探讨SD大鼠妊娠期子宫内慢性缺氧(CIH)后，其子代大鼠胰腺Pdx-1基因表达水平。

选择处于妊娠期的16只健康SD大鼠为研究对象，按照随机数字表法，将其随机分为CIH组及对照组，每组各8只。对CIH组孕鼠建立CIH模型，对照组孕鼠不进行CIH处理。CIH组孕鼠第1天缺氧1 h后，对2组孕鼠均进行动脉血气分析。2组SD大鼠各分娩子代大鼠192与208只。于日龄为56 d时，采用随机数字表法随机选取CIH组及对照组子代大鼠各24只，然后处死，摘取胰腺，制作胰腺组织标本，分别纳入CIH亚组与对照亚组。对CIH亚组与对照亚组的各6例子代大鼠胰腺标本，采用苏木精-伊红染色法(HE)染色及光镜观察子代大鼠胰岛细胞改变情况，并采用Image Pro Plus 6.0图像分析软件对胰岛β细胞面积进行定量分析。采用免疫组化、Western blotting法分别检测CIH亚组与对照亚组的各6例子代大鼠胰腺Pdx-1蛋白表达水平。采用亚硫酸氢盐测序法，检测CIH亚组与对照亚组的各6例子代大鼠胰腺Pdx-1基因启动子甲基化水平。采用统计学分析方法比较2组孕鼠动脉血气分析结果，以及子代大鼠胰岛β细胞面积，胰腺Pdx-1蛋白相对积分光密度(IOD)值及其表达水平，胰腺Pdx-1基因启动子甲基化水平。

①2组孕鼠动脉血气分析结果显示，CIH组孕鼠动脉血氧饱和度(SaO₂)、动脉血氧分压(PaO₂)水平，均较对照组显著下降，差异均有统计学意义(t＝9.345，P<0.001；t＝2.537，P＝0.028)。2组孕鼠动脉血二氧化碳分压(PaCO₂)及动脉血pH值分别比较，差异均无统计学意义(t＝0.228，P＝0.822；t＝0.100，P＝0.920)。②光镜下观察2组子代大鼠胰腺组织切片(HE染色)结果显示，CIH亚组子代大鼠胰岛细胞大小不一，多数胰岛细胞增生肥大；对照亚组子代大鼠胰岛细胞呈圆形或椭圆形的细胞团，胰岛细胞分布均匀。CIH亚组子代大鼠胰岛β细胞面积为(254 216±6 211)μm²，较对照亚组的(208 253±2 714) μm²明显增大，二者比较，差异有统计学意义(t＝10.313，P<0.001)。③免疫组化结果显示，CIH亚组子代大鼠胰腺Pdx-1蛋白的相对IOD值为(1.84±0.51)×10⁴，显著低于对照亚组的(3.61±0.71)×10⁴，二者比较，差异有统计学意义(t＝14.177，P<0.001)。Western blotting法检测结果显示，CIH亚组子代大鼠胰腺Pdx-1蛋白表达水平为(1.08±0.22)mg/(kg·d)，较对照亚组的(2.09±0.36) mg/(kg·d)低，二者比较，差异有统计学意义(t＝0.787，P＝0.040)。④2个亚组子代大鼠胰腺CpG岛1、2甲基化水平分别比较，差异均无统计学意义(P>0.05)。CIH亚组子代大鼠胰腺CpG岛3甲基化水平显著高于对照亚组，并且差异有统计学意义(t＝17.626，P<0.001)。

SD大鼠产前发生CIH，可导致子代大鼠胰Pdx-1基因启动子甲基化表达上调，Pdx-1基因表达下降，Pdx-1蛋白表达减少。

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

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.

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).

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.