CN113041261A - Method for improving asthenospermia sperm function by using exosome - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
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Abstract
The invention discloses a method for improving the function of asthenospermia sperm by using exosome, belonging to the technical field of medicine. The method comprises the following steps: separating normal human seminal plasma exosomes, screening and pretreating asthenospermia sperms, and intervening the asthenospermia sperms by the exosomes. The invention intervenes sperms in vitro by utilizing seminal plasma exosomes from a human body, improves the functions of sperm motility and the like, aims to intervene and treat asthenospermia by simple non-invasive means such as artificial fertilization and the like, and improves the birth outcome of patients.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a method for improving the function of asthenospermia sperm by using exosome.
Background
Male reproductive health has become a focus of intense concern in today's human society. According to WHO statistics, the infertility rate of the breeding age couples all over the world is 10-15%, wherein the infertility caused by male factors accounts for about half. Sperm quality problems are a major cause of male infertility. Epidemic disease surveys find that asthenospermia has become a large group of important factors causing male infertility and shows an increasing trend year by year. Asthenospermia refers to a type of infertility (WHO fifth edition) caused by a percentage of forward movement of sperm in semen lower than 32%. Meanwhile, asthenospermia not only has reduced forward motility, but also is accompanied by other sperm function abnormalities, such as capacitation, hyperactivation, acrosome reaction and the like, and the male infertility is caused by the comprehensive factors. However, because the etiology of the weak sperm is complex, the pathogenesis is not clear, the research model and means are relatively deficient, and an effective treatment means is not available at present. Although progeny may be produced by abnormal conception means (e.g., egg cytoplasm single sperm microinjection techniques, pre-embryo implantation genetic diagnosis/screening), there is still a significant risk to the health of test-tube infants. Therefore, the conventional treatment of the patients with the asthenospermia makes the patients to have the fertility in a natural mode or an artificial fertilization mode with small wound have important significance to human health.
Semen is composed of cells (sperm cells) and non-cellular components (seminal plasma). Seminal plasma is rich in various substances required by sperm life activities such as lipid, sugar, growth factors, transcription factors, proteins and the like, can provide stable and comfortable environment for sperm survival and movement, can also be used as a carrier for transporting the sperm to the female genital tract fertilization part, and has several important functions: providing nutrition for sperm transportation; can protect the sperm from being infected by harmful substances; the sperm is made to overcome the hostile chemical and immunological environment of the vagina. Research has shown that seminal plasma also contains a large number of exosomes, which are excreted with prostatic secretions during ejaculation and are involved in the maturation and functional regulation of sperm cells, and therefore, exosomes have great potential in the treatment of asthenospermia.
Currently, there is an urgent need to develop a method for treating asthenospermia with low toxicity and side effect and convenient clinical application to improve sperm function, so that patients can breed naturally after intervention or breed by artificial fertilization with less wound.
Disclosure of Invention
Aiming at the technical problems that the conventional treatment means of the asthenospermia in clinic is limited at present, and the asthenospermia with unknown etiology has no effective treatment means, the invention provides a method for improving the sperm function of the asthenospermia by utilizing exosome.
In order to achieve the above object, the present invention adopts the following technical means:
a method for improving asthenospermia sperm function by using exosome comprises the following steps:
step 2, screening and pre-treating asthenospermia sperms: placing a patient semen sample collected on the same day in a 37 ℃ constant-temperature water bath for liquefaction for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human sperms by using a computer-assisted semen analyzer, screening samples meeting the requirements of diagnosis parameters of the asthenospermia, and dyeing and removing the samples with remarkably abnormal sperm morphology;
step 3, intervention of exosome on asthenospermia sperm: washing asthenospermia sperm twice by using HS, wherein each time is 1000 g and 5 min, adding HS solution or HTF solution with 90% of semen volume to resuspend the sperm, then adding exosome with 10% of semen volume, and incubating for 10 min at 37 ℃.
Further, the sample satisfying the diagnostic parameter requirement for asthenospermia in step 2 is a sample with sperm motility < 32%.
The invention intervenes sperms in vitro by utilizing seminal plasma exosomes from a human body, improves the functions of sperm motility and the like, aims to intervene and treat asthenospermia by simple non-invasive means such as artificial fertilization and the like, and improves the birth outcome of patients.
Drawings
FIG. 1 shows the improvement of the sperm forward motility of asthenospermia by exosomes. Wherein: HS is a solvent control group, N-exosome is a normal exosome, A-exosome is an exosome derived from asthenospermia, and HSA is a positive control of human serum albumin.
FIG. 2 shows the effect of exosomes on sperm hyperactivation ability. Wherein: HTF was solvent control and Exosome was normal Exosome.
FIG. 3 shows the effect of exosomes on sperm motility. Wherein: HTF is a solvent control group, N-exosome is a normal exosome, A-exosome is an exosome derived from asthenospermia, and P4 is progesterone.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
The formula of the HS solution adopted in the invention is as follows: 135 mM NaCl, 5 mM KCl, 1 mM MgSO4，2 mM CaCl 220 mM HEPES, 5 mM sucrose, 10 mM lactic acid, 1 mM sodium pyruvate, pH adjusted to 7.4 using NaOH.
The formula of the HTF solution is as follows: 93.8 mM NaCl, 4.69 mM KCl, 0.2 mM MgSO4，0.37 mM KH2PO4，2.04 mM CaCl2，21.4 mM lactic acid，2.78 mM glucose，21 mM HEPES，4 mM NaHCO3The pH was adjusted to 7.4 and 330 mM Na-Pyruvate (1000: 1), HSA (25:1), 21 mM NaHCO, were added before use3。
Example 1
The specific separation method is as follows: collecting semen of normal fertile volunteers, centrifuging at 12000 g for 10 min, and removing cells such as sperm and other impurities; sequentially filtering the seminal plasma with 0.45 μm and 0.22 μm microporous filter membranes to remove cell debris, macromolecular aggregate impurities, etc.; 2.5 mL of the supernatant was added to 300. mu.L of a sucrose cushion solution containing 20 mM Tris/30% sucrose/deuterium oxide 100000 g, ultracentrifuged at 4 ℃ for 90 min; collecting supernatant, and ultracentrifuging at 4 deg.C for 5 hr at 100000 g in sucrose cushion solution containing 20 mM Tris/25% sucrose/deuterium oxide; the exosomes in the 30% and 25% sucrose pads were mixed and washed once with HS solution (100000 g, 4 ℃, ultracentrifugation for 90 min); dissolving the obtained exosome with HS; and finally, carrying out BCA protein quantification, adjusting the concentration to be 10 times of physiological concentration, and storing at-80 ℃ for later use.
Step 2, screening and pretreatment of asthenospermia sperms
The semen sample of the patient collected on the day is liquefied in a thermostatic water bath at 37 ℃ for 60 min. Blowing, uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human sperms by using a Computer Aided Sperm Analyzer (CASA), screening samples meeting the requirements of diagnosis parameters (sperm activity < 32%) of asthenospermia in the 5 th edition (WHO5) semen standard parameters of the handbook of human semen examination and treatment laboratories of world health organization, and dyeing to eliminate samples with obvious abnormal sperm morphology.
Step 3, intervention of exosome on asthenospermia sperm
Washing asthenospermia sperm twice with HS, 1000 g each time for 5 min; sperm were reselected with HS according to 90% of the starting semen volume (e.g., 2 mL of semen resuspended with 1.8 mL of HS or HTF solution), then 10% exosomes were added and incubated for 10 min at 37 ℃.
And then analyzing the sperm motility parameters and the hyperactivation level of the exosome treated by CASA, thereby evaluating the improvement level of the exosome on the sperm function. Hyperactivation is considered to occur when the sperm motility parameters VCL > =150um, LIN < =50%, ALH > =7 μm, the percentage of hyperactivation being the number of spermatozoa/total number of spermatozoa in which hyperactivation occurs.
The ability of sperm to penetrate viscous medium is detected by using a climbing experiment, and the specific method comprises the following steps:
the mucus penetrating capacity of the sperm simulates the capacity of the sperm to penetrate through the female reproductive tract, and 1% methylcellulose simulates the environment of the female reproductive tract, because the sperm can be combined with the ovum to finish fertilization only after penetrating through the female reproductive tract. And (3) treating exosomes and sperms in an HTF solution for 30 min, sucking in a capillary glass tube, putting a capillary tube containing 20 mu L of 1% methyl cellulose into the uniformly mixed sperm sample, and recording the number of sperms at 1, 2 and 3cm in the capillary tube under a microscope after 1 h.
As can be seen from fig. 1, normal exosomes at physiological concentrations can significantly increase the forward motility of sperm by about 60% relative to the non-exosome group, but exosomes from severe asthenozoospermia do not have this ability. As can be seen from fig. 2, exosomes can significantly increase the hyperactivated motility level of sperm (increase of about 60%). In addition, another parameter reflecting sperm fertilization ability, transmyxosis, was also significantly improved by the addition of exosomes (fig. 3, increase by about 70%), with an effect similar to that produced by 1 μ M progesterone. These data indicate that the addition of normal exosomes has the effect of significantly improving sperm function, and is expected to be used for the treatment of asthenospermia in the future.
Claims (2)
1. A method for improving the function of asthenospermia sperm by using exosome is characterized in that: the method comprises the following steps:
step 1, separating normal human seminal plasma exosomes: collecting normal fertile semen, centrifuging for 10 min at 12000 g, removing cells and impurities, filtering seminal plasma by using microporous filter membranes with the diameters of 0.45 mu m and 0.22 mu m in sequence, removing cell debris and macromolecular aggregate impurities, adding 2.5 mL of supernatant into 300 mu L of sucrose cushion solution containing 20 mM Tris, 30 wt.% sucrose and deuterium oxide, ultracentrifuging for 90 min at 4 ℃, collecting supernatant into 100000 g of sucrose cushion solution containing 20 mM Tris, 25 wt.% sucrose and deuterium oxide, ultracentrifuging for 5 h at 4 ℃, mixing exosomes obtained by two times of centrifugation, washing by using HS solution, dissolving the obtained exosomes by using the HS solution, adjusting the exosomes to be 10 times of physiological concentration, and storing at-80 ℃ for later use;
step 2, screening and pre-treating asthenospermia sperms: placing a patient semen sample collected on the same day in a 37 ℃ constant-temperature water bath for liquefaction for 60 min, blowing and uniformly mixing, taking 10 mu L of liquefied semen, analyzing various parameters of human sperms by using a computer-assisted semen analyzer, screening samples meeting the requirements of diagnosis parameters of the asthenospermia, and dyeing and removing the samples with remarkably abnormal sperm morphology;
step 3, intervention of exosome on asthenospermia sperm: washing asthenospermia sperm twice by using HS, wherein each time is 1000 g and 5 min, adding HS solution or HTF solution with 90% of semen volume to resuspend the sperm, then adding exosome with 10% of semen volume, and incubating for 10 min at 37 ℃.
2. The method of improving human sperm function utilizing seminal plasma exosomes according to claim 1, wherein: the sample meeting the diagnosis parameter requirement of the asthenospermia in the step 2 refers to a sample with sperm forward movement sperm motility of less than 32%.
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|---|---|---|---|---|
| CN113171378A (en) * | 2021-04-30 | 2021-07-27 | 奥启(深圳)创投科技有限公司 | Stem cell exosome preparation for preventing and treating male sexual dysfunction |
| CN114621337A (en) * | 2022-03-31 | 2022-06-14 | 南通大学 | Polypeptide containing IDER sequence for improving sperm function and application thereof |
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