Study on changes of pancreatic Pdx-1 gene epigenetic mechanism of offspring rats with chronic intrauterine hypoxia

doi:10.3877/cma.j.issn.1673-5250.2016.06.010

Objective

To study the pancreatic expression level of Pdx-1 gene of the offspring rats after SD pregnant rats suffering from chronic intrauterine hypoxia (CIH).

Methods

A total of 16 healthy SD pregnant rats were chosen as research objects, and they were randomly divided into CIH group and control group by random digits table, and 8 pregnant rats of each group. The SD pregnant rats in CIH group were established as CIH pregnant rats model, while the SD pregnant rats in control group did not receive any hypoxia treatment. After 1 h of hypoxia treatment on the first day of CIH group, arterial blood gas analysis was performed on two groups of SD pregnant rats. SD pregnant rats in CIH group and control group delivered 192 and 208 offspring rats, respectively. When the offspring rats of two groups were 56 days of age, random number table method was used to select 24 offspring rats in each group. Then they were killed and pancreases were obtained from them, and enrolled into CIH sub-group and control sub-group. Hematoxylin-eosin (HE) stain and light microscope were used to observe the changes of pancreatic islet of the offspring rats in CIH sub-group and control sub-group (6 offspring rats in each sub-group), and Image Pro Plus 6.0 image analysis software was used for quantitative analysis of the islet beta cells area. Immunohistochemical method and Western blotting method were used to detect the pancreatic Pdx-1 protein expression level of the offspring rats in two sub-groups (6 offspring rats in each sub-group). Bisulfite genomic sequencing method was used to detect the promoter methylation of pancreatic Pdx-1 gene of the offspring rats in two sub-groups (6 offspring rats in each sub-group). The arterial blood gas analysis results of SD pregnant rats in CIH group and control group, the islet beta cells area, relative integral optical density (IOD) value and expression level of pancreatic Pdx-1 protein, and promoter methylation level of pancreatic Pdx-1 gene of the offspring rats in two sub-groups were statistically analyzed by statistical methods.

Results

①The results of arterial blood gas analysis of SD pregnant rats in CIH group and control group showed that the saturation of oxygen in artery (SaO₂), partial pressure of oxygen in artery (PaO₂) levels in CIH group were significantly decreased compared with those in control group, and both the differences were statistically significant (t=9.345, P<0.001; t=2.537, P=0.028). There were no significant differences in partial pressure of oxygen in artery (PaCO₂) level and arterial blood pH value between CIH group and control group (t=0.228, P=0.822; t=0.100, P=0.920). ②Light microscopic examination of pancreatic biopsy HE stain of offspring rats in two groups showed that islet cells in CIH sub-group were multiformed in shape, and many islet cells were hypertrophy and hyperplasia. While the islet cells in CIH sub-group were round or oval shape and identical in distribution. The area of islet beta cells was (254 216±6 211) μm² in CIH sub-group, which was significantly higher than (208 253±2 714) μm² in control sub-group, and the difference was statistically significant (t=10.313, P<0.001). ③The results of immunohistochemical method showed that, the relative IOD value of Pdx-1 protein in pancreatic islet beta cells of offspring rats in CIH sub-group was (1.84±0.51)×10⁴ which was significantly lower than (3.61±0.71)×10⁴ in control sub-group, and the difference was statistically significant (t=14.177, P<0.001). Western blotting results showed that the expression level of pancreatic Pdx-1 protein of offspring rats in CIH sub-group was (1.08±0.22) mg/(kg·d), which was significantly lower than (2.09±0.36) mg/(kg·d) in control sub-group, and the difference was statistically significant (t=0.787, P=0.040). ④The methylation level of pancreatic Pdx-1 gene promoter of CpG island 3 of offspring rats in CIH sub-group was significantly higher than that in control sub-group, and the difference was statistically significant (t=17.626, P<0.001). There were no statistical differences among the methylation levels of pancreatic Pdx-1 gene promoter of CpG island 1 and 2 between two sub-groups (P>0.05).

Conclusions

SD pregnant rats suffer CIH during pregnancy can lead to the increase of methylation of pancreatic Pdx-1 gene promoter and the decrease of pancreatic Pdx-1 gene expression and the decrease of pancreatic Pdx-1 protein expression of the offspring rats.

Key words: Chronic intermittent hypoxia, Pancreas, Pdx-1 gene, rat, Rats, Sprague-Dawley, Methylation

表1 2组孕鼠动脉血气分析结果比较(±s)

组别	鼠数(只)	SaO₂(kPa)	PaO₂(kPa)	PaCO₂(kPa)	pH值
CIH组	6	75.2±3.2	7.2±2.7	5.9±2.2	7.3±2.0
对照组	6	92.4±3.1	11.2±2.7	6.2±1.1	7.4±0.9
t值	?	9.345	2.537	0.228	0.100
P值	?	<0.001	0.028	0.822	0.920

表1 2组孕鼠动脉血气分析结果比较(±s)

组别	鼠数(只)	SaO₂(kPa)	PaO₂(kPa)	PaCO₂(kPa)	pH值
CIH组	6	75.2±3.2	7.2±2.7	5.9±2.2	7.3±2.0
对照组	6	92.4±3.1	11.2±2.7	6.2±1.1	7.4±0.9
t值	?	9.345	2.537	0.228	0.100
P值	?	<0.001	0.028	0.822	0.920

图2 CIH亚组与对照亚组子代大鼠胰腺组织切片免疫组化结果(图2A：CIH亚组；图2B：对照亚组)(DAB染色，低倍)

表2 CIH亚组与对照亚组子代大鼠胰腺组织切片Pdx-1蛋白相对积分光密度值比较

组别	鼠数(只)	相对IOD值(×10^4, ±s)
CIH亚组	6	1.84±0.51
对照亚组	6	3.61±0.71
t值	?	14.177
P值	?	<0.001

表2 CIH亚组与对照亚组子代大鼠胰腺组织切片Pdx-1蛋白相对积分光密度值比较

组别	鼠数(只)	相对IOD值(×10^4, ±s)
CIH亚组	6	1.84±0.51
对照亚组	6	3.61±0.71
t值	?	14.177
P值	?	<0.001

图3 CIH亚组与对照亚组子代大鼠胰腺Pdx-1蛋白表达情况

表3 CIH亚组与对照亚组子代大鼠胰腺Pdx-1基因启动子甲基化水平比较(%, ±s)

组别	鼠数(只)	CpG岛1甲基化水平	CpG岛2甲基化水平	CpG岛3甲基化水平
CIH亚组	6	4.0±0.5	1.5±0.6	6.5±0.6
对照亚组	6	4.0±0.5	1.5±0.6	3.5±0.6
t值	?	－0.178	0.880	17.626
P值	?	0.869	0.610	<0.001

表3 CIH亚组与对照亚组子代大鼠胰腺Pdx-1基因启动子甲基化水平比较(%, ±s)

组别	鼠数(只)	CpG岛1甲基化水平	CpG岛2甲基化水平	CpG岛3甲基化水平
CIH亚组	6	4.0±0.5	1.5±0.6	6.5±0.6
对照亚组	6	4.0±0.5	1.5±0.6	3.5±0.6
t值	?	－0.178	0.880	17.626
P值	?	0.869	0.610	<0.001

图4 CIH亚组与对照亚组子代大鼠胰腺Pdx-1基因启动子甲基化水平的亚硫酸氢盐测序结果(图4A：CIH亚组CpG岛1；图4B：对照亚组CpG岛1；图4C：CIH亚组CpG岛2；图4D：对照亚组CpG岛2；图4E：CIH亚组CpG岛3；图4F：对照亚组CpG岛3)

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