【作者】 曹东华；

【导师】 金春莲；

【作者基本信息】 中国医科大学 ， 遗传学， 2010， 博士

【摘要】 GLI3基因与单纯性马蹄内翻足的相关性研究及其作用机制探讨前言单纯性马蹄内翻足(Idiopathic congenital talipes equinovarus, ICTEV)是常见的严重危害儿童健康的先天畸形之一,发病率为1～8‰。遗传因素在ICTEV的发病过程中发挥重要作用,遗传度为65%。但其遗传方式、外显率等均不清楚,易感基因尚未确定。目前研究较多的与ICTEV发病相关的基因主要集中在与足踝部的骨骼、软骨、肌肉和神经发育相关的基因,如COL9A1, CASP10, WNT7A, HOXD13,GLI3等。本课题组前期应用SNP关联分析的方法对84个ICTEV核心家系GLI3基因内的2个多态位点进行分析,提示CLI3基因可能是单纯性马蹄内翻足的易感基因。GLI基因家族是一个高度保守的转录因子家族,其中的GLI3是极化活性区内的信号分子之一,它可以沿肢体前后轴方向限定各个基因表达的区域边界,这为建立肢体不对称模式提供了必要的位置信息。国内外学者对GLI3基因的研究主要集中于它与指(趾)的发育畸形相关,其突变可以导致多种与指(趾)畸形相关的疾病,但CLI3基因是否与ICTEV的发病相关还未见报道。另有报道HOXD13基因与ICTEV的发生密切相关,本实验进一步探讨GLI3基因与ICTEV的相关性,以及在ICTEV发生过程中HOXD13基因如何调控CLI3基因的机制。方法标本：84例ICTEV患者外周静脉血和15例ICTEV患者肌肉组织标本由中国医科大学附属盛京医院小儿外科提供,9例同年龄组的正常人足部肌肉组织及肺组织由中国医科大学法医学院提供。所有标本使用均经患者知情同意。成年Wistar大鼠购自中国医科大学实验动物中心,所有的实验过程都遵照动物保护条例。1、变性梯度凝胶电泳技术检测GLI3基因编码区的突变PCR扩增84例患者GLI3基因10个外显子(另外4个外显子基因突变筛查工作前期已经完成),应用20%～80%的变性聚丙烯酰胺凝胶电泳筛查10个外显子的突变情况。2、RT-PCR方法研究GLI3基因在ICTEV患者下肢表达情况。分别提取ICTEV患者及正常人下肢足踝部拇长屈肌、正常人肺组织的总RNA,应用RT-PCR方法检测GLI3基因的表达。3、构建ICTEV大鼠模型Wistar大鼠于孕10天全反式维甲酸灌胃给药(剂量：135mg/kg体重)。4、Real Time PCR,免疫组织化学染色和Western Blotting方法研究Gli3基因在ICTEV模型鼠下肢肌肉组织中的表达分别提取ICTEV模型胎鼠及正常对照胎鼠下肢肌肉组织的总RNA,应用Real Time PCR检测Gli3基因的表达情况；分别提取ICTEV模型胎鼠及正常对照胎鼠下肢肌肉组织的蛋白质,应用Western Blotting检测Gli3蛋白的表达情况；分别取ICTEV模型胎鼠及正常对照胎鼠下肢,甲醛固定,石蜡包埋,按照免疫组化试剂盒操作说明检测Gli3蛋白表达情况。5、应用P-Match软件预测大鼠Gli3基因5′上游序列转录因子的结合位点获取大鼠Gli3基因上游1100bp 5′侧翼序列信息(http://www.ensembl.org),应用P-Match软件预测其转录因子结合位点。6、荧光素酶报告基因系统PCR扩增大鼠Gli3基因启动子区域依次截短的启动子序列,构建荧光素酶报告基因表达载体pGL3-Gli3。瞬时转染大鼠L6细胞,观察大鼠Gli3基因启动子区域活性。7、染色质免疫沉淀实验(ChIP)将孕14天胎鼠下肢肢芽直接匀浆处理,甲醛交联、酶切后应用Gli3抗体进行沉淀,沉淀下来DNA通过PCR扩增检测结果。8、凝胶迁移实验(EMSA)孕14天胎鼠下肢肢芽组织核蛋白和3′生物素标记的探针(含有位点2)在凝胶阻滞缓冲液中室温结合1小时,10%聚丙烯酰胺凝胶电泳,转膜、紫外交联后,应用化学发光检测系统检测结果。9、RNAi使Hoxdl3基因表达下调,观察Gli3基因的表达情况化学合成Hoxdl3基因的siRNA,转染大鼠L6细胞后,通过Real Time PCR的方法观察Gli3基因表达水平的改变。10、在大鼠L6细胞系中外源表达Hoxdl3后观察Gli3基因表达构建Hoxdl3的真核细胞表达载体PIRES2-EGFP-Hoxdl3,瞬时转染大鼠L6细胞后,通过Real Time PCR的方法观察Gli3基因表达水平的改变。结果1、在84例ICTEV患者外周静脉血中未发现GLI3基因外显子1～8、外显子13存在突变。2、在ICTEV患者及正常人下肢拇长屈肌中都未检测到GLI3基因的表达。3、不论在mRNA水平,还是在蛋白质水平,Gli3基因在ICTEV模型胎鼠下肢组织中的表达都要明显高于正常对照胎鼠。4、大鼠Gli3基因5′侧翼序列启动子区域存在不同的调控因子,利用P-Match软件预测后发现2个Hoxd13的可能结合位点,分别命名为Hoxdl3结合位点1(-667～-663)和Hoxd13结合位点2(-477～-473)。5、在大鼠胚胎肢体发育过程中Hoxd13可以和Gli3基因启动子区Hoxd13结合位点2直接结合。应用染色质免疫沉淀技术验证在体内Hoxdl3和Gli3基因上游序列的结合作用。以孕14天的大鼠胎鼠下肢肢芽组织为研究材料进行了染色质免疫沉淀实验,实验证实反胶联后提取的DNA上仅有Hoxdl3结合位点2的扩增,无Hoxdl3结合位点1的扩增。实验结果表明在大鼠胚胎肢体发育过程中Hoxdl3蛋白可以和Gli3启动子区的Hoxdl3结合位点2结合发挥其转录调节作用。6、在体外Hoxdl3蛋白可以和Gli3基因启动子区位点2直接结合。EMSA结果表明,当Hoxdl3蛋白质存在时出现阻滞的DNA-蛋白质复合体,当加入Hoxdl3抗体时出现超阻滞条带。证明在体外Hoxdl3和预测的Hoxdl3结合位点2可以直接结合。7、RNAi方法干扰HoxD13基因表达后,Gli3基因表达情况。将吉玛公司设计的三个Hoxdl3基因的siRNA瞬时转染大鼠L6细胞系中,发现其中一个siRNA能使Hoxdl3表达下调,同时检测到该标本中Gli3基因表达明显上调,说明Hoxdl3很可能是Gli3基因的负调控因子。8、大鼠L6细胞系中外源过量表达Hoxdl3后Gli3基因的表达情况。将Hoxdl3的真核细胞表达载体Hoxdl3瞬时转染大鼠L6细胞后,通过Real Time PCR的方法观察到Hoxdl3基因的表达水平明显上调,同时Gli3基因表达水平下调。结论1、GLI3基因的编码区突变可能不是ICTEV发病的原因。2、HOXDl3基因的表达下调并导致GLI3基因的表达上调可能与ICTEV畸形的发生有关。3、在大鼠胚胎肢体发育过程中Hoxdl3蛋白结合于Gli3基因启动子区域的Hoxdl3结合位点2上,并调控Gli3基因的表达。更多还原

【Abstract】 Study on the relationship between GLI3 and Idiopathic congenital talipes equinovarus and the pathogenesis mechanismIntroductionIdiopathic congenital talipes equinovarus (ICTEV) is a type of congenital limb deformity with an estimated incidence of 1-8%o live births. The mechanism by which ICTEV develops remains unclear even though the mechanical, neurological, muscular, bony, connective tissue, and vascular mechanisms have been proposed. Whilst both genetic and environmental factors are implicated, no specific genes have been identified. At present, investigations on human ICTEV mainly focus on the environmental factors at early stage of pregnancy and many syndromes with clubfoot malformations. Candidate genes and regions for ICTEV, such as COL9A1, CASP10, WNT7A, HOXD13 and GLI3 have been identified, respectively. However, little is known about the pathogenesis of human ICTEV.Our previous studies using the transmission disequilibrium test showed that the GLI3 gene maybe one of the predisposing genes in ICTEV. GLI family (GLI1, GLI2, and GLI3) is a highly conserved family. GLI3 is one of the signaling molecules in the zone of polarizing activity (ZPA). It limited the expression domain of some gene along the anterior-posterior (AP). Most GLI3 research focuses on digit abnormalities and deformities. GLI3 mutations cause limb development disorders about doigt. But it is still unknown whether GLI3 has relation with ICTEV. Some authors reported that HOXD13 has intimate relation with ICTEV. We further investigate the relation between GLI3 and ICTEV and how HOXD13 regulate GLI3. Samples:Veinous blood of 84 ICTEV patients and 15 ICTEV muscle tissues were obtained from the Department of Pediatric Orthopedic Surgery, Second Affiliated Hospital, China Medical University. Flexor hallucis longus and lung tissue were from nine normal cadavers provided by the institute of the forensic medicine of China medical university. Adult SD rats were obtained from the experimental animal center of our university. All procedures were carried out in accordance with an approved animal handling protocol.1. Mutation in the coding region of GLI3 in 84 ICTEV patients was detected by denaturing gradient electrophoresis.The total 10 exons (other 4 exons have been detected) of GLI3 gene were PCR from 84 ICTEV patients,20%-80%DGGE was used to detect the mutation in these 10 exons.2. The expression of GLI3 in the ICTEV patients’limb was detected by RT-PCRRNA was extracted from the flexor hallucis longus of ICTEV patients and normal controls and the lung of normal control, then tested the gene GLI3 expression.3. Make ICTEV model ratsThe ICTEV phenotype in rat embryos was induced by administration of 135 mg/kg all-trans-retinoic acid (ATRA) on gestation day 10 (GD10).4. The mRNA and protein levels of GLI3 were evaluated by Real Time-PCR and immunohistochemistry, Western Blotting, respectively.RNA and protein were extracted from the hindlimbs of the ICTEV model rat embryos and normal controls and detected GLI3 expression by Real Time PCR, Western Blotting and immunohistochemistry.5. P-Match software was used to analyze the sequence upstream of the transcription start site of the rat Gli3 gene.The 1100bp sequence uptream of rat GliS gene was obtained from http://www.ensembl.org, we used P-Match software to predict the binding sites of transcriptional factors on the sequence.6. Luciferase report vector assay.The promoter sequence in Gli3 were obtained by polymerase chain reaction, we constructed pGL3-Gli3 vectors. L6 cell were transfected pGL3-Gli3. After 24h, we analyzed the activity of Gli3 promotor.7. Chromatin immunoprecipitation assay.The chromatin was extracted from limb buds dissected from E14 wild-type embryos, sheared with an Enzymatic Shearing Kit to obtain 200～1000bp fragments. Gli3 antibody was used at the immunoprecipitation step. Eluted DNA from the sample and control was assessed for the presence of Gli3 DNA region by PCR.8. Electrophoretic mobility shift assay.Nucleoprotein was extracted from E14 rat embryonic limbs and incubated with the Gli3 upstream region-containingbinding site 2 labeled using a Biotin 3’End DNA labeling Kit for 60 min at room temperature in a gel shift buffer. Reactions were examined for nucleoprotein binding by electrophoretic mobility shift assays (EMSA) on a 10%nondenaturing polyacrylamide, transfered to the memberane, cross-linked. DNA binding bands were detected using a chemiluminescence system.9. Analysis Gli3 expression in L6 cell transfected Hoxd13 siRNAGli3 expression was analyzed after chemosynthesising Hoxd13 siRNA and transfecting it to L6 cell.10. Analysis Gli3 expression in L6 cell transfected HoxD13 expression vector. We constructed Hoxd13 expression vector, pcDNA-Hoxdl3, and transfected the expression vector in L6 cell. After 24h, the expression of Gli3 level was detected.Results1. No mutation was found in the exon 1～8,13 of GLI3 in 84 samples from patients with ICTEV.2. GLI3 is not expressed in the flexor hallucis longus of ICTEV patients and normal control.3. Expression of Gli3 in both mRNA and protein was higher in ICTEV model rat embryonic hindlimbs compared to normal control rat embryonic hindlimbs.4. The 5’region of the rat Gli3 gene contained two potential binding sites for the Hoxd13 protein designated Hoxd13 binding site 1(-667--663) and Hoxd13 binding site 2(-477--473).5. Hoxd13 binds with site 2 in Gli3 promoter region in the developing limb in rat.6. Hoxd13 directly binds to site 2 in Gli3 promoter region.Strong DNA binding was observed in the presence of the Hoxd13 protein. A competition experiment and supershift existence in the presence of the Hoxd13 antibody demonstrated the specificity of such binding. The result indicated Hoxd13 is bound to site 2 in vitro.7. Expression of Gli3 in mRNA was higher in L6 cell after Hoxd13 silence compared to control.8. The exogenous expression of Hoxdl3 down-regulated Gli3 transcription in L6 cells. Conclusions1. GLI3 gene mutation in coding region was not involved in outbreak in ICTEV.2. The decrease in HOXD13 expression led to an increase in the expression of GLI3, which maybe relate to ICTEV.3. Hoxdl3 directly controls the expression of Gli3 through Hoxd13 binding site 2 in the developing limb in rat embryo.更多还原