CN115847893B - Preparation method of letrozole slow-release preparation for constructing PCOS mouse model - Google Patents
Preparation method of letrozole slow-release preparation for constructing PCOS mouse model Download PDFInfo
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- CN115847893B CN115847893B CN202211603069.4A CN202211603069A CN115847893B CN 115847893 B CN115847893 B CN 115847893B CN 202211603069 A CN202211603069 A CN 202211603069A CN 115847893 B CN115847893 B CN 115847893B
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Abstract
The invention discloses a preparation method of a letrozole slow test tube for constructing a PCOS mouse model, which comprises the following steps: cutting section-shaped silica gel tube, semi-sealing section-shaped silica gel tube, pressing letrozole powder into the section-shaped silica gel tube, and fully sealing the section-shaped silica gel tube to obtain letrozole slow test tube; the method sequentially cuts the segment-shaped silica gel tube, semi-seals the segment-shaped silica gel tube, presses the letrozole powder into the segment-shaped silica gel tube, and fully seals the segment-shaped silica gel tube to prepare the letrozole slow-release tube, so that the defect of the domestic letrozole slow-release ball market is overcome, the preparation is simple and convenient, the length of the letrozole slow-release tube can be adjusted according to the actual situation of an experimenter, scientific researchers are greatly facilitated, the experimental operation time is saved, the economic cost is reduced, and the method is very worth popularizing.
Description
Technical Field
The invention relates to the technical field of slow test tube preparation, in particular to a preparation method of a letrozole slow test tube for constructing a PCOS mouse model.
Background
Polycystic ovary syndrome (PCOS) is a complex disorder caused by endocrine and metabolic abnormalities common to women of childbearing age, with overall prevalence of between 6% and 10%. In addition to having the typical clinical manifestations of thin hair with egg discharge or anovulation, hyperandrogenism, polycystic ovary-like changes, insulin resistance, hyperinsulinemia, and obesity are common clinical features of this syndrome and can exacerbate the severity of PCOS.
Regarding PCOS model selection, primates, such as rhesus monkeys, with physiological and reproductive functions most similar to those of humans should be selected in principle, but have long propagation period and high cost, and limited scientific research application. Rodents such as rats, mice and the like have stable genetic background, high sex hormone sensitivity, strong fertility, short estrus cycle, strong experimental operability, wide sources, low price and easy feeding, so that the model has short modeling cycle, high modeling rate and stable model with replicability, and is the PCOS animal species most selected at present. The rat estrus cycle is stable, the size and the organ are large, the blood volume is high, the operation and the sample collection are facilitated, and the defects of difficult replication, genetic engineering transformation and the like exist. The mice have multiple inbreeding, high variety consistency, easy replication and high experimental repeatability, and are more beneficial to the research of genetic engineering. The method for constructing the PCOS mouse model is mostly used for stomach infusion of letrozole for 21 days or subcutaneous injection of Dehydroepiandrosterone (DHEA), and the PCOS mouse model mode for subcutaneously implanting letrozole slow-release balls into the neck is also used at present, so that the application prospect is wider.
In the method for constructing the PCOS mouse model, the letrozole has long gastric lavage time, and continuous gastric lavage is needed during the drug intervention period, so that on one hand, the digestive system of the mouse is continuously damaged, the experimental result is affected, and on the other hand, the experimental operation time is greatly prolonged, and great inconvenience is caused to scientific researchers; the subcutaneous injection of DHEA needs to be used as a carrier, and the subcutaneous injection needs to be injected at regular time and quantity every day, so that subcutaneous injection of the mice can be accumulated, the life of the mice is greatly influenced, and the states of the mice are often poor, so that the experimental results are influenced; in addition, the existing letrozole slow-release ball special for PCOS modeling is extremely difficult to obtain, so that scientific researchers in the related field lack of raw materials for building a model, and the progress of scientific research is seriously influenced, and therefore, the invention provides a letrozole slow-release test tube preparation method for building a PCOS mouse model, so that the problems in the prior art are solved.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a preparation method of a letrozole slow test tube for constructing a PCOS mouse model, which solves the problems that the existing preparation method of the letrozole slow test tube has long experimental operation time, inaccurate experimental results and difficult acquisition of letrozole slow-release ball raw materials.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme: a preparation method of a letrozole slow test tube for constructing a PCOS mouse model comprises the following steps:
step one: firstly, obtaining a sufficient amount of silica gel tube raw materials according to actual preparation needs, then carrying out high-pressure sterilization on the obtained silica gel tube raw materials, then placing the silica gel tube raw materials subjected to high-pressure sterilization on an ultra-clean workbench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silica gel tube raw materials subjected to high-pressure sterilization into section-shaped silica gel tubes by utilizing the cutting blade subjected to sterilization and disinfection;
step two: firstly, according to the inner diameter specification of the segment-shaped silica gel tube prepared in the first step, obtaining a cylindrical wood rod matched with the inner diameter specification of the cylindrical wood rod, cutting the obtained cylindrical wood rod into cylindrical wood blocks, and then plugging the cut cylindrical wood blocks into one end of the segment-shaped silica gel tube to realize the sealing of one end of the segment-shaped silica gel tube, so as to obtain a semi-closed segment-shaped silica gel tube;
step three: pouring letrozole powder into a clean plastic culture dish, pressing letrozole powder into the opening end of the semi-closed section-shaped silicone tube prepared in the second step, and compacting the letrozole powder in the semi-closed section-shaped silicone tube by using an iron wire with the inner diameter being matched with that of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, thereby reaching the standard;
step four: the cylindrical wood block manufactured in the second step is plugged into the opening end of the semi-closed section-shaped silica gel tube with letrozole powder in the third step, so that the two ends of the semi-closed section-shaped silica gel tube are fully closed, and the fully closed letrozole silica gel tube is obtained;
step five: firstly, placing the fully-closed letrozole silica gel tube manufactured in the step four into a centrifuge tube, then adding sterile normal saline into the centrifuge tube, and carrying out water bath soaking on the fully-closed letrozole silica gel tube, thus obtaining the letrozole slow test tube for subcutaneous implantation after the soaking is completed.
The further improvement is that: in the first step, the inner diameter of the silica gel tube raw material is 0.04mm, the outer diameter of the silica gel tube raw material is 0.085mm, and the cut segment-shaped silica gel tube is 8mm long.
The further improvement is that: in the first step, the specific steps of high-pressure sterilization of the obtained silica gel tube are as follows: firstly, placing the silica gel tube raw material into an ultrahigh pressure container, and then, in the closed ultrahigh pressure container, applying 400-600 MPa pressure to the silica gel tube raw material by using water as a medium so as to kill bacteria on the silica gel tube raw material.
The further improvement is that: in the first step, the specific steps of sterilizing the cutting blade are as follows: firstly, soaking a cutting blade to be used in alcohol for sterilization, then wiping the cutting blade soaked in alcohol, and putting the cutting blade into an ultraviolet sterilizer 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 silica gel pipe, 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 silica gel pipe.
The further improvement is that: in the third step, the clean plastic culture dish is sterilized and disinfected at high temperature before being used, and the length of the letrozole powder is detected by a ruler after the letrozole powder is compacted, so that whether the letrozole powder reaches the standard or not is judged.
The further improvement is that: in the fifth step, the centrifuge tube is a 50ml centrifuge tube, the concentration of the sterile physiological saline is 0.9%, the dosage is 30ml, the temperature of the fully-closed letrozole silica gel tube for water bath soaking is 37 ℃, and the soaking time is 24 hours.
The further improvement is that: the cylindrical wood blocks are soaked in alcohol for sterilization, and the plastic culture dish is sterilized by a high-temperature sterilization kettle, and the sterilization temperature is set to be 100-120 ℃.
The beneficial effects of the invention are as follows: the invention sequentially cuts the segment-shaped silica gel tube, semi-seals the segment-shaped silica gel tube, presses letrozole powder into the segment-shaped silica gel tube, and fully seals the segment-shaped silica gel tube to prepare the letrozole slow test tube. The method overcomes the defect of the domestic letrozole sustained-release ball market, is simple and convenient to manufacture, can adjust the length of the letrozole sustained-release tube according to the actual situation of an experimenter, greatly facilitates scientific researchers, saves experimental operation time, reduces economic cost and is very worth popularizing.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is a flow chart of a preparation method of the present invention;
FIG. 2 is a schematic diagram of estrus cycle variation in an embodiment of the invention;
FIG. 3 is a schematic representation of ovarian morphology change in an embodiment of the present invention;
FIG. 4 is a schematic representation of serum hormone variation in an embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1, the present embodiment provides a method for preparing a letrozole slow test tube for constructing a PCOS mouse model, comprising the following steps:
step one: cut segment-shaped silica gel tube
Firstly, obtaining a sufficient amount of silica gel tube raw materials according to actual preparation requirements, wherein the inner diameter of the silica gel tube raw materials is 0.04mm, the outer diameter of the silica gel tube raw materials is 0.085mm, then performing high-pressure sterilization on the obtained silica gel tube raw materials, then placing the high-pressure sterilized silica gel tube raw materials on an ultra-clean workbench, then performing sterilization and disinfection on a cutting blade, and cutting the high-pressure sterilized silica gel tube raw materials into a section-shaped silica gel tube with the length of 8mm by using the cutting blade after sterilization and disinfection;
the method comprises the following specific steps of performing high-pressure sterilization on the obtained silica gel tube: firstly, placing a silica gel tube raw material into an ultrahigh pressure container, and then, in the closed ultrahigh pressure container, applying 400MPa pressure to the silica gel tube raw material by using water as a medium so as to kill bacteria on the silica gel tube raw material;
the specific steps of sterilizing the cutting blade are as follows: firstly, soaking a cutting blade to be used in alcohol for sterilization, then wiping the cutting blade soaked in alcohol, and putting the cutting blade into an ultraviolet sterilizer for further sterilization;
step two: semi-closed segment-shaped silica gel tube
Firstly, according to the inner diameter specification of the section-shaped silica gel tube prepared in the first step, acquiring a cylindrical wood rod, wherein the section diameter of the cylindrical wood rod is matched with the inner diameter of the section-shaped silica gel tube, then cutting the acquired 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 silica gel tube to realize the sealing of one end of the section-shaped silica gel tube, so as to obtain a semi-closed section-shaped silica gel tube, wherein the cylindrical wood blocks are soaked in alcohol for sterilization before being plugged into the section-shaped silica gel tube;
step three: pressing in letrozole powder
Pouring letrozole powder into a clean plastic culture dish, sterilizing the plastic culture dish at a high temperature of 100 ℃ through a high-temperature sterilizing kettle before using, pressing letrozole powder into the opening end of the semi-closed section-shaped silicone tube prepared in the second step, and compacting the letrozole powder in the semi-closed section-shaped silicone tube through an iron wire with the inner diameter being matched with that of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, namely reaching the standard, and detecting the length of the letrozole powder by using a ruler after the compaction of the letrozole powder is finished so as to judge whether the standard is reached;
step four: totally-enclosed segment-shaped silica gel tube
The cylindrical wood block manufactured in the second step is plugged into the opening end of the semi-closed section-shaped silica gel tube with letrozole powder in the third step, so that the two ends of the semi-closed section-shaped silica gel tube are fully closed, and the fully closed letrozole silica gel tube is obtained;
step five: test tube for preparing letrozole
Firstly, placing the fully-closed letrozole silica gel tube manufactured in the step four into a 50ml centrifuge tube, then adding 30ml of sterile normal saline into the centrifuge tube, soaking the fully-closed letrozole silica gel tube in water bath at 37 ℃, wherein the concentration of the sterile normal saline is 0.9%, and obtaining the letrozole slow test tube for subcutaneous implantation after 24 hours of soaking.
Example two
Referring to fig. 1, the present embodiment provides a method for preparing a letrozole slow test tube for constructing a PCOS mouse model, comprising the following steps:
step one: cut segment-shaped silica gel tube
Firstly, obtaining a sufficient amount of silica gel tube raw materials according to actual preparation requirements, wherein the inner diameter of the silica gel tube raw materials is 0.04mm, the outer diameter of the silica gel tube raw materials is 0.085mm, then performing high-pressure sterilization on the obtained silica gel tube raw materials, then placing the high-pressure sterilized silica gel tube raw materials on an ultra-clean workbench, then performing sterilization and disinfection on a cutting blade, and cutting the high-pressure sterilized silica gel tube raw materials into a section-shaped silica gel tube with the length of 8mm by using the cutting blade after sterilization and disinfection;
the method comprises the following specific steps of performing high-pressure sterilization on the obtained silica gel tube: firstly, placing a silica gel tube raw material into an ultrahigh pressure container, and then, in the closed ultrahigh pressure container, applying 600MPa pressure to the silica gel tube raw material by using water as a medium so as to kill bacteria on the silica gel tube raw material;
the specific steps of sterilizing the cutting blade are as follows: firstly, soaking a cutting blade to be used in alcohol for sterilization, then wiping the cutting blade soaked in alcohol, and putting the cutting blade into an ultraviolet sterilizer for further sterilization;
step two: semi-closed segment-shaped silica gel tube
Firstly, according to the inner diameter specification of the section-shaped silica gel tube prepared in the first step, acquiring a cylindrical wood rod, wherein the section diameter of the cylindrical wood rod is matched with the inner diameter of the section-shaped silica gel tube, then cutting the acquired 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 silica gel tube to realize the sealing of one end of the section-shaped silica gel tube, so as to obtain a semi-closed section-shaped silica gel tube, wherein the cylindrical wood blocks are soaked in alcohol for sterilization before being plugged into the section-shaped silica gel tube;
step three: pressing in letrozole powder
Pouring letrozole powder into a clean plastic culture dish, sterilizing the plastic culture dish at a high temperature of 120 ℃ through a high-temperature sterilizing kettle before using, pressing letrozole powder into the opening end of the semi-closed section-shaped silicone tube prepared in the second step, and compacting the letrozole powder in the semi-closed section-shaped silicone tube through an iron wire with the inner diameter being matched with that of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, namely, the letrozole powder reaches the standard, 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 silica gel tube
The cylindrical wood block manufactured in the second step is plugged into the opening end of the semi-closed section-shaped silica gel tube with letrozole powder in the third step, so that the two ends of the semi-closed section-shaped silica gel tube are fully closed, and the fully closed letrozole silica gel tube is obtained;
step five: test tube for preparing letrozole
Firstly, placing the fully-closed letrozole silica gel tube manufactured in the step four into a 50ml centrifuge tube, then adding 30ml of sterile normal saline into the centrifuge tube, soaking the fully-closed letrozole silica gel tube in water bath at 37 ℃, wherein the concentration of the sterile normal saline is 0.9%, and obtaining the letrozole slow test tube for subcutaneous implantation after 24 hours of soaking.
The letrozole slow test tube prepared in the example is implanted into a test mouse to evaluate the reproduction and metabolism functions subcutaneously, and the estrus cycle change condition, the ovary morphology change (HE staining) condition and the serum hormone change condition of the test mouse are obtained as follows:
1. estrus cycle variation
As shown in fig. 2, the mice of the Control group all showed normal estrus cycle (4-5 days), while the estrus cycle of the mice of the Model group was significantly disturbed, showing prolonged estrus cycle (> 5 days) or no estrus cycle change, always in estrus interval, where P represents pre-estrus, E represents estrus, M represents post-estrus, D represents estrus;
2. ovarian morphology changes
As shown in FIG. 3, no apparent corpus luteum was seen in the ovaries of the Model mice, and the number of sinus follicles was significantly increased, compared to the Control group;
3. serum hormonal changes
As shown in fig. 4, serum Dihydrotestosterone (DHT) and Fasting Insulin (FINS) levels were significantly elevated 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 test tube prepared in the embodiment is proved to successfully construct a PCOS mouse model, and has obvious hyperandrogenic symptoms and insulin resistance phenotypes.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. The preparation method of the letrozole slow-release preparation for constructing the PCOS mouse model is characterized by comprising the following steps of:
step one: firstly, obtaining a sufficient amount of silica gel tube raw materials according to actual preparation needs, then carrying out high-pressure sterilization on the obtained silica gel tube raw materials, then placing the silica gel tube raw materials subjected to high-pressure sterilization on an ultra-clean workbench, then carrying out sterilization and disinfection on a cutting blade, and cutting the silica gel tube raw materials subjected to high-pressure sterilization into section-shaped silica gel tubes by utilizing the cutting blade subjected to sterilization and disinfection;
step two: firstly, according to the inner diameter specification of the segment-shaped silica gel tube prepared in the first step, obtaining a cylindrical wood rod matched with the inner diameter specification of the cylindrical wood rod, cutting the obtained cylindrical wood rod into cylindrical wood blocks, and then plugging the cut cylindrical wood blocks into one end of the segment-shaped silica gel tube to realize the sealing of one end of the segment-shaped silica gel tube, so as to obtain a semi-closed segment-shaped silica gel tube;
step three: pouring letrozole powder into a clean plastic culture dish, pressing letrozole powder into the opening end of the semi-closed section-shaped silicone tube prepared in the second step, and compacting the letrozole powder in the semi-closed section-shaped silicone tube by using an iron wire with the inner diameter being matched with that of the semi-closed section-shaped silicone tube until the length of the letrozole powder reaches 4mm, thereby reaching the standard;
step four: the cylindrical wood block manufactured in the second step is plugged into the opening end of the semi-closed section-shaped silica gel tube with letrozole powder in the third step, so that the two ends of the semi-closed section-shaped silica gel tube are fully closed, and the fully closed letrozole silica gel tube is obtained;
step five: firstly, placing the fully-closed letrozole silica gel tube manufactured in the step four into a centrifuge tube, then adding sterile normal saline into the centrifuge tube, and carrying out water bath soaking on the fully-closed letrozole silica gel tube, thus obtaining the letrozole slow-release tube for subcutaneous implantation after the soaking is completed.
2. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the first step, the inner diameter of the silica gel tube raw material is 0.04mm, the outer diameter of the silica gel tube raw material is 0.085mm, and the cut segment-shaped silica gel tube is 8mm long.
3. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the first step, the specific steps of high-pressure sterilization of the obtained silica gel tube are as follows: firstly, placing the silica gel tube raw material into an ultrahigh pressure container, and then, in the closed ultrahigh pressure container, applying 400-600 MPa pressure to the silica gel tube raw material by using water as a medium so as to kill bacteria on the silica gel tube raw material.
4. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the first step, the specific steps of sterilizing the cutting blade are as follows: firstly, soaking a cutting blade to be used in alcohol for sterilization, then wiping the cutting blade soaked in alcohol, and putting the cutting blade into an ultraviolet sterilizer for further sterilization.
5. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the second step, the diameter of the section of the cylindrical wood rod is matched with the inner diameter of the section-shaped silica gel pipe, 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 silica gel pipe.
6. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the third step, the clean plastic culture dish is sterilized and disinfected at high temperature before being used, and the length of the letrozole powder is detected by a ruler after the letrozole powder is compacted, so that whether the letrozole powder reaches the standard or not is judged.
7. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: in the fifth step, the centrifuge tube is a 50ml centrifuge tube, the concentration of the sterile physiological saline is 0.9%, the dosage is 30ml, the temperature of the fully-closed letrozole silica gel tube for water bath soaking is 37 ℃, and the soaking time is 24 hours.
8. The method for preparing letrozole slow-release preparation for constructing a PCOS mouse model according to claim 1, wherein the method comprises the following steps: the cylindrical wood blocks are soaked in alcohol for sterilization, and the plastic culture dish is sterilized by a high-temperature sterilization kettle, and the sterilization 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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