| 摘要: |
| 将胎儿成纤维细胞悬浮于不同的冷冻保护液中，在不同的温度下进行冷冻保存试验。结果显示，当保护液中含有900 mL/L胎牛血清(FBS)时，-196 ℃下保存的山羊胎儿成纤维细胞解冻后的细胞贴壁率高于-80 ℃，且前者贴壁细胞增殖也较后者快，二者间差异显著；而在-20 ℃条件下只有一小部分细胞能够存活，与前两者相比差异极显著，说明-20 ℃不适合冷冻胎儿细胞。冷冻保护液中含有100 mL/L二甲基亚砜(DMSO)的保护效果明显好于50 mL/L DMSO，二者间差异显著。用组织块法培养的细胞，在两种培养基DMEM和M199中添加100 mL/L FBS和2 mmol/L 2-巯基乙醇(2-Me)，其组织块成活率及细胞生长速度均高于单纯添加100 mL/L FBS，同时证明DMEM和M199都能用于胎儿成纤维细胞的体外培养。以DMEM培养液为基础，分别添加2 mmol/L 2-Me，5 mL/L FBS，100 mL/L FBS和2 mmol/L 2-Me+100 mL/L FBS对山羊胎儿成纤维细胞进行体外培养，结果显示，细胞在100 mL/L FBS和2 mmol/L 2-Me+100 mL/L FBS培养液中生长迅速，两者间无明显差异，2-Me的存在促进了细胞的增殖；两者都与添加2 mmol/L 2-Me组存在显著差异；添加2 mmol/L 2-Me和5 mL/L FBS相比，前者也明显地起到了促进细胞增殖的作用，二者间差异显著。说明2-Me对细胞的促生长作用大于抗氧化作用，100 mL/L FBS能保证细胞的生长和增殖，5 mL/L FBS在培养液中长期存在不仅抑制细胞分裂，而且导致大量丢失。 |
| 关键词: 胎儿成纤维细胞 体外培养 2-巯基乙醇 二甲基亚砜 山羊 |
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| 基金项目:国家863计划项目“家畜体细胞克隆技术体系的建立和应用”(2001AA213081) |
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| Goat fetal fibroblasts frozen and in vitro culture |
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| Abstract: |
| Goat fetal fibroblasts were frozen at different temperature with various protecting solution in order to find a proper procedure to preserve its rare resources for further study both in transgenic and cloning practice.The result showed that fetal fibroblasts froze at -196 ℃ with 900 mL/L fetal bovine serum (FBS) grew faster than that at -80 ℃ after thawing (P<0.05).-20 ℃ was not an effective temperature in frozen cells.100 mL/L dimethyl sulfoxide (DMSO) contained in frozen solution was better than 50 mL/L in improving protection effectiveness (P<0.05).In vitro culture of fetal fibroblasts experiment showed that both DMEM and M199 can support cell growth and proliferation and that a supplementation of 2 mmol/L 2-mercaptoethanol (2-Me) was better than not in improving tissue vitality and growth speed.In cell culture procedure,2-Me could improve the effect of 100 mL/L FBS in stimulating cell proliferation.And individual 2-Me supplementation in the culture medium could stimulate cell growth.As a result,2-Me was more likely a cell proliferation stimulator than an oxidation retarding factor.High serum concentration 100 mL/L was vital for cell proliferation,serum starvation (5 mL/L FBS) could not only inhibit cell division but also cause cell loss during long term culture. |
| Key words: fetal fibroblasts frozen in vitro culture 2-mercaptoethanol dimethyl sulfoxide goat |
