【作者】 陈丽；

【导师】 孙晋虎；

【作者基本信息】 广西医科大学 ， 口腔临床医学， 2015， 硕士

【摘要】 目的本研究通过载体微珠将柠檬醛植入正常发育的20期鸡胚鼻窝组织中,建立鸡胚唇裂模型；制备地高辛标记的成纤维细胞生长因子8(FGF8)RNA探针,通过整胚原位杂交的方法检测鸡胚唇部发育和唇裂发生过程中FGF8基因时空表达的变化,探索FGF8在唇裂发生中机理,为早期发现预防唇裂提供理论依据。方法1. FGF8 RNA探针的制备本实验应用Marion WassefPhD惠赠的鸡FGF8/pBluescript KS Ⅱ (+)重组质粒,EcoRI、Xhol限制性内切酶酶切得到线性化DNA模板,利用pBluescrip tKSⅡ(+)载体中的T3、T7RNA聚合酶上的启动子,体外转录合成地高辛标记的FGF8正、反义RNA探针。探针的合成为后续的整胚原位杂交试验做好了准备。2.各个时期鸡胚的收集将正常发育的20期鸡胚随机分为3组,分别是实验组、阴性对照组、正常空白对照组。实验组(CT组)：20期鸡胚右侧鼻窝外胚层上植入携带0.6g/mL浓度的柠檬醛微珠；阴性对照组(DMSO组)：20期鸡胚右侧鼻窝外胚层植入浸泡于DMSO中的微珠；空白对照组：20期鸡胚未经任何处理。3组分别收集20期至28期每期5只鸡胚。3.整胚原位杂交检测FGF8基因mRNA的时空表达通过整胚原位杂交的方法检测鸡胚唇部发育和唇裂发生过程中FGF8基因的时空表达变化。结果1、体外成功转录获得正、反义鸡的FGF8 RNA探针。2、正常组、DMSO组和CT组正常侧三者唇部发育无明显差别。正常组、DMSO组和CT组正常侧鸡胚20期微珠植入1h后,鼻板凹陷形成浅窝；22期时面部各突起已经形成,由额鼻突、侧鼻突、上颌突围成鼻窝；24期内外侧鼻突与上颌突围成的鼻缝较宽,并与下颌突一起围成了口凹；随时间推移,各突起逐渐发育并接触融合,两侧对称边缘连续。20期+1hCT组实验侧未观察到明显异常；22期开始观察到上颌突、侧鼻突出现发育异常,上颌突及内外侧鼻突发育缺损,体积较正常组明显缩小,突起融合发生障碍。3、FGF8的表达在鸡胚颌面部呈动态性,正常组、DMSO组和CT组正常侧三者颌面部FGF8的表达无明显差别。正常组、DMSO组和CT组正常侧中可观察到20期+1h时FGF8在额鼻区和鼻窝内侧边缘广泛高表达,上颌突的下缘和尾端及下颌突的外侧上缘也呈高表达；随着颌面部的发育,22期FGF8的表达局限在额鼻突中央、内侧鼻突、上颌突下缘及下颌突的上缘；内侧鼻突的表达在24期扩展至外侧鼻突,额鼻突中央及下颌突表达消失；24期后FGF8的表达开始下降,28期FGF8在突起接触融合处表达明显降低甚至消失。20期+1hCT组实验侧FGF8的表达未观察到明显异常；22期至24期侧鼻突FGF8的表达较正常组、DMSO组和CT组正常侧下降；26期及28期侧鼻突与上颌突融合处FGF8的表达较正常组、DMSO组和CT组正常侧高。结论1、制备的FGF8反义RNA探针具有较高的特异性,能有效的检测FGF8基因的表达。2、柠檬醛可影响鸡胚唇部组织FGF8的表达,FGF8表达的变化影响鸡胚唇部的发育,提示FGF8在唇部发育和唇缺损畸形发生过程中具有重要作用。更多还原

【Abstract】 Objective Citral beads were implanted into the right nasal pit of chicken embryos at stage 20, to establish the model of chicken embryos cleft lip. Probe for FGF8 of chicken were labeled with digoxingenin-UTP, to detect the temporal and spatial expression of FGF8 in the chicken embryo cleft lip by the method of Whole-mount in situ hybridization. Then to investigate the mechanism of FGF8 in the development of chicken embryo’s lip, and provide the theretical evidence for preventive action.Methods1、Preparation of probe for FGF8 of chicken labeled with digoxingenin-UTPRecombinant plasmid were presentation by Marion Wassef PhD, then plasmids were linearized with the restriction enzymes of EcoRI and XhoI respectively. The sense and anti-sense DIG labeled RNA probes were synthetized in vitro by T3 and T7 RNA polymerase respectively, and RNA probes for Whole-mount in situ hybridization were prepared.2、Establishment the model of chicken embryo cleft lipAt the stage 20, normal development of chicken embryos were randomly divided into 3 groups:CT group, DMSO group and normal group. CT group:beads which were soaking in citral with concentration of 0.6 g/ml which were dissolved in the DMSO were implantation on the right side of the nasal pit under the ectoderm. DMSO group:beads which were soaking in DMSO were implantation on the right side of the nasal pit under the ectoderm. Normal group without any operation. Then the chicken embryos continued incubating until stage20+1h,22,24,26 and 28, and selected 5 embryos in different groups of each stage.3、Temporal and spatial expression pattern of the FGF8 genesThe temporal and spatial expression of FGF8 during the development of chicken embryo’s lip were detected by the the method of whole-mount in situ hybridization.Results1、The probes for FGF8 of chicken labeled with DIG were synthetized in vitro and positive signals were viewed in chick embryos.2、There was no significant difference of morphology between the Normal group, DMSO group and normal side of CT group. At the stage 20+1h of normal group and DMSO group and normal side of CT group, nasal placodes were became fossa. The nasal pit were formed with frontonasal prominences, lateral nasal prominences and maxillary prominences at stage 22. By the development of chicken embryos the facial process continued proliferation, growth and fusion with each other. While on the right side of CT group, it was not obviously abnormal at the stage20+1h, but was observed that the maxillary prominences and lateral nasal prominences defected lightly after stage22, and the right nasal pit was fusional impairment.3、The expression of FGF8 in the craniofacial of chicken embryos were dynamics, and there was no significant difference of the expression between the Normal group, DMSO group and normal side of CT group. At the stage 20+1h of normal group and DMSO group, FGF8 was high expression in the frontonasal prominences and the margin of median nasal prominences, lower edge and bottom of maxillary prominences, lateral superior border of mandibular prominences. With development the expression of FGF8 limitations in the middle of frontonasal prominences and median nasal prominences, lower edge of maxillary prominences, lateral superior border of mandibular prominences. At stage 24, the expression were disappear in the frontonasal prominences and maxillary prominences, and the median lateral nasal prominences extended to the lateral nasal prominences. After stage 24, the expression of FGF8 began to decline,and significantly reduced or even disappear after fusion contact of the prominences. While on the right side of CT group, the expression of FGF8 was not obviously abnormal with the normal group and the normal side of CT group at the stage20+1h, but was down obviously at stage 22 to 24. While after stage 24, it was high expression than the normal group.Conclusions1.The antisense probe of chicken has high specificity, and be effectively to detect the expression of FGF8 gene during chicken development.2. Citral can regulate the expression of FGF8, and cause the lip tissue defect of chicken embryos.更多还原