臺灣博碩士論文加值系統

本研究目的在探討如何利用細胞治療來預防骨質疏鬆症。SAM（senescence accelerated mice)老鼠在早期的研究已被證實為與骨骼老化及骨骼生理有關，本研究將利用SAMP8老鼠以去除性腺產生嚴重骨質疏鬆症作為實驗動物的模式。細胞材料利用具有類似間葉幹細胞特性之老鼠胚胎纖維母細胞NIH3T3先處理Platelet-Rich Plasma（PRP）促進其細胞增生與骨母細胞分化(osteoblastogenic differentiation)之能力，並移植到SAMP8體內觀察細胞治療的長期效果。探討治療後的SAMP8老鼠骨質密度的改變，是否有減少降低骨質流失的現象，再以組織切片染色。目前的實驗我們發現NIH3T3+PRP後的SAMP8老鼠在四個月後骨質有明顯的增加，且在免疫染色的結果發現我們的NIH3T3有移動到全身各地，並結合了光學影像（optical imaging）照影系統的模式追蹤細胞所在位置。在本實驗證實了移植到SAMP8老鼠NIH3T3＋PRP有治療骨質疏鬆症的現象。

Osteoporosis has became a major public health problem in all developed countries. Despite numerous investigations, treatment and prevention of osteoporosis remains unsolved issues. Many researches focus on age-related bone in experimental animals models in senescence accelerated mice (SAM). In this study we establish the ovariectomized mice by SAMP8 mice as osteoporosis animal model.
NIH3T3 was capable to differentiate into osteoblast followed by stimulation with platelet-rich plasma and injected into the knee joint . PRP also has been reportrd to promote osteogentic differentiation of stem cell. We have recently established a system to transplant stem cells into SAMP mice to prevent osteoporosis. Treated SAMP mice showed a marked increase in bone mineral density (BMD) and decrease bone loss. We also trace the stem cell migration into the whole body that could induce osteogenesis to prevention of osteoporosis. In this experiment we found that transplantation with NIH3T3 and PRP could obviously increase BMD in the 3 month time point and traced the stem cell migration into the whole body detected by using immunhistochemical staining and optical imaging. Therefore, this study examined the NIH3T3 ＋ PRP transplant into the SAMP8 mice to prevent the osteoporosis.

第一章 緒論 1
第二章 理論基礎 3
2.1 骨骼細胞的介紹 3
2.1.1 蝕骨細胞 3
2.1.2 成骨細胞 4
2.2 骨頭重朔作用 5
2.3 骨質疏鬆症老鼠動物模式建立 6

圖目錄
圖1、SAM老鼠樣品處理與SEM結果之處理與分析 18
圖2、實驗大綱流程圖 22
圖3、實驗設計流程圖 22
圖4、不同濃度PRP處理對NIH3T3細胞增生能力之影響 32
圖5、 0-4個月四種不同處理在SAMP8老鼠於Spine及Knee之BMD測定曲線圖 33
圖6、0-4個月未經處理之SAMR1老鼠及經二種不同處理之SAMP8老鼠於Spine及Knee之BMD測定曲線圖 34
圖7、0.5-4個月SAMP8老鼠Spine及Knee之IHC染色圖 35
圖8、 0-4個月SAMR、SAMP8老鼠不同部位之SEM及軟體分析圖 37
圖9、 0-4個月SAMR、SAMP8老鼠不同部位之SEM及軟體分析圖 39
圖10、 0-4個月SAMR、SAMP8老鼠不同部位之SEM及軟體分析圖 41
圖11、光學照影系統之細胞方面 42
圖12、光學照影系統之動物方面 43
圖13、免疫組織染色證實NIH3T3細胞分化能力 44
圖14、 0-4個月SAMR1及四種不同處理對於SAMP8老鼠之生存試驗統計圖 45


2.4 細胞背景說明 7
2.5 血小板濃厚液應用 8
2.6 細胞活性 9
2.7 骨質密度測定 9
2.8掃瞄式電子顯微鏡 10
2.9 非侵入性照影系統 10
第三章 材料與方法 12
3.1 實驗材料 12
3.1.1 藥品與試劑： 12
3.1.2 主要儀器： 13
3.1.3細胞株與動物： 13
3.2 實驗方法 14
3.2.1 骨質疏鬆症動物模式之建立 14
3.2.1.1 實驗動物 14
3.2.1.2 雌鼠結紮手術（Ovariectomy） 14
3.2.2 體外試驗 15
3.2.2.1 細胞培養 15
3.2.2.2 血小板濃厚液製備 15
3.2.3 建立研究細胞與動物模式 15
3.2.3.1細胞活性分析（MTT assay） 15
3.2.3.2 老鼠鑽骨手術 16
3.2.4 功能性評估（Function index） 16
3.2.4.1 骨礦物質密度測定（Bone mineral density, BMD） 16
3.2.4.2 掃描式電子顯微鏡觀察（Scanning electron microscope, SEM） 17
3.2.4.3 顯微數位相機軟體分析（Histomorphometrical evalation） 17
3.2.4.4 切片及免疫組織化學染色（Immunohistochemical , IHC） 18
3.2.4.5 體內照影系統（In vivo imaging system） 19
3.2.4.6 生存試驗（Survival assay） 20
第四章 結果 21
4.1 實驗設計 21
4.2 不同濃度PRP處理對NIH3T3細胞增生能力影響的結果 23
4.3 SAM老鼠不同部位之BMD變化 23
4.4 以免疫組織染色追蹤NIH3T3細胞的存在 24
4.5 掃描式電子顯微鏡觀察SAM老鼠其不同部位骨質變化 24
4.6 非侵入性照影模式 25
4.7 細胞分化的結果 26
4.8 生存曲線探討實驗中老鼠的存活率 26
第五章 討論 27
第六章 結論 31
第七章 參考文獻 46

1.JOSEPH M. LANE EHR, and PHILIP Z. WIRGANOWICZ. Osteoporosis: Diagnosis and Treatment. J. Bone Joint Surg. 1996;78:618-632.
2.Guyton AC. Guyton Human Physiology and Mechanism of Disease. WB Saunders Co. Penn USA. 1991;5th:590-612.
3.Ziegler EEaF, Jr., L.J. Present Knowledge in Nutrition. ILSI Press, Wash., D.C., USA. 1996;7th:245-255.
4.Whitney ENaR, R. Understanding Nutrition. Wadsworth Publishing Co., Calif., USA. 1999;8th:336-403.
5.Rolfes SR, DeBruyne, L.K. and Whitney, E.N. Life Span Nutrition, Conception through Life. 1998;2nd:385-413.
6.黃永彥. 骨質疏鬆症-基礎與臨床. 1997.
7.Jilka RL, Hangoc G, Girasole G, et al. Increased osteoclast development after estrogen loss: mediation by interleukin-6. Science. Jul 3 1992;257(5066):88-91.
8.Gowen M, Wood DD, Ihrie EJ, McGuire MK, Russell RG. An interleukin 1 like factor stimulates bone resorption in vitro. Nature. Nov 24-30 1983;306(5941):378-380.
9.Eriksen EF, Colvard DS, Berg NJ, et al. Evidence of estrogen receptors in normal human osteoblast-like cells. Science. Jul 1 1988;241(4861):84-86.
10.McKane WR, Khosla S, Burritt MF, et al. Mechanism of renal calcium conservation with estrogen replacement therapy in women in early postmenopause--a clinical research center study. J Clin Endocrinol Metab. Dec 1995;80(12):3458-3464.
11.Clemens TL, Zhou XY, Myles M, Endres D, Lindsay R. Serum vitamin D2 and vitamin D3 metabolite concentrations and absorption of vitamin D2 in elderly subjects. J Clin Endocrinol Metab. Sep 1986;63(3):656-660.
12.Zadik Z, Chalew SA, McCarter RJ, Jr., Meistas M, Kowarski AA. The influence of age on the 24-hour integrated concentration of growth hormone in normal individuals. J Clin Endocrinol Metab. Mar 1985;60(3):513-516.
13.C.Guyton A. 蓋統生理學. 1997.
14.Dominiczak B. 醫學生物化學. 2001.
15.Hauge EM, Qvesel D, Eriksen EF, Mosekilde L, Melsen F. Cancellous bone remodeling occurs in specialized compartments lined by cells expressing osteoblastic markers. J Bone Miner Res. Sep 2001;16(9):1575-1582.
16.Consensus development conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med. Jun 1993;94(6):646-650.
17.Cameron JR, Sorenson J. Measurement Of Bone Mineral In Vivo: An Improved Method. Science. Oct 11 1963;142:230-232.
18.侯淑真. 數種台灣民間藥在脛骨骨髓細胞培養系中對成熟蝕骨細胞之影響及細胞激素在其中扮演角色. 2003.
19.Tortora GJ. Principles of Human Anatomy. 1999(8th).
20.Lange C, Schroeder J, Stute N, Lioznov MV, Zander AR. High-potential human mesenchymal stem cells. Stem Cells Dev. Feb 2005;14(1):70-80.
21.Saadeh PB, Mehrara BJ, Steinbrech DS, et al. Transforming growth factor-beta1 modulates the expression of vascular endothelial growth factor by osteoblasts. Am J Physiol. Oct 1999;277(4 Pt 1):C628-637.
22.Aubin JE, Liu F, Malaval L, Gupta AK. Osteoblast and chondroblast differentiation. Bone. Aug 1995;17(2 Suppl):77S-83S.
23.Mundy GR. “Bone Remodeling and its Disorders. 1999;2nd.
24.Takeda T, Hosokawa M, Higuchi K, Hosono M, Akiguchi I, Katoh H. A novel murine model of aging, Senescence-Accelerated Mouse (SAM). Arch Gerontol Geriatr. Sep-Oct 1994;19(2):185-192.
25.Takeda T, Hosokawa M, Higuchi K. Senescence-accelerated mouse (SAM): a novel murine model of senescence. Exp Gerontol. Jan-Apr 1997;32(1-2):105-109.
26.Takeda T, Hosokawa M, Takeshita S, et al. A new murine model of accelerated senescence. Mech Ageing Dev. Oct 1981;17(2):183-194.
27.Takeda T. Senescence-accelerated mouse (SAM): a biogerontological resource in aging research. Neurobiol Aging. Mar-Apr 1999;20(2):105-110.
28.Duque G, Macoritto M, Dion N, Ste-Marie LG, Kremer R. 1,25(OH)2D3 acts as a bone-forming agent in the hormone-independent senescence-accelerated mouse (SAM-P/6). Am J Physiol Endocrinol Metab. Apr 2005;288(4):E723-730.
29.Ichioka N, Inaba M, Kushida T, et al. Prevention of senile osteoporosis in SAMP6 mice by intrabone marrow injection of allogeneic bone marrow cells. Stem Cells. 2002;20(6):542-551.
30.Ebeling PR, Thomas DM, Erbas B, Hopper JL, Szer J, Grigg AP. Mechanisms of bone loss following allogeneic and autologous hemopoietic stem cell transplantation. J Bone Miner Res. Mar 1999;14(3):342-350.
31.Kushida T, Inaba M, Hisha H, et al. Intra-bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice. Blood. May 15 2001;97(10):3292-3299.
32.Shui C, Scutt AM. Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro. J Cell Physiol. Nov 2002;193(2):164-172.
33.Sun WB, Wang J, Lu C, Tang GX. [Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells.]. Chin Med J (Engl). Oct 20 2005;118(20):1703-1709.
34.Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. Dec 30 1994;79(7):1147-1156.
35.Nishikawa M, Ozawa K, Tojo A, et al. Changes in hematopoiesis-supporting ability of C3H10T1/2 mouse embryo fibroblasts during differentiation. Blood. Mar 1 1993;81(5):1184-1192.
36.Shea CM, Edgar CM, Einhorn TA, Gerstenfeld LC. BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis. J Cell Biochem. Dec 15 2003;90(6):1112-1127.
37.Ogino Y, Ayukawa Y, Kukita T, Koyano K. The contribution of platelet-derived growth factor, transforming growth factor-beta1, and insulin-like growth factor-I in platelet-rich plasma to the proliferation of osteoblast-like cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. Jun 2006;101(6):724-729.
38.Weibrich G, Kleis WK, Hafner G, Hitzler WE. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg. Apr 2002;30(2):97-102.
39.Lane NE, Kelman A. A review of anabolic therapies for osteoporosis. Arthritis Res Ther. 2003;5(5):214-222.
40.Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T. Platelet-rich plasma enhances human osteoblast-like cell proliferation and differentiation. J Oral Maxillofac Surg. Mar 2005;63(3):362-369.
41.Lucarelli E, Beccheroni A, Donati D, et al. Platelet-derived growth factors enhance proliferation of human stromal stem cells. Biomaterials. Aug 2003;24(18):3095-3100.
42.Johnson DW, McIntyre HD, Brown A, Freeman J, Rigby RJ. The role of DEXA bone densitometry in evaluating renal osteodystrophy in continuous ambulatory peritoneal dialysis patients. Perit Dial Int. Jan-Feb 1996;16(1):34-40.
43.Chen H, Shoumura S, Emura S. Ultrastructural changes in bones of the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis. Histol Histopathol. Jul 2004;19(3):677-685.
44.Chen H, Emura S, Isono H, Shoumura S. Effects of traditional Chinese medicine on bone loss in SAMP6: a murine model for senile osteoporosis. Biol Pharm Bull. May 2005;28(5):865-869.
45.Mayer-Kuckuk P, Gade TP, Buchanan IM, et al. High-resolution imaging of bone precursor cells within the intact bone marrow cavity of living mice. Mol Ther. Jul 2005;12(1):33-41.
46.Minn AJ, Kang Y, Serganova I, et al. Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest. Jan 2005;115(1):44-55.
47.Jiang SD, Dai LY, Jiang LS. Osteoporosis after spinal cord injury. Osteoporos Int. Feb 2006;17(2):180-192.
48.Beaupre GS, Lew HL. Bone-density changes after stroke. Am J Phys Med Rehabil. May 2006;85(5):464-472.
49.Pinheiro MM, Castro CM, Szejnfeld VL. Low femoral bone mineral density and quantitative ultrasound are risk factors for new osteoporotic fracture and total and cardiovascular mortality: a 5-year population-based study of Brazilian elderly women. J Gerontol A Biol Sci Med Sci. Feb 2006;61(2):196-203.