不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

Effects of Different Cryoprotectants on Ovine Preadipocytes Cryopreservation

【目的】探讨绵羊前体脂肪细胞冷冻保存的最佳冻存保护剂种类和浓度。【方法】在含20%胎牛血清的DMEM/F12培养液中分别添加不同浓度的DMSO、EG、PG、G和PVP作为冻存液,对原代培养的绵羊前体脂肪细胞进行冷冻保存后,检测复苏细胞存活率、细胞增殖能力、细胞中脂肪沉积量,测量GPDH活性,并用实时荧光定量PCR检测PPARγ和LPL mRNA的表达水平,最后对复苏细胞进行染色体核型分析。【结果】各种冻存保护液中,10%DMSO和5%DMSO+5%PVP组细胞存活率最高,与对照组相比差异不显著(P>0.05),其它各试验组复苏细胞存活率均显著低于对照组(P<0.05);各试验组中10%DMSO组细胞增殖能力最强,且与对照组相比差异不显著(P>0.05);细胞复苏后培养6 d,各试验组脂肪沉积量与对照组之间没有显著差异(P>0.05),而第11天时,10%DMSO组脂肪沉积量显著高于其它试验组(P<0.05),且与对照组相比差异不显著(P>0.05);各试验组GPDH活性和LPL、PPARγmRNA的表达量与对照组之间没有显著差异(P>0.05);染色体核型分析发现,复苏细胞二倍体细胞占主体,与原代细胞没有显著差异(P>0.05)。【结论】含20%FBS的DMEM/F12培养液中添加10%DMSO或10%PVP、5%DMSO+5%PVP均能有效保存绵羊前体脂肪细胞,但以10%DMSO最佳。

【Objective】 The aim of this experiment is to investigate a suitable cryoprotectant and its optimal concentration for ovine preadipocytes from omental.【Method】Trypan blue exclusion test,MTT,oil red O staining and real-time PCR were used to test the effects of following cryoprotective agents（CPAs） with different concentrations on post-thaw survival,proliferation,differentiation capacity,PPARγ and LPL mRNA expression of ovine preadipocytes from omental,dimethyl sulphoxide（DMSO）,ethylene glycol（EG）,propylene glycol（PG）,glycerol（G） and polyvinylpyrrolidone（PVP）.In addition to the CPAs,the basic medium is DMEM/F12 medium plus 20% FBS.Then karyotype was analyzed.【Result】Trypan blue exclusion test showed that the highest survival rate,no significant difference with the primary cells,was obtained when cryopreserved with 10% DMSO or 5% DMSO plus 5% PVP among all the CPA treatments in this study.The highest survival rate（94.96%） and cell viability were obtained when cryopreserved with 10% PVP,and showed no significant difference compared with primary cells.Oil red O staining showed no significant difference in lipogenesis among all the CPAs groups and primary cells on 6 th day（P0.05）,while on 11th day,cells cryopreserved with 10% DMSO turned up significantly greater lipogenesis than other CPAs（P0.05）,but had no significant difference with primary cells（P0.05）.Activity of the GPDH,mRNA expression of LPL and PPARγ showed no significant difference among all the groups（P0.05）.Karyotype analysis showed that diploid cells were dominant post thaw and showed no significant difference compared with primary cells（P0.05）.【Conclusion】Results of the present study indicate that ovine preadipocytes could successfully be cryopreserved with DMEM/F12 medium containing 10% DMSO or 10 % PVP,5% DMSO plus 5% PVP,while 10% DMSO is a suitable CPA for ovine preadipocytes.