低氧对牛体细胞体外增殖的影响

本研究通过比较常氧(20%)和低氧(5%)环境下培养牛体细胞的生长效果,进而探讨低氧对牛体细胞体外增殖的影响。选用牛的3种常用核移植供体细胞(胎儿成纤维细胞、胎儿输卵管上皮细胞、卵丘颗粒细胞)分别在常氧和低氧2种培养环境下进行连续传代培养和细胞克隆培养,并对其倍增水平和细胞克隆形成效率作比较分析。结果显示,5%的低氧环境对这3种细胞的体外增殖均有促进效果,而且各细胞的增殖水平(50.61±2.47、16.35±0.43、43.38±0.84)均显著高于常氧组(27.42±0.23、12.14±0.83、32.76±1.53,P<0.01)。以500个·皿-1(直径100mm)的细胞浓度接种培养的情况下,低氧培养的胎儿输卵管上皮细胞的克隆形成效率((53.05±4.62)%)显著高于常氧组((36.68±5.68)%)(P<0.01),而低氧组胎儿成纤维细胞和卵丘颗粒细胞获得的细胞克隆数只是略多于常氧组,差异并不显著(P>0.05)。当以1个.孔-1的细胞浓度接种于96孔板培养时,低氧组各类细胞的单细胞克隆形成效率((21.60±2.37)%、(22.29±5.42)%、(27.92±3.69)%)显著高于常氧组((12.01±1.42)%、(7.92±2.86)%、(10.49±3.07)%)(P<0.01或P<0.05)。将体外培养条件的常氧含量(20%)调减至更接近体内生理状况的低氧含量(5%)可以延长牛体细胞增殖寿命和提高单细胞克隆形成效率,对细胞生长有很好的促进作用。

The Effect of Low Oxygen on Proliferation of Bovine Somatic Cells in vitro

In order to study the effect of low oxygen on cell proliferation in vitro by culturing bovine somatic cells under the normoxic(20%) and hypoxic(5%) conditions,three types of bovine donor cells commonly used for nuclear transfer(fetal fibroblasts,fetal oviduct epithelial cells,cumulus granulosa cells) were cultured in 20% and 5% oxygen content of the culture environment for continuous subculture and cell colony formation,at the same time,cell proliferation lifespan(be determined by Population doubling level) and colony formation efficiency.The results indicated that 5% oxygen environment promoted the three kinds of cells proliferation in vitro,and proliferation lifespan of cells in hypoxic group extended significantly(50.61±2.47,16.35±0.43,43.38±0.84 vs 27.42±0.23,12.14±0.83,32.76±1.53,P<0.01).In the case of culture at 500 cells·dish-1(diameter 100 mm),the cell colony formation efficiency of fetal oviduct epithelial cells cultured in low oxygen was much higher than that cultured in normal oxygen((53.05±4.62)% vs(36.68±5.68)%,P<0.01),however,fetal fibroblasts and cumulus granulosa cells in hypoxic group displayed only a litter more cell colonies than the normoxic group,the difference was not significant(P>0.05).When seeding at one cell·well-1 in 96-well plates,single-cell colony formation efficiency of hypoxic group was significantly higher than that of the normoxia group((21.60±2.37)%,(22.29±5.42)%,(27.92±3.69)% vs(12.01±1.42)%,(7.92±2.86)%,(10.49±3.07)%,P<0.01 or P<0.05).The result indicate that reducing the oxygen content(20%) of in vitro culture conditions to low oxygen(5%),which better approximate to the physiologic situation,can extend cell proliferation lifespan and improve the efficiency of single-cell colony formation,low oxygen has a good role in promoting cell growth.