共聚焦激光显微技术结合分子信标精准检测慢性胰腺炎早期纤维化效能评价

Chronic pancreatitis is a disease characterized by pancreatic inflammation and damages to the pancreatic duct and pancreatic parenchyma. Early diagnosis is particularly important for improving patient prognosis. Needle-based confocal laser endomicroscopy (nCLE) combines the advantages of endoscopic ultrasonography in the diagnosis of small lesions and the direct observation of cell morphology by confocal imaging, thereby improving the sensitivity of early diagnosis of chronic pancreatitis. But its sensitivity is still not satisfactory. If there was a molecular beacon which could identify the early markers of chronic pancreatitis and enhance the fluorescence effect, it will greatly improve the sensitivity of early diagnosis. Preliminary in vitro experiments verified the binding capacity of molecular beacons in normal pancreatic cell lines (hped6-c7) and PSC cell lines, confirming the applicability of this molecular beacon. Current studies have shown that one of the main features of pancreatic fibrosis is the large amounts of fibroblast proliferation and extracellular matrix deposition. Nanoparticles were used as a carrier to prepare fluorescent probes based on this molecular beacon. In combination with confocal laser endomicro technique, the enhancement effect was confirmed at the cell level and animal level, and the toxicity was evaluated by observing the proliferation, migration, apoptosis and invasion of cells in each group, laying a foundation for the early diagnosis of chronic pancreatitis with nCLE binding molecular beacon in the future.

慢性胰腺炎是以胰腺炎症、胰管和胰腺实质破坏为主要特点的疾病，早期诊断对改善患者预后尤为重要。穿刺针引导下的共聚焦激光显微内镜检查（nCLE）综合了超声内镜诊断小病变的优势和共聚焦显微镜直接观察细胞形态的特点，在一定程度上提高了慢性胰腺炎早期诊断的敏感性，但仍不尽如人意。若有一种分子信标能识别慢性胰腺炎早期标志性分子，增强荧光效应，将大大提高其早期诊断的敏感性。前期体外实验验证了分子信标在胰腺正常细胞系（HPED6-C7）、PSC细胞系的结合能力，印证了分子信标应用的可行性。目前研究表明，胰腺纤维化的主要特征之一是大量成纤维细胞增生和细胞外基质沉积,本研究以纳米粒子作为载体应用分子信标制备荧光探针，结合共聚焦激光显微技术于细胞水平、动物水平证实其增强效应，并通过对各组细胞的增殖、迁移、凋亡和侵袭的观察评估其毒性，为下一步运用nCLE结合分子信标进行慢性胰腺炎早期诊断奠定基础。

慢性胰腺炎是一种多病因导致的纤维炎症综合征，胰腺反复炎症发作导致广泛纤维组织替代，使胰腺内外分泌功能受损，影响患者生活质量，缩短预期寿命，严重者会发生癌变。慢性胰腺炎全球年发病率为10/10000，并且呈逐年上升趋势。然而除了全胰腺切除术之外仍然没有有效的治疗方法，因此早发现早诊断对于改善慢性胰腺炎患者的预后至关重要。早期慢性胰腺炎缺乏特异性的临床表现与形态特征，使得现有的诊断手段敏感性不高。穿刺针引导下的共聚焦激光显微内镜检查（nCLE）综合了超声内镜诊断小病变的优势和共聚焦显微镜直接观察细胞形态的特点，在胰腺的亚细胞层面就可以根据特定的显像特征做出“光学活检诊断”，在一定程度上提高了对胰腺病变的诊断效率，但仍存在非特异性的缺点。针对上述问题，我们首先用转化生长因子-β（transforming growth factor-β,TGF-β)造模多能干细胞（PSC）细胞系，模拟慢性胰腺炎病理条件下激活态PSCs，验证基质金属蛋白酶1（matrix metallopeptidase 1，MMP-1）在PSCs激活后的表达升高；用MMP-1荧光抗体与激活前后PSCs结合，共聚焦显微镜观察并量化比较其荧光强度差异；二丁基二氯化物（DBTC）尾静脉注射法造模大鼠慢性胰腺炎模型，解剖后验证胰腺纤维化情况，分离大鼠PSCs与MMP-1荧光抗体结合，验证PSCs激活情况；大鼠胰腺组织与MMP-1荧光抗体结合，共聚焦显微镜观察并量化比较其荧光强度差异；应用 19G 超声内镜穿刺针于猪主胰管内注射 3%浓度海藻酸钠溶液构建大动物慢性胰腺炎模型，解剖后观察胰腺纤维化情况以及炎性假瘤形成情况；猪胰腺标本MMP-1荧光染色，共聚焦显微镜观察并量化比较其荧光强度差异。穿刺针引导下的共聚焦激光显微内镜检查结合分子信标对于慢性胰腺炎早期纤维化的精准检测技术上可行，值得进一步研究及推广。