不同分离方法对鼠胚胎成纤维细胞生长影响的比较

Comparison of mouse embryonic fibroblasts through different isolation methods

目的:采用组织块培养法、单细胞悬液法、滤网过滤改良法分离培养鼠胚胎成纤维细胞,比较不同分离方法对其生长形态、生长曲线及细胞周期的影响。方法:实验于2006-03/09在辽宁医学院解剖实验室完成。①选取清洁级昆明种七八周龄雄性小鼠10只,4周龄雌性小鼠20只,雄雌鼠按1∶2合笼,次日晨检查有阴栓者记为孕0.5d,选择孕12.5～14.5d雌鼠断颈处死,取出胎鼠,去除胚胎头部、内脏和四肢,将躯干部用无菌眼科剪剪成1mm3以下的碎块,分别用以下方法进行分离培养。②组织块培养法:将剪碎的组织块吸置于离心管内,加入1mL消化液(含2.5g/L胰蛋白酶和0.4g/L乙二胺四乙酸的磷酸盐缓冲液),吹吸30s,加入等体积的完全培养基(含体积分数为0.1的胎牛血清)终止消化,弃上清液,留下鼠胚组织块沉淀物,按相互距离0.5cm放入培养瓶内,培养箱静置2～4h后,加入适量完全培养基,3d换液一次。③单细胞悬液法:将剪碎的组织块加入1mL消化液,吹打30s,室温下静置3～5min,吸取上层液体,再加入消化液,重复上述操作五六次,最后弃组织块,将收集的上层液离心,1000r/min离心5min,弃上清液,加入完全培养基5mL,吹打均匀后吸至培养瓶内培养。培养条件、换液时间与组织块培养法相同。④滤网过滤改良法:将剪碎的组织块加入1mL消化液,吹打30s,37℃水溶箱中轻摇3～5min,加入完全培养基1mL终止消化,室温下静置1.0～2.0min,用直径5cm、孔径为150目滤网过滤后,将过滤液以1000r/min离心5min;弃上清液,加入适量完全培养基,轻吹30～50下,吸至培养瓶内,37℃、体积分数为0.05的CO2孵箱中培养24h,半量换液,以后3d换液一次。⑤检测不同分离方法对鼠胚胎成纤维细胞生长形态、生长曲线及细胞周期的影响。结果:①鼠胚胎成纤维细胞生长形态观察:组织块培养法第2天可见少量成纤维细胞,细胞生长缓慢,需要七八天才能传代。单细胞悬液法第2天可见成纤维细胞生长,细胞量虽多,但死亡的细胞也较多,细胞活力减弱,四五天后传代。滤网过滤改良法第2天可见成纤维细胞呈长梭形,胞浆饱满,细胞生长迅速,三四天长满培养瓶,三四天传代。②细胞生长曲线检测:组织块培养法培养的细胞第4～8天进入对数生长期,单细胞悬液法培养的细胞第3～5天进入对数生长期,滤网过滤改良法培养的细胞第2～4天进入对数生长期。③细胞周期检测与增殖指数:培养第3天,滤网过滤改良法所得的鼠胚胎成纤维细胞增殖指数最高,达到54.12%,单细胞悬液法、组织块培养法细胞增殖指数分别为53.52%和53.15%,3种方法差异无显著性意义(P>0.05),表明其细胞周期基本相同。结论:滤网过滤改良法可以在较短时间内培养出优质高采的鼠胚胎成纤维细胞。

AIM： Tissue cultural method, single cell suspension method, and the reformed filter method are used to isolate and culture mouse embryonic fibroblasts （MEF）. To compare the different effects of three methods on cellular morphology, protracting curve of growth and cell cycle. METHODS： The experiment was conducted from March to September 2006 in Anatomy Lab of Liaoning Medical College. ①Totally 10 SPF Kunming male mice aged 7-8 weeks and 20 SPF Kunming female mice aged 4 weeks were selected. Male and female mice were put in the same cage together at 1：2. The mice were pregnant for 0.5 days when found the pessary in the following moming. The pregnant mice when marked 12.5-14.5 days were killed. Fetal mice were drawn out of the uterus, and then discarded the head, visceral organs and four legs of the fetal mouse. The trunk was cut into 1 mm^3 tissue masses. They were isolated and cultured as follows： ②tissue cultural method： The masses were removed into the pipette. The masses were trypsinized with 1 mL digestive of 2.5 g/L trypsin-0.4 g/L EDTA PBS. The masses were blown for 30 s. 1 mL of complete medium （with 0.1 FBS） were drawn into the pipette. The supematant was removed and precipitate was left. The masses were drawn into culture flasks each 0.5 cm. After incubating for 2-4 hours, sufficiently complete medium was added, and medium was refreshed after 3 days,③ single cell suspension method： The masses were trypsinized with 1 mL digestive, and blown for 30 s, quietly laid for 3-5 minutes at room temperature. Up liquid was took before digestive was added, Up-mentioned operation was done for 5-6 times. The masses were removed at last. Cells were centrifuged for 5 minutes at 1 000 r/min. Supematant was removed, and then 5 mL complete medium was added. After blowing uniformly dense, the cells were incubated in culture flask. Cultural condition, liquid-change time and tissue cultural method were the same. ④reformed filter method： The masses were trypsinized with 1 mL digestive, blown for 30 s, swayed for 3-5 minutes at 37 % tank. 1 mL complete medium was added to stop digestion, and then laid at room temperature for 1-2 minutes. After filtering with 150 wells filter with 5 cm diameter, filtered liquid was centrifuged for 5 minutes at 1 000 r/min. Supematant was discarded, and sufficiently complete medium was added into the pipette, and then blown for 30-50 times, inhaled into the culture flasks at 37 % /0.05 volume fraction of CO2 for 24 hours. Half medium was refreshed, and then refreshed after 3 days. ⑤The different effects of three methods was assessed by observing cellular morphology, curve of growth and cell cycle. RESULTS： ①MEF cellular morphology： MEF cells isolated by tissue cultural method appeared at day 2 and grew slowly. Cells could be passaged after 7-8 days. In the second day, we could find few flbroblasts with single cell suspension method. MEF cells isolated by cell suspend had more dead cells and different types. They should be passaged after 4-5 days. MEF cells isolated by reformed filter method had more fusiform fibroblasts with full kytopiasm and grew fast. They must be transferred and passaged within 3-4 days.②Curve of growth： The logarithmic phases of MEF cells isolated by tissue cultural method, single cell suspension method and reformed filter method were the 4^th-8th day, the 3^rd-5^th day and the 2^nd-4^th day, respectively.④Cell cycle and proliferation index （PI）： On the 3^rd day, the PI of cells isolated by reformed filter method was 54.12%, which was the highest. While the PI of cells isolated by single cell suspension method was 53.52% and tissue cultural method was 53.15%. There was no significant difference among the three methods （P 〉 0.05）, which indicated that cells isolated by different methods had the same cell cycle. CONCLUSION： Reformed filter method is proved to be the better method to isolate and culture MEFin short times.