GRIM-19在小鼠卵母细胞和植入前胚胎中表达及定位的研究

发布时间：2018-02-26 20:35

  本文关键词： GRIM-19 线粒体 植入前胚胎 定位　出处：《山东大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的 GRIM-19(干扰素/甲酸联合应用诱导细胞凋亡相关基因)是GRIMs (genes associated with retinoid interferon induced mortality)家族成员之一,属于IFN/RA诱导的细胞凋亡调节因子。在细胞的增殖和凋亡的调控过程,GRIM-19蛋白表达量的减少或基因位点突变都可能会引起正常细胞开始转向恶性增殖。 在胚胎的早期发育过程中,线粒体的数量、分布和活性都会随胚胎的发育过程而改变,线粒体功能状态的改变都会直接影响到植入前胚胎的生长和发育。而GRIM-19作为线粒体复合物Ⅰ的一个基本功能单位,在线粒体Ⅰ型呼吸过程中起着至关重要的作用,它的异常表达和缺失都会导致早期胚胎的异常发育和生长。但据研究表明,到目前为止,关于GRIM-19在小鼠卵母细胞和植入前胚胎细胞中的表达和定位情况的研究,尚无明确的报道,在小鼠卵母细胞和植入前胚胎生长和发育过程中,GRIM-19到底是如何发挥作用的我们还不是很清楚。本研究拟通过观察GRIM-19在小鼠卵母细胞和植入前各期胚胎中的表达和定位情况,探讨了GRIM-19对早期胚胎生长发育的作用,为进一步探讨胚胎的发育潜能和胚胎质量评估方法提供新的思路。 方法 1收集小鼠成熟卵细胞和胚胎 选取10只雌性性成熟的健康小鼠,每只通过腹腔注射法注射尿促性素10IU,间隔48小时后,每只再通过腹腔注射法注射绒促性素10IU,将这些雌性与雄性小鼠1：1比例合笼,第二天检查雌鼠有无阴栓。阴栓呈现阳性的,利用脱颈法处死小鼠,取雌鼠的双侧输卵管,在实体显微镜下,从输卵管中,取出其受精卵。将预先在37℃培养箱中平衡过的400μ1G1培养液加入到四孔皿中,受精卵转入四孔皿后,矿物油覆盖,置于37℃、6%C02培养箱内培养。分别在注射HCG48h后,每隔10h分别取2-细胞胚胎、4-细胞胚胎、8-细胞胚胎、桑葚胚及囊胚期的各胚胎。同时处死HCG处理过的雌鼠,取出成熟的卵母细胞。 2免疫荧光化学法检测GRIM-19在鼠胚的表达 分别将收集到的卵母细胞与各期胚胎细胞,PBS洗2-3次。移入新鲜配制的4%多聚甲醛室温固定15min, PBS洗3-4次。移入PBS,在室温下放置8min,用含10%的山羊血清的PBS封闭1小时。PBS洗2次,移入100μl一抗(1：100稀释),放入湿盒室温孵育1h或4℃过夜。PBS洗3次,移入200μl二抗(1：400稀释)室温作用1h。用PBS洗4次,每次5min,将卵母细胞与胚胎移入Mito tracker (invitrogen)(1：1000稀释),温育10min, PBS洗3-4次。PBS洗4次,移入DAPI温育5min,PBS洗3次。加15μl抗淬灭剂于多聚赖氨酸处理过的载玻片上,将卵母细胞与胚胎分别滴在载玻片上,盖上盖玻片,涂好指甲油。Zeiss激光共聚焦扫描显微镜观察小鼠卵母细胞与各期胚胎GRIM-19蛋白的表达及定位情况,激发光的波长为543nm。3囊胚△Ψm的分析 按照1：1的比例将预先配好的JC-1工作液,添加到含有培养胚胎的培养液中,并置于培养箱中染色25min,条件为37℃,6%的CO,用培养液(其中含10%SSS的mHTF)多次冲洗胚胎后,利用荧光显微镜的绿色荧光模块和红色荧光模块进行观察,拍照获取所需图像,同时测量了同一时期胚胎的红色荧光和绿色荧光强度值,两者荧光值进行比较,重复上面的操作,对不同时期囊胚进行测量,比较分析△Ψm。 结果 通过对细胞核和线粒体进行染色,利用免疫荧光组织化学法,在激光扫描共聚焦显微镜下观察GRIM-19在小鼠卵母细胞,2-细胞,4-细胞,8-细胞,桑椹胚,囊胚中的表达和定位情况,结果显示在鼠胚各期植入前胚胎中GRIM-19均存在表达,且主要分布于细胞浆中,细胞核内几乎无表达。同时通过检测小鼠囊胚线粒体膜电位变化,与GRIM-19的表达比较,发现二者都呈现升高的趋势。 结论 1. GRIM-19在小鼠植入前胚胎各期中持续表达,同时小鼠囊胚线粒体膜电位变化与GRIM-19的表达一致,显示GRIM-19在早期胚胎发育过程中具有一定的作用,其中具体的机制有待更深入的研究。 2GRIM-19主要分布在细胞浆中,细胞核内几乎无表达,推测GRIM-19可能在胞浆中调控其他细胞凋亡相关蛋白,至于在核中的特异性功能还需进一步的实验证实。
[Abstract]:objective
GRIM-19 (interferon / combined acid induced apoptosis related gene GRIMs (genes) is associated with retinoid interferon induced mortality) is one of the members of the family IFN/RA induced apoptosis regulatory factor. In the regulation of cell proliferation and apoptosis, the expression of GRIM-19 protein decreased or mutation may cause normal cells to start turn to malignant proliferation.
In the early development of embryos, the number of mitochondria, the activity and distribution will change with the process of embryonic development, mitochondrial function changes will directly affect preimplantation embryo growth and development. GRIM-19 is used as a basic unit of the mitochondrial complex I can, plays a crucial role in type I respiratory mitochondrial process, its deletion and abnormal expression will lead to abnormal embryonic development. It is reported that so far, the research on the expression and localization of embryonic cells in mouse oocytes and preimplantation GRIM-19, there is no clear reports in mouse oocytes and preimplantation embryo growth and development process, how to play the role of GRIM-19 in the end we are not very clear. This study observed GRIM-19 in mouse oocyte and preimplantation embryo in each stage The effect of GRIM-19 on the growth and development of early embryos was discussed, providing new ideas for further exploration of embryo development potential and embryo quality assessment methods.
Method
1 to collect mature egg cells and embryos of mice
A total of 10 female adult healthy mice, each by intraperitoneal injection Menotrophin 10IU, 48 hours after the interval, each by intraperitoneal injection of hCG injection of 10IU, the proportion of male and female 1:1 mice mated female rats second days, check without vaginal plug Yin. Bolt positive, the mice were killed by cervical dislocation, the female rats in the fallopian tube, the entity under the microscope, the fallopian tubes, remove the fertilized eggs. The pre 37 degrees in the training in the balance box 400 1G1 medium was added to the four hole plate, the fertilized egg into the four hole plate after the mineral oil covered at 37 DEG C, 6%C02 culture in the incubator. Respectively after injection of HCG48h, 10h were taken every 2- cell embryos, embryonic 4- cells, 8- cell embryo, the embryo morula and blastocyst. While female mice treated with HCG were sacrificed and removed the mature oocytes.
Detection of GRIM-19 expression in mouse embryos by 2 immunofluorescent chemical assay
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