机械法和胰酶法分离大鼠卵巢颗粒细胞的比较

Isolation of rat ovarian granulosa cells:a comparison between mechanical method and trypsin method

目的探讨胰酶法分离大鼠卵巢颗粒细胞的优势。方法取3~4周龄雌性SD大鼠的卵巢,分别采用机械法和胰酶法分离颗粒细胞并进行培养。机械法:于40倍解剖显微镜下用针头刺破卵巢成熟的卵泡,将含有卵巢剩余组织及卵泡液的培养液用200目滤网过滤;胰酶法:用无菌眼科剪将卵巢剪成小于2 mm3的小碎块,然后加入0.25%胰酶,室温消化1 h后,将含有卵巢碎块的消化液用200目滤网过滤。比较这2种分离方法获得的细胞数量、细胞活力,及雌二醇和孕酮的分泌情况。结果机械法和胰酶法这2种方法分离的原代大鼠卵巢颗粒细胞存活率均>90%。将细胞培养1~9 d,除24 h外,其余各时间点胰酶法分离的细胞增殖率均高于机械法;胰酶法细胞增殖率峰值出现时间早于机械法24 h,并且胰酶法分离的细胞最大增殖率[72 h:(210.09±0.95)%]高于机械法[96 h:(180.50±0.74)%],差异有统计学意义(P<0.05)。胰酶法分离的细胞激素的最大分泌量、每日平均分泌量及分泌总量均高于机械法,差异均有统计学意义(P均<0.05)。结论胰酶法分离的大鼠卵巢颗粒细胞在细胞活力和激素分泌功能方面均优于机械法,值得在卵巢早衰的研究中推广使用。

Objective To study the advantages of trypsin in isolating rat ovarian granulosa cells.Methods Ovarian granulosa cells of female SD rats aged 3-4 weeks were isolated and cultured by mechanical method and trypsin method,respectively.Mechanical method:under 40 folds anatomic microscope,mature follicles were punctured with a needle,and the culture solution containing the remaining tissue and follicular fluid of ovary was filtered with 200 mesh filter screen.Trypsin method:the ovary was cut into small pieces less than 2 mm3 with a sterile eye scissors,and then 0.25%trypsin was added.After digestion at room temperature for 1 h,the digestion solution containing the fragments of ovary was filtered with 200 mesh filter screen.The cell count,cell viability and secretion of estradiol and progesterone were compared.Results The survival rate of primary ovarian granulosa cells isolated by the two methods was both higher than 90%.When cultured for 9 d,the proliferation rate of the ovarian granulosa cells isolated by trypsin method was higher than that by mechanical method,except for 24 h.The peak time of cell proliferation rate of trypsin method was 24 h earlier than that of mechanical method,and the maximum cell proliferation rate of trypsin method was higher than that of mechanical method(72 h:[210.09±0.95]%vs 96 h:[180.50±0.74]%,P<0.05).The maximum secretion amount,total secretion amount and average daily secretion amount of estradiol and progesterone in ovarian granulosa cells isolated using trypsin method were significantly higher than that using mechanical method(all P<0.05).Conclusion Both cell viability and hormone secretion function of rat ovarian granulosa cells isolated by trypsin method are obviously superior to those by mechanical method.Trypsin method is worthy of further promotion in the study of premature ovarian failure.