Uteroplacental insufficiency alters liver and skeletal muscle branched-chain amino acid metabolism in intrauterine growth-restricted fetal rats

Uteroplacental insufficiency causes intrauterine growth restriction (IUGR) and decreases plasma levels of the branched-chain amino acids in both humans and rats. Increased fetal oxidation of these amino acids may contribute to their decline in the IUGR fetus. The rate-limiting step of branched-chain amino acid oxidation is performed by the mitochondrial enzyme branched-chain alpha-keto acid dehydrogenase (BCKAD), which is regulated by a deactivating kinase. We therefore hypothesized that uteroplacental insufficiency increases BCKAD activity through altered mRNA and protein levels of BCKAD and/or the BCKAD kinase. In IUGR fetal liver, BCKAD activity was increased 3-fold, though no difference in hepatic BCKAD protein or mRNA levels were noted. Hepatic BCKAD kinase mRNA and protein levels were significantly decreased in association with the increase in BCKAD activity. In IUGR fetal skeletal muscle, BCKAD mRNA levels were significantly increased. IUGR skeletal muscle BCKAD protein levels as well as BCKAD kinase mRNA and protein levels were unchanged. We also quantified mRNA levels of two amino acid transporters: LAT1 (system L) and rBAT (cysteine and dibasic amino acids). Both hepatic and muscle LAT1 mRNA levels were significantly increased in the IUGR fetus. We conclude that uteroplacental insufficiency significantly increases hepatic BCKAD activity in association with significantly decreased mRNA and protein levels of the deactivating kinase. We speculate that these changes contribute to the decreased serum levels of branched-chain amino acids seen in the IUGR fetus and may be an adaptation to the deprived milieu associated with uteroplacental insufficiency.     

谷氨酰胺对内毒素血症幼年大鼠小肠上皮细胞凋亡的作用及其机制探讨

目的：通过内毒素血症幼年大鼠小肠Bcl-2及BaxmRNA表达的变化,观察谷氨酰胺对小肠细胞凋亡的作用,探讨谷氨酰胺对内毒素血症幼年大鼠小肠的保护机制。方法：用腹腔注射内毒素(4mg/kg大肠杆菌脂多糖O55B5)方法制备18日龄大鼠内毒素血症模型(内毒素组);腹腔注射同等量生理盐水为对照组;腹腔注射内毒素同时注射N(2)-L-丙氨酰-L-谷氨酰胺(2g/kg)为谷氨酰胺干预组。分别在注射后2,4,6,24,72h取全部回肠,半定量RT-PCR法测Bcl-2,BaxmRNA的表达。结果：正常对照和内毒素组小肠各时间点的Bcl-2mRNA均无表达,谷氨酰胺组各时间点的Bcl-2mRNA表达增强。正常对照BaxmRNA表达较弱,内毒素组小肠各时间点的BaxmRNA表达均明显增强,而谷氨酰胺组2h亚组的表达较正常明显减弱,其余各时间点的BaxmRNA表达接近对照组。内毒素组Bax,Bcl-2mRNA表达的比值明显高于对照组,谷氨酰胺组Bax,Bcl-2mRNA表达的比值明显低于对照组和内毒素组,其中以2h亚组的变化最显著。结论：谷氨酰胺使内毒素血症小肠Bcl-2基因表达增强,明显高于正常;Bax基因表达减弱至接近正常;Bax,Bcl-2mRNA表达的比值明显下降,从而抑制细胞凋亡,维持肠屏障结构的完整,以对抗内毒素对肠黏膜的损害。     

Uteroplacental insufficiency alters cerebral mitochondrial gene expression and DNA in fetal and juvenile rats

Uteroplacental insufficiency increases the risk of perinatal and long-term neurologic morbidity by depriving the fetus of oxidative substrate and causing intrauterine growth retardation. Skeletal muscle and liver from growth retarded fetal and juvenile rats respond to this deprivation by altering mitochondrial gene expression and function. The objective of this study was to determine whether cerebral mitochondrial mRNA is similarly altered in fetal and juvenile growth retarded rats and to correlate these alterations with mitochondrial DNA and marker protein levels. To fulfill this objective, mRNA levels of four important mitochondrial proteins were quantified using RT-PCR in growth retarded and sham-operated control fetal and juvenile rat brains; these proteins were NADH-ubiquinone oxireductase subunit 4, subunit C of the F1F0-ATPase, and the adenine nucleotide transporters 1 and 2. Mitochondrial DNA/nuclear DNA ratios and mitochondrial 60 kD marker protein levels were also quantified in growth retarded and sham-operated control fetal and juvenile rat brains using PCR and Western Blotting, respectively. Cerebral mRNA levels of all four proteins were increased in the IUGR fetuses and decreased in the IUGR juvenile animals. Cerebral mitochondrial/nuclear DNA ratios and mitochondrial marker protein levels were not significantly altered in the IUGR fetuses; however, both were significantly diminished in IUGR juvenile pups. These studies suggest that the metabolic stresses associated with uteroplacental insufficiency in the rat cause altered fetal and postnatal cerebral mitochondrial mRNA and DNA levels.     

Mucosal expression of p21, p27, p53, Bcl-2, and bax after small bowel resection and autotransplantation in pigs

Massive small bowel resection increases ileal villus height as part of normal adaptation. However, despite no gut loss, autotransplantation of the entire small intestine also increases ileal villus height. Our aim was to test whether similar modulation of enterocyte proliferation and apoptosis underpin these comparable increases in villus height. Fifteen pigs were randomly assigned for laparotomy (n=5), 75% proximal small bowel resection (n=5), or jejunoileal autotransplantation (n=5). Eight weeks postoperatively, full-thickness small bowel sections underwent routine immunohistochemistry for cell cycle inhibitors (p53, p21, and p27), antiapoptotic Bcl-2, and proapoptotic bax. The specimens were analyzed semiquantitatively, and the number of intensively positive epithelial cells for each group was compared from 20 digital images (0.32 mm²/image). Compared with laparotomy, small bowel resection decreased the number of p27-positive enterocytes in both jejunum and ileum, increased the number of bax-expressing cells in ileum, but decreased the number of bax-expressing cells in jejunum. In contrast, compared with laparotomy, jejunoileal autotransplantation altered neither mucosal bax nor p27 expression. In all groups, Bcl-2 expression was similarly confined to inflammatory cells of the lamina propria, while both p53 and p21 were negative. We conclude that long-term alterations in the enterocytic expression of certain cell cycle and apoptosis markers (p27 and bax) accompany small bowel resection. These changes differ between the jejunum and the ileum and are not seen after whole small bowel autotransplantation. Therefore, increased ileal villus height after autotransplantation, despite resembling postresectional intestinal adaptation, is underpinned by different regulation of enterocyte proliferation and apoptosis.     

Underexpression of neural cell adhesion molecule and neurotrophic factors in rat brain following thromboxane A(2)-induced intrauterine growth retardation

Intrauterine growth retardation (IUGR) often results in clinical neurodevelopmental disorders. To clarify the influence of uteroplacental insufficiency on central nervous system development, we have created a model of IUGR in rats using maternal administration of synthetic thromboxane A(2). We investigated expression patterns of neural cell adhesion molecule (NCAM) and reelin in this model by semiquantitative competitive polymerase chain reactions. On postnatal day 2, NCAM expression was decreased in rat cerebral cortex, and reelin expression was decreased in hippocampus from levels in controls without maternal thromboxane exposure. No significant differences in NCAM expression were seen in hippocampus, nor did reelin expression differ in cerebral cortex between control and IUGR groups. We also examined expression of two neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). In cerebral cortex the IUGR group showed less BDNF and NT-3 expression than controls. Delay of neuronal migration and histological changes observed in our IUGR rats may be related to altered expression of these molecules.     

Intrauterine growth retardation: fetal glucose transport is diminished in lung but spared in brain.

"Uteroplacental insufficiency" often causes asymmetric fetal growth retardation. Glucose transporters control cell glucose utilization and thus may be critical in the control of fetal growth. We hypothesized that uteroplacental insufficiency might alter glucose transporter activity, protein, and gene expression and thereby affect discordant organ growth in small-for-gestational-age (SGA) fetuses. We performed bilateral uterine artery ligation in pregnant rats on d 19 of gestation (term-21.5 d) to cause uteroplacental insufficiency and obtained fetal brain and lung tissue on d 20. The brain mass of SGA fetuses did not differ from that of sham and normal fetuses, but lung mass was significantly diminished. Glucose transport, measured with [3H]2-deoxyglucose, was similar in glial cells and brain tissue of SGA, sham, and normal fetuses. In contrast, type II pneumocytes, lung fibroblasts, and lung tissue of SGA fetuses had significantly decreased glucose transport. The intrinsic activity of the glucose transporter (Km) was not altered in the brain or lung of SGA fetuses. Total glucose transporter protein measured by cytochalasin-B binding and glucose transporter 1 mRNA was diminished in SGA lung tissue and type II pneumocytes, but not in SGA brain tissue or glial cells. We could not detect glucose transporter 3 mRNA in significant quantity in any tissue. With uteroplacental insufficiency, glucose transport is differentially altered in lung and brain. Glucose transporter protein and gene expression are diminished in the lung and normal in the brain of SGA fetuses. These changes may contribute to fetal growth retardation and the phenomenon of "brain sparing."     

Effect of maternal oxygen inhalation on the fetus with growth retardation

Decreased nutrient and oxygen transfer to the fetus accounts for fetal growth retardation in pregnancies complicated by severe uteroplacental insufficiency. A model of uteroplacental insufficiency was produced by ligation of the uterine artery of one horn in pregnant rats at 17 days of gestation. The pregnant rats were assigned to environmental chambers containing a gas mixture of either an increased fractional inspired oxygen concentration of 0.40 (O2) or room air from day 17 through 21 of gestation. Supplemental oxygen inhalation resulted in increased survival of the fetuses from the ligated horn [34 +/- 6% in intrauterine growth retardation (IUGR)-room air versus 57 +/- 8% in IUGR-O2] and an increase in fetal weight, expressed as a percentage of nonligated appropriate for gestational age control littermates (67 +/- 2% in IUGR-room air versus 74 +/- 2% in IUGR-O2). A role for hypoxia in the suppression of fatty acid synthesis in IUGR fetal tissues had been postulated; however, maternal oxygen inhalation did not result in any increase in fatty acid content or specific activity in liver, lung, or carcass of IUGR fetuses. These data indicate that supplemental maternal oxygen inhalation improves survival and growth of fetuses compromised by uteroplacental insufficiency, but it has no apparent effect on lipogenesis at term.     

Altered placental and fetal expression of IGFs and IGF-binding proteins associated with intrauterine growth restriction in fetal sheep during early and mid-pregnancy

The insulin-like growth factors (IGFs) are postulated to be altered in association with the development of intrauterine growth restriction (IUGR). The present studies examined placental and fetal hepatic mRNA concentration of components of the IGF system at two time points (55 and 90 d gestational age, dGA; Term 147 dGA) in a hyperthermia (HT)-induced sheep model of placental insufficiency-IUGR. Maternal plasma insulin and IGF-I were constant at 55 and 90 dGA and were unaffected by treatment. Umbilical vein insulin concentrations tended to be reduced at 90 dGA following HT exposure. Caruncle IGF-I mRNA was increased at 90 dGA in HT placentae (p < 0.05), while cotyledon concentrations were constant over gestation and unaltered by treatment. In control cotyledons, IGF-II mRNA concentration increased (p < 0.01) and IGFBP-3 decreased between 55 and 90 dGA (p < 0.01). Cotyledon IGF-II and caruncle IGFBP-4 mRNA were elevated at 55 dGA in HT placentae compared with control (p < 0.01 and p < 0.05 respectively). Fetal hepatic IGF-I, IGFBP-2, -3 and -4 concentrations rose over gestation (p < 0.05), but there were no treatment effects. These data suggest that changes in placental IGF expression in early and mid gestation may predispose the pregnancy to placental insufficiency, resulting in inadequate substrate supply to the developing fetus later in gestation.     

Bax与p53、Bcl-2、PCNA在不同类型神经母细胞瘤中的表达及意义

目的探讨Bax与p53、Bcl-2、PCNA在不同神经母细胞瘤（组织结构不良型,UFH;组织结构良好型,FH）中的表达及意义。方法收集近6年来上海儿童医学中心保存的儿童神经母细胞瘤石蜡标本33例（UFH型10例,FH型23例;男17例,女16例;年龄1个月至15岁,平均年龄33．1个月）。利用组织芯片的高通量、低误差、经济省时等优点,将两组标本制作成组织芯片,再行免疫组化SP法检测Bax与p53、Bcl-2、PCNA在神经母细胞瘤中的表达。并采用荧光原位杂交法（FISH）检测神经母细胞瘤中p53、Bcl-2基因的表达。采用SAS统计软件对结果进行统计学处理,两样本比较采用Wilcoxon秩和检验。结果两组神经母细胞瘤（UFH型和FH型）中Bax与p53均呈低表达,差异无统计学意义（P〉0．05）;而Bcl-2和PCNA在两组肿瘤中表达均较高,差异无统计学意义（P〉0．05）。p53、Bcl-2基因检测结果与免疫组化染色结果一致。结论UFH型和FH型神经母细胞瘤在表征细胞增殖和凋亡的4个指标（Bax与p53、Bcl-2、PCNA）检测中显示小完全的一致性,表明不同组织结构类型对神经母细胞瘤的细胞增殖和凋亡无显著影响。在两组神经母细胞瘤中Bcl-2的高表达与Bax的低表达呈负相关,说明Bcl—2可能参与了儿童神经母细胞瘤的发生,而Bax可能对肿瘤的发生起抑制作用。两组神经母细胞瘤中p53基因发生突变的比例较低,而细胞增殖功能均较旺盛。     

Intrauterine growth restriction modifies the normal gene expression in kidney from rabbit fetuses

The aim of this work was to study the effect of intrauterine growth restriction (IUGR) on fetal kidneys. The IUGR was induced by uteroplacental vessels ligature in a model of pregnant rabbit. We centralized the study in the gene expression of essential proteins for fetal kidney development and kidney protection against hypoxia, osmotic stress, and kidney injury. The gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM were studied by qRT-PCR and Western blot in kidneys from control and IUGR fetuses. Experimental IUGR fetuses were significantly smaller than the control animals (39 vs. 48 g, p < 0.05). The number of glomeruli was decreased in IUGR kidneys, without morphological alterations. IUGR increased the gene expression of HIF-1α, NFAT5, IL-1β, NGAL, and ATM (p < 0.05) in kidneys of fetuses undergoing IUGR, suggesting that fetal blood flow restriction produce alterations in gene expression in fetal kidneys.     

高体积分数氧致慢性肺疾病早产鼠肺上皮细胞凋亡及其调控机制

目的 探讨高体积分数氧致慢性肺疾病(CLD)早产鼠肺泡上皮细胞(AEC)凋亡的动态变化规律及Caspase-3 mRNA、Bax和Bcl-2基因的调控作用.方法 将60只早产SD鼠生后1 d内随机分为高体积分数氧暴露组(高氧组)和空气组(对照组)各30只,制备高体积分数氧致CLD早产鼠模型,应用脱氧核苷酸末端转移酶介导的原位缺口末端标记(TUNEL)、反转录-聚合酶链反应(RT-PCR)技术及链霉亲和素-过氧化物酶复合物(SABC)免疫组织化学方法,检测生后1、3、7、14及21 d AEC的凋亡指数(AI)和肺组织Caspase-3 mRNA、Bax及Bcl-2基因蛋白的表达.相关分析采用Spearman分析.结果 与对照组比较,高氧组3 d AEC的AI、肺组织Caspase-3 mRNA水平及Bax基因蛋白表达开始升高,7～21 d明显增加,Caspase-3 mRNA水平在此期间持续于高水平;Bcl-2基因表达7 d开始降低,14～21 d明显降低.高氧组AEC的AI与肺组织Caspase-3 mRNA及Bax表达均呈正相关,与Bcl-2表达呈负相关.结论 高体积分数氧可致CLD早产鼠肺组织Caspase-3 mRNA基因表达增强,使Bax和Bcl-2介导Bax/Bcl-2基因表达比例失衡,是CLD早产鼠肺上皮细胞凋亡的重要机制之一.     

Altered expression and function of mitochondrial beta-oxidation enzymes in juvenile intrauterine-growth-retarded rat skeletal muscle.

Uteroplacental insufficiency and subsequent intrauterine growth retardation (IUGR) affects postnatal metabolism. In juvenile rats, IUGR alters skeletal muscle mitochondrial gene expression and reduces mitochondrial NAD(+)/NADH ratios, both of which affect beta-oxidation flux. We therefore hypothesized that gene expression and function of mitochondrial beta-oxidation enzymes would be altered in juvenile IUGR skeletal muscle. To test this hypothesis, mRNA levels of five key mitochondrial enzymes (carnitine palmitoyltransferase I, trifunctional protein of beta-oxidation, uncoupling protein-3, isocitrate dehydrogenase, and mitochondrial malate dehydrogenase) and intramuscular triglycerides were quantified in 21-d-old (preweaning) IUGR and control rat skeletal muscle. In isolated skeletal muscle mitochondria, enzyme function of the trifunctional protein of beta-oxidation and isocitrate dehydrogenase were measured because both enzymes compete for mitochondrial NAD(+). Carnitine palmitoyltransferase I, the trifunctional protein of beta-oxidation, and uncoupling protein 3 mRNA levels were significantly increased in IUGR skeletal muscle, whereas mRNA levels of isocitrate dehydrogenase and mitochondrial malate dehydrogenase were unchanged. Similarly, trifunctional protein of beta-oxidation activity was increased in IUGR skeletal muscle mitochondria, and isocitrate dehydrogenase activity was unchanged. Interestingly, skeletal muscle triglycerides were significantly increased in IUGR skeletal muscle. We conclude that uteroplacental insufficiency alters IUGR skeletal muscle mitochondrial lipid metabolism, and we speculate that the changes observed in this study play a role in the long-term morbidity associated with IUGR.     

高氧对新生大鼠肺caspase-3和p53基因表达及肺细胞凋亡的影响

目的探讨高氧对新生大鼠肺caspase3和p53基因表达及肺细胞凋亡的影响。方法采用SpraqueDawley新生大鼠95%氧气暴露建立高氧肺损伤模型。应用RTPCR技术检测肺组织caspase3mRNA和p53mRNA水平,凝胶电泳条带用成像系统照相分析结果。计算目的基因PCR产物条带与内参照βactin条带光密度值的比值,作为p53基因的相对表达量,结果以x±s标记,而caspase3mRNA表达量则以阳性表达或阴性表达为标记。应用脱氧核糖核酸转移酶介导的细胞凋亡标记技术(TUNEL)原位检测细胞凋亡。光镜下随机计算5个视野中500个肺细胞中的阳性细胞数,结果以x±s标记。结果新生大鼠暴露于95%氧浓度环境中24h后肺组织中p53mRNA表达中度增加(q=3.2305,P>0.05),48h后表达显著增加,与空气对照组相比差异有统计学意义(q=7.2941,P<0.05)。新生鼠高氧处理72h、96h后,肺组织p53mRNA表达又恢复到正常水平。在各空气对照组和各高氧处理组中个别新生鼠肺的caspase3mRNA有微量表达,绝大多数新生鼠肺的caspase3mRNA没有表达,差异无统计学意义。95%氧暴露7天的新生鼠肺细胞凋亡水平明显高于空气暴露组新生鼠肺细胞凋亡水平,两者比较差异有极显著的统计学意义(F=100,P<0.001)。结论在高浓度供氧下,肺组织通过暂时上调p53基因的表达,介导细胞周期停滞,阻止G0/G1期细胞进入S期,抑制细胞分裂、增殖,同时p53促进细胞凋亡,从而导致肺生长发育受阻和肺损伤。新生鼠暴露于95%氧环境中,肺组织caspase3基因基本上不表达,因此推测高氧肺细胞凋亡可能存在不经过caspase3的凋亡途径。     

Changes in the expression of hypothalamic lipid sensing genes in rat model of intrauterine growth retardation (IUGR)

Intrauterine growth retardation (IUGR) has been linked to the development of type 2 diabetes in later life. The mechanisms underlying this phenomenon are unknown. Recent data suggest that some of the molecular defects underlying type 2 diabetes reside in the CNS. The enzyme carnitine palmitoyltransferase-1 (CPT1) regulates long-chain fatty acid (LCFA) entry into mitochondria, where LCFA undergo beta-oxidation. Hypothalamic inhibition of CPT1 decreases food intake and suppresses endogenous glucose production. Our aim was to investigate the effects of uterine artery ligation, a procedure that mimics uteroplacental insufficiency, on the CNS expression of CPT1 and other key enzymes of LCFA metabolism. Bilateral uterine artery ligation was performed on d 19 of gestation in the pregnant rat; sham-operated pregnant rats served as controls. Hypothalamus, cerebellum, hippocampus, and cortex were dissected and analyzed at birth by real-time PCR. Nonesterified fatty acid (NEFA) serum levels were significantly higher in IUGR pups (p<0.0001). In IUGR rats, the hypothalamic expression of CPT1 isoform C (p=0.005) and acetyl-CoA carboxylase (ACC) isoforms alpha (p<0.05) and beta (p=0.005) were significantly decreased. The data presented here support the hypothesis that an abnormal intrauterine milieu can induce changes in hypothalamic lipid sensing.     

Uteroplacental insufficiency affects kidney VEGF expression in a model of IUGR with compensatory glomerular hypertrophy and hypertension

Low nephron endowment secondary to intrauterine growth restriction (IUGR) results in compensatory hypertrophy of the remaining glomeruli, which in turn is associated with hypertension. However, gender differences exist in the response of the kidney to injury, and IUGR female offspring seems protected from an unfavorable outcome. We previously reported differences in gender-specific gene expression in the IUGR kidney as well as increased circulating corticosterone levels following uteroplacental insufficiency (UPI). Vascular endothelial growth factor (VEGF), which is critical for renal development, is an important candidate in the IUGR kidney since its expression can be regulated by sex-steroids and glucocorticoids. We hypothesize that IUGR leads to altered kidney VEGF expression in a gender-specific manner. Following uterine ligation in the pregnant rat, UPI decreases renal VEGF levels in male and female IUGR animals at birth and through postnatal day 21. However, by day 120 of life, IUGR females have increased kidney VEGF expression, not present in the IUGR males. In addition, IUGR males exhibit increased serum testosterone levels as well as proteinuria. These findings are intriguing in light of the difference in glomerular hypertrophy observed: IUGR males show increased glomerular area when compared to IUGR females. In this model characterized by decreased nephron number and adult onset hypertension, UPI decreases renal VEGF expression during nephrogenesis. Our most intriguing finding is the increased renal VEGF levels in adult IUGR females, associated with a more benign phenotype. We suggest that the mechanisms underlying renal disease in response to IUGR are most likely regulated in a gender specific manner.     

Placental LPL gene expression is increased in severe intrauterine growth-restricted pregnancies

Intrauterine growth restriction (IUGR) is associated with reduced placental supply of nutrients to the fetus. Lipoprotein lipase (LPL) mediates the hydrolysis of triglycerides from maternal lipoproteins to obtain fatty acids. Here, we tested the hypothesis that placental LPL gene expression level is altered in pregnancies complicated by IUGR. To this purpose, 28 IUGR fetuses were identified during pregnancy and divided in two groups: 7 M-IUGR ["mild" IUGR, with normal umbilical artery pulsatility index (PI)] and 21 S-IUGR ("severe" IUGR, with abnormal PI). Moreover, 10 out of 28 IUGR pregnancies were associated with preeclampsia. Controls were 19 normal pregnancies delivering appropriate for gestational age (AGA) fetuses. Relative real-time quantification of LPL was carried out in RNA from placental chorionic villi by the DeltaDeltaCt method, using beta-actin as normalizing gene. Placental LPL mRNA expression levels were significantly higher in IUGR than in AGA. In particular, significantly higher values were observed in S-IUGR, independent from the concomitant association with preeclampsia. No significant relationship was observed between placental LPL mRNA expression levels or gestational age. In conclusion, placental LPL mRNA gene expression is increased in severe IUGR, characterized by enhanced vascular placental resistances and alterations of placental nutrient transport.     

Study of genetic expression of intrauterine growth factors IGF-I and EGFR in placental tissue from pregnancies with intrauterine growth retardation

To investigate the possibility of altered gene expression of growth factors in prenatal growth retardation, we assessed expression of the genes for insulin-like growth factor-I (IGF-I) and epidermal growth factor receptor (EGFR) by RT-PCR from human placentas at term delivery in two groups: appropriate for gestational age (AGA) and pregnancies complicated with IUGR. The placentas from IUGR gestations showed reduced IGF-I expression with a significance of p = 0.008, whereas we did not find any significant differences in EGFR gene expression.