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標題: | 使用全椎間盤培養系統之體外實驗探討外生性交聯療法對治療退化性椎間盤之可能性 Assessment of Exogenous Cross-Linking Therapy for Intervertebral Disc Degeneration - An ex vivo Study Using Whole Disc Culture System |
作者: | Ya-Ching Chang 張雅晴 |
指導教授: | 王兆麟(Jaw-Lin Wang) |
關鍵字: | 椎間盤,退化模型,外生性交聯,梔子素,體外椎間盤培養系統, intervertebral disc,degeneration model,exogenous cross-linking,genipin,ex vivo whole disc culture system, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 目的:利用體外椎間盤培養系統探討外生性交聯療法對於不同退化程度椎間盤之療效。
背景簡介:一般中老年人常見的脊椎退化性病徵為下背痛,許多文獻指出下背痛可能源自於椎間盤退化,而椎間盤退化是由許多影響因子共同調控的結果,其機制目前尚未完全明瞭,為了針對退化性椎間盤作全面性的治療,完整模擬椎間盤退化即為首要課題。現今研究中,用於治療椎間盤退化的藥物之一為天然胜肽交聯劑-梔子素,其生化作用機制為增加蛋白質或膠原蛋白之間的胺基鍵結,用以增加椎間盤整體結構穩定度、提高動態強度與緩衝能力,然而,尚未有研究對於梔子素於不同退化程度之椎間盤治療成效作詳細探討與比較。 材料與方法:實驗使用六個月大的年輕豬隻,試樣取用為胸椎骨第一到第六節(T1~T6)之間的椎間盤,為確保試樣新鮮將於豬隻屠宰後四個小時內進行實驗。本研究使用胰蛋白酶(trypsin)模擬椎間盤退化早期蛋白質變性的過程,模擬退化組中每個試樣於第一天處理完畢後打入0.5 ml, 0.25%的胰蛋白酶,模擬椎間盤晚期嚴重退化的組別將於第二天施加高強度之疲勞負載(平均力量420N, 負載頻率2.5Hz, 負載時間4hours, 負載次數約36000 cycles),而治療組則進一步於第二天打入1 ml, 0.33%的梔子素(genipin)作治療,各組試樣均放置於生物培養系統中,並於培養一週後作力學測試及生化測試。 結果: 模擬早期退化之蛋白質變性椎間盤,其靜態及動態力學性質皆有顯著性降低,染色切片結果顯示椎間盤組織結構鬆散、排列不一致;模擬晚期退化之蛋白質變性加破壞椎間盤,其聚合模數及勁度上升但衝擊緩衝能力及滲透度下降,染色切片結果顯示其組織破壞損傷嚴重、失去結構完整性,因此無法維持原本高度被壓得更緊緻。加入梔子素治療後,蛋白質變性椎間盤的椎間盤高度、聚合模數、循環負載後勁度值、循環負載前阻尼係數及椎間核水份含量,相對於治療前皆有顯著性回復;然而,梔子素治療對於蛋白質變性加破壞的椎間盤,其內部組織缺損破壞無法修復,但治療後組織強度仍然有回復的趨勢,而椎間核含水量大幅增加,可推測其相比於破壞嚴重的退化組有較好的緩衝能力,因此阻尼係數於循環負載前後皆有顯著地回復。 結論:天然胜肽交聯劑-梔子素可用於治療早期退化性椎間盤,並回復其生物力學性質;若椎間盤退化嚴重且伴隨組織缺損,則梔子素可以回復椎間盤喪失之部分動態力學性質,但無法完全修復缺損的組織,而這些組織破壞可能導致椎間盤退化持續進行。 Objective: To investigate the efficacy of exogenous cross-linking therapy in treating the degenerated intervertebral disc by using ex vivo whole disc culture system. Summary of background data: Disc degeneration induced low back pain syndrome is observed in elderly human. The disc degeneration can be induced by factors such as protein denaturation and micro-defect within disc fiber. A disc degeneration model can be effectively used for the screening of clinical strategy for disc degeneration treatment. The treatment based on this model can be studied to find the physiological effect. In recent studies, exogenous cross-linking therapy has been shown to enhance stability and dynamic properties of disc by increasing protein cross-linking. Nevertheless, the mechanical and biological effects of peptide cross-linking reagent on different degrees of disc degeneration shall be concerned to further assess the feasibility of exogenous cross-linking therapy. Methods: Total 40 porcine thoracic discs (T1~T6) were dissected from 6-month-old juvenile pigs within 4 hours after sacrifice. After specimen preparation, discs were equally assigned to five groups (n=8 for each group), and all specimens were incubated for 1 week before test. Groups were divided as follows: intact group, early degenerated group, early degenerated with treatment group, severely degenerated group, severely degenerated with treatment group. For all groups except intact group, 0.5 ml 0.25% trypsin solution was injected into specimen on Day 1 to produce protein denaturation, which is the simulation for early degenerated disc, and high strength fatigue loading (Frms= 420 N, frequency= 2.5 Hz, loading period= 4 hours) was given to produce micro-defects within disc fiber to resembling the severe disc degeneration on Day 2. For groups with treatment, specimens were injected with 1 ml 0.33% genipin solution after simulation of disc degeneration. Mechanical test and biological detection were performed after one week incubation. Result: In group of early degenerated disc, the results showed that the mechanical property of disc significantly decreased both in static and dynamic test, and also the disorganization of the laminate structure was found in histological stain. If the disc is damaged by fatigue loading after protein denaturation, the aggregate modulus and stiffness increase with height loss, but damping coefficient, permeability and water content decrease because of structural defects. This defect was also found in histological stain. After taking exogenous cross-linking therapy, the degenerated disc recover its mechanical property and restores the water content to normal condition, but the structural defects which occur in severely degenerated disc cannot be repair by cross-linker. Conclusion: Genipin-treated discs show the recovery of mechanical properties after protein denaturation which presumes to be process of early disc degeneration, and the efficacy maintains for at least one week; however, mechanical defects which occur in severely degenerated disc cannot be treated by protein cross-linking. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/10037 |
全文授權: | 同意授權(全球公開) |
顯示於系所單位: | 醫學工程學研究所 |
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