CN108378022A - A kind of human sperm's cryoprotector - Google Patents
A kind of human sperm's cryoprotector Download PDFInfo
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- CN108378022A CN108378022A CN201810435420.0A CN201810435420A CN108378022A CN 108378022 A CN108378022 A CN 108378022A CN 201810435420 A CN201810435420 A CN 201810435420A CN 108378022 A CN108378022 A CN 108378022A
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- Prior art keywords
- sperm
- cryoprotector
- milliliters
- human
- rate
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Classifications
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
A kind of cryoprotector suitable for human sperm's freezen protective, cryoprotector provided by the invention mainly consist of the following compositions：Sodium citrate, trishydroxymethylaminomethane, Pehanorm base ethanesulfonic acid, glycerine and trehalose.Cryoprotector provided by the invention avoids potential bio-safety hidden danger without animal derived materials such as yolk.Compared with prior art; freezen protective is carried out to human sperm using cryoprotector provided by the invention; better anabiosis rate can be obtained after defrosting; progressive sperm after recovery is more; propulsion ability is preferable, while the spontaneous acrosome reaction rate of the sperm after recovery and DNA fragment rates are lower, i.e. the complete sperm of acrosome is more; the nuclear damage of sperm is small, more preferable to the protective capability of sperm function.
Description
Technical field
Field is frozen the present invention relates to human cell, specifically, being related to a kind of human sperm's cryoprotector.
Background technology
The freezen protective of human sperm is one of the premise for ensureing auxiliary procreation technology and smoothly carrying out, freezen protective technology
It improves, helps to improve available sperm quality in clinically supplementary reproduction.The perfect of cryoprotection agent prescription of sperm be
Improve one of the key factor of human sperm's freezen protective.Ice crystal in the refrigerating process of ultralow temperature is formed, and the knot of sperm is damaged
Structure influences the function of sperm, such as the raising of spontaneous acrosome reaction rate and DNA fragment with spermatozoon rate, and then influences the fertilization work(of sperm
Energy.Therefore, not only demand protects Sperm motility to the cryoprotection of sperm, it is also necessary to improve the guarantor in terms of sperm function
Protect effect.The cryoprotector clinically used at present is broadly divided into two classes, and one kind is commercially available cryoprotector, and one kind is respectively to face
Bed application places make cryoprotector by oneself.The shortcomings that commercially available cryoprotector, is the cryoprotective effect of sperm, especially smart
Protecting effect in terms of subfunction is limited, and part makes cryoprotector by oneself containing animal derived materials such as lecithalities, exists certain
Bio-safety hidden danger.Application for a patent for invention 201301725422 discloses a kind of mankind's cryoprotector, the protection announced
Contain fresh yolk in agent ingredient, there are bio-safety hidden danger.Application for a patent for invention 2015110283406 discloses a kind of no ovum
Yellow cryoprotector has no although improving in terms of Cryopreservation rate for the guarantor in terms of sperm function
Shield.The present invention provides a kind of cryoprotector of the animal derived materials such as no yolk, and human sperm is frozen suitable for sequencing, and
Ideal Cryopreservation rate can be obtained after thawing and reduces damage of the sperm in refrigerating process in terms of function.
Invention content
The present invention provides a kind of cryoprotectors of human sperm, can reduce to a certain extent in freezing to sperm
The damage of function.
To achieve the above object, the present invention provides following human sperm's cryoprotector, and particular technique embodiment is as follows：
A kind of human sperm's cryoprotector, every 100 milliliters of protective agents contain following ingredient：
0.325 ± 0.095 gram of sodium citrate, 0.875 ± 0.255 gram of trishydroxymethylaminomethane (Tris), trihydroxy methyl
0.305 ± 0.075 gram of methylamino ethanesulfonic acid (TES), 13 ± 5 milliliters of glycerine, 5.5 ± 2.1 grams of trehalose.
Specific preparation method is as follows：
A, proper amount of sodium citrate, trishydroxymethylaminomethane and Pehanorm base ethanesulfonic acid are weighed, with 50 milliliter five
Water is steamed to stir to abundant dissolving, with citric acid regulating solution pH value to 7.5；
B, appropriate trehalose is weighed, above-mentioned solution is added, is slowly stirred to abundant dissolving；
C, qs glycerin is measured, above-mentioned solution is added, is slowly stirred to abundant dissolving；
D, five are added and steam water, be settled to 100 milliliters；
E, above-mentioned configuration solution passes through 0.45 micron and 0.25 zut filter respectively successively after fully dissolving；
F, filtered protective agent is dispensed, is put into ultra low temperature freezer and freezes；
Compared with prior art, it is an advantage of the present invention that without the animal derived materials such as yolk, behaviour in cryoprotector
Make simplicity, preparation time is short, and the freezen protective of human sperm is carried out using this protective agent, can obtain after thawing preferable multiple
Su Xiaoguo, the preceding tropism locomitivity of sperm is preferable after recovery, while compared with commercially available protective agent, spontaneous acrosome reaction rate and essence
Sub- DNA fragment rates reduce.
Specific implementation mode
Below the technical solution in implementation process of the present invention will in detail, completely describe, it is notable that institute
The scheme of description only represents some embodiments.The scientific worker of this field upon reading this disclosure, is based on the present invention
In embodiment, the every other embodiment that technological staff is obtained without making creative work,
Belong to protection scope of the present invention.
Embodiment 1：
A, clean volumetric flask after cancellation poison measures 50 milliliter of five steaming water, and 0.3 gram of sodium citrate of addition is added five and steams in water,
Stirring is dissolved to abundant；
B, 0.8 gram of trishydroxymethylaminomethane is weighed, 0.3 gram of Pehanorm base ethanesulfonic acid is added above-mentioned solution, stirs
It mixes to abundant dissolving；
C, above-mentioned solution is adjusted to pH7.5 with the citric acid solution of 0.1mol/L；
D, 5.5 grams of trehalose is weighed, above-mentioned solution is added, is slowly stirred, bubble is avoided to generate, until fully dissolving；
E, 10 milliliters of glycerine is measured, above-mentioned solution is added, it is slowly relatively slow, avoid bubble from generating, until fully dissolving；
F, the use of aperture is that 0.45 micron filter filters above-mentioned solution, solution is put into sterile cleaning container temporarily after filtering
It deposits；
G, above-mentioned solution filtering using 0.22 micron filter, solution carries out pathogen culture after taking Partial filtration, remaining
Solution packing freezes to -80 DEG C of refrigerators.After pathogen culture result is negative, packing solution can be applied to practice；
H, 0.5 milliliter of human seminal fluid after fresh abundant liquefaction is taken, 0.5 milliliter of above-mentioned configuration protective agent is added in cryopreservation tube
In, it mixes well.
I, above-mentioned sperm and protective agent mixed liquor are frozen according to the freezing procedure of Slow-rate freezing instrument preset value, completes freezing journey
After sequence, cryopreservation tube is frozen in liquid nitrogen container.
The refrigerating effect of sperm freezing protecting agent provided by the invention is detected below by way of Experimental comparison：
A, choose commercially available sperm freezing protecting agent (comparative example 1), with human sperm's cryoprotector provided by the invention into
Row comparison, specific embodiment are same as Example 1；
B, the 5th edition semen routine analysis standard of the World Health Organization is pressed, 10 normal human subject sperm are randomly selected；
C, after sperm fully liquefies, Semen routione parameter is analyzed and recorded using computer-aided analysis system, it will be every
Example sperm is randomly divided into four parts, and a copy of it is detected for detecting spontaneous acrosome reaction, portion for DNA fragment with spermatozoon rate, is remained
Remaining other two parts are used for freezen protective contrast experiment：Portion uses 1 protective agent of comparative example, is operated according to operation instructions；It is another
Part provides embodiment 1 using the present invention and configures protective agent；
D, sperm is pressed：Protective agent=1:Sperm and sperm freezing protecting agent are fully mixed after the ratio of 1 (volume ratio) will liquefy
It is even, i.e. in 1 milliliter of mixing liquid, contain 0.5 milliliter of sperm and 0.5 milliliter of protective agent；
E, it is freezed according to Slow-rate freezing instrument setting program, is put into liquid nitrogen and preserves 30 minutes after program；
F, 37 DEG C of shaking baths thaw, and the mixed liquor after abundant thaw are divided into three parts, wherein portion is used for computer aided manufacturing
Semen routione parameter after helping analysis system detection to thaw, portion are used for DNA fragment with spermatozoon for detecting spontaneous acrosome reaction, portion
Rate detects；
Using human sperm's cryoprotector provided by the invention, with commercially available human sperm's cryoprotector by mutually year-on-year
Example, same procedure carry out freezen protective, right using computer aided pass design after being thawed using identical defreezing method
Cryopreservation rate, sperm motility and progressive sperm sum than sperm compare and analyze, using commercial reagent box to certainly
Acrosome reaction rate and the detection of DNA fragment with spermatozoon rate are sent out, obtains that the experimental data are shown in the following table：
| Fresh semen | Comparative example 1 | Embodiment 1 | |
| Progressive sperm number | 46.94±17.3 | 36.02±16.31 | 39.72±16.11 |
| Curve speed | 80.77±12.18 | 60.34±10.36 | 66.89±10.88 |
| Cryopreservation rate | 70.31±16.44 | 77.16±18.68 | |
| Spontaneous acrosome reaction rate | 36.32±20.44 | 71.27±16.29 | 62.25±15.34 |
| DNA fragment with spermatozoon rate | 15.15±7.21 | 22.52±12.07 | 16.13±7.07 |
Sperm motility=Cryopreservation rate before sperm motility ÷ freezen protectives after defrosting.
The detection of spontaneous acrosome reaction rate is using Anhui peace section bioengineering (group) limited liability company perforatorium dye
Color reagent box (PSA-FITC decoration methods), by specification is operated and is counted.
The detection of DNA fragment with spermatozoon rate uses the Zhejiang biotech inc Xing Bo sperm nucleus integrality staining reagent
Box, by specification are operated and are counted.
From the above data, using cryoprotector provided by the present invention, compared with commercially available protective agent comparative example 1,
Progressive sperm number after thawing increases, and anabiosis rate increases；Spontaneous acrosome reaction rate reduces simultaneously, it is meant that uses this hair
After the protective agent freezing of bright offer, the complete sperm number of acrosome will be more than commercially available protective agent comparative example 1,；DNA fragment with spermatozoon rate
It reduces, it is meant that the DNA damage using sperm after protective agent provided by the invention freezing is small compared with comparative example 1, illustrates to use this hair
The cryoprotector of bright offer is preferable to the protectiveness of human sperm's function.
According to the embodiment given above, those skilled in the art or researcher can not depart from original of the invention
Embodiment is changed in the case of reason, change, is replaced, the scope of the present invention is limited by the following claims and their equivalents
It is fixed.
Claims (7)
1. a kind of human sperm's cryoprotector, which is characterized in that every 100 milliliters of protective agents contain following component：Sodium citrate,
Trishydroxymethylaminomethane, Pehanorm base ethanesulfonic acid, trehalose, glycerine and five steam water surplus.
2. according to sodium citrate described in claim 1, which is characterized in that every 100 milliliters of protective agents contain sodium citrate 0.325
± 0.095 gram.
3. according to trishydroxymethylaminomethane described in claim 1, which is characterized in that every 100 milliliters of protective agents contain three hydroxyls
0.875 ± 0.255 gram of aminomethane.
4. according to Pehanorm base ethanesulfonic acid described in claim 1, which is characterized in that every 100 milliliters of protective agents contain
0.305 ± 0.075 gram of Pehanorm base ethanesulfonic acid.
5. according to trehalose described in claim 1, which is characterized in that every 100 milliliters of protective agents contain trehalose 5.5 ± 2.1
Gram.
6. according to glycerine described in claim 1, which is characterized in that every 100 milliliters of protective agents contain 13 ± 5 milliliters of glycerine.
7. according to cryoprotector described in claim 1, which is characterized in that its pH value is 7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810435420.0A CN108378022A (en) | 2018-05-09 | 2018-05-09 | A kind of human sperm's cryoprotector |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810435420.0A CN108378022A (en) | 2018-05-09 | 2018-05-09 | A kind of human sperm's cryoprotector |
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| CN108378022A true CN108378022A (en) | 2018-08-10 |
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| CN201810435420.0A Pending CN108378022A (en) | 2018-05-09 | 2018-05-09 | A kind of human sperm's cryoprotector |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111202051A (en) * | 2020-02-26 | 2020-05-29 | 东蕴医疗科技(上海)有限公司 | Sperm refrigerating fluid for human assisted reproduction and preparation method thereof |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
Citations (4)
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|---|---|---|---|---|
| CA2391370A1 (en) * | 1999-11-24 | 2001-05-31 | Xy, Inc. | Method of cryopreserving selected sperm cells |
| CN104365583A (en) * | 2014-11-07 | 2015-02-25 | 郜鸿生物科技（上海）有限公司 | Extremely-low sperm freezing protective agent and application thereof |
| CN105532643A (en) * | 2015-12-31 | 2016-05-04 | 中信湘雅生殖与遗传专科医院有限公司 | Sperm cryoprotectant and preparation method thereof |
| CN105746494A (en) * | 2016-04-21 | 2016-07-13 | 山西省人口计生委科学研究所 | Human sperm cryoprotectant containing D-trehalose and preparation method of human sperm cryoprotectant containing D-trehalose |
-
2018
- 2018-05-09 CN CN201810435420.0A patent/CN108378022A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2391370A1 (en) * | 1999-11-24 | 2001-05-31 | Xy, Inc. | Method of cryopreserving selected sperm cells |
| CN104365583A (en) * | 2014-11-07 | 2015-02-25 | 郜鸿生物科技（上海）有限公司 | Extremely-low sperm freezing protective agent and application thereof |
| CN105532643A (en) * | 2015-12-31 | 2016-05-04 | 中信湘雅生殖与遗传专科医院有限公司 | Sperm cryoprotectant and preparation method thereof |
| CN105746494A (en) * | 2016-04-21 | 2016-07-13 | 山西省人口计生委科学研究所 | Human sperm cryoprotectant containing D-trehalose and preparation method of human sperm cryoprotectant containing D-trehalose |
Non-Patent Citations (2)
| Title |
|---|
| 朱伟杰等: "四种人类精液冷冻保护剂的比较研究", 《中华泌尿外科杂志》 * |
| 朱伟杰等: "无卵黄型人类精液冷冻保护剂试验与临床初步应用", 《暨南大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111202051A (en) * | 2020-02-26 | 2020-05-29 | 东蕴医疗科技(上海)有限公司 | Sperm refrigerating fluid for human assisted reproduction and preparation method thereof |
| CN111436421A (en) * | 2020-05-21 | 2020-07-24 | 新乡医学院三全学院 | Human sperm cryopreservation method |
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Application publication date: 20180810 |
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