GRIM-19在小鼠植入前胚胎中表达及作用的研究

发布时间：2018-08-16 18:08

【摘要】：目的线粒体是卵胞质中含量最为丰富的细胞器,其产生的ATP是卵子、受精卵以及胚胎主要的能量来源。研究证明在胚胎早期发育过程中,线粒体经历了一系列形态和位置的改变,其中,线粒体的结构尤其内膜的各种成分与线粒体的功能密切相关,因此,在此期间线粒体的功能状态改变直接影响植入前胚胎的生长和增殖。如果线粒体呼吸链受损,使得ATP产生减少,还能导致非整倍体的发生,并干扰正常的受精及胚胎发育而影响卵子的发育潜能。干扰素／维甲酸联合应用诱导细胞凋亡相关基因(gene associated with retinoid-interferon-induced mortality-19,GRIM-19)是线粒体呼吸链复合物Ⅰ的一部分,其对线粒体呼吸链复合体Ⅰ-Ⅳ的装配及线粒体呼吸链的电荷转移和电化学势能的维持都是必需的,GRIM-19缺乏会导致线粒体结构、分布、以致功能的异常。研究显示,缺乏GRIM-19的小鼠胚胎在9.5天死亡提示GRIM-19在胚胎发育中起着非常关键的作用,并且GRIM-19在心脏的早期发育中也起着重要的作用。目前尚无GRIM-19在植入前胚胎中表达情况的研究报道,GRIM-19在早期胚胎有何作用及作用机制仍不清楚。本研究通过检测GRIM-19基因在小鼠植入前胚胎的表达,观察GRIM-19在植入前胚胎的动态表达情况,探讨GRIM-19对早期胚胎生长发育的作用。方法 1、小鼠超排卵及胚胎获取雌性性成熟昆明系小鼠,腹腔注射HMG10IU/只,48h后腹腔注射hCG10IU/只,与性成熟雄性昆明系小鼠按1：1比例合笼过夜。次日晨检查雌鼠阴栓,阴栓阳性的(hCG后27h)脱颈法处死雌鼠,取双侧输卵管,在实体显微镜下,取出受精卵,转入四孔皿,每孔中加入400μl预先在培养箱中平衡的G1培养液,覆盖矿物油,37℃、5%CO2培养箱内培养备用。分别在注射hCG后的46-48h、55h、65h、75h、84-86h取2-细胞、4-细胞、8-细胞、桑葚胚及囊胚期的胚胎。 2、胚胎分级 8-细胞胚胎分级标准：1级,卵裂球等大、均质透明、无碎片；2级,卵裂球不完全等大、均质透明、无碎片；3级,卵裂球等大、均质但有少数碎片存在于卵间隙中；4级,卵裂球不等大、有碎片、颗粒不均变黑等。因有些级别的胚胎数太少为便于统计将1,2,3级胚胎列为优胚组(A组),4级为非优胚组(B组) 3、Western blot 分别将收集到的各期胚胎(500个)用PBS冲洗1-2次,加入20μl蛋白提取缓冲液,在超声波细胞粉碎机下超声20次(130watt,20khz,5s)。加入SDS样品缓冲液,100℃煮沸5min, SDS-PAGE电泳分离后将蛋白转至PVDF膜上,5%奶粉封闭,然后与兔抗鼠IgG/GRIM-19抗体(1：500),兔抗鼠p-actin抗体(1：4000)4℃孵育过夜。辣根过氧化物酶(HRP)偶联的羊抗兔IgG作为二抗(1：5000),常温孵育1h。ECL化学发光法显色,胶片于暗室中曝光。用Image J软件获得各条带光密度值,以目的基因与内参β-actin比值表示其含量 4、Real-time PCR i.配置胚胎裂解液和逆转录液(50μl体系,冰上配制)2μl NP-40,1μl RRI (Ribonuclease Inhibitor),10μl5×Prime ScriptTM Buffer (for Real Time),2.5μl Oligo dt Primer (50μM)×1,5μl Random6mers (100μM),22μl Rnase Free dH2O。 ii.胚胎的裂解通过上述方法获取的各期胚胎,用Tyrode’s酸去除透明带,经PBS冲洗,在体视镜下用微吸管吸取20个卵裂球,吸入体积控制在5μl以内,分别加至己含细胞裂解液的无RNA酶的PCR管中,空白对照管中加入无细胞的等量PBS。放置冰上2h,充分裂解细胞。 ⅲ.逆转录向上述各PCR管中加入3μl Prime ScriptTM RT Enzyme Mix I混匀,反应条件：第一步,37℃20min,第二步,85℃5sec,第三步,12℃forever,第四部,stop。 iv. Real Time PCR(20μl体系)10Oμl SYBR Premix Ex TagTM,0.2μl PCR F Primer,0.2μl PCR R Primer,2.0μl cDNA模板,7.6μl3dw灭菌。反应条件：95℃30sec,Holding stage；95℃5sec,60.0℃30sec,40cycles;95.0℃15sec,60.0℃1min,90℃15sec,Melt curve stage. 5、显微注射抗体小鼠促排卵获取受精卵(如上所述),随机分为两组,a组(注射抗体组)使用显微注射技术将抗GRIM.19抗体注射至受精卵胞质中,b组(空白对照组)将等量的G1培养液注射至受精卵胞质中,观察受精卵的发育情况。结果 1、GRIM-19蛋白在小鼠植入前各期胚胎中均有表达,其蛋白水平从2-细胞期逐渐增高,至8-细胞期达高峰,随后呈下降趋势,至囊胚期达到最低。 2、GRIM-19mRNA转录水平从2-细胞期逐渐增高,至8-细胞期达高峰,随后呈下降趋势,至囊胚期达到最低。 3、8-细胞胚胎中的优胚组GRIM-19mRNA水平明显高于非优胚组(P0.05)。 4、显微注射抗GRIM-19抗体组胚胎的成活率低于对照组,并且相对于对照组胚胎,实验组胚胎的生长发育受到不同程度的阻滞,胚胎不能正常发育至囊胚阶段。结论 1.GRIM-19在小鼠植入前胚胎中持续表达,其表达量随发育时程的变化而改变,提示GRIM-19在早期胚胎发育过程中具有一定的作用,并随胚胎增殖进程而有所变化,其中具体的机制有待更深入的研究。 2.GRIM-19的表达量与胚胎分级正相关,说明GRIM-19的表达与胚胎质量及发育潜能相关,提示GRIM-19可以作为评价早期胚胎发育潜能的一个潜在指标。 3.GRIM-19蛋白的阻断能影响胚胎的存活率,并阻滞小鼠早期胚胎的发育,提示GRIM-19在胚胎的早期发育中发挥重要作用。
[Abstract]:objective
Mitochondria are the most abundant organelles in the cytoplasm of eggs. The ATP produced by mitochondria is the main energy source of eggs, fertilized eggs and embryos. If the mitochondrial respiratory chain is damaged, ATP production will decrease, aneuploidy will occur, and normal fertilization and embryonic development will be interfered with, affecting the development potential of the egg.
Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) is a part of mitochondrial respiratory chain complex I. Its assembly of mitochondrial respiratory chain complex I-IV, charge transfer of mitochondrial respiratory chain and maintenance of electrochemical potential energy are also involved. Necessary, the lack of GRIM-19 leads to abnormal mitochondrial structure, distribution, and function. Studies have shown that the death of mouse embryos lacking GRIM-19 at 9.5 days suggests that GRIM-19 plays a crucial role in embryonic development, and GRIM-19 also plays an important role in the early development of the heart. The expression of GRIM-19 in mouse preimplantation embryos was detected to observe the dynamic expression of GRIM-19 in preimplantation embryos and to explore the effect of GRIM-19 on the growth and development of early embryos.
Method
1, superovulation and embryo acquisition in mice.
Female mature Kunming mice were intraperitoneally injected with HMG10IU/mice, 48 hours later, intraperitoneally injected with hCG10IU/mice, and sexually mature male Kunming mice were caged in a ratio of 1:1 overnight. The next morning, female rats were examined for pudendal suppository, and the female rats were killed by the method of pudendal suppository positive (27 hours after hCG). Bilateral fallopian tubes were taken out under the solid microscope, fertilized eggs were transferred into four-hole dishes. A 400 ml pre-balanced G1 medium was added into the hole and covered with mineral oil. The embryos were cultured in the incubator at 37 C and 5% CO2. The 2-cell, 4-cell, 8-cell, mulberry and blastocyst stage embryos were taken at 46-48 h, 55 h, 65 h, 75 h, 84-86 h after injection of hCG.
2, embryo grading
8-Cell embryo grading standard: 1 grade, large blastomere, homogeneous transparent, no fragments; 2 grade, blastomere incomplete large, homogeneous transparent, no fragments; 3 grade, blastomere large, homogeneous but a few fragments exist in the egg space; 4 grade, blastomere is not large, there are fragments, granular non-uniform black and so on. The 1,2,3 grade embryos were classified as the excellent embryo group (group A), and the 4 group was the non embryo group (group B).
3, Western blot
The collected embryos (500 embryos) were washed with PBS for 1-2 times, then added with 20 ml protein extract buffer, and ultrasonic 20 times (130 watt, 20 khz, 5 s). SDS sample buffer was added, boiled at 100 C for 5 min, SDS-PAGE was separated and the protein was transferred to PVDF membrane, then sealed with 5% milk powder, and then mixed with rabbit anti-mouse IgG/GRIM-19 antibody (1. Horseradish peroxidase (HRP) conjugated sheep anti-rabbit IgG as a second antibody (1:5000), incubated at room temperature for 1 h. ECL chemiluminescence method was used to develop the color. The film was exposed in darkroom. The light density values of each band were obtained by image J software, and the content was expressed by the ratio of target gene to internal reference beta-actin.
4, Real-time PCR
I. Configuration of embryo lysate and reverse transcription fluid (50 ml system, prepared on ice) 2 UL NP-40, 1 u l RRI (Ribonuclease Inhibitor), 10 U L 5 *Prime ScriptTM Buffer (for Real Time), 2.5 u l Oligo DT Primer (50 u M)*1,5 u l Random 6mers (100 u M), 22 u l Rnase Freed H2O.
Ii. Embryos obtained by the above-mentioned method were washed with PBS after removal of zona pellucida by Tyrode's acid. Twenty blastomeres were sucked by micropipette under stereoscopy. The volume of inhalation was controlled within 5 ml. The blastomeres were added to RNA-free PCR tubes containing cell lysate, and the blank control tubes were added with the same amount of acellular PBS. 2H, fully lysate cells.
I I I. Reverse transcription was performed by adding 3 ml Prime ScriptTM RT Enzyme Mix I into the PCR tubes. The reaction conditions were as follows: the first step, 37 20 min, the second step, 85 5 sec, the third step, 12 forever, the fourth part, stop.
IV. Real Time PCR (20 ull system) 10O_ L SYBR Premix Ex TagTM, 0.2_ L PCR F Primer, 0.2_ L PCR R Primer, 2.0_ L cDNA template, 7.6_ L 3 DW sterilization. Reaction conditions: 95 30sec, Holding stage; 95 5sec, 60.0 30sec, 40cycles; 95.0 15sec, 60.0 sec, 1 min, 90 15sec, Melt curve stage.
5, microinjection antibody.
Mice were randomly divided into two groups, group a (injection antibody group) was injected with anti-GRIM.19 antibody into the cytoplasm of fertilized eggs, group B (blank control group) was injected with the same amount of G1 culture medium into the cytoplasm of fertilized eggs to observe the development of fertilized eggs.
Result
1. GRIM-19 protein was expressed in mouse embryos at different stages before implantation. Its protein level gradually increased from 2-cell stage to 8-cell stage, reached the peak, then decreased, and reached the lowest level at blastocyst stage.
2. The transcription level of GRIM-19 mRNA increased gradually from 2-cell stage to 8-cell stage, reached a peak, then decreased to the lowest level in blastocyst stage.
The level of GRIM-19mRNA in 3,8- embryos was significantly higher than that in the non embryo group (P0.05).
4. The survival rate of embryos in the microinjection group was lower than that in the control group. Compared with the control group, the growth and development of embryos in the experimental group were blocked to some extent, and the embryos could not develop to blastocyst stage normally.
conclusion
1. GRIM-19 was continuously expressed in mouse preimplantation embryos. The expression of GRIM-19 changed with the development of mouse preimplantation embryos, suggesting that GRIM-19 plays a certain role in early embryonic development and changes with the process of embryo proliferation. The specific mechanism needs further study.
2. The expression of GRIM-19 was positively correlated with embryo grading, indicating that the expression of GRIM-19 was correlated with embryo quality and developmental potential, suggesting that GRIM-19 could be used as a potential indicator for evaluating the developmental potential of early embryos.
3. The blockade of GRIM-19 protein can affect the survival rate of embryos and block the development of mouse early embryos, suggesting that GRIM-19 plays an important role in the early development of embryos.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321-33

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