INSL3基因在单侧隐睾睾丸组织中表达的研究

咦岛素样因子3(INSL3)基因及其表达的多肽对于雄性性腺位置的改变和性腺的发育起到重要作用。在睾丸下降的第一阶段(经腹下降阶段)主要是由INSL3介导;成熟Leydig细胞分泌IN- SL3能减少生殖细胞凋亡。本研究旨在了解单侧隐睾对睾丸组织中INSL3基因转录水平的影响。     

婴幼儿血管瘤survivin基因启动子甲基化的研究

目的 采用亚硫酸氢盐修饰后测序法(BSP)检测不同病理阶段的婴幼儿血管瘤组织及正常皮肤组织中survivin基因启动子区域CpG岛的甲基化状态,探讨survivin基因启动子甲基化与婴幼儿血管瘤增生与退化的可能关系.方法 ①采用免疫组化S-P法检测增殖期婴幼儿血管瘤石蜡标本30例、消退期30例及正常包皮皮肤组织标本10例中survivin蛋白的表达;②提取石蜡包埋块组织基因组DNA并纯化后,采用亚硫酸氢盐修饰后测序法(BSP)分别检测增殖期血管瘤30例、消退期血管瘤30例及正常包皮组织10例中survivin基因启动子CpG岛甲基化情况.结果 ①survivin 蛋白在增殖期血管瘤、消退期血管瘤和正常包皮组织中阳性表达率分别为76.6％ (23/30)、33.3％(10/30)和20.0％(2/10);②10例正常包皮皮肤组织中1例(10.0％)survivin基因启动子CpG岛非甲基化,9例(90.0％)甲基化;30例消退期血管瘤标本中8例(26.6％)survivin基因启动子CpG岛非甲基化,22例(73.3％)甲基化;30例增殖期血管瘤标本中24例(80.0％) survivin基因启动子CpG岛非甲基化,6例(20.0％)甲基化;增殖期血管瘤survivin基因启动子CpG岛甲基化率明显低于消退期和正常包皮组织;③survivin蛋白表达阳性的血管瘤33例中31例survivin基因启动子CpG岛为非甲基化,survivin蛋白表达阴性的27例中26例survivin基因启动子CpG岛为甲基化状态.结论 ①增殖期血管瘤survivin蛋白表达明显高于消退期血管瘤;②survivin基因启动子CpG岛甲基化状态在增殖期血管瘤、消退期血管瘤中存在明显差异;血管瘤组织中survivin基因启动子的甲基化状态与survivin蛋白的表达具有相关性;survivin基因启动子区CpG岛异常甲基化在血管瘤的增殖与消退调控中可能起到了一定作用.     

己烯雌酚对大鼠腹腔内睾丸下降的影响及相关因子的变化

目的 观察不同剂量己烯雌酚(DES)致大鼠腹腔内隐睾及其对睾丸胰岛素样因子3(INSL3)、睾丸类固醇生成因子-1(SF-1)、引带同源盒基因-A10(HOXA10)表达的影响.方法 50只SD孕鼠随机分为5组,每组10只,在孕期第13.5天(E 13.5)分别皮下注射二甲基亚砜(对照组)和DES2.5、5、10、20 mg/kg,在E 19.5取出胚胎雄鼠,体视显微镜下观察后计算死胎率、睾丸下降程度;RT-PCR检测INSL3、SF-1及HOXA10 mRNA水平,Western blot检测INSL3蛋白水平.结果 ①与较对照组比较,DES处理各组的死胎率明显增高、睾丸下降程度明显较差(P＜0.01),且均呈剂量效应关系;②DES显著下调了INSL3、SF-1 mRNA和INSL3蛋白的表达,且呈剂量效应关系(P＜0.01);③2.5 mg/kg的DES未影响HOXA10的表达(P＞0.05),5、10、20 mg/kg的DES均下调HOXA10表达(P＜0.05).结论 DES通过下调大鼠睾丸INSL3表达致腹腔内睾丸下降受阻,且呈剂量效应关系.DES通过剂量效应下调SF-1来下调INSL3的表达.低剂量DES(2.5 mg/kg)未影响引带HOXA10表达,HOXA10在高剂量(5、10、20 mg/kg)DES诱导的大鼠腹腔内隐睾中发挥了作用.     

白细胞介素6/STAT3信号活化在急性免疫性血小板减少性紫癜患儿辅助性T淋巴细胞17/调节性T淋巴细胞失衡中的作用

目的探讨IL-6/STAT3信号活化在儿童急性免疫性血小板减少性紫癜(ITP)辅助性T淋巴细胞17(Th17)/调节性T淋巴细胞(Treg)失衡中的作用。方法选择急性ITP患儿30例,同年龄健康对照组30例。ELISA法检测其血浆IL-6水平;荧光定量PCR检测CD4+T淋巴细胞RORγt mRNA、FOXP3 mRNA、细胞因子信号转导抑制因子(SOCS)1 mRNA、SOCS3 mRNA表达;流式细胞术检测其外周血CD4+CD25+FOXP3+T淋巴细胞、CD4+IL-17A+T淋巴细胞比例及CD4+T淋巴细胞磷酸化STAT3(pSTAT3)蛋白平均荧光强度(MFI);甲基化特异性定量PCR检测CD4+T淋巴细胞SOCS1基因外显子2、SOCS3基因5’端非翻译区(5’-UTR)3个可能的STAT3结合位点CpG岛甲基化水平。结果 1.与健康对照组比较,ITP组患儿CD4+IL-17A+T淋巴细胞比例及RORγt mRNA表达水平显著上调(Pa<0.05),CD4+CD25+FOXP3+T淋巴细胞比例及FOXP3 mRNA表达显著降低(Pa<0.05),Th17/Treg比值明显升高(P<0.05);2.与健康对照组比较,ITP组患儿IL-6表达水平显著上调(P<0.05),且pSTAT3 MFI值亦明显增高(P<0.05);3.与健康对照组比较,ITP组患儿CD4+T淋巴细胞SOCS1和SOCS3 mRNA水平显著增加(Pa<0.05),与其Th17/Treg比值均呈负相关(r=-0.63、-0.70,Pa<0.05);健康对照组SOCS1基因外显子2、SOCS3基因5’-UTR区第3个STAT3结合位点的CpG岛完全去甲基化,急性ITP患儿呈低甲基化状态(Pa<0.05),其去甲基化水平与表达呈正相关(r=0.76、0.65,Pa<0.05)。各组SOCS3基因5’-UTR区第1、2个STAT3结合位点CpG岛均处于完全去甲基化状态(Pa>0.05)。结论 SOCS1和SOCS3基因低甲基化所致IL-6/STAT3信号异常活化可能是急性ITP患者Th17/Treg细胞失衡的因素之一。     

肾母细胞瘤RASSF1A基因启动子区甲基化研究

目的了解肾母细胞瘤RASSF1A基因启动子区甲基化情况。方法手术获取9例肾母细胞瘤及瘤旁残留肾组织,提取DNA后用亚硫酸氢钠处理,巢式PCR扩增RASSF1A基因启动子区DNA片段,测序,检测启动子区甲基化情况。结果发现1例16个CpG岛位点中有10个存在甲基化(10/16),相应瘤旁残留肾组织无甲基化(0/16);在另1例瘤组织中检测到12个CpG岛位点存在甲基化(12/16),但仅有8个CpG岛位点与上例相同,另外4个位点不同。结论肾母细胞瘤中RASSF1A基因启动子区存在甲基化,与瘤旁残留肾组织甲基化情况有差别。     

急性髓性白血病p15INK4B基因表达和基因甲基化状态的研究

p15^INK(p15)基因被认为是一个重要的肿瘤抑制基因,对细胞周期起负调控作用。p15基因的启动区中有一Cpg岛,许多血液系统恶性病变表现为CpG岛异常的高甲基化状态。虽然有关急性白血病p15基因启动区中CpG岛甲基化异常已有不少的研究报道,但主要见于成人,CpG岛异常甲基化与该基因表达水平的关系研究则较少见。本研究同时检测儿童急性髓性白血病(acute myeloid leukemia,AML)p15基因表达水平和甲基化状态,以探讨两者之间的相互关系以及在急性白血病发病中的作用。     

巢式PCR法检测肝母细胞瘤中p15和p16基因启动子异常甲基化

目的：DNA甲基化被认为是反映细胞内DNA转录状态的重要遗传学标记。该研究旨在对小儿肝母细胞瘤中p15和p16基因5′启动子区CpG岛异常甲基化的相关性进行评估,并分析其与小儿肝母细胞瘤发生发展的关系。方法：采用甲基化特异性PCR法,对30例小儿肝母细胞瘤、癌旁和远癌正常组织进行巢式PCR扩增,经凝胶电泳和测序方法检测目的片段。结果：30例小儿肝母细胞瘤组织中p15和p16基因5′CpG岛有30%(9/30)和67%(20/30)、癌旁组织中有20%(6/30)和57%(17/30)、远癌正常组织有13%(4/30)和33%(10/30)异常甲基化。在肝母细胞瘤中发现1例p15E2纯合缺失,缺失率为3%(1/30),E1和部分I1区域未见缺失;p16E2和部分I2区域中3例杂合缺失,缺失率为10%(3/30),E1和部分I1区域未见缺失。结论：p15和p16基因5′启动子区CpG岛的异常甲基化可能在肝母细胞瘤发生发展中扮演了重要角色,其主要机制可能是启动子区CpG岛甲基化抑制了基因的转录。     

内皮素受体B基因启动子上游CpG岛甲基化对神经嵴细胞迁移的影响

目的 探讨内皮素受体B(EDNRB)基因启动子上游CpG岛甲基化对肠神经系统(ENS)发育中神经嵴细胞迁移的影响.方法 采用甲基化特异性PCB(MSP)技术检测2007年9月-2008年12月华中科技大学同济医学院附属协和医院经病理检查等证实的16例先天性巨结肠症(HD)患儿挛缩段组织标本中EDNRB基因启动子上游cpG岛甲基化状态;采用荧光定量PCR(SYBRGreen法)检测HD患儿病变组织标本(包括扩张段、移行段和挛缩段3段组织)中EDNRB基因mRNA的相对表达量.并收集同期16例非HD患儿的正常结肠组织标本作为对照.结果 HD患儿挛缩段标本行MSP检测,甲基化率为12.5%(2/16例);非HD患儿正常组织均未发现甲基化.16例HD患儿病变组织标本(包括扩张段、移行段、挛缩段)荧光定量PCR检测发现2例甲基化病变组织标本(包括扩张段、移行段、挛缩段)中EDNRB基因表达水平较非甲基化组织明显下调,差异有统计学意义(P<0.05).结论 在HD发病机制中,EDNRB基因启动子上游CpG岛甲基化可能对ENS发育中神经嵴细胞的迁移有一定影响.     

邻苯二甲酸二(2-乙己基)酯导致隐睾症发生跨代遗传的机制研究

目的 探讨增塑剂邻苯二甲酸二(2-乙己基)酯(DEHP)于性腺发育的关键时期作用于孕鼠(0),研究F1-F3代隐睾跨代遗传的演变情况及各代睾丸基因组DNA甲基化转移酶水平的改变情况.方法 妊娠SD大鼠随机分为两组:正常对照组和DEHP实验组,实验组自妊娠第七天(GD7)到第十九天(GD19)持续经口予以DEHP 750 mg·kg-1 ·d-1灌胃,观察子代隐睾发生情况,雌鼠受孕率;记录大鼠体重和睾丸、附睾重量以及AGD值,观察精子数量和质量;观察连续三代大鼠睾丸组织形态的演变情况,检测DNA甲基化转移酶变化情况.结果 孕鼠在孕期(GD7-GD19)暴露于DEHP,第一代(F1)隐睾发生率为30％,第二代(F2)隐睾发生率为12.5％,第三代(F3)未见隐睾发生;交配实验F1代受孕率50％,F2代75％,F3代100％;HE染色发现F1代睾丸生精上皮明显萎缩,生精细胞少,F2代有所改善,F3代形态趋于正常.Real Time-PCR、免疫组化和Western Blot表明DNA甲基化转移酶的表达随着遗传代数的增加而上调,差异有统计学意义.结论 DEHP损伤大鼠雄性生殖功能,通过改变DNA甲基化转移酶的表达,继而导致基因组印记甲基化修饰模式改变并遗传给下一代,从而使子代雄性生殖系统发育的关键性印记基因作用失衡,并最终导致子代产生隐睾,可能是导致生殖系统损害的重要毒理机制之一.     

EDNRB基因CpG岛甲基化在先天性巨结肠发病机制中作用的初步探讨

目的 探讨内皮素受体B(EDNRB)基因启动子上游CpG岛甲基化在先天性巨结肠(Hirschsprung disease,HD)发病机制中的作用.方法 采用甲基化特异性PCR(MSP)技术检测2007年9月至2009年3月收集的经病检等证实的25例HD患儿病变组织标本EDNRB基因CpG岛甲基化状态, 以同期收集的16例非HD患儿正常结肠组织标本作为阴性对照, 采用荧光定量PCR(SYBR-Green 法)和Western-blot技术检测这25例HD患儿病变组织标本中EDNRB基因mRNA和蛋白的相对表达量.结果 25例标本MSP检测, 有2例发现甲基化, 甲基化率为2/25(8%); 16例阴性对照均未发现甲基化.荧光定量PCR发现: 甲基化组织EDNRB基因Ct值(扩张段0.13±0.08, 移行段0.10±0.06, 挛缩段0.09±0.05)均较非甲基化组织(扩张段0.59±0.24, 移行段0.57±0.32, 挛缩段0.48±0.30)有明显下调,差异具有统计学意义(P＜0.05).Western-blot发现:甲基化组织EDNRB基因蛋白相对灰度值(扩张段1.56±0.43, 移行段1.32±0.32, 挛缩段1.27±0.21)较非甲基化组织(扩张段1.72±0.36, 移行段1.52±0.62, 挛缩段1.48±0.49)有所下降,但差异无统计学意义(P＞0.05).结论 内皮素受体B(EDNRB)基因启动子上游CpG岛甲基化可能通过影响其mRNA的表达水平导致其表观遗传学的改变,进而导致HD的发生.     

儿童血浆促酰化蛋白的测定及意义

目的：介绍一种简易、快速、特异性高的人血浆促酰化蛋白(acylationstimulatingprotein,ASP)的检测技术,并阐明ASP测定意义。方法：161例研究对象,其中健康儿童98例,单纯性肥胖症患儿63例,年龄2～6岁,ELISA方法测定血浆ASP的浓度,波长490nm。结果：该方法最佳线性范围:ASP浓度0.5～10ng/mL,平均批内和批间变异系数(CV)分别为8.17%和9.72%。正常学龄前儿童血浆ASP浓度为69.14±25.58nM;单纯性肥胖症儿童血浆ASP浓度为95.64±36.24nM,明显高于健康儿童(P<0.001);ASP与BMI呈正相关。结论：该检测方法能简易、快速对血浆ASP进行准确定量,在ASP基础和临床相关疾病的研究中有重要的意义。     

SOCS低甲基化在儿童过敏性紫癜Th17/Treg细胞失衡中的作用研究

目的 通过研究细胞因子信号转导抑制因子（SOCS）低甲基化与过敏性紫癜（HSP）患儿Th17/Treg细胞失衡的关系,探讨HSP的免疫发病机制。方法 选取2014年5月至2015年1月32例急性期HSP住院患儿为研究对象,另选取行健康体检的28例儿童作为健康对照组。采用ELISA法检测血浆IL-6水平;流式细胞术检测外周血CD4⁺IL-17A⁺T细胞（Th17细胞）比例、CD4⁺CD25⁺调节性T细胞（Treg）比例和CD4⁺T细胞磷酸化STAT3（pSTAT3）蛋白平均荧光强度（MFI）;实时荧光定量PCR（RT-qPCR）技术检测CD4⁺T细胞SOCS1、SOCS3基因mRNA表达;高分辨率熔解曲线（HRM）分析法检测外周血单个核细胞SOCS1基因外显子2、SOCS3基因5'端非翻译区（5'-UTR）可能的STAT3结合位点CpG岛甲基化水平。结果 与健康对照组比较,HSP组血浆IL-6浓度、CD4⁺T细胞pSTAT3的MFI显著增加;HSP组Th17细胞比例显著上调,Treg细胞比例显著下调（P < 0.05）。HSP组患儿急性期外周血单个核细胞SOCS1 mRNA和SOCS3 mRNA水平均显著高于健康对照组（P < 0.05）;HSP组SOCS1 mRNA及SOCS3 mRNA表达均与Th17/Treg比值呈负相关（P < 0.05）。HSP组患儿急性期SOCS1基因外显子2、SOCS3基因5'-UTR区可能的STAT结合位点CpG岛呈低甲基化,而健康对照组呈完全去甲基化状态。结论 SOCS1、SOCS3基因低甲基化所致其相对表达不足可能是HSP患儿Th17/Treg失衡的因素之一。     

Upstream CpG island methylation of the PAX3 gene in human rhabdomyosarcomas

BACKGROUND: Adult tumors can be characterized by hypermethylation of CpG islands associated with 5'-upstream and coding regions of specific genes. This hypermethylation can also be part of the aging process. In contrast, much less is known about gene hypermethylation in childhood cancers, where methylation changes are not part of the aging process but likely represent developmental dysregulation. PAX3 is an important gene in muscle development and muscle-producing neoplasms such as rhabdomyosarcomas. PROCEDURES: We examined the methylation status of a PAX3 5'-CpG island in rhabdomyosarcoma subtypes and in normal fetal skeletal muscle. PAX3 methylation was analyzed in 15 embryonal rhabdomyosarcomas, 12 alveolar rhabdomyosarcomas, and in six normal skeletal muscle samples, using semi-quantitative PCR analysis of DNA digested with methyl-sensitive restriction enzymes. RESULTS: The CpG island in the upstream region of the human PAX3 gene was hypermethylated in the majority of ERMS examined (13 of 15 tumors, mean of 52% methylation), whereas most ARMS (9 of 12 tumors) and all normal muscle samples showed relative hypomethylation (both 18% mean methylation). Various CpG sites differ in contribution to overall PAX3 CpG island methylation, with methylation at a HaeII site being inversely correlated with PAX3 expression. CONCLUSIONS: PAX3 CpG island methylation appears to distinguish embryonal subtype of rhabdomyosarcoma from alveolar, and methylation at certain sites within this CpG island is inversely correlated with PAX3 expression. In addition to exemplifying developmental dysregulation, methylation of PAX3 has potential in the development of an epigenetic profile for the diagnosis of rhabdomyosarcoma.     

Genetic analysis of the human insulin-like 3 gene in pediatric patients with testicular torsion

Purpose

Testicular torsion (TT) mainly affects boys under 18 years old. To avoid orchiectomy, TT requires an immediate operative management. The etiology of TT is still controversial. Observed familiar recurrence suggests the presence of a genetic involvement. The INSL3 gene consists of two exons, and it is specifically expressed in fetal and adult Leydig cells. In transgenic mice, deletion of this gene was observed an increased testicular mobility and testicular torsion. We have hypothesized the possible involvement of the INSL3 gene as a predisposing factor of human TT.

Methods

We performed genetic analysis in 25 pediatric patients with unilateral and intravaginal TT (left, n?=?13, 56%; right, n?=?12, 48%). The age of the patients ranged from 1 to 16 years (median age n?=?10.4?±?5.46 years). In this study, we included two first male cousins affected by TT. Venous peripheral blood samples was obtained after parental written informed consent.

Results

The Thr60Ala polymorphism was detected in exon 1 of INSL3 gene and other 2 rarer variants (rs1047233 and rs1003887) were identified in the 3′ untranslated region. These variants are prevalent in patients with TT instead of healthy subjects.

Conclusions

Additional studies in a larger population are needed to better understand the clinical consequence of the INSL 3 variations founded. This would allow in the future to identify the patients at risk of TT to improve clinical management.

     

Hypermethylation as a potential prognostic factor and a clue to a better understanding of the molecular pathogenesis of medulloblastoma--results of a genomewide methylation scan

BACKGROUND: The molecular mechanisms controlling initiation and progression of medulloblastomas are largely unclear. Changes in DNA methylation of promoter regions have been shown to disturb the expression of growth regulatory genes. PATIENTS AND METHODS: We evaluated DNA methylation patterns in 17 medulloblastomas, 5 stPNETs and 5 medulloblastoma cell lines using Restriction Landmark Genomic Scanning (RLGS), a method displaying up to 2.000 potential gene loci in a single gene. To test whether previously characterized tumor suppressor genes are affected by hypermethylation we performed MS-PCR for p15INK4B, p16INK4A, VHL, TP53 and E-cadherin. RESULTS: The analysis of RLGS profiles from tumors revealed an abundance of hypermethylation in primary tumors and cell lines. Extrapolated to the human genome with its approximately 36,000 genes a total of 420 loci become hypermethylated in the tumor genomes. The previously characterized medulloblastoma breakpoint cluster in 17p11.2 appears to be a hotspot for aberrant methylation. Cox regression analysis of survival data identified seven CpG islands for which hypermethylation is suggestive of a poor prognosis. MS-PCR analysis of known genes demonstrated hypermethylation of p16INK4A in a limited number of tumors. The pattern of DNA hypermethylation was similar in medulloblastomas and stPNETs. However, some CpG islands were shown to be specific for a tumor type, while others were shared targets. CONCLUSIONS: Hypermethylation is a common abnormality in primary medulloblastomas and supratentorial PNETs. Several hundreds of CpG islands are potential targets for methylation in medulloblastomas including the breakpoint cluster in 17p11.2. The methylation status of certain gene sequences appears to be associated with the clinical outcome. Promoter hypermethylation has an outstanding potential as a marker for the identification of novel tumor suppressors as well as diagnostic and therapeutic targets in medulloblastomas.     

Epigenetic changes in the DAP-kinase CpG island in pediatric lymphoma

BACKGROUND: Hypermethylation of CpG islands in the promoter region of death-associated protein kinase (DAP-kinase) coupled with the loss of gamma-interferon-induced apoptosis have been reported in B-cell malignancies suggesting a role in pathogenesis or prognosis. Along with B-cell malignancies, pediatric lymphomas also include T-cell non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma, and Hodgkin disease, each with unique prognoses. The purpose of this study was to elucidate epigenetic changes in the DAP-kinase promoter region of pediatric cases to determine associations with aberrant hypermethylation. PROCEDURES: Thirty-nine cases of different lymphoid pathology [10 Burkitt lymphoma, 1 B-cell NHL; 7 T-cell lymphoblastic lymphoma; 4 anaplastic large cell lymphoma (LCL); 2 B-cell LCL; 14 nodular sclerosing Hodgkin disease (NSHD); and 1 B-cell acute lymphoblastic leukemia (ALL)] had methylation-specific polymerase chain reaction performed on bisulfite-treated DNA, which distinguishes the methylation status of the promoter region, and DAP-kinase mRNA expression assays performed on available specimens. RESULTS: In normal lymphocytes, the CpG islands in the promoter region were unmethylated, as were the T-cell lymphoblastic lymphoma and anaplastic LCL. In contrast, 100% of the Burkitt lymphoma (10/10) and B-cell ALL (1/1) were hypermethylated. Of the specimens with mRNA available, 7/8 Burkitt lymphoma had no DAP-kinase mRNA expression compared to normal expression in 3/3 and 4/4 T-cell lymphoblastic lymphoma and NSHD, respectively. CONCLUSIONS: In these pediatric lymphoid tumors, hypermethylation of the DAP-kinase promoter region with associated loss of DAP-kinase gene expression was associated with B-cell malignancies and thus may be important in the development and/or provide a prognostic tool in B- cell lymphomas.     

己烯雌酚和胰岛素样因子3对睾丸引带细胞中富含亮氨酸的G蛋白耦联受体8mRNA和蛋白表达水平的影响

目的 探讨不同水平的己烯雌酚(DES)和胰岛素样因子3(INSL3)对体外培养的睾丸引带细胞中富含亮氨酸的G蛋白耦联受体8(LGR8) mRNA和蛋白表达的影响.方法 将3日龄的雄性昆明小鼠的睾丸引带组织解剖取出,并进行细胞培养.传代后,随机分为正常对照组及实验组(不同浓度DES组:A～E组,不同浓度INSL3组:Ⅰ～Ⅳ组)共11组.其中,实验组加入的DES浓度分别是3.7×10-2 mmol·L-1、3.7×10-3 mmol·L-1、3.7×10-4 mmol·L-1、3.7×10-5 mmol·L-1和二甲基亚砜溶剂对照组;INSL3的浓度分别为3.3×10-3 μmol·L-1、3.3×10-4 μmol·L-1、3.3×10-5 μmol·L-1及3.3×10-6 μmol·L-1.加药持续作用48 h后,应用反转录PCR和流式细胞术分析睾丸引带细胞中LGR8 mRNA及其蛋白的表达情况.结果 A组和Ⅳ组LGR8的mRNA及蛋白水平显著高于正常对照组(P<0.01);与正常对照组相比,B、C、D组LGR8 mRNA及其蛋白表达均显著下降(Pa<0.05),而E组及Ⅰ、Ⅱ、Ⅲ组LGR8 mRNA和其蛋白的表达差异均无统计学意义(Pa>0.05).结论 高剂量的DES和极低剂量的INSL3均能上调睾丸引带细胞中LGR8 mRNA及其蛋白表达水平,推测DES和INSL3可能通过LGR8作用途径来影响睾丸引带的发育.     

Ala/Thr60 variant of the Leydig insulin-like hormone is not associated with cryptorchidism in the Japanese population

BACKGROUND: Leydig insulin-like hormone (Insl3), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of fetal and postnatal murine testis. Recently, the absence of the Insl3 gene has been reported to result in bilateral cryptorchidism in male mice and it has been suggested that mutations of the INSL3 gene may cause cryptorchidism in humans. METHODS: We sequenced the INSL3 gene from five Japanese patients with sporadic bilateral cryptorchidism. Patients' genome DNA was prepared from blood leukocytes. Two exons of the INSL3 gene were amplified by polymerase chain reaction and were sequenced directly. A restriction fragment length polymorphism assay was performed on 70 control samples for analysis of polymorphism. RESULTS: Three of five cases had a heterozygous single-base change, a G to A transition at position 178 of the INSL3 gene, which predicts an alanine (GCC) to threonine (ACC) change at codon 60 (designated A60T). However, the A60T mutation was also found in the normal Japanese population at an allele frequency of 26%, which suggests that this mutation is a common polymorphism and is not associated with the occurrence of cryptorchidism. CONCLUSIONS: No mutation has been found in the INSL3 gene from Japanese patients with idiopathic cryptorchidism. We did find the A60T polymorphism, which was not associated with the occurrence of cryptorchidism.