供体细胞处理方式对山羊核移植胚胎早期发育的影响
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摘要
要提高哺乳动物核移植的效率，不仅要提高卵母细胞体外成熟(IVM)质量，阐明成熟卵母细胞胞质内重编程因子的产生及作用机制；也要研究能使供体细胞经过处理后更易受重编程因子作用的方法。本文以布尔山羊胎儿成纤维细胞(BGFF)作为核移植供体细胞，研究了供体细胞处理方式对核移植胚胎早期发育的影响。
分别以DMEM或M199为基础培养液，对供体组织块进行培养，又以0.2％胶原酶Ⅱ与0.25％胰蛋白酶按2/3混合消化供体组织，结果两种方法都获得了体外培养的BGFF。细胞可在含5％DMSO(二甲亚砜)的冷冻液中于-70℃条件下短期冻存，也可在-196℃条件下长期保存。
研究了培养液中血清(胎牛血清FBS)或发情山羊血清(EGS)及促性腺素(FSH、LH和HMG(人血绒毛膜促性腺素))的来源及含量、表皮生长因子(EGF)的添加量和溶剂水的来源对山羊卵母细胞IVM的影响。结果显示：用TCM199+10％EGS+0.075IU/mL HMG+1μg/mLE_2对山羊卵母细胞进行IVM，卵母细胞成熟率为85.7％。EGS的效果不但比FBS好，而且与添加量正相关；用HMG获得的成熟率稍高于不同配比的FSH/L；EGF浓度低于30ng/mL不能显著提高卵母细胞体外成熟率，但能提高其孤雌激活胚胎的发育率。配培养液宜现制超纯水，商品水宜现买现用。
对比研究了乙醇和离子霉素(Ionomycine)分别与6-二甲基嘌啉(6-DMAP)联合激活山羊体外成熟卵母细胞的效果。结果表明：7％乙醇不能有效激活山羊体外成熟卵母细胞，而5μmol/L Ionomycine能明显地起到激活作用，且该方法激活的卵母细胞的孤雌发育囊胚率达23.5％，与乙醇处理组(0％囊胚率)相比差异极显著(P＜0.01)。
比较了处于第二次减数分裂中期(MⅡ)和末期(TⅡ)的山羊卵母细胞作为核移植受体的差异。结果表明：在TⅡ期去核效率明显高于MⅡ期，但二者间核移植胚的囊胚发育率差异不显著(P＞0.05)。
用四种方法对供体细胞作了G_0/G_1期诱导。免疫荧光检测显示：血清饥饿和G_0/G_1期抑制剂Roscovitine诱导能分别使79.5％和84.5％的BGFF进入G_0/G_1期，而作为对照组的对数生长期处理法(60.3％)的诱导效率明显低于前二者(P＜0.05)。高汇合度法的诱导效率虽然也较高(70.1％)，但与对照组相比差异不显著(P＞0.05)。
研究了供体细胞经热或诱导处理后用胞质内注射法构建核移植所得胚胎发育能力间的异同。将细胞水浴热变性后构建的核移植胚囊胚发育率为11.8％。G_0/G_1期诱导细胞构建的核移植胚的囊胚率依次为：血清饥饿(15.9％)、Roscovitine(11.9％)、高汇合度(7.5％)和对照组(0％)。结果表明热变性和G_0/G_1期诱导细胞构建的核移植胚胎囊胚发育率间无显著差异(P＞0.05)。
For the sake of enhancing the effectiveness of mammalian nuclear transplantation, it is necessary to research on how to improve the quality of oocyte after in vitro maturation(IVM), which is useful for explaining the mechanism of reprogram induced by factors in oocyte cytoplasm; and it is also necessary to study the method that could make the donor chromosome sensitive to the reprogram factors. Based on Bore Goat Fetal Fibroblast(BGFF) as the nuclear transfer donor cell, this paper researched influence of treatments on the donor cells to the cloned embryo developmental competency in early stage.
Fetal fibroblasts were obtained either by two basic culture media DMEM and M199 or were digested by a mixture of 0.2% collagenasell and 0.25% trypsin at the ratio of 2/3. The cells could be stored both at -196℃ for a long time and at -70 ℃ for a
short time under the condition that 5% DMSO was added into freezing media.
The influencing factors concerning goat oocyte IVM in culture medium were studied through 4 methods. That is: the origination and concentration of serum( both fetal bovine serum(FBS) and estrogenic goat serum(EGS)) and gonadotropins(FSH/ LH and human chorionic gonadotropin(HMG)), the concentrtion of epithelial growth factor(EGF) and the origination of water. The result showed that the ratio of goat oocyte IVM could be as high as 85.7% if they were cultured in the medium of TCM199+10% EGS+0.075IU/mL HMG+1 n g/mLE2. It showed that EGS was better than FBS in cuturing goat oocytes and the efficiency was improved alone with the rising concentration of EGS; HMG was slightly superior to FSH/LH; EOF could not inhance the maturating efficiency at a concentration of 30 ng/mL but was useful for early embryo development; it is advisable to use fresh water dissolve media, if purchase, a small package is recommended it should be used up as soon as possible.
The efficency of ethanol and ionomycin united with 6-dimethyladenine (6-DMAP) in stimulating goat IVM oocytes were studied. The result showed that ethanol was not fit for parthenogenetic development of goat oocytes while 5u.mol/L ionomycin did. The ratio of blastocysts between them were greatly different (P<0.01), which was 23.5% to 0% respectitively.
The influence of the cell cycle stage of receptor, Mil and Til, on goat nuclear transfer were studied. The result showed that Til stage method could enhance the enucleation efficiency significantlly (P<0.05) , however the blastocysts that obtained from both methods had no significant difference (P>0.05) .
Four methods were introduced to capture G0 /G1 stage fetal fibroblasts and the efficiency were examined by immuno-flourescent. The result demonstrated that 79.5% and 84.5% cells were at GO/G1 stage that were induced by serum deprivation and by Roscovitine, a inhibitor of G0/G1, respectively. Both of them had significantly higher effectiveness than control group(60.3%). High confluncy method had a inducing ratio of GO/G1 cells of 70.1%, which was also higher than control group but the difference was not significant (P>0.05) between them.
The influence of cell synchronization and heat denaturaton of donor cells on the early development of nuclear transfer embryo were evaluated. Heat denaturation donor cells were cloned by introcytoplasmic injection and the blastocysts developmental ratio was 11.8%. GO/G1 induced cells were also cloned and the blastocysts developmental tatio were 15.9%(serum deprivation), 11.9%(Roscovitine treatment), 7.5%(high confluency treatment)and 0%(control group). The result suggested that both heat denaturing method and GO/G1 stage incucement could be applied to goat cloning and there were no significant differences in blastocyst development (P>0.05) among them.
分别以DMEM或M199为基础培养液，对供体组织块进行培养，又以0.2％胶原酶Ⅱ与0.25％胰蛋白酶按2/3混合消化供体组织，结果两种方法都获得了体外培养的BGFF。细胞可在含5％DMSO(二甲亚砜)的冷冻液中于-70℃条件下短期冻存，也可在-196℃条件下长期保存。
研究了培养液中血清(胎牛血清FBS)或发情山羊血清(EGS)及促性腺素(FSH、LH和HMG(人血绒毛膜促性腺素))的来源及含量、表皮生长因子(EGF)的添加量和溶剂水的来源对山羊卵母细胞IVM的影响。结果显示：用TCM199+10％EGS+0.075IU/mL HMG+1μg/mLE_2对山羊卵母细胞进行IVM，卵母细胞成熟率为85.7％。EGS的效果不但比FBS好，而且与添加量正相关；用HMG获得的成熟率稍高于不同配比的FSH/L；EGF浓度低于30ng/mL不能显著提高卵母细胞体外成熟率，但能提高其孤雌激活胚胎的发育率。配培养液宜现制超纯水，商品水宜现买现用。
对比研究了乙醇和离子霉素(Ionomycine)分别与6-二甲基嘌啉(6-DMAP)联合激活山羊体外成熟卵母细胞的效果。结果表明：7％乙醇不能有效激活山羊体外成熟卵母细胞，而5μmol/L Ionomycine能明显地起到激活作用，且该方法激活的卵母细胞的孤雌发育囊胚率达23.5％，与乙醇处理组(0％囊胚率)相比差异极显著(P＜0.01)。
比较了处于第二次减数分裂中期(MⅡ)和末期(TⅡ)的山羊卵母细胞作为核移植受体的差异。结果表明：在TⅡ期去核效率明显高于MⅡ期，但二者间核移植胚的囊胚发育率差异不显著(P＞0.05)。
用四种方法对供体细胞作了G_0/G_1期诱导。免疫荧光检测显示：血清饥饿和G_0/G_1期抑制剂Roscovitine诱导能分别使79.5％和84.5％的BGFF进入G_0/G_1期，而作为对照组的对数生长期处理法(60.3％)的诱导效率明显低于前二者(P＜0.05)。高汇合度法的诱导效率虽然也较高(70.1％)，但与对照组相比差异不显著(P＞0.05)。
研究了供体细胞经热或诱导处理后用胞质内注射法构建核移植所得胚胎发育能力间的异同。将细胞水浴热变性后构建的核移植胚囊胚发育率为11.8％。G_0/G_1期诱导细胞构建的核移植胚的囊胚率依次为：血清饥饿(15.9％)、Roscovitine(11.9％)、高汇合度(7.5％)和对照组(0％)。结果表明热变性和G_0/G_1期诱导细胞构建的核移植胚胎囊胚发育率间无显著差异(P＞0.05)。
For the sake of enhancing the effectiveness of mammalian nuclear transplantation, it is necessary to research on how to improve the quality of oocyte after in vitro maturation(IVM), which is useful for explaining the mechanism of reprogram induced by factors in oocyte cytoplasm; and it is also necessary to study the method that could make the donor chromosome sensitive to the reprogram factors. Based on Bore Goat Fetal Fibroblast(BGFF) as the nuclear transfer donor cell, this paper researched influence of treatments on the donor cells to the cloned embryo developmental competency in early stage.
Fetal fibroblasts were obtained either by two basic culture media DMEM and M199 or were digested by a mixture of 0.2% collagenasell and 0.25% trypsin at the ratio of 2/3. The cells could be stored both at -196℃ for a long time and at -70 ℃ for a
short time under the condition that 5% DMSO was added into freezing media.
The influencing factors concerning goat oocyte IVM in culture medium were studied through 4 methods. That is: the origination and concentration of serum( both fetal bovine serum(FBS) and estrogenic goat serum(EGS)) and gonadotropins(FSH/ LH and human chorionic gonadotropin(HMG)), the concentrtion of epithelial growth factor(EGF) and the origination of water. The result showed that the ratio of goat oocyte IVM could be as high as 85.7% if they were cultured in the medium of TCM199+10% EGS+0.075IU/mL HMG+1 n g/mLE2. It showed that EGS was better than FBS in cuturing goat oocytes and the efficiency was improved alone with the rising concentration of EGS; HMG was slightly superior to FSH/LH; EOF could not inhance the maturating efficiency at a concentration of 30 ng/mL but was useful for early embryo development; it is advisable to use fresh water dissolve media, if purchase, a small package is recommended it should be used up as soon as possible.
The efficency of ethanol and ionomycin united with 6-dimethyladenine (6-DMAP) in stimulating goat IVM oocytes were studied. The result showed that ethanol was not fit for parthenogenetic development of goat oocytes while 5u.mol/L ionomycin did. The ratio of blastocysts between them were greatly different (P<0.01), which was 23.5% to 0% respectitively.
The influence of the cell cycle stage of receptor, Mil and Til, on goat nuclear transfer were studied. The result showed that Til stage method could enhance the enucleation efficiency significantlly (P<0.05) , however the blastocysts that obtained from both methods had no significant difference (P>0.05) .
Four methods were introduced to capture G0 /G1 stage fetal fibroblasts and the efficiency were examined by immuno-flourescent. The result demonstrated that 79.5% and 84.5% cells were at GO/G1 stage that were induced by serum deprivation and by Roscovitine, a inhibitor of G0/G1, respectively. Both of them had significantly higher effectiveness than control group(60.3%). High confluncy method had a inducing ratio of GO/G1 cells of 70.1%, which was also higher than control group but the difference was not significant (P>0.05) between them.
The influence of cell synchronization and heat denaturaton of donor cells on the early development of nuclear transfer embryo were evaluated. Heat denaturation donor cells were cloned by introcytoplasmic injection and the blastocysts developmental ratio was 11.8%. GO/G1 induced cells were also cloned and the blastocysts developmental tatio were 15.9%(serum deprivation), 11.9%(Roscovitine treatment), 7.5%(high confluency treatment)and 0%(control group). The result suggested that both heat denaturing method and GO/G1 stage incucement could be applied to goat cloning and there were no significant differences in blastocyst development (P>0.05) among them.
引文
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3． Bradley A, Evans M. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature, 1984; 309: 255-256．
4． Bradley A. Evans M. Formation of ferm-line chimeras from embryo-derived teratocarcinoma cell lines. Nature, 1984, 309: 255-256．
5． Burhans WC, Vasselev LT. Et al. Identification of an origin of bi-directional replication in mammalian chromosomes. Cell 1990; 62: 955-965．
6． Campisi J. Morreo G. et al. Kinetics of G1 transit following brief starvation for serum factors Espt Cell Res 1984; 152:459-462．
7． Carrel A. On the permanent life of tissue out side of the organism. J. Exp. Med. 1912; 15: 516-528．
8． Crissman HA. GT Hirons. Staining of DNA in live and fixed cell. Methods Cell Biol. 1994; 41: 195-209．
9． Darzynkiewicz Z. Mammalian cell-cycle analysis. Zin the cell cycle. A practical approach (ed. P. Fantes and R. Brooks), Oxford University Press.New York. 1993．
10． Dijkwel PA. Hamlin JL> Initiation of DNA in the DHFR locus is confined to the early S period in
CHO cells synchronized with the plant amino acid momosine. Mol Cell Biol 1992; 12: 3715-3722．
11． Eagle H. Nutrition needs of mammalian cells in tissue culture. Science, 1955; 122: 501-504．
12． Evans MJ. Kaufman M. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981,292:151-56．
13． Fraser RSS. Nurse P. Novel cell cycle control of RNA synthesis in yeasts. Nature, 1978; 27:726-730．
14． Graham CF. Morgan RW. Changes in the cell cycle during early amphibian development. Develop. Biol. 1966; 14:439-460．
15． Harris H, Watkins JF. Hybrid cells from mouse and man: Artificial heterokaryons of mammalian cells from different species Nature, 1965; 205:640-646．
16． Harrision R, Observations on the living developing nerve fiber. Proc Soc. Exp. Biol. Med. 1907; 4: 140-143．
17． Hartwell L.H. Weinert J. Checkpoints control that ensure the order of cell cycle events. Science, 1989,246: 629-634．
18． Heintz NH. Hamlin JL. An amplified chromosomal sequence that includes the gene for DHFR initiates replication within specific restriction fragments. Proc Natl Acad Sci USA 1982; 79: 4803-4087．
19． Ian Freshney, Culture of animal cells, a manual of basic technique. Second edi., Alan R. Liss, Inc. New York, 1987．
20． John G. et al. Enhanced survivability of cloned calves derived from Roscovitine-treated adult somatic cells. Biol. Of Reprod. 2002,; 66: 895-900．
21． Johnson RT. Rao PN. Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature, 1970; 226: 717-722．
22． Kohler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256: 495-497．
23． Lehmann AR. Carr AM. DNAdamage,reparir and the cell cycle. Trends in Cell Biol. 1994; 4: 24-26．
24． Li JJ. Deshaies RJ. Exercising self-restraint: Discouraging illicit acts of S and M in eukaryotes. Cell, 1993; 74: 223-226．
25． Littlefield JW. Selection of hubrids form matings of fibroblasts in vitro and their presumed recombinants. Science, 1964; 145:709-710．
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2． Baserga R. Rubin R. Cell cycleand growth control. Crit Rev Eukaryot Gene Expr. 1993;3(1) : 47-61．
3． Bradley A, Evans M. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature, 1984; 309: 255-256．
4． Bradley A. Evans M. Formation of ferm-line chimeras from embryo-derived teratocarcinoma cell lines. Nature, 1984, 309: 255-256．
5． Burhans WC, Vasselev LT. Et al. Identification of an origin of bi-directional replication in mammalian chromosomes. Cell 1990; 62: 955-965．
6． Campisi J. Morreo G. et al. Kinetics of G1 transit following brief starvation for serum factors Espt Cell Res 1984; 152:459-462．
7． Carrel A. On the permanent life of tissue out side of the organism. J. Exp. Med. 1912; 15: 516-528．
8． Crissman HA. GT Hirons. Staining of DNA in live and fixed cell. Methods Cell Biol. 1994; 41: 195-209．
9． Darzynkiewicz Z. Mammalian cell-cycle analysis. Zin the cell cycle. A practical approach (ed. P. Fantes and R. Brooks), Oxford University Press.New York. 1993．
10． Dijkwel PA. Hamlin JL> Initiation of DNA in the DHFR locus is confined to the early S period in
CHO cells synchronized with the plant amino acid momosine. Mol Cell Biol 1992; 12: 3715-3722．
11． Eagle H. Nutrition needs of mammalian cells in tissue culture. Science, 1955; 122: 501-504．
12． Evans MJ. Kaufman M. Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981,292:151-56．
13． Fraser RSS. Nurse P. Novel cell cycle control of RNA synthesis in yeasts. Nature, 1978; 27:726-730．
14． Graham CF. Morgan RW. Changes in the cell cycle during early amphibian development. Develop. Biol. 1966; 14:439-460．
15． Harris H, Watkins JF. Hybrid cells from mouse and man: Artificial heterokaryons of mammalian cells from different species Nature, 1965; 205:640-646．
16． Harrision R, Observations on the living developing nerve fiber. Proc Soc. Exp. Biol. Med. 1907; 4: 140-143．
17． Hartwell L.H. Weinert J. Checkpoints control that ensure the order of cell cycle events. Science, 1989,246: 629-634．
18． Heintz NH. Hamlin JL. An amplified chromosomal sequence that includes the gene for DHFR initiates replication within specific restriction fragments. Proc Natl Acad Sci USA 1982; 79: 4803-4087．
19． Ian Freshney, Culture of animal cells, a manual of basic technique. Second edi., Alan R. Liss, Inc. New York, 1987．
20． John G. et al. Enhanced survivability of cloned calves derived from Roscovitine-treated adult somatic cells. Biol. Of Reprod. 2002,; 66: 895-900．
21． Johnson RT. Rao PN. Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature, 1970; 226: 717-722．
22． Kohler G. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 1975, 256: 495-497．
23． Lehmann AR. Carr AM. DNAdamage,reparir and the cell cycle. Trends in Cell Biol. 1994; 4: 24-26．
24． Li JJ. Deshaies RJ. Exercising self-restraint: Discouraging illicit acts of S and M in eukaryotes. Cell, 1993; 74: 223-226．
25． Littlefield JW. Selection of hubrids form matings of fibroblasts in vitro and their presumed recombinants. Science, 1964; 145:709-710．
26． M．Pagano主编，张世馥主译，细胞周期一材料和方法．人民卫生出版社，2000． 1．
27． Marshall E. The business of stem cells, Science, 2000; 287: 1419-1421．
28． Masui Y. Marker CL. Cytoplasmic control of nuclear behavior during meiotic maturation frog oocytes. J. Exp. Zool. 1971; 177: 129-146．
29． O'Connor PM. Jackman J. et al. Role of the p53 tumor suppressor gene in cell cycle arrest and radiosensitivity of Burkitt's lymphoma cell lines. Cancer Res. 1993; 53::4776-4780．
30. O'Connor PM. Kohn KW. A fundamental role for cell cycle control in the chemosensitivity of cancer cells. Sem Cancer Biol. 1992; 3: 409-416.
31. Pamel J, et al. Stem cell branch out. Science, 2000, 287: 1417.
32. Pines J. Arresting development in cell-cycle control. Trends in Biochem. Sci., 1994; 19: 143-145.
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