5个品系小鼠胚胎干细胞系建立的方法学比较

以70％的大鼠心脏细胞条件培养基(RH-CM)为培养液，以小鼠胚胎成纤维细胞(PMEF)为饲养层，采用添加1％鸡血清的消化液和“连续离散法”作为小鼠Es细胞建系的改进方法，比较了5个品系小鼠ES细胞系建立的特点。与常规方法相比，3个近交系小鼠129／ter、C57BL/6J、BALB／c的ES细胞建系率分别由11．8％、3．7％和2．9％提高到33．3％、13．3％和19．4％，差异十分显著；直接采用改进的方法建立KM和ICR小鼠ES细胞系，建系率分别达12％和42．1％。讨论了ICM增殖的时间，即离散时机对ES集落形成及建系率的影响，结果显示：129／ter、C57BL/6J、BALB／c、KM和ICR小鼠品系ICM适宜的离散时机分别为增殖4～6d、3～3．5d、4d、4～5d和4～5d；同时，讨论了不同ES细胞建系所需最适宜的消化液浓度，其中BALB／c小鼠的ES细胞对高浓度的消化液十分敏感，0．05％Trypsin-0．008％EDTA是其比较理想的离散浓度。设计了两种离散方法，即“一次离散法”和“连续离散法”，用来离散增殖的ICM和ICM离散后出现的ES集落，结果表明：后者在建系过程中的作用明显优于前者。RH-CM与添加uF的常规ES细胞培养基相比，不但具有显著抑制小鼠ES细胞分化、维持其二倍体核型的作用，而且明显促进ES细胞的贴壁生长。新建细胞系鉴定结果表明，这一改进方法有效地维持了其作为多能性胚胎干细胞的一系列特征。

Comparison of the Method Establishing Embryonic Stem Cell Lines from Five Different Mouse Strains

We compared the characteristics of the method establishing embryonic stem cell lines from five different mouse strains using the medium containing 70% rat heart cell-conditioned medium (RH-CM) as ES cell culture medium,using the primary murine embryo fibroblast as feeder cells,and using the digestive enzyme buffer containing 1% chicken serum and "the series digestive method".We first reported new ES cell lines established from the outbred strain mice KM and ICR using the improved method in our lab and the ratio of establishment of ES cell lines from KM and ICR strain mice is up to 12% and 42.1% respectively.Compared with routine method of establishing ES cell lines,the improved method made distinct differences,increasing the ratio of ES cell line's establishment of 129/ter mouse from 11.8% to 33.3%,that of C57BL/6J mouse from 3.7% to 13.3%,that of BALB/c mouse from 2.9% to 19.4%.We tested the appropriate dispersing occasion,that is proliferating period of the ICM,affected the formation of ES clones and the ratios of ES cell lines established.It was shown that the most appropriate dispersed occasion for the ICM of 129/ter,C57BL/6J,BALB/c,KM and ICR mice was 4～6 d,3～3.5 d,4 d,4～5 d,4～5 d after ICM proliferation respectively.At the same time,the effects of the concentration of digestive enzyme buffer were discussed.It was found that the ES cells from BALB/c mice were sensitive to the high concentration of digestive enzyme buffer and the 0.05% Trypsin-0.008% EDTA is an ideal concentration for their establishment and maintenance.It was shown that ‘the series dispersed method' was much better than ‘the once dispersed method' on the aspect of dispersing the proliferating ICM and formation of ES clones.Compared with the routine ES cell culture medium containing mLIF,the RH-CM not only remarkably inhibited the differentiation of murine ES cells and maintained their diploid karyotype,but also promoted the attachment and growth of ES cells.This improved method of establishment and culture of ES cell lines effectively maintained a series of their characteristics of pluripotent embryonic stem cells.