适用于甲壳动物细胞的缓冲液配方优化

Optimization of buffer formulation for crustacean cells

目前,在对螯虾、对虾等甲壳动物细胞的研究中,广泛采用了适用于哺乳动物细胞或昆虫细胞的各类缓冲液和培养液。但是,甲壳动物体液的渗透压与哺乳动物等有很大差异,直接沿用哺乳动物等细胞的缓冲系统不合适。本研究首先测定了红螯螯虾(Cherax quadricarinatus)、克氏原螯虾(Procambarus clarkii),与饲养在盐度30海水中的凡纳滨对虾(Litopenaeus vannamei)的血清渗透压,其值分别为(409±12)、(465±8)、(704±15)mOsmol/kg。随后,通过调节NaCl的浓度来改变抗凝剂和磷酸盐缓冲液(PBS)的渗透压,使其与相应动物的血清渗透压相等。优化后适用于红螯螯虾的抗凝剂和PBS中,NaCl的浓度分别为70、200 mmol/L;适用于克氏原螯虾的抗凝剂和PBS中,NaCl的浓度分别为100、230 mmol/L;适用于凡纳滨对虾的抗凝剂和PBS中,NaCl的浓度分别为220、360 mmol/L。进一步比较了3种动物的血细胞在不同渗透压缓冲液中的存活情况。结果表明,红螯螯虾、克氏原螯虾以及凡纳滨对虾这3种甲壳动物的血细胞在未优化的PBS中处理15 min,死亡率就分别达到4.5%、2.0%以及10.0%左右;然而当使用优化后的PBS进行处理时,死亡率则分别降至1.0%、1.0%以及2.0%左右。而血细胞在抗凝剂中的死亡率则较低(<2.0%),优化前后没有显著差异。综上,在甲壳动物活细胞实验中采用优化后的缓冲液,能改善细胞的状态,提高实验的准确性。

Various buffers and culture mediums designed originally for mammalian cells or insect cells are widely used for the experiments of crustacean cells.However,the osmolality of crustacean humoral fluids is very different from that of mammals,it is not appropriate to directly use the buffer system of mammalian cells for crustacean cells.In this study,the osmolality of the serums of Cherax quadricarinatus,Procambarus clarkia and Litopenaeus vannamei(maintained in salinity 30),was measured as(409±12)mOsmol/kg,(465±8)mOsmol/kg and(704±15)mOsmol/kg,respectively.Subsequently,the osmolalities of anticoagulant and PBS buffer were changed by adjusting the concentration of NaCl according to the serum osmolality of the corresponding animal.After optimization,the concentrations of NaCl in anticoagulant and PBS for C.quadricarinatus were 70 mmol/L and 200 mmol/L,respectively,and for P.clarkii were 100 mmol/L and 230 mmol/L,separately.Meanwhile the concentrations of NaCl in anticoagulant and PBS for L.vannamei were 220 mmol/L and 360 mmol/L,respectively.We further compared the haemocyte survivals of these crustaceans in different osmolality buffers.It showed that the mortality rates of hemocytes of C.quadricarinatus,P.clarkii,and L.vannamei reached 4.5%,2.0%and 10.0%,respectively,when treated in PBS for 15 mins.While in optimized PBS,the rates were reduced to about 1.0%,1.0%and 2.0%,respectively.However,the mortality rate of haemocyte in anticoagulants was lower(<2.0%)and there was no significant difference before and after optimization.In conclusion,the optimized buffer solution can improve the state of crustacean cells and the exactness of experiments.