CN114190363A - Epsilon-polylysine microsphere carrier and application thereof in cryopreservation of a small amount of sperms - Google Patents
Epsilon-polylysine microsphere carrier and application thereof in cryopreservation of a small amount of sperms Download PDFInfo
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- CN114190363A CN114190363A CN202111602002.4A CN202111602002A CN114190363A CN 114190363 A CN114190363 A CN 114190363A CN 202111602002 A CN202111602002 A CN 202111602002A CN 114190363 A CN114190363 A CN 114190363A
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- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 claims description 12
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- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 claims description 5
- 206010003883 azoospermia Diseases 0.000 claims description 5
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 claims description 4
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
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- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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Abstract
The invention belongs to the technical field of assisted reproduction, and discloses an epsilon-polylysine microsphere carrier and application thereof in cryopreservation of a small amount of sperms. The epsilon-polylysine microsphere carrier is prepared from sodium alginate and epsilon-polylysine, the carrier is hollow microspheres, the diameter of the carrier is 50-180 mu m, the shell of the carrier is a polyelectrolyte membrane formed by the electrostatic complexation reaction of the sodium alginate and the epsilon-polylysine, the inner core of the carrier is a sodium alginate solution, and the epsilon-polylysine microsphere carrier can be applied to the cryopreservation of a small amount of sperms. The epsilon-polylysine microsphere carrier provided by the invention has the advantages of non-toxic materials, good biocompatibility, no influence on the activity of loaded sperms and the like, and is beneficial to maintaining the activity of the sperms.
Description
Technical Field
The invention belongs to the technical field of assisted reproduction, and particularly relates to an epsilon-polylysine microsphere carrier and application thereof in cryopreservation of a small amount of sperms.
Background
The testis of a patient with non-obstructive azoospermia (NOA) cannot produce or only produces a very small amount of sperms, so that it is difficult to obtain enough sperms by using a general sperm extraction technology, and clinical treatment is quite difficult, which is an important cause of male infertility and affects about 0.6% of males or 10.0% of infertile males. In recent years, along with the deep understanding and research on NOA, sperm extraction technologies such as testicular fine needle aspiration, testicular incision sperm extraction, microscopic testicular incision sperm extraction and the like have been developed, and the technology is combined with an intracytoplasmic sperm injection technology (ICSI), so that an originally sterile NOA patient can obtain own biological offspring. Clinically, the pregnancy rate for ICSI treatment cycles is 50.0%, and patients may need to repeat ICSI treatment several times in order to obtain a fraction of pregnancy. Clinically, testicle semen collection is an invasive operation, and each semen collection can damage the testicles, possibly causing testicle atrophy or function reduction, even completely losing the semen generation function. Therefore, the small amount of movable sperms (a few or more than ten) obtained by diagnostic puncture of the azoospermia patient or remained in the ICSI treatment period need to be frozen and stored, so that the sperms can be revived for ICSI treatment when the female takes eggs, and the flexibility and the reliability of the auxiliary fertility treatment of the non-obstructive azoospermia patient are greatly improved. Therefore, the cryopreservation of the trace sperms is a key technology for improving the success rate of pregnancy.
The cryopreservation of a small amount of sperms is a difficult problem in the field of assisted reproduction, and no ideal cryopreservation method is clinically used at present. The cryopreservation method of a small amount of sperms under study comprises a zona pellucida method, a slice method, a micromanipulation needle method, a micro-tube method and the like, and the methods all have the problems of difficult material source, ethics, pollution, complex operation and the like, and cannot be popularized and applied on a large scale clinically. The zona pellucida method is a better method for preserving a small amount of sperms in a freezing way, and the zona pellucida is a layer of glycoprotein wrapped outside oocytes and plays an important role in oogenesis, oosperm protection and polyspermia prevention. The content in the transparent band is removed by using a micromanipulation technology, the transparent band can be used as a cryopreservation carrier, and a small amount of sperms are injected into the empty transparent band, so that a good cryopreservation effect can be obtained. Although the recovery rate and activity of the sperm preserved by the zona pellucida method are both above 70.0 percent, the method has some problems that firstly the zona pellucida of mammal or human ova is used, the material source is difficult (the zona pellucida of the human ova is difficult to obtain), secondly the ethical dispute (the host DNA is in danger of being injected into the human ova together with the sperm during the ICSI treatment) exists, and in addition, the risk of the infection of the virus of the human and the livestock exists, so the method is difficult to be widely applied in the assisted reproduction clinic.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the epsilon-polylysine microsphere carrier and the application thereof in cryopreservation of a small amount of sperms.
The above purpose of the invention is realized by the following technical scheme: an epsilon-polylysine microsphere carrier is prepared from sodium alginate and epsilon-polylysine, the carrier is a hollow microsphere with the diameter of 50-180 mu m, the shell is a polyelectrolyte membrane formed by the electrostatic complexation reaction of the sodium alginate and the epsilon-polylysine, and the inner core is a sodium alginate solution with certain viscosity.
Further, the molecular weight of the sodium alginate is 70.0-200.0 kD, and the molecular weight of guluronic acid in the sodium alginate is as follows: the mass ratio of mannuronic acid is 1.0-3.0, the solution concentration is 0.8-2.0 wt%, and the solution viscosity is 50.0-150.0 mPas.
Furthermore, the molecular weight of the epsilon-polylysine is 5.0-30.0 kD, and the solution concentration is 0.01-0.5 wt%.
The molecular weight of the sodium alginate is 10.0-160.0 kD, and guluronic acid in the sodium alginate is: the mannuronic acid content is 1.2-2.4, the solution concentration is 1.0-1.6 wt%, and the solution viscosity is 60.0-120.0 mPas.
In a further preferred embodiment of the present invention, the polylysine has a molecular weight of 5.0 to 24.0kD and a solution concentration of 0.05 to 0.3 wt%.
Further, the preparation method of the epsilon-polylysine microsphere carrier comprises the following steps:
(1) adding sodium alginate into normal saline, stirring at high speed to dissolve completely;
(2) dripping sodium alginate solution into 0.1M calcium chloride solution by electrostatic liquid drop method, and calcification for 30min to form calcium alginate gel beads;
(3) adding calcium alginate gel beads into an epsilon-polylysine solution (the volume ratio of the calcium alginate gel beads to the epsilon-polylysine solution is 1:10), oscillating a universal shaking table to enable the calcium alginate gel and the epsilon-polylysine to carry out a crosslinking reaction, finishing the crosslinking reaction after 10min, and washing with deionized water to remove the epsilon-polylysine solution;
(4) and (3) adding the product obtained in the step (3) into a 55.0mM sodium citrate solution for liquefaction for 5min, liquefying the inner core of the microsphere to form a hollow structure, and washing with normal saline for 3 times to obtain the hollow epsilon-polylysine microsphere carrier.
The invention also protects the application of the epsilon-polylysine microsphere carrier in the cryopreservation of a small amount of sperms.
Furthermore, in the application of the epsilon-polylysine microsphere carrier in cryopreservation of a small amount of sperms, the preserved sperms are testicular puncture sperms, epididymal puncture sperms and sperms of patients with severe oligospermia and asthenospermia, when the epsilon-polylysine microsphere carrier is used, the sperms are preserved in a microsphere cavity, and the number of the loaded sperms of each carrier is 1-20, preferably 1-10.
Further, in the application of the epsilon-polylysine microsphere carrier in cryopreservation of a small amount of sperms, the microsphere carrier is preserved in a cryopreservation tube or a McLee tube, the number of the microsphere carriers preserved in each cryopreservation tube or McLee tube is 1-20, preferably 1-10, and the cryopreservation tubes or the McLee tubes are preserved in liquid nitrogen.
Compared with the prior art, the invention has the beneficial effects that:
1. the epsilon-polylysine microsphere carrier provided by the invention is non-toxic in material, good in biocompatibility, free of influence on the activity of loaded sperms and beneficial to keeping the activity of the sperms.
2. The epsilon-polylysine microsphere carrier provided by the invention has small volume, can easily find and grab sperms during micromanipulation, and is beneficial to keeping the activity of the sperms.
3. The shell of the epsilon-polylysine microsphere carrier provided by the invention is a polyelectrolyte complex membrane, the mobility is good, the puncture of an ICSI operation needle is easy, the epsilon-polylysine microsphere carrier can be quickly healed after the micromanipulation, the needle eye is not formed, and the sperm loss is not caused.
4. The epsilon-polylysine microsphere carrier provided by the invention has higher strength, unchanged shape after cryopreservation, and can keep integrity, thereby avoiding the loss of sperms in the processes of cryopreservation and recovery and having high sperm recovery rate.
5. The epsilon-polylysine microsphere carrier provided by the invention has mild preparation conditions, does not use organic solvent in the preparation process, and can avoid the influence on the activity of frozen sperm caused by the residual organic solvent.
6. The raw material for preparing the microsphere carrier provided by the invention is epsilon-polylysine, and because the epsilon-polylysine has a bacteriostatic action, the microbial pollution can be effectively prevented.
7. The artificial transparent belt provided by the invention has a simple preparation process, and is easy to amplify and industrially produce.
Drawings
The invention will be further explained with reference to the drawings and the detailed description
FIG. 1 is a lower structural view of a microscope showing the epsilon-polylysine microsphere carrier prepared in example 1;
FIG. 2 is a diagram showing the structure of a sperm-loaded epsilon-polylysine microsphere carrier (FIG. 2A shows sperm loading, and FIG. 2B shows sperm removal).
Detailed Description
The invention is described in more detail below with reference to specific examples, without limiting the scope of the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be obtained from commercial sources.
Example 1
1. The embodiment provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
(1) adding sodium alginate into normal saline, stirring at high speed to dissolve completely;
(2) dripping sodium alginate solution into 0.1M calcium chloride solution by electrostatic liquid drop method, and calcification for 30min to form calcium alginate gel beads;
(3) adding calcium alginate gel beads into an epsilon-polylysine solution (the volume ratio of the calcium alginate gel beads to the epsilon-polylysine solution is 1:10), oscillating a universal shaking table to enable the calcium alginate gel and the epsilon-polylysine to carry out a crosslinking reaction, finishing the crosslinking reaction after 10min, and washing with deionized water to remove the epsilon-polylysine solution;
(4) and (3) adding the microsphere carrier into 55.0mM sodium citrate solution for liquefying for 5min, completely liquefying the microsphere core to form a hollow structure, and cleaning with normal saline for 3 times to obtain the hollow microsphere carrier.
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 153.0kD, the G/M ratio is 1.63, the solution concentration is 1.0 wt%, and the solution viscosity is 64.8 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.2 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 110 μm;
film thickness: and 30 μm.
Comparative example 1
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 153.0kD, the G/M ratio is 1.63, the solution concentration is 1.0 wt%, and the solution viscosity is 64.8 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.1 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 110 μm;
film thickness: 20 μm.
Comparative example 2
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 153.0kD, the G/M ratio is 1.63, the solution concentration is 1.0 wt%, and the solution viscosity is 64.8 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.05 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 110 μm;
film thickness: 10 μm.
Example 2
1. The embodiment provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight was 153k.0D, the G/M ratio was 1.63, the solution concentration was 1.2% by weight, and the solution viscosity was 91.5 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.2 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 160 mu m;
film thickness: and 30 μm.
Comparative example 3
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 153.0kD, the G/M ratio is 1.63, the solution concentration is 1.2 wt%, and the solution viscosity is 91.5 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.1 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 160 mu m;
film thickness: 20 μm.
Comparative example 4
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 153.0kD, the G/M ratio is 1.63, the solution concentration is 1.2 wt%, and the solution viscosity is 91.5 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.05 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 160 mu m;
film thickness: 10 μm.
Example 3
1. The embodiment provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight was 153.0kD, the G/M ratio was 1.63, the solution concentration was 1.5% by weight, and the solution viscosity was 138.3 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.2 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: and 30 μm.
Comparative example 5
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight was 153.0kD, the G/M ratio was 1.63, the solution concentration was 1.5% by weight, and the solution viscosity was 138.3 mPas;
(2) epsilon-polylysine: the molecular weight is 16kD, and the solution concentration is 0.1 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: 20 μm.
Comparative example 6
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight was 153.0kD, the G/M ratio was 1.63, the solution concentration was 1.5% by weight, and the solution viscosity was 138.3 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.05 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: 10 μm.
Example 4
1. The embodiment provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 167.0kD, the G/M ratio is 0.67, the solution concentration is 1.0 wt%, and the solution viscosity is 74.9 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.2 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: and 30 μm.
Comparative example 7
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 167.0kD, the G/M ratio is 0.67, the solution concentration is 1.0 wt%, and the solution viscosity is 74.9 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.1 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: 20 μm.
Comparative example 8
1. The comparative example provides an epsilon-polylysine microsphere carrier, and the preparation method comprises the following steps:
same as example 1
2. The property and preparation condition of the microsphere carrier preparation material are as follows:
(1) sodium alginate: the molecular weight is 167.0kD, the G/M ratio is 0.67, the solution concentration is 1.0 wt%, and the solution viscosity is 74.9 mPas;
(2) epsilon-polylysine: the molecular weight is 16.0kD, and the solution concentration is 0.05 wt%.
3. The property of the microsphere carrier is as follows:
the particle size of the microspheres is as follows: 120 μm;
film thickness: 10 μm.
Example 5
Sperm cryopreservation experimental study
1. Sperm cryopreservation
Sucking 1 drop of sperm culture solution, placing the sperm culture solution into a culture dish to form a drop (about 0.5 mu l), then adding 10.0ml of mineral oil to cover the drop, sucking a hollow microsphere carrier, placing the hollow microsphere carrier into the drop of the sperm culture solution, grabbing 1 movable sperm by using a micro-injection needle, placing the sperm into 1 cryopreservation carrier, sucking the cryopreservation carrier by using a suction tube, placing the cryopreservation carrier into a cryopreservation tube added with the sperm cryopreservation solution, precooling and cooling by liquid nitrogen steam with the height of 4.0cm above the liquid nitrogen surface for 3min (the cooling speed is 20 ℃/min), and then putting the cryopreservation tube into liquid nitrogen for rapid freezing and preserving.
2. Cryopreservation sperm resuscitation
The cryopreservation tube was taken out of the liquid nitrogen tank, thawed quickly at 37 ℃, and the cryopreservation solution containing the microsphere carrier was taken out of the cryopreservation tube with a pipette and added quickly to a petri dish (5.0 ml of sperm culture solution containing 37 ℃). The sperm is microscopically sought in the cryopreservation carrier and is grasped from the cryopreservation carrier by a micro-needle micromanipulator and transferred to another dish containing sperm medium.
3. Experiment grouping
The experiment was divided into a blank group, an example 1 group, a comparative example 2 group, an example 2 group, a comparative example 3 group, a comparative example 4 group, an example 3 group, a comparative example 5 group, a comparative example 6 group, an example 4 group, a comparative example 7 group, and a comparative example 8 group. 100 cryopreservation vehicles were selected per group, each loaded with 1 sperm.
4. Detection method
(1) Frozen carrier integrity test
Observing the shape of the carrier after cryopreservation by a microscope, wherein the shape of the carrier is not changed and is complete, the deformation or the rupture of the carrier is form rupture, and the integrity rate (%) of the carrier is (the number of the complete carriers before cryopreservation-the number of the complete carriers after cryopreservation)/the number of the complete carriers before cryopreservation.
(2) Sperm recovery assay
The number of sperm before cryopreservation and after cryopreservation was counted under a microscope, and the sperm recovery (%) was (number of sperm before cryopreservation-number of sperm after cryopreservation)/number of sperm before cryopreservation.
(3) Sperm motility assay
Observing the sperm motility under a microscope, counting the number of the sperms before and after cryopreservation, and obtaining the sperm motility rate (%) after cryopreservation (the total number of the recovered sperms after cryopreservation-the number of the recovered sperm after cryopreservation)/the total number of the recovered sperms after cryopreservation.
5. Results of the experiment
TABLE 1
From the experimental results shown in table 1, the carrier strength and the particle diameter are related to the film thickness, the smaller the particle diameter is, the larger the film thickness is, the higher the carrier strength is, the higher the carrier integrity rate after cryopreservation is, the sperm recovery rate is positively related to the carrier integrity rate, the higher the carrier integrity rate is, the higher the sperm recovery rate is, the sperm activity rate is not related to the carrier integrity rate and the sperm recovery rate, and the sperm activity rate of each experimental group is about 80%. From the view of the preparation conditions of the carrier, the particle size of the carrier is positively correlated with the concentration and viscosity of the sodium alginate solution, the lower the concentration and viscosity, the smaller the particle size, but the concentration and viscosity cannot be too low, and the gel beads cannot be formed by too low viscosity, so the gel beads should be prepared by adopting the proper concentration and viscosity of the sodium alginate; the thickness of the carrier film is positively correlated with the dosage of epsilon-polylysine, and the more the dosage of epsilon-polylysine is, the larger the film thickness is, the higher the carrier strength is. The carrier strength is also related to the G/M ratio of sodium alginate, the larger the G/M ratio, the higher the carrier strength and the higher the integrity of the carrier after freezing storage, and the analysis reasons are mainly that epsilon-polylysine can only carry out complexation reaction with carboxyl in guluronic acid in sodium alginate molecules to form a polyelectrolyte membrane and does not react with carboxyl in mannuronic acid molecules, the higher the content of guluronic acid in sodium alginate molecules is, the more carboxyl can participate in the reaction, the higher the strength of the formed polyelectrolyte complexation membrane is, and the higher the integrity of the carrier after freezing storage is. In conclusion, the carrier has higher carrier integrity rate, sperm recovery rate and sperm mobility when used for freezing and storing a small amount of sperms.
The embodiments described above are merely preferred embodiments of the invention, rather than all possible embodiments of the invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.
Claims (8)
1. The epsilon-polylysine microsphere carrier is characterized by being prepared from sodium alginate and epsilon-polylysine, the carrier is hollow microspheres with the diameter of 50-180 mu m, the shell is a polyelectrolyte membrane formed by the electrostatic complexation reaction of the sodium alginate and the epsilon-polylysine, and the inner core is a sodium alginate solution.
2. The epsilon-polylysine microsphere carrier of claim 1, wherein the sodium alginate has a molecular weight of 70.0-200.0 kD, and the molecular weight of guluronic acid in sodium alginate is as follows: the mass ratio of mannuronic acid is 1.0-3.0, the solution concentration is 0.8-2.0 wt%, and the solution viscosity is 50.0-150.0 mPas.
3. The epsilon-polylysine microsphere carrier of claim 1, wherein the epsilon-polylysine has a molecular weight of 5.0-30.0 kD and a solution concentration of 0.01-0.5 wt%.
4. The method of preparing an epsilon-polylysine microsphere carrier of claim 1, comprising the steps of:
(1) adding sodium alginate into normal saline, stirring at high speed to dissolve completely;
(2) dripping sodium alginate solution into 0.1M calcium chloride solution by electrostatic liquid drop method, and calcification for 30min to form calcium alginate gel beads;
(3) adding calcium alginate gel beads into an epsilon-polylysine solution, wherein the volume ratio of the calcium alginate gel beads to the epsilon-polylysine solution is 1:10, oscillating a universal shaking table to enable the calcium alginate gel and the epsilon-polylysine to carry out a crosslinking reaction, finishing the crosslinking reaction after 10min, and washing with deionized water to remove the epsilon-polylysine solution;
(4) and (3) adding the product obtained in the step (3) into a 55.0mM sodium citrate solution for liquefaction for 5min, liquefying the inner core of the microsphere to form a hollow structure, and washing with normal saline for 3 times to obtain the hollow epsilon-polylysine microsphere carrier.
5. Use of an epsilon-polylysine microsphere carrier of claim 1 for cryopreservation of small numbers of sperm.
6. The application of the epsilon-polylysine microsphere carrier in cryopreservation of a small number of sperms, which is characterized in that the preserved sperms are testicular-punctured sperms, epididymal-punctured sperms and sperms of patients with severe oligospermia and asthenospermia, when in use, the sperms are preserved in the microsphere cavity, and the number of the sperms loaded on each carrier is 1-20.
7. The use of the epsilon-polylysine microsphere carrier in cryopreservation of a small number of spermatozoa as claimed in claim 5, wherein the microsphere carrier is stored in cryopreservation tubes, the number of the microsphere carriers stored in each cryopreservation tube is 1-20, and the cryopreservation tubes are stored in liquid nitrogen.
8. The use of the epsilon-polylysine microsphere carrier in cryopreservation of a small number of spermatozoa as claimed in claim 5, wherein the microsphere carrier is stored in Mcjeldahl tubes, wherein the number of microsphere carriers stored in each Mcjeldahl tube is 1-20, and the Mcjeldahl tubes are stored in liquid nitrogen.
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