黏附分子在生殖道黏膜免疫淋巴细胞归巢过程中的作用

发布时间：2018-06-03 04:07

本文选题：重组乳酸杆菌 + 阴道黏膜免疫　；参考：《复旦大学》2013年硕士论文

【摘要】：黏膜免疫系统(mucosal immune system, MIS)----指广泛分布于呼吸道、胃肠道、泌尿生殖道黏膜下及一些外分泌腺体处的淋巴组织,在结构和功能上均有别于系统性免疫系统。黏膜免疫系统中的黏膜相关淋巴组织(mucosal associated lymphoid tissues, MALT)包括：肠相关淋巴组织(GALT)、支气管相关淋巴组织(BALT)、鼻相关淋巴组织(NALT)以及泌尿生殖道黏膜相关淋巴组织等。MALT中的淋巴细胞主要是在黏膜淋巴系统内进行再循环,这与其表达的归巢分子有关。MIS按功能不同可分为诱导部位和效应部位。在诱导部位和效应部位之间,主要通过淋巴细胞归巢发生联系。从黏膜诱导部位归巢到效应部位这一功能上相关联的系统统称为共同黏膜免疫系统(common mucosal immune system)=共同黏膜免疫系统在黏膜免疫中有共同的效应,但对不同部位的黏膜,其免疫机制又有各自的特殊性,这与各器官的解剖和功能不同有关。在黏膜免疫机制的研究中,胃肠道黏膜免疫系统研究较深入。女性生殖道是一个特殊的黏膜免疫系统同时又有内分泌功能,所以生殖道黏膜免疫更为复杂。近几年生殖道黏膜免疫系统的作用也慢慢被大家所关注。我们实验室前期成功构建了分泌hCGβ及携分子佐剂C3d3的hCGβ-C3d3融合蛋白重组乳酸杆,并已研究证明,通过黏膜接种BALB/c小鼠,能产生抗hCGP体液免疫应答,但是对于这些参与免疫反应的淋巴细胞是如何归巢及居留在生殖道的研究还很少。因此本实验着重于研究生殖道黏膜免疫后淋巴细胞归巢相关粘附分子的表达及生殖道局部微环境对淋巴细胞归巢的影响,解析生殖道黏膜免疫的部分机制。第一部分阴道黏膜免疫后淋巴细胞归巢相关黏附分子的表达目的探讨经野生型及重组乳酸杆菌疫苗经阴道黏膜接种BALB/c小鼠后淋巴细胞归巢相关黏附分子的表达方法用磷酸盐缓冲液(PBS),野生型乳酸杆菌(Lb.)及重组乳酸杆菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010剂量(50u1)经阴道滴注接种6～8周龄雌性BALB/c小鼠,每组10只。3周后用相同剂量加强免疫一次。加强免疫2周末处死小鼠,取各组子宫及阴道。部分标本放入液氮储存,待组织RNA提取,行实时定量PCR检测黏附分子基因表达水平和组织蛋白提取,行Western Blot实验,进行蛋白定量。部分标本用4%多聚甲醛固定24h,石蜡包埋,切片(厚度5μm),行免疫组织化学染色对黏附分子的表达进行定位。另外应用流式细胞术检测各免疫组生殖道局部淋巴细胞表面黏附分子配体的表达。结果加强免疫后,生殖道黏膜黏附分子的表达发生改变,黏附分子E-selectin, ICAM, VCAM, VAP-1and E-cadherin均可以在基因和蛋白水平检测到表达。并且绝大部分黏附分子在重组乳酸杆菌免疫后的子宫表达量增加。结论黏附分子E-selectin, ICAM, VCAM, VAP-1and E-cadherin参与了生殖道局部淋巴细胞的归巢。第二部分生殖道粘膜免疫中归巢淋巴细胞表面高表达黏附分子相应配体的表达目的探讨经野生型及重组乳酸杆菌疫苗经阴道黏膜接种BALB/c小鼠后淋巴细胞归巢相关黏附分子配体的表达方法用磷酸盐缓冲液(PBS),野生型乳酸杆菌(Lb.)及重组乳酸杆菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010剂量(50ul)经阴道滴注接种6～8周龄雌性BALB/c小鼠,每组10只。3周后用相同剂量加强免疫一次。加强免疫2周末处死小鼠,取各组子宫及阴道。原代分离出脾脏及生殖道局部淋巴细胞应用流式细胞术检测各免疫组生殖道局部淋巴细胞表面黏附分子配体的表达。应用ELISA检测各实验组子宫内膜间质细胞培养上清中TGF-β1量的表达。结果分子佐剂C3d3能够显著的增加生殖道淋巴细胞归巢相关黏附分子配体αEβ7, α4β7, LFA-1, VLA-4, PSGL-1的表达。结论加强免疫之后,分子佐剂C3d3可以增加黏附分子配体αEβ7,α4β7, LFA-1, VLA-4, PSGL-1的表达,促进生殖道局部T,B淋巴细胞的归巢。加强免疫之后,重组乳酸杆菌免疫组可以显著促进子宫内膜间质细胞TGF-β1的表达。第三部分阴道黏膜免疫后局部微环境对淋巴细胞归巢的影响目的探讨各免疫组加强免疫后,生殖道局部微环境对淋巴细胞归巢的影响。方法用磷酸盐缓冲液(PBS),野生型乳酸杆菌(Lb.)及重组乳酸杆菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010剂量(50ul)经阴道滴注接种6～8周龄雌性BALB/c小鼠,每组10只。3周后用相同剂量加强免疫一次。加强免疫2周末处死小鼠,无菌条件下取出各组子宫及阴道。原代分离子宫内膜间质细胞,收集培养上清,将各组培养上清种transwell小室的下室(600ul),上室种各免疫组脾脏淋巴细胞(1×106/100ul),37℃培养4h,收集迁移到下室的淋巴细胞经流式细胞术检测迁移至下室淋巴细胞的数量和淋巴细胞的比例。另外应用流式细胞术检测各免疫组脾脏及生殖道T淋巴细胞表型的改变。结果免疫之后,各实验组培养上清较对照组相比可以显著的增加淋巴细胞的迁移,尤其是重组乳酸杆菌免疫组,并且生殖道的微环境更倾向于增加T细胞的比例,并促进Tcell尤其是γδT细胞向下室的迁移。结论经阴道黏膜加强免疫后,局部的微环境能够显著的增加T淋巴细胞的比例,尤其是γδT细胞。
[Abstract]:The mucosal immune system ( MALT ) is a special mucosal immune system .

The expression of intercellular adhesion molecule in lymphocytes of the first part of vaginal mucosa after immunization

Objective To investigate the expression of lymphocyte homing - related adhesion molecules in BALB / c mice immunized with wild - type and recombinant Lactobacillus vaccine via vaginal mucosa .

Methods BALB / c mice were inoculated with phosphate buffer ( PBS ) , wild - type lactobacillus ( Lb . ) and recombinant lactobacillus ( Lb . hCG尾 and Lb . hCG尾 - C3d3 ) at 1010 doses ( 50u1 ) for 6 - 8 week old female BALB / c mice .

Results After strengthening immunity , the expression of adhesion molecules in the genital tract was changed , and the expression of E - selectin , ICAM , VCAM , vap - E - cadherin could be detected at the level of gene and protein . Most of the adhesion molecules increased after immunization with recombinant lactobacillus .

Conclusion E - selectin , ICAM - 1 , VCAM - 1 and E - cadherin are involved in the homing of local lymphocytes in the reproductive tract .

The expression of adhesion molecule - related ligands on the surface of homing lymphocytes in the second part of reproductive tract mucosal immunity

Objective To investigate the expression of lymphocyte homing related adhesion molecule in BALB / c mice immunized with wild type and recombinant Lactobacillus vaccine via vaginal mucosa .

Methods BALB / c mice were inoculated with phosphate buffer ( PBS ) , wild - type lactobacillus ( Lb . ) and recombinant lactobacillus ( Lb . hCG尾 and Lb . hCG尾 - C3d3 ) at 1010 doses ( 50ul ) for 6 - 8 week old female BALB / c mice .

Results The molecular adjuvant C3d3 was able to significantly increase the expression of 伪E尾7 , 伪4尾7 , lfa - 1 , leukocyte - 4 , PSGL - 1 in reproductive tract lymphocytes .

Conclusion After strengthening immunity , the molecule adjuvant C3d3 can increase the expression of the adhesion molecule ligand 伪E尾7 , 伪4尾7 , lfa - 1 , leukocyte - 4 , PSGL - 1 , promote the homing of local T and B lymphocytes in the reproductive tract . After strengthening immunity , the recombinant Lactobacillus immunization group can significantly promote the expression of TGF - 尾1 in the endometrial stromal cells .

Effect of local microenvironment on lymphocyte homing in the third part of vaginal mucosa

Objective To investigate the effect of local microenvironment on lymphocyte homing in different immune groups .

Methods BALB / c mice were inoculated with phosphate buffer ( PBS ) , wild - type lactobacillus ( Lb . ) and recombinant lactobacillus ( Lb . hCG尾 and Lb . hCG尾 - C3d3 ) at 1010 doses ( 50ul ) for 6 - 8 week old female BALB / c mice .

Results After immunization , the culture supernatant of each experimental group can significantly increase the migration of lymphocytes , especially the recombinant Lactobacillus immune group , and the microenvironment of the reproductive tract tends to increase the proportion of T cells and promote the migration of Tcell , especially 纬未T cells .

Conclusion The local microenvironment can significantly increase the proportion of T lymphocytes , especially 纬未T cells .
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

【参考文献】