| 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.
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