CN108441465B - Mouse embryo transplantation glue and preparation method thereof - Google Patents
Mouse embryo transplantation glue and preparation method thereof Download PDFInfo
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- CN108441465B CN108441465B CN201810258476.3A CN201810258476A CN108441465B CN 108441465 B CN108441465 B CN 108441465B CN 201810258476 A CN201810258476 A CN 201810258476A CN 108441465 B CN108441465 B CN 108441465B
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Abstract
The invention discloses a mouse embryo transfer glue and a preparation method thereof, wherein the mouse embryo transfer glue is prepared by adding pregnant horse serum, chorionic gonadotropin, amniotic membrane substance, mouse plasma and streptomycin into HEPES buffer solution with pH of 7.0-7.2 and concentration of 20 mmol/L; the amounts of each substance added per mL of HEPES buffer were as follows: 0.01-0.05 mu g of pregnant horse serum, 100-375 units of chorionic gonadotropin, 50-70 mu g of streptomycin, 20 mu L of amniotic membrane substance and 8-10 mu L of mouse plasma. The mouse embryo transfer glue provided by the invention not only can adhere the embryo to the uterus and improve the implantation rate of the embryo, but also provides nutrition for the growth of the implanted embryo and greatly improves the survival rate of the mouse embryo.
Description
Technical Field
The invention belongs to the technical field of reproduction, and particularly relates to mouse embryo transplantation glue and a preparation method thereof.
Background
The technology of mammalian embryo transplantation is an important technology in experimental embryology, genetics and developmental biology research, and embryo transplantation, namely, an early embryo of one female animal is taken out from an oviduct or uterus and transplanted into the oviduct or uterus of the other female animal, so that the embryo is continuously developed into a fetus. The mouse is the most important model mammal in the embryo transplantation technology, and the early embryo of the mouse is a common experimental material for researching the early occurrence mechanism of the mammal and the human. Mouse embryo transfer, i.e., transfer of embryos obtained by in vitro fertilization or other means to pseudopregnant recipients, allows the embryos to develop and live in vivo. In the mouse embryo transfer process, the embryo can be deformed rapidly, the implantation rate of the embryo transfer is low, and the development after the embryo transfer is unstable, so that the abortion rate is high.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides the mouse embryo transfer glue and the preparation method thereof, and the provided mouse embryo transfer glue can obviously improve the implantation rate and the development stability of mouse embryos and greatly improve the success rate of mouse embryo transfer.
The invention aims to provide mouse embryo transfer glue, which is prepared by adding pregnant horse serum, chorionic gonadotropin, amniotic membrane substances, mouse plasma and streptomycin into HEPES buffer solution with the pH value of 7.0-7.2 and the concentration of 20 mmol/L;
the amounts of each substance added per mL of HEPES buffer were as follows: 0.01-0.05 mu g of pregnant horse serum, 100-375 units of chorionic gonadotropin, 30-50 mu g of streptomycin, 20 mu L of amniotic membrane substance and 8-10 mu L of mouse plasma;
the amniotic membrane substance is prepared by the following method:
taking amnion after mouse parturition as raw material, obtaining acellular amnion after cell removal treatment of trypsin combined with Triton X-100, then carrying out high-speed grinding under the condition of liquid nitrogen freezing, and carrying out irradiation disinfection by cobalt 60 to obtain granular amnion; inoculating umbilical cord mesenchymal stem cells on the granular amnion, performing combined culture for 5-8 h, and resuspending with normal saline to obtain the amnion substance.
Preferably, every 1mg of the granular amnion is inoculated with 1-1.5 × 105Umbilical cord mesenchymal stem cells.
The second purpose of the invention is to provide a preparation method of the mouse embryo transfer glue, which comprises the following steps:
s1: preparation of amniotic Material
Taking amnion after mouse parturition as raw material, and obtaining acellular amnion after cell removal treatment by combining trypsin with Triton X-100; grinding the obtained acellular amnion at high speed under the condition of liquid nitrogen freezing to obtain particle fragments, irradiating and sterilizing the obtained particle fragments by cobalt 60,obtaining a granular amnion; 1-1.5 × 10 of granular amnion is inoculated per 1mg5According to the proportion of the umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells are inoculated on the granular amnion, the combined culture is carried out for 5-8 h, a stem cell-granular amnion mixture is obtained, and the mixture is sieved by a 120 mu m sieve and then is resuspended by normal saline to obtain the amnion substance;
s2: preparation of mouse plasma
Taking a fresh blood sample isolated from a mouse, obtaining plasma rich in platelets through centrifugation, and storing the plasma at 4 ℃ for later use within 5 days;
s3: and (2) respectively taking pregnant mare serum, chorionic gonadotropin, amniotic membrane substances, mouse plasma, streptomycin and HEPES (high efficiency electron transfer) buffer solution with the pH of 7.0-7.2 and the concentration of 20mmol/L according to the proportion, and uniformly mixing the substances to obtain the mouse embryo transfer gel.
Compared with the prior art, the invention has the beneficial effects that:
(1) the mouse embryo transfer glue provided by the invention can adhere an embryo to the uterus, improve the implantation rate of the embryo and provide nutrition for the growth of the embryo after implantation, the amniotic membrane substance in the mouse embryo transfer glue is prepared from the amniotic membrane of the mouse during delivery, contains a large amount of different collagens, fibronectin and laminin, has good adhesive force and excellent biocompatibility, and umbilical cord mesenchymal stem cells inoculated in the amniotic membrane substance have strong anti-inflammatory, anti-immune rejection and proliferation promoting functions; the mouse plasma is rich in various nutritional factors, can promote fibroblasts to secrete collagen, and can release a large amount of growth factors after being mixed with the amniotic membrane substance to promote the proliferation of embryonic cells.
(2) The mouse embryo transfer glue provided by the invention can well transfer mouse embryos into mouse bodies to form a gel state, so that the mobility of the implanted mouse embryos is reduced; the stem cells can be induced into fiber cells at the implantation part to secrete collagen, so that the degraded amniotic membrane particles are compensated, the slow release function after implantation in the body is realized, and the survival rate of mouse embryos is greatly improved.
(3) Because the raw materials of the mouse embryo transfer glue are the mouse autologous substances, the biocompatibility and the anti-inflammation are good, the antibacterial property in the mouse embryo transfer process can be realized by adding a small amount of streptomycin, and the influence of antibacterial drugs on the development of mouse embryos is reduced.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. Unless otherwise specifically stated, the various starting materials, reagents, instruments and equipment used in the following examples of the present invention are either commercially available or prepared by conventional methods.
Example 1
The mouse embryo transfer glue of the embodiment is prepared by the following specific steps:
taking amnion after mouse parturition as raw material, and obtaining acellular amnion after cell removal treatment by combining trypsin with Triton X-100; grinding the obtained acellular amnion at high speed under the condition of liquid nitrogen freezing to obtain particle fragments, and irradiating and sterilizing the obtained particle fragments by using cobalt 60 to obtain granular amnion; inoculating granular amnion at a dose of 1.0 × 105According to the proportion of the umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells are inoculated on the granular amnion and are subjected to combined culture for 5 hours to obtain a stem cell-granular amnion mixture, and the mixture passes through a 120 mu m screen and then is resuspended by using normal saline to obtain the amnion substance; taking a fresh blood sample isolated from a mouse, obtaining plasma rich in platelets through centrifugation, and storing the plasma at 4 ℃ for later use within 5 days; taking pregnant mare serum, chorionic gonadotropin, amniotic membrane substances, mouse plasma, streptomycin and HEPES buffer solution with pH of 7.0 and concentration of 20mmol/L according to the proportion, wherein the addition amount of each substance in each mL of HEPES buffer solution is as follows: pregnant horse serum 0.01 mug, 200 units of chorionic gonadotropin, 30 mu g of streptomycin, 20 mu L of amniotic membrane substance and 8 mu L of mouse plasma; and uniformly mixing the substances to obtain the mouse embryo transfer glue.
Example 2
The mouse embryo transfer gel of this example was prepared in the same manner as in example 1, except that the amount of each substance added per mL of HEPES buffer was as follows: 0.05 μ g of pregnant horse serum, 375 units of chorionic gonadotropin, 50 μ g of streptomycin, 20 μ L of amniotic membrane substance and 10 μ L of mouse plasma.
Example 3
The mouse embryo transfer gel of this example was prepared in the same manner as in example 1, except that the amount of each substance added per mL of HEPES buffer was as follows: pregnant horse serum 0.035 μ g, chorionic gonadotropin 100 units, streptomycin 42 μ g, amniotic membrane substance 20 μ L, mouse plasma 8 μ L.
Comparative example 1
The mouse embryo transfer glue of the comparative example is prepared by the following specific steps:
taking pregnant mare serum, chorionic gonadotropin, polyacrylamide hydrogel, streptomycin and HEPES buffer solution with pH of 7.0 and concentration of 20mmol/L according to the proportion, wherein the addition amount of each substance in each mL of HEPES buffer solution is as follows: 0.01 mu g of pregnant mare serum, 200 units of chorionic gonadotropin, 30 mu g of streptomycin and 30 mu L of polyacrylamide hydrogel; and uniformly mixing the substances to obtain the mouse embryo transfer glue.
The following tests show the excellent effects of the mouse embryo transfer glue provided in embodiments 1 to 3 of the present invention, and the specific method is as follows:
selecting 16 sexually mature female Kunming mice, dividing the mice into an experimental group and a control group, wherein each group comprises 8 mice, feeding the experimental mice in an SPF animal room, controlling the temperature at 25-28 ℃, carefully sucking about 10 mu L of embryo-carrying embryo transfer glue provided in example 1 by a transfer tube under the auxiliary observation of a microscope during the embryo transfer of the experimental group, blowing the embryo transfer glue into a uterus, and transferring 5 embryos to each mouse; the control group aspirates about 10. mu.L of the embryo transfer gel provided in comparative example 1 with embryos using a transfer tube.
After the mice are naturally produced, 31 mice are produced by 8 mice in the experimental group, the embryo transplantation success rate reaches about 77.5%, 8 mice are produced by 8 mice in the control group, each mouse produces at most 2 mice, one of the 2 mice does not produce any mouse, and the embryo transplantation success rate of the control group is only 20%. As can be seen from the comparison of the results of the experimental group and the control group, the mouse embryo transfer glue provided in the embodiments 1-3 of the present invention has a significant effect compared with the mouse embryo transfer glue provided in the comparative example 1, mainly because the mouse embryo transfer glue provided in the present invention can significantly improve the implantation rate of the embryo and can provide nutrients for the development of the embryo. The polyacrylamide hydrogel used in comparative example 1 has good biocompatibility and adhesive property, but is degraded quickly and has poor persistence, and the degradation product hydroxy acid is locally aggregated in the degradation process, so that the pH is reduced, the embryo is poisoned and even killed, and the embryo survival rate is low.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations. The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of protection is not limited thereto. The equivalents and modifications of the present invention which may occur to those skilled in the art are within the scope of the present invention as defined by the appended claims.
Claims (2)
1. A mouse embryo transfer glue is characterized in that pregnant horse serum, chorionic gonadotropin, amniotic membrane substances, mouse plasma and streptomycin are added into HEPES buffer solution with pH of 7.0-7.2 and concentration of 20mmol/L to prepare the glue;
the amounts of each substance added per mL of HEPES buffer were as follows: 0.01-0.05 mu g of pregnant horse serum, 100-375 units of chorionic gonadotropin, 30-50 mu g of streptomycin, 20 mu L of amniotic membrane substance and 8-10 mu L of mouse plasma;
the mouse embryo transfer glue is prepared by the following method:
s1: preparation of amniotic Material
Taking amnion after mouse parturition as raw material, and obtaining acellular amnion after cell removal treatment by combining trypsin with Triton X-100; grinding the obtained acellular amnion at high speed under the condition of liquid nitrogen freezing to obtain particle fragments, and irradiating and sterilizing the obtained particle fragments by using cobalt 60 to obtain granular amnion; 1-1.5 × 10 of granular amnion is inoculated per 1mg5According to the proportion of the umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells are inoculated on the granular amnion, the combined culture is carried out for 5-8 h, a stem cell-granular amnion mixture is obtained, and the mixture is sieved by a 120 mu m sieve and then is resuspended by normal saline to obtain the amnion substance;
s2: preparation of mouse plasma
Taking a fresh blood sample isolated from a mouse, obtaining plasma rich in platelets through centrifugation, and storing the plasma at 4 ℃ for later use within 5 days;
s3: and (2) respectively taking pregnant mare serum, chorionic gonadotropin, amniotic membrane substances, mouse plasma, streptomycin and HEPES (high efficiency electron transfer) buffer solution with the pH of 7.0-7.2 and the concentration of 20mmol/L according to the proportion, and uniformly mixing the substances to obtain the mouse embryo transfer gel.
2. The mouse embryo transfer glue according to claim 1, wherein 1-1.5X 10 of the particulate amniotic membrane is inoculated per 1mg of the particulate amniotic membrane5Umbilical cord mesenchymal stem cells.
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| CN201810258476.3A CN108441465B (en) | 2018-03-27 | 2018-03-27 | Mouse embryo transplantation glue and preparation method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779989A (en) * | 2010-01-19 | 2010-07-21 | 广东温氏食品集团有限公司 | Method for pig in-vitro fertilization and embryo transplantation |
| CN107254433A (en) * | 2017-05-24 | 2017-10-17 | 瑞柏生物（中国）股份有限公司 | A kind of embryo transfer glue and preparation method thereof |
| CN107583109A (en) * | 2017-09-22 | 2018-01-16 | 吉林大学 | A kind of corium deep layer filler and its preparation method and application |
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2018
- 2018-03-27 CN CN201810258476.3A patent/CN108441465B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779989A (en) * | 2010-01-19 | 2010-07-21 | 广东温氏食品集团有限公司 | Method for pig in-vitro fertilization and embryo transplantation |
| CN107254433A (en) * | 2017-05-24 | 2017-10-17 | 瑞柏生物（中国）股份有限公司 | A kind of embryo transfer glue and preparation method thereof |
| CN107583109A (en) * | 2017-09-22 | 2018-01-16 | 吉林大学 | A kind of corium deep layer filler and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| The potential of amniotic membrane/amnion-derived cells for regeneration of various tissues;Ayaka Toda，et al;《J Pharmacol Sci》;20071106;第105卷(第3期);215-28 * |
| 小鼠孤雌胚、体外培养胚与体内胚H3K9乙酰化模式的比较;陈利 等;《遗传》;20150131;第37卷(第1期);77-83 * |
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Inventor after: Liu Yong Inventor after: Wang Wenying Inventor after: Li Wenyong Inventor after: Xin Jing Inventor after: Kong Feng Inventor after: Wu Qiaoqin Inventor after: Wu Xiaoqing Inventor before: Liu Yong Inventor before: Li Wenyong Inventor before: Xin Jing Inventor before: Kong Feng Inventor before: Wang Wenying Inventor before: Wu Qiaoqin Inventor before: Wu Xiaoqing |
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