自体外周血淋巴细胞经泪腺注射与静脉回输两种方式诱导兔自身免疫性干眼模型的对比分析

Comparison of the rabbit autoimmune dry eye model established by lacrimal gland injection and intravenous transfusion of autologous peripheral blood lymphocytes

目的 基于泪液学、眼表组织及Th17/Treg相关指标比较自体外周血淋巴细胞(PBLs)经泪腺注射与静脉回输两种方式诱导兔自身免疫性干眼模型的异同。方法 将雌性新西兰大白兔随机分为正常对照组(NC组)、生理盐水泪腺注射组(NSI组)、自体PBLs泪腺注射组(ALI组)、生理盐水静脉回输组(NST组)、自体PBLs静脉回输组(ALT组),每组8只;自体PBLs通过与泪腺上皮细胞共培养以激活;ALI组予泪腺注射激活的PBLs 200μL,ALT组予耳缘静脉回输同等细胞量的PBLs 1 mL,NSI组予泪腺注射生理盐水200μL,NST组予耳缘静脉回输生理盐水1 mL;造模前与造模后4周检测各组兔泪液分泌量(SIT)、泪膜破裂时间(BUT)、角膜荧光素染色(FL)评分、泪液中白细胞介素17(IL-17)和白细胞介素35(IL-35)浓度,泪腺HE染色观察组织形态学改变,免疫组织化染色检测ROR-γt、Foxp3蛋白表达。结果 造模后4周,ALI组、ALT组兔眼均观察到SIT降低、BUT缩短、FL评分增加、泪液IL-17浓度上升和IL-35浓度下降(均为P<0.05),泪腺组织HE染色见腺体萎缩及淋巴细胞浸润等炎症改变;免疫组织化学染色结果显示,与NC组相比,ALI组、ALT组兔眼泪腺中ROR-γt蛋白表达增加及Foxp3蛋白表达减少(均为P<0.05);ALI组与ALT组兔眼间BUT、FL评分、泪液IL-17浓度、泪腺ROR-γt蛋白表达相比差异均有统计学意义(均为P<0.05)。结论 激活的自体PBLs通过泪腺注射和静脉回输两种方式均可建立兔自身免疫性干眼模型,静脉回输法获得的动物模型更稳定、持久。

Objective To compare the similarities and differences of the rabbit autoimmune dry eye model established by injection of autologous peripheral blood lymphocytes(PBLs) via the lacrimal gland and intravenous transfusion of PBLs based on lachrymology, ocular surface tissue, T helper cell 17(Th17)/regulatory cell(Treg) and other related indicators. Methods Female New Zealand rabbits were randomly divided into the normal control group(Group NC), the group of normal saline injection via the lacrimal gland(Group NSI), the group of autologous PBL injection via the lacrimal gland(Group ALI), the group of normal saline intravenous transfusion(Group NST) and the group of autologous PBLs intravenous transfusion(Group ALT), with 8 rabbits in each group. Autologous PBLs were activated by co-culture with lacrimal epithelial cells. In Group ALI, 200 μL of activated PBLs were injected into the lacrimal gland;in Group ALT, 1 mL of PBLs with the same amount of cells were reinfused through the marginal ear vein;in Group NSI, 200 μL of normal saline was injected into the lacrimal gland;in Group NST, 1 mL of normal saline was reinfused into the marginal ear vein. Before modeling and 4 weeks after modeling, Schirmer I test(SIT), tear break-up time(BUT), fluorescein staining(FL) score, concentrations of interleukin 17(IL-17) and interleukin 35(IL-35) in tears were tested, hematoxylin-eosin(HE) staining of the lacrimal gland was carried out to observe the histomorphological changes, and immunohistochemical staining was used to detect the expressions of retinoic acid-related orphan receptor gamma t(ROR-γt) and forkhead box P3(Foxp3). Results Four weeks after modeling, SIT decrease, BUT shortening, increase in FL score, increase in tear IL-17 concentration and decrease in IL-35 concentration were observed in rabbit eyes in Group ALI and Group ALT(all P<0.05), and HE staining of lacrimal gland tissue showed inflammatory changes such as gland atrophy and lymphocyte infiltration. The results of immunohistochemical staining showed that compared with Group NC, the expression of ROR-γt increased and the expression of Foxp3 decreased in the lacrimal gland of rabbits in Group ALI and Group ALT(all P<0.05). There were statistically significant differences in BUT, FL score, IL-17 concentration in tears and ROR-γ expression in the lacrimal gland between Group ALT and Group ALT(all P<0.05). Conclusion The model of autoimmune dry eye can be established by lacrimal gland injection and intravenous transfusion of activated autologous PBLs. The model established through intravenous transfusion is more stable and durable.