第四章 結果
第一節 雙側卵巢切除與電針足三里及三陰交對大鼠體重影響 37
大鼠隨機分為三組，控制組(CON)實施假手術，卵巢切除組(OVX)及 電針組 (OVX+EA) 實施雙側卵巢切除術，開始進行實驗後每週測量大鼠 體重一次。實驗進行中各組體重逐漸上升，卵巢切除組大鼠體重於第四 週及第五週明顯較控制組及電針組大鼠來的重，而電針組自第三週起至 第五週，體重明顯較卵巢切除組來的輕。
大鼠體重(g) CON(n=6) OVX(n=6) OVX+EA(n=6) 第一週 146.7±8.4 151.3±11.1 0145.5±1.8 第二週 197.2±13.4 201.7±14.7 200.8±12.8 第三週 225.0±12.2 251.7±2.0 209.2±9.2#
第四週 239.2±12.0 274.8±5.3* 253.0±1.5#
第五週 256.3±11.7 294.8±15.9* 273.8±3.9#
表 4.1.1 大鼠體重變化，*p<0.05 vs CON group(student＇s t-test)，#p<0.05 (one-way ANOVA).
Time (week)
0 1 2 3 4 5
Body w e ight (g)
6 切除組來的輕。*p<0.05 vs CON group(student＇s t-test)，#p<0.05 (one-way ANOVA).
第二節 雙側卵巢切除與電針足三里及三陰交對大鼠成骨作用影響
實驗進行中，每週抽取大鼠血清，測量與成骨作用相關指標。利用 ELISA 方法測定大鼠血清中鹼性磷酸酶(ALP)活性，於第四、五週時卵巢 切除組大鼠 ALP 含量明顯下降，而電針組大鼠於第四、五週血清中 ALP 含量明顯高於卵巢切除組。
ALP activity
CON(n=6) OVX(n=6) OVX+EA(n=6)
第一週 0.237±0.015 0.236±0.040 0.245±0.019 第二週 0.273±0.039 0.257±0.049 0.277±0.059 第三週 0.284±0.045 0.227±0.033 0.295±0.069 第四週 0.277±0.044 0.216±0.025* 0.297±0.048#
第五週 0.286±0.047 0.202±0.009* 0.293±0.050#
表 4.2.1 大鼠血清 ALP 活性變化, *p<0.05 vs CON
group(student＇s t-test)，#p<0.05 (one-way ANOVA).
Time (week)
0 1 2 3 4 5
Serum A L P a c ti v it y
6
group(student＇s t-test)，#p<0.05 (one-way ANOVA).
第三節 雙側卵巢切除與電針足三里及三陰交對大鼠骨顯微構造影響
實驗結束後，將大鼠脛骨固定、切片，並利用 H/E stain 染色後，
可觀察到卵巢切除組大鼠骨顯微構造有空洞化的情況，利用 CT 測量大 鼠骨質密度及骨質含量，卵巢切除鼠骨質密度及骨質含量明顯低於控制 組，而電針組骨質含量明顯高於卵巢切除組。
Tibia CON(n=6) OVX(n=6) OVX+EA(n=6) BMD (g/cm3) 0.137±0.005 0.121±0.005* 0.130±0.006 BMC (g) 0.385±0.016 0.346±0.020* 0.370±0.014#
表 4.3.1 大鼠脛骨 BMD 及 BMC 比較。*p<0.05 vs CON
group(student＇s t-test)，#p<0.05 (one-way ANOVA).
BMD (g/cm3 ) 明顯較 OVX 組高。 *p<0.05 vs CON group(student＇s t-test)，#p<0.05 (one-way ANOVA).
第四節 雙側卵巢切除與電針足三里及三陰交對大鼠脛骨重量及長度及 力學特性影響
實驗結束後，將大鼠脛骨分離，將肉剔除後測量長度及重量，各組 長度並無顯著差異，而卵巢切除組重量明顯低於控制組，電針組重量則 明顯高於卵巢切除組。
Tibia CON(n=6) OVX(n=6) OVX+EA(n=6)
Length (mm)
3.73±0.01 3.74±0.02 3.72±0.03Wet weight (g)
761.8±15.9 707.8±18.4* 755.5±32.6#表 4.4.1 大鼠脛骨長度及重量比較*p<0.05 vs CON group (student＇s t-test)，#p<0.05 (one-way ANOVA).
圖 4.4.1 各組大鼠近端脛骨骨幹骺端切片(H/E stain)，可見卵巢切除 組骨小樑數目減少，空洞化明顯，而電針組則無此現象。
Tibia length (mm)
Tibia Wet weight (g)
600
*p<0.05 vs CON group(student＇s t-test)，#p<0.05 (one-way ANOVA).
第五章 討論
由於經濟的繁榮及醫藥的進步，國人平均壽命大幅延長，人口老化 的結果，慢性病已成為國人最主要的健康問題 。自 1993 年台灣 65 歲 以上老年人口已超過百分之七，符合 WHO 之“高齡化國家(aging
society)＂定義，已邁入老年化社會；2007 年老人人口比例更達到 10%
以上[32]，為目前衛生署「2020 健康國民白皮書」的焦點族群之一[33]。
世界衛生組織(World Health Organization, WHO)列出八大類未來全球 化的慢性疾病重要課題，其中第六類「慢性風濕病況 Chronic Rheumatic Condition」包括風濕關節炎(rheumatic arthritis)，退化性骨關節炎 (osteoarthritis)、骨質疏鬆症(osteoporosis)、及脊椎疾患(spinal disorders)、重大肢體創傷(severe limb trauma)[34]，可見骨質疏鬆 疾病是未來一個重要的課題。
顯高於控制組(CON)，這種情況和停經後婦女體重增加情形類似，而電 針組(OVX+EA)於第三週後，體重即明顯低於卵巢切除組（OVX），代表電 針可以有效避免大鼠卵巢切除後體重增加。
實驗進行中每週抽取大鼠血清，並測量相關骨生化指標，骨生化中 和成骨作用相關指標如總 ALP 及骨特異性 ALP 或血清中 osteocalcin、
procollagen type I C- 或 N-terminal propeptides[35]。由圖 4.2.1 可知，卵巢切除組大鼠於第四週後血清中 ALP 含量明顯少於控制組，而
實驗動物均飼養在一個高度控制的環境中，除了環境外，並餵食相 同飼料，但是人類因為種族、年齡及生活方式的不同，並無法確定是否 電針可以造成相同的結果，而實驗動物只能呈現相關血清、顯微組織的 變化，並無法模擬停經後婦女骨折風險增加的情況。
由以上討論我們可以知道，大鼠卵巢切除後經電針刺激足三里及三 陰交，的確可以改善骨質流失情形，也許是透過刺激骨母細胞分泌相關 造骨因子如 ALP 所形成，但是詳細作用機轉需要透過進一步的實驗才能 更加明瞭。
第六章 結論
總結來說，我們的實驗呈現了電針卵巢切除大鼠雙側足三里及三陰 交穴可以改善卵巢切除後骨質流失的情形並且增加骨質顯微構造及強 度，可能是透過刺激骨母細胞分泌造骨相關因子如 ALP，這提供了未來 發展電針方式治療停經後婦女骨質疏鬆並預防骨折的一個證據。
參考文獻
1. Kanis JA, Melton LJ 3RD, Christiansen C, Johnston CC, Khaltaev N, The diagnosis of osteoporosis. J Bone Miner Res, 1994; 9(8): 1137-1141.
2. Who are candidates for prevention and treatment for osteoporosis? Osteoporos Int, 1997; 1-6.
3. IOF. Facts and statistics about osteoporosis and its impact. Available
at:http://www.iofbonehealth.org, 2008.
4. Yang NP, Deng CY, Chou YJ, Chen PQ, Lin CH, Chou P, Chang HJ. Estimated prevalence of osteoporosis from a Nationwide Health Insurance database in
Taiwan. Health Policy, 2006; 75(3): 329-37.
5. Heckman JD, Ryaby JP, McCabe J, Freym JJ, Kilcoyne RF. Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound. J Bone Joint Surg, 1994; 76: 26-34.
6. 吳明霞, 周琳瑛, 陳文列,吳炳煌,劉獻祥. 針灸對去卵巢大鼠骨密度、強度及 超微結構的影響.中國臨床康復雜誌,2004; 8(18): p. 3546-7.
7. 張華, 馮新送, 莊洪,劉慶思,魏合偉. 中醫對原發性骨質疏鬆症的認識及辨證 施治研究,中醫正骨, 2001; 13(3): p. 55-56.
8. Consensus Development Conference. Consensus development conference:
646-649.
9. Chie WC, Yang RS, Liu JP, Tsai KS. High incidence rate of hip fracture in Taiwan:
estimated from a nationwide health insurance database. Osteoporos Int, 2004; 15:
998-1002.
10. 行政院衛生署食品衛生處.台灣地區老人營養健康狀況調查 1999-2000 調查結 果老年人骨質健康狀況。2004.
11. Lin JD, Chen JF, Chang HY, Ho C. Evaluation of bone mineral density by quantitative ultrasound of bone in 16862 subjects during routine health
examination. Br J Radiol, 2001; 74: 302-306.
12. Weitzmann MN, Pacifici R. The role of T lymphocytes in bone metabolism.
Immunol Rev, 2005; 208: 154-168.
13. Weitzmann MN, Pacifici R. Estrogen deficiency and bone loss: an inflammatory tale. J Clin Invest, 2006; 116(5): 1186-1194.
14. Chapuy MC, Arlot ME, Duboeuf F, Brun J, Crouzet B, Arnaud S, Delmas PD,
Meunier PJ. Vitamin D3 and calcium to prevent hip fractures in the elderly women. N Engl J Med 1992; 327(23): 1637-1642.
15. Vehmanen L, Elomaa L, Blomqvist C, Saarto T. Tamoxifen treatment after
adjuvant chemotherapy has opposite effects on bone mineral density in
premenopausal patients depending on menstrual status. Clin Oncol, 2006; 24(4):
675-680.
16. Powles TJ, Hickish T, Kanis JA, Tidy A, Ashley S. Effect of tamoxifen on bone
mineral density measured by dual-energy x-ray absorptiometry in healthy premenopausal and postmenopausal women. J Clin Oncol, 1996; 14(1): 78-84.
17. Arnala I, Saastamoinen J, Alhava EM. Salmon calcitonin in the prevention of
bone loss at perimenopause. Bone, 1996; 18(6): 629-632.
18. Liberman UA, Weiss SR, Broll J, Minne HW, Quan H, Bell NH,
Rodriguez-Portales J, Downs RW Jr, Dequeker J. Favus M Effect of oral alendronate on bone mineral density and the incidence of fractures in
postmenopausal osteoporosis. The Alendronate Phase III Osteoporosis Treatment Study Group. N Engl J Med 1995; 333(22): 1437-1443.
19. Harris ST, Gertz BJ, Genant HK, Eyre DR, Survill TT, Ventura JN, De Brock J,
Ricerca E, Chesnut CH 3d. The effect of short term treatment with alendronate on vertebral density and biochemical markers of bone remodeling in early
postmenopausal women. . J Clin Endocrinol Metab, 1993; 76(6): 1399-1406.
20. Cranney A, Wells G, Willan A, Griffith L, Zytaruk N, Robinson V, Black D, Adachi J, Shea B, Tugwell P, Guyatt G. Meta-analyses of therapies for
postmenopausal osteoporosis. II. Meta-analysis of alendronate for the treatment of
postmenopausal women. . Endocr Rev 2002; 23(4): 508-516.
21. Bischoff-Ferrari HA, Willett WC, Wong JB, Giovannucci E, Dietrich T,
ameta-analysis of randomized controlled trials. Jama, 2005; 293(18): 2257-2264.
22. 丁桂芝, 李榕. 從骨礦含量變化規律看腎主骨理論的科學性. 1991; 13(2): 27.
23. 許書亮, 張永保. 骨質疏鬆症中醫證型的初步臨床報告. 中醫正骨, 1995; 3(4):
30-31.
24. 張賢, 蔡建平. 單味中藥防治骨質疏鬆症的實驗研究進展. 時珍國醫國藥,
2008; 19(7): 1701-1702.
25. 高明, 華元, 穆敬平. 針灸治療骨質疏鬆症的現狀與思考. 上海針灸雜誌,
2007; 26(2): 40-42.
26. Thompson DD, Simmons HA, Pirie CM, Ke HZ. FDA Guidelines and animal models for osteoporosis. Bone, 1995; 17: 125S-133S.
27. Turner RT, Maran A, Lotinun S, Hefferan T, Evans GL, Zhang M, Sibonga JD.
Animal models for osteoporosis. Rev Endocr Metab Disord, 2001; 2(1): 117-127.
28. 賈經漢, 邱新建., 陳志堅, 骨質疏鬆動物模型的研究進展. 中國康復醫學雜 誌, 2007; 22(8): 765-768.
29. Horton JA, Murray GM, Spadaro JA, Margulies BS, Allen MJ, Damron TA.
Precision and accuracy of DXA and pQCT for densitometry of the rat femur. J Clin Densitom, 2003; 6(4): 381-390.
30. 李忠仁. 實驗針灸學:附錄一常用實驗動物針灸穴位. 中國中醫藥出版社.
2007; pp. 257.
31. 楊文達, 林昭庚, 劉旭然. 以醋酸扭體實驗在小鼠觀察不同針尖規格之鎮痛
效果. J Chin Med, 2003; 14(1): 69-74.
32. 衛生署：衛生統計重要指標。http://www.doh.gov.tw/statistics/index.htm。引用 2008/1/2。
33. 衛生署企劃處： 2020 健康國民白皮書。http://www.doh.gov.tw。引用 2008/1/2。
34. WHO. Chronic diseases and health promotion. Available at:
http//:www.who.int/chp/topics. Accessed January 2, 2008.
35. Seibel MJ, Eastell R, Gundberg CM. Biochemical markers of bone metabolism. In:
Principles of Bone Biology (Bilezikian JP, Raisz LG, Rodan GA, eds). 2nd edn.
San Diego: Academic Press, 2002; pp1543–71
英文摘要
In normal bone, there is constant matrix remodeling of bone;
up to 10% of all bone mass may be undergoing remodeling at any point in time. Bone is resorbed by osteoclast cells, after which new bone is deposited by osteoblast cells. Mechanical or
electrical stimuli can influence bone formation and resorption in vitro and in vivo, suggesting that these modalities can be used clinically to inhibit or reverse osteopenia.
Considering the high incidence of pharmacological treatments, electroacupuncture treatment with best cost benefit rates for osteoporosis were investigated in this study. Eighteen 6-week old SD female rats (BioLASCO Taiwan Co., Ltd.) weighing 180-220 g were used in this study. The rats were randomly divided into three groups, a control group (n=6), a model group (n=6) and an electroacupuncture(EA) group (n=6). The rats in Model group and EA group were received ovariectomy operation while the rats in control group received sham operation. The rats in EA group were then given EA on ST36 and SP6 bilaterally, fifteen mins per day, five days per week during four weeks (total twenty times), under
isoflurane anesthesia (in flow of oxygen and nitrous oxide mixture;
3% for induction and 1.5% for maintenance). The positive electrode was connected to ST36, and the negative electrode to SP6.
Electrical stimulation were provided by a pulse stimulator (Trio 300, Ito Co., Japan), which produced a biphasic square wave with the following characteristics: 2 Hz frequency, 1-2 mA duration pulses. The current intensity were adjusted until localized muscle contractions were observed. In this study, EA increased the bone volume of secondary spongiosa in tibia. EA on ST36 and SP6 could prevent ovariectomized (OVX) rats＇ tibia from
osteopenia change and increased serum level of alkaline
phosphatase (ALP). The EA also avoided the body weight gain of rats in EA group compared to Model group. Taken together, our results might provide the evidence that EA can enhance bone formation by elevate serum ALP level in OVX rats.
Index Words: electroacupuncture, osteoporosis, ALP, bone formation