoplakia, lichen planus and the like, and the combination of oral cavity cleaning and triple therapy for treating helicobacter pylori infection patients can improve the success rate of eradicating gastric helicobacter pylori infection and reduce the recurrence rate. The oral cavity is the main colonization site and important reservoir for helicobacter pylori, however, the existing pharmaceutical treatment methods lack topical drugs effective in preventing and treating oral helicobacter pylori infection. The most commonly used treatment after helicobacter pylori infection is drug treatment typified by antibiotics, such as triple therapy of a proton pump inhibitor with two antibiotics or quadruple therapy of a proton pump inhibitor with a bismuth agent combined with two antibiotics. Although new methods of combination therapy or sequential therapy of gastric protectants with antibiotics are being used clinically, reports on helicobacter pylori antibiotic resistance are still increasing, and eradication of helicobacter pylori is difficult to achieve with current methods of treatment. The eradication rate of the triple therapy is 62-66% and the eradication rate of the quadruple therapy is 84-91% according to statistics. In addition, poor use of antibiotics tends to cause disturbance of host flora and thus disease development. Therefore, development of new anti-helicobacter pylori medicines for preventing and treating helicobacter pylori infection and reducing antibiotic resistance thereof is urgent.
DMAEM (dodecylmethylaminoethyl methacrylate) is a novel tertiary amine monomer with pH sensitive antibacterial property, which can be protonated in an acidic environment to have strong antibacterial capability, and can raise the pH value of the environment to inhibit bacterial growth. The pH sensitive antibacterial property of DMAEM is that the DMAEM has the capability of adapting to pH changes of the environment of the oral cavity and the stomach, and can effectively inhibit helicobacter pylori in the oral cavity and the stomach.
Through the above analysis, the problems and defects existing in the prior art are as follows: the prior art lacks topical drugs effective in the prevention and treatment of oral helicobacter pylori infection; the drug resistance of helicobacter pylori is rising year by year, and the poor use of antibiotics is easy to cause the disorder of host flora and further cause the occurrence and development of diseases.
The difficulty of solving the problems and the defects is as follows: the world health organization indicates that the development of new antibiotics is not optimistic. Most of the existing medicines are still in the early stage of research and development, and the effectiveness and safety of the medicines still need to be proved; meanwhile, the new medicine has little effect compared with the existing treatment method, and the development of the new medicine aiming at gram-negative bacteria, which are key drug-resistant bacteria, is little. There is a lack of development of new drugs effective against helicobacter pylori infection.
The meaning of solving the problems and the defects is as follows: helicobacter pylori is a human class I carcinogen and has a high infection rate, and more than half of people worldwide are infected, so that its infection is highly dangerous. The technology provides a novel medicine for effectively resisting helicobacter pylori, and reduces oral transmission of helicobacter pylori; meanwhile, the whole body use of antibiotics is reduced, and bacterial drug resistance, flora disturbance, occurrence and development of host diseases and the like caused by poor use of antibiotics are reduced.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides the application of a novel tertiary amine monomer with pH sensitive antibacterial property in preparing medicines for inhibiting helicobacter pylori in the oral cavity and stomach.
The invention is realized in this way, a new tertiary amine monomer (DMAEM, dodecylmethylaminoethyl methacrylate) with pH sensitive antibacterial property is used in preparing the medicine for inhibiting helicobacter pylori in the mouth and stomach.
Further, the MIC of the novel tertiary amine monomer after acting on helicobacter pylori is 8 μg/mL; MBC for H.pylori was 64. Mu.g/mL. Namely, the tertiary amine has the effect concentration of more than 8 mug/mL, can effectively inhibit the growth and reproduction of helicobacter pylori and pathogenic effect, and can effectively kill helicobacter pylori when the tertiary amine has the effect concentration of more than 64 mug/mL.
It is another object of the present invention to provide a method for detecting the efficacy of a novel tertiary amine monomer for use as a novel material against helicobacter pylori, the method comprising:
step one, culturing a flat plate and a liquid culture medium, and determining the minimum inhibitory concentration and the minimum bactericidal concentration of a medicine;
step two, measuring bacterial quantity change under the action of the drug and measuring bacterial virulence of the drug:
and thirdly, detecting the safety of the cells.
Further, in the first step, the specific process of plate culture is as follows:
uniformly smearing helicobacter pylori on a CBA solid culture medium by using a 10 mu L disposable inoculating loop, and culturing for 24 hours under a microaerophilic condition at a temperature of 37 ℃ in a carbon dioxide constant-temperature incubator;
the microaerophilic conditions are: 5% oxygen, 10% carbon dioxide and 85% nitrogen.
The minimum bacteriostasis concentration and the minimum bacteriostasis concentration of the medicine are determined, and the specific process is as follows:
scraping fresh thalli from a CBA culture medium by using a 10 mu L disposable inoculating loop, placing the thalli in a BHI serum culture medium, and fully and uniformly mixing by alternately using a gun tip for blowing and a vortex oscillator;
adjusting the OD value of the bacterial liquid to OD600 nm=0.3 by using a multifunctional enzyme-linked detector SpectraMax iD5, and diluting 10 times, wherein the concentration of the bacterial liquid is about 1×10 5 CFU/mL; adding the bacterial liquid into a sterile 96-well plate with the system of 100 mu L, adding 98 mu L of bacterial suspension and 2 mu L of 2-time serial diluted DMAEM to ensure that the drug concentration is 0, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 mu g/mL, and placing the mixture into a incubator for culturing for 24 hours; the lowest drug concentration group with clear bacterial liquid is the MIC of the drug, each drug action concentration is absorbed by 2 mu L of the drug to be vertically suspended and dripped into a CBA culture medium, after 24 hours of culture, the minimum drug concentration generated by aseptic colony is the MBC of the drug, and three groups of parallel samples are arranged in the experimentThe cost is high.
Further, in the second step, the bacterial amount determination process under the action of the drug is as follows: preparing a standard curve of the bacterial load of the living bacteria and measuring the bacterial load;
the preparation method of the viable bacteria standard curve comprises the following specific steps:
the bacterial cells on the CBA solid culture medium are scraped off by using a sterile 10 mu L disposable inoculating loop and placed in PBS, evenly mixed, and the OD600 nm=1 is regulated, and the bacterial liquid concentration is about 1×10 8 CFU/mL; sequentially 10-fold gradient dilution to obtain 1×10 8 CFU/mL、1×10 7 CFU/mL、1×10 6 CFU/mL、1×10 5 CFU/mL and 1X10 4 CFU/mL concentration bacterial solutions are 1mL each; the experiment sets three groups of parallel samples, and the operation is performed according to the instruction book of the bacterial genome DNA extraction kit; centrifuging the sample obtained in the above step with 5000g for 10 min, and using TB Green as template TM Premix Ex Taq TM II, a kit adopts a 12.5 mu L System, runs on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument and detects the helicobacter pylori 16S rDNA gene; drawing a regression curve by taking the logarithmic value of the bacterial concentration as an x value of an abscissa and the CT value obtained in the last step as a y value of an ordinate to obtain a standard equation y=kx+b, and completing the statistical analysis of the obtained experimental data by using software SPSS 16.0;
the bacterial strain measuring process comprises the following steps: detecting the OD value of bacteria by using a multifunctional enzyme-linked detector SpectraMax iD 5; taking 1mL of bacterial liquid with each drug concentration, extracting bacterial DNA, obtaining CT value through qPCR, and measuring the bacterial quantity of living bacteria through the obtained formula;
the specific process of determining the bacterial virulence of the medicine is as follows:
after measuring the MIC of DMAEM to helicobacter pylori to be 8 mug/mL, taking the medicine with the concentration of 0, 0.5, 1, 2, 4 and 8 mug/mL to act on helicobacter pylori below the MIC and incubating for 24 hours; 1mL of the enriched bacterial liquid is centrifuged for 10 minutes at 5000rpm, and Trizol is resuspended;
extracting bacterial RNA: transferring into a wall breaking tube with zirconia magnetic beads, cooling with liquid nitrogen, and breaking cells in a high-speed tissue refiner; ice bath for 2-5 min, and gradually eliminating bubbles generated in the wall breaking process; 350. Mu.L of "chloroform" was added: isoamyl alcohol (24:1) "solution, vortex oscillator shake for 15 seconds, stand for 10 minutes; 13000rpm,4 ℃, centrifugation for 15 minutes; pipette 400. Mu.L of the supernatant clear liquid and transfer to a new enzyme-free EP tube; sequentially adding pre-cooled 350 mu L of high-salt solution and 250 mu L of isopropanol, reversing the EP tube up and down for a plurality of times to fully mix, and standing for 10 minutes; 13000rpm,4 ℃, centrifugation for 15 minutes; the supernatant was discarded, centrifuged at 13000rpm at 4℃for 2 minutes, and the supernatant was carefully removed; 1mL of a pre-chilled 70% ethanol solution was added to the pellet, the EP tube was inverted up and down several times, centrifuged at 13000rpm at 4℃for 5 minutes; discarding the supernatant, and naturally air-drying the bottom sediment; adding 40-70 mu L of enzyme-free water according to the amount of sediment at the bottom; carrying out water bath at 65 ℃ for 10 minutes, and blowing and uniformly mixing by using an enzyme-free gun tip; the concentration and purity of RNA was determined using a NanoDrop 2000 spectrophotometer;
RNA purification and reverse transcription: using PrimeScript TM RT reagent Kit with gDNA Eraser, removing DNA from RNA sample, purifying and performing subsequent RNA reverse transcription.
Further, the real-time fluorescent quantitative reverse transcription polymerase chain reaction: setting 4 multiple wells per sample using reverse transcribed cDNA as template, using TB Green TM Premix Ex Taq TM II, a kit adopts a 20 mu L system; operating on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument to determine reaction conditions; the expression detection of H.pylori 16S rRNA and VacA, cagA, ureaB, flaA, luxS was carried out by using H.pylori housekeeping gene 16S rRNA as an internal reference gene and a 2-DeltaCT method.
Further, in the third step, the cell safety detection process is as follows:
inoculating 1x10 in 96-well plate 4 HOK cells or L929 cells in 5% CO 2 Incubating the cells in a cell incubator at 37 ℃ overnight; after cell adhesion, the original culture medium is sucked, fresh 100 mu L of DMEM culture medium containing DMAEM with different concentrations is added, and the cells are incubated in a cell incubator at 37 ℃; after 24h, 6 duplicate wells per group were used for cytotoxicity assays using CCK8 kit.
Further, the cell safety detection process further comprises: the original culture medium is sucked, PBS is used for washing three times, 100 mu L of DMEM culture medium containing 10% of CCK8 reagent is added, and the culture medium is incubated in a 37 ℃ cell incubator in a dark place; after 1-2 hours, the supernatant was transferred to a new 96-well plate and the light absorption value of A450 was read under an microplate reader.
It is another object of the present invention to provide a kit for inhibiting helicobacter pylori in the oral cavity and the stomach, which comprises the novel tertiary amine monomer.
The invention also aims to provide an application of the novel tertiary amine monomer in preparing medicines for resisting helicobacter pylori and related diseases such as chronic gastritis, gastric ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma caused by helicobacter pylori. The invention can also be applied to the addition of tertiary amine in dental materials (such as resin filling materials, pit and groove sealing materials, root canal filling materials and the like).
By combining all the technical schemes, the invention has the advantages and positive effects that:
(1) The invention has pH sensitive characteristic, can effectively inhibit and kill helicobacter pylori in the oral cavity and the stomach, can effectively resist the oral cavity and stomach infection colonization of the helicobacter pylori, and reduces the probability of suffering stomach and systemic diseases caused by helicobacter pylori infection.
(2) The invention can effectively inhibit helicobacter pylori when the action concentration is more than 8 mug/mL, can effectively kill helicobacter pylori when the action concentration is more than 64 mug/mL, and has stronger helicobacter pylori resistance. Can be applied to oral cavity, environment, etc. to achieve local effective antibacterial effect, and reduce helicobacter pylori infection risk from source.
(3) The method for detecting the drug effect of the novel material by using the novel tertiary amine monomer to act on helicobacter pylori comprises the steps of culturing a flat plate and a liquid culture medium, and determining the minimum antibacterial concentration and the minimum antibacterial concentration of the drug; measuring bacterial load under the action of a drug and measuring the influence of the drug on the bacterial virulence level; cell safety detection and the like, the methods can effectively detect the killing and inhibiting effects of the new drug on helicobacter pylori, embody the cell safety of the drug and provide guidance for the new drug against helicobacter pylori.
(4) The technical effect or experimental effect of comparison. The novel pH sensitive tertiary amine monomer can effectively resist helicobacter pylori in the oral cavity and the stomach, can realize protonation-deprotonation intelligent response according to the pH change of the environment in different environments, has good biocompatibility, and is a safe and effective helicobacter pylori resistant medicament.
The invention has stronger anti-helicobacter pylori growth and proliferation effects, thereby reducing the bad use of antibiotics, and further reducing the problems of antibiotic resistance and host flora disorder caused by the antibiotic resistance.
Drawings
FIG. 1 is a flow chart of a novel anti-helicobacter pylori material detection method provided by the embodiment of the invention.
FIG. 2 is a graph showing the effects of DMAEM provided in the examples of the present invention after acting on helicobacter pylori.
FIG. 3 is a graph showing the detection of OD in DMAEM against H.pylori according to an example of the present invention.
FIG. 4 is a schematic diagram showing quantitative detection of qPCR bacteria after DMAEM provided in the examples of the present invention acts on helicobacter pylori.
FIG. 5 is a schematic diagram showing the effect of DMAEM provided in the examples of the present invention on H.pylori detection at concentrations below MIC.
Fig. 6 is a schematic diagram showing CCK8 detection of DMAEM acting on oral epithelial cells HOK provided in the examples of the present invention.
FIG. 7 is a schematic diagram showing CCK8 detection of DMAEM on mouse fibroblast L929 provided in the examples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In view of the problems existing in the prior art, the present invention provides a novel method for preparing a material against helicobacter pylori, and the present invention is described in detail below with reference to the accompanying drawings.
Other steps may be performed by those skilled in the art to prepare the novel helicobacter pylori resistant material of the present invention, and the novel helicobacter pylori resistant material preparation method of the present invention shown in fig. 1 is merely one specific example.
The invention provides an application of a novel tertiary amine monomer (DMAEM, dodecylmethylaminoethyl methacrylate) with pH sensitive antibacterial property in preparing medicines for inhibiting helicobacter pylori in the oral cavity and the stomach.
After the novel tertiary amine monomer acts on helicobacter pylori, the minimum concentration of the culture medium is MIC of 8 mug/mL; MBC for H.pylori was 64. Mu.g/mL.
As shown in FIG. 1, the novel anti-helicobacter pylori material detection method provided by the embodiment of the invention comprises the following steps: s101: culturing the plate, and determining the minimum antibacterial concentration and the minimum sterilizing concentration of the medicine;
s102: determination of bacterial amount under the action of a drug and determination of bacterial virulence of the drug:
s103: and (5) detecting the cell safety.
In S101 provided by the embodiment of the present invention, the specific procedure of plate culture is:
helicobacter pylori was uniformly smeared on a CBA solid medium using a 10. Mu.L disposable inoculating loop, and placed in a carbon dioxide constant temperature incubator at 37℃for cultivation under microaerophilic conditions for 24 hours.
The microaerophilic conditions are: 5% oxygen, 10% carbon dioxide and 85% nitrogen.
In S101 provided by the embodiment of the present invention, the minimum inhibitory concentration and the minimum bactericidal concentration of the drug are determined, and the specific process is as follows:
scraping fresh thalli from a CBA culture medium by using a 10 mu L disposable inoculating loop, placing the thalli in a BHI serum culture medium, and fully and uniformly mixing by alternately using a gun tip for blowing and a vortex oscillator;
adjusting the OD value of the bacterial liquid to OD600 nm=0.3 by using a multifunctional enzyme-linked detector SpectraMax iD5, and diluting 10 times, wherein the concentration of the bacterial liquid is about 1×10 5 CFU/mL; adding the bacterial liquid into a sterile 96-well plate with a system of 100 mu L, adding 98 mu L of bacterial suspension and 2 mu L of 2-fold serial diluted DMAEM to ensure that the drug concentration is 0, 0.5, 1, 2, 4, 8 and 16. 32, 64, 128 μg/mL, placed in incubator for 24 hours; and (3) observing that the minimum drug concentration group with clear bacterial liquid is the MIC of the drug, sucking 2 mu L of each drug action concentration, vertically suspending and dripping the drug into a CBA culture medium, and culturing for 24 hours, wherein the minimum drug concentration generated by the sterile colony is the MBC of the drug. The experiment was set up with three parallel sets of samples.
In S102 provided by the embodiment of the present invention, the bacterial load change determination process under the action of the drug is: preparing a standard curve of the bacterial load of the living bacteria and measuring the bacterial load;
the preparation method of the viable bacteria standard curve comprises the following specific steps:
the bacterial cells on the CBA solid culture medium are scraped off by using a sterile 10 mu L disposable inoculating loop and placed in PBS, evenly mixed, and the OD600 nm=1 is regulated, and the bacterial liquid concentration is about 1×10 8 CFU/mL; sequentially 10-fold gradient dilution to obtain 1×10 8 CFU/mL、1×10 7 CFU/mL、1×10 6 CFU/mL、1×10 5 CFU/mL and 1X10 4 CFU/mL concentration bacterial solutions are 1mL each; the experiment sets three groups of parallel samples, and the operation is performed according to the instruction book of the bacterial genome DNA extraction kit; centrifuging the sample obtained in the above step with 5000g for 10 min, and using TB Green as template TM Premix Ex Taq TM II, a kit adopts a 12.5 mu L System, runs on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument and detects the helicobacter pylori 16S rDNA gene; and drawing a regression curve by taking the logarithmic value of the bacterial concentration as an x value on the abscissa and taking the CT value obtained in the last step as a y value on the ordinate to obtain a standard equation y=kx+b. Statistical analysis of the resulting experimental data was done using software SPSS 16.0.
The bacterial strain measuring process comprises the following steps: detecting the OD value of bacteria by using a multifunctional enzyme-linked detector SpectraMax iD 5; taking 1mL of bacterial liquid with each drug concentration, extracting bacterial DNA, obtaining CT value through qPCR, and measuring the viable bacteria amount through the obtained formula.
In S102 provided by the embodiment of the present invention, the specific process of determining bacterial virulence by the drug is:
after measuring the MIC of DMAEM to helicobacter pylori to be 8 mug/mL, taking the medicine with the concentration of 0, 0.5, 1, 2, 4 and 8 mug/mL to act on helicobacter pylori below the MIC and incubating for 24 hours; 1mL of the enriched bacterial liquid is centrifuged for 10 minutes at 5000rpm, and Trizol is resuspended;
extracting bacterial RNA: transferring into a wall breaking tube with zirconia magnetic beads, cooling with liquid nitrogen, and breaking cells in a high-speed tissue refiner; ice bath for 2-5 min, and gradually eliminating bubbles generated in the wall breaking process; 350. Mu.L of "chloroform" was added: isoamyl alcohol (24:1) "solution, vortex oscillator shake for 15 seconds, stand for 10 minutes; 13000rpm,4 ℃, centrifugation for 15 minutes; pipette 400. Mu.L of the supernatant clear liquid and transfer to a new enzyme-free EP tube; sequentially adding pre-cooled 350 mu L of high-salt solution and 250 mu L of isopropanol, reversing the EP tube up and down for a plurality of times to fully mix, and standing for 10 minutes; 13000rpm,4 ℃, centrifugation for 15 minutes; the supernatant was discarded, centrifuged at 13000rpm at 4℃for 2 minutes, and the supernatant was carefully removed; 1mL of a pre-chilled 70% ethanol solution was added to the pellet, the EP tube was inverted up and down several times, centrifuged at 13000rpm at 4℃for 5 minutes; discarding the supernatant, and naturally air-drying the bottom sediment; adding 40-70 mu L of enzyme-free water according to the amount of sediment at the bottom; carrying out water bath at 65 ℃ for 10 minutes, and blowing and uniformly mixing by using an enzyme-free gun tip; RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer.
RNA purification and reverse transcription: using PrimeScript TM RT reagent Kit with gDNA Eraser kit (DNA in RNA sample is removed for purification and subsequent RNA reverse transcription process).
Real-time fluorescent quantitative reverse transcription polymerase chain reaction: setting 4 multiple wells per sample using reverse transcribed cDNA as template, using TB Green TM Premix Ex Taq TM II, a kit adopts a 20 mu L system; operating on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument to determine reaction conditions; the expression detection of H.pylori 16S rRNA and VacA, cagA, ureaB, flaA, luxS was carried out by using H.pylori housekeeping gene 16S rRNA as an internal reference gene and a 2-DeltaCT method.
In S103 provided by the embodiment of the present invention, the specific process of cell safety detection is:
inoculating 1x10 in 96-well plate 4 The HOK cells or L929 cells were incubated in a 5% CO2 cell incubator at 37℃overnight; after cell adhesion, the original medium was aspirated and fresh was added100. Mu.L of DMEM medium containing DMAEM at different concentrations was incubated in a 37℃cell incubator; after 24h, 6 duplicate wells per group were used for cytotoxicity assays using CCK8 kit.
The specific process is as follows: the original culture medium is sucked, PBS is used for washing three times, 100 mu L of DMEM culture medium containing 10% of CCK8 reagent is added, and the culture medium is incubated in a 37 ℃ cell incubator in a dark place; after 1-2 hours, the supernatant was transferred to a new 96-well plate and the light absorption value of A450 was read under an microplate reader.
The technical scheme of the present invention will be described in detail with reference to specific embodiments.
Standard strain G27 of helicobacter pylori.
Columbia blood agar (Columbia blood agar, CBA) medium: 15.6g of CBA powder (Oxoid, basingstoke, UK) was dissolved in 400mL of sterilized deionized water and sterilized at high temperature under high pressure. Heating in a microwave oven to completely melt, heating in a water bath at 60deg.C for 30 min, cooling to 50deg.C, adding 100mL sterilized defibrinated sheep blood (Solarbio, china), and mixing. Pouring into sterile plate rapidly, and cooling and solidifying completely. Sealing with sealing film, storing at 4deg.C, and using within 1 month.
Brain heart infusion broth (Brian Heart Infusion, BHI) serum medium: BHI powder (Becton, dickinson and Company, usa) 14.8g was dissolved in 400mL of sterile deionized water, filtered through a 0.22 μm microporous filter, added with 100mL of sterile fetal bovine serum (Gibco, australia), mixed well and stored at 4 ℃.
Anti-helicobacter pylori activity test of novel materials
1. Plate culture: helicobacter pylori was uniformly smeared onto CBA solid medium using a 10 μl disposable inoculating loop, and placed in a carbon dioxide incubator (Thermo, usa) for 24 hours under microaerophilic conditions (5% oxygen, 10% carbon dioxide and 85% nitrogen) at 37 ℃.
2. Minimum inhibitory concentration of drug (minimum inhibitory concentration, MIC) and minimum bactericidal concentration (minimum bactericidal concentration, MBC) were determined: fresh cells were scraped from CBA medium using a 10. Mu.L disposable inoculating loop, placed in BHI serum medium, and alternately blown with a gun tip and vortexedThe rotary oscillator is fully and evenly mixed. Adjusting the OD value of the bacterial liquid to OD600 nm=0.3 by using a multifunctional enzyme-linked detector SpectraMax iD5 (Molecular Devices, U.S.), and diluting 10 times, wherein the concentration of the bacterial liquid is about 1×10 5 CFU/mL. The bacterial solution is added into a sterile 96-well plate, the system is 100 mu L, 98 mu L of bacterial suspension and 2 mu L of DMAEM which is diluted 2 times continuously are added, so that the drug concentration is 0, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 mu g/mL, and the bacterial solution is placed in a incubator for culturing for 24 hours. The lowest drug concentration group at which the bacteria liquid was observed to be clear was the MIC of the drug. Each drug action concentration is absorbed by 2 mu L of the vertical suspension drop-added to the CBA culture medium, and after 24 hours of culture, the minimum drug concentration generated by the sterile colony is MBC of the drug. The experiment was set up with three parallel sets of samples.
3. Bacterial amount change determination under the action of the drug:
(1) Preparation of a standard curve of viable bacteria
The cells on the CBA solid medium were scraped off with a sterile 10. Mu.L disposable inoculating loop and placed in PBS (Gibco, australia), mixed well, and the OD600 nm=1 was adjusted to give a bacterial solution concentration of about 1X10 8 CFU/mL. Sequentially 10-fold gradient dilution to obtain 1×10 8 CFU/mL、1×10 7 CFU/mL、1×10 6 CFU/mL、1×10 5 CFU/mL and 1X10 4 CFU/mL concentration bacterial solutions were 1mL each. The experiment was set up with three parallel sets of samples. The procedure was as per the bacterial genomic DNA extraction kit (TIANGEN, china). The sample obtained in the above was centrifuged at 5000g for 10 minutes. Using TB Green with the extracted DNA as a template TM Premix Ex Taq TM II kit (Tli RNase H Plus, takara, japan) was run on a LightCycler 480 II System (Forrenstrase 2,6343 Rotkreuz, switzerland) real-time fluorescence quantitative PCR apparatus using a 12.5. Mu.L System. Detecting the helicobacter pylori 16S rDNA gene. And drawing a regression curve by taking the logarithmic value of the bacterial concentration as an x value on the abscissa and taking the CT value obtained in the last step as a y value on the ordinate to obtain a standard equation y=kx+b. Statistical analysis of the experimental data obtained was done using software SPSS 16.0 (SPSS inc., usa).
(2) Bacterial mass measurement
Bacterial OD values were measured using a multifunctional enzyme-linked detector SpectraMax iD5 (Molecular Devices, USA); taking 1mL of bacterial liquid with each drug concentration, extracting bacterial DNA, obtaining CT value through qPCR, and measuring the viable bacteria amount through the obtained formula.
4. Drug toxicity to bacteria assay: the MIC of DMAEM for H.pylori was determined in step 2 to be 8. Mu.g/mL. Below MIC, drug concentrations of 0, 0.5, 1, 2, 4, 8 μg/mL were applied to helicobacter pylori and incubated for 24 hours. The enriched bacterial solution was centrifuged at 1mL at 5000rpm for 10 minutes and Trizol (Invitrogen, USA) was resuspended.
(1) Extracting bacterial RNA: transferring into a wall breaking tube (Fastrep tube, betin Co., ltd., china) with zirconia magnetic beads (diameter of 0.1 mm), cooling with liquid nitrogen, and breaking cells in a high-speed tissue homogenizer (Bertin Co., france); ice bath for 2-5 min, and gradually eliminating bubbles generated in the wall breaking process; 350. Mu.L of "chloroform" was added: isoamyl alcohol (24:1) "solution (Solarbio company, china), vortex shaker shake for 15 seconds, stand for 10 minutes; 13000rpm,4 ℃, centrifugation for 15 minutes; mu.L of the supernatant clarified liquid (without touching the white disrupted tissue pellet) was aspirated and transferred to a new enzyme-free EP tube. Sequentially adding pre-cooled 350 μl high-salt solution (Takara, china) and 250 μl isopropanol, inverting the EP tube several times to mix thoroughly, and standing for 10 min; 13000rpm,4 ℃, centrifugation for 15 minutes; the supernatant was discarded, centrifuged at 13000rpm at 4℃for 2 minutes, and the supernatant was carefully removed; 1mL of a pre-chilled 70% ethanol solution was added to the pellet, the EP tube was inverted up and down several times, centrifuged at 13000rpm at 4℃for 5 minutes; discarding the supernatant, and naturally air-drying the bottom sediment; adding 40-70 mu L of enzyme-free water according to the amount of sediment at the bottom; carrying out water bath at 65 ℃ for 10 minutes, and blowing and uniformly mixing by using an enzyme-free gun tip; RNA concentration and purity were determined using a Nanodrop 2000 spectrophotometer (Gene Company Limited, china).
(2) RNA purification and reverse transcription: using PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (Taraka Bio Inc, otsu, japan) removes DNA from the RNA sample for purification and subsequent RNA reverse transcription.
(3) Real-time fluorescent quantitative reverse transcription polymerase chain reaction (Real-time Quantitative Polymerase Chain Reaction, RT-qPCR): in reverse transcribed cDNA was used as template, 4 wells were set per sample using TB Green TM Premix Ex Taq TM II kit (Tli RNase H Plus, takara, japan) 20. Mu.L system was used. The reaction was run on a LightCycler 480 II System (Forrenstrase 2,6343 Rotkreuz, switzerland) real-time fluorescent quantitative PCR apparatus under the reaction conditions shown in tables 2-4. The expression detection of H.pylori 16S rRNA and VacA, cagA, ureaB, flaA, luxS was carried out by using H.pylori housekeeping gene 16S rRNA as an internal reference gene and a 2-DeltaCT method.
5. Cell safety detection: inoculating 1x10 in 96-well plate 4 HOK cells or L929 cells in 5% CO 2 The cells were incubated in an incubator overnight at 37 ℃. After cell attachment, the original medium was aspirated, fresh 100. Mu.L of DMEM medium containing varying concentrations of DMAEM (0-100. Mu.g/mL) was added and incubated in a 37℃cell incubator. Each group had 6 duplicate wells. After 24h, the cytotoxicity assay was performed using CCK8 kit (APExBIO). The specific operation steps are as follows: the original medium was aspirated, washed three times with PBS, 100. Mu.L of DMEM medium containing 10% CCK8 reagent was added, and incubated in a 37℃cell incubator protected from light. After 1-2 hours, the supernatant was transferred to a new 96-well plate and the light absorption value of A450 was read under an microplate reader.
The invention combines the pH sensitivity characteristic of DMAEM for the first time, acts the DMAEM on helicobacter pylori, discovers that the DMAEM has obvious bactericidal effect on helicobacter pylori, and can effectively prevent and treat helicobacter pylori infection, because the helicobacter pylori infection in the oral cavity and the stomach has different pH environments, and the situation that the local medicine for effectively treating the helicobacter pylori infection is lack of the traditional condition and the helicobacter pylori antibiotic resistance rate rises year by year; and simultaneously, the detection of DMAEM has good biological safety.
The technical scheme of the invention is further described in the following in connection with experiments.
FIG. 2 shows that the minimum concentration of DMAEM at which the medium is clear by visual inspection after action on H.pylori is MIC, 8. Mu.g/mL. Bacteria coated plates were produced aseptically at 64. Mu.g/mL, indicating that the DMAEM drug had an MBC of 64. Mu.g/mL for H.pylori;
FIG. 3 is a graph showing that the OD of bacteria at a concentration of 8. Mu.g/mL was greatly reduced, as measured by the OD of DMAEM against H.pylori, indicating that the MIC was now that the drug was acting on the bacteria;
FIG. 4 shows the qPCR bacterial quantification performed after the action of DMAEM on H.pylori, the bacterial concentration was greatly reduced at drug concentrations of 8. Mu.g/mL and 64. Mu.g/mL, indicating that these two concentrations are MIC and MBC of drug on H.pylori, respectively;
FIG. 5 shows the extent of expression of the major virulence factors of H.pylori by DMAEM acting on H.pylori at concentrations below the MIC. Since DMAEM has a strong inhibitory effect on bacteria, the expression level of bacterial virulence factors is greatly increased at concentrations lower than MIC. And at MIC concentrations, the amount of virulence factor expression decreases;
FIG. 6 is a CCK8 assay of DMAEM against oral epithelial cells HOK, showing that DMAEM has good biosafety;
fig. 7 is a CCK8 assay of DMAEM acting on mouse fibroblast L929, indicating that DMAEM has good biosafety.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (8)
1. Use of a tertiary amine monomer dodecylmethylaminoethyl methacrylate having pH sensitive antibacterial properties for the preparation of a medicament for inhibiting helicobacter pylori in the mouth and stomach.
2. The use according to claim 1, wherein the MIC after the tertiary amine monomer has been reacted with helicobacter pylori is 8 μg/mL; MBC for H.pylori was 64. Mu.g/mL; the tertiary amine has the effect concentration of more than 8 mug/mL, can effectively inhibit the growth and reproduction of helicobacter pylori and pathogenic effect, and can effectively kill helicobacter pylori when the tertiary amine has the effect concentration of more than 64 mug/mL.
3. A method for detecting the efficacy of a tertiary amine monomer for use according to any one of claims 1 to 2 as a helicobacter pylori-resistant material for non-diagnostic or therapeutic purposes, characterized in that the method comprises:
step one, culturing a flat plate and a liquid culture medium, and determining the minimum inhibitory concentration and the minimum bactericidal concentration of a medicine;
step two, measuring bacterial quantity change under the action of the drug and measuring the influence of the drug on the bacterial virulence level;
and thirdly, detecting the safety of the cells.
4. The method according to claim 3, wherein in the first step, the plate and the liquid medium are cultured in the following steps:
uniformly smearing helicobacter pylori on a CBA solid culture medium by using a 10 mu L disposable inoculating loop, and culturing for 24 hours under a microaerophilic condition at a temperature of 37 ℃ in a carbon dioxide constant-temperature incubator;
the microaerophilic conditions are: 5% oxygen, 10% carbon dioxide and 85% nitrogen;
the minimum bacteriostasis concentration and the minimum bacteriostasis concentration of the medicine are determined, and the specific process is as follows:
scraping fresh thalli from a CBA culture medium by using a 10 mu L disposable inoculating loop, placing the thalli in a BHI serum culture medium, and fully and uniformly mixing by alternately using a gun tip for blowing and a vortex oscillator;
adjusting the OD value of the bacterial liquid to OD600 nm=0.3 by using a multifunctional enzyme-linked detector SpectraMax iD5, and diluting 10 times, wherein the concentration of the bacterial liquid is about 1×10 5 CFU/mL; adding the bacterial liquid into a sterile 96-well plate with the system of 100 mu L, adding 98 mu L of bacterial suspension and 2 mu L of tertiary amine monomer with 2 times of serial dilution, so that the concentration of the medicine is 0, 0.5, 1, 2, 4, 8, 16, 32, 64 and 128 mu g/mL, and placing the mixture into a incubator for culturing for 24 hours; and (3) observing that the lowest drug concentration group with clear bacterial liquid is the MIC of the drug, sucking 2 mu L of bacterial liquid from each hole with the drug action concentration, vertically suspending and dripping the bacterial liquid into a CBA culture medium, culturing for 24 hours, and setting three groups of parallel samples according to the experiment, wherein the minimum drug concentration generated by the sterile colony is MBC of the drug.
5. The method according to claim 3, wherein in the second step, the bacterial load change measurement under the action of the drug is performed by: preparing a standard curve of the bacterial load of the living bacteria and measuring the bacterial load;
the preparation method of the viable bacteria standard curve comprises the following specific steps:
the bacterial cells on the CBA solid culture medium are scraped off by using a sterile 10 mu L disposable inoculating loop and placed in PBS, evenly mixed, and the OD600 nm=1 is regulated, and the bacterial liquid concentration is about 1×10 8 CFU/mL; sequentially 10-fold gradient dilution to obtain 1×10 8 CFU/mL、1×10 7 CFU/mL、1×10 6 CFU/mL、1×10 5 CFU/mL and 1X10 4 CFU/mL concentration bacterial solutions are 1mL each; the experiment sets three groups of parallel samples, and the operation is performed according to the instruction book of the bacterial genome DNA extraction kit; centrifuging the obtained sample for 10 minutes by 5000g, taking the extracted DNA as a template, using a TB Green ™ Premix Ex Taq ™ II kit, adopting a 12.5 mu L System, and running on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument to detect helicobacter pylori 16S rDNA genes; drawing a regression curve by taking the logarithmic value of the bacterial concentration as an x value of an abscissa and the CT value obtained in the last step as a y value of an ordinate to obtain a standard equation y=kx+b, and completing the statistical analysis of the obtained experimental data by using software SPSS 16.0;
the bacterial strain measuring process comprises the following steps: detecting the OD value of bacteria by using a multifunctional enzyme-linked detector SpectraMax iD 5; taking 1mL of bacterial liquid with each drug concentration, extracting bacterial DNA, obtaining CT value through qPCR, and measuring the bacterial quantity of living bacteria through the obtained formula;
the specific process of determining the bacterial virulence of the medicine is as follows:
when the MIC of the tertiary amine monomer to helicobacter pylori is 8 mug/mL and below the MIC, the medicine concentration is 0, 0.5, 1, 2, 4 and 8 mug/mL, the tertiary amine monomer acts on helicobacter pylori and incubate for 24 hours; 1mL of the enriched bacterial liquid is centrifuged for 10 minutes at 5000rpm, and Trizol is resuspended;
extracting bacterial RNA: transferring into a wall breaking tube with zirconia magnetic beads, cooling with liquid nitrogen, and breaking cells in a high-speed tissue refiner; ice bath for 2-5 min, and gradually eliminating bubbles generated in the wall breaking process; 350. Mu.L of "chloroform" was added: isoamyl alcohol, 24:1' solution, vortex oscillator shake for 15 seconds, and stand for 10 minutes; 13000rpm,4 ℃, centrifuging for 15 minutes; pipette 400. Mu.L of the supernatant clear liquid and transfer to a new enzyme-free EP tube; sequentially adding pre-cooled 350 mu L of high-salt solution and 250 mu L of isopropanol, reversing the EP tube up and down for a plurality of times to fully mix, and standing for 10 minutes; 13000rpm,4 ℃, centrifuging for 15 minutes; the supernatant was discarded, centrifuged at 13000rpm at 4℃for 2 minutes, and the supernatant was carefully removed; adding 1mL pre-cooled 70% ethanol solution into the precipitate, reversing the EP tube upside down for several times, centrifuging at 13000rpm and 4 ℃ for 5 minutes; discarding the supernatant, and naturally air-drying the bottom sediment; adding 40-70 mu L of enzyme-free water according to the amount of sediment at the bottom; carrying out water bath at 65 ℃ for 10 minutes, and blowing and uniformly mixing by using an enzyme-free gun tip; the concentration and purity of RNA was determined using a NanoDrop 2000 spectrophotometer;
RNA purification and reverse transcription: the PrimeScript ™ RT reagent Kit with gDNA Eraser kit was used to remove DNA from the RNA samples for purification and subsequent RNA reverse transcription.
6. The method of claim 5, wherein the real-time fluorescent quantitative reverse transcription polymerase chain reaction: setting 4 compound holes in each sample by taking the cDNA after reverse transcription as a template, and adopting a 20 mu L system by using a TB Green ™ Premix Ex Taq ™ II kit; operating on a LightCycler 480 II System real-time fluorescence quantitative PCR instrument to determine reaction conditions; helicobacter pylori housekeeping gene16S rRNAHelicobacter pylori as an internal reference gene using the 2- ΔΔCT method16S rRNA、VacA、CagA、UreaB、 FlaA、LuxSExpression detection of (c).
7. The method according to claim 3, wherein in the third step, the cell safety detection process comprises:
inoculating 1x10 in 96-well plate 4 HOK cells or L929 cells, at 5% CO 2 Incubating the cells in a cell incubator at 37 ℃ overnight; after cell adhesion, the original culture medium is sucked, fresh 100 mu L of DMEM culture medium containing tertiary amine monomers with different concentrations is added, and the cells are incubated in a cell incubator at 37 ℃; after 24h of each group of 6 compound holes, the method usesThe CCK8 kit is used for detecting the cytotoxicity of the cell proliferation.
8. The assay of claim 7, wherein the cell safety assay further comprises: the original culture medium is sucked, PBS is used for washing three times, 100 mu L of DMEM culture medium containing 10% of CCK8 reagent is added, and the culture medium is incubated in a 37 ℃ cell incubator in a dark place; after 1-2 hours, the supernatant is sucked and transferred to a new 96-well plate, and the light absorption value of A450 is read under an enzyme label instrument.
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