臺灣博碩士論文加值系統

摘要
拜醫學科技躍進所賜人壽命日趨年長，骨質疏鬆症(Osteoporosis)已經嚴重威脅人體健康主要的骨骼疾病，美國醫學會指出：「老人股骨骨折後，大約5% ~ 20%的患者一年內死亡，50%以上的人行動不便。」在中醫治療方面以補腎療法為主，其理論基礎源於中醫“腎主骨生髓”學說，目前臨床及動物實驗顯示使用補腎中藥可使骨質疏鬆患者血鈣上升。因此本研究利用人類脂肪所萃取出間葉系幹細胞能分化成硬骨特性，以加牛黃清心丸及續斷，探討人類脂源性間葉幹細胞是否對骨質形成有所助益。研究結果得知添加續斷培養二十日對骨質誘發促進新生有明顯成效，其理想濃度分別為150ppm、100ppm，比control組多增加3~16%；但牛黃清心丸(非補腎中藥)培養二十日後觀察，其對骨質細胞誘發增殖卻有抑制及減緩的現象並無增益之效用。
而另一個研究以MTT assay、Gap-Junctional intercelluler communication (GJIC)添加四種砷化物(Arsenite、Arsenate、Disodium methyl arsenate、Dimethyl arsenate acid)，並以四種不同細胞株(PLA、WB、MSU及Mahlaun)進行試驗。研究結果顯示，WB添加arsenite、arsenate、disodium methyl arsenate dimethyl arsenate acid測試一、三、七天，砷化物將WB本身positive轉換negative，使細胞間隙連接通訊由傳遞變不傳遞現象，因此研究證實砷化物可使WB細胞株產生不穩定現象，而其不影響細胞活性最理想濃度分別為3ppm、8ppm、12ppm、6ppm。Mahlaun為肝癌細胞株添加arsenite濃度0.25ppm後，細胞本身為negative並無轉變現象。而MSU的arsenite濃度在0.25ppm，細胞有positive轉換negative，與WB細胞株的表現相似。PLA幹細胞arsenite濃度為8ppm，其他會造成細胞negative轉換positive濃度為(arsenate 12ppm；disodium methyl arsenate 15ppm；dimethyl arsenate acid 10ppm)，比起WB、MSU及Mahlaun肝癌細胞株對砷化物濃度高許多，故研究證實PLA幹細胞可比一般cell line細胞株對砷化物方面耐受強度較好。

Abstract
As the older population increases, the global has changed gradually into elder society. Due to the growth of age, the incidence of osteoporotic fractures is expected to dramatically rise during the next decades. Adipose tissue has several clear advantages as an initial material for harvesting stem cell, as it is abundant and relatively easy to procedure. Therefore, the objective of this study discusses whether it is helpful for formation of osteoid after adding Dipsacus japonicus mag. and nious huang cing sin wan. The method is to use the characteristics of being differentiated osteogenesis by adipose mesenchymal stem cell.

The significant concentrations of Dipsacus japonicus mag.is 150 ppm and 100 ppm respectively. There is significantly increasing cell proliferations around in 1, 3 and 7 days. The result of von kossa stain in the cells is not significant differently by utilising Dipsacus japonicus mag. between the experiment group and the negative control group. After incubated 14 days, it is found that the medium of cadmium concentration and the differentiated osteogensis with tea polyphenols and cadmium is much lower 51.9%, 51.9% and 52.8% than the control group. In differentiated osteogensis with nious huang cing sin wan., there is no tremendous difference von kossa stain between the control group after 20 days incubated. As a result, the consequence indicates that Dipsacus japonicus mag. would stimulate adipose mesenchymal stem cell to differentiate bone cell rather than nious huang cing sin wan.
Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the four arsenic compounds(arsenite, arsenate, dimethyl arsenate acid, disodium methyl arsenate) in 4 different cell types. In 4 type cells, using the dye transfer assay, we observe a time-dependent stimulation of GJIC by arsenic compounds at non-cytotoxic concentrations. Arsenite induced GJIC from positive to negative in WB, in 3-12 ppm, and MSU, in 0.25 ppm, cell lines. There is no effect of GJIC in hepatoma cell line, Mahlaun. Arsenic compound induced GJIC from negative to positive in meschymal stem cell.

目錄
謝誌……………………………………………………………………………..3
摘要…………………………………………………………………………..…9
Abstract………………………………………………………………………...11
壹、 緒論
一、 幹細胞醫學…………………………………………………………13
二、 幹細胞發展史………………………………………………………22
三、 研究動機……………………………………………………………23
四、 材料與方法(materials and methods)……………………….............25
貳、 砷化物
一、 前言(introduction)………………………………………….............29
二、 材料與方法(materials and methods)………………………............37
參、 中藥(續斷、牛黃清心丸)
一、 前言(introduction)………………………………………….............39
二、 材料與方法(materials and methods)……………………………….40
肆、 細胞活性試驗 ( MTT assay )
一、 前言(introduction)………………………………………….............42
二、 材料與方法(materials and methods)………………………............42
三、 結果與討論(results and discussion)……………………………….43
伍、 Gap-Junctional intercelluler communication (GJIC)
一、 前言(introduction)………………………………………................46
二、 材料與方法(materials and methods)………………………............46
三、 結果與討論(results and discussion)…………………………….…47
陸、 人類脂源性成體間葉幹細胞之骨質誘發試驗
一、 前言(introduction)…………………………………………….…...48
二、 材料與方法(materials and methods)……………………………...48
三、 結果與討論(results and discussion)………………………………49
柒、 鈣化節點染色法 (Von kossa stain)
一、 前言(introduction)………………………………………………....51
二、 材料與方法(materials and methods)………………………...........51
三、 結果與討論(results and discussion)………………………………51
捌、 結論(conclusions)……………………………………………………..53
玖、 圖表及附錄…………………………………………………………....55
圖一、 Stem cell分化樹狀.………………………………………………...56
圖二、 多能力性幹細胞(pluripotent stem cells)…………………………...56
圖三、 多潛能幹細胞(multipotent stem cells)……………………………..57
圖四、 間葉幹細胞(Mesenchymal stem cells, MSCs)……………………..57
圖五、 幹細胞之應用………………………………………........................58
圖六、 台灣砷暴露地區……………………………………………………58
圖七、 野生續斷………………………………………………………...….59
圖八、 續斷生藥材……………………………………………………...….59
圖九、 Gap-Junctional Inbercelluler Communication(GJIC)……………….60
表一、 Medicinal use of arsenic……………………………..........................61
MTT assay………………………………………………………………….…62
Table 1 A-C.(WB&Arsenite)……………………………………..………..….63
Table 2 A-C.(WB&Arsenate)…………………………………….…………...63
Table 3 A-C.(WB&Disodium methyl arsenate)………………………………64
Table 4 A-C.(WB&Dimethyl arsenate acid)…………………………………..64
Table 5 A-C.(PLA&Arsenite)………………………………………………....65
Table 6 A-C.(PLA&Arsenate)………………………………………………...65
Table 7 A-C.(PLA&Disodium methyl arsenate)…...………………………....66
Table 8 A-C.(PLA&Dimethyl arsenate acid)………...……………………….66
Table 9 A-C.(MSU&Arsenite)………………………………………………..67
Table 10 A-C.(Malhare&Arsenite)…………...……………………….............67
Table 11 A-C.(PLA&DAG)：A、牛黃清心丸；B、續斷………….............68
Fig 10、WB & Arsenite……………………………………………………....69
Fig 11、WB & Arsenate……………………………………………………...70
Fig 12、WB&Disodium methyl arsenate…………………………………….71
Fig 13、WB&Dimethyl arsenate acid………………………………………..72
Fig 14、PLA&Arsenite………………………………………………………73
Fig 15、PLA&Arsenate……………………………………………………...74
Fig 16、PLA&Disodium methyl arsenate……………………………………75
Fig 17、PLA&Dimethyl arsenate acid……………………………………….76
Fig 18、MSU&Arsenite……………………………………………………...77
Fig 19、Malhare&Arsenite……………………………………………..........78
Fig 20、PLA&DAG&牛黃清心丸…………………………………………..79
Fig 21、PLA&DAG&續斷…………………………………………………..80
Gap-Junctional Inbercelluler Communication………………………………..81
Figs22.A-D. (WB & Arsenite)………………………………..………………82
Figs23.A-D. (WB & Arsenate)……………………………………………….83
Figs24.A-D. (WB & Disodium methyl arsenate)…………………………….84
Figs25.A-D. (WB & Dimethyl arsenate acid)………………………………..85
Figs26.A-D. (PLA & Arsenite)……………………………………………….86
Figs27.A-D. (PLA & Arsenate)………………………………………………87
Figs28.A-D. (PLA & Disodium methyl arsenate)……………………………88
Figs29.A-D. (PLA & Dimethyl arsenate acid)……………………………….89
Figs30.A-F. (Malhara & Arsenite)……………………………………………90
骨質細胞誘發試驗…………………………………………………………...91
Figs31.A-F. (MSU & DAG)………………………………………………….92
Figs32.A-C. (PLA & D medium)…………………………………………….93
Figs33.A-F. (PLA & DAG)…………………………………………………..94
Figs34.A-F. (PLA&DAG&牛黃清心丸)…………………………………….95
Figs35.A-C. (PLA&DAG&續斷, 80000ppm)……………………………….96
Figs36.A-F. (PLA&DAG&續斷, 3200ppm)...…………………….…………97
Figs37.A-F. (PLA&DAG&續斷, 150ppm)………………………….……….98
Von kossa stain……………………………………………………………….99
Figs38.A-C: (A) DAG；(B)DAG&牛黃清心丸；(C) DAG&續斷………100
Figs39.A-C: (A) D medium；(B) DAG；(C) DAG&續斷………………..101
儀器設備與試劑……………………………………………………..……..102
壹拾、 參考文獻(references)…………………………..….………………104

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