以VIP之免疫組織化學染色定量評估自主神經病變之汗腺神經支配

自主神經系統對於身體內不同器官以及組織正常功能的維持，例如心跳、血壓、體溫等，扮演著重要之調節角色，當發生自主神經病變 (Autonomic neuropathy, AN) 時，會使得心血管系統、排汗功能、消化道系統、泌尿系統、性功能等正常生理功能受到影響，而排汗功能的評估可直接自皮膚表皮觀察，較不具侵入性且方便施行，但目前臨床上檢測方法皆以生理功能之測試為主，對於造成排汗功能降低的可能原因，如 : 汗腺細胞萎縮、支配汗腺的自主神經纖維退化等，無法單純的就功能性測試結果得知，所以本研究利用皮膚切片進行免疫組織化學染色，從形態觀察汗腺神經支配情形，並且加以定量。吾人選擇三種分子的免疫化學染色以評估汗腺之神經支配，Na+ K+ Cl- cotransporter isoform1 (NKCC1) 為分布在汗腺分泌細胞側邊及基底膜上，與汗液製造相關的輸送蛋白 (transporter)；vasoactive intestinal peptide (VIP) 會表現於汗腺周圍的自主神經纖維上，對於汗腺而言，VIP 為一種神經調節物(neuromodulator)，雖不直接引發汗腺發生排汗反應，但有促進排汗反應的效果。Protein gene product 9.5 (PGP 9.5) 為一種神經細胞內普遍存在的碳氧基水解?，本研究主要包含三種定量系統，分別為 VIP / NKCC1 雙抗體螢光標定、PGP 9.5 / NKCC1 雙抗體螢光標定、PGP 9.5 單一抗體標定定量系統。研究結果發現，無論在何種定量系統中，女性的汗腺神經支配指數皆比男性來的高，其中在 VIP / NKCC1 及 PGP 9.5 / NKCC1 這兩種雙抗體螢光標定定量系統中都具有顯著性差異；在年齡大小方面，我們發現無論在何種定量系統中，汗腺神經支配指數並不會受到年紀的不同而有差異；將三種不同定量系統之汗腺神經支配指數做相關性比較發現，彼此間皆具有顯著的關連性。比較自主神經病變患者及正常人的汗腺神經支配指數，研究發現無論在何種染色方式中，纏繞在汗腺周圍的神經纖維皆會減少許多甚至消失，進一步定量發現，在所有定量系統中，自主神經病變患者的汗腺神經支配指數皆較一般正常人的汗腺神經支配指數低，且在 PGP 9.5 單一抗體標定定量系統及 VIP / NKCC1 雙抗體螢光標訂定量系統中具有顯著性差異。為了評估本研究所建立的汗腺神經支配指數定量方法，能否當作臨床上判定自主神經病變的依據，所以我們分別計算出三種定量系統的敏感性，VIP / NKCC1 雙抗體螢光標定定量系統的敏感性最高為 79.3 %，在 PGP 9.5 單一抗體染色定量系統中為 25.0%，在 PGP 9.5 / NKCC1 定量系統中則為 20.7%。由於 VIP / NKCC1 雙抗體螢光標定定量系統其敏感性最高，且在自主神經病變患者及正常人的汗腺神經支配指數比較中，差異性最大，因此我們利用此定量系統，探討糖尿病患者對汗腺神經支配指數所造成的影響，研究發現糖尿病患者且無自主神經病變症狀者與正常人的汗腺神經支配指數並無顯著性差異，而 VIP / NKCC1 雙抗體螢光標定定量系統的專一性為 63.2%；在糖尿病患者且有自主神經病變症狀者與正常人的汗腺神經支配指數間，則是具有顯著性差異。根據以上的結果，我們認為利用 VIP 與 NKCC1 這兩種與排汗功能較有直接關係的蛋白進行免疫組織螢光染色，更能有效的反應出汗腺神經支配是否發生異常；並且以其高敏感性及專一性，希望能作為臨床上判定為汗腺神經支配異常造成的自主神經病變之依據。

關鍵字

自主神經病變；汗腺神經支配指數；腸血管活性多胜；鈉鉀氯離子運輸蛋白；糖尿病病變

並列摘要

In the present study, we explored the possibility of designing quantitative systems of sweat gland innervation based on skin biopsies to assess autonomic neuropathy. Most tests of autonomic neuropathy are based on functional studies and there are only limited on the pathology. There are three quantitative approaches in the current study: (1) vasoactive intestinal peptide (VIP) / Na+ K+ Cl- cotransporter isoform1 (NKCC1) double labeling with fluorescence, (2) protein gene product 9.5 (PGP 9.5) / NKCC1 double labeling with fluorescence, and (3) PGP 9.5 single labeling. NKCC1 located on the basolateral plasma membranes of secretory cells of sweat glands is related to sudomotor function. For sweat glands, VIP, a neuromodulator, is expressed in sympathetic nerve fibers that innervate sweat glands. It is considered that VIP modulates the sudomotor function. PGP 9.5, an ubiquitin hydrolase, is a common neuronal marker. Sweat gland innervation index of females was higher than that of males in all three quantified approaches. In all three quantified approaches, sweat gland innervation index was not affected by ages. There were significant correlations of sweat gland innervation indexes among different approaches of quantification. VIP (+) and PGP 9.5 (+) nerve fibers innervated sweat glands were reduced in autonomic neuropathy. In all three quantified approaches, sweat gland innervation indexes of autonomic neuropathy were lower than those of control. The sensitivity of VIP / NKCC1 was highest (79.3%), compared to PGP 9.5 (25.0%) and PGP 9.5 / NKCC1 (20.7%). Because the index of VIP / NKCC1 showed the highest sensitivity and the most significant difference between autonomic neuropathy and control, we applied this approach to evaluate the sweat gland innervation of diabetic mellitus. Diabetic patients with autonomic neuropathy showed significant reduction of sweat gland innervation index compared to those patients without autonomic neuropathy. In conclusion, the sweat gland innervation index of VIP / NKCC1 provides a new pathological approach to diagnose autonomic neuropathy.

並列關鍵字

autonomic neuropathy ； vasoactive intestinal peptide ； protein gene product 9.5 ； Na+ K+ Cl- cotransporter isoform1 ； diabetic mellitus ； sweat gland innervation index

參考文獻

Abdel-Rahman, T. A., K. J. Collins, et al. (1992). "Immunohistochemical, morphological and functional changes in the peripheral sudomotor neuro-effector system in elderly people." J Auton Nerv Syst 37(3): 187-97.

Boulant, J. A. (1981). "Hypothalamic mechanisms in thermoregulation." Fed Proc 40(14): 2843-50.

Chien, H. F., T. J. Tseng, et al. (2001). "Quantitative pathology of cutaneous nerve terminal degeneration in the human skin." Acta Neuropathol (Berl) 102(5): 455-61.

Chowdhury, D. and N. Patel (2006). "Approach to a case of autonomic peripheral neuropathy." J Assoc Physicians India 54: 727-32.

Eedy, D. J., C. Shaw, et al. (1994). "The regional distribution of neuropeptides in human skin as assessed by radioimmunoassay and high-performance liquid chromatography." Clin Exp Dermatol 19(6): 463-72.

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以VIP之免疫組織化學染色定量評估自主神經病變之汗腺神經支配

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