CN116875507A - Wessella-J4-1 fusion and application of extracellular polysaccharide thereof in prevention and treatment of colon cancer - Google Patents
Wessella-J4-1 fusion and application of extracellular polysaccharide thereof in prevention and treatment of colon cancer Download PDFInfo
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- CN116875507A CN116875507A CN202310916117.3A CN202310916117A CN116875507A CN 116875507 A CN116875507 A CN 116875507A CN 202310916117 A CN202310916117 A CN 202310916117A CN 116875507 A CN116875507 A CN 116875507A
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- weissella
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- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a Weissella fusion J4-1 and application thereof. The preservation number of the Wessezia J4-1 is CCTCC NO: M20221189, and the Wessezia J4-1 is preserved in China center for type culture Collection. The Weissella fusion J4-1 can secrete exopolysaccharide, and the secreted exopolysaccharide can obviously inhibit proliferation of colon cancer cells, inhibit G0/G1 phase cell cycle progress of the colon cancer cells, and effectively play a role in resisting colon cancer; the extracellular polysaccharide has no influence on the weight and growth state of the mice, has no damage to important organs of the mice, and has high safety. Therefore, the Weissella J4-1 fusion and the exopolysaccharide secreted by the Weissella J4-1 fusion can be used for preparing medicines for preventing, relieving or/and treating colon cancer.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of fusion Weissella J4-1 and extracellular polysaccharide thereof in prevention and treatment of colon cancer.
Background
Colorectal cancer is the third leading fatal cancer worldwide, the next leading cause of cancer death to lung cancer. Colorectal cancer currently has various treatment means for colon cancer, and local treatment comprises endoscope, operation and metastasis ablation. Systemic treatment such as radiotherapy, chemotherapy, targeted therapy immunotherapy, palliative chemotherapy, and the like. The existing treatment schemes and screening means enable the total survival time of patients with colorectal cancer advanced diseases to reach 3 years, but the death rate of the patients is not reduced yet, and the survival rate of Chinese patients, especially male patients, is still lower in 5 years. In addition, the main treatment means such as radiotherapy and targeted therapy have obvious toxic and side effects at present. Therefore, it is of great importance to find new colorectal cancer treatment methods with smaller toxic and side effects. More and more studies indicate that the occurrence and development of colorectal cancer are closely related to intestinal microorganisms. In recent years, it has been gradually confirmed that some intestinal microorganisms can inhibit the growth of colorectal cancer, and thus it has become a research hotspot as to whether colorectal cancer can be treated by supplementing intestinal microorganisms. However, because the living microorganism has high requirements on storage environment, and whether the living microorganism which inhibits colorectal cancer is safe or not, whether the living microorganism opportunistically infects a tumor patient or not has reduced immunity, the demonstration is insufficient, and the clinical application of the living microorganism is limited.
Lactic acid bacteria exopolysaccharide is a carbohydrate macromolecular compound that is excreted outside the cell wall during the bacterial growth and metabolism. The extracellular polysaccharide has high safety, and has the natural advantages of biodegradability, innocuity, good biocompatibility and the like. Extracellular polysaccharide can directly inhibit colorectal cancer by inducing apoptosis, inducing cell cycle arrest, etc. In addition, it can reduce cell canceration by anti-mutation, anti-oxidation, anti-inflammatory, regulating tumor related signal path, etc. In addition, extracellular polysaccharides have an effect of modulating immunity, and can be used as immunomodulators for modulating innate and adaptive immunity. Extracellular polysaccharides promote cell viability and proliferation of various immune cell types including macrophages, dendritic cells, NK cells and lymphocytes, and induce cytokines, enhancing host immune defenses. At present, research on strains producing exopolysaccharide is mainly focused on lactobacillus, but the yield of the exopolysaccharide of the lactobacillus is lower, and the mechanism of inhibiting colorectal cancer is not deep enough, so that the industrialization and clinical application of the exopolysaccharide are limited. Therefore, lactic acid bacteria with high extracellular polysaccharide yield are developed, and the inhibition of colorectal cancer by extracellular polysaccharide is deeply discussed, so that the method has important significance in the application of the method in colorectal cancer prevention and treatment.
Fusion of WeissellaWeissella confusa) Belongs to one of lactic acid bacteria, occupies an important ecological position in nature and is widely used in saliva, breast milk, human gastrointestinal tracts and traditional fermented foods. The extracellular polysaccharide of the fusion Weissella is high in yield and is mainly used as a thickening agent, a stabilizing agent and a gel agent to be applied to foodProcessing, cosmetic and pharmaceutical processing, and the like. The study of the influence of the fusion of Weissella exopolysaccharides on human health has started later and the probiotic properties in human health have yet to be explored further. The reports prove that the extracellular polysaccharide fused with the Weissella has certain effects of resisting oxidation, inhibiting pathogenic bacteria, promoting the growth of lactic acid bacteria and bifidobacteria and regulating immunity, but the application of the Weissella and the extracellular polysaccharide thereof in resisting tumors is not reported at present, and the inhibition of colorectal cancer and related mechanisms by the extracellular polysaccharide fused with the Weissella are not clear.
Disclosure of Invention
The invention aims to provide an application of fusion Weissella J4-1 and extracellular polysaccharide thereof in preventing and treating colon cancer.
In order to achieve the aim of the invention, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a method for fusing Weissella J4-1%Weissella confusaJ4-1), wherein the preservation number of the fusion Weissella J4-1 is CCTCC NO: M20221189, the preservation organization is China Center for Type Culture Collection (CCTCC), and the preservation organization address is the eight 299-path China center for type culture collection of Wuhan university in Wuhan district of Wuhan, hubei province; the date of preservation is 2022, 7 and 27.
The screening and identifying process of the Wessezia J4-1 fusion comprises the following steps: adding healthy infant feces into sterile PBS, shaking for resuspension, collecting supernatant, and gradient diluting to 10 -5 The diluted solution was added to a lactobacillus growth medium MRS liquid medium, and the culture was allowed to stand at 37 ℃ for 48 h. Sucking the bacterial liquid and gradient diluting to 10 -5 The dilutions were then blotted into MRS solid medium and incubated at 37℃until distinct single colonies developed. Single colony is picked into MRS liquid culture medium, after standing culture is carried out for 24 hours at 37 ℃, bacterial liquid of 0.5 ml is taken, centrifugation is carried out for 5 minutes at 4 ℃ and 5000 g, and after supernatant is removed, bacteria sediment is resuspended by adding sterile PBS. Bacteria were subjected to bacterial suspension 16S DNA sequence amplification by using primers (27F: 5 '-AGAGTTTGATCCTGGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'), and the amplified sequences were sent to the department of Biotechnology, inc. for sequencing. Sequencing of 16S rDNAThe result is input into a BLAST database of NCBI for identification, and finally, the separated strain is confirmed to be the fusion Weissella according to homologous sequence analysis, named as the fusion Weissella J4-1, and is preserved in China Center for Type Culture Collection (CCTCC), and the strain preservation number is CCTCC NO: M20221189.
The 16S rDNA sequence fused with Weissella J4-1 is shown as a sequence 1.
In a second aspect, the invention provides the use of a Weissella fusion J4-1 or a Weissella fusion J4-1 culture according to the first aspect for the preparation of a product for the prevention, alleviation or/and treatment of colon cancer.
In a third aspect the present invention provides an extracellular polysaccharide secreted by the above-described Weissella fusion J4-1 of the first aspect.
According to the above extracellular polysaccharide, preferably, the extracellular polysaccharide is obtained by fermenting and culturing the fusion Weissella J4-1, and separating and purifying the fermentation culture solution.
According to the above extracellular polysaccharide, preferably, the extracellular polysaccharide is prepared by: inoculating the Weissella fusion J4-1 to a culture medium for fermentation culture to obtain a culture solution, and removing thalli and protein from the culture solution to obtain a supernatant; and (3) carrying out alcohol precipitation treatment on the supernatant, collecting precipitate, and carrying out dialysis impurity removal and freeze-drying treatment on the precipitate to obtain the extracellular polysaccharide.
According to the extracellular polysaccharide described above, preferably, the medium is an MRS medium or an MRS medium.
According to the extracellular polysaccharide described above, preferably, the mrs medium has a composition of: 10g/L of Peptone (pepone), 10g/L of Beef extract (Beef extract), 5g/L of Yeast extract (Yeast extract), 1ml/L of Tween 80 (Tween 80), 2g/L of dipotassium hydrogen phosphate, 5g/L of diammonium citrate, 5g/L of anhydrous sodium acetate, 0.2g/L of magnesium sulfate, 0.05g/L of manganese sulfate, 100g/L of sucrose and the balance of water; the fermentation culture temperature is 30-37 ℃. More preferably, the culture temperature is 37 ℃.
According to the above extracellular polysaccharide, preferably, the dialysis cut-off molecular weight is 8000 to 14000 Da.
According to the above extracellular polysaccharide, preferably, the alcohol precipitation treatment is to add absolute ethanol into the supernatant, mix them uniformly and then stand at 4 ℃ for 12-18 hours.
According to the extracellular polysaccharide described above, preferably, the specific operation of protein removal is: adding trichloroacetic acid into the culture solution after removing thalli, uniformly mixing, standing at 4 ℃ for 12-18 h, centrifuging, and removing precipitate. More preferably, the trichloroacetic acid is used in an amount of: the mass volume percentage concentration of trichloroacetic acid in the culture solution is 4%.
According to the above extracellular polysaccharide, preferably, the specific operation of removing the bacterial cells is: centrifuging the culture solution to remove the thalli. More preferably, the centrifugation conditions are: centrifuge at 12000g for 30min at 4 ℃.
In a fourth aspect, the present invention provides the use of an exopolysaccharide according to the third aspect above in the manufacture of a product for preventing, alleviating or/and treating colon cancer.
In a fifth aspect, the present invention provides a medicament for preventing, treating and/or alleviating colon cancer, the medicament comprising an active ingredient which is a culture of the above-described first aspect of Weissella fusion J4-1 or an extracellular polysaccharide as described in the above-described second aspect.
According to the above medicament, preferably, the medicament further comprises pharmaceutically acceptable excipients.
According to the above-mentioned drugs, preferably, the drugs are administered by oral administration or parenteral administration. More preferably, the parenteral administration is intravenous, subcutaneous, intramuscular, intraperitoneal, topical, intratumoral or intrarectal.
Compared with the prior art, the invention has the positive beneficial effects that:
(1) The invention screens and obtains the fusion WeissellaWeissella confusa J4-1（W. confusaJ4-1) can secrete exopolysaccharide, the secreted exopolysaccharide can obviously inhibit proliferation of colon cancer cells, inhibit G0/G1 phase cell cycle progress of colon cancer cells, and effectively developThe effect of resisting colon cancer is achieved; the extracellular polysaccharide has no influence on the weight and growth state of the mice, has no damage to important organs of the mice, and has high safety.
(2) The Weissella food D-2 is screened from the intestinal tract of a healthy infant, and the extracellular polysaccharide of the metabolite is also a natural component generated in the metabolic process of lactic acid bacteria, so that compared with other antitumor drugs, the extracellular polysaccharide fused with Weissella J4-1 is safer from the clinical application point of view.
Drawings
FIG. 1 is a schematic view ofW. confusaScreening a J4-1 fermentation medium and extracting extracellular polysaccharide; wherein A isW. confusaJ4-1 exopolysaccharide secretion photograph on MRS solid culture plate; b isW. confusa J4-1 exopolysaccharide secretion photographs on mMRS solid culture plates, C is exopolysaccharide freeze-dried sample photographs;
FIG. 2 is a graph showing the result of scanning electron microscopy fused with Weissella exopolysaccharide;
FIG. 3 is a graph showing the results of ultraviolet spectral scanning analysis of an extracellular polysaccharide fused with Weissella;
FIG. 4 is a graph showing the results of an infrared spectroscopic scan analysis of an extracellular polysaccharide fused with Weissella J4-1;
FIG. 5 is a graph showing the results of HPLC analysis of the extracellular polysaccharide fused with Weissella J4-1;
FIG. 6 is a graph showing the results of weight measurement of mice in each experimental group;
FIG. 7 is a graph showing the results of tumor volume change detection in mice of each experimental group; wherein, A is a tumor physical image of 5 mice in each experimental group; b is a tumor volume change data statistical result graph;
FIG. 8 is a graph of H & E staining of heart, liver, spleen, lung and kidney tissue of mice of each experimental group;
FIG. 9 is a CCK8 experimental studyW. confusaExperimental results of inhibition of colon cancer cell proliferation by J4-1 exopolysaccharide;
FIG. 10 is a diagram ofW. confusaCell clone formation result experimental graphs and data statistical graphs after J4-1 exopolysaccharide treatment of human colon cancer cells HT29 (A) and SW480 (B);
FIG. 11 is FIG. 11W. confusaJ4-1 extracellular polypResults of studies of the effect of sugar on colon cancer cell cycle HT29 (a), SW480 (B); wherein, the Control group represents a Control group, and EPSJ4-1 represents a J4-1 exopolysaccharide treatment group;
FIG. 12 is a graph of Western blot experiment results; wherein, the Control group represents a Control group, EPSJ4-1 represents a J4-1 exopolysaccharide treatment group, A is a protein detection result of a mutant P53, B is a protein detection result of a P21, C is a protein detection result of CDK2, and D is a protein detection result of cyclin E1.
Description of the embodiments
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
The following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. Furthermore, it should be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, components, and/or groups thereof.
The experimental methods in the following examples, in which specific conditions are not specified, are all conventional in the art or according to the conditions suggested by the manufacturer; the reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: fusion of WeissellaW. confusaAcquisition and characterization of J4-1
Adding healthy infant feces into sterile PBS, shaking for resuspension, collecting supernatant, and gradient diluting to 10 -5 The diluted solution was added to a lactobacillus growth medium MRS liquid medium, and the culture was allowed to stand at 37 ℃ for 48 h. Sucking the bacterial liquid and gradient diluting to 10 -5 The dilutions were then blotted into MRS solid medium and incubated at 37℃until distinct single colonies developed. Single colony was picked to MRAnd (3) in the S liquid culture medium, standing at 37 ℃ for culturing for 24 hours, taking bacterial liquid of 0.5 ml, centrifuging at 4 ℃ for 5 minutes at 5000 g, removing the supernatant, and adding sterile PBS to resuspend bacterial sediment.
Bacteria were subjected to bacterial suspension 16S DNA sequence amplification by using primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'), and the amplified sequences were sequenced by the same were sent to the department of Prinsepia Biotechnology Co. Sequencing the obtained fusion WeissellaW. confusaThe 16S rDNA sequence of J4-1 is shown in SEQ ID NO. 1.
Inputting the sequencing result of 16S rDNA into BLAST database of NCBI for identification, and finally confirming the separated strain to be the fusion Weissella according to homologous sequence analysis, named as the fusion Weissella J4-1%W. confusaJ4-1), and preserving the strain in China Center for Type Culture Collection (CCTCC), wherein the strain is preserved under the CCTCC NO: M20221189.
Example 2:W. confusaacquisition of J4-1 extracellular polysaccharide
1、W. confusaOptimization of J4-1 fermentation Medium and extracellular polysaccharide acquisition:
by means of flat scribingW. confusaJ4-1 is respectively streaked on an MRS solid culture plate and an mMRS solid culture plate, and then the solid culture plate is put at 37 ℃ for culturing 24-48 hours for observationW. confusaExopolysaccharide secretion of J4-1 on MRS solid culture plates, mMRS solid culture plates. The experimental results are shown in figure 1 as A, B.
As can be seen from FIG. 1, A, B, the fusion Weissella J4-1 exopolysaccharide grown in mMRS medium was more viscous than the fusion Weissella J4-1 exopolysaccharide grown in MRS medium, indicating an increased yield of fusion Weissella J4-1 exopolysaccharide. Therefore, mMRS pairs are preferredW. confusaJ4-1 is fermented.
Will beW. confusaThe J4-1 freezing tube is placed in a 37 ℃ water bath for melting, a sterile inoculating loop is dipped with 1-2 drops of bacteria liquid, the three-partition streak method is used for inoculation, the mixture is placed in a 37 ℃ constant temperature incubator for 48 hours, and single colonies can be observed. The single colony obtained by picking the micro gun head is inoculated into 5ml of liquid MRS culture medium for culture, the culture condition is that the culture is kept stand for 24 hours at 37 ℃,the bacterial solution was observed to become cloudy. Inoculating the bacterial liquid with an inoculum size of 2% (v/v) into a conical flask of an improved MRS liquid culture medium (mMRS culture medium), and standing at 30 ℃ for culturing for 48 hours until the culture medium is sticky, thus obtaining bacterial fermentation liquor. Boiling the bacterial liquid in a water bath at 100 ℃ for 15min to achieve the aim of inactivating bacteria and enzymes, and cooling to room temperature. If the bacterial liquid is viscous, the sterilized deionized water can be used for diluting the bacterial liquid. Centrifuging the boiled bacterial liquid in a high-speed refrigerated centrifuge at 12000g and 4 ℃ for 30min, and discarding the precipitate such as bacterial cells to obtain supernatant. Adding 80% (m/v) trichloroacetic acid (TCA) to the supernatant to obtain final concentration of trichloroacetic acid of 4% (m/v) to remove protein, standing in a refrigerator at 4deg.C for 12h, centrifuging at 12000g for 30min at 4deg.C with a high-speed refrigerated centrifuge, collecting supernatant, and repeating the steps for 2-3 times to remove protein impurities as much as possible. The absolute ethyl alcohol is pre-cooled at 4 ℃ in advance, three times of volume (v/v) of cold absolute ethyl alcohol is added into supernatant fluid after impurities such as protein and the like are removed, the supernatant fluid is kept stand for 12h in a 4 ℃ refrigerator, and the purpose is that extracellular polysaccharide is precipitated by alcohol, and precipitation is obtained by centrifugation for 30min at 4 ℃ and 12000 g. And re-dissolving the precipitate in pure water, and repeating the alcohol precipitation step for 2-3 times. After the alcohol precipitation is finished, the extracellular polysaccharide obtained by the alcohol precipitation is redissolved, and then is dialyzed (dialysis cut-off molecular weight is 8000-14000 Da) for 48-72 h under the environment of 4 ℃ to remove impurities, and water is changed every 8h. And (3) further performing vacuum freeze drying on the dialyzed polysaccharide solution to obtain extracellular polysaccharide freeze-dried powder (shown as C in figure 1). As can be seen from FIG. 1C, the extracellular polysaccharide lyophilized powder is a fluffy dry white flocculent solid.
Example 3:Weissella confusacharacterization of J4-1 extracellular polysaccharide
Dissolving proper amount of the extracellular polysaccharide lyophilized powder fused with Weissella with ethanol, ultrasonic cleaning, sampling, infrared drying, metal spraying (10 nm), sizing, and the like, and fixing under an electron microscope for observation. The surface microscopic morphology of the extracellular polysaccharide lyophilized powder was observed at 1000, 2000, 5000, 10000 times magnification under an accelerating voltage of 5.0 kV. The scanning electron microscope results are shown in fig. 2.
As can be seen from FIG. 2, the extracellular polysaccharide fused with Weissella J4-1 forms a porous compact lamellar.
Accurately weighing a proper amount of the frozen powder of the extracellular polysaccharide fused with the Weissella, dissolving the frozen powder in ultrapure water, preparing samples with different concentrations of 200 mug/ml, 400 mug/ml and 1000 mug/ml, then scanning characteristic absorption peaks in a wavelength range of 200-700 nm by using a spectrophotometer, and observing the ultraviolet absorption condition of the extracellular polysaccharide derived from the extracellular polysaccharide fused with the Weissella at different wavelengths. The results are shown in FIG. 3.
As can be seen from FIG. 3, the ultraviolet spectrum scanning result shows that the ultraviolet absorption spectrum curve of the extracellular polysaccharide fused with the Weissella J4-1 is smooth and flat, no obvious absorption peaks are found at the wavelengths of 260 nm and 280nm, and no protein and no nucleic acid exist in the extracellular polysaccharide sample fused with the Weissella J4-1.
Accurately weighing a proper amount of lyophilized powder of extracellular polysaccharide fused with Weissella, mixing with potassium bromide according to a certain proportion, compressing the mixed particles by a potassium bromide conventional (KBr) tabletting method, and measuring a sample at 4000cm by using a Fourier infrared spectrometer -1 -500cm -1 Absorption spectrum in the range. The results are shown in FIG. 4.
As can be seen from FIG. 4, the flow rate is 4000-500cm −1 Spectra in the wavelength range revealed a vibration of functional groups in the extracellular polysaccharide fused with Weissella J4-1. 3420cm −1 The broad and strong peak is the characteristic peak of the stretching vibration of the hydroxyl O-H in the saccharide component, and is the typical characteristic of saccharide substances. 2923cm −1 And 1655cm −1 The sharp peaks at the absorption peaks at the positions correspond to the stretching vibration of the c—h single bond and the stretching vibration of the c=o double bond in the sugar ring, respectively. 1424cm −1 And 1346cm −1 The absorption peak in between is due to the variable angle vibration of the CH bond. At 1000 cm −1 And 1200 cm −1 The absorption peak in between is considered as the fingerprint region of the polysaccharide, and the broad absorption peak at 1157 and 1023 of the polysaccharide is caused by C-OH linkage stretching vibration and C-O-C glycosidic linkage stretching vibration of the sugar ring, which also indicates that the polysaccharide contains a pyranose residue. At 912cm −1 The peak at this point was caused by asymmetric ring stretching vibration of the pyran ring, again demonstrating the presence of the polysaccharide pyran ring saccharide. At 619cm −1 The peak at 890 cm indicates the presence of a glycosidic bond −1 No peak was present at this point, confirming thatThe beta-configuration of the glycosidic bond exists at 846cm -1 Is caused by stretching vibration of the alpha-anomeric carbon, indicating the alpha configuration of the glycosidic bond in the polysaccharide. Thus, FT-IR pattern indicated that the extracellular polysaccharide fused with Weissella J4-1 had a pyranose residue and an alpha-glycosidic bond.
And separating different monosaccharide components through a chromatographic column, analyzing the monosaccharide components in different samples, and quantitatively analyzing the monosaccharide content through an external standard. And taking a proper amount of reference substances such as mannose, arabinose, ribose, rhamnose, galacturonic acid, glucuronic acid, N-acetyl-glucosamine, glucose, N-acetyl-galactosamine, xylose, fucose, galactose and the like, adding water to dissolve and dilute the reference substances, and mixing the reference substances with the concentration of 50 mug/ml respectively. Mu.l of the mixed control solution was pipetted into a 5ml EP tube, 250. Mu.l of 0.6 mol/l NaOH and 0.4mol/l PMP-methanol 500. Mu.l at 70℃for 1h. Ice water bath for 10 minutes; 500. mu.l (0.3 mol/l) HCl was neutralized, 1ml chloroform was swirled for 1 minute, centrifuged at 3000r for 10 minutes, and the supernatant was extracted 3 times. The supernatant was used for HPLC analysis.
The extracellular polysaccharide freeze-dried powder which is derived from the fusion Weissella J4-1 is precisely weighed, 5.0mL of TFA (2 mol/l) is added into about 5mg to 10mL of ampere, a tube is sealed, and acidolysis is carried out at 120 ℃ for 4 hours. The TFA was removed by blowing with methanol nitrogen and redissolved in 5.0ml of water. Mu.l of the sample solution was taken into a 5mL EP tube. The following is the same.
The chromatographic conditions for HPLC analysis were: the instrument is Shimadzu LC-20AD, the chromatographic column is Xtime C18.6 x 200mm 5 mu M, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the detection wavelength is 250nm, the sample injection amount is 20ul, and the mobile phase is 0.05M potassium dihydrogen phosphate solution (pH is regulated to 6.70 by sodium hydroxide solution) -acetonitrile.
The detection results are shown in FIG. 5.
As can be seen from FIG. 5, the result of high performance liquid chromatography shows that the extracellular polysaccharide fused with Weissella J4-1 is dextran.
Example 4: animal experiment verificationW. confusaAnti-colorectal cancer efficacy of J4-1 exopolysaccharides
SPF grade 4-5 week female BALB/C nudle female nude mice were selected and weighing about 14-17g, and the experimental groups were an intratumoral injection PBS group (NC group), an intratumoral injection Low-dose extracellular polysaccharide group (Low-dose group) and an intratumoral injection High-dose extracellular polysaccharide group (High-dose group), each group of 5 mice.
And (3) digesting the HT29 colon cancer cell line by using pancreatin, centrifuging at 1000rpm for 5min, collecting cells, and re-suspending the HT29 colon cancer cells by using PBS to obtain HT29 colon cancer cell suspension. 100. Mu.L of HT29 colon cancer cell suspension (5X 10) was subcutaneously injected in the right armpit of BALB/C nudle nude mice 6 Individual cells/individual) into tumors.
NC group: after the nude mice subcutaneous tumor model is constructed, the tumor volume reaches 200mm 3 About, 100 μl of PBS was injected intratumorally every 4 days, and continued until the end of the experiment.
Low-dose group and High-dose group: after the nude mice subcutaneous tumor model is constructed, the tumor volume reaches 200mm 3 About, both groups were injected intratumorally with 100 μl of extracellular polysaccharide solution every 4 days. Wherein, each nude mouse in the Low-dose group is injected with 1.5 mg of extracellular polysaccharide in each tumor, and each nude mouse in the high-dose group is injected with 2.5. 2.5 mg of extracellular polysaccharide in each tumor.
4、W. confusaEffect of J4-1 exopolysaccharide on mouse growth conditions:
after the nude mice subcutaneous tumor model is molded, feeding, mental state, activity response and the like of the mice in the NC group, the Low-dose group and the High-dose group are closely observed until the experimental end point.
As a result, NC mice were found to be normal in feeding and defecating, good in activity, maintained or slightly reduced in body weight during the experiment, and not dead; the Low-dose mice have normal feeding and defecation, good activity, and no death due to weight maintenance or slight decrease during the experiment; mice in the High-dose group were fed and defecating normally, were well-behaved, remained weight-maintaining or slightly declined during the experiment, and did not die.
5、W. confusaEffect of J4-1 exopolysaccharide on mouse body weight and tumor volume:
the weight and tumor size of the mice were weighed every other day after the molding of the subcutaneous tumor model of the nude mice, and the weight and tumor volume of the mice were measured daily after each experimental group administration.
The calculation formula of the tumor volume is: tumor volume (mm++3) = (l×s++2)/2, where: l is the tumor mass length; s is the short diameter of the tumor mass.
The results of the measurement of the body weight of the mice are shown in FIG. 6.
As can be seen from FIG. 6, at the end of the experiment, there was no significant difference between the weights of the Low-dose group and the High-dose group compared with the NC group, indicating that the Low-dose and High-dose of the Wissella J4-1 exopolysaccharide fusion had no significant effect on the weights of the mice.
The results of the detection of tumor volumes in mice are shown in FIG. 7.
As can be seen from fig. 7, the tumor volume of NC mice increased from 263 cubic millimeters prior to administration to about 1149 cubic millimeters; the average tumor volume of the Low-dose mice before administration is 369 cubic millimeters, the average tumor volume at the time of sacrifice is 787 cubic millimeters, and the growth speed is obviously lower than that of the NC mice; the tumor volume of the High-dose mice increases from 408 cubic millimeters to 700 cubic millimeters, and with the increase of the number of doses, the cumulative dose is 5 mg/dose after the 7 th or 2 nd dose, the tumor volume stops increasing and is maintained, the cumulative dose is 7.5 mg/dose after the 3 rd dose, the tumor volume starts to shrink, and the tumor volume at the time of sacrifice is 469 cubic millimeters. Therefore, compared with NC group, the tumor volume of the mouse with tumor is obviously reduced when the Weissella J4-1 extracellular polysaccharide group is fused by the Low-dose group and the High-dose group, and the well tumor inhibiting effect of the Weissella J4-1 extracellular polysaccharide is proved.
6、W. confusaDamage condition research of J4-1 exopolysaccharide on heart, liver, spleen, lung and kidney tissues of mice:
after 4 times of intratumoral injection, the mice are sacrificed, heart, liver, spleen, lung and kidney tissues of the mice are collected and treated, and the mice are placed in tissue fixing liquid for fixing for 24-48 hours. The tissue structure changes of important organs such as heart, liver, spleen, lung, kidney and the like are analyzed through H & E staining. The results are shown in FIG. 8.
As can be seen from FIG. 8, at the end of the experiment, the mice in the Low-dose group and the High-dose group had normal morphology of heart, liver, spleen, lung and kidney tissues, and were not significantly different from those in the NC group. In combination with the above weight data, it is thus demonstrated that the low and high doses of the fusion Weissella J4-1 exopolysaccharide have no significant toxic or side effects on the weight and tissue organs of mice.
Example 5: cell experiment verificationW. confusaAnti-colorectal cancer efficacy of J4-1 exopolysaccharides
Cell line: colon cancer cell lines HT29 and SW480 cells, SW480 cells were cultured in DMEM high sugar complete medium, HT29 was cultured in RPMI 1640 complete medium.
The experimental grouping included: experimental group (EPSJ 4-1 group) and Control group (Control group). The experimental group (EPSJ 4-1 group) cells are added with J4-1 exopolysaccharide, the J4-1 exopolysaccharide group is added with exopolysaccharide with different concentrations according to different functional tests, and the control group is not added with exopolysaccharide.
2、W. confusaStudy of inhibition of proliferation of colon cancer cells by J4-1 exopolysaccharide:
(1) CCK8 experiment:
the experimental method specifically comprises the following steps: SW480 cells and HT29 cells with good growth state were taken, and when the monolayer of cells reached more than 80% of the bottom area, the resuspended cells were digested and counted. 100 μl of cell suspension is added to each well of a 96-well plate, 1000 cells/well is incubated, after observing the cell wall, the 96-well plate is removed and old culture medium is removed by pipetting, 100 μl of complete culture medium containing J4-1 extracellular polysaccharide at corresponding sample concentrations (0.2, 0.4, 0.8, 1.0) is added to each well, and 3-6 multiplex wells are made for each concentration. Incubating for 72 hours in an incubator; mu.L of CCK8 solution was added to each well and incubated in the incubator for 1-3 hours in the dark. Absorbance at 450nm was measured with a full wavelength microplate reader.
The experimental results are shown in FIG. 9.
As can be seen from FIG. 9, SW480 cell proliferation rate showed a concentration-dependent inhibition effect when treated with the extracellular polysaccharide of Wessezia J4-1 for 72 hours, wherein the cell proliferation rate was 57.13% when the extracellular polysaccharide of Wessezia J4-1 was 1mg/mL, which is significantly lower than that of the control group. In the HT29 cell line, when the concentration of extracellular polysaccharide of the fusion Weissella J4-1 is 1mg/mL, the cell proliferation rate is 71.29% which is obviously lower than that of the control group. Thus, it is demonstrated that the fusion of Weissella J4-1 exopolysaccharide significantly inhibits proliferation of colorectal cancer cells SW480, HT29, exhibiting a degree of concentration dependence and time dependence.
(2) Cloning experiments:
taking SW480 and HT29 colon cancer cells growing in log phase and having good state, spreading 6 pore plates after pancreatin digestion, growing for 12h by adherence, adding 2ml polysaccharide solution with working concentration of 1mg/ml into each pore of experimental group, adding 2ml complete culture medium into control group cells, and placing in 5% CO 2 In the incubator, the culture was carried out at 37℃for 72 hours. After 72h, each well of cells was essentially confluent with 80% of the bottom of the well, pancreatin digested, cell counted, EPS treated colon cancer cell line (HT 29, SW 480) was inoculated into 12 well plates as experimental group, complete medium treated cells as control group, each well containing 1000 cells, and the experimental and control groups of cells were cultured with complete medium for 2 weeks. After the cells grew into single clones (. Gtoreq.50 cells), the old culture solution was aspirated, washed once with PBS, 1ml of 4% paraformaldehyde was added, and the mixture was fixed for 20min. The fixative was discarded, washed once with PBS, 1ml of crystal violet was added and stained for 15min. Recovering crystal violet, flushing with flowing water and drying. The 12-well plate was back-snapped onto a white background plate, photographed, and the number of clones counted. Image J was used to count the number of clones. Clone formation rate = number of clones/number of inoculated cells x 100%.
The experimental results are shown in FIG. 10.
As can be seen from FIG. 10, the fusion of Weissella J4-1 exopolysaccharide significantly inhibited the clonogenic capacity of SW480 and HT29 cells compared to the complete medium treated control group.
3、W. confusaStudy of the effect of J4-1 exopolysaccharide on colon cancer cell cycle:
SW480 cells and HT29 cells with good states are taken, 2mL of complete medium containing 1mg/mL of J4-1 exopolysaccharide is added as an experimental group, and 2mL of complete medium is added to cells of a control group, and the cells are co-cultured for 72 hours. Pancreatin digestion was performed to collect 6-well plate cells, 1ml of pre-chilled ethanol was added to resuspend the cells, standing was performed at 4℃for 12h, and centrifugation was performed at 1200 rpm for 5min to pellet the cells. The supernatant was discarded and the fixed cells were washed 1-2 times with 1ml pre-chilled PBS. To each sample was added 500. Mu.l of propidium iodide staining solution to resuspend the cells, stained at 4℃for 30min in the absence of light, and detected on-line within 24 hours.
The experimental results are shown in FIG. 11.
As can be seen from FIG. 11, after 1mg/mL of J4-1 exopolysaccharide was used to treat HT29 cells and SW480 cells for 72 hours, the ratio of G0/G1 phase of the control group of HT29 cells was 58.72%, the experimental group was 63.09%, and the exopolysaccharide blocked the G0/G1 phase of HT29 cells. Among SW480 cells, the proportion of cells in the G0/G1 phase of the experimental group was 53.07% higher than 33.84% of the control group, the S phase of the control group was 47.33%, the 35.44% of the experimental group, the G2 phase of the control group was 18.72% of the experimental group, and the 11.42% of the experimental group. Thus, J4-1 exopolysaccharides block mainly the G0/G1 phase of SW 480. Thus, it was demonstrated that the fusion of Weissella J4-1 exopolysaccharide can inhibit proliferation of cancer cells by blocking the G0/G1 phase of colorectal cancer cells.
4. Western blot experimental studyW. confusaEffect of related protein expression in J4-1 extracellular polysaccharide cell cycle arrest mechanism:
taking HT29 cells with good state, adding 2mL of complete culture medium containing 1mg/mL J4-1 exopolysaccharide as an experimental group, adding 2mL of complete culture medium into cells of a control group, and co-culturing for 72h. According to 99:1 mixing RIPA and PMSF in proportion to prepare a lysate, adding 500 mu L of the lysate into each well, reacting on ice for 30min to fully lyse the protein, centrifuging at 12000rpm for 30min at 4 ℃, and taking the supernatant. The BCA method detects the protein concentration and levels, and after adding 1/4 (5×) loading buffer, mixing well, heating at 100deg.C for 5min, and thoroughly denaturing the protein. The expression of related proteins (mutant P53, P21, CDK2 and cyclin E1) in the anti-colon cancer cell proliferation mechanism was detected by Western blot assay at-20 ℃. The specific operation of the Western blot experiment is a conventional technology in the field, and is not described in detail herein.
The experimental results are shown in FIG. 12.
As can be seen from FIG. 12, 1mg/mL of J4-1 exopolysaccharide was used to treat HT29 cells for 72 hours, and J4-1 group cell mutans P53, P21, CDK2 and cyclin E1 were highly expressed compared to control group HT29 cells. Thus, it was demonstrated that the fusion of Weissella J4-1 exopolysaccharide inhibited cell cycle and thus colon cancer cell proliferation via the P53/P21/CDK2 axis.
In conclusion, the invention effectively overcomes the defects in the prior art and has high industrial utilization value. The above-described embodiments are provided to illustrate the gist of the present invention, but are not intended to limit the scope of the present invention. It will be understood by those skilled in the art that various modifications and equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A fusion Weissella J4-1, which is characterized in that the nucleic acid sequence of the fusion Weissella J4-1 contains a sequence shown in a sequence 1.
2. The Weissella fusion J4-1 of claim 1, wherein the Weissella fusion J4-1 has a preservation number of CCTCC NO: M20221189.
3. An extracellular polysaccharide secreted by the fusion weissella J4-1 of claim 1 or 2.
4. The extracellular polysaccharide according to claim 3, wherein the extracellular polysaccharide is obtained by fermenting and culturing the fusion Weissella J4-1, and separating and purifying the fermentation culture solution.
5. The extracellular polysaccharide according to claim 4, wherein the extracellular polysaccharide is prepared by a method comprising: inoculating the Weissella fusion J4-1 of claim 1 to a culture medium for fermentation culture to obtain a culture solution, and removing thalli and removing proteins from the culture solution to obtain a supernatant; and (3) carrying out alcohol precipitation treatment on the supernatant, collecting precipitate, and carrying out dialysis impurity removal and freeze-drying treatment on the precipitate to obtain the extracellular polysaccharide.
6. The extracellular polysaccharide according to claim 5, wherein the culture medium is MRS medium or MRS medium; the fermentation culture temperature is 30-37 ℃; the dialysis cut-off molecular weight is 8000-14000 Da.
7. The extracellular polysaccharide according to claim 5, wherein the alcohol precipitation treatment is to add absolute ethanol into the supernatant, mix them uniformly and then stand at 4 ℃ for 12-18 h; the specific operation of protein removal is as follows: adding trichloroacetic acid into the culture solution after removing thalli, uniformly mixing, standing at 4 ℃ for 12-18 h, centrifuging, and removing precipitate.
8. Use of a Weissella fusion J4-1 according to claim 1 or 2 or a Weissella fusion J4-1 culture or an extracellular polysaccharide according to any one of claims 3 to 7 for the preparation of a product for the prevention, alleviation or/and treatment of colon cancer.
9. A medicament for preventing, treating and/or alleviating colon cancer, which comprises an active ingredient and a pharmaceutically acceptable auxiliary material, wherein the active ingredient is the culture of the Weissella fusion J4-1 or Weissella fusion J4-1 according to claim 1 or 2 or the extracellular polysaccharide according to any one of claims 3 to 7.
10. The medicament of claim 9, wherein the medicament is administered by oral administration or parenteral administration.
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