宫内TCDD暴露对子代印记基因及空间学习记忆影响的跨代遗传研究
发布时间：2018-08-01 18:42
【摘要】：2,3,7,8-四氯代二苯并-对-二VA英(TCDD)可以通过胎盘和乳汁向下一代传递,在直接暴露的个体及其子代中引起严重的异常生理和功能性改变。已有研究证实了胎儿发育中的环境因素刺激可诱导基因组的表观遗传调控的长期改变,造成表型的跨代遗传效应。目前宫内TCDD暴露对大鼠体细胞组织印记基因改变的跨代遗传效应尚未有报道;宫内TCDD暴露是否能够造成子代的空间学习记忆损伤,该效应是否能够跨代传递,亦尚未有文献报道。目的探索性腺性别决定期宫内TCDD暴露对F1-F3代生长发育,以及F1代和F3代雄鼠肝脏印记基因Igf2的影响,进一步研究相关的表观遗传跨代调控机制;并对宫内TCDD暴露对F1-F3代成年大鼠空间学习记忆能力的影响进行研究。方法建立跨代遗传大鼠模型,孕F0代雌性SD大鼠在性腺性别决定期(孕8-14天,GD 8-14)灌胃染毒TCDD,剂量分别为0,200,800 ng/kg bw。F1-F3代仔鼠围产期间测量其顶臀长和尾长;对肛门生殖孔距离(AGD)和体重增长情况进行测定。使用亚硫酸氢钠测序法(BSP)法对F1和F3代雄鼠肝脏组织中Igf2/H19位点两个差异性甲基化区域(ICR,DMR2)的甲基化状态进行测定;使用Real-time PCR法对F1代和F3代雄鼠肝脏印记基因胰岛素样生长因子2(Igf2),DNA甲基转移酶(DNMT1,DNMT3A,DNMT3B)的m RNA表达水平进行测定;使用Morris水迷宫实验测试F1-F3代成年大鼠的空间学习记忆能力。结果1宫内性腺性别决定期TCDD暴露未对F1-F3代窝大小和出生性别比例造成影响,但是造成了F1代雌鼠和雄鼠的出生存活率和哺乳存活率的显著下降,围产期顶臀长和尾长的缩短,围产期和成年期的AGD缩短;这些效应在F2代和F3代子代中仍持续,但随着传代的进行,效应逐渐减弱;2宫内性腺性别决定期TCDD暴露可显著造成F1和F2代的低出生体重以及围产期的体重增长缓慢。体重增长趋势上,高剂量(800 ng/kg bw)TCDD宫内暴露造成F1代大鼠体重持续低于对照组,并随着传代的进行,直至F3代仍出现不同程度的体重增长缓慢现象;而在低剂量(200 ng/kg bw)TCDD宫内暴露后代中则出现了不同程度的体重增长高于同期对照组的现象,并持续到F3代;3宫内TCDD暴露可引起F1-F3代雄鼠(睾丸,附睾,前列腺)和雌鼠(卵巢)的生殖系统脏器重量和脏器系数的不同程度的改变,并随着传代该效应衰减;同时也观察到了宫内TCDD暴露对F1-F3代一般脏器(脾脏,肾脏,心脏,肝脏)的重量和脏器系数的长期影响;4宫内TCDD暴露可造成子F1代和F3代雄鼠肝脏印记基因Igf2的m RNA表达下降。200和800 ng/kg bw TCDD组的F1代雄鼠Igf2的m RNA表达分别为对照组的0.81倍和0.39倍(P0.05);经过两代雄性生殖系的传递后,F3代Igf2的表达水平仍显示了明显的降低趋势,200和800 ng/kg bw TCDD组F3代雄鼠Igf2的m RNA表达分别为对照组的0.25和0.33倍(P0.05);5对印记基因Igf2的两个差异性甲基化区域的甲基化程度的分析结果显示,F1代ICR区域甲基化程度随染毒剂量增加而上升,DMR2区域甲基化程度则随剂量增加而下降;并且随着传代的进行,宫内TCDD暴露诱导的ICR和DMR2的甲基化模式的改变在F3代仍呈现相似的趋势;6伴随着印记基因Igf2差异性甲基化区域甲基化模式的改变,建立和维持印记基因表达的DNMTs也受宫内TCDD暴露的影响:F1代DNMT1,DNMT3A和DNMT3B的m RNA表达呈现非单调改变模式,并且该效应在F3代仍凸显;7 Morris水迷宫获得性训练阶段,TCDD宫内暴露组F1-F3代雌雄成年大鼠的潜伏期和游泳路程与对照组相比,具有不同程度的延长现象,该效应持续到F3代仍显示与对照组有微弱的差异;8 Morris水迷宫探查测试中,F1代的800 ng/kg bw TCDD组的雄鼠穿越原平台的次数明显减少,但是该效应并没有在F2,F3代中凸显出来,F1-F3代雌性后代也未出现差异;F1代800 ng/kg bw TCDD组的雌雄成年大鼠在目标象限花费的时间多于对照组;9 Morris水迷宫获得性训练阶段,宫内TCDD作用造成的子代学习记忆能力具有明显的性别差异。F2代TCDD组雄鼠相对于同剂量组的雌鼠花费更长的时间找到隐匿平台;并且在F1代和F2代中,TCDD组雄鼠表现差于对照组的雌鼠,显示了一定程度的雌性化。上述性别差异经历两代传递后衰减。结论1宫内性腺性别决定期TCDD暴露对F1-F3代子代的一般生长发育和AGD造成一定的影响,并随着传代的进行,效应逐渐减弱;2宫内TCDD暴露对子代体重增长的长期影响可能呈现“倒U”现象:TCDD宫内暴露子代体重的增长可能在低剂量作用下得到刺激,出现代偿性生长;而在较高剂量的作用下体重增长受到抑制,该种现象可能会跨代传递;3性腺性别决定期TCDD宫内暴露具有广泛的发育毒性作用,可能扰乱器官形成,对子代生殖系统,免疫系统造成影响,产生肾脏毒性,心脏毒性和肝脏毒性,一些效应还能够持续影响多代,增加疾病风险;4宫内TCDD暴露可能并不直接造成不良妊娠结局,相反的可能对于生长和发育指标产生长久的细微的效应或损伤。靶器官的跨代效应影响可能也涉及表观遗传因素的影响,对于以后的TCDD跨代遗传研究靶标选择上具有一定的提示作用;5性腺性别决定期TCDD宫内暴露可通过扰乱印记基因Igf2的表观遗传修饰,从而造成印记基因表达的改变,并且该印记基因的异常甲基化模式可在传代中逃避基因组的重新甲基化过程,进行跨代遗传;DNA甲基转移酶在TCDD造成的印记基因甲基化模式的跨代遗传传递中起一定的作用;6宫内TCDD暴露可造成F1-F3代雌雄成年大鼠空间学习记忆能力的损伤,具有跨代遗传效应;宫内TCDD暴露诱导的空间学习记忆能力的改变具有性别差异。TCDD子代雄鼠显示了“雌性化”表现,提示TCDD可能通过激素紊乱效应造成雄性子代的神经行为的“去雄化”表现,但是该效应经过两代传递后逐渐衰减。
[Abstract]:2,3,7,8- four chloro two benzo benzo - two VA e (TCDD) can be transmitted through the placenta and milk to the next generation, causing severe abnormal physiological and functional changes in directly exposed individuals and their progenies. The intergenerational genetic effect of the intrauterine TCDD exposure has not yet been reported on the intergenerational effect of intrauterine exposure to the imprinted gene of the somatic tissue of the rat. Whether the intrauterine TCDD exposure can cause the spatial learning and memory damage of the offspring, whether the effect can be transmitted across the generation, and not yet be reported in the literature. Purpose to explore the intrauterine TCDD storm in the sex determination period of the gonadal gland. The effect of exposure on the growth and development of F1-F3 generation, as well as the liver imprinting gene Igf2 of F1 and F3 male rats, further study the related epigenetic cross generation regulation mechanism, and study the effect of TCDD exposure on the spatial learning and memory ability of the adult rats in the F1-F3 generation. Methods a cross generation rat model was established, and the female SD rats of F0 generation were in the gonads. The sex determination period (8-14 days of pregnancy, GD 8-14) was administered to the stomach of TCDD, and the dose of 0200800 ng/kg bw.F1-F3 offspring was measured in the perinatal period. The distance of the anal genital pore (AGD) and the weight growth were measured. The Igf2/H19 loci in the liver tissues of F1 and F3 male rats were two difference using the Sodium Bisulfite sequencing method (BSP) method. The methylation status of ICR (DMR2) was measured and the Real-time PCR method was used to determine the expression level of the expression level of insulin like growth factor 2 (Igf2), DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) of the liver imprinted gene of the F1 and F3 male mice, and the spatial learning of adult rats in the water maze test was tested. Results in 1 intrauterine gonadal sex determination, TCDD exposure did not affect the size of F1-F3 nests and the proportion of birth sex, but resulted in a significant decrease in the birth and lactation survival rates of the F1 generation female rats and male rats, the shortening of the perinatal length and tail length, and the shortening of the perinatal and adult AGD; these effects were in the F2 and F3 generations. The offspring still continued, but with the passage of the passage, the effect gradually weakened; the sex determination period of the 2 intrauterine sex gland TCDD exposure could significantly cause the low birth weight of the F1 and F2 generation and the slow growth of the perinatal body weight. In the trend of weight growth, Gao Jiliang (800 ng/kg BW) TCDD intrauterine exposure resulted in the weight of the F1 generation rats lower than the control group, and along with the transmission. On behalf of the F3 generation, there were still different degrees of slow weight growth, while in the low dose (200 ng/kg BW) TCDD intrauterine offspring, the weight gain of different degrees was higher than that of the same control group, and continued to the F3 generation, and the 3 intrauterine TCDD exposure could cause the F1-F3 male male rats (testis, epididymis, prostate) and the female rats (ovaries). The changes in the organ weight and organ coefficient of the reproductive system were changed to varying degrees, and the effect of this effect was attenuated with the passage. The long-term effect of intrauterine TCDD exposure on the weight and organ coefficient of the F1-F3 generation general organs (spleen, kidney, heart, liver) was also observed; 4 intrauterine TCDD exposure could cause the liver imprinting gene Igf2 of the offspring F1 and F3 generations of the male rats. The expression of M RNA expression of F1 generation male rat Igf2 in M RNA expression and 800 ng/kg BW TCDD group was 0.81 times and 0.39 times (P0.05), respectively. After the transmission of male reproductive lines in two generations, the expression level of F3 generation was still obviously decreasing. 200 and 800 were the control group. 0.25 and 0.33 times (P0.05); 5 Analysis of the methylation degree of two differential methylation regions of the imprinting gene Igf2 showed that the degree of methylation in the F1 generation ICR region increased with the increase of the dose, and the degree of methylation in the DMR2 region decreased with the increase of the dose; and as the transmission was carried out, the TCDD exposure induced ICR and DMR2 in the intrauterine exposure. The changes in the F3 generation are similar in the F3 generation; with the alteration of the methylation pattern in the differential methylation region of the imprinting gene Igf2, the establishment and maintenance of the DNMTs of the imprinted gene expression is also affected by the intrauterine TCDD exposure: the F1 generation DNMT1, the m RNA expression of DNMT3A and DNMT3B present a non monotonic pattern, and the effect is still convex in the F3 generation. 7 Morris water maze acquired training stage, the incubation period and swimming distance of the F1-F3 generation adult rats in the TCDD intrauterine exposure group had different degrees of prolongation compared with the control group, and the effect continued until the F3 generation still showed a weak difference with the control group; in the 8 Morris test, the 800 ng/kg BW TCDD group of the F1 generation was male. The number of mice passed through the original platform significantly decreased, but the effect was not highlighted in the F2, F3 generation, and the female offspring of the F1-F3 generation did not differ. The time spent in the female and male adult rats of the F1 800 ng/kg BW TCDD group was more than the control group; the 9 Morris water maze acquired the sexual training stage and the offspring learning caused by the intrauterine TCDD action. The male rats of.F2 generation TCDD took longer time to find concealed platforms than the female rats in the same dose group; and in the F1 and F2 generations, the male rats in the group TCDD showed a certain degree of feminization. The above sex difference experienced two generations of transmission attenuation. Conclusion the 1 intrauterine gonads TCDD exposure in the sex determination period has a certain effect on the general growth and development of the F1-F3 generation and AGD, and the effect gradually diminished with the passage of the passage. 2 the long-term effect of TCDD exposure on the offspring's body weight may show "inverted U" phenomenon: the growth of TCDD intrauterine exposure progeny weight may be stimulated under the effect of low dose. Modern compensatory growth; and the growth of body weight under the high dose of inhibition, the phenomenon may pass through the generation; 3 sex determination of sex in the gonad period TCDD intrauterine exposure has extensive developmental toxicity, may disturb the formation of organs, the reproductive system of the progeny, the immune system, and produce renal toxicity, heart toxicity, and liver toxicity. Some effects can also continue to affect multiple generations and increase the risk of disease; 4 intrauterine TCDD exposure may not directly cause adverse pregnancy outcomes. On the contrary, it may have long and subtle effects or damage to growth and development indicators. The effect of the cross generation effect of target organs may also involve epigenetic factors, for the subsequent TCDD cross generation. The selection of genetic research targets has a certain suggestive effect; 5 the intrauterine exposure to TCDD in the sex determination period of sex glands can cause the epigenetic modification of the imprinted gene Igf2 to cause changes in the expression of the imprinting genes, and the abnormal methylation pattern of the imprinted gene can escape the process of the re methylation of the genome in the passage and carry out the cross generation. Heredity; DNA methyltransferase plays a certain role in the transgenerational transmission of the imprinted gene methylation pattern caused by TCDD; 6 intrauterine TCDD exposure can cause damage to the spatial learning and memory ability of the adult male and female adult rats of F1-F3 generation, and has a cross generation genetic effect; the changes of spatial learning and memory ability induced by intrauterine TCDD exposure have sex differences. The TCDD progeny male mice showed "feminization", suggesting that TCDD may result in the "derangement" of the neurobehavioral behavior of the male offspring through the hormonal disorder effect, but the effect gradually attenuates after two generations of transmission.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.5
[Abstract]:2,3,7,8- four chloro two benzo benzo - two VA e (TCDD) can be transmitted through the placenta and milk to the next generation, causing severe abnormal physiological and functional changes in directly exposed individuals and their progenies. The intergenerational genetic effect of the intrauterine TCDD exposure has not yet been reported on the intergenerational effect of intrauterine exposure to the imprinted gene of the somatic tissue of the rat. Whether the intrauterine TCDD exposure can cause the spatial learning and memory damage of the offspring, whether the effect can be transmitted across the generation, and not yet be reported in the literature. Purpose to explore the intrauterine TCDD storm in the sex determination period of the gonadal gland. The effect of exposure on the growth and development of F1-F3 generation, as well as the liver imprinting gene Igf2 of F1 and F3 male rats, further study the related epigenetic cross generation regulation mechanism, and study the effect of TCDD exposure on the spatial learning and memory ability of the adult rats in the F1-F3 generation. Methods a cross generation rat model was established, and the female SD rats of F0 generation were in the gonads. The sex determination period (8-14 days of pregnancy, GD 8-14) was administered to the stomach of TCDD, and the dose of 0200800 ng/kg bw.F1-F3 offspring was measured in the perinatal period. The distance of the anal genital pore (AGD) and the weight growth were measured. The Igf2/H19 loci in the liver tissues of F1 and F3 male rats were two difference using the Sodium Bisulfite sequencing method (BSP) method. The methylation status of ICR (DMR2) was measured and the Real-time PCR method was used to determine the expression level of the expression level of insulin like growth factor 2 (Igf2), DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) of the liver imprinted gene of the F1 and F3 male mice, and the spatial learning of adult rats in the water maze test was tested. Results in 1 intrauterine gonadal sex determination, TCDD exposure did not affect the size of F1-F3 nests and the proportion of birth sex, but resulted in a significant decrease in the birth and lactation survival rates of the F1 generation female rats and male rats, the shortening of the perinatal length and tail length, and the shortening of the perinatal and adult AGD; these effects were in the F2 and F3 generations. The offspring still continued, but with the passage of the passage, the effect gradually weakened; the sex determination period of the 2 intrauterine sex gland TCDD exposure could significantly cause the low birth weight of the F1 and F2 generation and the slow growth of the perinatal body weight. In the trend of weight growth, Gao Jiliang (800 ng/kg BW) TCDD intrauterine exposure resulted in the weight of the F1 generation rats lower than the control group, and along with the transmission. On behalf of the F3 generation, there were still different degrees of slow weight growth, while in the low dose (200 ng/kg BW) TCDD intrauterine offspring, the weight gain of different degrees was higher than that of the same control group, and continued to the F3 generation, and the 3 intrauterine TCDD exposure could cause the F1-F3 male male rats (testis, epididymis, prostate) and the female rats (ovaries). The changes in the organ weight and organ coefficient of the reproductive system were changed to varying degrees, and the effect of this effect was attenuated with the passage. The long-term effect of intrauterine TCDD exposure on the weight and organ coefficient of the F1-F3 generation general organs (spleen, kidney, heart, liver) was also observed; 4 intrauterine TCDD exposure could cause the liver imprinting gene Igf2 of the offspring F1 and F3 generations of the male rats. The expression of M RNA expression of F1 generation male rat Igf2 in M RNA expression and 800 ng/kg BW TCDD group was 0.81 times and 0.39 times (P0.05), respectively. After the transmission of male reproductive lines in two generations, the expression level of F3 generation was still obviously decreasing. 200 and 800 were the control group. 0.25 and 0.33 times (P0.05); 5 Analysis of the methylation degree of two differential methylation regions of the imprinting gene Igf2 showed that the degree of methylation in the F1 generation ICR region increased with the increase of the dose, and the degree of methylation in the DMR2 region decreased with the increase of the dose; and as the transmission was carried out, the TCDD exposure induced ICR and DMR2 in the intrauterine exposure. The changes in the F3 generation are similar in the F3 generation; with the alteration of the methylation pattern in the differential methylation region of the imprinting gene Igf2, the establishment and maintenance of the DNMTs of the imprinted gene expression is also affected by the intrauterine TCDD exposure: the F1 generation DNMT1, the m RNA expression of DNMT3A and DNMT3B present a non monotonic pattern, and the effect is still convex in the F3 generation. 7 Morris water maze acquired training stage, the incubation period and swimming distance of the F1-F3 generation adult rats in the TCDD intrauterine exposure group had different degrees of prolongation compared with the control group, and the effect continued until the F3 generation still showed a weak difference with the control group; in the 8 Morris test, the 800 ng/kg BW TCDD group of the F1 generation was male. The number of mice passed through the original platform significantly decreased, but the effect was not highlighted in the F2, F3 generation, and the female offspring of the F1-F3 generation did not differ. The time spent in the female and male adult rats of the F1 800 ng/kg BW TCDD group was more than the control group; the 9 Morris water maze acquired the sexual training stage and the offspring learning caused by the intrauterine TCDD action. The male rats of.F2 generation TCDD took longer time to find concealed platforms than the female rats in the same dose group; and in the F1 and F2 generations, the male rats in the group TCDD showed a certain degree of feminization. The above sex difference experienced two generations of transmission attenuation. Conclusion the 1 intrauterine gonads TCDD exposure in the sex determination period has a certain effect on the general growth and development of the F1-F3 generation and AGD, and the effect gradually diminished with the passage of the passage. 2 the long-term effect of TCDD exposure on the offspring's body weight may show "inverted U" phenomenon: the growth of TCDD intrauterine exposure progeny weight may be stimulated under the effect of low dose. Modern compensatory growth; and the growth of body weight under the high dose of inhibition, the phenomenon may pass through the generation; 3 sex determination of sex in the gonad period TCDD intrauterine exposure has extensive developmental toxicity, may disturb the formation of organs, the reproductive system of the progeny, the immune system, and produce renal toxicity, heart toxicity, and liver toxicity. Some effects can also continue to affect multiple generations and increase the risk of disease; 4 intrauterine TCDD exposure may not directly cause adverse pregnancy outcomes. On the contrary, it may have long and subtle effects or damage to growth and development indicators. The effect of the cross generation effect of target organs may also involve epigenetic factors, for the subsequent TCDD cross generation. The selection of genetic research targets has a certain suggestive effect; 5 the intrauterine exposure to TCDD in the sex determination period of sex glands can cause the epigenetic modification of the imprinted gene Igf2 to cause changes in the expression of the imprinting genes, and the abnormal methylation pattern of the imprinted gene can escape the process of the re methylation of the genome in the passage and carry out the cross generation. Heredity; DNA methyltransferase plays a certain role in the transgenerational transmission of the imprinted gene methylation pattern caused by TCDD; 6 intrauterine TCDD exposure can cause damage to the spatial learning and memory ability of the adult male and female adult rats of F1-F3 generation, and has a cross generation genetic effect; the changes of spatial learning and memory ability induced by intrauterine TCDD exposure have sex differences. The TCDD progeny male mice showed "feminization", suggesting that TCDD may result in the "derangement" of the neurobehavioral behavior of the male offspring through the hormonal disorder effect, but the effect gradually attenuates after two generations of transmission.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.5
【共引文献】
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