以實驗性熱中風之小鼠模式下，比較人類乳牙幹細胞和臍帶血幹細胞的抗凋亡作用

Comparison analysis of anti-apoptotic effects between stem cells from human exfoliated deciduous teeth and human umbilical cord blood cells in experimental heatstroke mice model

曾令澍(Ling-Shu Tseng)

指導教授 ： 林英助

高雄醫學大學/口腔醫學院/牙醫學系博士班/博士(2018年)

摘要

研究目的：乳牙幹細胞 (SHEDs) 與臍帶血幹細胞 (HUCBCs) 均被提出具有分化及修復的功能，對神經退化具有療效。已知熱中風之生成與下視丘溫控功能 (例如體溫降低) 與神經退化極其相關。動物模式顯示HUCBCs對熱中風有治療作用，但乳牙幹細胞對於熱中風後是否減緩下視丘溫控功能之抑制，以及對熱壓力引起的下視丘體神經退化之作用甚少討論，尤其對於SHEDs是否可減少熱中風後的影響還不得而知。在實驗性小鼠誘發熱中風後注入SHEDs和HUCBCs，因為降低全身性炎症反應和下丘腦神經元凋亡進而改善多重器官的細胞凋亡反應。我們的目標是比較兩者幹細胞的抗凋亡反應。
研究材料與方法：ICR 雄小鼠曝置於41.2。C的環境，60分鐘後移至環境溫度26℃，觀察四天後其存活率。動物隨機分為四大組: (1) 未經過熱處置(WBH)小鼠接受靜脈注射生理食鹽水 (non-WBH＋vehicle)； (2) 經過熱處置小鼠接受靜脈注射生理食鹽水 (WBH＋vehicle) ； (3) 經過熱處置小鼠接受靜脈注射臍帶血幹細胞 (WBH＋HUCBCs) 以及 (4) 經過熱處置小鼠接受靜脈注射乳牙幹細胞 (WBH＋SHEDs)。在熱處置後不同時間點將部分小鼠犧牲後，取出血液及組織進行生化及組織學鑑定。
結果：以乳牙幹細胞和臍帶血幹細胞注射後，兩者均可延長實驗性熱中風小鼠之存活率，但無統計學之差異，熱中風小鼠表現低體溫與神經功能異常，隨伴著下視丘體的神經細胞退化與凋亡、血液中glutamate濃度提升、lactate /pyruvate比例增加、氧化壓力提升 (例如氫氧化自由基提升) ，血中促發炎細胞激素濃度提升，以及引起全身性器官的細胞凋亡，而以上之熱中風症狀均可被乳牙幹細胞或臍帶血幹細胞大幅度的減弱。同時經由乳牙幹細胞或臍帶血幹細胞注射後均可提升動物之熱耐力 (例如血中ACTH及corticosterone之提升) 以及在熱壓力下之存活率。
注射幹細胞後提升小鼠之熱耐受力進而減緩熱中風症狀的保護作用，可能與幹細胞的下列作用有關:降低熱中風後的下視丘體的細胞退化、凋亡與氧化壓力，進而提升小鼠熱中風後的熱耐受力，以及減緩多重器官凋亡及炎症反應。在減少細胞凋亡方面，有觀察到在肺臟，肝臟，腎臟，脾臟等組織在乳牙幹細胞處置後，減少細胞凋亡的效果更勝於臍帶血幹細胞處置後的效果。
結論：乳牙幹細胞與臍帶血幹細胞均可自人體經醫療處理過後不再需要的組織中分離出來，兩種幹細胞在處置實驗性熱中風小鼠的減緩效果是前者優於後者，且乳牙幹細胞取得更為容易，未來具有潛力應用在醫學領域。

關鍵字

熱中風 ； 乳牙幹細胞 ； 臍帶血幹細胞

並列摘要

Objectives：Stem cells from human exfoliated deciduous tooth (SHEDs) and Human umbilical cord blood cell (HUCBCs) present the potential of differentiation and repair function. Animal model showed therapeutic effects of HUCBCs on heat stroke. However, the ability of SHEDs to decrease the impact of heat stroke is unknown. Our aims were to compare the abilities of SHEDs and HUCBCs to reduce the systemic inflammatory responses and hypothalamic neuronal apoptosis after heat stroke of mice.
Materials and methods：Unanesthetized and unrestraint male mice, immediately after the initiation of heat stress (41.2。C for 60 minutes) were divided into four groups：whole body heating (WBH) mice received normal saline (WBH＋NS) , WBH mice received SHEDs (WBH＋SHEDs) or WBH mice received HUCBCs (WBH＋HUCBCs), and mice received NS and without WBH as normal controls. The NS group was given 0.3 ml normal saline, and the SHEDs or HUCBCs group were given in a numbers of 1×105 cells /0.3ml intravenously.
WBH mice were followed up to 4 days , and the datas were collected for statistical analysis. Part of the mice were sacrificed at 4 hour post-WBH, and their organs were analyzed using histochemical and biochemical evaluation. Ischemic indicators in the hypothalamus were determined by microdialysis, where as oxidative stress indicators in the hypothalamus were determined by histochemical analysis. Apoptotic cells in the hypothalamus were determined by immunofluorescence method. Both body temperature and neurological function were determined by rectal probe placement and modified neurological severity score (mNSS), respectively.
Results: The SHEDs or HUCBCs groups after WBH were exhibited the following therapeutic benefits after heat stroke：(a) reduction of neurologic and thermoregulatory deficits; (b) reduction of ischemic, hypoxic, and oxidative damage to the hypothalamus; (c) reduction of plasma overproduction of TNF-; (d) reduction of hypothalamic–pituitary–adrenocortical (HPA) axis impairment as reflected by increasing plasma levels of both adrenocorticotrophic hormone (ACTH) and corticosterone ; and (e) attenuation of multiple organ apoptosis as well as lethality.
Conclusion：Treatment with SHEDs or HUSBCs can improve heat tolerance or lethality by the increase of HPA axis activity,and reduction of hypothalamic neuron apoptosis, oxidative stress, systemic inflammation and multiple organ apoptosis following the WBH injury.SHEDs transplantation may have higher potential to prevent the occurrence of heatstroke than HUCBCs have, especially in the anti-apoptotic effects. SHEDs are more easily acquired, which means they have great application in the medical field.

並列關鍵字

heatstroke ； SHEDs ； HUCBCs

參考文獻

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