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目的:探讨改良人早孕绒毛外细胞滋养细胞原代分离培养方法。方法:建立改良的复合酶消化梯度离心法,分离培养绒毛外细胞滋养细胞,通过免疫细胞化学、Giemsa染色和细胞侵袭实验检测细胞纯度、数量及生物学活性(侵袭性),并与低浓度胰蛋白酶消化方法进行比较。结果:低浓度胰蛋白酶消化方法和改良的复合酶消化梯度离心法所得贴壁细胞量分别为(11.69±1.26)×104和(17.21±0.51)×104,两者比较有统计学差异(P<0.05);绒毛外细胞滋养细胞纯度分别为(63.20±7.12)%和(92.10±5.11)%,两者比较有统计学差异(P<0.05),穿膜细胞数分别为(36.10±3.28)和(66.20±5.17),两者比较有统计学差异(P<0.05),提示该实验室改良的复合酶消化梯度离心法所得细胞总量、绒毛外细胞滋养细胞纯度及数量均显著高于低浓度胰蛋白酶消化方法。结论:该实验室改良的复合酶消化梯度离心法获得绒毛外细胞滋养细胞纯度和数量显著提高。
Objective: To explore the method of primary isolation and culture of extracytokine of human chorionic villi in early pregnancy. Methods: A modified multiplex enzyme digestion gradient centrifugation method was used to isolate and culture villous cytotrophoblast cells. The purity, quantity and biological activity (invasiveness) of cells were detected by immunocytochemistry, Giemsa staining and cell invasion assay. Protease digestion methods were compared. Results: The number of adherent cells obtained by low concentration trypsin digestion and modified enzyme digestion gradient centrifugation were (11.69 ± 1.26) × 104 and (17.21 ± 0.51) × 104, respectively, with statistical significance (P < (63.20 ± 7.12)% and (92.10 ± 5.11)%, respectively (P <0.05). The numbers of transmembrane cells were (36.10 ± 3.28) and (66.20 ± 5.17). There was a significant difference between the two groups (P <0.05), suggesting that the total cell volume, the purity and number of extravillous cytotrophoblast cells in the modified enzyme digestion gradient centrifugation method in the laboratory were significantly higher than those in the low concentration Trypsin digestion method. Conclusion: The purity and number of extracytosynovial cytotrophoblast cells obtained by the modified enzyme digestion gradient centrifugation in this laboratory were significantly increased.