自然流產(碩士)

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自然流產(碩士)(論文35000字)
中文摘要
女性在孕期前20周內發生三次以上的自然流產而導致的不孕不育現象稱為反復自然流產(Recurrent Spontaneous Abortion, RSA),自然反復流產引起的不孕不育占所有不孕女性的10%左右,目前,由于該病的患病原因比較復雜,并且致病機理尚未清楚,在全世界都缺少相應有效的治療方法和治療藥物。因此,關于RSA的發病機制以及治療的研究,是當前醫學的熱門研究問題之一。
成功妊娠的建立和維持主要取決于滋養層細胞,它是參與早期胎盤發育的主要胎兒細胞。滋養層細胞像腫瘤細胞一樣具有增殖和侵襲的共同特征。 胎盤滋養細胞浸潤不足和血管重塑不良導致妊娠相關并發癥,包括RSA,先兆子癇和胎兒宮內發育遲緩。一些RSA研究者發現了許多導致滋養層細胞功能紊亂的危險因素,包括滋養層細胞毒性細胞免疫應答,滋養層細胞抗原和抗成粒細胞抗體,DNA損傷,顆粒溶素和異常的血管生成。由此可見,絨毛外滋養層細胞的侵襲能力與反復流產的發生密切相關。
MicroRNAs是由19-24個核苷酸長度組成的一類非編碼小分子RNA,它們通過與3'UTR中的特定位點結合抑制信使RNA(mRNA)功能來調控基因表達。 越來越多的證據表明大量的miRNA在人胎盤中表達,并且miRNA異常表達可能參與URSA發病機制。然而,URSA中miRNA的臨床意義還沒有得到充分的探索。在本研究中,已發現miRNA-24在URSA患者的胎盤中異常表達,但其在流產發病機制中的作用仍不清楚。
本研究結果主要分為如下兩個部分:
第一部分:MicroRNA-106a對人類滋養層細胞侵襲和遷移的影響。
本研究中,利用qPCR方法檢測了URSA患者的胎盤組織中以及滋養層細胞中的MicroRNA-106a表達水平,結果發現URSA患者的胎盤組織中miRNA表達水平與健康人群比較存在較大的差異,接下來,構建了miR-34a過表達和低表達模型來研究miR-106a對JEG-3細胞侵襲和遷移調控的影響。 Western blotting和RT-qPCR分別應用于mRNA和蛋白表達的定量檢測。MTT法檢測JEG-3細胞的侵襲和遷移能力。結果:我們的研究表明,microRNA-106a(miR-106a)在人滋養層細胞和組織中相對于正常胎盤細胞和組織增加,并且轉染miR-106a模擬物在體外抑制細胞增殖并促進細胞凋亡。此外,發現ZBTB7A 3'-UTR直接被miR-106a下調,表明ZBTB7A是滋養細胞系中miR-106a的重要靶點。當miR-106a在滋養層細胞中過度表達時,ZBTB7A的mRNA和蛋白質水平都被抑制,而miR-106a的抑制導致滋養層細胞系中ZBTB7A表達水平的增加。 ZBTB7A的敲低能顯著促進滋養層細胞增殖,促進細胞凋亡,說明ZBTB7A可能作為滋養層細胞的抑制劑。ZBTB7A表達的恢復可以抵消miR-106a對滋養層細胞增殖的抑制作用,抑制滋養細胞的凋亡。總之,這些結果表明,miR-106a是ZBTB7A的一個新的調控因子,miR-106a和ZBTB7A在自然流產的發病機制中都起著重要的作用。
第二部分:MicroRNA-24-3p對人類滋養層細胞侵襲和遷移的影響。
本研究中,利用qPCR方法檢測了URSA患者的胎盤組織中以及滋養層細胞中的miR-24-3p表達水平,結果發現URSA患者的胎盤組織中miR-24-3p表達水平與健康人群比較存在較大的差異。結果顯示miR-24-3p在URSA胎盤中過表達。另外,通過提高JEG-3滋養層細胞系中的miR-124-3p水平來評估miR-24-3p的功能。發現JEG-3細胞的增殖,遷移和侵襲受到miR-124-3p表達的抑制。此外,我們發現ZNF654 mRNA是JEG-3細胞中使用雙熒光素酶報告基因測定法的miR-24-3p的直接靶標。另外,通過提高miR-24-3p水平,通過ZNF654的低表達來鑒定JEG-3細胞的侵襲。我們的研究結果表明miR-24-3p通過直接靶向ZNF654在URSA中發揮非常重要的作用。
綜上所述,我們的研究表明,MicroRNA-106a和MicroRNA-24-3p在調控滋養層細胞的侵襲和遷移功能方面具有著重要的調節作用。

關鍵詞:滋養層細胞、MicroRNA-106a、MicroRNA-24-3p、侵襲、遷移
 
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

目錄
中文摘要    1
Abstract    3
目錄    6
前言    8
1.1自然流產的發生率    8
1.2自然流產的診斷    9
1.3病因和風險因素    9
1.3.1 一般風險因素    9
1.3.2 遺傳因素    10
1.3.3 解剖學因素    10
1.3.4 抗磷脂綜合癥    11
1.3.5內分泌因素    12
1.3.6其他免疫相關因素和自然殺傷細胞的作用    12
1.3.7感染的影響    14
1.4 RSM的治療    14
1.4.1懷孕前的治療    14
1.4.2在懷孕期間的治療    16
1.4.3 抗磷脂綜合癥    16
1.5 miRNAs 在調控胎盤發育與功能方面起到重要的作用    17
實驗材料和方法    19
2.1實驗材料    19
2.1.1 實驗組織樣本    19
2.1.2實驗細胞    19
2.1.3實驗菌種    19
2.1.4主要實驗試劑    19
2.1.5 實驗儀器設備    22
2.1.6主要試劑的配制    23
2.2實驗方法    25
2.2.1細胞培養    25
2.2.2實時熒光定量PCR法    26
2.2.3 Western Blot檢測    28
2.2.4 MTT 法檢測細胞黏附能力    32
2.2.5 Transwell 法測細胞侵襲能力    32
2.2.6 劃痕實驗    33
2.2.7 組織基因組的提取    34
2.2.9質粒的提取    35
2.2.10 質粒的化學轉化    36
2.2.11 DNA瓊脂糖凝膠電泳    36
2.2.12 載體構建    36
2.2.13重組質粒轉染    38
2.2.14雙熒光素酶報告基因分析    38
2.2.3甲醛變性瓊脂糖凝膠電泳檢查總RNA的完整性    39
2.2.4數據處理和統計學方法分析    40
結果    41
3.1 MicroRNA-106a對人類滋養層細胞侵襲和遷移的影響    41
3.1.1 miR-106a在胎盤中的表達水平    41
3.1.2 敲低miR-106a促進JEG-3細胞的增殖,遷移和侵襲    41
3.1.3 miR-106a的過度表達抑制了JEG-3細胞的增殖,遷移和侵襲    42
3.1.4 ZBTB7A是miRNA-106a的靶基因    43
3.2 MicroRNA-24-3p對人類滋養層細胞侵襲和遷移的影響    45
3.2.1 URSA臨床樣品中miR-24-3p和ZNF654的表達    45
3.2.2 MiR-24-3p抑制JEG-3細胞的增殖,遷移和侵襲    46
3.2.3 MiR-24-3p通過靶向ZNF654來抑制JEG-3細胞的侵襲和遷移    47
討論    49
4.1 MicroRNA-106a對人類滋養層細胞侵襲和遷移的影響    50
4.2 MicroRNA-24-3p對人類滋養層細胞侵襲和遷移的影響    51
結論    54
參考文獻    55

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