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研究生:吳家瀅
研究生(外文):Wu, Chia-Ying
論文名稱:探討中藥X對免疫細胞生成的影響
論文名稱(外文):Study on the Effect of Traditional Chinese Medicine Herb X in Immune Cell Production
指導教授:張原翊張原翊引用關係
指導教授(外文):Chang, Yuan-I
口試委員:涂玉青簡采汝
口試日期:2023-06-12
學位類別:碩士
校院名稱:國立陽明交通大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:95
中文關鍵詞:中藥表兒茶素免疫系統幹細胞分化髓源抑制性细胞
外文關鍵詞:Traditional Chinese medicineEpicatechinImmune systemHaematopoiesisMyeloid-derived suppressor cells (MDSCs)
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中藥X 是一種藥用的豆科植物,在傳統中國上用於治療貧血、風濕病、月經異常和其他疾病,過去的研究表明中藥X的指示性成分具有調節血液系統的藥理活性、抗腫瘤、抗氧化、抗病毒、抗菌及抑制黑色素沉積等功能。然而,中藥X是否透過調節造血幹細胞和前驅細胞進而影響免疫細胞的生成仍然未知。因此本研究旨在探討中藥X與其主要成分—表兒茶素 (Epicatechin, EC) 在先天和適應性免疫系統中所扮演的角色與潛在之分子機制。在此研究中,我們應用滲透壓控制的藥物輸送系統(滲透壓幫浦)來減輕動物不適並提高生物利用度,以研究中藥X或EC在造血系統當中的生物效應。我們的結果顯著表明,中藥X或EC不僅增加造血組織中造血幹細胞和前驅細胞,並且影響先天性和適應性免疫細胞的生成。在先天免疫系統中,中藥X增加周邊血中單核球,而EC增加嗜中性球的數量。同時,中藥X及EC皆提高了造血系統中髓源細胞與髓源抑制細胞 (MDSCs) 的生成以調節免疫作用。在適應性免疫系統中,我們發現中藥X減少了T細胞中輔助和毒殺型T細胞的產生,與中藥X相反,EC則促使T細胞增生。我們的研究結果提供了中藥X在體內免疫調節功能方面的系統觀點,這將有助於在未來開發其新的治療應用。
The immune system plays a critical role in protecting the human body from infectious diseases and cancer. Its two main contributors include innate and adaptive immunity responses. Herb X is a medicinal plant (Leguminosae) traditionally used in China for the management of anemia, rheumatism, abnormality in menstruation and other disorders. Past studies denoted that the indicative ingredients in herb X exert pharmacological activities on regulation of the blood system, anti-tumor, anti-oxidation, anti-virus, anti-bacteria, and inhibition of melanin deposition. However, whether herb X modulate functions of hematopoietic stem and progenitor cells to influence immune cell production is still unknown. On the other hand, the main ingredient of herb X is Epicatechin (EC), which is one of major catechins. Therefore, the underlying molecular mechanisms of herb X and its main compound in innate and adaptive immune systems were intendedly studied in this research.
Here, we applied the osmotic pressure-controlled drug delivery system (osmotic pumps) to mitigate animal upset and improve the bioavailability to study the biological effect of herb X or EC in hematopoietic system. Our results significantly demonstrated that herb X or EC not only increase the compartments of hematopoietic stem and progenitor cells in hematopoietic tissues, but also influence innate and adaptive immune cell production. In innate immune system, herb X increase the numbers of monocytes, and EC increase the numbers of neutrophil in the peripheral blood. Further analysis indicated that herb X or EC increase myeloid cell production, and promoted the production of myeloid-derived suppressor cells (MDSCs), which exhibit immunomodulatory effects in various pathophysiological processes. In adaptive immune system, our study found that herb X reduced T cell production, including helper and cytotoxic T cells. Surprisingly, EC was shown to have the opposite effect, which increased T cell production. In conclusion, our results provide a systemic view of herb X in immunomodulatory functions in the body, which will contribute to develop its new therapeutic application in the future.
中文摘要 i
英文摘要 ii
第一章、 緒論 1
1.1 免疫系統 1
1.2 T細胞發育與分化 2
1.3 造血作用 3
1.4 髓源抑制細胞與其分化途徑 4
1.5 髓外造血作用 5
1.6 中藥X對於免疫系統 5
第二章、 研究目的 7
第三章、 材料與方法 8
3.1 材料 8
3.1.1 抗體 8
3.1.2 引子探針 (Primer probe) 8
3.1.3 藥物與試劑 8
3.1.4 儀器 8
3.2 方法 8
3.2.1 動物模型 8
3.2.2 血液常規檢驗分析 (Complete blood count, CBC analysis) 9
3.2.3 螢光抗體染色樣品處理 9
3.2.4 流式細胞儀分析 10
3.2.5 髓源性抑制細胞分化實驗 10
3.2.6 骨髓、脾臟與胸腺細胞分離 11
3.2.7 利用磁珠收集小鼠骨髓和脾臟中之CD11b+ 細胞 11
3.2.8 RNA萃取、反轉錄與即時定量聚合酶鏈式反應 (RNA extraction and reverse transcription and quantitative PCR analysis) 11
3.2.9 蛋白質磷酸化分析 (Phospho-Flow Cytometry) 12
3.2.10 細胞分選 12
3.2.11 統計分析 (Statistical analysis) 13
第四章、 結果 14
4.1 給予中藥X會增加小鼠脾臟重量但不影響體重 14
4.2 給予中藥X影響周邊血中免疫細胞,並增加髓源抑制細胞 (MDSCs) 14
4.3 給予中藥X會促進脾臟中髓源前驅細胞分化 15
4.4 給予中藥X不影響骨髓中巨核細胞比例 15
4.5 給予小鼠中藥X會影響淋巴群系細胞組成,並降低T細胞比例 15
4.6 給予中藥X降低骨髓中髓源抑制細胞分化之關鍵轉錄因子的基因表現量 16
4.7 給予中藥X降低骨髓中DNA甲基化轉移酶基因表現量 16
4.8 利用體外實驗驗證了中藥X會增加骨髓中髓源抑制細胞分化 17
4.9 給予中藥X會降低骨髓中髓源抑制細胞STAT3磷酸化路徑 17
4.10 給予中藥X降低胸腺中雙陰性T細胞ERK1/2磷酸化路徑 18
4.11 給予EC會降低小鼠脾臟與胸腺重量 18
4.12 給予EC會增加周邊血中嗜中性、單核球細胞且降低淋巴球比例 18
4.13 給予EC會增加髓源抑制細胞比例,並增加上游祖細胞分化 19
4.14 給予EC不影響巨核細胞 19
4.15 給予EC會影響淋巴細胞比例,並增加T細胞分化 19
4.16 給予EC降低CD11b+細胞中MDSCs分化路徑相關基因表現量,增加功能性基因表現量 20
4.17 給予EC不影響骨髓中AKT與MEK途徑相關基因表達量 20
4.18 給予EC會降低胸腺中T細胞分化關鍵基因 21
4.19 體外給予EC影響骨髓中髓源抑制細胞分化 21
4.20 給予EC降低骨髓中髓源抑制細胞STAT3磷酸化路徑 22
4.21 給予EC不影響胸腺中雙陰性與雙陽性T細胞STAT3磷酸化路徑 22
第五章、 結論 23
第六章、 討論 24
第七章、 參考文獻 28
第八章、 圖 33
第九章、 表 87
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