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鸡胚原始生殖细胞的冷冻保存和体外培养 总被引：2，自引：1，他引：1
采用Ficoll密度梯度离心,提取第19期(孵化72h)鸡受精蛋性腺中的原始生殖细胞(primordialgermcells,PGCs),用不同的冷冻保护液对PGCs采用提纯后直接冷冻保存和在培养体系中培养24h后再进行冷冻保存,7d后复苏,用台盼蓝染色检测其存活率,并用PAS染色对冷冻后的PGCs进行特异性鉴定。复苏后的PGCs在培养体系中培养5d后,更换为培养体系继续培养,进行体外分化试验。结果如下分离后直接进行冷冻保存的PGCs复苏后存活率最高为(85.93±1.24)%;复苏后的PGCs在培养体系中培养48h后进行了PAS染色鉴定,结果PGCs呈PAS阳性(细胞核不着色,细胞质呈红色),表明PGCs的染色特性没有因冷冻保存和体外培养而发生改变;复苏后的PGCs在培养体系中培养可形成细胞克隆,当把PGCs更换到培养体系培养5d后,PGCs克隆有的仍保持克隆状态,但体积缩小,克隆中细胞排列紧密,颜色较深,有的克隆由鸟巢状变为不规则形,且在克隆周围形成类神经细胞样细胞。而经体外培养后再进行冷冻保存的PGCs,复苏后存活率最高为(42.26±1.36)%。 相似文献
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本研究对发育至第19期的鸡胚性腺原始生殖细胞(primordial germ cells，PGCs)分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖、PVP（聚乙烯吡咯烷酮）分别组成6种慢速冷冻保护液和6种玻璃化冷冻保护液，进行超低温冷冻保存。复苏后苔盼蓝染色测定细胞存活率，体外接种培养、传代。结果：①慢速冷冻保存中，PGCs在慢速冷冻液V（10％EG+10% FBS+0.1 mol/L蔗糖）条件下复苏后存活率最高（92.20%），且与慢速冷冻液I（10％ DMSO+10% FBS）存活率之间差异显著（P<0.05）。②玻璃化冷冻保存中，PGCs在玻璃化冷冻液I（10% DMSO+10% EG+20% FBS+10% PVP）条件下复苏后存活率最高（84.15%），且与其余5种玻璃化冷冻液下复苏后存活率之间差异均极显著（P<0.01）。③培养传至第3代的慢速冷冻复苏后PGCs细胞和培养传至第2代的玻璃化冷冻复苏后PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。 相似文献
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发育至第19期和第28期的鸡胚性腺原始生殖细胞（PGCs）分离提纯后，在DMEM中添加冷冻保护剂DMSO（二甲基亚砜）、EG（乙二醇）、蔗糖进行超低温冷冻保存，比较冷冻保护剂在单独或联合使用条件下对PGCs的冷冻保护效果，复苏后台盼蓝染色测定细胞存活率，体外接种培养、传代。结果表明：（1）第19期PGCs冷冻保存中，10％EG＋10％FBS＋0．1mol／L。蔗糖条件下细胞存活率最高为（92．20±2．18）％，且与10％DMSO＋10％FBS的存活率差异显著（P〈0．05），其余各冷冻保护液间存活率差异不显著；第28期PGCs冷冻保存中，5％DMsO＋5％EG＋10％FBS＋0．1mol／L。蔗糖条件下细胞存活率最高为（92．41±2．82）％，但各冷冻保护液间存活率差异均不显著（P〉0．05）。（2）解冻后体外培养的PGCs，在饲养层和3种细胞因子（I。IF、SCF、bFGF）的共同作用下，可持续增殖，传至第3代的PGCs，PAS染色、AKP染色均呈阳性并保持完整的二倍体核型，仍处于未分化状态。 相似文献
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采用PBS作为冷冻基础液,分别用甘油和二甲基亚砜(DMSO)作为冷冻保护液,在程序化冷冻保存和玻璃化冷冻保存条件下,研究小鼠生发泡期(GV期)卵母细胞的抗冻能力。结果表明,2种冷冻方法对小鼠GV期卵母细胞解冻后形态正常率和存活率无显著影响(P>0.05)。冷冻保护剂种类对小鼠GV期卵母细胞解冻后形态正常率无显著影响(P>0.05);但对存活率有显著影响,玻璃化冷冻采用二甲基亚砜作为冷冻保护液效果极显著优于甘油(P<0.01)。以冷冻效果较好的二甲基亚砜作为冷冻保护液,采用玻璃化冷冻不同发育阶段(GV期和MⅡ期)的小鼠卵母细胞,解冻后形态正常率无显著差异(P>0.05),但存活率GV期要显著优于MⅡ期卵母细胞(P<0.05)。 相似文献
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《中国家禽》2006,28(19):102-102
目的：研究鸡胚胎原始生殖细胞（EPGCs）对不同冷冻体系的适合性。方法：对发育至第19期和第28期胚胎性腺PGCs，采用二甲基亚砜（DMSO）、乙二醇、聚乙二醇为冷冻保护液，在不同组合、不同浓度、不同的冷冻程序下进行超低温冷冻保存。结果：①采用同一种冷冻保护液，10%浓度与15％浓度之间，除DMSO＋乙二醇（冷冻保护液Ⅳ）外，其他类型的冷冻液对EPGCs保存后存活率的影响差异不显著（P〉0．05）；20%浓度的各种冷冻保护液对EPGCs保存后的存活率最低，且与10%和15％浓度相比差异显著（P〈0．05）或极显著（P〈0．01）；②采用同一种冷冻保护液，浓度为10%时， 相似文献
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通过比较不同冷冻保存方法和冷冻保护剂对小鼠耳皮肤成纤维细胞冷冻-解冻复苏后细胞存活率、48h贴壁率和细胞生长曲线的影响,筛选适宜的小鼠耳皮肤成纤维细胞冷冻保存方法和冷冻保护剂。结果表明,以100mL/L二甲基亚砜(DMSO)作为冷冻保护剂进行小鼠耳皮肤成纤维细胞冷冻保存时,方法3处理组解冻复苏后细胞存活率和培养48h细胞贴壁率均高于方法2处理组(P>0.05),并分别极显著高于方法1和方法4。采用方法3进行冷冻保存时,以100mL/L DMSO作为冷冻剂的细胞贴壁率显著高于100mL/L甘油(GL)组(P<0.05),且冷冻解冻后的细胞呈现正常的分裂增殖生长模式。因此,宜选择100mL/L胎牛血清(FBS)+100mL/L DMSO+DMEM作为冷冻保护液,采用方法3进行小鼠耳皮肤成纤维细胞的冷冻保存。 相似文献
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对发育至第19期和第28期鸡胚性腺原始生殖细胞（Primordial germ cells，PGCs），用6种玻璃化冷冻液1：10％DMS0＋10％EG＋10％PVP，Ⅱ：20％EG＋10％PVP，Ⅲ：20％DMSO＋10％PVP，Ⅳ：10％DMSO＋10％EG＋0．5mol／L Surcose，Ⅴ：20％EG＋0．5mol／LSurcose，Ⅵ：20％DMSO＋0．5mol／L Surcose进行冷冻保存。结果：第19期鸡胚PGCs玻璃化冷冻复苏后存活率，在冷冻液Ⅱ与Ⅳ之间差异不显著（P〉0．05），Ⅴ与Ⅵ之间差异显著（P〈0．05），其余各冷冻液之间差异均极显著（P〈0．01）。第28期鸡胚PGCs玻璃化冷冻复苏后存活率，在冷冻液Ⅳ与Ⅴ之间差异显著（P〈0．05），Ⅲ与Ⅳ、Ⅴ之间差异不显著（P〉0．05），Ⅱ与Ⅲ、Ⅳ之间差异不显著（P〉0．05），其余各玻璃化冷冻液之间差异均极显著（P〈0．01）。复苏后接种培养传至第2代的鸡胚PGCs细胞，PAS染色、AKP染色呈阳性并保持完整的二倍体核型。 相似文献
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原始生殖细胞（Primordial Germ Cells：PGCs）是指能够发育为性细胞的前体细胞,可作为胚胎干细胞（ES）分离与克隆的一种新型材料,其形态、表面标志、体内外分化潜能及体外培养条件均类似于胚胎干细胞.体外培养过程中,在培养液中加入一定的诱导分化剂,PGCs细胞将发生定向分化.长期以来神经系统损伤和神经退行性病变是临床上难以解决的问题,由于原始生殖细胞具有发育全能性,可以在体外通过对其进行定向诱导,得到特定类型的细胞,这将使原始生殖细胞成为今后细胞替代疗法和组织器官移植的来源.本试验采用不同的诱导剂对PGCs进行诱导,以探讨PGCs细胞向神经细胞分化的适宜条件. 相似文献
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Yoshiaki NAKAMURA Mariko TASAI Kumiko TAKEDA Keijiro NIRASAWA Takahiro TAGAMI 《The Journal of reproduction and development》2013,59(6):580-587
The Japanese quail (Coturnix japonica) is a valuable bird as both
an experimental animal, for a wide range of scientific disciplines, and an
agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs
would be a feasible strategy for the conservation of both male and female fertility
cells in Japanese quail. However, the effects of freeze-thaw treatment on viability,
migration ability and germline transmission ability of quail PGCs still remain
unclear. In the present study, male and female PGCs were isolated from the blood of
2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196
C for 77–185 days, respectively. The average recovery rate of PGCs after
freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly
lower than that of the control group (P<0.05) (85.5% vs. 95.1%).
Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into
recipient embryos migrated toward and were incorporated into recipient gonads,
although the number of PGCs settled in the gonads was 48.5% lower in the frozen group
than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that
one female and two male recipients produced live progeny derived from the
frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than
that of unfrozen controls, but the difference was not significant (4.0
vs. 14.0%). These results revealed that freeze-thaw treatment
causes a decrease in viability, migration ability and germline transmission ability
of PGCs in quail. 相似文献
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在鸡胚孵化的 19期以Ficoll密度梯度离心和酶解离两种方法分离生殖嵴中的原始生殖细胞 (PGCs)。探索在生殖嵴中PGCs分离、培养的适宜方法 ,以获得较多数量 ,较高活力的PGCs作介导生产转基因鸡。在倒置显微镜下进行形态观察 ,台盼兰染色比较存活时间 ,PAS特异染色法识别鉴定PGCs。结果表明两种分离方法均能分离到一定数量的PGCs细胞。与Ficoll密度梯度离心法相比 ,酶解离法分离到的PGCs的相对数量较多 ,存活时间较长 ,是一种较可行的分离方法。在鸡胚孵化的第 19期 ,PGCs大量聚集在肢体后端的生殖嵴原基处 ,此时的生殖嵴大小已达一定程度 ,分离其中的PGCs操作简便 ,有较强的可操作性 ;提取出的PGCs为转基因鸡的生产提供了介导材料 相似文献
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The objective of this study was to investigate the effects of beta‐mercaptoethanol (β‐ME) on post‐thaw embryo developmental competence and implantation rate of mouse pronuclear (PN) embryos that were cryopreserved after slow freezing, solid surface vitrification (SSV) or open‐pulled straw (OPS) vitrification methods. Mouse PN embryos were cryopreserved by using slow freezing, SSV and OPS methods. After cryopreservation, freeze–thawed PN embryos were cultured up to blastocyst stage in a defined medium supplemented without or with 50 μm β‐ME. The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (40.0%) or vitrified by OPS method (18.3%) were lower than those vitrified by SSV method (55.6%) and fresh embryos (61.9%) in the absence of 50 β‐ME in the culture media (p < 0.05). The blastocyst formation rate of embryos that were cryopreserved by slow freezing method (53.1%) or by OPS method (41.9%) were lower than those vitrified by SSV method (79.5%) and that of fresh (85.7%) in the presence of β‐ME in the culture media (p < 0.05). The embryos transfer results revealed that the implantation rate of blastocyst derived from mouse PN embryos vitrified by SSV method (31.9% vs 51.2%) was similar to that of the control (39.0% vs 52.5%), but higher than those cryopreserved by slow freezing (28.2% vs 52.0%) and by OPS method (0.0% vs 51.2%) (p < 0.05). In conclusion, supplementation of β‐ME in an in vitro culture medium was shown to increase survival of embryo development and implantation rate of frozen–thawed mouse PN embryos after different cryopreservation protocols. 相似文献
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Nakamura Y Usui F Miyahara D Mori T Ono T Kagami H Takeda K Nirasawa K Tagami T 《The Journal of reproduction and development》2012,58(4):432-437
Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens. 相似文献
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ABSTRACT1. In order to increase the efficiency of generating transgenic chicken, this trial focused on two points: primordial germ cells (PGCs）transfection in vivo and a germline-specific promoter.2. In order to transfect PGCs in vivo, two plasmids (pZB-CAG-GFP, pCMV-ZB）were co-injected into chicken embryos via the subgerminal cavity at Hamburger and Hamilton (HH) stage 2–3 or via blood vessel at HH stage 13–14. Results showed that the percentage of GFP+ embryos, viability and hatching rate of embryos injected at HH stage 13–14 were significantly higher than that at HH stage 2–3.3. Two plasmid transposon systems were used for chicken embryo micro-injections. The donor plasmid, with a green fluorescent protein (GFP) reporter gene, was mediated by the ZB transposon. The helper plasmid was a transposase expression vector driven by the promoter of the chicken vasa homologue (Cvh) gene or Human cytomegalovirus (CMV) promoter. Results showed that 60.98% of gonads in Cvh group expressed GFP, which was 52.50% higher than seen in the CMV group. Only gonad tissue from the Cvh group showed any GFP signal, whereas both gonads and other tissues in the CMV group showed green fluorescence.4. The data suggested that ZB transposon-mediated gene transfer was efficient for transfecting PGCs in vivo; the Cvh promoter drove the transposase gene specifically in the germline and increased the efficiency of germline transmission. Blood vessels injection at HH stage 13–14 may be a more efficient route for PGCs transfection in vivo. 相似文献
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试验对绵羊精液分别采用液氮熏蒸冷冻法和冷冻仪冷冻法进行冷冻,通过测定精液解冻后精子活率、顶体完整率、质膜完整率和谷草转氨酶活性及乳酸脱氢酶活性来比较不同的冷冻方法对解冻后精液品质的影响;通过测定精液稀释后、平衡后、冷冻仪法冷冻后的酶活性来比较谷草转氨酶与乳酸脱氢酶在不同阶段的释放量。结果表明,采用冷冻仪法冷冻后的精子活率和质膜完整率极显著高于液氮熏蒸冷冻法（P<0.01）;冷冻仪法冷冻后的谷草转氨酶和乳酸脱氢酶活性极显著低于液氮熏蒸冷冻法（P<0.01）;精液稀释后的谷草转氨酶和乳酸脱氢酶活性极显著低于精液冷冻后的活性（P<0.01）。表明采用冷冻仪冷冻法的绵羊精液品质好于液氮熏蒸冷冻法。精子中谷草转氨酶主要在平衡阶段释放,而乳酸脱氢酶主要在冷冻阶段释放。 相似文献