移植低能量雷射刺激之脂肪幹細胞治療大白鼠缺血性腦中風之研究

Transplantation of low-energy laser stimulation on adipose tissue-derived stem cells treatment of focal cerebral ischemia in rats

謝復順(Fu-Shun Hsieh)

指導教授 ： 劉百栓

中臺科技大學/健康科學院/醫學影像暨放射科學系暨研究所/碩士(2012年)

https://doi.org/10.6822/CTUST.2012.00052

摘要

本研究主要探討大面積低功率雷射對於誘導朝向神經細胞分化之脂肪幹細胞的增生與分化之影響，並進行大鼠大腦缺血性中風治療以探討相關治療機轉。首先進行大面積低功率雷射對於誘導朝向神經細胞分化之脂肪幹細胞的增生評估：經由DAPI stain觀察後顯示，細胞的數量並未因為照射雷射而增多；而經MTT assay分析後，亦發現照射大面積低功率雷射之細胞與未照射組的細胞數量無明顯差異，顯示了大面積低功率雷射對於誘導朝向神經細胞分化之脂肪幹細胞的增生是無影響的；接著進行大面積低功率雷射對於誘導朝向神經細胞分化之脂肪幹細胞的分化評估：經由免疫螢光染色觀察發現，誘導朝向神經細胞分化之脂肪幹細胞經大面積低功率雷射照射後，其神經幹細胞標定蛋白Nestin有明顯之增加，另外，神經膠細胞標定蛋白GFAP及神經元前體細胞蛋白DCX並無顯著之差異，原因為，本研究所使用之分化劑為誘導分化成為神經細胞，因此不會朝向神經膠細胞分化，而神經元前體細胞蛋白DCX則是因為此蛋白是神經幹細胞構成神經元之後才可發現到的，屬於較晚期之蛋白，所以有無照射雷射對於此蛋白的表現量並無影響；而經由西方墨漬法分析也發現，誘導朝向神經細胞分化之脂肪幹細胞經大面積低功率雷射照射後，其神經幹細胞標定蛋白Nestin有顯著的表現。由細胞實驗結果得知，雖然大面積低功率雷射對於誘導朝向神經分化之脂肪幹細胞的增生無特別之影響，但對於誘導朝向神經細胞分化是有明顯的幫助。
接著進行幹細胞移植治療中風大鼠之實驗：在運動功能恢復評估結果顯示，中風大鼠移植i-ADSCs（LS+）治療後的第14天開始，其跑步及抓力功能已開始恢復，相較於移植i-ADSCs（LS-）治療組，仍呈現功能低下之狀態，在治療後的第28天發現，i-ADSCs（LS+）治療組之運動功能已與Sham組相差不遠，而i-ADSCs（LS-）治療組的運動功能則依然略顯低落；而經由大鼠腦組織的H&E免疫染色結果顯示，i-ADSCs（LS+）治療組的腦組織修復的相當完整，幾乎無腦組織壞死，反觀i-ADSCs（LS-）治療組的腦組織則出現許多壞死斑痕及空洞；另外，在西方墨漬法分析結果顯示，i-ADSCs（LS+）治療組的腦部與Sham組一樣，含有大量中樞神經髓鞘寡突膠細胞蛋白Oligo-2的表現，藉此可判斷移植i-ADSCs（LS+）治療能較有效的修復因缺血而損傷之大腦，以此實驗結果證明了大面積低功率雷射對於誘導脂肪幹細胞分化是有正面影響，且可以有效地治療大鼠缺血性腦中風，恢復運動功能。

關鍵字

缺血性腦損傷 ； 大面積低功率雷射 ； 脂肪幹細胞 ； 幹細胞 ； 幹細胞治療

並列摘要

This study investigated the influence of a large-area irradiation of low-level laser on the proliferation and differentiation of inductions of adipose-derived stem cells (i-ADSCs) into nerve cells. We then applied this approach to treat ischemic stroke in rats in the evaluation of various treatment mechanisms. DAPI staining showed that the number of cells did not increase as a result of irradiation; MTT assays indicated no significant difference between the number of laser-treated and non-laser-treated cells. Our results demonstrate that wide-area irradiation using a low power laser did not have a significant impact on the differentiation of i-ADSCs. Immunofluorescent staining indicated a significant increase in the neural stem cell marker Nestin following exposure to low power laser irradiation. No significant difference was observed between the glial cell marker GFAP and neuronal precursor cell protein DCX because the differentiation agent used in this study induced differentiation into nerve cells, rather than glial cells. Moreover, DCX can only be detected after neural stem cells form neurons in later stages; therefore, we conclude that the irradiation had no influence on the expression of this protein. Western blotting also showed that following irradiation, the expression of Nestin was significantly increased. The experiment results indicate that a large-area irradiation of low-level laser had no particular influence on the proliferation of i-ADSCs but still facilitated the differentiation of i-ADSCs considerably.
This study employed stem cell implantation to treat rats following stroke. In the recovery of motor functions, the rats implanted with i-ADSCs (LS+) began regaining running and gripping functions on the 14th day following treatment. In comparison, stroke rats implanted with i-ADSCs (LS-) presented symptoms of dysfunction. At 28 days post treatment, the motor functions of the rats treated with i-ADSCs (LS+) did not differ greatly from those in the sham group, while the motor functions of the rats treated with i-ADSCs (LS-) remained somewhat dysfunctional. H&E-stained brain tissue samples from the rats treated with i-ADSCs (LS+) exhibited near-complete recovery with almost no brain tissue damage, whereas tissue from the rats treated with i-ADSCs (LS-) displayed necrotic scarring and voids. Western blotting revealed a significant expression of Oligo-2, a protein produced by the myelin-forming oligodendrocytes of the central nervous system, in the rats treated with i-ADSCs (LS+) as well as the sham group. From this, we can determine that treatment involving the implantation of i-ADSCs (LS+) is capable of repairing brain tissue damaged by ischemia. The results of this experiment also demonstrate that a large-area irradiation of low-level laser has a positive influence on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.

並列關鍵字

adipose tissue-derived stem cells ； stem cell ； large-area irradiation of low-level laser ； stem cell treatment

參考文獻

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