自然流产(硕士)

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自然流产(硕士)(论文35000字)
中文摘要
女性在孕期前20周内发生三次以上的自然流产而导致的不孕不育现象称为反复自然流产(Recurrent Spontaneous Abortion, RSA),自然反复流产引起的不孕不育占所有不孕女性的10%左右,目前,由于该病的患病原因比较复杂,并且致病机理尚未清楚,在全世界都缺少相应有效的治疗方法和治疗药物。因此,关于RSA的发病机制以及治疗的研究,是当前医学的热门研究问题之一。
成功妊娠的建立和维持主要取决于滋养层细胞,它是参与早期胎盘发育的主要胎儿细胞。滋养层细胞像肿瘤细胞一样具有增殖和侵袭的共同特征。 胎盘滋养细胞浸润不足和血管重塑不良导致妊娠相关并发症,包括RSA,先兆子痫和胎儿宫内发育迟缓。一些RSA研究者发现了许多导致滋养层细胞功能紊乱的危险因素,包括滋养层细胞毒性细胞免疫应答,滋养层细胞抗原和抗成粒细胞抗体,DNA损伤,颗粒溶素和异常的血管生成。由此可见,绒毛外滋养层细胞的侵袭能力与反复流产的发生密切相关。
MicroRNAs是由19-24个核苷酸长度组成的一类非编码小分子RNA,它们通过与3'UTR中的特定位点结合抑制信使RNA(mRNA)功能来调控基因表达。 越来越多的证据表明大量的miRNA在人胎盘中表达,并且miRNA异常表达可能参与URSA发病机制。然而,URSA中miRNA的临床意义还没有得到充分的探索。在本研究中,已发现miRNA-24在URSA患者的胎盘中异常表达,但其在流产发病机制中的作用仍不清楚。
本研究结果主要分为如下两个部分:
第一部分:MicroRNA-106a对人类滋养层细胞侵袭和迁移的影响。
本研究中,利用qPCR方法检测了URSA患者的胎盘组织中以及滋养层细胞中的MicroRNA-106a表达水平,结果发现URSA患者的胎盘组织中miRNA表达水平与健康人群比较存在较大的差异,接下来,构建了miR-34a过表达和低表达模型来研究miR-106a对JEG-3细胞侵袭和迁移调控的影响。 Western blotting和RT-qPCR分别应用于mRNA和蛋白表达的定量检测。MTT法检测JEG-3细胞的侵袭和迁移能力。结果:我们的研究表明,microRNA-106a(miR-106a)在人滋养层细胞和组织中相对于正常胎盘细胞和组织增加,并且转染miR-106a模拟物在体外抑制细胞增殖并促进细胞凋亡。此外,发现ZBTB7A 3'-UTR直接被miR-106a下调,表明ZBTB7A是滋养细胞系中miR-106a的重要靶点。当miR-106a在滋养层细胞中过度表达时,ZBTB7A的mRNA和蛋白质水平都被抑制,而miR-106a的抑制导致滋养层细胞系中ZBTB7A表达水平的增加。 ZBTB7A的敲低能显着促进滋养层细胞增殖,促进细胞凋亡,说明ZBTB7A可能作为滋养层细胞的抑制剂。ZBTB7A表达的恢复可以抵消miR-106a对滋养层细胞增殖的抑制作用,抑制滋养细胞的凋亡。总之,这些结果表明,miR-106a是ZBTB7A的一个新的调控因子,miR-106a和ZBTB7A在自然流产的发病机制中都起着重要的作用。
第二部分:MicroRNA-24-3p对人类滋养层细胞侵袭和迁移的影响。
本研究中,利用qPCR方法检测了URSA患者的胎盘组织中以及滋养层细胞中的miR-24-3p表达水平,结果发现URSA患者的胎盘组织中miR-24-3p表达水平与健康人群比较存在较大的差异。结果显示miR-24-3p在URSA胎盘中过表达。另外,通过提高JEG-3滋养层细胞系中的miR-124-3p水平来评估miR-24-3p的功能。发现JEG-3细胞的增殖,迁移和侵袭受到miR-124-3p表达的抑制。此外,我们发现ZNF654 mRNA是JEG-3细胞中使用双荧光素酶报告基因测定法的miR-24-3p的直接靶标。另外,通过提高miR-24-3p水平,通过ZNF654的低表达来鉴定JEG-3细胞的侵袭。我们的研究结果表明miR-24-3p通过直接靶向ZNF654在URSA中发挥非常重要的作用。
综上所述,我们的研究表明,MicroRNA-106a和MicroRNA-24-3p在调控滋养层细胞的侵袭和迁移功能方面具有着重要的调节作用。

关键词:滋养层细胞、MicroRNA-106a、MicroRNA-24-3p、侵袭、迁移
 
Abstract
Infertility caused by spontaneous abortion more than three times during the first 20 weeks of pregnancy is called Recurrent Spontaneous Abortion (RSA). Infertility caused by recurrent miscarriage accounts for 10% of all infertile women %, At present, due to the complex causes of the disease, and the pathogenesis is not yet clear, the world is the lack of appropriate effective treatment and treatment of drugs. Therefore, the study on the pathogenesis and treatment of RSA is one of the most popular medical problems.
The establishment and maintenance of a successful pregnancy depends mainly on the trophoblast cells, which are the primary fetal cells involved in early placental development. Trophoblast cells share the same hallmarks of proliferation and invasion as tumor cells. Inadequate placental trophoblast infiltration and poor vascular remodeling lead to pregnancy-related complications including RSA, pre-eclampsia and intrauterine growth retardation. Some RSA researchers have identified a number of risk factors that contribute to trophoblastic dysfunction including trophoblast immune responses, trophoblastic and anti-granulocyte antibodies, DNA damage, granulysin, and aberrant angiogenesis. Thus, villi trophoblast cells invasion and recurrent abortion are closely related.
miRNAs are a group of non-coding small RNAs consisting of 19-24 nucleotides in length that regulate gene expression by inhibiting messenger RNA (mRNA) function by binding to specific sites in the 3'UTR. There is growing evidence that a large number of miRNAs are expressed in human placenta, and abnormal miRNA expression may be involved in the pathogenesis of URSA. However, the clinical significance of miRNAs in URSA has not been sufficiently explored. In this study, miRNA-24 has been found to be abnormally expressed in the placenta of URSA patients, but its role in the pathogenesis of miscarriage remains unclear.
The results of this study are mainly divided into the following two parts:
Part I: Effect of MicroRNA-106a on Invasion and Migration of Human Trophoblast Cells.
In this study, qRT-PCR was used to detect the expression level of MicroRNA-106a in placenta and trophoblast cells of URSA patients. The results showed that miRNA expression level in placenta of URSA patients was significantly different from that of healthy people , MiR-34a overexpression and low expression models were constructed to study the effect of miR-106a on invasion and migration regulation of JEG-3 cells. Western blotting and RT-qPCR were applied to the quantitative detection of mRNA and protein expression respectively.MTT assay was used to detect the invasion and migration of JEG-3 cells. Results: Our study shows that microRNA-106a (miR-106a) is increased in human trophoblast cells and tissues relative to normal placental cells and tissues, and transfection of miR-106a mimetics inhibits cell proliferation and promotes apoptosis in vitro . In addition, the 3'-UTR of ZBTB7A was found to be down-regulated directly by miR-106a, indicating that ZBTB7A is an important target for miR-106a in the trophoblast cell line. When miR-106a was overexpressed in trophoblast cells, both mRNA and protein levels of ZBTB7A were inhibited, whereas inhibition of miR-106a resulted in an increase in ZBTB7A expression in trophoblast cells. ZBTB7A knockdown can significantly promote trophoblast proliferation and promote apoptosis, indicating that ZBTB7A may act as an inhibitor of trophoblast cells. The recovery of ZBTB7A expression can counteract the inhibitory effect of miR-106a on the proliferation of trophoblast cells and the inhibition of trophoblast apoptosis. Taken together, these results suggest that miR-106a is a novel regulator of ZBTB7A, both of which play an important role in the pathogenesis of spontaneous abortion.
Part II: Effect of MicroRNA-24-3p on Invasion and Migration of Human Trophoblast Cells.
In this study, qPCR method was used to detect the expression of miR-24-3p in placenta and trophoblast cells of URSA patients. The results showed that the expression level of miR-24-3p in URSA patients placenta compared with healthy people Big difference. The results show that miR-24-3p is overexpressed in URSA placenta. In addition, the function of miR-24-3p was assessed by increasing miR-124-3p levels in JEG-3 trophoblast cell lines. The proliferation, migration and invasion of JEG-3 cells were found to be inhibited by miR-124-3p expression. In addition, we found that ZNF654 mRNA is the direct target of miR-24-3p using dual luciferase reporter assay in JEG-3 cells. In addition, JEG-3 cell invasion was identified by low expression of ZNF654 by increasing miR-24-3p levels. Our results indicate that miR-24-3p plays a very important role in URSA by targeting ZNF654 directly.
Taken together, our study shows that MicroRNA-106a and MicroRNA-24-3p play an important regulatory role in the regulation of trophoblast cell invasion and migration.

Keywords: Trophoblast cells, MicroRNA-106a, MicroRNA-24-3p, Invasion, Migration

目录
中文摘要    1
Abstract    3
目录    6
前言    8
1.1自然流产的发生率    8
1.2自然流产的诊断    9
1.3病因和风险因素    9
1.3.1 一般风险因素    9
1.3.2 遗传因素    10
1.3.3 解剖学因素    10
1.3.4 抗磷脂综合症    11
1.3.5内分泌因素    12
1.3.6其他免疫相关因素和自然杀伤细胞的作用    12
1.3.7感染的影响    14
1.4 RSM的治疗    14
1.4.1怀孕前的治疗    14
1.4.2在怀孕期间的治疗    16
1.4.3 抗磷脂综合症    16
1.5 miRNAs 在调控胎盘发育与功能方面起到重要的作用    17
实验材料和方法    19
2.1实验材料    19
2.1.1 实验组织样本    19
2.1.2实验细胞    19
2.1.3实验菌种    19
2.1.4主要实验试剂    19
2.1.5 实验仪器设备    22
2.1.6主要试剂的配制    23
2.2实验方法    25
2.2.1细胞培养    25
2.2.2实时荧光定量PCR法    26
2.2.3 Western Blot检测    28
2.2.4 MTT 法检测细胞黏附能力    32
2.2.5 Transwell 法测细胞侵袭能力    32
2.2.6 划痕实验    33
2.2.7 组织基因组的提取    34
2.2.9质粒的提取    35
2.2.10 质粒的化学转化    36
2.2.11 DNA琼脂糖凝胶电泳    36
2.2.12 载体构建    36
2.2.13重组质粒转染    38
2.2.14双荧光素酶报告基因分析    38
2.2.3甲醛变性琼脂糖凝胶电泳检查总RNA的完整性    39
2.2.4数据处理和统计学方法分析    40
结果    41
3.1 MicroRNA-106a对人类滋养层细胞侵袭和迁移的影响    41
3.1.1 miR-106a在胎盘中的表达水平    41
3.1.2 敲低miR-106a促进JEG-3细胞的增殖,迁移和侵袭    41
3.1.3 miR-106a的过度表达抑制了JEG-3细胞的增殖,迁移和侵袭    42
3.1.4 ZBTB7A是miRNA-106a的靶基因    43
3.2 MicroRNA-24-3p对人类滋养层细胞侵袭和迁移的影响    45
3.2.1 URSA临床样品中miR-24-3p和ZNF654的表达    45
3.2.2 MiR-24-3p抑制JEG-3细胞的增殖,迁移和侵袭    46
3.2.3 MiR-24-3p通过靶向ZNF654来抑制JEG-3细胞的侵袭和迁移    47
讨论    49
4.1 MicroRNA-106a对人类滋养层细胞侵袭和迁移的影响    50
4.2 MicroRNA-24-3p对人类滋养层细胞侵袭和迁移的影响    51
结论    54
参考文献    55