抑制OGR1表現可減緩慢性神經性疼痛藉由減少顆粒性白血球數及非IB4神經元之鈣訊號

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姓名

秦茵(Yin Chin) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

抑制OGR1表現可減緩慢性神經性疼痛藉由減少顆粒性白血球數及非IB4神經元之鈣訊號
(OGR1 knockdown reduces granulocyte number and calcium signals of IB4-negative neurons to relieve chronic neuropathic pain)

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摘要(中)

神經病變性疼痛是由中樞或周邊神經系統病變或受損引起的疼痛，症狀包含自發性疼
痛、感覺異常和痛覺過敏，並且還會嚴重干擾病人的生活品質。神經損傷往往伴隨著
發炎反應。當發炎發生時，受損部位會釋放細胞因子並誘導免疫細胞聚集。此外，受
損部位的組織酸化也在神經損傷中有著重要作用。卵巢癌G 蛋白偶聯受體1（OGR1）
是一種在pH6.8 環境下會被活化並且表達於小直徑背根神經節（DRG）神經元上的酸
敏感受體，除此之外還有表達在巨噬細胞和T 淋巴細胞等免疫細胞上。細胞因子增加
免疫細胞中的OGR1 表達以調節免疫細胞的功能。但是，目前尚不清楚OGR1 在神經
性疼痛中的作用
為了解決這些問題，我使用坐骨神經慢性壓迫性損傷模型（Chronic constriction injury,
CCI），並以shRNA 抑制周邊神經元上OGR1 表達以研究其影響。靜默OGR1 基因表
達改善CCI 誘導的機械痛覺過敏。在DRG 神經元中也發現抑制OGR1 基因表達可以
在手術後第4 周抑制其鈣訊號。這些結果顯示OGR1 可以調節細胞內神經元中的鈣信
號反轉CCI 誘導的機械痛覺過敏。

摘要(英)

Neuropathic pain is one of the chronic pain that causes severe disturbance to quality of life.
Inflammation accompanied with the nerve injury is associated with induction of neuropathic
pain. While inflammation happened, cytokines release and induce immune cells recruitment.
Furthermore, the local tissue acidosis accompanied with the nerve injury also plays an
important role in neuropathic pain. Ovarian Cancer G Protein-Coupled Receptor 1 (OGR1),
a proton-sensing GPCR fully activated at pH6.8 environment is expressed in small-diameter
dorsal root ganglion (DRG) neurons, macrophages and lymphocytes. Cytokines increase
OGR1 expression in immune cells to regulate the function of immune cells. However, it
remains unclear which role OGR1 plays in neuropathic pain. To address these questions,
used shRNA to knockdown OGR1 gene expression in peripheral afferents before chronic
constriction injury (CCI) of the sciatic nerve. Knockdown of OGR1 gene expression
reversed CCI-induced mechanical hyperalgesia from 4 weeks to 14weeks post CCI surgery.
Intracellular calcium signals recorded in DRG neurons were upregulated post CCI surgery,
and further increased at least 4w. Suppression of OGR1 gene expression inhibited calcium
signals at 4w after CCI, maining in IB4(-) neurons. Accordingly, these results suggested that
OGR1 may regulate intracellular calcium signals in neuron to reverse CCI-induced
mechanical hyperalgesia.

關鍵字(中)

★ 疼痛
★ G蛋白耦合受體
★ 坐骨神經
★ 痛覺過敏
★ 神經性疼痛

關鍵字(英)

論文目次

第一章
緒論 1
1.1神經性疼痛 (Neuropathic pain) 2
1.2神經發炎反應 (Neuroinflammation) 3
1.2.1巨噬細胞 4
1.2.2 顆粒性白血球 4
1.3 組織酸化 (Tissue acidosis) 5
1.3.1 酸敏感 G蛋白偶合受體卵巢癌 G 蛋白偶合受體 1 (ovarian cancer G protein coupled receptor 1, OGR1) 6
1.4 研究動機與目的 7
第二章
材料與方法 9
2.1 實驗材料 10
2.1.1 細胞株 (Cell lines) 10
2.1.2 實驗動物 (Experimental animal) 10
2.1.3 核糖核酸干擾 (RNA interference) 10
2.1.4 實驗藥品與手術器具 10
2.2 實驗方法 (Experimental methods) 11
2.2.1 大腸桿菌勝任細胞之轉型作用 (Transformation) 11
2.2.2 質體中量製備 (Midi prep.) 11
2.2.4 陽離子明膠注射（ Cationized Gelation injection 12
2.2.5 背根神經節基因表現與定量 (Gene expression) 13
2.2.6 圓形玻片前處理 (Coverslides coating) 14
2.2.7 細胞轉染 (Transfection) 14
2.2.8 鈣離子成像分析 (Calcium images) 14
2.2.9背根神經節初代培養 (Primary DRG cultures) 15
2.2.10神經慢性壓迫損傷模型 (Chronic contraction injury model, CCI model) 16
2.2.11 機械性痛覺行為測試 (Mechanical behavioral test) 17
2.2.12 蘇木素伊紅染色 17
2.2.13免疫組織化學染色法 (Immunohistochemistry Paraffinstain, IHC P stain)18
2.2.14免疫組織化學螢光染色法 (Immunohistochemistry stain, IHC stain) 19
2.2.15統計分析 (Statistics) 20
第三章
結果 21
3.1 shOGR1 質體結構與抑制方法 22
3.2 cherry shOGR1可在體外實驗中抑制酸誘導的 OGR1所引發之鈣訊號 22
3.3 注射 shOGR1後與小直徑背根神經節神經元共表達情形 23
3.4坐骨神經慢性壓迫損傷誘導長期機械性痛覺並且抑制 OGR1基因表達可抑制
該疼痛 23
3.5 抑制背根神經節 OGR1基因表現後改變坐骨神經損傷部位免疫細胞的表現 24
3.6 OGR1參與調控坐骨神經損傷部位 M1及 M2巨噬細胞的數量 25
3.7 CCI手術會增加背根神經節對酸的敏感並引發細胞內鈣離子增加 27
3.8 抑制 OGR1表達可抑制 CCI手術引起的鈣離子增加 28
第四章
討論 30
4.1抑制背根神經節OGR1基因表現改變坐骨神經損傷部位 M1 / M2 巨噬細胞數量 31
4.2抑制背根神經節OGR1基因表現改變坐骨神經損傷部位顆粒性白血球數量 32
4.3 OGR1參與在坐骨神經慢性壓迫性損傷所增強的胞內鈣離子變化 33
第五章
參考資料 34

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指導教授

孫維欣(Wei-Hsin Sun)

審核日期

2019-8-26

推文