有關豬皮膠原蛋白膜之組織整合性及細胞性免疫的研究

A study of tissue integration and cellular immunity associated with PDCM

李長祐(Lee Chang-Yu)

指導教授 ： 呂炫堃 ； 吳銘芳

臺北醫學大學/口腔學院/牙醫學系碩博士班/碩士(1998年)

摘要

本研究的目的是要來探討將豬皮膠原蛋白(PDCM)於非活體或植入活體之模式中，是否 會與結締組織產生黏連的作用，以評估其是否具有組織整合性；並觀察3%GA-PDCM於 老鼠牙齦下所引起之細胞性免疫反應表現，以進一步瞭解膜片於組織中被吸收之機轉。 本實驗分成兩個階段，a)首先利用不同濃度戊二醛(glutaraldehyde; GA)處理過之PDCM與 手術取下之人體顎側牙齦一起於非活體狀況下培養，培養1,3,5天，共30個標本。冷凍包 埋後以免疫染色 ABC 法（Avidin-Biotin Peroxidase Complex Method；ABC Method）染色， 觀察牙齦與膜片介面之CD44，ICAM，integrinα2，integrinα3，integrinα5，E-cadherin， actin 等分子的變化。b)第二階段實驗直接採用3%GA-PDCM植入老鼠牙齦下，以每組3隻 老鼠分10組，經由1,2,3,5,7,10,14,21,28,42天，犧牲老鼠，共30組。取出標本之後，五分之 一的標本用福馬林固定，石蠟包埋；其餘部分直接急速冷凍包埋。再利用H&E stain、 Alcian blue-PAS stain，觀察形態及組織中基質的變化及利用免疫染色 ABC 法染色觀察 fibronectin、integrinα2、α3、α6β1 、CD4、CD8、CD11b的分佈，另外針對CD4、CD8 、CD11b利用grid screen加以計數，觀察其消長情形。在非活體實驗中發現，牙齦結締組 織與膜片接觸良好，具有組織整合的現象。在活體實驗中發現PDCM於牙齦下，於第一 到三天時多為neutrophils的浸潤，實驗期間fibronectin、integrinα2、α3於組織中呈現陽性 反應，促使細胞、血管往膜片移動，使得膜片漸漸被纖維囊包圍。於第十四天時integrin α6β1有明顯出現於組織中表示血管伴隨組織侵入膜片，且根據Alcian blue-PAS stain 發 現組織中不僅細胞外基質多另外還有新生成之膠原蛋白堆積，CD4、CD8、CD11b+細胞 之染色發現CD4、CD8+細胞於第七、十天細胞計數增多而且呈現約為2：1的比例。 CD11b+細胞隨著CD4+細胞之增加而增加，表示PDCM所引起之免疫反應機轉為APCs吞 噬膜片後，將具有抗原性之部份與ClassⅡMHC分子結合後，呈現給CD4+細胞中之Th1 cells，Th1 cells經活化後再分泌cytokines刺激活化macrophages，由於PDCM結構太大， macrophages便融合成giant cells，利用macrophages及 giant cells一起分解吞噬PDCM。於第 十四天時CD4、CD8、CD11b+細胞開始減少，經過第四十二天，仍然有CD11b+細胞存 在，但是周圍多被新生成的纖維組織取代。由本實驗的結果可以發現於第十四天時， CD4、CD8、CD11b+細胞開始減少，表示炎症反應下降，同時組織中有新生成之膠原蛋 白堆積，血管開始明顯增加，表示3%GA-PDCM與結締組織於此時開始形成組織整合性 ；於細胞性免疫方面，3%GA-PDCM引起之免疫反應推論是經由ClassⅡMHC分子之路徑 活化macrophages，導致PDCM被分解吸收。雖然3%GA-PDCM於六週後未被完全分解仍 存在於組織中刺激延長慢性炎症反應，但是其引發之慢性炎症反應輕微，且3%GA- PDCM未被分解存在於組織中可繼續保持細胞分隔之作用，假使應用於GTR術式中，將 可避免外圍組織過早侵入骨缺損。綜合以上結論，3%GA-PDCM確實符合引導組織再生 膜於臨床應用的要求。

關鍵字

豬皮膠原蛋白膜 ； 黏連 ； 組織整合性

並列摘要

The purposes of this study were to study the cellular immunity and tissue integration associated with porcine dermal collagen membrane (PDCM) in vitro and in vivo. The study contains two parts. In the first part, surgical explant of gingiva was harvested from patients during their distal wedge operation. Four different GA-conditioned PDCMs were co-cultured with the explants for 1, 3, and 5 days. The interfaces between PDCM and the explant was investigated by using Avidin-Biotin Peroxidase Complex Method (ABC method) in order to dictate the existence of specific adhesion molecules (CD44，ICAM，integrinα2，integrinα3，integrinα5，E-cadherin，actin). In the second part of the study, 3% GA-PDCM was implanted in the upper jaw of Wistar rats. The specimens were harvested 1, 2, 3, 5, 7, 10, 14, 21, 28,and 42 days after the surgery. One-fifth of the specimens were processed for H&E stain、 Alcian blue-PAS stain, and the rest of the specimens were frozen immediately and processed for immunohistaochemical stain (ABC method) to localize the distribution of integrinα2、integrinα3、integrinα6β1、fibronectin and CD4、CD8、CD11b positive cells. In order to understand the cellular immunity induced by 3%GA-PDCM, the number of CD4、CD8、 CD11b positive cells were counted by using grid screen. The results of in vitro study shows that the cultured ginigiva connective tissue presents good contact with the PDCM and prevented the epithelial cell to move downward. There were positive reaction of integrinα2、integrinα3 and fibronectin in the specimens during the period of in vivo study. There were more CD4+ cells than CD8+ cells in the local tissue and the ratio between these two cells was 2:1. The counting of CD11b+ cells increases following the increasing counting of CD4+ cells. On fourteenth days, there were obvious positive reaction of integrinα6β1 and PAS stain, however, the intensity of and the numbers of CD4+、CD8+ 、CD11b+ cells decreased. It indicated that 3%GA-PDCM starts to achieve good tissue integration in connective tissue on the day of fourteenth. There were no significant pathologic reaction and evidence of tissue damage. In conclusion, these results indicated that PDCM possesses a good quality of tissue in tegration with adjacent connective tissue. The CD4/CD8/CD11b counting also shows that the immunological response induced by PDCM is a chronic inflammation through the pathway of ClassⅡ MHC restricted cascade.

並列關鍵字

porcine dermal collagen membrane (PDCM) ； adhesion ； tissue integration