空间细胞电融合关键技术的地基研究——烟草细胞的电融合

本文针对建立空间细胞电融合技术存在的三个主要问题进行了研究。结果表明,用低温(4℃)、融合介质(0.55 mol/L甘露醇)并添加0.1%纤维素酶保存原生质体,72 h内可以使约94%细胞维持无壁状态,同时并未使细胞丧失再生能力,基本满足从地面制备亲本细胞到在微重力条件下进行电融合,对亲本细胞保持无壁状态的要求。为减少剪切力环境对亲本细胞造成的损伤,一方面用超速离心方法对亲本细胞之一去液泡,另一方面用电泳代替蠕动泵混合亲本细胞。而且,由于原生质体壁生长与其膜电位之间存在负相关性,因此利用电泳方法可以有效地富集和优化亲本细胞。根据地面实验结果推测,空间有/无液泡亲本细胞电融合的较适合参数可能为:交流电场强度90V/cm,频率0.8 MHz,排列时间20 s,直流脉冲1.0—1.3 kV/cm,幅宽40μs,两次脉冲。

THE GROUND-BASED RESEARCH FOR KEY TECHNIQUES OF ELECTROFUSION OF CELLS IN SPACE

Three main problems about developing electrofusion technology of cells in space were studied using tobacco protoplasts. The results showed that, about 94% plant protoplasts preserved in the electro-fusion medium (0.55mol/L mannitol) with 0.1% cellulase were viable and wall-less after 72 h under lower temperature. This satisfied the demands of keeping the parent cells without wall during the period from preparing these cells on the ground to making electrofusion of cells in space.In order to resolve the harmful effect of shearing stress on the parent cells, we studied on two respects: first, preparing evacuolate protoplasts as one of the parentcells: second, driving the parent cells into fusion chamber from storage unit with electrophoresis instead of by peristaltic pump. As there was an oppositive relationship between the cell wall growth and the cell membrane potential, the electro-phoretic method was effective in collecting and optimizing the parent cells. According to our experimental results on the ground, it was suggested that the possibly suitable parameters for the fusion of va-cuolate and evacuolate protoplasts are: alternative field 90 V/cm, 0.8MHz (peak to peak), cell align time 20s, fusion pulse 1.0-1.3KV/cm, pulse wide 40 us, two pulse.