【作者】 林婷婷；

【导师】 何彦津；

【作者基本信息】 天津医科大学 ， 眼科学， 2010， 博士

【摘要】 目的总结分析目前眼眶泪腺腺样囊性癌的临床治疗方法和预后,评估可能影响预后的因素。利用纳米技术及局部化疗减少全身化疗缺乏选择性、副作用大的问题。制备连接有叶酸(folic acid,FA)的长春新碱(vinscristine, VCR)靶向缓释纳米微球(nanopaticles, NPs),简称为FA-PLGA(VCR)-NPs,观察其理化特征；评估该药物对腺样囊性癌细胞株(ACC-2)及裸鼠眼眶移植瘤的抑制作用。进一步寻找眼眶腺样囊性癌治疗的新靶标,即肿瘤干细胞。观察肿瘤干细胞相关标志蛋白CD44、CD133和ABCG2在泪腺腺样囊性癌中的表达,研究它们与病理分型、预后的关系,为进一步开展针对肿瘤干细胞的靶向治疗奠定基础。方法1.采用回顾性系列病例研究,分析75例眼眶腺样囊性癌患者相关临床资料,包括患者的手术记录、病理分型及随访记录。2.采用改良的复乳法制备FA-PLGA(VCR)-NPs;MTT比色法观察空白微球PLGA-NPs的毒性,比较VCR原药、载药微球PLGA(VCR)-NP和FA-PLGA (VCR)-NPs在不同时间和不同浓度条件下对ACC-2细胞的影响；FITC标记微球,荧光显微镜观察PLGA-NPs与FA-PLGA-NPs对肿瘤细胞的作用；瘤细胞悬液接种法建立裸鼠眼眶腺样囊性癌移植瘤,分别予以瘤内注射生理盐水、VCR、PLGA-NPs、PLGA(VCR)-NPs和FA-PLGA(VCR)-NPs,局部给药一次,隔天测量肿瘤体积变化,计算体积抑制率,高效液相色谱法测定给药后不同时间肿瘤组织内残余VCR浓度,透射电镜及HE染色观察肿瘤组织标本。3.采用二步法免疫组织化学检测人眼眶腺样囊性癌肿瘤组织标本33例,复发切除标本5例及6例荷瘤裸鼠移植瘤标本的CD44、CD133和ABCG2的表达情况,分析其与病理分型及预后的关系。结果1.眼眶实体型腺样囊性癌2年复发率为85%(17／20)、5年复发率为100%(19／19),而腺样-管状型分别为23.53%(8／34)和64.52%(20／31),差异有统计学意义(2年,p=0.000；5年,p=0.003)。前者发生局部蔓延和远处转移例数亦多于后者。肿瘤切除术后联合放射治疗的5年复发率为70%(14／20),低于单纯手术切除的复发率92.86%(13／14)(p=0.198)。首次手术行眶内容物剜除术的5年复发率为25%(1／4),低于复发后再行眶内容物剜除术的病例的75%(6／8)(p=0.222),γ刀、粒子刀、化疗及生物治疗的效果不能确定。局部蔓延主要是至颅内、副鼻窦和颞窝,远处转移可到达肺、骨、肝、耳前淋巴结。5年远处转移率为25.71%(9／35),肺转移和骨转移各占33.33%(3／9)。5年生存率74.29%(26／35),死亡率25.71%(9／35),无瘤生存率37.14%(13／35),10年无瘤生存率17.14%(6／35)。最常见的死亡原因是颅内蔓延。肿瘤切除联合放射治疗可以使5年生存率提高到80%(16／20)。2.FA-PLGA(VCR)-NPs呈规则球形,平均粒径249.2nm,载药率4.53%,体外释放时间达14天；PLGA-NPs与ACC-2细胞共培养5天,细胞存活率达80%以上；给药后第4、5天FA-PLGA(VCR)-NPs对ACC-2细胞的抑制率显著高于VCR,呈时间和浓度依赖性；微球附着于肿瘤细胞表面,这种识别可以被FA竞争性抑制；FA-PLGA(VCR)-NPs组及PLGA(VCR)-NPs组对裸鼠肿瘤的体积抑制率显著高于VCR组(前者p=0.016,后者p=0.029),FA-PLGA(VCR)-NPs组高于PLGA(VCR)-NPs组,但无统计学差异(p=0.376)；给药后第1、7、14天肿瘤组织中残留药物浓度存在差异(p=0.000)；透射电镜观察14天肿瘤细胞内仍有高电子密度的纳米颗粒聚集,肿瘤细胞坏死明显,注射周围组织结构形态正常。3.CD44阳性表达率为54.5%(18／33)。实体型阳性率76.9%(10／13),多呈片状散在分布于肿瘤边缘浸润灶,而在肿瘤细胞密集处常常没有着染；筛网型阳性率为40%(8／20),大多分布在腺管样结构的外层细胞,即肌上皮细胞,二者差异不具有统计学意义(p=0.072)；在非炎症及恶性肿瘤性疾病切除的正常泪腺组织中CD44亦有阳性表达。CD133阳性表达率为57.6%(19／33),在实体型和筛网型中分别为76.92%(10／13)、45%(9／20),差异不具有统计学意义(p=0.087)；表达产物定位于细胞膜和细胞浆,呈淡黄色至棕褐色,部分病例同时表达于胞浆和胞核,其中实体型标本有46.15%(6／13),均属于发生转移或死亡的病例,筛网型有40%(8／20),其中4例发生复发或转移,4例四年内无复发；在非恶性肿瘤性疾病切除的正常泪腺组织中均无表达。ABCG2阳性表达率为21.2%(7／33),在实体型和筛网型中分别为30.77%(4／13)、15%(3／20),差异不具有统计学意义(p=0.393)；ABCG2阳性表达细胞具有沿血管分布的倾向。CD44、CD133及ABCG2在预后较好组(4年无复发)的阳性表达率均低于预后差组,无复发组低于发生转移或死亡组,但差异均不具有统计学意义(p<0.05)。Spearman相关分析提示CD44阳性表达和CD133的阳性表达之间存在正相关关系,(rs=0.416,p=0.016)。在6例荷瘤裸鼠肿瘤组织标本中,CD44阳性1例,CD133表达阳性1例,ABCG2表达阳性4例。结论1.腺样囊性癌是高度恶性的眼眶肿瘤,复发率和死亡率均较高,病理分型、治疗方法均影响预后。采取综合治疗方法,可以减少复发,提高生存率。2.叶酸靶向长春新碱纳米缓释微球具有稳定的载药率和体外释放行为,具有良好的靶向识别力,体外细胞学实验及荷瘤裸鼠体内实验均证实具有优于原药的抗肿瘤能力。3.在眼眶腺样囊性癌肿瘤组织中存在CD44、CD133及ABCG2阳性细胞,分别表达于腺样囊性癌肿瘤组织的不同部位,其表达随着病程进展而变化,可能会影响预后,但并不能作为评估预后的因素。更多还原

【Abstract】 Objective1.To summarize the treatment outcomes of orbital adenoid cystic carcinoma and to evaluate prognostic factors.2.To prepare and observe the character of Folate Receptor-mediated VCR-loaded Nanoparticles, which is abbreviated to FA-PLGA (VCR)-NPs. Study the inhibitive effect on ACC-2 cells in vitro and the xenograft tumor in BALB/c-nu mice.3.To observe the expression and distribution of CD44, CD133,and ABCG2 in orbital ACC and investigate their correlations with pathological type and prognosis.Method1.A retrospective case series study was performed on 75 patients with orbital adenoid cystic carcinoma.2.The modified W/O/W extraction-evaporation technique was chosen to prepare FA-PLGA (VCR)-NPs. These particles were characterized for size, modality, encapsulation efficiency, and release in vitro.The cytotoxicity of high concentration blank particles PLGA-NPs towards tumor cells was evaluated by MTT colorimetry. The tumor cells were divided into three groups:VCR、PLGA(VCR)-NPs and FA-PLGA(VCR)-NPs.Seven doses of VCR were tested: 0.05μg/ml,0.25μg/ml,0.5μg/ml,1μg/ml,5μg/ml, 10μg/ml,30μg/ml.After 1day, 2day,3day,4day,5day, the cells growth inhibition ratio were evaluated also by MTT colorimetry. PLGA-NPs and FA-PLGA-NPs were labeled by FITC. Conditions of those particles uptaked by ACC-2 cells with or without free FA were observed with fluorescence microscope. ACC xenograft tumor of nude mice orbit was built by injecting ACC cell suspension and divided into four groups: VCR, PLGA(VCR)-NPs, FA-PLGA(VCR)-NPs, and control group.The inhibition ratio of gross tumor volume was observed every two days. Residual concentrations of VCR in tumors were evaluated by HPLC.The feature of histopathology was observed by electron microscope and HE stain.3.Two stages method of immunohistochemical staining was employed in 33 cases paraffin embedded surgical specimens of human orbital ACC,5 cases recurrence samples,3 cases excisional lachrymal gland caused by neither inflammation nor tumor diseases, and 6 cases xenografts tumor in nude mice.Results1.The 2- and 5- year local recurrence rate of solid type orbital adenoid cystic carcinoma was significantly higher than that of the adeno-tubiform type. [2-year, 85%(17/20) vs 23.53%(8/34),χ2=19.14, p=0.000; 5-year,100%(19/19) vs 64.52% (20/31),Fisher’s exact test, p=0.003] The regional extension and distant metastasis of solid type were more than those of adeno-tubiform type. The 5-year local recurrence rate treated by postoperative radiation was lower than that treated only by surgical excision [70%(14/20) vs 92.86%(13/14);Fisher’s exact test, p=0.198].The 5-year local recurrence rate in patients initially treated by orbital evisceration during the first time was lower than that of cases which evisceration procedure was used after the recurrence. [25% (1/4)) vs 75% (6/8), Fisher’s exact test, p=0.222].Tumors always extend into intracalvarium, nasal cavity and temporal fossa. They may spread to the lung, bone, liver and lymph node. The 5-year metastasis rate was 25.71% (9/35).Both of the lung and bone metastasis rates were 33.33% (3/9).The overall 5-year accumulative survival was 74.29% (26/35), mortality was 25.71% (9/35), and rate of survival without tumor recurrence was 37.14%(13/35).The 10-year disease free survival rate was 17.14% (6/35).Patients were most likely to die with intracranial extension. Surgical excision with postoperative radiation improved the 5-year survival rate to 80%(16/20).2.FA-PLGA (VCR)-NPs were smooth and spherical with a mean particle size 249.2nm. The drug loading rate was 4.53%.The release of VCR from PLGA nanoparticles persists for 14d. After blank particles PLGA-NPs and ACC-2 cells co-cultured for 5 days, cell viability had remained at more than 80 percent. The inhibitive effect of FA-PLGA (VCR)-NPs was more effective than VCR at 4th and 5th day. They were both in dose-dependent and time-dependent manner. Targeting particles could attach to tumor surface, via folate receptor. FA was competitive inhibitor of this recognization. The volume inhibition ratios of FA-PLGA(VCR)-NPs and PLGA(VCR)-NPs were significant higher than VCR. (p=0.016,p=0.029) The inhibition ratio of FA-PLGA(VCR)-NPs was higher than PLGA(VCR)-NPs. (p=0.376) There was signigicant different between residual concentrations of VCR at the 1st day,7th day and 14th day. (p=0.000) TEM pictures showed lots of electron-dense microspheres in tumor cells at the 14th day. Tumor necrosis was obviously, while surrounding tissues were normal.3.The positive rate of CD44 was 54.5% (18/33),with 76.9%(10/13)in solid type and 40% (8/20) in adeno-tubiform type. There wasn’t statistically different between them (p=0.072).In solid type the positive expression part often located at the marginal part of the cancer nest. While in the adeno-tubiform type positive cells often located at the outer layer of the tubiform structure, which were the myoepithelial cells. It was also expressed in some normal tissues.The positive rate of CD133 was 57.6%(19/33),with 76.9%(10/13)in solid type and 45% (9/20) in adeno-tubiform type. There wasn’t statistically different between them (p=0.087).Parts of the expression of CD133 antigen were both on the cytoplasm and the nucleus. The positive rate of ABCG2 was 21.2%(7/33),with 30.77% (4/13) in solid type and 15%(3/20) in adeno-tubiform type. There wasn’t statistically different between them (p=0.393). Lots of positive cells surrounded vessels in tumor tissues.There were no significant difference between different prognosis groups of there surface phenotypes. The correlative analysis results of three surface phenotypes showed that CD44+ cells have positive correlation with CD133+ cells (Spearman, r,=0.416,p=0.016). Besides 1 case CD44+,1 case CD133+ and 4 cases ABCG2+ were detected in transplanted tumor of nude mice.Conclusions1.Orbital adenoid cystic carcinoma is one of the most malignant tumors in the orbit. They have a high local recurrence rate and low survival rate. Tumor histological types and treatment procedure can influence the prognosis. Combined therapy may decrease the recurrence and increase the survival rate.2.FA-PLGA(VCR)-NPs are stable and have high drug entrapment efficiency and high effect of growth inhibition in vitro and vivo.It is targeted and can be proposed as a potential controlled and targeted delivery system for the treatment of ACC.3.The expression of CD44, CD133 and ABCG2 in ACC should have some clinical significance in the progress of ACC.But it couldn’t be regarded as one of the factors to evaluate the prognosis.更多还原