不同冻存保护剂对绵羊前体脂肪细胞冻存效果的影响

【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、PPARγ mRNA 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。

Effects of Different Cryoprotectants on Ovine Preadipocytes Cryopreservation

【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(P>0.05),while on 11th day,cells cryopreserved with 10% DMSO turned up significantly greater lipogenesis than other CPAs(P<0.05),but had no significant difference with primary cells(P>0.05).Activity of the GPDH,mRNA expression of LPL and PPARγ showed no significant difference among all the groups(P>0.05).Karyotype analysis showed that diploid cells were dominant post thaw and showed no significant difference compared with primary cells(P>0.05).【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.