本文關鍵詞： GRIM-19 線粒體 植入前胚胎 定位　出處：《山東大學》2013年碩士論文　論文類型：學位論文

【摘要】：目的 GRIM-19(干擾素/甲酸聯(lián)合應用誘導細胞凋亡相關基因)是GRIMs (genes associated with retinoid interferon induced mortality)家族成員之一,屬于IFN/RA誘導的細胞凋亡調節(jié)因子。在細胞的增殖和凋亡的調控過程,GRIM-19蛋白表達量的減少或基因位點突變都可能會引起正常細胞開始轉向惡性增殖。 在胚胎的早期發(fā)育過程中,線粒體的數(shù)量、分布和活性都會隨胚胎的發(fā)育過程而改變,線粒體功能狀態(tài)的改變都會直接影響到植入前胚胎的生長和發(fā)育。而GRIM-19作為線粒體復合物Ⅰ的一個基本功能單位,在線粒體Ⅰ型呼吸過程中起著至關重要的作用,它的異常表達和缺失都會導致早期胚胎的異常發(fā)育和生長。但據(jù)研究表明,到目前為止,關于GRIM-19在小鼠卵母細胞和植入前胚胎細胞中的表達和定位情況的研究,尚無明確的報道,在小鼠卵母細胞和植入前胚胎生長和發(fā)育過程中,GRIM-19到底是如何發(fā)揮作用的我們還不是很清楚。本研究擬通過觀察GRIM-19在小鼠卵母細胞和植入前各期胚胎中的表達和定位情況,探討了GRIM-19對早期胚胎生長發(fā)育的作用,為進一步探討胚胎的發(fā)育潛能和胚胎質量評估方法提供新的思路。 方法 1收集小鼠成熟卵細胞和胚胎 選取10只雌性性成熟的健康小鼠,每只通過腹腔注射法注射尿促性素10IU,間隔48小時后,每只再通過腹腔注射法注射絨促性素10IU,將這些雌性與雄性小鼠1：1比例合籠,第二天檢查雌鼠有無陰栓。陰栓呈現(xiàn)陽性的,利用脫頸法處死小鼠,取雌鼠的雙側輸卵管,在實體顯微鏡下,從輸卵管中,取出其受精卵。將預先在37℃培養(yǎng)箱中平衡過的400μ1G1培養(yǎng)液加入到四孔皿中,受精卵轉入四孔皿后,礦物油覆蓋,置于37℃、6%C02培養(yǎng)箱內培養(yǎng)。分別在注射HCG48h后,每隔10h分別取2-細胞胚胎、4-細胞胚胎、8-細胞胚胎、桑葚胚及囊胚期的各胚胎。同時處死HCG處理過的雌鼠,取出成熟的卵母細胞。 2免疫熒光化學法檢測GRIM-19在鼠胚的表達 分別將收集到的卵母細胞與各期胚胎細胞,PBS洗2-3次。移入新鮮配制的4%多聚甲醛室溫固定15min, PBS洗3-4次。移入PBS,在室溫下放置8min,用含10%的山羊血清的PBS封閉1小時。PBS洗2次,移入100μl一抗(1：100稀釋),放入濕盒室溫孵育1h或4℃過夜。PBS洗3次,移入200μl二抗(1：400稀釋)室溫作用1h。用PBS洗4次,每次5min,將卵母細胞與胚胎移入Mito tracker (invitrogen)(1：1000稀釋),溫育10min, PBS洗3-4次。PBS洗4次,移入DAPI溫育5min,PBS洗3次。加15μl抗淬滅劑于多聚賴氨酸處理過的載玻片上,將卵母細胞與胚胎分別滴在載玻片上,蓋上蓋玻片,涂好指甲油。Zeiss激光共聚焦掃描顯微鏡觀察小鼠卵母細胞與各期胚胎GRIM-19蛋白的表達及定位情況,激發(fā)光的波長為543nm。3囊胚△Ψm的分析 按照1：1的比例將預先配好的JC-1工作液,添加到含有培養(yǎng)胚胎的培養(yǎng)液中,并置于培養(yǎng)箱中染色25min,條件為37℃,6%的CO,用培養(yǎng)液(其中含10%SSS的mHTF)多次沖洗胚胎后,利用熒光顯微鏡的綠色熒光模塊和紅色熒光模塊進行觀察,拍照獲取所需圖像,同時測量了同一時期胚胎的紅色熒光和綠色熒光強度值,兩者熒光值進行比較,重復上面的操作,對不同時期囊胚進行測量,比較分析△Ψm。 結果 通過對細胞核和線粒體進行染色,利用免疫熒光組織化學法,在激光掃描共聚焦顯微鏡下觀察GRIM-19在小鼠卵母細胞,2-細胞,4-細胞,8-細胞,桑椹胚,囊胚中的表達和定位情況,結果顯示在鼠胚各期植入前胚胎中GRIM-19均存在表達,且主要分布于細胞漿中,細胞核內幾乎無表達。同時通過檢測小鼠囊胚線粒體膜電位變化,與GRIM-19的表達比較,發(fā)現(xiàn)二者都呈現(xiàn)升高的趨勢。 結論 1. GRIM-19在小鼠植入前胚胎各期中持續(xù)表達,同時小鼠囊胚線粒體膜電位變化與GRIM-19的表達一致,顯示GRIM-19在早期胚胎發(fā)育過程中具有一定的作用,其中具體的機制有待更深入的研究。 2GRIM-19主要分布在細胞漿中,細胞核內幾乎無表達,推測GRIM-19可能在胞漿中調控其他細胞凋亡相關蛋白,至于在核中的特異性功能還需進一步的實驗證實。
[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
鍒嗗埆灝嗘敹闆嗗埌鐨勫嵉姣嶇粏鑳?yōu)涓庡悇鏈熻儦鑳幘l嗚優(yōu),PBS媧，

本文編號：1539624