桂枝茯苓丸藉由蛻膜細胞、巨噬細胞、血管內皮細胞的交互作用在促發炎因子的刺激下，對於改善血管內皮功能的影響

The improvement of endothelial function by Guizhi Fuling Wan via its effects on IL-1β-treated first trimester decidual cells and subsequent interaction with macrophages

方郁婕(Yu-Chieh Fang)

指導教授 ： 蔡金川

共同指導教授 ： 黃瑟德(Joseph Huang)

義守大學/醫學院/學士後中醫學系/碩士(2022年)

摘要

成功的胚胎著床、胎盤形成與順利的孕程皆需仰賴充足的母胎介面中血管生成以及健全的內皮細胞功能。在母體側，子宮內膜上健康的內皮細胞對於胚胎着床與發育的順利與否扮演重要且關鍵的角色。胎兒與母體間的血流改變將對氣體、養份與代謝廢物的交換造成重大的影響。血管生成不良及功能障礙將造成多種妊娠併發症，如：子癇前症、流產與胎兒生長發育遲滯。遺憾的是，醫學界至今尚未有直接且有效率的方法可以進行治療或預防。桂枝茯苓丸，由桂枝、茯苓、牡丹皮、赤芍、桃仁五個成分所組成，出典於<金匱要略>，具有活血化瘀、消積散癥的作用。桂枝溫血通脈、消散瘀血，茯苓益心脾之氣而能滲利下行，桃仁破血散結，牡丹皮及赤芍清熱破瘀。實驗室先前研究已證明，將桂枝茯苓丸應用於具自然流產與胎兒發展遲緩特徵的小鼠模型時，可改善其胎兒與胎盤發育，並抑制蛻膜細胞發炎和促進胎兒生長。再者，巨噬細胞抑制血管增生的作用被發炎的蛻膜細胞增強，而在發炎的蛻膜細胞加入桂枝茯苓丸後則改善了此狀況。由於血管生成需倚賴充足且健康的內皮細胞。因此，本研究中兩具體目標將用來檢驗實驗假說”桂枝茯苓丸藉由緩解發炎的蛻膜細胞對巨噬細胞的影響，進而促進內皮細胞增生及改善內皮功能障礙。”
目標一、評估桂枝茯苓丸對於受到促發炎因子刺激後的蛻膜細胞影響之巨噬細胞所造成血管內皮細胞的增生效應。巨噬細胞將會先用促發炎因子刺激的蛻膜細胞之細胞培養液培養，然後與血管內皮細胞共同培養。進而用細胞計數試劑來檢視內皮細胞的增生效應。
目標二、檢視在有無桂枝茯苓丸的情況下，評估在受促發炎因子刺激的蛻膜細胞影響下的巨噬細胞對內皮細胞血管內皮功能異常的調控。定量聚合酶連鎖反應與酵素免疫分析法將被用來評估血管新生與內皮細胞功能異常的標記。
本研究結果將呈現桂枝茯苓丸對於子宮之蛻膜細胞、巨噬細胞及血管內皮細胞的交互作用的調控，進而對促進胎盤形成提供新的選擇。

關鍵字

胎兒生長發育遲滯 ； 自發性流產 ； 子癇前症 ； 桂枝茯苓丸 ； 血管內皮功能異常 ； 內皮細胞增生

並列摘要

Adequate angiogenesis and endothelial function on both sides of maternal-fetal interface are essential for successful implantation, placentation and subsequent gestation. On maternal side, healthy endothelial cells in endometrium are critical for the acceptance of implanting embryo and imminent maintenance of an uneventful pregnancy. Changes in blood flow in the maternal-fetal interface are believed to exert a dominant effect on gas, nutrient and waste exchange. Aberrant angiogenesis and endothelial dysfunction result in abnormal implantation and leads to adverse pregnancy outcomes, such as preeclampsia (PE), spontaneous abortion (SA) and intrauterine growth restriction (IUGR). However, no effective therapeutic or preventative intervention is currently available. Guizhi Fuling Wan (GFW) is a traditional Chinese herbal formula used to treat vital energy (qi) and blood stagnation as well as threatened abortion, which initially appeared in Chung-Ching Chang’s “Synopsis of Golden Chamber” about 1,800 years ago. GFW is derived from a traditional formula, Guizhi Tang, which consists of Poria cocos Wolf, Cinnamomum cassia Presl, Paeonia suffruticosa Andrews, Prinus persica (L.) Batsch, and Paeonia lactiflora Pall. Our previous results showed that GFW improved fetal-placental development and modulated the pro-inflammatory micro-environment in the fetal-maternal interface using SA/IUGR-prone mouse model. Moreover, the ability of macrophages in inhibiting angiogenesis was also enhanced by pro-inflammatory cytokine-treated first trimester decidual cells (FTDCs), whereas such inhibition of angiogenesis was reduced by treating FTDCs with GFW. However, treating endothelial cells with GFW did not enhance angiogenesis. Taken together, these findings suggest that GFW indirectly promotes angiogenesis. Since a sufficient number of healthy endothelial cells plays a primary role in angiogenesis, two specific aims will test the hypothesis that GFW promotes the proliferation of endothelial cells and ameliorates endothelial dysfunction under the influence of pro-inflammatory FTDCs-affected macrophages:
1) To evaluate the direct and indirect effects of GFW on the proliferation of endothelial cells: CCK8 assay will be used to examine the effects of GFW on proliferation of endothelial cells co-cultured with macrophages treated by conditioned media derived from IL-1β-treated FTDCs.
2) To evaluate the regulation of dysfunction of endothelial cells under the influence of macrophages affected by pro-inflammatory stimulus-treated FTDCs in the presence or absence of GFW: The expression of various markers of angiogenesis and endothelial dysfunction in endothelial cells in response to macrophages affected by IL-1β-treated FTDCs in the presence or absence of GFW will be assessed by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay.

並列關鍵字

fetal growth restriction ； spontaneous abortion ； pre-eclampsia ； Guizhi Fuling Wan ； endothelial dysfunction ； proliferation

參考文獻

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