妊娠糖尿病之IL-10甲基化研究

DNA methylation change of IL-10 gene in Gestational Diabetes Mellitus

許凱涵(Kai-Han Hsu)

指導教授 ： 李建南 ； 林芯伃

國立臺灣大學/醫學院/分子醫學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201704108

摘要

妊娠糖尿病在全國發生率約為3-9 %。對妊娠糖尿病而言，會提高2-4倍妊娠期高血壓疾病，妊娠糖尿病產前羊水過多，發生率較非妊娠糖尿病孕婦多10倍、子癲癇症、產下巨嬰、肩難產等的發生率也顯著增高。對於長期待在高血糖環境下的胎兒而言，較容易發生巨嬰症、新生兒死亡、新生兒呼吸窘迫症候群、新生兒低血糖症或低血鈣症等。若沒有持續追蹤，妊娠糖尿病患者35-60 %的機率在未來十到二十年內容易發展成糖尿病。
妊娠糖尿病是產科常見的併發症，他的病因除了由遺傳造成之外，種族、經濟文化、飲食習慣、糖尿病家族史等等都可能是其他要素，真正導致妊娠糖尿病的因子至今並不清楚，目前認為表觀遺傳學中DNA甲基化的改變程度對於胎兒長期暴露在高血糖的環境下和長大後容易造成肥胖、糖尿病、心血管疾病甚至是癌症之間有相關聯。
造成妊娠糖尿病的主因是因為身體對胰島素產生抵抗作用導致血糖無法分解給與身體吸收而產生高血糖的現象。文獻指出造成胰島素產生抵抗作用是因為胎盤產生的賀爾蒙和細胞激素相互作用的關係。其中一項影響的細胞激素稱為Interleukin-10 (IL10)，IL-10是免疫調節和抗發炎細胞激素。根據動物實驗顯示在骨骼肌肉中IL-10 Signaling和調節肥胖的發炎反應及胰島素拮抗作用有相關。在人體實驗身上，針對第二型妊娠糖尿病患者抽取血液做IL-10分析，發現第二型糖尿病患者的IL-10濃度皆明顯升高，因而我們了解到IL-10細胞激素的濃度在高血糖上扮演重要的角色之外，根據已經發表的研究到DNA甲基化的程度和母親及胎兒之間有相互關聯，因此，我們假設妊娠糖尿病患者之產前母血、臍帶血、胎盤組織中，其IL-10 DNA甲基化程度與非妊娠糖尿病患者有所差異。
本實驗分為兩個部分，第一部份為妊娠糖尿病產前母血、臍帶血、胎盤組織中妊娠糖尿病和IL-10甲基化的關係，實驗結果發現8位妊娠糖尿病產前母血中IL-10甲基化平均值比24位非妊娠糖尿病的甲基化程度低(兩組IL-10甲基化平均值分別0.23和0.26，p=0.036)。9位妊娠糖尿病臍帶血、胎盤組織之IL-10甲基化平均值比和27位非妊娠糖尿病的甲基化程度沒有差異(臍帶血、胎盤組織IL-10甲基化平均值分別0.30和0.33、0.51和0.5，p=0.314、0.350)。另外，針對產前母血做IL-10酵素免疫分析，顯示產前母血妊娠糖尿病IL-10濃度比非妊娠糖尿病高。(兩組間IL-10蛋白質濃度為8.57 pg / ml和3.08 pg /m L，p=0.002)。
實驗第二部分為24-28週妊娠糖尿病和非妊娠糖尿病IL-10甲基化程度，結果發現17位妊娠糖尿病IL-10甲基化平均值比39位非妊娠糖尿病的甲基化程度低(兩組間IL-10甲基化平均值分別為0.25和0.29，p=0.017)。24-28週母血IL-10酵素免疫分析，顯示24-28週母血妊娠糖尿病IL-10濃度和非妊娠糖尿病沒有顯著差異。(兩組間IL-10蛋白質濃度為7.97 pg / ml和7.45 pg /m L，p=0.07)。最後，利用THP-1細胞培養，隨著不同時間下，我們將THP-1細胞培養分為高葡萄糖與低葡萄糖環境做IL-10濃度比較，結果顯示在高濃度葡萄糖下的THP-1細胞其IL-10濃度在30分鐘、2、6、24小時時的IL-10濃度比在低濃度葡萄糖下的THP-1細胞要來的高。(其值為15.4 pg/ml和4.0 pg/ml、16.5 pg/ml和5.0 pg/ml、15.2 pg/ml和7.5 pg/ml、17.1 pg/ml和9.2 pg/ml，p值分別為0.0008、0.018、0.001、0.005)。
此實驗顯示，妊娠糖尿病孕婦血清呈現IL-10甲基化低下，預測DNA甲基化異常可能與妊娠糖尿病的原因相關，而由ELISA分析和細胞實驗也證實IL-10濃度會在妊娠糖尿病患者身上比非妊娠糖尿病中高，值得日後進一步探討其因果關係。

關鍵字

妊娠糖尿病 ； 表觀遺傳學 ； IL10甲基化

並列摘要

In general, the national incidence of gestational diabetes mellitus (GDM) is about 3-9%. It is associated with a much higher incidence of perinatal comorbidities and complications such as gestational hypertension、pre-eclampsia、macrosomia、shoulder dystocia, etc. Babies born under hyperglycemic environment are more prone to fetal macrosomia, and are also more likely to suffer from perinatal mortality, respiratory distress syndrome, hypoglycemia or hypocalcemia, among others. Without proper monitoring and treatment, those diagnosed with gestational diabetes mellitus have a 35-60% chance of developing into full-fledged diabetic patients in the coming 10 to 20 years.
Gestational diabetes mellitus is a common complication in obstetrics. In addition to genetic inheritance, it is associated with ethnicity, dietary preference, and family history. However, the etiologies and pathogenesis of gestational diabetes mellitus are not yet fully understood. Currently, epigenetic regulation, more specifically, the degree of DNA methylation, is thought to be the missing link between the babies with extended prenatal hyperglycemic exposure and future obesity, diabetes, cardiovascular disease and even cancer.
Gestational diabetes mellitus is defined by the body’s impaired ability to respond to the hormone insulin, with its onset or first recognition during pregnancy. Insulin resistance results in abnormal metabolism of carbohydrates and elevated levels of serum glucose. It has been established that the interaction between placental hormones and cytokines plays a role in insulin resistance. One of the cytokines involved is Interleukin-10 (IL-10), which is an immune regulatory and anti-inflammatory cytokine. The results of various animal studies showed that IL-10 signaling in skeletal muscles cells is involved in regulation of obesity-mediated inflammation and insulin resistance. In human studies, it has been documented that the concentration of serum IL-10 was significantly higher in patients with gestational diabetes mellitus. This suggests a relationship between the concentration of IL-10 cytokine and hyperglycemia. Furthermore, the extent of DNA methylation between the fetus and the mother is interrelated. Therefore, our study aimed to investigate the difference in IL10 gene methylation in maternal GDM blood, umbilical cord blood, and placenta tissue. We hypothesize that DNA methylation in IL-10 gene may contribute to the development of gestational diabetes mellitus.
Our study comes in two parts. The first part is to establish the methylation of IL-10 gene in maternal blood、umbilical cord blood、placenta tissue, and their association with GDM. Our results showed that the IL-10 gene methylation degree in maternal blood before delivery was significant lower in the GDM (n=8) group compared to the Non-GDM (n=24) group (p= 0.03). The IL-10 gene methylation degree in placenta tissue and umbilical cord blood were not significant different between the GDM (n=9) group and the Non-GDM (n=27) group (p=0.31, p=0.35 respectively). In addition, higher level of IL-10 cytokine concentration in the maternal plasma was confirmed in the GDM group.
The second part of our study established the variation in methylation of IL-10 gene in maternal blood of pregnant women at 24-28 weeks of gestational age. The IL-10 gene methylation degree in the maternal blood of GDM was significantly lower than that of Non-GDM group (p=0.017). The total concentration of IL-10 cytokine in maternal blood was not significant different between the GDM (n=17) group and the Non-GDM (n=39) group (p=0.07). Furthermore, in vitro study of GDM, THP-1 cells also showed a significant higher total IL-10 protein expression in the hyperglycemic environment (P < 0.05).
Our experiments showed that there is hypo-methylation of IL10 gene in maternal blood of women with gestational diabetes. This finding sheds some light on the pathogenesis of gestation diabetes mellitus, suggesting that GDM is associated with aberrant DNA methylation. Furthermore, the total concentration of IL-10 cytokine in gestational diabetes mellitus patients is higher, compared to that in the normal group. The relationship between IL-10 and gestational diabetes mellitus is worthy of further investigation.

並列關鍵字

Gestational diabetes mellitus ； epigenetics ； IL10 methylation

參考文獻

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