基因工程玉米Bt799对大鼠胚胎中脑细胞微团及腭板发育的影响

Effect of Bt799 maize on development of embryos midbrain cell cultures and palate in rats

目的通过体外培养大鼠中脑细胞微团和胚胎腭板,研究基因工程玉米Bt799及所表达Cry1Ac蛋白发育毒性。方法实验用大鼠选用Wistar大鼠,购自北京大学医学部实验动物部,年龄9周,体重(200±20)g,常规饲养于SPF级动物房。第一部分:于雌鼠孕13 d提取胚胎中脑细胞,以即刻离心血清替代培养基中胎牛血清,在不同条件下培养中脑细胞5 d,并观察其增殖分化情况。中脑细胞微团培养分八个组进行,分别对应八种不同培养环境。将制备ICS的Wistar大鼠随机分为六组,其中NM组、TM3组、TM1M组、TM3M组、PCB组为前五组大鼠,BP组、BC组、PCA组因大鼠未经直接干预且不影响组间比较,故共同使用第六组大鼠的血清。其中TM3、TM1M、TM3M组使用经Bt799玉米干预3 d、1个月、3个月大鼠的血清,BP组使用添加Cry1Ac蛋白的血清,PCA使用添加双酚A的血清,PCB组使用经双酚A灌胃3 d大鼠的血清,NM组使用经亲本玉米郑58干预3 d大鼠的血清、BC组使用经AIN-93G饲料喂养3 d大鼠的血清。第二部分:于雌鼠孕12.5 d摘取大鼠胚胎腭板,以加入Cry1Ac蛋白(BP组)或全反式视黄酸(RA组)条件旋转培养腭板72 h,根据腭板融合情况计算融合率,判断Cry1Ac蛋白对大鼠胚胎腭板发育的影响。结果第一部分:TM3、TM1M、TM3M、BP组细胞增殖率、分化率与NM组、BC组相比差异均无统计学意义。PCA组和PCB组细胞增殖率和分化率低于NM组、BC组且差异有统计学意义。第二部分:BP组腭板融合率与BC组相比差异无统计学意义。RA组腭板融合率低于BC组,且差异有统计学意义。结论本研究未观察到Bt799玉米及其表达蛋白Cry1Ac的胚胎发育毒性。

Objective To study the developmental toxicity of genetically modified maize Bt799 and the Cry1Ac protein in it by culturing rat embryos palatal plate and midbrain cells in vitro.MethodsWistar rats,aged 9 weeks,weighing(200±20)g,were purchased from the Experimental Animal Department of Peking University Health Science Center and raised in SPF animal room.The first part:The embryos midbrain cells were extracted on the 13th day of gestation,after 5 days of culture under different conditions(the fetal bovine serum was replaced by immediate centrifugation serum),the proliferation and differentiation of midbrain cells were observed.The culture of midbrain cells was divided into eight groups,corresponding to eight different culture environments.As source of ICS,the Wistar rats were randomly divided into six groups.The first five groups included NM group,TM3 group,TM1M group,TM3M group and PCB group;BP group,BC group and PCA group used the serum of the sixth group together because the rats had no direct intervention and it did not affect the comparison between groups.The TM3,TM1M and TM3M groups were treated with the serum of adult rats after intervention with Bt799 corn for different duration(1 day,1 month,3 months),BP group with the serum supplemented with Cry1Ac protein,PCA group with the serum supplemented with bisphenol A,PCB group with the serum extracted from rats perfused with bisphenol A for 3 days,NM group with the serum of the rats Intervened by the parent maize Zheng 58 for 3 days,and BC group with the serum of rats fed with AIN-93G diet for 3 days.The second part:On the 12.5th day of gestation,the palatal plates of rat embryos were extracted and then cultured for 72 hours by adding Cry1Ac protein(BP group)or all-trans retinoic acid(RA group).The palatal plate fusion rate was calculated to determine the effect of Cry1Ac protein on the development of the palatal plate of the rat embryos.ResultsThe first part:The proliferation rate and differentiation rate in TM3,TM1M,TM3M and BP groups were not significantly different from those in NM group and BC group(P>0.05).Compared with NM group and BC group,the proliferation rate and differentiation rate in PCA group and PCB group significantly decreased(P<0.05).The second part:There was no significant difference in palatal fusion rate between BP group and BC group(P>0.05).Compared with BC group,the palatal fusion rate of RA significantly decreased(P<0.05).ConclusionNo embryo-developmental toxicity of Bt799 maize and Cry1Ac protein was observed in this study.