精子中DNA甲基化修饰研究进展

DNA甲基化是表观遗传学的重要内容之一,精子在发生过程中经历了甲基化和去甲基化过程,精子DNA甲基化修饰的稳定性依赖于鱼精蛋白,不育男性精液质量的降低与精子基因组甲基化水平改变有关,但目前大家研究基因位点多而杂,这些位点甲基化模式的改变是否有相互联系尚不清楚,研究热点集中在某一些生精相关的印记基因的甲基化水平改变比较多,但其机制仍然模糊,精子DNA甲基化修饰改变是否影响辅助生殖结果目前也有争论,各种理化环境因素对精子中DNA甲基化修饰影响已被越来越多的研究者发现,但具体机制有待进一步研究。     

DNA甲基化对男性精液质量影响的研究进展

DNA甲基化是表观遗传学修饰的主要形式之一,对个体发育具有至关重要的作用.近年来,大量研究表明不育男性的精子中存在不同基因异常甲基化.本文主要阐述了印记基因(H19、FAM50B、GNAS、MEST、KCNQ1OT1、SNRPN)和其他相关基因(P16、LINE-1、MTHFR、SLC9B1、DDR1)异常甲基化会对精液质量(包括精子数目、活力和形态)产生不同程度的影响,从而导致男性不育.因此,检测精子特定基因的DNA甲基化程度有可能成为针对男性不育的新型辅助诊断手段.     

DNA异常甲基化在卵巢癌中的研究进展

卵巢癌是死亡率最高的妇科恶性肿瘤,严重威胁着女性的生命健康,其发生与多种分子变化有关。表观遗传学改变与人类多种肿瘤有着密切的关系,而肿瘤抑制基因的DNA甲基化则是卵巢癌中常见的表观遗传学现象,与卵巢癌的发生、发展有着密切的联系。DNA甲基化属于基因表观遗传调控的重要方式之一。新的研究发现,DNA异常甲基化,尤其是基因启动子CpG岛高甲基化,使肿瘤抑制基因——乳腺癌易感基因1(BRCA1)、Ras相关区域1A基因(RASSF1A)等失活,在卵巢癌发生、发展中起重要作用。综述DNA异常甲基化在卵巢癌中的研究进展,可为卵巢癌发病机制、早期诊断、治疗及判断预后提供新的方法。     

表观遗传学与卵巢癌耐药的关系研究

卵巢癌在妇科恶性肿瘤中致死率最高,目前其临床治疗手段主要是手术切除和辅助性化疗,但治疗后其易复发、易转移和易耐药的特性,使得很多抗肿瘤药对其作用仅限于初期治疗。研究表明肿瘤的发生、发展及其耐药机制与表观遗传学关系密切,而相关研究也证实卵巢癌的发生发展与耐药受表观遗传学的调控,如DNA甲基化,包括Ras相关区域1A基因(RASSF1A)和乳腺癌易感基因1(BRCA1)异常甲基化;组蛋白修饰,包括甲基化、乙酰化、磷酸化、泛素化等多种方式;微小RNA(mi RNA)调控相关基因等。同时研究表明通过调控表观遗传学如DNA甲基转移酶抑制剂、组蛋白去乙酰化酶抑制剂等与传统抗肿瘤药物进行联合治疗能够显著提高卵巢癌的疗效,因此研究表观遗传学与卵巢癌的耐药机制有望为临床治疗卵巢癌提供新的思路与研究基础。     

血清DNA定量及基因甲基化状态检测在卵巢上皮性癌诊断中的应用

卵巢恶性肿瘤是病死率最高的女性生殖系统肿瘤,其中90%为卵巢上皮性癌(卵巢癌),而70%的患者诊断时已达Ⅲ期或Ⅳ期,5年生存率仅为15%～20%[1].因此发现新的既敏感又特异的肿瘤标志物,对卵巢癌的早期诊断、提高生存率具有重要意义.近年的研究发现,作为表观遗传的主要方式之一,DNA异常甲基化是肿瘤发生的早期事件,可早于基因及其表达产物的改变[2].而且,肿瘤患者血清DNA含量明显升高,虽然其来源尚未十分明确,但其具有与肿瘤DNA类似的遗传学和表观遗传学改变[3].因此,血清DNA的异常甲基化用于肿瘤的早期诊断具有明显的优势和极大的潜力.本研究通过定量检测卵巢癌患者血清DNA含量,以及基因EGFLAM、CDKN2A和LSM2的启动子区胞嘧啶-磷酸-鸟嘌呤二核苷酸岛(CpG island,CGI)甲基化状态,探索新的可用于辅助卵巢癌早期诊断的血清DNA甲基化标志物.     

HPV相关宫颈癌DNA甲基化的研究进展

宫颈癌是最常见的妇科肿瘤之一，高危型人乳头瘤病毒(hrHPV)持续感染是宫颈恶性肿瘤的重要因素。表观遗传学是指非基因序列改变导致患者基因表达水平改变的一种可遗传现象，其改变形式包括DNA甲基化、染色体失活、基因组印迹、非编码RNA调控等。研究表明，表观遗传修饰影响细胞的生长、分化、凋亡、转化等与肿瘤发生发展相关的生物进程。其中，DNA甲基化是目前研究最清楚、最重要的表观遗传修饰形式。本文重点关注HPV相关宫颈癌中DNA甲基化的改变，对其致癌机制、临床应用的研究进展作一综述，以更好地了解HPV相关宫颈癌的发生发展过程，有助于预防与治疗宫颈癌。     

超促排卵和体外成熟人卵子中印迹基因H19和PEG1的甲基化状态

目的:探讨超促排卵(controlled ovarian hyperstimulation,COH)和未成熟卵母细胞体外培养(in vitro maturation,IVM)是否引起印迹基因甲基化异常。方法:采用单细胞低熔点琼脂糖包埋亚硫酸氢钠处理、半巢式PCR扩增及克隆测序的方法,检测人单个卵子印迹基因H19和PEG1的甲基化状态。结果:共检测分析了94个(58个来自COH,36个来自IVM)处于不同成熟阶段的卵子中H19基因和PEG1(MEST)基因的甲基化状态,分别有6个COH卵子和2个IVM卵子出现H19或PEG1的1～2个CpG位点甲基化异常。绝大多数COH卵子的H19(91.4%,53/58)和PEG1(91.7%,33/36)的所有检测CpG位点甲基化正常,H19和PEG1所有CpG位点的甲基化率分别为0.8%(8/1044)和99.4%(859/864)。结论:经超促排卵和体外成熟培养卵子的印迹基因H19和PEG1甲基化状态是正常的,1～2个CpG位点甲基化异常的卵子是否导致胚胎的基因印迹异常需进一步研究。     

宫颈癌中表观遗传学的研究进展

表观遗传学(epigenetics)是分子生物学的一个重要分支,重点研究基因甲基化,组蛋白修饰等引起的基因表达的改变。近来研究显示,人乳头状瘤病毒(HPV)和细胞内基因组在宫颈癌各阶段都发生了改变,包括:整个基因的低甲基化,关键抑癌基因的高甲基化及组蛋白修饰,其表观改变发生在致癌过程的早期阶段。该研究进展有助于宫颈癌的早期诊断,也为分子靶向治疗宫颈癌提供了可能。     

宫颈癌中表观遗传学的研究进展

表观遗传学(epigenetics)是分子生物学的一个重要分支,重点研究基因甲基化,组蛋白修饰等引起的基因表达的改变.近来研究显示,人乳头状瘤病毒(HPV)和细胞内基因组在宫颈癌各阶段都发生了改变,包括:整个基因的低甲基化,关键抑癌基因的高甲基化及组蛋白修饰,其表观改变发生在致癌过程的早期阶段.该研究进展有助于宫颈癌的早期诊断,也为分子靶向治疗宫颈癌提供了可能.     

印迹基因在产科领域的研究进展

基因印迹是指不同亲代来源的一对等位基因呈现差异性表达,仅一方等位基因选择性地表达,其另一方等位基因不表达或表达极弱,具有这种现象的基因被称作印迹基因.近年来,表观遗传学已成为各种疾病遗传学领域探讨的热点,其中印迹基因在产科疾病中的相关性及重要性越来越受到关注.研究表明,子痫前期、胎儿生长受限的发病与细胞周期蛋白酶抑制剂编码蛋白P57kip2、生长调节基因H19、胰岛素样生长因子2和生长调节基因H19等多种印迹基因的印迹状态和(或)表达异常有关.     

胰岛素样生长因子2基因印迹状态及其启动子活性在巨大儿胎盘组织中的变化

目的通过检测巨大儿的胎盘组织中胰岛素样生长因子2（IGF2）基因印迹状态及其mRNA表达水平和不同启动子活性,探讨巨大儿的发生机理。方法根据IGF2基因第9外显子ApaⅠ酶切位点多态性,应用RT-PCR结合限制性片段长度多态性技术,对83例分娩巨大儿产妇的胎盘组织（巨大儿组）和同期分娩正常体重儿的85例产妇的胎盘组织（正常体重儿组）进行杂合子筛选,对筛选出的杂合子进行IGF2基因印迹状态分析,用RT—PCR半定量检测两组胎盘组织IGF2 mRNA表达水平及不同启动子的活性。结果两组胎盘组织中各有30例杂合子。在这些杂合子中,IGF2基因均为单等位基因表达,呈维持印迹状态。巨大儿组胎盘组织中IGF2 mRNA表达水平为2．2±1．2,明显高于正常体重儿组的1．6±0．6,两组比较,差异有统计学意义（P〈0．05）;IGF24个启动子（P1、P2、P3、P4）在两组胎盘组织中均表现为：P4活性最强,P3次之,P2较弱,P1最弱,仅在两例（巨大儿组和正常体重儿组各1例）中检测到P1起始的转录本;两组胎盘组织IGF2不同启动子活性检测分别为：正常体重儿组P2：0．19±0．17、P3：0．98±0．80、P4：2．05±1．27;巨大儿组P2：0．20±0．20、P3：0．99±0．72、P4：2．06±1．26,两组分别比较,差异均无统计学意义（P〉0．05）。结论胎盘组织中IGF2 mRNA表达水平的增加可能与巨大儿的形成有关,而IGF2基因印迹状态和启动子活性与巨大儿的形成无明显关系。     

胰岛素相关微环境因子对子宫内膜癌细胞MAPK/ERK通路活化的时间和浓度效应研究

目的检测胰岛素样生长因子(IGF)及胰岛素(insulin)在子宫内膜癌Ishikawa及HEC-1-A细胞中激活MAPK/ERK通路的最佳时间和浓度。方法采用蛋白免疫印迹技术分别检测IGF-1、IGF-2及Insulin不同时间(浓度为10 nmol/L时,分别刺激5 min、10 min、20 min、30 min和60 min)和不同浓度(2 nmol/L、5 nmol/L、10 nmol/L、20 nmol/L和100 nmol/L)作用后,子宫内膜腺癌Ishikawa及HEC-1-A细胞中MAPK通路关键分子ERK1/2磷酸化水平的变化,以确定各因子的最佳刺激时间和最佳刺激浓度。结果①IGF-1、IGF-2及Insulin均能刺激Ishikawa及HEC-1-A细胞ERK通路的激活。②在Ishikawa细胞中,IGF-1浓度为2 nmol/L,刺激20 min时,ERK磷酸化水平最高;IGF-2浓度为10 nmol/L,刺激30 min时,ERK磷酸化水平最高;胰岛素浓度在10 nmol/L刺激5 min作用最强。③在HEC-1-A细胞中,IGF-1浓度为10 nmol/L,刺激10 min时,ERK磷酸化水平最高;IGF-2浓度为10 nmol/L,刺激30 min作用最强;胰岛素浓度在10 nmol/L,刺激10 min作用最强。结论 IGF-1、IGF-2及Insulin能够激活Ishikawa及HEC-1-A细胞的ERK通路,且各种微环境因子的作用均有时间和浓度依赖性。     

Steroid-modulated proliferation of human endometrial carcinoma cell lines: any role for insulin-like growth factor signaling?

OBJECTIVES: Estrogen-stimulated proliferation of the normal and malignant human endometrium is balanced by the differentiating properties of progesterone. This study evaluated the role of insulin-like growth factor (IGF) signaling in steroid-induced modulation of endometrial cancer cell proliferation. METHODS: We used the human endometrial, estrogen-responsive ECC-1 and progesterone-responsive PRAB-36 cell lines. Proliferation studies with IGFs in combination with either estrogen or progesterone were conducted. Furthermore, the mRNA and protein expression of insulin-like growth factor-binding proteins (IGFBPs) was evaluated. RESULTS: Using the ECC-1 cell line, we observed that estrogen-induced proliferation is modulated via the IGF-receptor signaling pathway, and that IGF-1-induced stimulation of proliferation does not influence estrogen receptor signaling. Furthermore, expression of the main modulators of IGF action, the IGFBPs, was found to be regulated by estrogen and progesterone in both cell lines. IGFBP-4 was up-regulated by estrogen in the ECC-1 cell line, and IGFBP-3 and IGFBP-6 were down-regulated by progesterone in the PRAB-36 cell line. CONCLUSION: Estrogen-induced stimulation of proliferation of ECC-1 endometrial cancer cells is partly achieved via IGF signaling. Furthermore, the IGFBPs are regulated by estrogens as well as progestagens and could potentially play a role in the modulation of endometrial cancer cell proliferation.     

Maternal and fetal insulin-like growth factors 1 and 2 (IGF-1, IGF-2) and IGF BP-3, and their relationship to fetal acidosis at delivery

OBJECTIVE: To investigate the relationship between levels of insulin-like growth factors 1 and 2 (IGF-1, IGF-2), and insulin-like growth factor binding protein 3 (IGFBP-3) in antenatal maternal serum and in fetal cord blood at delivery. METHODS: Prospective cohort study of 1650 low-risk Caucasian women in a University teaching hospital in London. Statistical analysis was performed using commercial software (SPSS for Windows, version 6.1, SPSS, Chicago, Illinois, USA), with p<0.05 as significant. Maternal IGF 1, IGF 2 and IGF BP-3 were assessed on maternal blood at booking and in fetal blood by cord blood analysis at delivery. Cord pH was also recorded. RESULTS: There was no significant correlation between maternal IGF-1, IGF-2, or IGFBP-3 levels and fetal acidosis. However, a significant correlation does exist between cord IGF-1 levels and fetal acidosis. CONCLUSION: Fetal cord IGF-1 has a significant correlation with fetal acidosis at delivery.     

Altered imprinting,promoter usage,and expression of insulin-like growth factor-II gene in gestational trophoblastic diseases

OBJECTIVE: We aimed to understand the involvement of imprinted genes in the pathogenesis of gestational trophoblastic diseases (GTD) such as hydatidiform mole (H-mole) and gestational trophoblastic tumors (GTT). METHODS: An allelic-typing assay was performed using a PCR-RFLP-based method for identification of heterozygous informative cases. The usage of insulin-like growth factor-II (IGF2) promoters was examined by RT-PCR using promoter-specific primers. The mRNA expression of IGF2 and H19 was quantified using a densitometer. RESULTS: The imprinting of IGF2 and H19 was maintained in all normal placenta tissues (n = 15) but relaxed in GTD (n = 47). Loss of imprinting (LOI) of IGF2 was in the order of GTT (57%) > complete H-mole (43%) > partial H-mole (25%). Similarly, LOI of H19 was in the order of GTT (40%) > complete H-mole (18%) > partial H-mole (0%). Promoter usage pattern of IGF2 changed with gestation stage of normal placentae and GTD. In normal placentae, the usage of promoter P1 was higher than that of P4 in the first trimester but lowered in the full term. H-mole and GTT predominantly used promoter P1 with relative silencing of promoter P4. Although normal early placenta and various GTD tissues showed the similar usage of IGF2 promoter P1, GTT tissues revealed the higher expression levels of IGF2 but a down-regulation of H19 relative to the normal early placentae. CONCLUSIONS: These results suggest that LOI, deregulation of IGF2 promoters, and the altered expression levels of IGF2 and H19 genes might be associated with the progression of GTD.     

Maternal insulin-like growth factors 1 and 2 (IGF-1, IGF-2) and IGF BP-3 and the hypertensive disorders of pregnancy

Objective.?To investigate the relationship between levels of insulin-like growth factors 1 and 2 (IGF-1, IGF-2) and insulin-like growth factor binding protein 3 (IGFBP-3) in antenatal maternal serum and gestational hypertension and pre-eclampsia (PET).

Methods.?Prospective cohort study of 1650 low-risk Caucasian women in a University teaching hospital in London. Statistical analysis was performed using commercial software (SPSS for Windows, version 6.1, SPSS, Chicago, IL), with P?<?0.05 as significant. Maternal IGF 1, IGF 2 and IGF BP-3 were assessed on maternal blood at booking. Blood pressure was checked at each visit in conjunction with urine analysis. The Davey & MacGillivray 1988 classification system was used in making the diagnosis of PET.

Results.?There was no significant correlation between maternal IGF-1 or IGFBP-3 levels and gestational hypertension or PET. However, a significant positive correlation does exist between maternal IGF-2 levels and PET.

Conclusions.?Maternal IGF-2 has a significant positive correlation with PET.     

米非司酮对子宫肌瘤胰岛素样生长因子-1的影响

目的观察米非司酮对子宫肌瘤组织中胰岛素样生长因子-1(IGF-1)表达及比不同治疗效果间肌瘤组织IGF—1表达的差异,探讨疗效的个体差异原因。方法子宫肌瘤患者手术前每天服用米非司酮10mg,共3个月,于治疗前后B超测定最大肌瘤三维径线,判定肌瘤体积变化,另采用免疫组化法(ABC法)对米非司酮治疗组和对照组不同月经期的子宫肌瘤组织中IGF-1的表达进行分析,以观察不同组间IGF—1水平的差别。结果①对照组肌瘤IGF-1的染色在增生期和分泌期无明显差异,均呈强阳性;②治疗组肌瘤IGF-1的染色呈不同程度减弱或阴性,与对照组差异显著(P<0.05);③治疗组中肌瘤缩小明显者IGF-1的染色弱于肌瘤缩小不明显者,并有显著差异(P<0.05)。结论①米非司酮可能通过抑制IGF-1的合成使肌瘤细胞生长受抑,肌瘤体积缩小。②不同治疗效果与IGF-1表达的受抑程度有关。     

Gene expression patterns of insulin-like growth factor 1, 2 (IGF-1, IGF-2) and insulin-like growth factor binding protein 3 (IGFBP-3) in human placenta from preterm deliveries: influence of additional factors

Objective

To compare patterns of human placental gene expression of IGF from pregnancies that ended with preterm delivery vs. full term pregnancies as controls.

Study design

Real-time PCR was used to assess gene expression of IGF in human placental samples from 104 preterm and 140 full term pregnancies.

Results

In the preterm delivery group, the proportion of smokers was significantly higher than in the control group. A history of preterm delivery was more common in the preterm delivery group compared to the control group. In the preterm delivery group, placental samples showed an underexpression of the IGF-1 gene compared to controls. In cases of male fetal gender an overexpression of both the IGF-2 and the IGFBP-3 genes was observed.

Conclusion

Among environmental factors influencing preterm delivery, smoking was the most significant in our study. In the majority of cases, preterm delivery was induced by intrauterine infection leading to a decreased activity of the IGF system. This mechanism may also play a role in the development of neurological sequelae and in decreased tolerance to fetal distress. The overexpression of the IGF-2 gene observed in the placenta with male fetal gender can be explained by its physiological role in the development of the male phenotype.