小鼠子宫内膜的组织培养

In vitro culture of mouse endometrium

目的:建立小鼠子宫内膜的体外培养方法:用挤压法获得子宫内膜,在添加200mL/L胎牛血清(FBS),17. 5nmol/L胰岛素, 63. 5nmol/L孕酮(P4 ), 7. 14nmol/L雌二醇(E2 )的F12 /DMEM(1∶1 )培养液和50mL/LCO2 + 950mL/L空气界面进行培养,比较分析20μg/L表皮生长因子(EGF)和50mL/LCO2 + 950mL/LO2 气体条件对子宫内膜维持的影响. HE染色,图像分析系统检测样本坏死率,并用SP法检测培养前后子宫内膜中白血病抑制因子(LIF)及类肝素结合样表皮生长因子(HB EGF)的表达. 结果:三组子宫内膜的坏死率(% )分别为19. 40±3. 38, 16. 41±3. 40和13. 71±2. 89,其差异有显著性(P=0. 000 );LIF及HB EGF在培养前后小鼠子宫内膜的阳性表达率均为100%,且同一指标在培养前后子宫内膜的阳性强弱分布无显著性差异(P分别为0. 237, 0. 224).结论:添加200mL/LFBS, 63. 5nmol/LP4, 7. 14nmol/LE2, 17. 5nmol/L胰岛素和20μg/LEGF的F12 /DMEM(1∶1)培养液和50mL/LCO2 +950mL/LO2 的气体环境最有利于小鼠子宫内膜的体外培养;子宫内膜在上述条件下培养24h后,对囊胚仍具有容受性.

AIM: To develop an in vitro culture model of mouse endometrium. METHODS: Mouse endometrium squeezed from the uterus horns were cultured at the gas-liquid interface between F12/DMEM (1∶1) including 200 mL/L FBS, 17.5 nmol/L insulin, 63.5 nmol/L progesterone, 7.14 nmol/L estradiol, and 50 mL/L CO 2 plus 950 mL/L air. The effects of 20 μg/L epidermal growth factor (EGF) and those of mixed gas containing 50 mL/L CO 2 plus 950 mL/L O 2 on the maintenance of the cultured endometrium were compared. The necrosis rate of the samples was measured after hematoxylin-eosin staining by using the image analysis system. The expressions of leukemia inhibitory factor (LIF) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in the mouse endometrium were detected by immunohistochemistry to determine whether the endometrium cultured for 24 h was still receptive to the blastocysts. RESULTS: The necrosis rate (%) of the endometrium in the three groups was 19.40± 3.38 , 16.41±3.40 and 13.71±2.89 respectively, showing a significant difference ( P =0.000). The positively rates of LIF and HB-EGF proteins in both cultured and controlled mouse endometrium were 100% unexceptionally and there was no significant difference in the staining intensity for each marker before and after the culture ( P =0.237, 0.224) . CONCLUSION: The culture condition: F12/DMEM (1∶1), 200 mL/L FBS, 17.5 nmol/L insulin, 63.5 nmol/L progesterone, 7.14 nmol/L estradiol, 20 μg/L EGF and 50 mL/L CO 2 plus 950 mL/L O 2, proves to be most optimal for the growth of mouse endometrium in vitro . The endometrium cultured for 24 hours is still receptive to the blastocysts.