细胞培养基MLM-45及培养条件对不同发育时期柞蚕卵巢细胞生长的影响

Effect of MLM-45 Medium and Culture Condition on Growth of Pupal Ovarian Cells of Antheraea pernyi at Different Developmental Stages

选择柞蚕5龄幼虫以及前滞育期、滞育期和解除滞育后发育前期、中期、后期的柞蚕蛹分离卵巢细胞,利用新研制的柞蚕细胞培养基MLM-45,在不同温度、pH值及渗透压条件下进行柞蚕卵巢细胞的体外培养,探究最佳培养条件及卵巢细胞取材的最佳发育时期。用台盼蓝染色活细胞计数方法,测定不同条件下用MLM-45培养基培养的不同发育时期柞蚕卵巢细胞的活力,当在27℃、pH 6.3、渗透压325 mOsm/kg的条件下培养30 d时,上述各发育时期柞蚕卵巢细胞的存活率最高,并且5龄幼虫、前滞育期和解除滞育后发育前期的卵巢细胞出现了增殖,其增殖率分别为54%、64%和2%,滞育期及解除滞育后发育中期和后期的卵巢细胞存活率也分别达到99.6%、96.6%和92.4%。在此最佳培养条件下,各发育时期柞蚕卵巢细胞培养2个月后其形态以圆形和椭圆形为主,尤以5龄幼虫和前滞育期蛹卵巢细胞的培养效果最好,细胞增殖7～8倍,是适于以MLM-45培养基培养的最佳发育时期柞蚕卵巢细胞。

The pupal ovarian cells of Antheraea pernyi at various developmental stages, namely larvae of the 5th instar （5L） and pupae at the pre-dispause （ PD）, diapause （ D）, post-diapause termination devetopment-prophase （ PDT-P）, post-diapause termination development-metaphase （PDT-M） and post-diapause termination development-anaphase （PDT-A） stages were cultured in vitro with a newly developed Antheraea pernyicell culture medium （MLM-45） under different culture temperatures, pH values, and osmotic pressures to investigate the optimal culture condition and best developmental stage for isolating ovarian cells. Trypan Blue staining was used to determine vitality of ovarian cells from dif- ferent developmental stages cultured in MLM-45 medium under different culture conditions. The highest survival rates of ovarian cells from each developmental stage were observed under culture condition of 27 ℃, pH 6.3 and 325 mOsm/kg of osmotic pressure for 30 days, Nevertheless, under this culture condition, ovarian cells from 5L, PD and PDT-P stageshad a proliferation rate of 54%, 64% and 2% respectively, while ovarian cells from D, PDT-M and PDT-A stages had a survival rate as high as 99.6%, 96, 6% and 92.4% respectively. Most of the ovarian cells from each developmental stage were round and oval after being cultured under this optimal condition for 2 months. Moreover, the cultured ovarian cells from 5L and PD stagesproliferated by 7 -8 times, indicating that 5L and PD stages are the optimal period to isolate ovarian cells of Antheraea pernyi for culture with MLM-45 medium.