類別:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39
2024-03-06T03:27:09Z龐貝氏症患者及帶原者酵素活性差異性之原因
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35908
標題: 龐貝氏症患者及帶原者酵素活性差異性之原因; Etiology of enzyme activity variability in Pompe’s disease
作者: Cheng-Ni Liu; 劉正暱
摘要: 龐貝氏症(Pompe’s disease)是一種肝醣儲積症(Glycogen storage disease, GSD),又稱為肝醣儲積症第二型(GSD II)。是由於先天缺乏溶小體酵素-酸性麥芽糖酶(Acid Maltase,又稱acid α-glucosidase, GAA),使得進入溶小體的肝醣無法被分解而持續堆積,進而影響到細胞的功能。臨床上依發病年齡分為嬰兒型、青少年型及成人型。龐貝氏症確定診斷的方法為測定皮膚纖維芽細胞或血液中GAA酵素活性。通常患者的酵素活性很低(<5%),而帶原者的活性會介於正常人和患者之間。臨床上發現,部分帶原者的酵素活性偏低,約只有正常人5 ~ 10%,可能會增加檢測困難度。因此,我們認為可能存在某些因數會影響酸性麥芽糖酶的表現,如基因多型性或其他機轉,影響到酸性麥芽糖酶的蛋白質穩定性。在本實驗室近年來的研究,發現熱休克蛋白Hsp27對於突變蛋白GTP cyclohydrolase I的dominant-negative effect會有影響。Hsp27是一種small heat shock蛋白,和蛋白質摺疊、細胞骨架之穩定性,以及細胞凋亡有關。突變酸性麥芽糖酶可能在某些情況下,如Hsp27存在下,其蛋白質穩定性會有所改變,也可能會受到其他因數的影響,導致細胞酵素活性降到如此的低。
本研究進行的方法(1)分析所有個案的GAA酵素活性、mRNA與蛋白質表現量。(2)藉著基因序列分析找尋是否有基因多型性的存在,與突變蛋白GAA酵素活性具有關聯性。(3)以表現Hsp27的質體轉染龐貝氏症患者或是帶原者的皮膚纖維芽細胞,瞭解Hsp27對於突變蛋白GAA酵素活性是否具有調控的能力。
本實驗結果證實GAA酵素活性表現與蛋白質表現量有正相關性。也證實GAA突變蛋白的存在。RNA實驗結果顯示,帶原者的GAA蛋白表現量和其mRNA間並無絕對相關性存在。以蛋白結構推測,GAA突變蛋白與正常蛋白具有差異性。雖然基因多型性可能會影響GAA突變蛋白的穩定性,在基因序列分析中,也發現許多序列多型性,並形成三個區塊,但並無特殊的基因多型性與酵素活性間有關聯性。Hsp27 -S3D實驗中發現Hsp27-S3D會降低患者與帶原者的酵素活性。
影響GAA酵素活性的因數,還是有其他基因多型性,或其他分子伴隨者調控的可能。本研究主要目的希望能應用於未來的臨床診斷及治療,在臨床上提供更精確診斷,治療上能有其他新藥物。我們的研究指出蛋白質的穩定性是影響酵素活性的重要因數。可能在基因的其他位置,例如是intron仍存在著有影響的序列多型性。或是細胞內的其他熱休克蛋白會影響GAA蛋白的穩定性,這些都還待進一步之研究。; Pompe’s disease is a lysosomal storage disease involving the storage of glycogen. It is used to be called type II glycogenosis. The etiology of this disease is the deficiency of acid maltase (or acid alpha-glucosidase, GAA). The deficiency of GAA leads to a progressive storage of glycogen in the lysosome, which affects the function of the cells. According to the onset clinically, there are three subtypes – infantile, juvenile, and adult type. The diagnosis of Pompe’s disease depends on the measurement of GAA activity in either skin fibroblast or in peripheral blood mononuclear cells. Theoretically, GAA activities in obligatory carriers should be 50% of normal. However, we frequently met carriers with GAA activities as low as 5~10% of normal. The current hypothesis is the presence of some important factors, ex. polymorphism or some other mechanisms that will alter the stability of GAA.
In our previous studies, Hsp27 can alter the stability of the GTP-cyclohydrolase I protein. Hsp27 is a member of the small HSP that is involved in protein folding, stability of the cytoskeleton, and cell apoptosis. It is possible that the stability of GAA will be changed under certain circumferences, and then causes the excessive low GAA activity in some individuals.
In this study, we are going to perform the assays: (1) the GAA activity, mRNA, and protein expression; (2) the gene polymorphism analysis; (3) to overexpress Hsp27 in skin fibroblasts from either Pompe’s patients or the carriers. We will observe the changes of GAA protein by the western blot analysis, and also GAA activities by enzyme assays, to see if these parameters will be changed by the expression of Hsp27. Special interests will be on Pompe’s carriers with excessive low GAA activities.
In the study, there is a positive correlation between the GAA activity and the protein expression. From the RNA study, there is no correlation between the expression of the GAA protein and of the mRNA. From the structure analysis, the polymorphism (V816I) may influence the stability of mutant GAA protein. We also find some polymorphisms and form three blocks in the gene polymorphism study. But, no relation between the polymorphisms and the GAA activity. The data of the Hsp27 study shows Hsp-S3D will decrease the GAA activity, either in the patients or carriers.
There still exists the possibility of some other factors that will influence the GAA activity, ex. Polymorphisms, or heat shock proteins. This study will contribute significantly the knowledge of the pathogenesis of disease, the mechanism of changes in protein stability, the diagnostic technology, and also deeply to the future treatment of the diseases, including those with similar molecular mechanisms. Our data showed the stability of GAA protein is one important factor to the GAA activity. Maybe some polymorphisms exists in some position of the GAA gene, ex. Intron, that will play some roles. Or, the other Hsp in the cells may affect the stability of the GAA protein. Those all will be needed further study.2005-01-01T00:00:00Z鼠鬚刺激系統之開發與眶下神經重合之術後功能性回復評估
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51289
標題: 鼠鬚刺激系統之開發與眶下神經重合之術後功能性回復評估; Stimulation system for rat whisker and functional recovery after infraorbital nerve transection
作者: Ji-Lin Chen; 陳吉麟
摘要: 臨床上許多狀況會造成周邊感覺神經的截斷,如刀傷、車禍碰撞、撕裂傷、移植手術等。少數神經重建術接合斷裂神經的患者在經過長時間復原後可達到部分的感覺回復,究竟這些重拾感覺輸入的患者其大腦發生了哪些轉變是我們所好奇的課題。本研究以大鼠的觸鬚-桶狀皮質系統作為模型,截斷與重新接合支配觸鬚感受器的眶下神經模擬臨床情況,建立出刺激參數與神經反應的調性方程式以評估手術前後感覺皮質發生的變化。
本研究開發了定量化且精確的觸鬚刺激系統以應用於動物實驗。兩組軟體產生的訊號經類比轉換及電壓放大後控制雙軸向壓電致動元件,利用黏附其上的夾具帶動鼠鬚產生雙自由度的撓動。藉由夾具的設計以及軟體的校正,本系統達到高精度的振幅刺激,此外,系統加入了應變量測裝置對輸出振幅作即時監測以減低實驗誤差。
於感受域分析結果,超過半數的樣本在術後可以重新獲得周邊觸鬚受器與皮質感受域的對應關係。其中約三成樣本的感受域分布在手術前後具有相似性。針對感受域回復的樣本,刺激速度辨識能力在手術之後立即降低,但會隨著復原時間增長而回升。另外,皮質神經的刺激後反應時間在手術後增加,但會隨著復原之過程而逐漸減少。本研究呈現了大鼠觸鬚系統在受損的感覺神經功能性回復期間所伴隨的生理變化。; Functional loss of peripheral nerve, such as cutting injury, blunt injury, laceration injury and nerve transplantation, is a common clinical condition. For example, the sensation of the finger will take a long time to recover following a nerve reconstruction surgery for some patients with nerve injury. Therefore, the neuroplasticity mechanisms in the primary somatosensory cortex by a nerve reconstruction surgery were investigated. The whisker-to-barrel model was used to evaluate the change of neuronal tuning following neurotomy and neurorrhaphy surgery, an approach that simulates a clinical nerve reconstruction surgery.
We first developed a whisker stimulator that can present precise whisker bending at various directions and speeds. The whiskers were bended by the piezoelectric actuator that has two degrees of freedom, each of which was driven by amplified voltage signals controlled by the software. We designed the whisker holder and completed the software calibration to provide whisker movement with precise amplitude and phase control. Furthermore, we developed a measuring device to monitor the amplitude movement of stimulator in real time.
Multi-unit neuronal activities were recorded before and after the neurotomy and neurorrhaphy surgery, and the follow-up period reached 4-6 weeks post-surgery. The results showed that all units lost their sensory responses immediately after the surgery. Surprisingly, the sensory responses could recover as early as 7 days. In the long-term follow-up, one third of the unit regained whisker tuning that was analogous to that before the surgery. Also, their tuning strength to stimulus speed was weakened in the early recovery period and then gradually increased in the late recovery period. Similarly, the onset latency of a cortical response to whisker stimulation also prolonged in the early recovery period and gradually decreased in the late recovery period. In conclusion, the present study reveals the neuronal mechanisms that account for the functional recovery following nerve reconstruction in the whisker-to-barrel model.2016-01-01T00:00:00Z黏性補給與外生性交聯對早期退化性椎間盤的動態生物力學性質影響
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45905
標題: 黏性補給與外生性交聯對早期退化性椎間盤的動態生物力學性質影響; Changes of Biodynamic Properties of Mild Degenerated Intervertebral Disc due to Viscosupplementation and Crosslinking
作者: Li-Chiang Hsu; 徐禮強
摘要: Objective: To evaluate the biodynamic properties of the mildly degenerated intervertebral disc after viscosupplementation and crosslinking.
Summary of Background Data: The matrix denaturation in early degenerated intervertebral disc compromise the hydraulic retention mechanism to resist compressive loading and thus degrade disc dynamic properties. Hyaluronic acid (HA) is one of primary compositions of extracellular matrix and characterized to absorb abundant water to increase the viscosity of synovial fluid. Whether the interaction of HA and the degenerated disc matrix could resume the disc dynamic properties by similar mechanism remains unclear. The dynamic properties of the degenerated discs were partially recovered by genipin-induced crosslinking. The recovery degree may be enhanced by increasing the genipin concentration and reaction time. The recovery efficiency of HA-mediated viscosupplementation and genipin-induced crosslinking on the dynamic properties of the mildly degenerated disc has not been compared yet.
Methods: A total of 36 porcine lumbar body-disc-body constructs were assigned to an “Intact disc” group (n=9) and a “Degenerated disc” group (n=27). The specimens of the “Intact disc“ were injected with 1 ml saline, while the specimens of the “Denatured disc” were injected with 1 ml 0.5% trypsin solution. After a 24 hr saline bath, an impact test was performed before and after a 30 min fatigue loading (peak to peak: 190-490 N) to obtain the dynamic properties, i.e. stiffness modulus (K, N/s) and damping coefficient (C, Ns/mm). The specimens of the “Intact disc” and 9 specimens of the “Denatured disc” were injected with 1 ml HA (“Viscosupplemented disc”), while the other 9 discs of the “Denatured disc” were injected with 1 ml 3.3% genipin solution (“Crosslinked disc”). Then the “Intact disc” and the “Viscosupplemented disc” rehydrated in saline solution for 24 hr, while the “Crosslinked disc” rehydrated for 72 hr. After that, the aforementioned protocols of rehydration, fatigue loading and the impact test were repeated to obtain the disc dynamic properties.
Results: For the intact disc, the stiffness modulus was 720.2 (75.4) N/mm and the damping coefficient was 0.60 (0.06) Ns/mm. After the fatigue loading, the stiffness modulus increased to 937.7 (16.4) N/mm and the damping coefficient decreased to 0.53 (0.03) Ns/mm. HA-mediated Viscosupplementation increased the stiffness modulus and the damping coefficient, but the effect was decreased for the stiffness modulus by the fatigue loading. The disc stiffness modulus and damping coefficient were degraded by the trypsin-induced degeneration, but fully recovered by the HA-mediated viscosupplementation and genipin-induced crosslinking. However, the stiffness modulus and damping coefficient of the viscosupplemented disc were degraded by the fatigue loading, while only the stiffness modulus of the crosslinked disc degraded after the fatigue loading. Compared to the previous data, the recovery level of the genipin solution on the stiffness modulus and damping coefficient of the degenerated disc were elevated when the genipin concentration was 10 folds increased and the crosslinking time was 3 times elongated.
Conclusion: The recovery efficiency of the genipin-induced crosslinking on the dynamic properties of the degenerated disc, especially on the damping coefficient, was more sustainable to the fatigue loading when compared to that of the HA-mediated viscosupplementation, and could be enhanced by increasing the genipin solution concentration and reaction time.2010-01-01T00:00:00Z黏性補充劑與胜肽交聯劑回復退化性椎間盤生物力學功能之可行性研究
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48681
標題: 黏性補充劑與胜肽交聯劑回復退化性椎間盤生物力學功能之可行性研究; Feasibility Analysis of Viscosupplement and Crosslinker in Recovering the Biomechanical Functions of Degenerated Intervertebral Disc
作者: Ya-Wen Kuo; 郭雅雯
摘要: 椎間盤為人體內負責承重與吸收衝擊的主要器官,椎間盤退化疾病將不可避免地降低患者生活品質,是人口高齡化社會所必須正視的醫療問題。椎間盤的功能表現取決於其固體結構機械性質以及液體流動機制,可由靜態流變性質與動態力學性質加以評估。深入了解影響椎間盤靜態流變性質與動態力學性質的各種機制,將有助於增進前瞻性退化性椎間盤治療方法的效益。本研究的第一部份,探討不同負載條件以及退化程度對椎間盤靜態流變性質的影響,探討的流變參數分別代表椎間盤於靜態負載時,整體勁度與內部液體流動性。實驗結果顯示:於潛變期間,椎間盤勁度增加,液體流動性降低;於鬆弛期間,椎間盤勁度降低,液體流動性不變;當負載力量增加,椎間盤勁度上升而液體流動性下降;疲勞負載時間越長,椎間盤勁度不變,但液體流動性降低;隨著退化程度增加,椎間盤勁度升高,液體流動性降低。本研究結果另可推論椎間盤勁度與含水量為負向相關,液體滲透度與椎間盤孔隙度為正向相關。
本研究的第二部份以胰蛋白脢降解椎間核模擬退化初期的椎間盤,比較黏性補充劑(玻尿酸)與胜肽交聯劑(梔子素)對於回復退化椎間盤的細部結構性質與整體動態力學性質的效益,並分析椎間盤細部結構性質對整體動態力學性質的影響程度。實驗中所探討的椎間盤細部結構性質包含椎間核含水量、椎間核黏彈性、椎間環結構完整性;椎間盤動態力學性質包含椎間盤受衝擊負載時,整體勁度與能量緩衝性。結果顯示:玻尿酸可回復退化椎間核的含水量,提升退化椎間盤於疲勞負載後的能量緩衝性;梔子素可回復退化椎間核的含水量,提升退化椎間核的彈性、聚合剥離的椎間環纖維,回復退化椎間盤於疲勞負載後的能量緩衝性。玻尿酸與梔子素皆無法改變退化椎間核的黏性與椎間盤於疲勞負載後的勁度。與玻尿酸相較之下,梔子素較能回復退化性椎間盤的結構性質與生物力學功能,但梔子素會將水分限制於椎間核內,使水分無法隨疲勞負載而外流,並改變椎間盤動態性質在疲勞負載下的變化趨勢,因此梔子素本身在回復退化性椎間盤生物力學功能上的可行性並不高。在椎間盤細部結構性質與整體動態力學性質的迴歸分析上,結果顯示椎間核彈性對椎間盤疲勞負載前的勁度有較大的相關性,椎間核黏性對椎間盤疲勞負載後的能量緩衝性有較大的相關性。
本論文推測椎間盤流變性質受負載條件與退化程度而改變的機制在於含水量與椎間盤內部孔徑的變化,當退化產生時,椎間盤勁度升高且內部水分的流通性降低。黏性補充劑與胜肽交聯劑皆無法完全回復退化椎間盤的生物力學功能,因此在未來的研究裡,仍需繼續尋找合適的生物性相容材料,以應用於退化性椎間盤疾病的治療策略上。; The major biomechanical functions of intervertebral disc are to resist the external load and to dissipate the shock energy imposed on the human body during daily activities. The disc functions depend on the internal fluid dynamics and compositional integrity of disc matrix. The clarifications of effect of loading conditions and degenerations on the biomechanical functions of disc will improve the treatment efficacy for the degenerated disc diseases. Two biomechanical models, i.e., rheological model and the dynamic model, have been used to simulate the disc medium term and short term behaviors. The rheological model is traditionally described by a linear biphasic theory model, and the dynamic model is described by a 1-D spring and dashpot model. The rheological and dynamic properties derived from the both models can indicate the disc stiffness and internal fluid flow capability of disc matrix and different time scale.
The first part of this dissertation studied the effect of loading history, loading magnitude, fatigue loading and degeneration on the disc rheology. The results showed that the disc stiffness increased and the fluid flow capability decreased during the creep loading, the disc stiffness decreased and the fluid flow capability unchanged during the relaxation loading. The disc stiffness increased and the fluid flow capability decreased with the loading magnitude. After the fatigue loading time, the disc stiffness unchanged but the fluid flow capability decreased. The degeneration increased the disc stiffness but decreased the fluid flow capability. The results suggested that the disc stiffness was negatively related to the disc hydration level, and the fluid flow capability was positively related to the matrix pore size.
In the second part of this study, the recovery efficiency of viscosupplement (i.e., hyaluronic acid) and crosslinker (i.e., genipin) on the degenerated disc was evaluated by changes of the water content and viscoelasticity of nucleus pulposus, structural integrity of the anular fibrosus, and dynamic properties of the whole disc responding to the fatigue loading. The results revealed that the intervention of hyaluronic acid and genipin could recover the water content of the degenerated nucleus pulposus after rest, but block the water outflow during the fatigue loading. The genipin could slightly increase the elasticity of the degenerated nucleus pulposus and condense the delaminated anular fibrosus. Neither the intervention of hyaluronic acid nor the genipin recovered the viscoelasticity of the degenerated nucleus pulposus. The intervention of hyaluronic acid and genipin could recover the disc stiffness and energy dissipation capability of the degenerated disc. However, these effects are degraded by the fatigue loading. The results indicated that the crosslinker could better recover the structural and mechanical properties of the degenerated disc than the viscosupplement does. However, the susceptibility to the fatigue loading casts the doubt on the feasibility of crosslinker to treat the degenerated disc diseases.
In conclusion, this dissertation proves that the disc mechanical functions are affected by the loading conditions and degeneration due to the change of disc hydration level and matrix pore size. The disc stiffness increased and the internal fluid flow capability decreased with degeneration. Neither the viscosupplement nor the crosslinker is feasible to reverse the full aspects of disc degenerative changes. New materials for treating degenerated disc should be studied in the future.2010-01-01T00:00:00Z