REGENERATION OF SALIVARY GLAND USING TISSUE ENGINEERING AND GROWTH FACTORS

唾液腺萎缩是老年人和接受放射治疗的患者面临的严重临床问题。然而，唾液腺的再生是不可行的。在这个项目中，我们研究了与唾液腺再生潜在相关的基因。此外，还探讨了细胞治疗萎缩性唾液腺的可能性。雄性Wistar大鼠(9周龄)麻醉，结扎一侧颌下腺导管。导管结扎后，唾液腺立即萎缩。切除结扎物后，腺体会再生。1周后摘除结扎，分别于摘除后12小时、36小时和6天取腺。DNA芯片分析结果显示，解除结扎后，Clusterin的表达立即增加，6天后几乎恢复到基础水平。逆转录-聚合酶链式反应和免疫组织化学分别检测到聚集素基因和蛋白的表达。在正常腺体中，簇状蛋白均匀分布，但仅局限于大部分实质的胞浆中。导管结扎摘除后，Clusterin立即在管腔内堆积，并沿萎缩腺泡的管腔表面持续强表达至3d，6d后表达减弱，分布恢复至正常腺体。这项研究的结果证实了该蛋白在大鼠下颌下腺再生过程中的动态变化，尽管其可能的作用尚不清楚。接下来，我们建立了一个小鼠的辐射唾液功能减退模型。通过离心法从骨髓中获取单核细胞，移植到萎缩的颌下腺。本研究对影响移植细胞贴壁和存活的各种因素进行了研究。此外，还评估了萎缩唾液腺的功能恢复情况。

Salivary gland atrophy is a serious clinical problem for elderly and patients who undergo irradiation. However, regeneration of salivary gland is not feasible. In this project, we have investigated the genes potentially relating to the regeneration of salivary gland. Furthermore, a possibility of cell therapy on atrophic salivary gland was investigated. Male Wistar rats (nine weeks old) were anesthetized and the unilateral duct of submandibular gland was ligated. After ductal ligation, salivary gland immediately becomes atrophic. The gland regenerates after the ligation removal. The ligation was removed after one week and the gland was harvested at 12 hours, 36 hours, and 6 days after removal of ligation. The results from DNA microarray analyses showed immediate increase of clusterin expression after removal of ligation, which returned to almost basal level after 6 days. The gene and protein expression of clusterin was confirmed by RT-PCR and immunohistochemistry, respectively. In the normal gland, clusterin was equally distributed but limited in the cytosol of most of the parenchyma. After ductal ligation and removal, clusterin immediately accumulated intra-luminal space and strong expression was continuously observed along with the luminal surface of the atrophic acini until day 3. The expression was decreased after day 6 and the distribution returned to that of the normal gland. The results from this study demonstrated the dynamic change of this protein during regeneration process of rat submandibular gland, though the possible roles yet to be known. Next, we have generated a mouse model of salivary hypofunction by irradiation. Monocytes from bone marrow were harvested by centrifugation and transplanted to the atrophic submanidbular gland. In this study, various factors which affect the attachment and survival of transplanted cells were investigated. Furthermore, functional recovery of the atrophic salivary gland was also evaluated.