CN113801841A - Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells - Google Patents

Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells Download PDF

Info

Publication number
CN113801841A
CN113801841A CN202010552714.9A CN202010552714A CN113801841A CN 113801841 A CN113801841 A CN 113801841A CN 202010552714 A CN202010552714 A CN 202010552714A CN 113801841 A CN113801841 A CN 113801841A
Authority
CN
China
Prior art keywords
insulin
adiporon
cells
group
novel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202010552714.9A
Other languages
Chinese (zh)
Inventor
肖敏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wenzhou Medical University
Original Assignee
Wenzhou Medical University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wenzhou Medical University filed Critical Wenzhou Medical University
Priority to CN202010552714.9A priority Critical patent/CN113801841A/en
Publication of CN113801841A publication Critical patent/CN113801841A/en
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Abstract

Method for detecting the insulin sensitivity of AdipoRon on mouse skeletal muscle cells, observing the influence of the AdipoRon on the insulin sensitivity of C2C12, inducing and differentiating C2C12 into myoblasts by using horse serum, and dividing the myoblasts into 6 groups: a blank control group, an adiplon high dose group, an adiplon low dose group, an insulin group, and an adiplon low dose + PI3K inhibitor group and an insulin + PI3K inhibitor group, acted for 12 hours, and supernatants were collected to measure glucose consumption. C2C12 is induced to differentiate into myotube cells in a six-well plate, the drug is added for 12 hours, and the mRNA level of GLUT4 is detected by an RT-PCR method. In the method, after the PI3K inhibitor is added into the AdipoRon group and the insulin group, the GLUT4mRNA level is reduced, and the sugar consumption is also reduced, which indicates that the action of the AdipoRon is possibly similar to that of insulin, the action of an insulin signal molecule is exerted, a PI3K/Akt pathway is activated, and glucose is taken into cells. At present, the method is not reported at home and abroad, and the influence of the AdipoRon on the sensitivity of the C2C12 insulin is observed, and the influence of the AdipoRon on myoblasts is used, so that the AdipoRon can improve the sensitivity of the insulin, and a new direction is provided for the treatment of diabetes.

Description

Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells
Technical Field
The effect of adiponectin receptor agonist adiplon on insulin sensitivity of mouse myoblast cell line (C2C12) and the mechanism of action thereof was investigated. Increased glucose consumption in cases where the adiplon is capable of cell proliferation. This is probably due to activation of PI3K, which in turn affects the PI3K/Akt pathway, the function of the glucose transporter GLUT4, and uptake of glucose into cells.
Background
Diabetes Mellitus (DM) is an endocrine disease caused by insufficient absolute or relative secretion of insulin in the body and causing metabolic disorders such as sugar, fat, protein and the like, can cause a series of complications, and is one of chronic diseases seriously harming the society and human health. Insulin resistance (myotube) is an important factor and a remarkable characteristic of the onset of type ii diabetes, and the efficiency of insulin in the body to promote glucose uptake and utilization is reduced (mainly liver, muscle and fat), so that the body secretes excessive insulin complementarily. Insulin sensitivity is used to describe the degree of insulin resistance, and generally the lower the insulin sensitivity, the less effective the unit of insulin acting, and the lower the ability to break down carbohydrates. Adiplon (adiponectin receptor agonist) increases the oxidation of free fatty acids and the transport rate of glucose in muscle tissue through multiple signaling pathways, increases insulin sensitivity, and thereby reduces the biological effects of insulin resistance. The C2C12 cell is from the skeletal muscle satellite cell of C3H mouse, and has homogeneous cell shape and characteristic, capacity of being cultured indefinitely, and excellent differentiation capacity, and obvious structural and functional change of differentiated cell. Through researching the process of glucose uptake of the AdipoRon to myotube cells, the glucose reduction mechanism of the AdipoRon is preliminarily discussed.
Disclosure of Invention
Diabetes mellitus is a common endocrine metabolic disease, and is mainly classified into type I diabetes mellitus caused by absolute deficiency of insulin due to beta cell destruction of pancreatic islets, and type II diabetes mellitus caused by relative insufficiency of insulin. Adiponectin receptor agonists (adiploron) are now found to have anti-diabetic biological activity. The method is used for researching the insulin sensitizing and blood sugar reducing effects of the insulin at the cellular level.
At the cellular level, insulin resistance is manifested as a failure in the process of taking up and utilizing glucose by insulin target tissue cells (such as skeletal muscle cells, fat cells and the like), skeletal muscle (the largest insulin target organ) utilizes glucose to generate ATP, and insulin resistance occurs when the glucose taking up and utilizing of skeletal muscle cells is disabled and no external action is exerted to stimulate the glucose taking up and utilizing of cells. Most tissue cells in the human body (such as skeletal muscle cells) can take up glucose by the action of glucose transporters (GLUTs) on cell membranes, and GLUT4 improves insulin resistance of skeletal muscle cells by transporting glucose.
The experiment selects and originates from a mouse myoblast cell strain C2C12, successfully induces the myocardial cells into mature myoblast cells through the action of low-concentration horse serum, detects the sugar consumption of the cells by a gop-pod method, compares the glucose consumption degree of a culture medium after the cells are added with high and low doses of AdipoRon and insulin, and finds that the AdipoRon increases the sugar consumption of the cells under the condition of not influencing the activity of the cells, thereby influencing the sugar metabolism of skeletal muscle cells. The results show that: the two drugs, adiplon and insulin, increased myotube cell glucose consumption and were statistically significant compared to the control blank.
The purpose of detecting the sugar consumption of cells by using the gop-pod method is to observe how drugs influence the GLUT4 function of insulin-sensitive cells such as skeletal muscle, fat and the like. The result shows that the AdipoRon can promote glucose uptake of well-differentiated C2C12 cells and improve the GLUT4 gene expression level of the cells, compared with a control blank group, after the AdipoRon and insulin are added, the GLUT4mRNA level is increased, and the statistical significance is achieved compared with the blank control group. Under normal physiological conditions, the target organ of insulin action is mediated by a step-by-step protein binding signal activation during glucose uptake, i.e., a series of signal transduction processes from insulin receptor (InsR), insulin receptor substrate (IRS-1), PI3K, and finally glucose transporter (GLUT4) are reported. LY294002 is a commonly used PI3K inhibitor. LY294002 can penetrate cells, specifically inhibit PI3K, and inhibit PI3K/Akt signaling pathway, including common inhibition of Akt phosphorylation. In the method, after the PI3K inhibitor is added into the AdipoRon group and the insulin group, the GLUT4mRNA level is reduced, and the sugar consumption is also reduced, which indicates that the action of the AdipoRon is possibly similar to that of insulin, the action of an insulin signal molecule is exerted, the AdipoRon is combined with an insulin receptor subunit, a PI3K/Akt pathway is activated, and glucose is taken into cells.
The method shows that the AdipoRon can promote the glucose uptake of mouse skeletal muscle cells, and shows that the AdipoRon can improve the insulin sensitivity.
Detailed Description
1. Method of producing a composite material
1.1 differentiation of C2C12
And (2) culturing the cells, growing the cells to 80% in a 10cm ^2 cell culture dish, digesting the cells, counting the cells, inoculating the cells into a six-hole plate, keeping the number of the inoculated cells in each hole consistent, growing the cells to 70% in the six-hole plate, differentiating by using 2% horse serum, adding 2ml of DMEM medium containing 2% horse serum into each hole, changing the solution every other day, and after 4 days, differentiating and culturing the mouse myoblasts into mature myotube cells.
1.2 glucose oxidase method for detecting glucose level in cell culture Medium
The differentiated C2C12 cells were seeded into a 96-well plate and then divided into two groups, an administration group and a control group, the administration group was added with 2% HS DMEM medium containing a high dose group (50. mu.g/ml of AdipoRon), a low dose group (20. mu.g/ml of AdipoRon), insulin group (20. mu.g/ml of insulin) and low dose drug plus PI3K inhibitor group (20. mu.g/ml of AdipoRon and 20. mu.g/ml of LY294002(PI3K inhibitor)) and insulin plus PI3K inhibitor group (20. mu.g/ml of AdipoRon and 20. mu.g/ml of LY294002), respectively, the control group (medium alone and no drug), and 9 replicate wells per group were set. After incubation for 12 hours, the cell culture broth was centrifuged to leave the supernatant, and the amount of glucose in the medium was measured by the gop-pod method, and the amount of glucose in each of the remaining wells was subtracted from the average of the medium in which only the DMEM group was added, to obtain the amount of glucose consumed by each group of cells. After washing the cells with PBS for 2 times, 100 μ L of the prepared CCK-8 solution was added to each well, incubated for 2h, and the absorbance at 450nm was measured with a microplate reader.
1.3 RT-PCR method for detecting GLUT4mRNA expression
Total RNA of cells is extracted by a Trizol method, 1 mu g of total RNA is taken as a template to be reversely transcribed into cDNA, and 2ul of cDNA is taken as a template to carry out RT-PCR. GLUT4 and the β -actin primers were synthesized by Shanghai.
2 results
2.1C 2C12 cell differentiation results
In the experiment, cells are differentiated by 2% horse serum when the cells are merged to 70% after passage of C2C12 cells, the cells start to fuse multinucleated cells on the 1 st day after differentiation, the cells still maintain the fibroblast morphology, multinucleated myotubes are formed on the 3 rd day, more than 90% of the cells are equally divided into mature myotube cells on the 4 th day, and the cells are used as drug treatment cells. See figure 2
2.2 Effect of AdipoRon on glucose consumption in myotube cell culture Medium
The results show that after 12h of intervention, glucose consumption of the adiplon high-dose group and the adiplon low-dose group is obviously increased, but cell proliferation is not influenced, and the cell sugar consumption of the adiplon high-dose group is obviously increased compared with that of the adiplon low-dose group, so that the method has statistical significance. The glucose consumption of the insulin group is obviously increased, and the cell proliferation can be promoted, so that the method has statistical significance. However, after the addition of the PI3K inhibitor, the sugar consumption of cells in the adiplon low dose + PI3K inhibitor group (both 20 μ g/ml) was significantly reduced compared to the adiplon low dose group. The insulin + PI3K inhibitor group had a significant decrease in sugar consumption and a significant decrease in cell viability. The high and low dose of Adiponron can increase the sugar consumption and does not affect the cell viability.
Figure RE-GSB0000189335370000031
Tab..1 Comparison of glu consumption and cell proliferation between six groups
NC：Normal control；H：High AdipoRon；L：Low AdipoRon；L+P：Low AdipoRon with PI3K inhibitors；I：Insulin；
L+P：Insulin with PI3K inhibitors
*P＜0.05，**P＜0.01，***P＜0.001，vs NC groupΔP＜0.05，vs L group
*P＜0.05，**P＜0.01，***P＜0.001，vs I group
2.3 Effect of AdipoRon on myocyte GLUT4mRNA expression
Insulin and AdipoRon intervention myotube cells 12h results show that the expression level of GLUT4mRNA in the AdipoRon high-dose group and the AdipoRon low-dose group is remarkably increased, and the expression level of GLUT4mRNA in the AdipoRon high-dose group is remarkably increased compared with that in the AdipoRon low-dose group. The expression level of insulin group GLUT4mRNA is increased remarkably. However, when LY294002 was added, the GLUT4mRNA expression level was significantly reduced in the AdipoRon low dose + PI3K inhibitor group (both at 20. mu.g/ml) compared to the AdipoRon low dose. The insulin + PI3K inhibitor group (both 20 μ g/ml) was significantly reduced compared to the insulin group.
Description of the drawings: FIG.1 is an experimental scheme; FIG.2 is a morphological diagram of C2C12 myoblasts observed microscopically for different days in differentiation; FIG.3 is a graph showing comparison of the expression amounts of GLUT4mRNA in six groups. FIG.1 an Experimental roadmap, FIG.2 Views of C2C12 cells isolated in the biogenic differentiation medium under phase contrast microscope, FIG. 3a Comparison of GLUT4 expression in the tissues beta plus groups.

Claims (5)

1. A novel experimental mode for differentiating and culturing mouse myoblasts into mature myotube cells for experiments.
2. The novel myoblast assay format of claim 1 wherein in an assay demonstrating the effect and mechanism of action of adiponectin receptor agonists, adiponectin receptor agonists improve insulin sensitivity in C2C12 cells, measurement of GLUT4mRNA expression in C2C12 cells; comprehensively detects the influence of the adiponectin receptor agonist on a PI3K/Akt signal pathway.
3. The novel myoblast assay of claim 1 wherein myoblasts are detected at the molecular level and at the cellular level, respectively. In the method, after the PI3K inhibitor is added into the AdipoRon group and the insulin group, the GLUT4mRNA level is reduced, and the sugar consumption is also reduced, which indicates that the action of the AdipoRon is possibly similar to that of insulin, the action of an insulin signal molecule is exerted, the AdipoRon is combined with an insulin receptor subunit, a PI3K/Akt pathway is activated, and glucose is taken into cells.
4. The novel myoblast assay format of claim 1, which simplifies the assay for the efficacy of adiponectin receptor agonists in the treatment of obesity-related disorders (e.g., type 2 diabetes). The method shows that the AdipoRon can promote the glucose uptake of mouse skeletal muscle cells, and shows that the AdipoRon can improve the insulin sensitivity.
5. The novel myoblast experimental mode of claim 1, which provides a novel and simple method and system for the validation experiment of the effect of adiponectin receptor agonist, and has good popularization and feasibility.
CN202010552714.9A 2020-06-12 2020-06-12 Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells Withdrawn CN113801841A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010552714.9A CN113801841A (en) 2020-06-12 2020-06-12 Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010552714.9A CN113801841A (en) 2020-06-12 2020-06-12 Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells

Publications (1)

Publication Number Publication Date
CN113801841A true CN113801841A (en) 2021-12-17

Family

ID=78892640

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010552714.9A Withdrawn CN113801841A (en) 2020-06-12 2020-06-12 Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells

Country Status (1)

Country Link
CN (1) CN113801841A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113350528A (en) * 2021-05-25 2021-09-07 四川大学华西医院 Application of adiponectin-modified islet cells in improvement or improvement of islet transplantation effect

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103622992A (en) * 2012-08-26 2014-03-12 复旦大学 Application of hydrogen sulfide and donor thereof sodium hydrosulfide to preparation of medicament for treating diabetes
CN108671223A (en) * 2018-06-11 2018-10-19 周口师范学院 FHL3 is being prepared for treating the purposes in insulin resistance drug

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103622992A (en) * 2012-08-26 2014-03-12 复旦大学 Application of hydrogen sulfide and donor thereof sodium hydrosulfide to preparation of medicament for treating diabetes
CN108671223A (en) * 2018-06-11 2018-10-19 周口师范学院 FHL3 is being prepared for treating the purposes in insulin resistance drug

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
肖敏: "AdipoRon对小鼠骨骼肌细胞胰岛素敏感性的影响及其机制", 中国应用生理学杂志, vol. 33, no. 04, pages 321 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113350528A (en) * 2021-05-25 2021-09-07 四川大学华西医院 Application of adiponectin-modified islet cells in improvement or improvement of islet transplantation effect

Similar Documents

Publication Publication Date Title
CN101809443A (en) Use of genomic testing and ketogenic compounds for treatment of reduced cognitive function
KR20170123665A (en) Composition for preventing or treating valvular calcification comprising DPP-4 inhibitor
CN113801841A (en) Method for detecting insulin sensitivity of AdipoRon to mouse skeletal muscle cells
EP3444266A2 (en) Peptide with anti-obesity and anti-diabetic efficacy and use thereof
KR20100059302A (en) Compositions for skin external application containing extracts of hisbiscior cortex
KR20100054428A (en) Extracts for improving glucose homeostasis fermented using lactobacillus gasseri kctc 3163 from ginseng or ginseng extract
CN116763928A (en) Application of miR-682 in treatment or diagnosis of diabetes
EP3574912B1 (en) Composition for treating diabetic disease
CN113181167B (en) Application of curatin in preparation of medicine for treating myocardial hypertrophy
CN1939310A (en) Composition for losing weight
CN101411781A (en) Use of pu'er tea in preparing medicament for treating or preventing diabetes
CN114053298A (en) Medicine for inhibiting myocardial hypertrophy and construction method of model
US7785585B2 (en) Compositions comprising sclareol or derivatives thereof and uses thereof
CN111773324A (en) Application of fresh dendrobium aqueous extract in preparing medicine for treating metabolic diseases
CN111773323A (en) Application of dendrobium water extract in treating type 2 diabetes
CN114376983B (en) Natural extract composite granule suitable for high uric acid population
CN115715779A (en) Application of digitonin D in preparation of products for regulating PI3K-AKT pathway
CN110123823B (en) Application of poria cocos oligosaccharide and pharmaceutical composition for treating glycolipid metabolic disorder diseases
YATIN et al. Polyamine synthesis and transport inhibition in a human anaplastic thyroid carcinoma cell line in vitro and as xenograft tumors
CN115804769A (en) Application of dihydroberberine in preparing product for regulating PI3K-AKT and/or MAPK pathway
CN108096261B (en) Application of stevioside in preparing health-care food and medicine for preventing or treating myocardial fibrosis
KR20110049592A (en) Composition comprising ganoderma lucidum extracts for dpp-iv inhibition
Qiu et al. Effect of Propylene Glycol Alginate Sodium Sulfate Nanoparticles on Myocardial Injury in Diabetic Rats via Silent Information Regulator 1/Hypoxia-Inducible Factor-1 Alpha Pathway
KR100709342B1 (en) Composition containing 9Z, 11E conjugated linoleic acid as a effective ingredient with a usage as prevention of disease mediated with gap junction damage
CN101884794B (en) Application of S100A16 gene in preparing medicaments for treating insulin resistance

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication
WW01 Invention patent application withdrawn after publication

Application publication date: 20211217