采用原位末端标记法检测人植入前胚胎中的凋亡征象

目的建立原位末端标记法（TdT-mediated nick end labeling TUNEL）检测人植入前胚胎凋亡征象的方法,阐明凋亡与人类植入前胚胎发育的关系。方法取试管婴儿助孕技术后异常受精的三原核（3PN）胚胎,于2-10细胞期进行固定,采用TUNEL法检测3PN胚胎中的凋亡征象。结果在发育正常3PN胚胎中,未检测到凋亡征象;在20%有碎片的3PN胚胎中检测到TUNEL阳性信号。结论采用TUNEL法可以有效检测人植入前胚胎中的凋亡征象,植入前胚胎中的细胞碎片可能与凋亡有关。     

人植入前胚胎细胞超薄电镜切片制作

目的改进人植入前胚胎细胞超薄切片的制备方法,观察不同时期人早期胚胎细胞超微结构的变化。方法选取我院生殖中心试管婴儿助孕技术后异常受精的3原核胚胎,于2~8细胞期进行固定,改进透射电镜的常规操作方法,使用3%~4%琼脂进行预包埋,制成超薄切片后在镜下观察植入前胚胎细胞凋亡的超微结构。结果使用本方法可以对少量胚胎进行超薄切片的制作。结论使用3%~4%琼脂预包埋的方法可以成功的制作人植入前胚胎细胞的超薄电镜切片。     

产前诊断与胚胎植入前论断

“产前”是指胎儿出生之前，而“植入前”是指受精卵分裂之卵裂球和囊胚种植入子宫内膜之前。为什么要在出生前早期再早期进行疾病诊断呢？这里有三个重要理由：１．生后很难治，绝大多数先天性和遗传性疾病，已证明有数千种，一旦出生后，除极少数疾病外，均不能治或很难治。因此必须在出生前或发育前进行早诊断，早预防。２．一个细胞就可能诊断遗传病的本质是遗传物质—基因，发生异常改变，主要表现在DNA和染色体发生突变或畸变，人类大约有５～１０万个结构基因，它们都蜷缩在每一个细胞核内，个别的基因在核外线粒体内。只要能得到…     

细胞凋亡的原位显示技术及应用

原位末端标记法是利用末端转移酶作为催化酶，在适当条件下将底物接合于ＤＮＡ双链钝端，再用免疫组化放大标记信号，然后ＤＡＢ显色，苏木素衬染，能结构完好地原位显示凋亡细胞     

胚胎植入前非整倍体筛查

染色体非整倍体改变在体外受精胚胎中十分多见 ,是导致人类辅助生殖失败的常见原因。胚胎细胞染色体的非整倍体筛查 ,已成为胚胎植入前遗传学诊断的主要内容。体外受精后胚胎活检 ,进行荧光原位杂交 ,避免非整倍体胚胎移入母体宫腔 ,能起到显著提高着床率和改善受孕率的效果。     

双重标记法定性检测AS斑块中的凋亡细胞

长期以来 ,人们一直认为动脉粥样硬化(ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ ,ＡＳ)是一种以平滑肌细胞 (ｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌｌ ,ＳＭＣ)增殖和变性为主要病变的疾病 ,但近年的研究表明 ,细胞凋亡作为一种生理性死亡形式不仅在正常的血管内存在 ,而且以高于正常情况下的凋亡率参与了ＡＳ的发病过程 ,并调节着动脉管壁中ＳＭＣ的数量[1] 。目前 ,细胞凋亡与ＡＳ关系的研究已成为探索ＡＳ发病机制的一条新途径。尽管已有大量的形态学证据支持细胞凋亡参与ＡＳ发生发展的观点 ,但由于ＡＳ斑块组成的相对复杂性 ,要阐明ＡＳ血管壁中凋亡…     

染色体易位携带者的胚胎植入前遗传学诊断研究进展

染色体易位是常见的染色体结构异常。应用荧光原位杂交技术，染色体易位携带者可在胚胎植入前遗传学诊断的帮助下增加正常妊娠的机会。现就相互易位减数分裂的情况进行综述，并讨论应用荧光原位杂交技术对染色体易位携带者进行胚胎植入前遗传学诊断的策略。     

哺乳动物胚胎植入前发育的基因表达

本文总结了哺乳动物植入前发育过程中特异基因的表达。对小鼠来说，在４－细胞期大部分检测的特异基因已经开始表达，有部分在２－细胞期就已开始表达。基因转录一旦激活将连续地进行下去，直至囊胚期，在此过程中基因转录产物ｍＲＮＡ不断累积。由于大部分基因表达的时间与３个遗传学形态转变期（紧贴期、成腔期、扩张囊胚期）的时间之间没有明确的相关，因此认为遗传形态改变的调控是在转录和转译的下游进行的。     

胚胎植入前遗传诊断技术的应用分析

过去20年间,胚胎植入前遗传检测(PGD)主要通过卵裂球活检结合聚合酶反应(PCR)或荧光原位杂交(FISH),对胚胎的单基因遗传异常,以及有限的染色体异常进行检测。近年来PGD的活检技术以及遗传检测技术都有巨大的提高。由于囊胚球分裂稳定性高,对胚胎干扰很小以及可提供的检测细胞多等特点,囊胚球活检在PGD中的应用逐渐受到重视。新兴的遗传检测方法,微阵列-比较基因组杂交(Array-CGH)以及单核苷酸多态性微阵列(SNP-array)技术使得同时检测24条染色体成为可能,并能以更高的精度检测小片段染色体的拷贝数变化、以及结构改变等。这使得PGD应用价值不仅在于可排除遗传异常胚胎,更可用于提高大龄生育、反复流产等不孕不育人群的受孕率。     

FISH技术在胚胎植入前遗传学诊断中的应用及前景

随着体外受精-胚胎移植技术的快速发展和进步，以及单细胞水平上基因诊断技术的成功，使得在体外受精过程中，胚胎移植入母体子宫前进行遗传学诊断成为可能。本文综述荧光原位杂交（FISH）技术在胚胎植入前遗传学诊断（PGD）中的应用和研究进展。     

兔心肌缺血性损伤致心肌细胞凋亡的定位及定量研究

目的探讨实验性急性心肌梗塞时心肌细胞凋亡的部位及凋亡率。方法结扎左冠状动脉的左室支建立心肌缺血模型,采用原位末端探针标记、透射电镜、自动图像分析仪和统计处理,对不同损伤区心肌细胞的损伤情况进行研究。结果中心缺血区心肌组织呈现缺血坏死征象,未见心肌凋亡细胞;在梗塞交界区存在心肌细胞凋亡现象,平均凋亡率为25.51％。结论急性心梗时,中心缺血区心肌损伤以坏死为主,心肌细胞凋亡参与梗塞交界区心肌的损害。这为进一步研究心肌梗塞的治疗提供一个新的参考依据。     

原位缺口平移法检测睾丸组织中凋亡细胞的应用

目的：研究原位缺口平移技术在显示睾丸细胞凋亡的应用。方法：采用原位缺口平移技术检测大鼠睾丸组织中ＤＮＡ断裂，显示细胞凋亡。结果：在睾丸组织中出现阳性标记细胞，在１０μｍ厚的切片上，每条精曲小管可见０～３个阳性标记细胞。结论：该方法可用于不同生理和病理条件下睾丸细胞凋亡的研究。     

肝细胞癌凋亡病变的原位末端标记显示

原位末端标记（ＩＳＥＬ）是显示凋亡的新技术。按Ｇａｖｒｉｅｌｉ法对４６例未接受过化疗与放疗的人肝细胞癌（ＨＣＣ）作回顾性（ＩＳＥＬ）研究，结果显示在对照系列中阴性与阳性均与理论相符，４６例ＨＣＣＩＳＥＬ（＋）可分为：①单个细胞（＋）；②点状（＋）；③小片状（＋）；④大片（＋）．Ⅰ级ＨＣＣ以单细胞（＋）较多；Ⅱ、Ⅲ级以小片状（＋）较多（Ｐ＜０．０５）．单个细胞（＋）为凋亡经典概念，而片状（＋）报道还不多。本文小片状（＋）很普遍，常位于癌巢中央，其ＨＥ形态以核浓缩为主，此结果表明，通常ＨＣＣ所见小片状＂变性坏死样改变＂可能为凋亡而非坏死。     

Developmental and cytogenetic assessments of preimplantation embryos derived from in-vivo or in-vitro matured human oocytes

Aneuploidy is of great relevance to embryo selection, as it represents one of the important causes of implantation failure. Furthermore, immature oocytes, retrieved during gonadotrophin-stimulated IVF cycles, are generally discarded in clinics; whereas, there was no detectable comprehensive evidence on higher rates of aneuploidy based on maturity status on the day of oocyte retrieval. As well, the correlation between embryo morphology on aneuploidy remains unclear. The aim was to evaluate the developmental and genetic integrity of human preimplantation embryos from rescue in-vitro matured MII stage oocytes as well as in vivo matured oocytes. 541 rescue in-vitro matured oocytes as case as well as 659 in-vivo matured oocytes as control were used for the developmental assay. Finally, 121 cleaved embryos with good quality were analyzed by FISH technique for the detection of chromosomes X, Y, 13, 15, 16, 18, 21 and 22. The fertilization rates were 61.62% and 61.76% in case and control groups, respectively. Also, embryo formation rates of 89.1% vs. 92.2% were recorded for case and control groups, respectively. Good quality embryos on day 3 were 62.54% in case and 68.36% in control groups. There were insignificant differences in fertilization, embryo formation and quality between the groups. Total abnormality in 35 of the 60 embryos was 58.5% in case and 62.3% in control (p < 0.05). There were significant differences between aneuploidy rates of embryos using only sex chromosome preimplantation genetic screening (PGS) and sex chromosome in combination with autosomal chromosomes PGS in case (58.5% vs 28.3%, p = 0.000) and control groups (62.3% vs 21.3%; p = 0.000). The results demonstrated that a high proportion of good quality embryos were aneuploid in both patient groups with no obvious increase in aneuploidies as a result of rescue IVM application. Furthermore, the morphological characteristics of embryos do not completely consistent with chromosomal content. Despite the Rescue IVM is currently not a routine procedure in association with IVF, our finding suggested a viable option for young infertile women facing cancellation of their IVF treatment due to ovarian over-response or resistance factors as well as patients with low functional ovarian reserve considering good quality of embryos from rescue IVM-MII oocytes.     

Caspase activity in preimplantation human embryos is not associated with apoptosis

BACKGROUND: Previous studies on mammalian preimplantation embryos have suggested an association between caspase activation, blastomere fragmentation and apoptosis. However, some reports on human embryos questioned the causal relationship between blastomere fragmentation and apoptosis, and information about the presence and activity of caspases in human embryos is lacking. METHODS: A fluorochrome-labelled universal caspase inhibitor was used to visualize active caspases in blastomeres and fragments of preimplantation human embryos. RESULTS: Caspase activity was detected only after fertilization, and was rare in blastomeres but frequent in fragments. The incidence of caspase activity in blastomeres and fragments was stable between the 2-cell and 12-cell stages. Caspase-positive blastomeres were only seen in poor-morphology embryos. The percentage of caspase-positive fragments was increased in embryos with multinucleated blastomeres but was unrelated to embryo morphology. Moreover, caspase-positive fragments detached from healthy blastomeres that were isolated by embryo biopsy and subsequently underwent mitotic division in culture. CONCLUSIONS: These data suggest that caspases in preimplantation human embryos are involved in developmental processes unrelated to cell death.     

可溶性糖基化终末产物受体抑制动物缺血再灌注导致的心功能障碍及心肌细胞凋亡

目的 建立动物心脏缺血再灌注模型,探讨可溶性糖基化终末产物受体（sRAGE）对缺血再灌注诱导的心功能及心肌细胞凋亡的作用。 方法 复制C57BL/6J小鼠心脏缺血再灌注模型,利用超声检测心功能;通过TUNEL染色及Caspase-3活性检测,评价心肌细胞凋亡的程度。 结果 与sham组比较,I/R组左室射血分数降低［sham组为（72.4±2.1）%,I/R组为（30.9±3.2）%,P<0.05］,短轴缩短率降低［sham组为(40.7±1.6)%, I/R组为（15.1±2.0）% , P<0.05］,TUNEL阳性心肌细胞数目增加［sham组为(1.0±0.2)%, I/R组为（20.0±1.6）% , P<0.05］, Caspase-3活性升高［（sham组(1.00±0.2）%比I/R组（2.64±0.4）%,P<0.05）。与I/R组相比较,sRAGE预处理能够明显升高左室射血分数［（46.5±2.0）% P<0.05］,增加短轴缩短率［（23.0±1.1）%,P<0.05］,同时降低TUNEL阳性心肌细胞数目［（9.2%±1.0）% P<0.05］,和Caspase-3活性［（1.94%±0.1）% P<0.05］。 结论 sRAGE能够明显改善缺血再灌注诱导的心功能降低,并抑制心肌细胞的凋亡。     

Increased apoptosis in human amnion is associated with labor at term

PROBLEM: To characterize whether increased apoptosis in human amnion was associated with labor at term. METHOD OF STUDY: Human amnion were obtained from term patients with vaginal delivery (n = 5) or who underwent elective Cesarean section (C/S) without labor (n = 5). Apoptosis was performed by the TUNEL (Terminal dUTP Nuclear End Labeling) assay. All nucleated cells stained with propidium iodide in the amnion epithelial cells were identified in red fluorescence. TUNEL positive apoptotic nuclei were identified in green fluorescence. Five random fields of each specimen were blindly counted by investigators. The percentage of apoptotic nuclei of total nuclei (apoptotic index) was calculated and compared between the two groups (25 microscopic fields for each group, respectively). RESULTS: Patients with term labor had a significantly higher mean apoptotic index in amnion epithelial cells than that with elective C/S without labor (27.3 +/- 4.1% versus 3.6 +/- 1.6%, P < 0.001). CONCLUSIONS: Our data indicate that apoptosis in human amnion is significantly increased and associated with labor at term.