CN115847893A - Letrozole slow test tube preparation method for constructing PCOS mouse model - Google Patents
Letrozole slow test tube preparation method for constructing PCOS mouse model Download PDFInfo
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- CN115847893A CN115847893A CN202211603069.4A CN202211603069A CN115847893A CN 115847893 A CN115847893 A CN 115847893A CN 202211603069 A CN202211603069 A CN 202211603069A CN 115847893 A CN115847893 A CN 115847893A
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Abstract
The invention discloses a letrozole buffer test tube preparation method for constructing a PCOS mouse model, which comprises the following steps: cutting a segment silicone tube, a semi-closed segment silicone tube, pressing letrozole powder, a fully-closed segment silicone tube and preparing a letrozole slow test tube; the letrozole slow-release test tube is prepared by cutting the segment silicone tube in sequence, semi-closing the segment silicone tube, pressing letrozole powder into the segment silicone tube and totally closing the segment silicone tube, so that the letrozole slow-release test tube makes up the domestic deficiency of letrozole slow-release ball market, is simple and convenient to prepare, can adjust the length of the prepared letrozole slow-release tube according to the actual condition of an experimenter, greatly facilitates scientific research personnel, saves the experimental operation time, reduces the economic cost at the same time, and is very worthy of popularization.
Description
Technical Field
The invention relates to the technical field of slow test tube preparation, in particular to a preparation method of letrozole slow test tube for constructing a PCOS mouse model.
Background
Polycystic ovary syndrome (PCOS) is a disease caused by a complex endocrine and metabolic abnormality common to women of childbearing age, the overall prevalence is between 6% and 10%. In addition to having the typical clinical manifestations of rare or no ovulation, hyperandrogenism, ovarian polycystic changes, insulin resistance, hyperinsulinemia and obesity are also common clinical features of the syndrome and can exacerbate the severity of PCOS.
Regarding PCOS model selection, primates with physiological and reproductive functions most similar to those of human beings, such as rhesus monkeys, should be selected in principle, but the breeding period is long, the cost is high, and scientific research and application are limited. Rodents such as rats, mice and the like have stable genetic background, high sensitivity to sex hormones, strong fecundity, short estrus cycle, strong experimental operability, wide sources, low price and easy feeding, so that the rodents have short molding cycle, high molding rate and stable model and have replicability, and are the most commonly selected PCOS animal species at present. The rat has stable oestrus cycle, large body size and organs, high blood volume, and is beneficial to operation and sample collection, but has the defects of difficult replication, genetic engineering modification and the like. The mouse has multiple inbreeding, high species consistency, easy replication and high experimental repeatability, and is more favorable for the research in the aspect of genetic engineering. A mouse model for constructing the PCOS is mostly a method for intragastric administration of letrozole for 21 days or subcutaneous injection of Dehydroepiandrosterone (DHEA), and a PCOS mouse model-making mode of implanting a letrozole slow-release ball subcutaneously in the neck is also available at present, so that the application prospect is wider.
In the mouse model construction method of PCOS, the gastric perfusion time of letrozole is long, and the continuous gastric perfusion is needed during the drug intervention period, so that on one hand, the digestive system of a mouse is continuously damaged, the experimental result is influenced, on the other hand, the experimental operation time is greatly prolonged, and great inconvenience is brought to scientific research personnel; the subcutaneous injection of the DHEA requires a mixed oil agent to be used as a carrier, and the DHEA needs to be injected regularly and quantitatively every day, so that the subcutaneous oil agent of the mouse is accumulated, the life of the mouse is greatly influenced, and the state of the mouse is often poor, so that the experimental result is influenced; in addition, the existing letrozole slow-release ball special for PCOS (prestressed concrete System) modeling is extremely difficult to obtain, so that scientific researchers in related fields lack raw materials for establishing models and seriously influence the progress of scientific research.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a letrozole slow test tube preparation method for constructing a PCOS mouse model, and solves the problems that the existing letrozole slow test tube preparation method is long in experimental operation time and inaccurate in experimental result, and letrozole slow release ball raw materials are difficult to obtain.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme: a preparation method of letrozole slow test tube for constructing a PCOS mouse model comprises the following steps:
the method comprises the following steps: firstly, obtaining sufficient silicone tube raw materials according to actual preparation requirements, then carrying out high-pressure disinfection on the obtained silicone tube raw materials, then placing the silicone tube raw materials subjected to high-pressure disinfection on a superclean workbench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silicone tube raw materials subjected to high-pressure disinfection into a segmented silicone tube by using the cutting blade subjected to sterilization and disinfection;
step two: firstly, according to the inner diameter specification of the segmented silicone tube prepared in the first step, obtaining a cylindrical wood rod matched with the inner diameter specification of the segmented silicone tube, cutting the obtained cylindrical wood rod into cylindrical wood blocks, and then plugging the cylindrical wood blocks obtained by cutting into one end of the segmented silicone tube to seal one end of the segmented silicone tube to obtain a semi-closed segmented silicone tube;
step three: firstly, pouring letrozole powder into a clean plastic culture dish, pressing letrozole powder into the open end of the semi-closed section-shaped silicone tube prepared in the step two, and compacting the letrozole powder by using an iron wire matched with the inner diameter of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, thus reaching the standard;
step four: plugging the cylindrical wood block prepared in the step two into the open end of the semi-closed section-shaped silicone tube with the letrozole powder in the step three, so as to realize the full sealing of two ends of the semi-closed section-shaped silicone tube and obtain a full-closed letrozole silicone tube;
step five: placing the fully-closed letrozole silicone tube prepared in the fourth step into a centrifuge tube, adding sterile normal saline into the centrifuge tube to soak the fully-closed letrozole silicone tube in a water bath, and obtaining a letrozole slow test tube for subcutaneous implantation after soaking.
The further improvement lies in that: in the first step, the inner diameter of the silicone tube raw material is 0.04mm, the outer diameter of the silicone tube raw material is 0.085mm, and the length of the cut segment-shaped silicone tube is 8mm.
The further improvement is that: in the first step, the specific steps of carrying out high-pressure disinfection on the obtained silicone tube are as follows: the raw material of the silicone tube is firstly put into an ultrahigh pressure container, and then the pressure of 400-600 MPa is applied to the raw material of the silicone tube by using water as a medium in the sealed ultrahigh pressure container so as to kill bacteria on the raw material of the silicone tube.
The further improvement lies in that: in the first step, the specific steps of sterilizing and disinfecting the cutting blade are as follows: firstly, a cutting blade to be used is soaked in alcohol for sterilization, then the cutting blade soaked in the alcohol is wiped dry, and the cutting blade is placed into an ultraviolet ray disinfection machine for further sterilization.
The further improvement is that: in the second step, the diameter of the section of the cylindrical wood rod is matched with the inner diameter of the section-shaped silicone tube, the length of the cylindrical wood block is 2mm, and the cylindrical wood block is sterilized at high temperature before being plugged into the section-shaped silicone tube.
The further improvement lies in that: in the third step, the clean plastic culture dish is subjected to high-temperature sterilization and disinfection before use, and the length of the letrozole powder is detected by using a ruler after the letrozole powder is compacted so as to judge whether the letrozole powder reaches the standard.
The further improvement is that: in the fifth step, the centrifugal tube is a 50ml centrifugal tube, the concentration of the sterile normal saline is 0.9%, the using amount of the sterile normal saline is 30ml, the temperature of the totally-enclosed letrozole silicone tube for water bath soaking is 37 ℃, and the soaking time is 24h.
The further improvement lies in that: the cylindrical wood blocks are soaked and disinfected by alcohol, the plastic culture dish is disinfected and disinfected by a high-temperature disinfection kettle, and the disinfection temperature is set to be 100-120 ℃.
The invention has the beneficial effects that: the letrozole slow test tube is prepared by cutting a segment silicone tube in sequence, semi-closing the segment silicone tube, pressing letrozole powder into the segment silicone tube and fully closing the segment silicone tube. The preparation method makes up the deficiency of the domestic letrozole slow release ball market, is simple and convenient to prepare, can adjust the length of the letrozole slow release tube according to the actual conditions of experimenters, greatly facilitates scientific research personnel, saves the experimental operation time, reduces the economic cost at the same time, and is very worthy of popularization.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a flow chart of the production process of the present invention;
FIG. 2 is a schematic diagram illustrating the variation of estrus cycles in an embodiment of the present invention;
FIG. 3 is a schematic representation of ovarian morphosis in an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the change in serum hormones in an example of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
Referring to fig. 1, this example provides a letrozole slow tube preparation method for constructing PCOS mouse model, comprising the following steps:
the method comprises the following steps: cutting segment shaped silicone tube
Firstly, obtaining sufficient silicone tube raw materials according to actual preparation requirements, wherein the inner diameter of the silicone tube raw materials is 0.04mm, and the outer diameter of the silicone tube raw materials is 0.085mm, then carrying out high-pressure sterilization on the obtained silicone tube raw materials, then placing the silicone tube raw materials subjected to high-pressure sterilization on a superclean workbench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silicone tube raw materials subjected to high-pressure sterilization into a section-shaped silicone tube with the length of 8mm by using the cutting blade subjected to sterilization and disinfection;
the specific steps of carrying out high-pressure disinfection on the obtained silicone tube are as follows: firstly, putting a silicone tube raw material into an ultrahigh pressure container, and then applying 400MPa pressure to the silicone tube raw material by using water as a medium in a closed ultrahigh pressure container so as to kill bacteria on the silicone tube raw material;
the specific steps of sterilizing and disinfecting the cutting blade are as follows: soaking a cutting blade to be used in alcohol for sterilization, wiping the cutting blade soaked in the alcohol, and placing the cutting blade into an ultraviolet sterilizer for further sterilization;
step two: semi-closed section-shaped silicone tube
Firstly, according to the specification of the inner diameter of the section-shaped silicone tube prepared in the first step, obtaining a cylindrical wood rod, wherein the section diameter of the cylindrical wood rod is matched with the inner diameter of the section-shaped silicone tube, then cutting the obtained cylindrical wood rod into cylindrical wood blocks with the length of 2mm, and then plugging the cylindrical wood blocks obtained by cutting into one end of the section-shaped silicone tube to seal one end of the section-shaped silicone tube to obtain a semi-closed section-shaped silicone tube, wherein the cylindrical wood blocks are soaked and sterilized by alcohol before being plugged into the section-shaped silicone tube;
step three: pressed letrozole powder
Firstly, pouring letrozole powder into a clean plastic culture dish, performing high-temperature sterilization on the plastic culture dish at the temperature of 100 ℃ through a high-temperature sterilization kettle before the plastic culture dish is used, pressing letrozole powder into the open end of the semi-closed section-shaped silicone tube prepared in the step two, compacting the letrozole powder by using an iron wire matched with the inner diameter of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, and detecting the length of the letrozole powder by using a ruler after the letrozole powder is compacted so as to judge whether the letrozole powder reaches the standard or not;
step four: totally-enclosed segment-shaped silicone tube
Plugging the cylindrical wood block prepared in the step two into the open end of the semi-closed section-shaped silicone tube with the letrozole powder in the step three, and completely closing two ends of the semi-closed section-shaped silicone tube to obtain a fully-closed letrozole silicone tube;
step five: prepared letrozole slow test tube
Placing the fully-closed letrozole silicone tube prepared in the fourth step into a 50ml centrifugal tube, adding 30ml of sterile normal saline into the centrifugal tube, and soaking the fully-closed letrozole silicone tube in water bath at the temperature of 37 ℃, wherein the concentration of the sterile normal saline is 0.9%, and after soaking for 24h, obtaining the letrozole slow test tube for subcutaneous implantation.
Example two
Referring to fig. 1, this example provides a letrozole buffered tube preparation method for constructing PCOS mouse model, comprising the following steps:
the method comprises the following steps: cutting segment shaped silicone tube
Firstly, obtaining sufficient silicone tube raw materials according to actual preparation requirements, wherein the inner diameter of the silicone tube raw materials is 0.04mm, and the outer diameter of the silicone tube raw materials is 0.085mm, then carrying out high-pressure sterilization on the obtained silicone tube raw materials, then placing the silicone tube raw materials subjected to high-pressure sterilization on a superclean workbench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silicone tube raw materials subjected to high-pressure sterilization into a section-shaped silicone tube with the length of 8mm by using the cutting blade subjected to sterilization and disinfection;
the specific steps of carrying out high-pressure disinfection on the obtained silicone tube are as follows: firstly, putting a silicone tube raw material into an ultrahigh pressure container, and then applying 600MPa pressure to the silicone tube raw material by using water as a medium in a closed ultrahigh pressure container so as to kill bacteria on the silicone tube raw material;
the specific steps of sterilizing and disinfecting the cutting blade are as follows: soaking a cutting blade to be used in alcohol for sterilization, wiping the cutting blade soaked in the alcohol, and placing the cutting blade into an ultraviolet sterilizer for further sterilization;
step two: semi-closed section-shaped silicone tube
Firstly, according to the specification of the inner diameter of the section-shaped silicone tube prepared in the first step, obtaining a cylindrical wood rod, wherein the section diameter of the cylindrical wood rod is matched with the inner diameter of the section-shaped silicone tube, then cutting the obtained cylindrical wood rod into cylindrical wood blocks with the length of 2mm, and then plugging the cylindrical wood blocks obtained by cutting into one end of the section-shaped silicone tube to seal one end of the section-shaped silicone tube to obtain a semi-closed section-shaped silicone tube, wherein the cylindrical wood blocks are soaked and sterilized by alcohol before being plugged into the section-shaped silicone tube;
step three: pressed letrozole powder
Firstly, pouring letrozole powder into a clean plastic culture dish, performing high-temperature sterilization on the plastic culture dish at the temperature of 120 ℃ through a high-temperature sterilization kettle before using the plastic culture dish, pressing letrozole powder into the open end of the semi-closed section-shaped silica gel tube prepared in the step two, compacting the letrozole powder by using an iron wire matched with the inner diameter of the semi-closed section-shaped silica gel tube until the length of the letrozole powder reaches 4mm, and detecting the length of the letrozole powder by using a ruler after the letrozole powder is compacted so as to judge whether the letrozole powder reaches the standard;
step four: totally-enclosed segment-shaped silicone tube
Plugging the cylindrical wood block prepared in the step two into the open end of the semi-closed section-shaped silicone tube with the letrozole powder in the step three, so as to realize the full sealing of two ends of the semi-closed section-shaped silicone tube and obtain a full-closed letrozole silicone tube;
step five: prepared letrozole slow test tube
Placing the fully-closed letrozole silicone tube prepared in the fourth step into a 50ml centrifugal tube, adding 30ml of sterile normal saline into the centrifugal tube, and soaking the fully-closed letrozole silicone tube in water bath at the temperature of 37 ℃, wherein the concentration of the sterile normal saline is 0.9%, and after soaking for 24h, obtaining the letrozole slow test tube for subcutaneous implantation.
The letrozole slow tube prepared in the example is implanted into a test mouse to carry out the evaluation of reproductive and metabolic functions, and the estrus cycle change condition, the ovary form change (HE staining) condition and the serum hormone change condition of the test mouse are obtained as follows:
1. periodic variation of estrus
As shown in fig. 2, the mice in the Control group all showed normal estrus cycles (4-5 days), while the estrus cycles of the mice in the Model group were significantly disordered and showed prolonged (5 days) or no change in the estrus cycles and were always in the estrus interval, wherein P represents the prophase of estrus, E represents the estrus, M represents the anaphase of estrus, and D represents the estrus interval;
2. ovarian morphological changes
As shown in FIG. 3, no significant corpus luteum was observed in the ovaries of the Model group mice and the number of antral follicles was significantly increased compared to the Control group;
3. changes in serum hormones
As shown in fig. 4, serum Dihydrotestosterone (DHT) and Fasting Insulin (FINS) levels were significantly increased in the Model group compared to the Control group, while total testosterone (T) was not significantly different.
Through the evaluation of the reproductive and metabolic functions, the letrozole slow tube prepared in the embodiment is proved to successfully construct a PCOS mouse model, and has obvious hyperandrogenism and insulin resistance phenotype.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A letrozole slow test tube preparation method for constructing a PCOS mouse model is characterized by comprising the following steps:
the method comprises the following steps: firstly, obtaining sufficient silicone tube raw materials according to actual preparation requirements, then carrying out high-pressure sterilization on the obtained silicone tube raw materials, then placing the silicone tube raw materials subjected to high-pressure sterilization on a superclean bench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silicone tube raw materials subjected to high-pressure sterilization into a segmented silicone tube by using the cutting blade subjected to sterilization and disinfection;
step two: firstly, according to the inner diameter specification of the segmented silicone tube prepared in the first step, a cylindrical wood rod matched with the inner diameter specification of the segmented silicone tube is obtained, the obtained cylindrical wood rod is cut into cylindrical wood blocks, and then the cylindrical wood blocks obtained through cutting are plugged into one end of the segmented silicone tube to seal one end of the segmented silicone tube, so that a semi-closed segmented silicone tube is obtained;
step three: firstly, pouring letrozole powder into a clean plastic culture dish, pressing letrozole powder into the open end of the semi-closed section-shaped silicone tube prepared in the step two, and compacting the letrozole powder by using an iron wire matched with the inner diameter of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, thus reaching the standard;
step four: plugging the cylindrical wood block prepared in the step two into the open end of the semi-closed section-shaped silicone tube with the letrozole powder in the step three, so as to realize the full sealing of two ends of the semi-closed section-shaped silicone tube and obtain a full-closed letrozole silicone tube;
step five: placing the totally-enclosed letrozole silicone tube prepared in the fourth step into a centrifugal tube, adding sterile normal saline into the centrifugal tube to soak the totally-enclosed letrozole silicone tube in a water bath, and obtaining a letrozole slow test tube for subcutaneous implantation after soaking.
2. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: in the first step, the inner diameter of the raw material of the silicone tube is 0.04mm, the outer diameter of the raw material of the silicone tube is 0.085mm, and the length of the cut segment-shaped silicone tube is 8mm.
3. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: in the first step, the specific steps of carrying out high-pressure disinfection on the obtained silicone tube are as follows: the raw material of the silicone tube is firstly put into an ultrahigh pressure container, and then the pressure of 400-600 MPa is applied to the raw material of the silicone tube by using water as a medium in the sealed ultrahigh pressure container so as to kill bacteria on the raw material of the silicone tube.
4. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the first step, the specific steps of sterilizing and disinfecting the cutting blade are as follows: firstly, a cutting blade to be used is soaked in alcohol for sterilization, then the cutting blade soaked in the alcohol is wiped dry, and the cutting blade is placed into an ultraviolet ray disinfection machine for further sterilization.
5. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: in the second step, the diameter of the section of the cylindrical wood rod is matched with the inner diameter of the section-shaped silicone tube, the length of the cylindrical wood block is 2mm, and the cylindrical wood block is sterilized at high temperature before being plugged into the section-shaped silicone tube.
6. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: in the third step, the clean plastic culture dish is subjected to high-temperature sterilization and disinfection before use, and the length of the letrozole powder is detected by using a ruler after the letrozole powder is compacted so as to judge whether the letrozole powder reaches the standard.
7. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: in the fifth step, the centrifugal tube is a 50ml centrifugal tube, the concentration of the sterile normal saline is 0.9%, the using amount of the sterile normal saline is 30ml, the temperature of the totally-enclosed letrozole silicone tube for water bath soaking is 37 ℃, and the soaking time is 24h.
8. The method for preparing letrozole buffer tube for constructing PCOS mouse model according to claim 1, wherein: the cylindrical wood blocks are soaked and disinfected by alcohol, the plastic culture dish is disinfected and disinfected by a high-temperature disinfection kettle, and the disinfection temperature is set to be 100-120 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5601835A (en) * | 1987-04-29 | 1997-02-11 | Massachusetts Institute Of Technology | Polymeric device for controlled drug delivery to the CNS |
| CN101612390A (en) * | 2008-06-27 | 2009-12-30 | 天津市中宝制药有限公司 | The preparation method of insulin subdermal implantation long-acting slow-release preparation |
| CN106573133A (en) * | 2014-08-19 | 2017-04-19 | 加利福尼亚大学董事会 | Implants for localized drug delivery and methods of use thereof |
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- 2022-12-13 CN CN202211603069.4A patent/CN115847893B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5601835A (en) * | 1987-04-29 | 1997-02-11 | Massachusetts Institute Of Technology | Polymeric device for controlled drug delivery to the CNS |
| CN101612390A (en) * | 2008-06-27 | 2009-12-30 | 天津市中宝制药有限公司 | The preparation method of insulin subdermal implantation long-acting slow-release preparation |
| CN106573133A (en) * | 2014-08-19 | 2017-04-19 | 加利福尼亚大学董事会 | Implants for localized drug delivery and methods of use thereof |
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