鼻腔递送IGF-1对HIBD大鼠大脑的作用及FOXG1基因的表达

发布时间：2018-05-09 01:33

本文选题：胰岛素样生长因子-1 + 缺氧缺血性脑损伤　；参考：《郑州大学》2012年硕士论文

【摘要】：目的应用新生7日龄SD大鼠建造缺氧缺血性脑损伤(HIBD)动物模型,经鼻腔递送胰岛素样生长因子-1(IGF-1),免疫组化法检测内源性神经干细胞增殖、迁移、分化情况,探索IGF-1对神经的营养保护作用；RT-PCR法检测HIBD后各组大鼠FoxglmRNA表达变化情况,探索IGF-1对其表达的影响。。材料与方法取7日龄SD新生大鼠,单纯随机分为假手术组、模型对照组、IGF-1干预组。每组按处死时间点不同,又分为HIBD后1d、3d、5d、7d、14d等5个亚组,共15个亚组。采用Rice法制作新生大鼠HIBD动物模型。模型对照组于HI后1h鼻腔递送生理盐水0.1ml；IGF-1干预组于HI后1h鼻腔递送IGF-12.5μg；假手术组仅分离颈总动脉,不结扎不缺氧,不鼻腔灌注。选用5’-嗅尿嘧啶(Brdu)标记新增殖细胞,巢蛋白(Nestin)标记神经干细胞,神经元特异性烯醇化酶(NSE)标记成熟神经元,胶质纤维酸性蛋白(GFAP)标记星形胶质细胞,Brdu-Nestin标记新增殖的神经干细胞,Brdu-NSE标记增殖细胞分化的神经元,Brdu-GFAP标记增殖细胞分化的星形胶质细胞。分别于术后1、3、5、7、14d处死大鼠。用于免疫组化实验的大鼠,在处死前一天被腹腔注射Brdu(50mg/kg/次,共3次),最后一次注射4小时后断头取脑,固定、切片,HE染色观察脑损伤组织形态结构变化；单、双标免疫组化染色法检测侧脑室室管膜下区(SVZ)、海马区以及皮层区Brdu阳性细胞、Brdu-Nestin、Brdu-NSE、Brdu-GFAP双阳性细胞数；用于RT-PCR实验的大鼠,常规断头取脑,半定量RT-PCR法检测各组大鼠脑组织FoxglmRNA的表达。结果 1模型鉴定大鼠制模前行为能力均正常,制模后翻身不能,夹尾不叫,夹尾左旋,常规HE染色显示结扎侧出现明显的病理变化,主要为神经元坏死,出现“红色神经元”。 2免疫组化结果 ①Brdu阳性细胞、Brdu-Nestin双阳性细胞：模型对照组术后第1天开始增多,3天达高峰,7天减少,14天明显减少,1d、3d、5d、7d、14d的Brdu阳性细胞数分别为：321.00±23.02、368.00±26.07、384.00±27.93、342.00±20.25、157.00±20.25; Brdu-Nestin双阳性细胞分别为：10.00±2.28、24.00±5.66、21.00±3.41、15.00±3.41、8.00±2.83；IGF-1干预组术后第1天增加明显,3天显著增多,5天达高峰期,7天稍减少,’14天明显减少,Brdu阳性细胞数值分别为：357.00±11.40、438.00±20.25、476.00±27.39、385.00±11.83、204.00±22.80；Brdu-Nestin双阳性细胞分别为：18.00±3.41、43.00±7.07、85.00±7.07、56.00±7.07、37.00±3.41；假手术组无明显增殖现象,随着时间的延长渐渐恢复到静息状态,Brdu阳性细胞数分别为240.00±21.21、200.00±7.07、120.00±7.07、60.00±10.00；Brdu-Nestin几无表达。在同一时间点各组间比较,IGF-1干预组高于模型对照组高于假手术组,差异具有统计学意义(P0.05)；②Brdu-NSE、Brdu-GFAP双标阳性细胞：模型对照组术后第7天开始出现,但胞体小,突起细、短,第14天细胞数增多,且胞体增大,突起增粗、增长,Brdu-NSE每个时间点数值为：15.00±2.27、25.00±3.96; Brdu-GFAP数值分别为：35.00±3.96、45.00±4.72；IGF-1干预组术后第5天就有少量阳性细胞,第7天在脑部有明显增加,14天增多达高峰,每个时间点数值分别为：Brdu-NSE:40.00±10.06、50.00±6.39; BrdU-GFAP:20.00±3.42、26.00±6.28。假手术组无表达。同一时间点两组比较,差异具有统计学意义俨0.05)。 3半定量RT-PCR结果假手术组Foxg1mRNA的表达基本稳定,无明显的变化,第1、3、5、7、14d的结果分别为0.77±0.046、0.65±0.134、0.76±0.041、0.68±0.047、0.72±0.024；模型对照组：前3天Foxg1mRNA的表达减少,3天后开始增加,7天时接近假手术组,结果分别为0.60±0.037、0.53±0.056、0.62±0.054、0.74±0.053、0.78±0.043；IGF-1干预组：Foxg1mRNA的表达趋势与模型对照组相同,但表达量高于同时间的对照组,差异有统计学意义(P0.05),结果分别为0.65±0.054、0.64±0.059、0.73±0.051、0.80±0.051、0.84±0.051。结论鼻腔递送IGF-1能促进HIBD后内源性神经干细胞的增殖、分化；IGF-1也能促进Foxg1基因的表达,使其更好地发挥神经调控作用。
[Abstract]:Purpose

To establish an animal model of hypoxic - ischemic brain damage ( HIBD ) in neonatal 7 - day - old SD rats . Insulin - like growth factor - 1 ( IGF - 1 ) was delivered through nasal cavity . Immunohistochemical method was used to detect the proliferation , migration and differentiation of endogenous neural stem cells .
The effects of IGF - 1 on the expression of FoxglmRNA in rats after HIBD were detected by RT - PCR .

Materials and Methods

SD neonatal rats were randomly divided into sham operation group , model control group and IGF - 1 intervention group . Each group was divided into 5 subgroups of 1d , 3d , 5d , 7d , 14d after HIBD .
IGF - 1 intervention group delivered IGF - 12.5 渭g in nasal cavity after HI .
The neural stem cells were labeled with 5 ' - sniffing uracil ( Brdu ) , neural stem cells were marked by Nestin , neural stem cells were marked by Brdu - NSE . Brdu - Nestin - labelled proliferating cells were injected with Brdu - NSE ( 50mg / kg / time , totally 3 times ) .
The number of Brdu - positive cells , Brdu - Nestin , Brdu - NSE , Brdu - GFAP double positive cells in the lateral ventricle of lateral ventricle ( SVZ ) , hippocampus and cortical area were detected by immunohistochemistry staining .
The expression of FoxglmRNA was detected by RT - PCR and semi - quantitative RT - PCR .

Results

1 Model Identification

Normal HE staining showed obvious pathological changes on the ligation side , mainly neuronal necrosis , and " red neurons " appeared .

2 Immunohistochemical results

( 1 ) Brdu - positive cells , Brdu - Nestin - positive cells : the number of Brdu - positive cells increased at 1 day after operation , peaked at 3 days , decreased in 7 days , and the number of Brdu - positive cells in 1d , 3d , 5d , 7d and 14d were 321.00 卤 23.02 , 362.00 卤 20.25 , 157.00 卤 20.25 ; Brdu - Nestin - positive cells were 10.00 卤 2.28 , 24.00 卤 5.66 , 21.00 卤 3.41 , 15.00 卤 3.41 , 8.00 卤 2.83 , respectively ;
The number of Brdu positive cells was 357.00 卤 11.40 , 4380.00 卤 20.25 , 476.00 卤 27.39 , 385.00 卤 11.83 , 204.00 卤 22.80 , respectively .
Brdu - Nestin - positive cells were 18.00 卤 3.41 , 43.00 卤 7.07 , 85.00 卤 7.07 , 56.00 卤 7.07 , 37.00 卤 3.41 , respectively .
The number of Brdu positive cells was 240.00 卤 21.21 , 200.00 卤 7.07 , 120.00 卤 7.07 , 60.00 卤 10.00 , respectively .
There was no expression of Brdu - Nestin . In the same time , the IGF - 1 intervention group was higher than that in the sham operation group ( P0.05 ) .
( 2 ) Brdu - NSE , Brdu - GFAP double labeled positive cells : the model control group began to appear on the 7th day after operation , but the cell body was small , the protrusion was thin , short , the number of cells in the 14th day increased , and the number of cells increased , the protrusion increased , and the Brdu - NSE value was 15 . 00 卤 2 . 27 , 25 . 00 卤 3.96 ; Brdu - GFAP value was 35.00 卤 3.96 , 45.00 卤 4.72 ;
There was a small number of positive cells in the IGF - 1 intervention group on the 5th day after operation . On the 7th day , there was a marked increase in the brain . The number of each time point was : Brdu - NSE : 40.00 卤 10.06 , 50.00 卤 6.39 ; and no expression of the sham operation group . The difference was statistically significant ( 0.05 ) at the same time point .

3 Semi - quantitative RT - PCR Results

The expression of Foxg1mRNA in sham operation group was stable without significant changes . The results were 0.77 卤 0.046 , 0.65 卤 0.134 , 0.76 卤 0.041 , 0.68 卤 0.047 , 0.72 卤 0.024 , respectively .
The expression of Foxg1mRNA was decreased in the first 3 days and increased after 3 days . The results were 0.60 卤 0.037 , 0.53 卤 0.056 , 0.62 卤 0.054 , 0.74 卤 0.053 , 0.78 卤 0.043 , respectively .
The expression of Foxg1mRNA was the same as that in the control group , but the expression level was higher than that in the control group with the same time ( P0.05 ) . The results were 0.65 卤 0.054 , 0.64 卤 0.059 , 0.73 卤 0.051 , 0.80 卤 0.051 , 0.84 卤 0.051 , respectively .

Conclusion

The nasal delivery of IGF - 1 can promote the proliferation and differentiation of endogenous neural stem cells after HIBD .
IGF - 1 can also promote the expression of Foxgl gene , and make it better play the role of neuromodulation .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R651.1

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