CN111718903A - Method for screening medicine capable of preventing and/or treating osteoporosis - Google Patents
Method for screening medicine capable of preventing and/or treating osteoporosis Download PDFInfo
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Abstract
The invention discloses a method for screening a medicament capable of preventing and/or treating osteoporosis, and belongs to the technical field of microorganisms and medicines. The invention provides a method for screening a medicament capable of preventing and/or treating osteoporosis, which comprises the steps of incubating human colon cancer epithelial cells Caco-2 treated by tumor necrosis factor alpha (TNF-alpha) in a cell culture solution added with the medicament to be screened to obtain an incubated osteoporosis cell model, detecting the relative change of transmembrane resistance of the incubated osteoporosis cell model, and screening the medicament capable of preventing and/or treating osteoporosis.
Description
Technical Field
The invention relates to a method for screening a medicament capable of preventing and/or treating osteoporosis, and belongs to the technical field of microorganisms and medicines.
Background
Osteoporosis (osteoporotis) is a bone metabolic disease characterized by a lower than normal bone mass per unit volume, and is mainly manifested by a decrease in bone mass, a deterioration in bone microstructure, an increase in bone fragility, and susceptibility to fracture. The biggest harm of osteoporosis is osteoporosis fracture, which causes pain and seriously reduces the life quality of osteoporosis patients, and in severe cases, osteoporosis fracture can cause disability, which makes patients restricted in activity and unable to take care of themselves, and brings heavy mental and economic burden to individuals, families and society.
At present, the drugs for clinically treating osteoporosis mainly comprise bisphosphonate, alendronic acid, selective estrogen receptor modulator, hormone substitute and the like, and the drugs can effectively promote the formation of osteoblasts and inhibit the formation of osteoclasts, and have better clinical curative effect on treating osteoporosis. However, in the process of using these drugs to treat osteoporosis, the potential complications and side effects of some drugs are also highlighted. For example, bisphosphonates may cause severe bone, joint or muscle discomfort in some patients, and an increased risk of osteonecrosis of the jaw bone in a few patients suffering from dental diseases or undergoing invasive dental treatment. Long-term use of alendronic acid has also been associated with the occurrence of sub-trochanteric and diaphyseal fractures of the femur. The selective estrogen receptor modulator raloxifene increases the risk of venous thromboembolism and stroke. In hormone replacement therapy, estrogen can increase endometrial hyperplasia and cancer risk of patients with intact uterus, and the incidence of gallstone disease and venous thromboembolism can be increased by 2-3 times by estrogen. Therefore, there is an urgent need to find a drug which can effectively alleviate osteoporosis without causing complications and side effects to patients in long-term use.
Probiotic drugs are novel drugs widely studied at present, and in recent years, various probiotics have been proved to have excellent curative effects on various diseases, for example, in Kangwenquan, Pajian cloud and the like, bifidobacterium is found to be used for treating constipation digestive system diseases (Lizhu Changle for treating slow-transmission constipation clinical curative effect observation [ J ] Chinese J. Med and Western medicine in combination for digestion, 2003,11 (6): 353-355); bernini et al have discovered that Bifidobacterium animalis is useful in the treatment of type II diabetes (Benedical effects of Bifidobacterium lactis on lipid profile and cytokine substrates with metabolic syndrome: A random tertiary. effects of probiotic metabolic syndrome [ J ] Nutrition, 2016, 32(6): 716-). Moreover, the probiotic medicaments naturally have the advantages of high safety and no complication or side effect, so that the screened probiotic medicaments capable of effectively relieving osteoporosis are very key to overcoming the defects of the existing medicaments for treating osteoporosis.
At this stage, the screening of drugs effective in alleviating osteoporosis is mainly accomplished using an osteoporosis model. The osteoporosis model mainly comprises two models, namely an animal model and a cell model, and the osteoporosis cell model mainly comprises an osteoblast model, an osteoclast model and a co-culture model of osteoblasts and osteoclasts. The osteoporosis animal model is complex in experimental operation, long in experimental period, high in cost and not suitable for large-scale screening, and the experimental animal is more difficult to use in the future along with increasingly strict ethical requirements of the animal; the osteoporosis cell model has the defects of difficult separation and culture, complex preparation process and the like.
Therefore, it is urgently needed to construct an osteoporosis cell model which is simple to prepare, and has the advantages of simple operation, short experimental period and low cost when the osteoporosis cell model is used for screening the medicines capable of preventing and/or treating osteoporosis.
Disclosure of Invention
[ problem ] to
The invention aims to provide an osteoporosis cell model which is simple to prepare, and has the advantages of simple operation, short experimental period and low cost when the osteoporosis cell model is used for screening medicines capable of preventing and/or treating osteoporosis.
[ solution ]
In order to solve the problems, the invention provides an osteoporosis cell model which is human colon cancer epithelial cells Caco-2 treated by tumor necrosis factor alpha (TNF-alpha).
In one embodiment of the invention, the preparation method of the osteoporosis cell model comprises the steps of firstly culturing the human colon cancer epithelial cells Caco-2 in a cell culture solution to be adherent, and then incubating the adherent human colon cancer epithelial cells Caco-2 in a cell culture solution added with tumor necrosis factor alpha.
In one embodiment of the present invention, the concentration of TNF-gamma in the cell culture medium is 1.8-2.2 ng/mL.
In one embodiment of the invention, the incubation temperature is 36.5-37.5 ℃ and the incubation time is 3.8-4.2 h.
The invention also provides a method for screening the medicine capable of preventing and/or treating osteoporosis, which comprises the steps of firstly incubating the osteoporosis cell model in a cell culture solution added with the medicine to be screened to obtain an incubated osteoporosis cell model, then detecting the relative change of transmembrane resistance of the incubated osteoporosis cell model, if the relative change of the transmembrane resistance of the incubated osteoporosis cell model is a positive value, the medicine to be screened corresponding to the detection result is the medicine capable of preventing and/or treating osteoporosis, and the larger the positive value is, the stronger the capacity of preventing and/or treating osteoporosis is.
In one embodiment of the invention, the drug is a fungus or a bacterium.
In one embodiment of the invention, the bacterium is a lactobacillus.
In one embodiment of the invention, the cellThe concentration of Lactobacillus in the culture solution is 0.8 × 108～1.2×108CFU/mL。
In one embodiment of the present invention, the concentration of lactobacillus in the cell culture solution is 1 × 108CFU/mL。
In one embodiment of the invention, the incubation temperature is 36.5-37.5 ℃ and the incubation time is 3.8-4.2 h.
The invention also provides a product for screening a medicament for preventing and/or treating osteoporosis, wherein the product contains the osteoporosis cell model.
In one embodiment of the invention, the product is a kit.
The invention also provides the application of the osteoporosis cell model or the method or the product in screening drugs for preventing and/or treating osteoporosis.
Has the advantages that:
(1) the invention provides an osteoporosis cell model, which can be obtained by only culturing human colon cancer epithelial cells Caco-2 in a cell culture solution to be adherent and then incubating the adherent human colon cancer epithelial cells Caco-2 in the cell culture solution added with tumor necrosis factor alpha, and is simple to prepare.
(2) The invention provides a method for screening a medicament capable of preventing and/or treating osteoporosis, which comprises the steps of incubating human colon cancer epithelial cells Caco-2 treated by tumor necrosis factor alpha (TNF-alpha) in a cell culture solution added with the medicament to be screened to obtain an incubated osteoporosis cell model, detecting the relative change of transmembrane resistance of the incubated osteoporosis cell model, and screening the medicament capable of preventing and/or treating osteoporosis.
Biological material preservation
A strain of Lactobacillus casei (Lactobacillus casei) CCFM1127 is classically named as Lactobacillus casei, is preserved in Guangdong province microorganism strain preservation center at 06 months 2020, and has the preservation number of GDMCCNo.61021 and the preservation address of No. 59 building of Dazhou No. 100 Jie of Jieli Zhonglu in Guangzhou.
A Lactobacillus fermentum (Lactobacillus fermentum) CCFM1126, which is classified and named Lactobacillus fermentum, is deposited in Guangdong province microorganism strain collection center in 2020 and 06 months, with the deposit number of GDMCC No.61020 and the deposit address of Guangzhou Mielizhou No. 100, large building No. 59, building 5.
Drawings
FIG. 1: clustering analysis of in vitro cell model and animal model conclusions.
FIG. 2: correlation analysis of in vitro cell model and animal model conclusions.
Detailed Description
Animal model of postmenopausal osteoporosis was first established in 1969 by Saville in ovariectomized rats and was repeatedly later confirmed to have now become a classic animal model for studying postmenopausal osteoporosis, and therefore, in the following examples, ovariectomized rats were used to simulate osteoporotic rats.
Human colon cancer epithelial cells, Caco-2, referred to in the examples below were purchased from shanghai institute of bioscience cell banks; tumor necrosis factor alpha (TNF-alpha) referred to in the following examples was purchased from Shanghai Biochemical Agents, Inc.; the type 3640 permeable nested cell culture dishes referred to in the examples below were purchased from the tin-free lefrax biological laboratory instruments ltd; the phosphate buffer solutions referred to in the following examples were purchased from national pharmaceutical group chemical agents; lactobacillus rhamnosus gg (lgg) referred to in the examples below was purchased from american type culture collection bank; lactobacillus plantarum CCFM8610, referred to in the following examples, is described in patent application publication No. CN102827796A, accession No. CGMCC No. 6077; SPF grade SD female rats referred to in the examples below were purchased from slaick laboratory animals ltd; the skim milk powder referred to in the following examples was purchased from nieuruiz food ltd; DMEM cell culture media referred to in the following examples were purchased from semer feishell instruments ltd; the ELISA kits described in the following examples are purchased from Wuhan Elley Biotechnology GmbH for detecting anti-tartaric acid phosphatase in rat serum, ELISA for detecting type I collagen cross-linked carboxyl terminal peptide in rat serum, ELISA for detecting estradiol in rat serum, ELISA for detecting endotoxin in rat serum, and ELISA for detecting alkaline phosphatase in rat serum bone.
The media involved in the following examples are as follows:
MRS solid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L, agar 20g/L, cysteine hydrochloride 0.5g/L, and pH 6.8.
MRS liquid medium (g/L): 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO40.05 g/L, Tween 801mL/L, cysteine hydrochloride 0.5g/L, and pH 6.8.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
The preparation of the Lactobacillus strains and suspensions referred to in the following examples is as follows:
streaking lactobacillus on MRS solid culture medium, culturing at 37 deg.C for 48h to obtain single colony, inoculating the single colony in MRS liquid culture medium, culturing at 37 deg.C for 18h for activation, continuously activating for two generations to obtain activated liquid, inoculating the activated liquid in MRS liquid culture medium according to the inoculum size of 2% (v/v), culturing at 37 deg.C for 18h to obtain bacterial liquid, centrifuging the bacterial liquid at 8000g for 10min to obtain lactobacillus thallus, washing the lactobacillus thallus with physiological saline, and suspending in 130g/L skimmed milk powder solution to obtain the thallus with bacterial concentration of 1 × 109CFU/mL to obtain lactobacillus suspension, and storing the suspension at-80 deg.C.
Example 1: construction of osteoporosis cell model
The method comprises the following specific steps:
spreading human colon cancer epithelial cells Caco-2 in a 3640 type permeable nested cell culture dish chamber added with cell culture solution, adding 400 mu L of DMEM cell culture solution containing 5% (v/v) fetal calf serum, 1% (v/v) penicillin and 1% (v/v) streptomycin into an inner chamber, adding 1mL of DMEM cell culture solution without serum and antibiotics into an outer chamber, and culturing at 37 ℃ until a single layer of cells cover the surface of an inner chamber membrane of the whole 3640 type permeable nested cell culture dish chamber to obtain the human colon cancer epithelial cells Caco-2 which are paved with the inner chamber membrane; adding tumor necrosis factor alpha (TNF-alpha) into an inner chamber of a cell culture solution until the final concentration is 2ng/mL, and then incubating human colon cancer epithelial cells Caco-2 which are paved on an inner chamber membrane in the cell culture solution added with the tumor necrosis factor alpha (TNF-alpha) for 4h at 37 ℃ to obtain the human colon cancer epithelial cells Caco-2 treated by the tumor necrosis factor alpha (TNF-alpha); after aspirating the cell culture fluid in the 3640 type permeable nested cell culture dish chamber with a pipette gun, human colon cancer epithelial cells Caco-2 treated by tumor necrosis factor alpha (TNF-alpha) are washed three times by phosphate buffer solution to obtain an osteoporosis cell model.
Example 2: acquisition of Lactobacillus casei and Lactobacillus fermentum
The method comprises the following specific steps:
respectively taking feces of healthy people, feces of infants, pickles and yoghourt as samples, sucking 0.5mL of the samples, adding the samples into 5mL of MRS liquid culture medium, culturing at 37 ℃ for 18-24 h, and enriching to obtain enriched samples; 0.5mL of the enriched sample was aspirated and added to 4.5mL of sterile physiological saline to obtain 10-1The dilution was then pipetted 0.5mL 10-1The dilution was taken in 4.5mL of physiological saline to give 10-2The dilution was carried out in this order to give 10-3，10-4，10-5，10-6Diluting the solution; draw 100. mu.L of the gradient diluent and spread on MRS solid medium, 10-4，10-5，10-6Culturing each gradient 1 plate at 37 ℃ for 48h to obtain bacterial colonies; selecting MRS solid according to colony shape, size, edge, transparency and the likeColonies with typical characteristics of lactobacillus casei or lactobacillus fermentum on the culture medium are picked by an inoculating loop and streaked on an MRS solid culture medium, and are cultured for 48 hours at 37 ℃ to obtain a purified single colony; selecting purified single colonies, respectively inoculating the single colonies into 5mL of MRS liquid culture medium, and culturing at 37 ℃ for 18-24 h to obtain a bacterial liquid; after numbering the strains corresponding to the respective bacterial liquids, the strains having typical characteristics of Lactobacillus casei or Lactobacillus fermentum were selected by performing experiments such as strain identification, gram staining, physiological and biochemical experiments, etc. according to the procedures described in textbook "microbiology" (Shen, Chengdong eds.), 67 strains were obtained through experiments, wherein 17 of the strains were Lactobacillus casei, which were named as Lactobacillus casei 2, 6, 8, 13, 14, 15, 16, 17, 32, 42, 45, 56, 61, 76, 82, 83, 84 (wherein Lactobacillus casei 61 was renumbered as CCFM1127, Lactobacillus casei 61 was deposited at Guangdong provincial microorganism culture Collection on 06 days in 2020, and GDMCC No.61021 was deposited), 12 of the strains were named as Lactobacillus fermentum, which were named as Lactobacillus fermentum 10, 34, 35, 36, 37, 39, 44, 46, 53, 61, 62, 58 (Lactobacillus fermentum 10 was renumbered as CCFM1126, and lactobacillus fermentum 10 has been deposited with the guangdong province collection of microorganisms at 2020, 05/06/2020, with deposit number GDMCC No. 61020);
wherein the strain identification process is as follows:
extracting genomes of Lactobacillus casei 2, 6, 8, 13, 14, 15, 16, 17, 32, 42, 45, 56, 61, 76, 82, 83, 84 and Lactobacillus fermentum 10, 34, 35, 36, 37, 39, 44, 46, 53, 61, 62, 58, amplifying and sequencing the 16S rDNA of Lactobacillus casei 6, 8, 13, 14, 15, 16, 17, 32, 42, 45, 56, 61, 76, 82, 83, 84 and Lactobacillus fermentum 10, 34, 35, 36, 37, 39, 44, 46, 53, 61, 62, 58 (completed by Shanghai Bioengineering GmbH), and sequencing the sequence of Lactobacillus casei 2, 6, 8, 13, 14, 15, 16, 17, 32, 42, 45, 56, 61, 76, 82, 83, 84 and Lactobacillus casei 10, 34, 35, 36, 37, 39, 44, 46, 53, 61, 62, 58 (SEQ ID NO: 1) The 16S rDNA sequence of Lactobacillus fermentum 10 is shown in SEQ ID No. 2) was aligned in GenBank.
Example 3: application of osteoporosis cell model
The method comprises the following specific steps:
cells of Lactobacillus rhamnosus GG, cells of Lactobacillus plantarum CCFM8610, cells of Lactobacillus casei 2, 6, 8, 13, 14, 15, 16, 17, 32, 42, 45, 56, 61, 76, 82, 83, 84 obtained in example 2, and cells of Lactobacillus fermentum 10, 34, 35, 36, 37, 39, 44, 46, 53, 61, 62, 58 obtained in example 2 were each resuspended in a cell culture medium to a cell concentration of 1 × 108CFU/mL to obtain bacterial suspension containing different lactobacilli; the human colon cancer epithelial cells Caco-2 cultured to be adherent obtained in example 1 are used as a blank control, an osteoporosis cell model added into a cell culture solution without lactobacillus for incubation is used as a model control, bacterial suspensions containing different lactobacilli are respectively added into a 3640 type permeable nested cell culture dish chamber filled with the osteoporosis cell model obtained in example 1, and the cells are incubated for 4 hours at 37 ℃, so that the osteoporosis cell model treated by different lactobacilli is obtained.
Cell culture fluid in a 3640 type permeable nested cell culture dish chamber is sucked by a pipette gun, and a blank control, a model control and an osteoporosis cell model treated by different lactobacilli are washed three times by phosphate buffer solution and then are measured by a cell resistance meter
ERS-2 measures the transmembrane resistance values of a blank control, a model control, an osteoporosis cell model before being treated by different lactobacilli and the transmembrane resistance value of the osteoporosis cell model after being treated by different lactobacilli, and calculates the relative change (change in TEE) of the transmembrane resistance of intestinal cells of the blank control, the model control and the osteoporosis cell model after being treated by different lactobacilli (the transmembrane resistance value of a single layer of cells is one of the standards for evaluating the change of the intestinal barrier), and the calculation results are shown in Table 1; wherein, the calculation formula is: relative change in intestinal cell transmembrane resistance ═ of model osteoporotic cells treated with different lactobacilliTransmembrane resistance value-transmembrane resistance value of osteoporosis cell model before treatment with different lactobacillus)/transmembrane resistance value of osteoporosis cell model before treatment with different lactobacillus]×100％。
As can be seen from Table 1, Lactobacillus rhamnosus GG, Lactobacillus plantarum CCFM8610, Lactobacillus casei 61, Lactobacillus fermentum 10 may have the ability to alleviate osteoporosis, and Lactobacillus casei 15, Lactobacillus fermentum 37, Lactobacillus fermentum 46 should not have the ability to alleviate osteoporosis.
TABLE 1 relative changes in intestinal cell transmembrane resistance for placebo, model control and model of osteoporosis cell model treated with different Lactobacillus
Example 4: verification of osteoporosis cell model
In order to verify the accuracy of the result obtained in example 3, lactobacillus rhamnosus GG, lactobacillus plantarum CCFM8610, lactobacillus casei 15, lactobacillus casei 61, lactobacillus fermentum 10, lactobacillus fermentum 58 and lactobacillus fermentum 37 in example 3 were selected, and their ability to alleviate osteoporosis was verified by an osteoporosis animal model, specifically including the following steps:
taking 50 SPF-grade SD female rats with the weight of 250 +/-20 g, randomly dividing the rats into 10 groups, wherein each group comprises 5 rats, and the 10 groups respectively comprise: the test results comprise a sham operation group, a model control group, a positive drug group, a lactobacillus rhamnosus GG, a lactobacillus plantarum CCFM8610, a lactobacillus casei 15, a lactobacillus casei 61, a lactobacillus fermentum 10, a lactobacillus fermentum 58, an LGG group of lactobacillus fermentum 37, a CCFM8610 group, a lactobacillus casei 15 group, a lactobacillus casei 61 group, a lactobacillus fermentum 10 group, a lactobacillus fermentum 58 group and a lactobacillus fermentum 37 group.
The experiment took 9 weeks: the first week is the adaptation period of rats, the rats in the adaptation period are raised in an environment with the temperature of 22 +/-2 ℃, the humidity of 40-70% and 12h alternating day and night, the used feed is the rat breeding compound feed purchased from Suzhou Shuangshi experimental animal feed science and technology limited, and the experiment is carried out after the ordinary diet is adapted for one week.
After the first week, rats of a model control group, a positive drug group, an LGG group, a CCFM8610 group, a lactobacillus casei 15 group, a lactobacillus casei 61 group, a lactobacillus fermentum 10 group, a lactobacillus fermentum 58 group and a lactobacillus fermentum 37 group are subjected to intraperitoneal injection of 10% (v/v) chloral hydrate for anesthesia according to a dose of 3.3mL/kg, the abdomen is disinfected conventionally, the abdominal cavity is opened through a central abdominal incision, the ovaries on both sides are cut off, and the incisions are sutured in two layers; injecting 10% chloral hydrate into abdominal cavity of a rat in a sham operation group according to the dose of 3.3mL/kg for anesthesia, carrying out conventional disinfection on the abdominal cavity, opening the abdominal cavity at the middle incision of the abdominal cavity, cutting off partial adipose tissues around the ovary, not removing the ovary, and suturing the incision in two layers; injecting penicillin into rats of each group for anti-infection according to the dosage of 20000U/100g for 3 days continuously after operation;
starting gavage at the 4 th week of the experiment until the end of the experiment, gavage the positive control group rats with 1 mg/(kg. d) of alendronate sodium solution (the alendronate sodium solution is obtained by dissolving alendronate sodium in skim milk powder solution with the concentration of 130 g/L), the LGG group, the CCFM8610 group, the Lactobacillus casei 15 group, the Lactobacillus casei 61 group, the Lactobacillus fermentum 10 group, the Lactobacillus fermentum 58 group and the Lactobacillus fermentum 37 group rats with 1.5 mL/day of dose of gastric suspension, and the sham operation group and the model control group with 1.5 mL/day of skim milk powder solution with the concentration of 130 g/L; gavage for 4 weeks.
Killing all rats after the completion of the gavage, uniformly sawing off the left femurs of all the rats from the long diameter midpoint, placing the distal ends of the femurs on the micro CT according to requirements, and measuring the bone density (BMD, g/cm) of the metaphysis part of the femurs near about 4mm away from the intercondylar fossa3) Cortex lycii volume (ct.v, mm)3) And bone volume fraction (BV/TV,%); measuring estradiol (E) in each group of rat serum by ELISA kit2pg/mL), tartrate phosphatase (TRACP, ng/mL), bone alkaline phosphatase (BALP, ng/mL) and endotoxin (ET, EU/mL); the colon is taken and qPCR is adopted to determine the relative expression quantity of the closely-connected related protein genes Occludin, ZO-2 and Claudin-1 in the colon of each group of rats (the detection result is shown in a table 2-3).
As can be seen from tables 2 to 3, the ability of each Lactobacillus to alleviate osteoporosis measured using an animal model for osteoporosis is consistent with the results obtained in example 3.
Further, the ability of each lactobacillus to alleviate osteoporosis, which was tested using an osteoporosis animal model, was subjected to cluster analysis and correlation analysis by the principle component analysis method, together with the results obtained in example 3, and the analysis results are shown in fig. 1-2.
As can be seen from FIGS. 1-2, the correlation between the ability of each Lactobacillus to alleviate osteoporosis, which was tested using an osteoporosis animal model, and the results obtained in example 3 was as high as 0.86, and the animal experimental results were significantly and positively correlated with the cell model.
TABLE 2 ability of each lactobacillus to alleviate osteoporosis tested using an osteoporosis animal model
TABLE 3 ability of each lactobacillus to alleviate osteoporosis tested using an osteoporosis animal model
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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gcttaaccga ggaagcgcat cggaaactgg gaaacttgag tgcagaagag gacagtggaa 660
ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa caccagtggc gaaggcggct 720
gtctggtctg taactgacgc tgaggctcga aagcatgggt agcgaacagg attagatacc 780
ctggtagtcc atgccgtaaa cgatgaatgc taggtgttgg agggtttccg cccttcagtg 840
ccgcagctaa cgcattaagc attccgcctg ggggagtacg accgcaaggt tgaaactcaa 900
ggaattgacg ggggcccgca caagcggtgg agcatgtggt tta 943
<210>2
<211>953
<212>DNA
<213> Lactobacillus fermentum
<400>2
gtctatcatg caagtcgacg cgttggccca attgattgat ggtgcttgca cctgattgat 60
tttggttgcc aacgagtggc ggacgggtga gtaacacgta ggtaacctgc ccagaagcgg 120
gggacaacat ttggaaacag atgctaatac cgcataacag cgttgttcgc atgaacaacg 180
cttaaaagat ggcttctcgc tatcacttct ggatggacct gcggtgcatt agcttgttgg 240
tggggtaacg gcctaccaag gcgatgatgc atagccgagt tgagagactg atcggccaca 300
atgggactga gacacggccc atactcctac gggaggcagc agtagggaat cttccacaat 360
gggcgcaagc ctgatggagc aacaccgcgt gagtgaagaa gggtttcggc tcgtaaagct 420
ctgttgttaa agaagaacac gtatgagagt aactgttcat acgttgacgg tatttaacca 480
gaaagtcacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc 540
cggatttatt gggcgtaaag agagtgcagg cggttttcta agtctgatgt gaaagccttc 600
ggcttaaccg gagaagtgca tcggaaactg gataacttga gtgcagaaga gggtagtgga 660
actccatgtg tagcggtgga atgcgtagat atatggaaga acaccagtgg cgaaggcggc 720
tacctggtct gcaactgacg ctgagactcg aaagcatggg tagcgaacag gattagatac 780
cctggtagtc catgccgtaa acgatgagtg ctaggtgttg gagggtttcc gcccttcagt 840
gccggagcta acgcattaag cactccgcct ggggagtacg accgcaaggt tgaaactcaa 900
aggaattgac gggggcccgc acaagcggtg gagctgtggt ttaattcgaa gct 953
Claims (10)
1. The osteoporosis cell model is characterized in that the osteoporosis cell model is human colon cancer epithelial cells Caco-2 treated by tumor necrosis factor alpha.
2. The osteoporosis cell model of claim 1, wherein the osteoporosis cell model is prepared by culturing the human colon cancer epithelial cells Caco-2 to be adherent in a cell culture solution, and then incubating the adherent human colon cancer epithelial cells Caco-2 in a cell culture solution supplemented with tnf α.
3. The osteoporosis cell model of claim 2, wherein the concentration of tnf α in the cell culture fluid is 1.8-2.2 ng/mL.
4. The model of claim 2 or 3, wherein the incubation is at a temperature of 36.5-37.5 ℃ for 3.8-4.2 hours.
5. A method for screening drugs for preventing and/or treating osteoporosis, which comprises the steps of incubating the osteoporosis cell model of any one of claims 1 to 4 in a cell culture solution to which a drug to be screened is added to obtain an incubated osteoporosis cell model, detecting a relative change in transmembrane resistance of the incubated osteoporosis cell model, and if the relative change in transmembrane resistance of the incubated osteoporosis cell model is a positive value, determining that the drug to be screened corresponding to the detection result is a drug for preventing and/or treating osteoporosis, wherein the greater the positive value, the stronger the ability of preventing and/or treating osteoporosis is.
6. The method of claim 5, wherein the drug is a fungus or a bacterium.
7. The method for screening drugs for prevention and/or treatment of osteoporosis of claim 5 or 6, wherein the concentration of Lactobacillus in the cell culture solution is 0.8 × 108～1.2×108CFU/mL。
8. The method for screening drugs for preventing and/or treating osteoporosis as claimed in any one of claims 5 to 7, wherein the incubation temperature is 36.5-37.5 ℃ and the incubation time is 3.8-4.2 h.
9. A product for screening drugs for preventing and/or treating osteoporosis, which comprises the osteoporosis cell model according to any one of claims 1 to 4.
10. Use of the osteoporosis cell model of any one of claims 1 to 4 or the method of any one of claims 5 to 8 or the product of claim 9 for screening a medicament for the prevention and/or treatment of osteoporosis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624520A (en) * | 2017-03-16 | 2018-10-09 | 景岳生物科技股份有限公司 | Promote the lactobacillus plantarum strain GMNL-662 and combinations thereof of inostosis |
| CN111718873A (en) * | 2020-06-19 | 2020-09-29 | 江南大学 | Lactobacillus fermentum with effect of relieving osteoporosis and application thereof |
| CN118207141A (en) * | 2024-05-21 | 2024-06-18 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving osteoporosis and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694468A (en) * | 2013-12-10 | 2015-06-10 | 中国科学院大连化学物理研究所 | Cell co-culture model for screening antiosteoporosis drugs and application of cell co-culture model |
| CN104755091A (en) * | 2012-06-04 | 2015-07-01 | 生命大地女神有限公司 | Selection and use of lactic acid bacteria preventing bone loss in mammals |
| CN105063211A (en) * | 2015-08-13 | 2015-11-18 | 黑龙江省乳品工业技术开发中心 | Method for rapidly evaluating capacity of LAB (lactic acid bacteria) in relieving cellular oxidation damage and application |
| CN105188723A (en) * | 2013-04-03 | 2015-12-23 | 普罗比公司 | Probiotic strains for use in treatment or prevention of osteoporosis |
-
2020
- 2020-06-24 CN CN202010595843.6A patent/CN111718903A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104755091A (en) * | 2012-06-04 | 2015-07-01 | 生命大地女神有限公司 | Selection and use of lactic acid bacteria preventing bone loss in mammals |
| CN105188723A (en) * | 2013-04-03 | 2015-12-23 | 普罗比公司 | Probiotic strains for use in treatment or prevention of osteoporosis |
| CN104694468A (en) * | 2013-12-10 | 2015-06-10 | 中国科学院大连化学物理研究所 | Cell co-culture model for screening antiosteoporosis drugs and application of cell co-culture model |
| CN105063211A (en) * | 2015-08-13 | 2015-11-18 | 黑龙江省乳品工业技术开发中心 | Method for rapidly evaluating capacity of LAB (lactic acid bacteria) in relieving cellular oxidation damage and application |
Non-Patent Citations (4)
| Title |
|---|
| KOJI MATSUHISA ET AL.: "Lignosulfonic acid attenuates NF-κB activation and intestinal epithelial barrier dysfunction induced by TNF-α/IFN-γ in Caco-2 cells", 《JOURNAL OF NATURAL MEDICINES》 * |
| SHAN-WEN CHEN AT AL.: "Protective effect of hydrogen sulfide on TNF-α and IFN-γ-induced injury of intestinal epithelial barrier function in Caco-2 monolayers", 《INFLAMMATION RESEARCH》 * |
| 刘丽娜等: "槲皮素改善 TNF-α诱导的肠上皮 Caco-2 细胞功能障碍", 《中国药科大学学报》 * |
| 彭江 等: "植物乳杆菌CCFM8610对去卵巢大鼠骨质疏松症的改善作用", 《食品与发酵工业》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624520A (en) * | 2017-03-16 | 2018-10-09 | 景岳生物科技股份有限公司 | Promote the lactobacillus plantarum strain GMNL-662 and combinations thereof of inostosis |
| CN108624520B (en) * | 2017-03-16 | 2022-01-25 | 景岳生物科技股份有限公司 | Lactobacillus plantarum strain GMNL-662 for promoting bone regeneration and composition thereof |
| CN111718873A (en) * | 2020-06-19 | 2020-09-29 | 江南大学 | Lactobacillus fermentum with effect of relieving osteoporosis and application thereof |
| CN118207141A (en) * | 2024-05-21 | 2024-06-18 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving osteoporosis and application thereof |
| CN118207141B (en) * | 2024-05-21 | 2024-09-17 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving osteoporosis and application thereof |
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Application publication date: 20200929 |