牛磺酸调节cAMP-PKA-CREB信号通路活性促进生长受限胎鼠神经干细胞增殖的体内外实验研究

发布时间：2018-07-20 12:51

【摘要】：背景:宫内生长受限(Intrauterine growth restriction,IUGR)是指各种因素致胎儿在宫内的生长受到限制,不能发挥最佳潜能,出生体重明显低于正常。母亲、胎儿以及胎盘等方面的多种病理因素均可导致IUGR的发生。IUGR可导致多脏器功能受累,对脑发育的不良影响尤为突出,严重影响远期神经功能。牛磺酸在脑发育过程中发挥重要作用,但机体自身合成较少,胎儿依靠胎盘转运获取足够的牛磺酸。本课题组前期的实验研究显示:孕鼠补充牛磺酸可改善IUGR鼠脑发育,如改善脑超微结构、增加神经细胞数量等,这些作用可能是通过调节cAMP-PKA-CREB信号通路活性实现的。牛磺酸可调节神经干细胞(Neural Stem Cells,NSC)的增殖、存活、粘附能力,但牛磺酸对IUGR胎鼠NSC增殖的影响以及机制尚未清楚。因此,本研究将通过体内外实验探讨IUGR对胎鼠NSC增殖的影响、产前补充牛磺酸对IUGR胎鼠NSC增殖的影响以及牛磺酸调节IUGR胎鼠NSC增殖的可能机制。方法:采用全程限制饮食的方法建立IUGR模型,即IUGR组孕鼠自受孕之日起仅给予对照组正常饮食的40%量喂养;体内实验分对照组、IUGR组、牛磺酸组(IUGR孕鼠自孕7天起加300mg/kg/d牛磺酸),自然分娩后称取体重及脑重;免疫组织化学染色法、RT-PCR、Western Blot的方法检测各组脑组织内NSC数量以及cAMP-PKA-CREB信号通路关键因子(PKA,cAMP,CREB,p-CREB,BDNF,GDNF,c-fos,c-jun,CaMK II)的表达;体外分离对照组及IUGR组胎鼠NSC,免疫荧光细胞化学染色法检测细胞FABP-7和Nestin的表达,CCK8试验以及细胞计数法检测不同浓度牛磺酸对IUGR胎鼠NSC增殖的影响,RT-PCR方法检测不同浓度牛磺酸以及信号通路抑制剂H89处理NSC后各组cAMP-PKA-CREB信号通路关键因子的表达。SPSS20.0软件对数据进行统计。结果:全程限制饮食的方法可成功建立IUGR新生鼠以及胎鼠模型;孕鼠补充牛磺酸可明显降低IUGR的发生,减少死亡率,使脑重增加;IUGR组胎鼠脑内NSC的数量明显减少,补充牛磺酸后IUGR组NSC数量明显增加(P0.05);IUGR胎鼠脑内cAMP-PKA-CREB信号通路关键因子的表达与对照组相比有显著差异,牛磺酸组关键因子的表达较IUGR组明显改变,差异有统计学意义(P0.05)。IUGR胎鼠NSC体外增殖能力及细胞数量明显降低,牛磺酸体外干预可使IUGR胎鼠NSC增殖能力明显增加,且与浓度有关(P0.05);IUGR胎鼠NSC体外培养条件下cAMP-PKA-CREB信号通路关键因子的表达受到抑制,部分因子呈代偿性增加,与对照组相比有明显差异,牛磺酸干预后相关因子的表达发生明显变化,差异有有统计学意义(P0.05)。结论:IUGR胎鼠脑内NSC数量减少,孕鼠补充牛磺酸后IUGR胎鼠NSC数量增加;IUGR胎鼠NSC体外增殖能力降低,牛磺酸可促进IUGR胎鼠NSC体外增殖;牛磺酸可能通过上调cAMP-PKA-CREB信号通路活性来促进IUGR胎鼠NSC增殖、改善脑发育。
[Abstract]:Background: intrauterine growth restriction (IUGR) refers to the fact that the fetal growth in the uterus is restricted due to various factors, and the birth weight is significantly lower than normal. Many pathological factors such as mother, fetus and placenta can lead to the occurrence of IUGR. IUGR can cause multiple organ function involvement, especially the adverse effects on brain development, which seriously affect long-term nerve function. Taurine plays an important role in brain development, but the body itself is less synthesised. Fetus relies on placental transport to obtain sufficient taurine. Our previous experimental study showed that the supplementation of taurine could improve the brain development of IUGR, such as improving the brain ultrastructure and increasing the number of nerve cells. These effects may be achieved by regulating the activity of cAMP-PKA-CREB signaling pathway. Taurine can regulate the proliferation, survival and adhesion of neural stem cells (NSC), but the effect of taurine on the proliferation of NSC in IUGR fetal mice and its mechanism are not clear. Therefore, the effects of IUGR on the proliferation of fetal NSC, the effect of prenatal taurine supplementation on the proliferation of NSC of IUGR fetal mice and the possible mechanism of taurine regulating the proliferation of NSC of IUGR fetal mice were studied in this study. Methods: the IUGR model was established by the method of whole course restriction diet. The pregnant rats in the IUGR group were fed only 40% of the normal diet of the control group from the day of conception, and were divided into the control group and the IUGR group. The 300mg/kg/d taurine was added to the pregnant rats from the 7th day of gestation, and the body weight and brain weight were measured by natural delivery. The expression of NSC in brain tissue and the expression of cAMP-PKA-CREB signal pathway key factor (PKAcAMPCREBp-CREBp-CREBN-BDNFfosc-juncCaMK II) were detected by immunohistochemical staining. Fetal rat NSCs were isolated from control group and IUGR group in vitro. The expression of FABP-7 and nestin was detected by immunofluorescence cytochemical staining and the effect of taurine at different concentrations on the proliferation of NSC in IUGR fetal mice was detected by cell counting method. RT-PCR was used to detect the proliferation of NSC in IUGR fetal mice. The expression of key factors of cAMP-PKA-CREB signal pathway in each group was analyzed by SPSS 20.0 software after treatment with taurine and signal pathway inhibitor H89. Results: the neonatal and fetal rat models of IUGR could be established successfully by the method of diet restriction, and the incidence and mortality of IUGR were significantly decreased by taurine supplementation in pregnant rats, and the number of NSC in the fetal brain of rats with increased brain weight was significantly decreased in IUGR group. The number of NSC in IUGR group was significantly increased after supplementation of taurine (P0.05) the expression of key factors of cAMP-PKA-CREB signal pathway in IUGR fetal brain was significantly different from that in control group, and the expression of key factor in taurine group was significantly different from that in IUGR group. The difference was statistically significant (P0.05). The proliferation ability and cell number of NSC of IUGR fetal mice were significantly decreased, and the proliferation ability of NSC of IUGR fetal mice was significantly increased by taurine intervention in vitro. In addition, the expression of key factors of cAMP-PKA-CREB signaling pathway was inhibited and some factors were compensatory, which were significantly different from those of the control group (P0.05). The expression of related factors changed obviously after taurine intervention. The difference was statistically significant (P0.05). Conclusion the number of NSC in the brain of fetal mice with 1% IUGR decreased, and the number of NSC in fetal mice of IUGR increased after supplementation of taurine. Taurine could promote the proliferation of NSC of IUGR fetal mice in vitro. Taurine may increase the activity of cAMP-PKA-CREB signaling pathway to promote the proliferation of IUGR fetal NSC and improve brain development.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.5

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