REGENERATION OF SALIVARY GLAND USING TISSUE ENGINEERING AND GROWTH FACTORS

唾液腺萎缩是老年人和放射治疗患者的严重临床问题。然而，唾液腺的再生是不可行的。在本项目中，我们研究了可能与唾液腺再生有关的基因。此外，细胞治疗萎缩的涎腺的可能性进行了调查。将雄性Wistar大鼠（9周龄）麻醉并结扎单侧下颌下腺导管。导管结扎后，唾液腺立即萎缩。腺体在结扎移除后再生。一周后去除结扎，并在去除结扎后12小时、36小时和6天收获腺体。DNA微阵列分析结果显示，clusterin表达在去除连接后立即增加，6天后几乎恢复到基础水平。用RT-PCR和免疫组化方法检测丛生素基因和蛋白的表达。在正常腺体，clusterin均匀分布，但局限于大多数的实质细胞质中。在导管结扎和去除后，clusterin立即积聚在管腔内空间，并且沿着萎缩腺泡的管腔表面连续沿着观察到强表达，直到第3天。第6天后表达减少，分布恢复到正常腺体。本研究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，但其可能的作用尚不清楚。接下来，我们通过照射产生了唾液功能减退的小鼠模型。通过离心从骨髓收获单核细胞并移植到萎缩的下颌下腺。在这项研究中，影响移植细胞的附着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。

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.