兔胚胎干细胞的分离和培养
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摘要
本研究分别从胚胎处理方法、培养液中成分以及不同饲养层及采用大鼠心肌条件培养基等对兔胚胎干细胞培养时的影响进行比较,发现胚胎分割法和pronase E处理粘蛋白及部分透明带的胚胎容易贴壁和增值,分离出的内细胞团易生长于小鼠成纤维细胞饲养层上,消化后能够生长出ES细胞样的细胞集落。添加白血病抑制因子和胰岛素利于抑制ES细胞的分化和促进ICM的增值。SD大鼠心肌条件培养基在兔胚胎干细胞分离培养过程中效果较好,所分离的兔胚胎干细胞集落具有岛屿状生长特征,碱性磷酸酶染色为强阳性,体外分化可形成类胚体状结构,贴壁的类胚体周边会出现许多分化的上皮样细胞或单个散在的细胞,经过常规冻存后再传代的胚胎干细胞集落具有较为一致的生长特征,并皇现胚胎干细胞集落所特有的岛屿状生长形态。试验结果如下:
1.分别采用pronase E对不同胚龄(3.5d、4d、4.5d及5d)的兔胚胎消化掉粘蛋白及部分透明带后进行了培养(MEF饲养层)。结果表明,3.5d、4d、4.5d的胚胎处理后,ICM在24～72h孵出并贴壁,其中,4d的囊胚内细胞团贴壁最早,且细胞增值良好。5d胚胎冲出时已经扩张,24h之内ICM就已孵出,但贴壁和增值情况较差。
2.分别在不同饲养层上对兔胚胎进行培养。结果显示,无饲养层中培养的胚胎脱带率较低,在兔原代成纤维细胞和小鼠原代成纤维细胞饲养层上,胚胎的脱带率差别不大。而不同的饲养层上胚胎的贴壁率差别较大,无饲养层中ICM几乎不能贴壁,且贴壁的少量胚胎,ICM增值较慢且容易分化。在兔胎儿原代成纤维细胞饲养层上培养的胚胎,贴壁后增值较慢。而在小鼠原代成纤维细胞饲养层上兔胚胎易扩展,脱带率和贴壁率都较高,而且贴壁后ICM增值较快,生长良好。故此后的实验均采用小鼠胚胎成纤维细胞作饲养层。
3.对兔胚胎进行不同方法处理。结果显示,所得不同状态的胚胎,其贴壁率不同。兔囊胚不经处理直接培养,经7-8d才有少数胚胎贴壁,贴壁率极低。去除黏蛋白及部分透明带的胚胎24h后ICM就能部分孵出,72h后完全孵出。经胚胎切割后,ICM可在48h内贴壁,能保持较快的增殖速率,贴壁率提高。ICM集落呈扁平圆形单层集落,细胞之间界限不清。用胰酶消化传代后,ES集落较小,呈微隆起椭圆状集落、颜色较暗。
4.本实验中分别在培养液中添加不同浓度的LIF因子(0.01、0.1、1、10ng/ml),结果显示,在饲养层上培养时,ES细胞贴壁、增值和抑制分化方面差异不显著;而在无饲养层培养液中培养时,0.1ng/ml和1ng/ml两种浓度的LIF使ES细胞生长良好。
5.添加胰岛素(64%)与对照组(63%)相比,ES细胞贴壁率差异不显著(P＞0.05).
6.采用大鼠心肌细胞条件培养液培养兔胚胎可明显提高胚胎的ICM贴壁率,最高可达59%,与对照组(ES培养液,添加10μg/mL牛胰岛素)差异显著(P＜0.05)。传代后24h可出现ES集落,而对照组(ES培养液,添加10μg/mL牛胰岛素)需48h以上,而且使用大鼠心肌细胞条件培养液后ES细胞的连续传代能力与对照组相比增强。
7.对兔类胚胎干细胞进行鉴定,结果显示,对第1、3、5代ES集落进行碱性磷酸酶染色,ES集落均呈强阳性。对稳定传代的第5代ES集落随机取样,4个样本的二倍体核型正常率分别是78%、81%、76%和85%,平均值为80%,二倍体核型正常率大于75%,三个为XY型,1个为XX型。
By analyzing effect of the treatment methods of embryos, components in cultural medium, different feeder layers and rat cardiomyocyte conditioned medium on rabbit embryonic stem cell, this experiment found that the embryos which were treated with pronase E, cutting and part of zona pellucida(ZP) attached and proliferated easily, the sperated inner cell mass(ICM) grew easily on mouse fiberocyte feeder layer, and it could develope into ES-like cell colony after treatment of pronase E. Added with LIF and insulin were good for inhibiting differentiation of ES cell and promoted proliferation of ICM. SD rat cardiomyocyte conditioned medium had better effect on culture of rabbit embryonic stem cell, the separated rabbit embryonic stem cell colonies had island-like growing characteristic, alkaline phosphatase stain showed positive strongly, they could differentiate into embryo-like structure in vitro, and there were many differentiated epithelium-like cells or single scattered cells around the attached embryo-like body, the embryonic stem cell colonies which were passed down by normal freezed preservation had identical growing characteristic, and showed island-like characteristic which specially belonged to the embryonic stem cell colony. The experimental results showed as follows:
1. Cultured 3.5d, 4d, 4.5d and 5d rabbit embryos with no membrane and part of ZP with treatment of pronase E. The results showed, ICM of the 3.5d, 4d, 4.5d embryos hatched out and attached during 24～72h, the 4d blastocyst ICM attached firstly, and cells proliferated well. The 5d embryos had already expanded after washing out, the ICM hatched out in24h, but the attachment and proliferation were bad.
2. Cultured rabbit embryos on different feeder layers. The results showed, the cultured embryos without feeder layer were hard to get rid of membrane, there were no significant differences between the embryos which cultured on rabbit primary fiberocyst and mouse primary fiberocyst feeder layer. While there were significant differences among the attachment rate of different feeder layers. The ICM cultured without feeder layer had lower attachment rate, the attached ICM proliferated slowly and differentiated easily. The embryos cultured on rabbit primary fiberocyst feeder layer proliferated slowly, while the rabbit embryos cultured on mouse primary fiberocyst feeder layer expanded easily, membrane-off rate and attachment rate were higher, and the attached ICM proliferated fast, grew better.
3. Treated rabbit embryos with different methods. The results showed, the embryos in different condition had different attachment rate. The rabbit blastocysts without culturing directly had extremely lowest attachment rate. The ICM of embryos with part of ZP and without membrane hatched out after 24h, completely hatched after 72h. The ICM of cutting embryos attached in 48h, proliferated fast and the attachment rate promoted.
4. Added with different concentration LIF factor(0.01, 0.1, 1, 10ng/ml), The results showed, there were no significant difference among the attachment rate, proliferation and differentiation restrained of ES when cultured on feeder layer; the LIF of 0.1ng/ml and 1ng/ml improved the growth of ES while cultured without feeder layer.
5. Compared the attachment rate of insulin-added group(64%) with that of the control group(63%), the results showed that there was no significant difference between them(p>0.05).
6. The rabbit embryos cultured in rat cardiomyocyte conditioned medium improved the attachment rate of ICM significantly, the highest rate reached to 59%, there was significant difference between this group and the control group(ES medium, 10μg/ml cattle insulin added)(p<0.05). This group grew up ES cell colony in 24h after passage, but it needed over 48h in control group, besides compared with the control group, the ability of ES cell continual passage after cultured in rat cardiomyocyte conditioned medium got promoted.
7. Identified the rabbit embryonic stem cell, the results showed, stained the 1,3,5 generation ES colony with IKP all showed positive strongly. Sampled the 5 generation ES colony which passed down steady at random, the nomal rate of diploid nuclear type of four samples were 78%,81% and 85% respectively, the average was 80%, the nomal rate of diploid nuclear type was more than 75%, three were XY, one was XX.
1.分别采用pronase E对不同胚龄(3.5d、4d、4.5d及5d)的兔胚胎消化掉粘蛋白及部分透明带后进行了培养(MEF饲养层)。结果表明,3.5d、4d、4.5d的胚胎处理后,ICM在24～72h孵出并贴壁,其中,4d的囊胚内细胞团贴壁最早,且细胞增值良好。5d胚胎冲出时已经扩张,24h之内ICM就已孵出,但贴壁和增值情况较差。
2.分别在不同饲养层上对兔胚胎进行培养。结果显示,无饲养层中培养的胚胎脱带率较低,在兔原代成纤维细胞和小鼠原代成纤维细胞饲养层上,胚胎的脱带率差别不大。而不同的饲养层上胚胎的贴壁率差别较大,无饲养层中ICM几乎不能贴壁,且贴壁的少量胚胎,ICM增值较慢且容易分化。在兔胎儿原代成纤维细胞饲养层上培养的胚胎,贴壁后增值较慢。而在小鼠原代成纤维细胞饲养层上兔胚胎易扩展,脱带率和贴壁率都较高,而且贴壁后ICM增值较快,生长良好。故此后的实验均采用小鼠胚胎成纤维细胞作饲养层。
3.对兔胚胎进行不同方法处理。结果显示,所得不同状态的胚胎,其贴壁率不同。兔囊胚不经处理直接培养,经7-8d才有少数胚胎贴壁,贴壁率极低。去除黏蛋白及部分透明带的胚胎24h后ICM就能部分孵出,72h后完全孵出。经胚胎切割后,ICM可在48h内贴壁,能保持较快的增殖速率,贴壁率提高。ICM集落呈扁平圆形单层集落,细胞之间界限不清。用胰酶消化传代后,ES集落较小,呈微隆起椭圆状集落、颜色较暗。
4.本实验中分别在培养液中添加不同浓度的LIF因子(0.01、0.1、1、10ng/ml),结果显示,在饲养层上培养时,ES细胞贴壁、增值和抑制分化方面差异不显著;而在无饲养层培养液中培养时,0.1ng/ml和1ng/ml两种浓度的LIF使ES细胞生长良好。
5.添加胰岛素(64%)与对照组(63%)相比,ES细胞贴壁率差异不显著(P＞0.05).
6.采用大鼠心肌细胞条件培养液培养兔胚胎可明显提高胚胎的ICM贴壁率,最高可达59%,与对照组(ES培养液,添加10μg/mL牛胰岛素)差异显著(P＜0.05)。传代后24h可出现ES集落,而对照组(ES培养液,添加10μg/mL牛胰岛素)需48h以上,而且使用大鼠心肌细胞条件培养液后ES细胞的连续传代能力与对照组相比增强。
7.对兔类胚胎干细胞进行鉴定,结果显示,对第1、3、5代ES集落进行碱性磷酸酶染色,ES集落均呈强阳性。对稳定传代的第5代ES集落随机取样,4个样本的二倍体核型正常率分别是78%、81%、76%和85%,平均值为80%,二倍体核型正常率大于75%,三个为XY型,1个为XX型。
By analyzing effect of the treatment methods of embryos, components in cultural medium, different feeder layers and rat cardiomyocyte conditioned medium on rabbit embryonic stem cell, this experiment found that the embryos which were treated with pronase E, cutting and part of zona pellucida(ZP) attached and proliferated easily, the sperated inner cell mass(ICM) grew easily on mouse fiberocyte feeder layer, and it could develope into ES-like cell colony after treatment of pronase E. Added with LIF and insulin were good for inhibiting differentiation of ES cell and promoted proliferation of ICM. SD rat cardiomyocyte conditioned medium had better effect on culture of rabbit embryonic stem cell, the separated rabbit embryonic stem cell colonies had island-like growing characteristic, alkaline phosphatase stain showed positive strongly, they could differentiate into embryo-like structure in vitro, and there were many differentiated epithelium-like cells or single scattered cells around the attached embryo-like body, the embryonic stem cell colonies which were passed down by normal freezed preservation had identical growing characteristic, and showed island-like characteristic which specially belonged to the embryonic stem cell colony. The experimental results showed as follows:
1. Cultured 3.5d, 4d, 4.5d and 5d rabbit embryos with no membrane and part of ZP with treatment of pronase E. The results showed, ICM of the 3.5d, 4d, 4.5d embryos hatched out and attached during 24～72h, the 4d blastocyst ICM attached firstly, and cells proliferated well. The 5d embryos had already expanded after washing out, the ICM hatched out in24h, but the attachment and proliferation were bad.
2. Cultured rabbit embryos on different feeder layers. The results showed, the cultured embryos without feeder layer were hard to get rid of membrane, there were no significant differences between the embryos which cultured on rabbit primary fiberocyst and mouse primary fiberocyst feeder layer. While there were significant differences among the attachment rate of different feeder layers. The ICM cultured without feeder layer had lower attachment rate, the attached ICM proliferated slowly and differentiated easily. The embryos cultured on rabbit primary fiberocyst feeder layer proliferated slowly, while the rabbit embryos cultured on mouse primary fiberocyst feeder layer expanded easily, membrane-off rate and attachment rate were higher, and the attached ICM proliferated fast, grew better.
3. Treated rabbit embryos with different methods. The results showed, the embryos in different condition had different attachment rate. The rabbit blastocysts without culturing directly had extremely lowest attachment rate. The ICM of embryos with part of ZP and without membrane hatched out after 24h, completely hatched after 72h. The ICM of cutting embryos attached in 48h, proliferated fast and the attachment rate promoted.
4. Added with different concentration LIF factor(0.01, 0.1, 1, 10ng/ml), The results showed, there were no significant difference among the attachment rate, proliferation and differentiation restrained of ES when cultured on feeder layer; the LIF of 0.1ng/ml and 1ng/ml improved the growth of ES while cultured without feeder layer.
5. Compared the attachment rate of insulin-added group(64%) with that of the control group(63%), the results showed that there was no significant difference between them(p>0.05).
6. The rabbit embryos cultured in rat cardiomyocyte conditioned medium improved the attachment rate of ICM significantly, the highest rate reached to 59%, there was significant difference between this group and the control group(ES medium, 10μg/ml cattle insulin added)(p<0.05). This group grew up ES cell colony in 24h after passage, but it needed over 48h in control group, besides compared with the control group, the ability of ES cell continual passage after cultured in rat cardiomyocyte conditioned medium got promoted.
7. Identified the rabbit embryonic stem cell, the results showed, stained the 1,3,5 generation ES colony with IKP all showed positive strongly. Sampled the 5 generation ES colony which passed down steady at random, the nomal rate of diploid nuclear type of four samples were 78%,81% and 85% respectively, the average was 80%, the nomal rate of diploid nuclear type was more than 75%, three were XY, one was XX.
引文
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