CN110840884A - Application of prodigiosin in preparation of cell proliferation inhibitor for lymphangiomatosis - Google Patents
Application of prodigiosin in preparation of cell proliferation inhibitor for lymphangiomatosis Download PDFInfo
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- CN110840884A CN110840884A CN201911219524.9A CN201911219524A CN110840884A CN 110840884 A CN110840884 A CN 110840884A CN 201911219524 A CN201911219524 A CN 201911219524A CN 110840884 A CN110840884 A CN 110840884A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
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Abstract
The invention discloses application of prodigiosin in preparation of a lymphangiomyomatosis cell proliferation inhibitor, and relates to the field of biological medicines. The method prepares the prodigiosin through a biological engineering technology, observes the change of cell morphology by using a microscope, measures the change of cell activity by a cck-8 method, observes the inhibition effect of the prodigiosin on the proliferation of a lymphangiosarcomatosis cell Tsc2null, and discovers that within 24h, the inhibition effect of the prodigiosin on the Tsc2null is obviously higher than that within Tsc2wt and 2h, the cell activity of the Tsc2null is in inverse proportion to the concentration of the prodigiosin, the higher the concentration of the prodigiosin is, the lower the cell activity is, the prodigiosin has obvious inhibition effect on the proliferation of the lymphangiomatosis cell, and the prodigiosin can be widely applied to the preparation of a proliferation inhibitor of the lymphangiomatosis cell or a proliferation medicine for resisting the lymphangiomatosis cell.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to application of prodigiosin in preparation of a cell proliferation inhibitor for lymphangiomyomatosis.
Background
Lymphangiomyomatosis (LAM) is a rare disease mainly caused by diffuse cystic lung disease, mainly occurs in women in the reproductive age, and the pathology is mainly characterized by abnormal proliferation and infiltration of smooth muscle-like cells. LAM has various clinical manifestations, and can affect kidney, lymphatic vessels, lymph nodes, spleen, liver, etc. besides characteristic cystic changes in the lung. Studies have shown that LAM is derived from mutations in the TSC1/TSC2 gene in patients, with TSC2 being the more common. TSC is a tumor suppressor gene, a TSC-LAM patient has mutation of a TSC gene at one site in nature, and after birth, the TSC gene at the second site is obtained again to cause disease. After the 2 allelic TSC (particularly TSC2) genes are completely inactivated, the inhibition is lost, and then the downstream mTOR pathway is abnormally activated, so that the LAM cell is stimulated to abnormally grow, proliferate, migrate and metastasize, and the structural damage and the dysfunction of the organ systems in the lung and outside the lung are caused.
Prodigiosin (Prodigiosin) is a red antibiotic found in various bacteria and actinomycetes, and the skeleton of the Prodigiosin is characterized by containing a large conjugated system of a three-pyrrole ring, so that the Prodigiosin has various activities such as cancer resistance, fungus resistance, bacteria resistance, protozoa resistance, immunosuppression and the like. The antitumor activity of prodigiosin has been of particular interest in recent years, and the average IC of prodigiosin has been found by the national cancer institute (NSI) Melvin et al50At 2.1. mu. mol/L, it was found to be resistant to 57 different human cancer cells and induce apoptosis. At present, the inhibitory effect of prodigiosin on the proliferation of LAM disease cells Tsc2null has not been studied.
Disclosure of Invention
Aiming at the technical defects, the invention aims to provide the application of prodigiosin in preparing a cell proliferation inhibitor for lymphangiomyopathy, and the prodigiosin has obvious inhibition effect on the proliferation of LAM disease cells Tsc2 null.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
application of prodigiosin in preparing cell proliferation inhibitor for lymphangiomatosis is provided.
Further, the lymphangiosarcomatosis cell is a Tsc2null cell.
Further, the concentration of the rubicin in the inhibitor is 0.01-100 mu mol/L.
Application of prodigiosin in preparing medicines for inhibiting cell proliferation of lymphangiomatosis is provided.
Further, the preparation method of the prodigiosin comprises the following steps: centrifuging the serratia marcescens culture solution, and separating supernatant and precipitate; leaching the lower layer precipitate with methanol, and centrifuging to obtain haematochrome; rotary evaporating with rotary evaporator to obtain dark purplish red pigment methanol solution, and processing with evaporator to obtain filtrate C18And (4) performing reversed-phase semi-preparative HPLC (high performance liquid chromatography) repeated purification, dissolving the finally obtained pigment in absolute ethyl alcohol, and performing vacuum low-temperature high-speed freeze-drying.
Has the advantages that: the invention prepares the prodigiosin by a biological engineering technology, observes the change of cell morphology by using a microscope, measures the change of cell activity by a cck-8 method, observes the inhibition effect of the prodigiosin on the proliferation of a LAM disease cell Tsc2null, and discovers: within 24h, the inhibition effect of prodigiosin on Tsc2null is obviously higher than that of Tsc2wt, within 2h, the cell activity of Tsc2null is in inverse proportion to the concentration of prodigiosin, the higher the concentration of prodigiosin, the lower the cell activity, the obvious inhibition effect on the proliferation of lymphatic vessel sarcoidosis cells is achieved, and the prodigiosin can be widely applied to the preparation of proliferation inhibitors of lymphatic vessel sarcoidosis cells or proliferation drugs for resisting the lymphatic vessel sarcoidosis cells.
Drawings
FIG. 1 is a graph showing the inhibitory effect of prodigiosin at various concentrations for 24h on Tsc2null cells;
FIG. 2 is a graph showing the inhibitory effect of prodigiosin at various concentrations of 48h on Tsc2null cells;
FIG. 3 is a graph showing the inhibitory effect of prodigiosin at various concentrations for 72h on Tsc2null cells;
FIG. 4 is a graph showing the inhibitory effect of prodigiosin at various concentrations for 24h on Tsc2wt cells;
FIG. 5 is a graph showing the inhibitory effect of prodigiosin at various concentrations for 48h on Tsc2wt cells;
FIG. 6 is a graph showing the inhibitory effect of prodigiosin at various concentrations for 72h on Tsc2wt cells;
FIG. 7 is a graph showing absorbance values of Tsc2null cells measured by the CCK-8 method after various treatments;
FIG. 8 is a graph showing the absorption values of Tsc2wt cells measured by the CCK-8 method after various treatments.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
Example 1
1) Separation, purification and preparation of prodigiosin
Serratia marcescens (S.marcocens) NJZT-1 is picked from a glycerol storage tube, coated on LB solid medium by using a plate marking method for activation, and cultured in an incubator at 26 ℃ for 3 days. Activated serratia marcescens single colonies are picked and transferred to another solid culture medium, and cultured in a thermostat at 26 ℃ for 3 days. Repeating the transfer for 2-3 times to obtain pure culture. A loop of the culture was picked from the solid medium, inoculated into a liquid medium, and cultured at 26 ℃ for 3 days with shaking at 180 r/min. Centrifuging the culture solution, and separating supernatant and precipitate; leaching the lower layer precipitate with methanol, centrifuging at 9000r/min for 10min, and separating to obtain haematochrome. And (5) carrying out rotary evaporation by using a rotary evaporator to obtain the dark purplish red element methanol solution. Posterior warp of C18And (3) performing reversed phase semi-preparative HPLC (high performance liquid chromatography) repeated purification, dissolving the finally obtained pigment in absolute ethyl alcohol, performing vacuum low-temperature high-speed freeze-drying, and storing at the temperature of minus 20 ℃ in a dark place.
2) Cell culture
Tsc2null (mutant)/Tsc 2 WildType (wild type) cells were cultured in DMEM basal medium (10% FBS, 1% penicillin-streptomycin diabody) (DMEM medium, penicillin-streptomycin diabody, trypsin, Fetal Bovine Serum (FBS) were purchased from Gibco, USA), and passed through digestion with 0.25% trypsin under 5% CO2Incubator (SANYO), 37 ℃ culture.
3) CCK-8 method cytotoxicity assay
Cells in logarithmic growth phase were seeded in 96-well plates in 100. mu.L cell suspension per well in 5% CO2The culture was carried out in an incubator at 37 ℃ for 24 hours. Adding 0.01-100 μmol/L prodigiosin to treat, each group has 6 parallel holes, and the two groups have different concentrationsAnd adding anhydrous ethanol and DMEM basic culture medium into the control group respectively in the same amount, adding 10 mu LCCK-8 reagent (CCK-8 kit purchased from Nanjing institute of bioengineering) into each hole after culturing for 72h, incubating in an incubator for 1h, and measuring absorbance at the wavelength of 450 nm.
As shown in fig. 1 to 6, the inhibitory effect of different concentrations of prodigiosin on Tsc2null cells and Tsc2wt cells was observed using inverted phase contrast microscopy (Leica) at 24h, 48h, and 72h, respectively. With increasing prodigiosin concentration, the inhibitory effect on Tsc2null cells and Tsc2wt cells was also increased. Meanwhile, the solvent absolute ethyl alcohol of the prodigiosin also has certain inhibiting effect on Tsc2null cells and Tsc2wt cells.
The CCK-8 method measures absorbance of differently treated Tsc2null cells as shown in FIG. 7: after 24h, each treatment can inhibit the Tsc2null cells to different degrees, and DMSO is used as a candidate solvent of prodigiosin, so that the inhibition effect on the Tsc2null cells is stronger than that of absolute ethyl alcohol; after 48 hours, the inhibitory effect of the prodigiosin with different concentrations on the Tsc2null cells reaches more than half; after 72h, the inhibitory effect of prodigiosin on Tsc2null cells tends to be stable.
The CCK-8 method measures absorbance of Tsc2wt cells after various treatments, as shown in FIG. 8: after 24h, each treatment does not produce obvious inhibition on the Tsc2wt cells, and DMSO has stronger inhibition effect on the Tsc2wt cells as an alternative solvent of prodigiosin than absolute ethyl alcohol; after 48 hours, the inhibitory effect of the prodigiosin with different concentrations on the Tsc2wt cells reaches more than half; after 72h, the inhibitory effect of prodigiosin on Tsc2wt cells was still increased.
In conclusion, the inhibitory effect of prodigiosin on Tsc2null is obviously higher than that of Tsc2wt within 24 h. Taking 1 mu mol/L as an example, the inhibitory effect of prodigiosin on Tsc2null is 2.1 times that of Tsc2wt cells. After 24h, the prodigiosin has different degrees of inhibition on the two cells, the inhibition effect is obvious, and the difference between the two cells is small. Within 72h, the activity of the Tsc2null and Tsc2wt cells is inversely proportional to the concentration of prodigiosin, and the higher the concentration of prodigiosin, the lower the activity of the cells. The cell morphology is observed under a microscope, and the higher the concentration of the prodigiosin, the fewer the attached cells are, and the number of round cells and death is increased.
It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (5)
1. Application of prodigiosin in preparing cell proliferation inhibitor for lymphangiomatosis is provided.
2. The use of claim 1, wherein the lymphangiosarcomatosis cells are Tsc2null cells.
3. The use as claimed in claim 1, wherein the concentration of the rubicin in the inhibitor is 0.01-100 μmol/L.
4. Application of prodigiosin in preparing medicines for inhibiting cell proliferation of lymphangiomatosis is provided.
5. The use according to any one of claims 1 to 4, characterized in that the prodigiosin is prepared by a process comprising: centrifuging the serratia marcescens culture solution, and separating supernatant and precipitate; leaching the lower layer precipitate with methanol, and centrifuging to obtain haematochrome; rotary evaporating with rotary evaporator to obtain dark purplish red pigment methanol solution, and processing with evaporator to obtain filtrate C18And (3) performing reverse phase semi-preparative HPLC (high performance liquid chromatography) repeated purification, dissolving the pigment in absolute ethyl alcohol, and performing vacuum low-temperature high-speed freeze-drying to obtain the pure prodigiosin.
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| CN201911219524.9A CN110840884A (en) | 2019-11-29 | 2019-11-29 | Application of prodigiosin in preparation of cell proliferation inhibitor for lymphangiomatosis |
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| CN201911219524.9A CN110840884A (en) | 2019-11-29 | 2019-11-29 | Application of prodigiosin in preparation of cell proliferation inhibitor for lymphangiomatosis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115093356A (en) * | 2022-06-21 | 2022-09-23 | 广东医科大学 | Preparation method and application of iron death inducer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115093356A (en) * | 2022-06-21 | 2022-09-23 | 广东医科大学 | Preparation method and application of iron death inducer |
| CN115093356B (en) * | 2022-06-21 | 2024-02-23 | 广东医科大学 | Preparation method and application of iron death inducer |
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