低蛋白饮食、尼古丁皮下注射和自然选择形成的胎儿生长受限大鼠模型胎盘细胞凋亡研究

A study of apoptosis in placental cells in fetal growth restriction rat models induced by low-protein,subcutaneous injection of nicotine and natural selection factors

目的探讨低蛋白饮食喂养、尼古丁皮下注射及自然选择3种方法形成的胎儿生长受限(FGR)大鼠模型胎盘细胞凋亡情况及所致大鼠FGR可能的致病机制。方法2018年6月至2019年1月于南方医科大学南方医院动物实验中心将26只Wistar雌鼠随机分为非干预组(n=14)、低蛋白饮食组(n=6)和尼古丁注射组(n=6)。非干预组大鼠正常饲养,低蛋白饮食组及尼古丁注射组分别采用相应干预手段建立FGR模型。非干预组共获子鼠132只,有12只出生体重低于同组平均体重2个标准差的FGR弱仔,收集弱仔胎盘作为研究组1(自然选择组);低蛋白饮食组共获子鼠67只,其中FGR弱仔20只,随机选择18只弱仔胎盘作为研究组2(低蛋白饮食组)。尼古丁注射组共获子鼠60只,其中FGR弱仔21只,随机选择18只弱仔胎盘作为研究组3(尼古丁注射组)。在非干预组随机选择42只适龄幼仔胎盘作为正常对照组。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色检测各组胎盘细胞凋亡率,逆转录聚合酶链反应法(RT-PCR)检测细胞凋亡因子(Bax、Bcl-2、p53、Fas和Fas-L)mRNA表达水平,免疫组织化学染色检测蛋白表达水平。结果低蛋白饮食和尼古丁皮下注射均成功建立FGR大鼠模型,其FGR发生率明显高于非干预组(正常对照组)(29.89%,35%vs.9.09%,P<0.05)。自然选择组和尼古丁注射组胎盘细胞凋亡率均显著高于正常对照组[(66.120±14.611)%,(73.046±13.666)%vs.(28.740±27.184)%,P<0.01];低蛋白饮食组胎盘细胞凋亡率稍高于正常对照组,差异无统计学意义。与正常对照组相比,研究组1中Bcl-2、Fas-L表达水平显著降低[mRNA表达水平:Bcl-2(0.257±0.043)vs.(1.000±0.260),P<0.01;Fas-L(0.285±0.065)vs.(1.000±0.196),P<0.01;蛋白表达水平:Bcl-234.60 vs.54.63,P<0.01;Fas-L 31.93 vs.64.65,P<0.01];研究组2中Bcl-2、Fas-L表达水平显著降低[m RNA表达水平:Bcl-2(0.261±0.029)vs.(1.000±0.260),P<0.01;Fas-L(0.423±0.032)vs.(1.000±0.196),P<0.01;蛋白表达水平:Bcl-240.05 vs.54.63,P<0.01;Fas-L 32.45 vs.64.65,P<0.01];p53表达水平显著升高[m RNA表达水平:(1.433±0.151)vs.(1.000±0.242),P<0.01;蛋白表达水平:48.00 vs.29.10,P<0.01];研究组3中Bax、p53、Fas表达水平显著升高[mRNA水平:Bax(1.438±0.177)vs.(1.000±0.113),P<0.01;p53(2.176±0.356)vs.(1.000±0.0.242),P<0.01;Fas(1.335±0.076)vs.(1.000±0.090),P<0.01;蛋白表达水平:Bax 52.10 vs.36.45,P<0.05;p5352.90 vs.29.10,P<0.01;Fas 49.03 vs.30.35,P<0.05];Bcl-2、Fas-L表达水平显著降低[mRNA水平:Bcl-2(0.213±0.024)vs.(1.000±0.0.260,P<0.01);Fas-L(0.503±0.100)vs.(1.000±0.0.196),P<0.01;蛋白表达水平:Bcl-232.73 vs.54.63,P<0.01;Fas-L 32.98 vs.64.65,P<0.01]。结论3种因素所致大鼠FGR胎盘细胞凋亡率均显著升高,说明胎盘细胞凋亡与FGR形成存在关系。低蛋白饮食、尼古丁及自然因素所致FGR大鼠胎盘细胞凋亡途径、凋亡相关因子各不相同,说明不同因素诱发FGR的机制也不完全一致。

Objective To explore the influence and the possible mechanism of apoptosis in placental cells in fetal growth restriction(FGR)rats induced by lowprotein,subcutaneous injection of nicotine and natural selection factors.Methods In Laboratory Animal Research Center of Southern Medical University from June 2018 to January 2019 female Wistar mice(n=26)were randomly divided into three groups:the non-intervention group(n=14),the low-protein diet group(n=6)and the injection of nicotine group(n=6),among which the non-intervention group was fed and the FGR models of the other two groups were successfully established by corresponding methods.The total number of litter of the non-intervention mice was 132,from which 12 FGR placentas was selected as the Group 1(we called the natural selection group)whose birth weight were less than 2 standard deviations of average body weight in the same group.Totally 42 placentas were selected from appropriate-for-gestational-age neonates of the non-intervention mice as the normal control group.The total number of litter of the low-protein diet group was 67 and 20 of them were FGR;18 placentas were selected from the FGR mice as Group 2(we called the low-protein diet group).The total number of litter of the subcutaneous-injection-ofnicotine group was 60 and 21 of them were FGR;18 placentas were selected from the FGR mice as the Group 3(we called the nicotine group).Adopt TUNEL staining,RT-PCR and immunohistochemical techniques to detect the apoptosis index,the mRNA expression of Bax,Bcl-2,p53,Fas and Fas-L and the expression of protein,respectively.Results The FGR rat models were successfully established.The incidence of induced FGR were 29.89%(the low protein diet group)and 35%(the subcutaneous-injection-of-nicotine group),and were both apparently higher than that of the non-intervention group(FGR were 9.09%)(P<0.05).The apoptosis index of Group 3 and the Group 1 were significantly higher than that of the normal control group[(66.120±14.611)%,(73.046±13.666)%vs.(28.740±27.184)%,P<0.01],but Group 1 was slightly higher.The expression level of Bcl-2 and Fas-L in Group 1 was significantly lower than that in the normal control group[expression level of mRNA:Bcl-2(0.257±0.043)vs.(1.000±0.260),P<0.01;Fas-L(0.285±0.065)vs.(1.000±0.196),P<0.01;expression level of protein:Bcl-234.60 vs.54.63,P<0.01;Fas-L 31.93 vs.64.65,P<0.01].The expression level of Bcl-2 and Fas-L in Group 2 were significantly lower than that in the normal control group[expression level of mRNA:Bcl-2(0.261±0.029)vs.(1.000±0.260),P<0.01:Fas-L(0.423±0.032)vs.(1.000±0.196),P<0.01:expression level of protein:Bcl-240.05 vs.54.63 P<0.01;Fas-L32.45 vs.64.65,P<0.01].The expression level of p53 was significantly higher[expression level of mRNA:(1.433±0.151)Vs.(1.000±0.242),P<0.01;expression level of protein:48.00 vs.29.10,P<0.01].The expression level of Bax,p53 and Fas in Group 3 was significantly higher than that in the normal control group[expression level of mRNA:Bax(1.438±0.177)vs.(1.000±0.113),P<0.01;p53(2.176±0.356)vs.(1.000±0.0.242),P<0.01;Fas(1.335±0.076)vs.(1.000±0.090),P<0.01;expression level of protein:Bax 52.10 vs.36.45,P<0.05;p5352.90 vs.29.10,P<0.01;Fas49.03 vs.30.35,P<0.05].The expression level of Bcl-2 and Fas-L were significantly lower[expression level of mRNA:Bcl-2(0.213±0.024)vs.(1.000±0.0.260),P<0.01;Fas-L(0.503±0.100)vs.(1.000±0.0.196),P<0.01;expression level of protein:Bcl-232.73 vs.54.63,P<0.01;Fas-L 32.98 vs.64.65,P<0.01].Conclusion The apoptosis index of FGR placental cells in rats induced by low-protein diet,subcutaneous injection of nicotine and natural selection are significantly increased,which shows that apoptosis of placenta cells is related to the formation of FGR.The apoptosis pathways and apoptosis related factors are different which indicates that the mechanism of inducing FGR by different factors is different.