CN114085792A - Lactobacillus paracasei for preventing and treating colon cancer and application thereof - Google Patents
Lactobacillus paracasei for preventing and treating colon cancer and application thereof Download PDFInfo
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- CN114085792A CN114085792A CN202111363912.1A CN202111363912A CN114085792A CN 114085792 A CN114085792 A CN 114085792A CN 202111363912 A CN202111363912 A CN 202111363912A CN 114085792 A CN114085792 A CN 114085792A
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Abstract
The lactobacillus paracasei is lactobacillus paracasei PC-H1 with the preservation number of CGMCC NO.22285 and is preserved in China general microbiological culture Collection Center (CCM) in 2021, 5 months and 7 days. The extracellular vesicles prepared by the lactobacillus paracasei PC-H1 provided by the invention have the capacity of inhibiting migration and invasion of HCT116 cells, SW1116 cells and SW620 cells, effectively induce colon cancer cells to generate apoptosis, obviously inhibit the growth of nude mouse transplanted tumors, and have good clinical application prospects.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to lactobacillus paracasei for preventing and treating colon cancer and application thereof.
Background
Colon cancer is a common malignancy, and the risk of developing colon cancer tends to increase due to increased intake of high-fat and high-sugar foods, decreased intake of dietary fiber, and the increasing year by year of overweight people. Surgery and chemotherapy remain the most effective treatment strategies as traditional anti-cancer therapies, but have poor clinical effects on colon cancer, and most patients receiving chemotherapy develop drug resistance. Therefore, there is an urgent need to find alternative anti-cancer drugs to improve future treatment of colon cancer.
Probiotics, especially lactobacilli, play an important role in maintaining the micro-ecological balance, preventing and treating various diseases. Probiotics belong to natural intestinal microbiota and can be planted in human intestinal tracts and have a health effect on human intestinal tracts. In addition, probiotics can prevent and treat colon cancer by promoting the balance of intestinal microbiota, producing anti-cancer substances, reducing intestinal inflammation and reducing the production of harmful enzymes.
Lactobacillus paracasei, a common probiotic, has adhesive properties that inhibit the proliferation of colon cancer cells in a time and dose dependent manner. In addition, the peptidoglycan component of the cell wall of lactobacillus paracasei can induce apoptosis of colon cancer cells to exert anticancer effect. Bacterial extracellular vesicles are membrane vesicles that are released from cells to the extracellular matrix, have diameters in the range of 20-400nm, and interact with immune cells to modulate inflammatory responses; modulating expression of endothelial cell adhesion molecules; stimulates the expression of the tight junction protein in the intestinal epithelial cells to enhance the intestinal barrier function and participates in intercellular communication as a natural nano-carrier. The bacterial extracellular vesicles are widely applied to a tool for delivering antitumor drugs due to the modifiability, high-efficiency loading capacity and natural tumor targeting property of the bacterial extracellular vesicles. The extracellular vesicles released by the probiotic bacteria may interact with the host to allow for cell-to-cell communication. It has been found that extracellular vesicles of lactobacillus paracasei reduce the extent of inflammation in mice with acute colitis induced by dextran sodium sulfate by means of increasing endoplasmic reticulum stress. Current research on lactobacillus paracasei extracellular vesicles has also focused primarily on the treatment of gastrointestinal inflammation.
Chinese patent 201910799557.9 discloses Lactobacillus paracasei (MIT-16) and its application in canine tumor immunotherapy. 1 lactobacillus paracasei MIT-16 with higher acid-producing capability is separated and screened from intestinal tracts of mice, has better acid resistance and stronger bacteriostatic capability, has obvious immunoregulation function, and especially has important effect on immunoregulation in tumor treatment. The strains are useful as immunomodulatory biotherapeutics. However, when the strain provided by the invention is applied to tumor inhibition, the strain needs to be combined with CAR-T cells, so that the application difficulty is increased.
Chinese patent 201980087214.8 discloses a pharmaceutical composition for preventing or treating inflammatory diseases or cancer, which comprises extracellular vesicles derived from lactobacillus paracasei as an active ingredient. But the drug effect of the compound is poor in the anti-cancer effect experiment of mice, so that the compound has a further optimized space.
Disclosure of Invention
In order to solve the problems, the invention separates 120 strains of lactic acid bacteria from the intestinal tract of a healthy person, and screens out lactobacillus paracasei which can resist colon cancer activity; the lactobacillus living preparation developed by the lactobacillus paracasei has the advantages of safety, reliability, less adverse reaction, good effect and the like, can inhibit the proliferation, migration and invasion of colon cancer cells, and is beneficial to human health.
In one aspect, the invention provides a microorganism.
The microorganism is Lactobacillus paracasei PC-H1, which is classified and named as Lactobacillus paracasei PC-H1 with the preservation number of CGMCC NO.22285, is preserved in China general microbiological culture Collection center of China Committee for Culture Collection of Microorganisms (CCM) within 5 months and 7 days in 2021, and the preservation address is the institute of microbiology of China academy of sciences No.3 of the North American district, West Lu No.1 of the Beijing city.
The lactobacillus paracasei PC-H1 provided by the invention is screened from healthy human fecal samples without signs of intestinal inflammation, tumor and the like in clinical detection, and the results are as follows after the separation and the colony morphology identification: the size is about 0.5-1.0mm, the colony edge is neat and has a bulge, gram staining is positive, the cell shape under a microscope is mostly short rod-shaped, and the size and the shape are stable.
The 16S rDNA sequence of the Lactobacillus paracasei PC-H1 provided by the invention is shown in SEQ ID NO.1, and the classification and the name of the Lactobacillus paracasei PC-H1 are confirmed to be Lactobacillus paracasei (Lactobacillus paracasei).
The invention also provides a culture method of the lactobacillus paracasei PC-H1, which comprises the steps of activating the lactobacillus paracasei PC-H1 and then inoculating the activated lactobacillus paracasei PC-H1 to a culture medium.
In another aspect, the invention provides a culture of lactobacillus paracasei.
The culture is obtained by inoculating lactobacillus paracasei PC-H1 in a culture medium for culture.
The culture medium includes but is not limited to MRS liquid culture medium, MC liquid culture medium, BCP culture medium.
The culture can be fermentation liquor of lactobacillus paracasei PC-H1.
The invention also protects the extract obtained by extracting the lactobacillus paracasei culture.
In yet another aspect, the present invention provides a product.
Specifically, the product comprises the viable bacteria of the lactobacillus paracasei PC-H1 and/or a preparation thereof.
More specifically, the preparation is the dead bacteria, culture extract, cell disruption product, fermentation supernatant, fermentation sediment, extracellular vesicles, modified bacteria, mutant bacteria and/or mutant bacteria of the lactobacillus paracasei PC-H1.
Preferably, the product is a live bacterial preparation.
In some embodiments, the viable bacteria preparation is a solid preparation, and the content of the lactobacillus paracasei PC-H1 is 1.0 x 105-9.9×1010CFU/g；
Preferably, the content of the lactobacillus paracasei PC-H1 is 1.0X 105-1.0×106CFU/g、1.0×106-1.0×107CFU/g、1.0×107-1.0×108CFU/g、1.0×108-1.0×109CFU/g、1.0×109-1.0×1010CFU/g、1.0×105-1.0×109CFU/g or 1.0X 1010-9.9×1010CFU/g; more preferably 2X 106CFU/g。
When the living bacteria reagent is a liquid or semi-solid preparation, the content of the lactobacillus paracasei is 1.0 multiplied by 105-9.9×1010CFU/mL；
Preferably, the content of the lactobacillus paracasei PC-H1 is 1.0 x 105-1.0×106CFU/mL、1.0×106-1.0×107CFU/mL、1.0×107-1.0×108CFU/mL、1.0×108-1.0×109CFU/mL、1.0×109-1.0×1010CFU/mL、1.0×105-1.0×109CFU/mL or 1.0X 1010-9.9×1010CFU/mL; more preferably 2X 106CFU/mL。
In yet another aspect, the invention provides an extracellular vesicle.
Specifically, the extracellular vesicles are prepared by the aforementioned Lactobacillus paracasei PC-H1.
In yet another aspect, the present invention provides a gene encoding a protein.
In particular, the gene can encode a protein in the aforementioned extracellular vesicles.
In another aspect, the invention provides a method for preparing extracellular vesicles of lactobacillus paracasei.
Specifically, the preparation method comprises the following steps:
(1) inoculating and culturing the lactobacillus paracasei PC-H1 bacterial liquid;
(2) centrifuging the cultured bacterial liquid at a low speed, collecting the precipitate, resuspending the precipitate with PBS, and inoculating the resuspended bacterial liquid into a culture medium for culturing;
(3) centrifuging the bacterial liquid cultured in the step (2) at a low speed and collecting supernatant;
(4) centrifuging the collected supernatant at low speed again to remove larger impurities;
(5) taking the supernatant obtained in the step (4) and filtering bacteria;
(6) transferring the filtered supernatant into an ultracentrifuge tube in batches, discarding the supernatant after ultracentrifugation, and collecting the precipitate obtained after each ultracentrifugation;
(7) after ultracentrifuging all the supernatant, collecting the collected liquid containing all the precipitates in an ultracentrifuge tube for secondary ultracentrifugation;
(8) discarding the supernatant, adding PBS to wash the precipitate from the ultracentrifuge tube, and ultracentrifuging again;
(9) the PBS was discarded and the resulting pellet was the extracellular vesicle of Lactobacillus paracasei PC-H1.
Specifically, the low-speed centrifugation conditions in the steps (2), (3) and (4) are that the rotating speed is less than or equal to 8000r/min or the relative centrifugal force is less than or equal to 10000 g.
Preferably, the rotation speed of the low-speed centrifugation is 3000g, 3500g, 3800g, 4000g, 4500g, 5000g, 5500g, 5700g, 6000g, 6500g, 7000g, 7500g or 8000 g.
In some embodiments, the low speed centrifugation in steps (2), (3) and (4) is performed at 4500g at 4 ℃ for 15 min.
Specifically, the ultracentrifugation conditions in the steps (6), (7) and (8) are not less than 60000 r/min.
Preferably, the ultracentrifugation conditions are: 60000r/min, 65000r/min, 68000r/min, 70000r/min, 75000r/min, 76000r/min, 80000r/min, 90000r/min, 95000r/min, 100000r/min, 105000r/min, 110000r/min, 115000r/min, 118000r/min, or 120000 r/min.
In some embodiments, the ultracentrifugation in steps (6), (7) and (8) is performed under conditions of 100000r/min and 60 minutes at 4 ℃.
In some embodiments, the preparation method comprises the following steps:
(1) respectively inoculating 1mL of lactobacillus paracasei PC-H1 bacterial liquid into 8 45mL of MRS culture media, putting the MRS culture media into a constant temperature oscillator at 37 ℃, and performing shake culture for 12 hours at 210 rpm;
(2) centrifuging the cultured bacterial liquid at 4500g and 4 ℃ for 15min, collecting the precipitate, resuspending the precipitate with 10mL of PBS, respectively taking 1mL of the resuspended bacterial liquid, inoculating the bacterial liquid into 10 MRS culture media with 300mL, and carrying out constant-temperature oscillation culture at 37 ℃ and 210rpm for 12 h;
(3) respectively putting 3000mL of lactobacillus paracasei PC-H1 bacterial liquid into 50mL centrifuge tubes, centrifuging at 4500g4 ℃ for 15min, and collecting supernatant;
(4) centrifuging the collected supernatant again at 4500g and 4 deg.C for 15min to remove larger impurities;
(5) after two times of low-speed centrifugation, a syringe absorbs the supernatant fluid and filters the supernatant fluid into a sterile conical flask by using a 0.45 mu m filter membrane, meanwhile, 20 mu L of the filtered supernatant fluid is smeared on an MRS agar plate and is put into a constant-temperature incubator at 37 ℃ for 12 hours, and whether bacteria are filtered out or not is detected by observing the growth of the bacteria on the MRS agar plate;
(6) transferring the filtered supernatant into an ultracentrifuge tube in batches, centrifuging at 100,000r/min and 4 ℃ for 60 minutes, removing the supernatant, and collecting the precipitate obtained after each ultracentrifuge;
(7) after ultracentrifugation of all supernatants, the collected liquid containing all precipitates was collected in an ultracentrifugation tube for final ultracentrifugation;
(8) discarding the supernatant, adding 2mL PBS to wash the precipitate from the ultracentrifuge tube, filling to 38mL with PBS, 100,000r/min, and centrifuging at 4 ℃ for 60 minutes;
(9) the PBS is discarded, the obtained precipitate is the extracellular vesicle of the lactobacillus paracasei PC-H1, the obtained extracellular vesicle is resuspended by 200 microliter PBS buffer solution, and the suspended extracellular vesicle is stored in a refrigerator at the temperature of minus 80 ℃ after being subpackaged.
In yet another aspect, the invention provides a lyophilized powder.
Specifically, the freeze-dried powder comprises embedding medium and the thallus of the lactobacillus paracasei PC-H1.
Specifically, the embedding medium comprises 7-10% of yeast extract powder, 8-9% of skim milk powder, 7-8% of soybean protein isolate, 6-7% of trehalose and 0.5-2% of sodium D-isoascorbate.
Preferably, the embedding medium comprises 9 mass percent of yeast extract powder, 8.4 mass percent of skim milk powder, 7.4 mass percent of isolated soy protein, 6.8 mass percent of trehalose and 1 mass percent of sodium D-isoascorbate.
Specifically, the bacterium content in the freeze-dried powder is 2 multiplied by 108-2×1012CFU/g。
Preferably, the bacterium content in the freeze-dried powder is 2 x 108-2×109CFU/g、2×109-2×1010CFU/g、2×1010-2×1011CFU/g、2×1011-2×1012CFU/g、2×108-2×1010CFU/g、2×109-2×1011CFU/g、2×1010-2×1012CFU/g。
In still another aspect, the present invention provides an application of the aforementioned microorganisms, products, extracellular vesicles, genes and/or lyophilized powder in the preparation of drugs, health products, foods, nutriments, medical devices and daily chemical products for preventing, treating, assisting in treating and/or prognostically nursing tumor diseases and/or for gastrointestinal conditioning.
In particular, the tumors include, but are not limited to: digestive system tumor, blood system tumor, craniocerebral tumor, respiratory system tumor, urinary system tumor, endocrine system tumor, reproductive system tumor, circulatory system tumor, skeleton tumor, skin tumor, and soft tissue tumor.
Preferably, the tumors of the digestive system include, but are not limited to: intestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, hepatocarcinoma, oral cancer, and pharyngeal cancer.
Further preferably, the intestinal cancer includes, but is not limited to: rectal cancer, colon cancer, duodenal cancer, cecum cancer, and appendiceal cancer.
In yet another aspect, the present invention provides a medical configuration.
Specifically, the pharmaceutical preparation comprises the microorganism, the product, the extracellular vesicles and/or the lyophilized powder.
Preferably, the pharmaceutical formulation is an oral drug.
Such oral pharmaceutical dosage forms include, but are not limited to: capsule, tablet, powder, granule, emulsion, suspension, pill, and powder.
Optionally, the medicinal preparation is solid, and the content of Lactobacillus paracasei PC-H1 in the medicinal preparation is 1.0 × 105-9.9×1010CFU/g；
Preferably, the content of the lactobacillus paracasei PC-H1 in the medical preparation is 1.0 x 105-1.0×106CFU/g、1.0×106-1.0×107CFU/g、1.0×107-1.0×108CFU/g、1.0×108-1.0×109CFU/g、1.0×109-1.0×1010CFU/g、1.0×105-1.0×109CFU/g or 1.0X 1010-9.9×1010CFU/g; more preferably 2X 106CFU/g。
Optionally, the pharmaceutical preparation is liquid or semisolid, and the content of Lactobacillus paracasei in the pharmaceutical preparation is 1.0 × 105-9.9×1010CFU/mL；
Preferably, the content of Lactobacillus paracasei PC-H1 in the medical preparation is 1.0 x 105-1.0×106CFU/mL、1.0×106-1.0×107CFU/mL、1.0×107-1.0×108CFU/mL、1.0×108-1.0×109CFU/mL、1.0×109-1.0×1010CFU/mL、1.0×105-1.0×109CFU/mL or 1.0X 1010-9.9×1010CFU/mL; more preferably 2X 106CFU/mL。
In some embodiments, the pharmaceutical formulation further comprises other pharmaceutically acceptable carriers or excipients.
The pharmaceutical carriers or excipients include, but are not limited to: adhesive, filler, disintegrating agent, thickening agent, preservative, antioxidant, flavoring agent, aromatic, cosolvent, emulsifier, solubilizer, osmotic pressure regulator and colorant.
Preferably, the pharmaceutical preparation is an injection, and the injection comprises the extracellular vesicles in an amount of 200 μ g/mL.
In another aspect, the invention provides a health product.
Specifically, the health care product comprises the microorganisms, the products, the extracellular vesicles and/or the freeze-dried powder.
The health care product can be used for daily gastrointestinal conditioning.
In yet another aspect, the present invention provides a food product.
The food comprises the microorganisms, products, extracellular vesicles and/or freeze-dried powder.
The food includes but is not limited to dairy products, beverages and snack foods.
In still another aspect, the present invention provides a daily chemical article.
The daily chemical product comprises the microorganisms, products, extracellular vesicles and/or freeze-dried powder.
The daily chemical articles comprise but are not limited to cleaning articles and maintenance articles.
The invention has the beneficial effects that:
the extracellular vesicle prepared by the lactobacillus paracasei PC-H1 provided by the invention has the capacity of inhibiting the migration and invasion of HCT116 cells, SW1116 cells and SW620 cells, effectively induces colon cancer cells to generate apoptosis, and obviously inhibits the growth of nude mouse transplanted tumors. Compared with the existing lactobacillus paracasei CGMCC1.9089 and CGMCC1.121, the extracellular vesicle concentration of 200 mug/mL can obviously inhibit cell proliferation of three colon cancer cells, namely HCT116, SW1116 and SW620, and the activity of the colon cancer cells is obviously reduced.
Deposit description
Chinese academic name: lactobacillus paracasei PC-H1
Latin literature name: lactobacillus paracasei PC-H1
The preservation number is: CGMCC NO.22285
Preservation time: 2021, 5 months and 7 days
The preservation unit: china general microbiological culture Collection center
The preservation address is as follows: beijing, Chaoyang district, Beichen Xilu institute of academy of sciences of China, No.3
Drawings
FIG. 1 is a diagram showing the result of morphological identification of Lactobacillus paracasei PC-H1, wherein A is a colony of Lactobacillus paracasei PC-H1, and B is the result of gram-staining of Lactobacillus paracasei PC-H1.
FIG. 2 is a transmission electron micrograph of Lactobacillus paracasei PC-H1 extracellular vesicles.
FIG. 3 is a graph showing the distribution of the diameter size of the extracellular vesicle particles of Lactobacillus paracasei PC-H1.
FIG. 4 shows the extracellular vesicle entry of Lactobacillus paracasei PC-H1 into colon cancer cells.
FIG. 5 is a graph showing the effect of Lactobacillus paracasei PC-H1 extracellular vesicles on the inhibition of colon cancer cell proliferation.
FIG. 6 shows that Lactobacillus paracasei PC-H1 extracellular vesicles inhibited colon cancer cell migration and invasion.
FIG. 7 is a graph showing that Lactobacillus paracasei PC-H1 extracellular vesicles induce apoptosis of colon cancer cells HCT 116.
FIG. 8 is a graph showing that Lactobacillus paracasei PC-H1 extracellular vesicles induce apoptosis of colon cancer cells SW 1116.
FIG. 9 is a graph showing that Lactobacillus paracasei PC-H1 extracellular vesicles induce apoptosis of colon cancer cells SW 620.
FIG. 10 is a graph showing the effect of Lactobacillus paracasei PC-H1 extracellular vesicles on inhibiting the growth of transplanted tumors in nude mice with human colon cancer.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Basic Experimental example 1 isolation of Lactobacillus paracasei Strain
The MRS medium comprises the following components: peptone 10.0 g; 10.0g of beef extract; 5.0g of yeast extract; 2.0g of diammonium hydrogen citrate; 20.0g of glucose; tween 80, 1.0 mL; sodium acetate (CH)3COONa·3H2O)5.0 g; dipotassium hydrogen phosphate (K)2HPO4·3H2O)2.0 g; magnesium sulfate (MgSO)4·7H2O)0.58 g; manganese sulfate (MnSO)4·H2O)0.25 g; 1000mL of distilled water; adjusting pH to 6.2-6.6.
The separation steps are as follows:
(1) 120 samples of feces of healthy people without intestinal inflammation and tumor signs in clinical detection are collected, numbered in sequence, inoculated in MRS liquid culture medium and subjected to anaerobic culture at 37 ℃ for 24 hours.
(2) Randomly selecting 5 single colonies from each sample, culturing and enriching the single colonies in 2mL of MRS liquid culture medium for 24h, and numbering the colonies in sequence.
(3) The amplified bacteria are used for bacterial conservation and DNA preparation. Identifying strains of hundreds of separated bacteria respectively; and (3) detecting the capability of the supernate for inhibiting the colon cancer cells, and screening one strain of lactobacillus paracasei with the strongest colon cancer cell inhibition, namely PC-H1.
Basic Experimental example 2 identification of Lactobacillus paracasei Strain
The strain PC-H1 selected in basic Experimental example 1 was identified as follows:
(1) morphological identification: on MRS lactobacillus culture medium plate, the separated strain PC-H1 shows about 0.5-1.0mm in size, regular colony edge, raised, positive gram staining, and short rod-like cell shape under microscope, as shown in A in FIG. 1; gram staining positive, short rod shape, can be connected into chain, see figure 1B.
(2)16S rDNA sequence homology analysis
The strain PC-H1 obtained by the separation is cultivated by a conventional method, the total DNA of the strain is extracted to be used as a gene amplification template, and the conserved region of the 16S rDNA gene of the bacteria is amplified by adopting universal primers 27F (SEQ ID NO.2) and 1492R (SEQ ID NO. 3).
The amplification system (25. mu.L) was: 1 XPCR reaction buffer, 200 mu mol/L dNTPs, upstream and downstream primers of 0.2 mu mol/L, 1U Taq DNA polymerase, 1 mu L template DNA. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 40s, and extension at 72 ℃ for 30s for 25 cycles; extension at 72 ℃ for 10 min. The PCR product was detected by 1% gel electrophoresis, and positive results were subjected to bidirectional sequencing using universal primers 27F and 1492R. Sequence splicing and similarity analysis are completed by using DNAStar software, and sequence alignment is completed on line by NCBI database (http:// www.ncbi.nlm.nih.gov) of the national center for biotechnology information and is determined to be lactobacillus paracasei.
The sequence of 16S rDNA of the strain PC-H1 is shown in SEQ ID NO. 1.
Through the identification, the PC-H1 is preserved in the China general microbiological culture Collection center (CGMCC NO. 22285) at the 5 th and 7 th months of 2021, and is classified and named as Lactobacillus paracasei (Lactobacillus paracasei).
Example 1A method for preparing Lactobacillus paracasei PC-H1 extracellular vesicles
The method comprises the following steps:
(1) respectively inoculating 1mL of lactobacillus paracasei PC-H1 bacterial liquid into 8 45mL of MRS culture media, putting the MRS culture media into a constant temperature oscillator at 37 ℃, and carrying out shaking culture for 12H at 210 rpm;
(2) centrifuging the cultured bacterial liquid at 4500g and 4 ℃ for 15min, collecting precipitates, re-suspending the precipitates by using 10mL PBS, respectively inoculating 1mL of the re-suspended bacterial liquid into 10 300mL MRS culture media, and carrying out constant-temperature oscillation culture at 37 ℃ and 210rpm for 12 h;
(3) 3000mL of lactobacillus paracasei PC-H1 bacterial liquid is respectively put into 50mL centrifuge tubes, centrifuged for 15min at the temperature of 4500g4 ℃, and supernatant fluid is collected;
(4) centrifuging the collected supernatant again at 4500g4 deg.C for 15min to remove larger impurities;
(5) after two times of low-speed centrifugation, a syringe absorbs the supernatant fluid and filters the supernatant fluid into a sterile conical flask by using a 0.45 mu m filter membrane, meanwhile, 20 mu L of the filtered supernatant fluid is smeared on an MRS agar plate and is put into a constant-temperature incubator at 37 ℃ for 12 hours, and whether bacteria are filtered out or not is detected by observing the growth of the bacteria on the MRS agar plate;
(6) transferring the filtered supernatant into an ultracentrifuge tube in batches, centrifuging at 100,000r/min and 4 ℃ for 60 minutes, removing the supernatant, and collecting the precipitate obtained after each ultracentrifuge;
(7) after ultracentrifugation of all supernatants, the collected liquid containing all precipitates was collected in an ultracentrifugation tube for final ultracentrifugation;
(8) discarding the supernatant, adding 2mL PBS to wash the precipitate from the ultracentrifuge tube, filling to 38mL with PBS, 100,000r/min, and centrifuging at 4 ℃ for 60 minutes;
(9) the PBS is discarded, the obtained precipitate is the extracellular vesicle of the lactobacillus paracasei PC-H1, the obtained extracellular vesicle is resuspended by 200 microliter PBS buffer solution, and the suspended extracellular vesicle is stored in a refrigerator at the temperature of minus 80 ℃ after being subpackaged.
Example 2 identification of Lactobacillus paracasei PC-H1 extracellular vesicles
Culturing lactobacillus paracasei PC-H1 in a liquid culture medium, centrifuging at low speed to obtain a bacterium culture supernatant, filtering the bacterium supernatant by using a filter membrane to remove thalli, and separating extracellular vesicles from the bacterium culture supernatant by using an ultracentrifugation method. The extracted extracellular vesicles were resuspended in 10-20 μ L PBS, the resuspended extracellular vesicles were fixed with 2.5% glutaraldehyde and placed on a 300 mesh copper mesh, the copper mesh was stained with 2% uranyl acetate, and morphological characteristics of the extracellular vesicles were observed under a transmission electron microscope. Morphological features of the extracellular vesicles were observed under transmission electron microscopy at next 50,000, 100,000 and 150,000 magnifications, see fig. 2.
Diluting the extracellular vesicles with 1 XPBS, performing on-machine detection on a sample by using a ZetaView nanoparticle tracking analyzer, tracking the Brownian motion of single extracellular vesicles through ZetaView 8.04.02 software, and calculating the hydrodynamic diameter and concentration of the extracellular vesicles by combining a Stockes-Einstein equation. The diameter size distribution of the extracellular vesicle particles was characterized by nanoparticle tracer analysis and the concentration of the particles was found to peak at approximately 200nm, indicating that the diameter size of the extracellular vesicle particles was mainly concentrated in the range of approximately 200nm and substantially coincided with the results observed under a transmission electron microscope, see fig. 3.
Example 3 Lactobacillus paracasei PC-H1 extracellular vesicles are able to enter colon cancer cells
The Lactobacillus paracasei PC-H1 extracellular vesicles used in this example were prepared by the method described in example 1.
HCT116 cells used in this example were purchased from ATCC and received CCL-247; SW1116 cells were purchased from ATCC with the accession number CCL-233; SW620 cells were purchased from ATCC under the accession number CCL-227.
The general experimental procedure for the three different cells was as follows:
(1) completely digesting the cells in the culture bottle, adding DMEM culture solution, repeatedly blowing and beating the digested cells to make the cells detached and made into cell suspension, and centrifuging at 800r/min for 5 min.
(2) The supernatant was discarded, an appropriate amount of medium was added for resuspension, and 20. mu.L of cells were aspirated to the counting area and counted in a cell counter. Adjusting the cell density to 7X 104one/mL.
(3) The cells were allowed to settle in a confocal cuvette by adding 1mL of the cell suspension adjusted to densityUniformly distributing in a small dish, putting the confocal small dish into a container with the temperature of 37 ℃ and the content of CO of 5 percent2The cell culture chamber of (1) was cultured overnight.
(4) And adding 50-100 mu g of extracellular vesicles into 300 mu L of incubation buffer solution, and uniformly mixing.
(5) To the mixture was added 3. mu.M to 10. mu.M of ExoGlow RNA probe (purchased from System Biosciences, USA, under the trade name EXOGR800A-1), mixed well, and incubated at 37 ℃ for 1 hour in the absence of light.
(6) Free probe was removed using a PD SpinTrap G-25 exchange column as follows:
a. the liquid suspended in PD SpinTrap G-25 was vortexed.
b. Loosening screw cap and unscrewing bottom closing device, placing exchange column into collection tube, centrifuging at 800g for 1min, and discarding storage solution in collection tube.
c. Add 400. mu.L PBS to the exchange column, centrifuge at 800g for 1min, replace the new collection tube, repeat this step 4 times.
d. 100-180. mu.L of the sample was slowly added to the middle of the packed bed of the exchange column, centrifuged at 800g for 2min, and the liquid in the collection tube was collected.
(7) The cell culture medium in the confocal dish was discarded, 600. mu.L of the cell culture medium was added to the mixture of labeled extracellular vesicles, mixed uniformly and slowly added to the confocal dish.
(8) The confocal dish was placed in a cell culture chamber for 48h and observed under a confocal microscope.
The results showed that fluorescently labeled extracellular vesicles were taken up by HCT116 cells, SW1116 cells and SW620 cells, and that extracellular vesicles of lactobacillus paracasei PC-H1 were able to enter colon cancer cells, see fig. 4.
Example 4 Lactobacillus paracasei PC-H1 extracellular vesicles inhibit colon cancer cell migration and invasion
The Lactobacillus paracasei PC-H1 extracellular vesicles used in this example were prepared by the method described in example 1.
1. The cell migration detection comprises the following specific steps:
(1) digesting the cells in the culture bottle completely, and centrifuging to removeRemoving the cell culture fluid. Resuspend cells with serum-free medium and adjust cell density to 8X 104one/mL.
(2) 200 μ L of cell suspension was slowly added to the upper chamber of the transwell chamber. Control and experimental groups were set, and the experimental group treated the cells with 200. mu.g/mL of extracellular vesicles.
(3) 600 μ L of DMEM cell culture medium containing 10% FBS was added to the lower chamber of the transwell plate. The transwell plate was placed in a cell incubator for 48 h.
(4) After 48h incubation, the chamber was removed, the liquid in the upper chamber was blotted with a cotton swab and fixed with 4% paraformaldehyde at room temperature for 20-30 min.
(5) The chamber was soaked with PBS wash and the upper chamber was stained with 0.1% crystal violet for 5-10 min. After staining, the chamber was washed three times with PBS, removed and the cells inside the chamber were gently wiped with a cotton swab.
(6) After the chamber was dried, 5-10 fields were randomly selected under an optical microscope for photographing and counting.
2. The cell invasion detection method specifically comprises the following steps:
(1) the Matrigel stored at-20 ℃ was allowed to stand overnight at 4 ℃ in advance to become liquid.
(2) The Matrigel was diluted on ice at a ratio of 1:5 in serum-free cell DMEM medium in advance, and after mixing, 50 μ L of the mixture was spread evenly in the upper chamber of a transwell chamber, and the plate was placed in a 37 ℃ incubator until the Matrigel polymerized into a gel.
(3) The cells full of the culture flask were digested completely, and after the digestion was terminated, the cell culture solution was removed by centrifugation. Resuspend cells with serum-free medium and adjust cell density to 8X 104one/mL.
(4) 150 μ L of cell suspension was slowly added to the upper chamber of the transwell chamber. Control and experimental groups were set, and the experimental group treated the cells with 200. mu.g/mL of extracellular vesicles.
(5) 600 μ L of DMEM cell culture medium containing 10% FBS was added to the lower chamber of the transwell plate. The transwell plate was placed in a cell incubator for 48 h.
(6) After 48h incubation, the chamber was removed, the liquid in the upper chamber was blotted with a cotton swab and fixed with 4% paraformaldehyde at room temperature for 20-30 min.
(7) The chamber was soaked with PBS wash and the upper chamber was stained with 0.1% crystal violet for 5-10 min. After staining, the chamber was washed three times with PBS, and the cells inside the chamber were gently wiped with a cotton swab.
(8) After the chamber was dried, 5-10 fields were randomly selected under an optical microscope for photographing and counting.
The results showed that the lactobacillus paracasei PC-H1 extracellular vesicles had the ability to inhibit migration and invasion of HCT116 cells, SW1116 cells and SW620 cells, see fig. 6.
Example 5 Lactobacillus paracasei PC-H1 extracellular vesicles induce apoptosis of colon cancer cells
The Lactobacillus paracasei PC-H1 extracellular vesicles used in this example were prepared by the method described in example 1.
Annexin V/PI double staining flow cytometry is adopted, and the specific steps are as follows:
(1) completely digesting the cells in the culture bottle, adding DMEM culture solution, repeatedly blowing and beating the digested cells to make the cells detached and made into cell suspension, and centrifuging at 800r/min for 5 min.
(2) The supernatant was discarded, an appropriate amount of medium was added for resuspension, and 20. mu.L of cells were aspirated to the counting area and counted in a cell counter. Adjusting the cell density to 5X 104One per mL.
(3) Adding 500 μ l of the cell suspension with the adjusted density into each well, uniformly distributing the cells in the well plate, and putting the 24-well plate into a cell culture box for culture.
(4) After the cells were attached to the wall, the cell culture medium in the wells of the 24-well plate was aspirated away. To the experimental wells 500 μ L of cell culture medium containing extracellular vesicles at a concentration of 200 μ g/mL was added, while to the control wells fresh cell culture medium was added. The 24-well plate was placed into a cell incubator for 48 h.
(5) After 48h of culture, cells are digested by trypsin without EDTA, when adherent cells can be blown down by gentle blowing, cell culture solution is added, the cells are blown down by gentle blowing and transferred into a centrifugal tube, and the cells are collected by centrifugation for 5min at 500-1000 g.
(6) After the cells were collected, a precooled PBS solution was added and gently blown to wash, and the cells were collected by centrifugation and washed twice in total.
(7) Adding 1 × Binding buffer working solution into the cell sediment, and resuspending the cells to make the cell concentration reach 1 × 106one/mL.
(8) Pipette 100. mu.L of cell suspension into a new tube, add 5. mu.L of Annexin V-FITC and 5-10. mu.L of PI, mix gently, incubate at room temperature in the dark for 15 min. A normal cell group, a PI single staining group and an Annexin V-FITC single staining group are set.
(9) After staining incubation, 400. mu.L of 1 XBinding buffer working solution was added to each tube, mixed well and detected using a flow cytometer.
The results showed that extracellular vesicles were able to efficiently induce apoptosis in colon cancer cells, see figures 7-9.
Example 6 Lactobacillus paracasei PC-H1 extracellular vesicles inhibit the growth of human colon cancer in nude mice transplanted tumors
The Lactobacillus paracasei PC-H1 extracellular vesicles used in this example were prepared by the method described in example 1.
Establishing a nude mouse transplantation tumor model by subcutaneous injection, which comprises the following specific steps:
(1) preparation of HCT116 cell suspension: HCT116 cells in good cell state and in logarithmic growth phase were selected. When the cell density reaches 80% -90%, the original culture solution in the cell culture bottle is discarded, and PBS is added for washing twice. Digesting cells with pancreatin, centrifuging at 800r/min for 5min, discarding supernatant, resuspending the obtained precipitate with PBS, adjusting cell density to 1 × 107one/mL.
(2) Grouping and subcutaneous injection: all nude mice were randomly divided into two groups of 5 mice each, and 200 μ Ι _ of PBS contained: (a) HCT116 cells (1.5X 10)60.2 mL); (b) HCT116 cells (1.5X 10)60.2mL) and extracellular vesicles (200. mu.g/mL) were injected subcutaneously into two groups of nude mice, respectively. The mid-posterior axilla of nude mice was selected for subcutaneous injection. Before injection, the cell suspension was mixed well and placed on ice. In operation, the nude mouse is grasped, the skin of the injection site is disinfected conventionally, the skin is lifted, the needle is inserted about 1cm under the skin, and the suspension is injected slowlyAnd (6) adding. In addition, in order to better distinguish each nude mouse, the ear number marking method is selected to carry out numbering marking on the nude mice.
(3) Observation of general conditions of nude mice and measurement of size of periodically transplanted tumor: the state of nude mice was closely observed every day, including: hair color, diet, defecation, mobility and mental status. After neoplasia, longest diameter (a) and shortest diameter (b) of the transplanted tumor were measured every three days with a vernier caliper according to the formula: 0.5X longest diameter (mm) X shortest diameter2(mm2) The volume of the nude mouse graft tumor was calculated. The average value of the volume of the transplanted tumor of each group of nude mice is used for drawing a transplanted tumor growth curve.
(4) Stripping of nude mouse transplanted tumor: after 30 days of tumor formation, the nude mice were anesthetized with ether, placed on an ultraclean bench, the tumor site was exposed and photographed. After the nude mice were sacrificed, the nude mouse graft tumor was peeled off, the peeled nude mouse graft tumor was placed on an ultra-clean bench to take a picture, the longest diameter (a) and the shortest diameter (b) of the graft tumor were measured with a vernier caliper, and the graft tumor was weighed with an electronic balance.
The results show that: the lactobacillus paracasei PC-H1 extracellular vesicles significantly inhibited the growth of the transplanted tumor in nude mice, as shown in fig. 10.
Example 7A lyophilized powder of Lactobacillus paracasei
The method comprises the following steps: embedding medium and thallus of Lactobacillus paracasei PC-H1.
The embedding medium comprises 9% of yeast extract powder, 8.4% of skim milk powder, 7.4% of isolated soy protein, 6.8% of trehalose and 1% of sodium D-isoascorbate.
The content of bacteria in lyophilized powder is 2 × 108-2×1012CFU/g。
Comparative example comparative experiment on inhibition of colon cancer cell proliferation by Lactobacillus paracasei PC-H1 extracellular vesicles and other Lactobacillus paracasei extracellular vesicles
In order to better reflect the capability of the lactobacillus paracasei PC-H1 extracellular vesicles to inhibit colon cancer cells, the separated other lactobacillus paracasei (H6, B35) and publicly obtained lactobacillus paracasei strains (CGMCC1.9089, CGMCC1.121) are used as controls in the research.
The method comprises the following specific steps:
(1) completely digesting HCT116 cells, SW1116 cells, SW620 cells and NCM460 cells (CVCL _0460) in a full culture flask, adding DMEM culture solution, repeatedly blowing the digested cells to remove walls and prepare cell suspension, and centrifuging at 800r/min for 5 min.
(2) The supernatant was discarded, an appropriate amount of medium was added for resuspension, and 20. mu.L of cells were aspirated to the counting area and counted in a cell counter. Adjusting the cell density to 1X 104One per mL.
(3) Adding 100 μ L of cell suspension with adjusted density into 96-well plate, uniformly distributing cells in the plate, placing 96-well plate at 37 deg.C and containing 5% CO2Cultured in a cell culture box.
(4) After the cells were attached, the cell culture medium in the wells of the 96-well plate was discarded. Extracellular vesicles at a concentration of 200. mu.g/mL were added to each experimental well and fresh medium was added to the control wells. The 96-well plate was placed into the cell incubator for 48 h.
(5) After the extracellular vesiculation was completed, 10. mu.L of WST-1 solution was added to each well and incubated for 1-2h in a cell incubator. Absorbance at 450nm was measured using a microplate reader.
The result shows that the lactobacillus paracasei PC-H1 extracellular vesicles (200 mug/mL) and three colon cancer cells of HCT116, SW1116 and SW620 can obviously inhibit cell proliferation after acting for 48 hours, and the activity of the colon cancer cells is obviously reduced. The other lactobacillus paracasei (H6, H35) and the publicly obtained lactobacillus paracasei strains (CGMCC1.9089, CGMCC1.121) have no obvious inhibition effect on the three tumor cells at the same concentration (200 mug/mL), and the effect is obviously lower than that of the lactobacillus paracasei PC-H1. All lactobacillus paracasei extracellular vesicles (200. mu.g/mL) used were found to have no inhibitory effect on NCM460 cells after coculture with human normal colonic epithelial cells NCM460 cells.
Sequence listing
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HARBIN MEIHUA BIOTECHNOLOGY Co.,Ltd.
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Claims (13)
1. The microorganism is lactobacillus paracasei PC-H1 with the preservation number of CGMCC NO.22285, and is preserved in China general microbiological culture Collection center (CGMCC) at 2021, 5 and 7 days.
2. A product comprising a live bacterium of lactobacillus paracasei according to claim 1 and/or a preparation thereof; the preparation is the dead bacteria, culture extract, cell disruption product, fermentation supernatant, fermentation precipitate, extracellular vesicle, modified bacteria, mutant bacteria and/or mutant bacteria of the lactobacillus paracasei of claim 1.
3. The product of claim 2, wherein the product is a live bacterial preparation, and the content of lactobacillus paracasei is 1.0 x 105-9.9×1010CFU/g or 1.0X 105-9.9×1010CFU/mL。
4. An extracellular vesicle produced by the microorganism according to claim 1.
5. A gene encoding a protein, which is capable of encoding the protein in the extracellular vesicle of claim 4.
6. A method for preparing an extracellular vesicle, comprising the steps of:
(1) inoculating and culturing the microbial strain solution of claim 1;
(2) centrifuging the cultured bacterial liquid at a low speed, collecting the precipitate, resuspending the precipitate with PBS, and inoculating the resuspended bacterial liquid into a culture medium for culturing;
(3) centrifuging the bacterial liquid cultured in the step (2) at a low speed and collecting supernatant;
(4) centrifuging the collected supernatant at low speed again to remove larger impurities;
(5) taking the supernatant obtained in the step (4) and filtering bacteria;
(6) transferring the filtered supernatant into an ultracentrifuge tube in batches, discarding the supernatant after ultracentrifugation, and collecting the precipitate obtained after each ultracentrifugation;
(7) after ultracentrifuging all the supernatant, collecting the collected liquid containing all the precipitates in an ultracentrifuge tube for secondary ultracentrifugation;
(8) discarding the supernatant, adding PBS to wash the precipitate from the ultracentrifuge tube, and ultracentrifuging again;
(9) the PBS was discarded and the resulting pellet was the extracellular vesicle of Lactobacillus paracasei PC-H1.
7. The method according to claim 6, wherein the low-speed centrifugation in the steps (2), (3) and (4) is performed at 4500g at 4 ℃ for 15 min; the ultracentrifugation conditions in the steps (6), (7) and (8) are 100000r/min and 60 minutes of centrifugation at 4 ℃.
8. A lyophilized powder of Lactobacillus paracasei comprising an embedding medium and the microbial biomass of claim 1.
9. Use of the microorganism of claim 1, the product of claims 2-3, the extracellular vesicle of claim 4, the gene of claim 5 and/or the lyophilized powder of claim 8 for the preparation of a medicament, a nutraceutical, a food, a nutraceutical, a medical device, a daily chemical product for the prevention, treatment, co-treatment and/or prognostic care of a tumor disease, and/or for gastrointestinal conditioning.
10. The use of claim 9, wherein the tumor is a tumor of the digestive system, a tumor of the hematological system, a tumor of the cranium, a tumor of the respiratory system, a tumor of the urinary system, a tumor of the endocrine system, a tumor of the reproductive system, a tumor of the circulatory system, a tumor of the bone, a tumor of the skin and/or a tumor of the soft tissue.
11. The use according to claim 10, wherein the tumor of the digestive system is intestinal cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, liver cancer, oral cancer and/or pharyngeal cancer.
12. Use according to claim 11, wherein the intestinal cancer is cancer of the rectum, colon, duodenum, caecum and/or appendix.
13. A pharmaceutical preparation, health product, food or chemical product comprising the microorganism of claim 1, the product of claims 2-3, the extracellular vesicle of claim 4, the gene of claim 5 and/or the lyophilized powder of claim 8.
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| CN115161216A (en) * | 2022-03-16 | 2022-10-11 | 中国农业大学 | Novel biological preservative and application thereof |
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| CN117603885A (en) * | 2024-01-18 | 2024-02-27 | 东北农业大学 | Lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage |
| CN117603885B (en) * | 2024-01-18 | 2024-04-05 | 东北农业大学 | Lactobacillus paracasei LP-116 and application thereof in preparation of products for regulating intestinal barrier damage |
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