發(fā)布時(shí)間：2018-06-03 04:07

  本文選題：重組乳酸桿菌 + 陰道黏膜免疫��； 參考：《復(fù)旦大學(xué)》2013年碩士論文

【摘要】：黏膜免疫系統(tǒng)(mucosal immune system, MIS)----指廣泛分布于呼吸道、胃腸道、泌尿生殖道黏膜下及一些外分泌腺體處的淋巴組織,在結(jié)構(gòu)和功能上均有別于系統(tǒng)性免疫系統(tǒng)。黏膜免疫系統(tǒng)中的黏膜相關(guān)淋巴組織(mucosal associated lymphoid tissues, MALT)包括：腸相關(guān)淋巴組織(GALT)、支氣管相關(guān)淋巴組織(BALT)、鼻相關(guān)淋巴組織(NALT)以及泌尿生殖道黏膜相關(guān)淋巴組織等。MALT中的淋巴細(xì)胞主要是在黏膜淋巴系統(tǒng)內(nèi)進(jìn)行再循環(huán),這與其表達(dá)的歸巢分子有關(guān)。MIS按功能不同可分為誘導(dǎo)部位和效應(yīng)部位。在誘導(dǎo)部位和效應(yīng)部位之間,主要通過(guò)淋巴細(xì)胞歸巢發(fā)生聯(lián)系。從黏膜誘導(dǎo)部位歸巢到效應(yīng)部位這一功能上相關(guān)聯(lián)的系統(tǒng)統(tǒng)稱為共同黏膜免疫系統(tǒng)(common mucosal immune system)=共同黏膜免疫系統(tǒng)在黏膜免疫中有共同的效應(yīng),但對(duì)不同部位的黏膜,其免疫機(jī)制又有各自的特殊性,這與各器官的解剖和功能不同有關(guān)。在黏膜免疫機(jī)制的研究中,胃腸道黏膜免疫系統(tǒng)研究較深入。女性生殖道是一個(gè)特殊的黏膜免疫系統(tǒng)同時(shí)又有內(nèi)分泌功能,所以生殖道黏膜免疫更為復(fù)雜。近幾年生殖道黏膜免疫系統(tǒng)的作用也慢慢被大家所關(guān)注。我們實(shí)驗(yàn)室前期成功構(gòu)建了分泌hCGβ及攜分子佐劑C3d3的hCGβ-C3d3融合蛋白重組乳酸桿,并已研究證明,通過(guò)黏膜接種BALB/c小鼠,能產(chǎn)生抗hCGP體液免疫應(yīng)答,但是對(duì)于這些參與免疫反應(yīng)的淋巴細(xì)胞是如何歸巢及居留在生殖道的研究還很少。因此本實(shí)驗(yàn)著重于研究生殖道黏膜免疫后淋巴細(xì)胞歸巢相關(guān)粘附分子的表達(dá)及生殖道局部微環(huán)境對(duì)淋巴細(xì)胞歸巢的影響,解析生殖道黏膜免疫的部分機(jī)制。 第一部分陰道黏膜免疫后淋巴細(xì)胞歸巢相關(guān)黏附分子的表達(dá) 目的探討經(jīng)野生型及重組乳酸桿菌疫苗經(jīng)陰道黏膜接種BALB/c小鼠后淋巴細(xì)胞歸巢相關(guān)黏附分子的表達(dá) 方法用磷酸鹽緩沖液(PBS),野生型乳酸桿菌(Lb.)及重組乳酸桿菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010劑量(50u1)經(jīng)陰道滴注接種6～8周齡雌性BALB/c小鼠,每組10只。3周后用相同劑量加強(qiáng)免疫一次。加強(qiáng)免疫2周末處死小鼠,取各組子宮及陰道。部分標(biāo)本放入液氮儲(chǔ)存,待組織RNA提取,行實(shí)時(shí)定量PCR檢測(cè)黏附分子基因表達(dá)水平和組織蛋白提取,行Western Blot實(shí)驗(yàn),進(jìn)行蛋白定量。部分標(biāo)本用4%多聚甲醛固定24h,石蠟包埋,切片(厚度5μm),行免疫組織化學(xué)染色對(duì)黏附分子的表達(dá)進(jìn)行定位。另外應(yīng)用流式細(xì)胞術(shù)檢測(cè)各免疫組生殖道局部淋巴細(xì)胞表面黏附分子配體的表達(dá)。 結(jié)果加強(qiáng)免疫后,生殖道黏膜黏附分子的表達(dá)發(fā)生改變,黏附分子E-selectin, ICAM, VCAM, VAP-1and E-cadherin均可以在基因和蛋白水平檢測(cè)到表達(dá)。并且絕大部分黏附分子在重組乳酸桿菌免疫后的子宮表達(dá)量增加。 結(jié)論黏附分子E-selectin, ICAM, VCAM, VAP-1and E-cadherin參與了生殖道局部淋巴細(xì)胞的歸巢。 第二部分生殖道粘膜免疫中歸巢淋巴細(xì)胞表面高表達(dá)黏附分子相應(yīng)配體的表達(dá) 目的探討經(jīng)野生型及重組乳酸桿菌疫苗經(jīng)陰道黏膜接種BALB/c小鼠后淋巴細(xì)胞歸巢相關(guān)黏附分子配體的表達(dá) 方法用磷酸鹽緩沖液(PBS),野生型乳酸桿菌(Lb.)及重組乳酸桿菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010劑量(50ul)經(jīng)陰道滴注接種6～8周齡雌性BALB/c小鼠,每組10只。3周后用相同劑量加強(qiáng)免疫一次。加強(qiáng)免疫2周末處死小鼠,取各組子宮及陰道。原代分離出脾臟及生殖道局部淋巴細(xì)胞應(yīng)用流式細(xì)胞術(shù)檢測(cè)各免疫組生殖道局部淋巴細(xì)胞表面黏附分子配體的表達(dá)。應(yīng)用ELISA檢測(cè)各實(shí)驗(yàn)組子宮內(nèi)膜間質(zhì)細(xì)胞培養(yǎng)上清中TGF-β1量的表達(dá)。 結(jié)果分子佐劑C3d3能夠顯著的增加生殖道淋巴細(xì)胞歸巢相關(guān)黏附分子配體αEβ7, α4β7, LFA-1, VLA-4, PSGL-1的表達(dá)。 結(jié)論加強(qiáng)免疫之后,分子佐劑C3d3可以增加黏附分子配體αEβ7,α4β7, LFA-1, VLA-4, PSGL-1的表達(dá),促進(jìn)生殖道局部T,B淋巴細(xì)胞的歸巢。加強(qiáng)免疫之后,重組乳酸桿菌免疫組可以顯著促進(jìn)子宮內(nèi)膜間質(zhì)細(xì)胞TGF-β1的表達(dá)。 第三部分陰道黏膜免疫后局部微環(huán)境對(duì)淋巴細(xì)胞歸巢的影響 目的探討各免疫組加強(qiáng)免疫后,生殖道局部微環(huán)境對(duì)淋巴細(xì)胞歸巢的影響。 方法用磷酸鹽緩沖液(PBS),野生型乳酸桿菌(Lb.)及重組乳酸桿菌(Lb.hCGβ和Lb.hCGβ-C3d3)以1010劑量(50ul)經(jīng)陰道滴注接種6～8周齡雌性BALB/c小鼠,每組10只。3周后用相同劑量加強(qiáng)免疫一次。加強(qiáng)免疫2周末處死小鼠,無(wú)菌條件下取出各組子宮及陰道。原代分離子宮內(nèi)膜間質(zhì)細(xì)胞,收集培養(yǎng)上清,將各組培養(yǎng)上清種transwell小室的下室(600ul),上室種各免疫組脾臟淋巴細(xì)胞(1×106/100ul),37℃培養(yǎng)4h,收集遷移到下室的淋巴細(xì)胞經(jīng)流式細(xì)胞術(shù)檢測(cè)遷移至下室淋巴細(xì)胞的數(shù)量和淋巴細(xì)胞的比例。另外應(yīng)用流式細(xì)胞術(shù)檢測(cè)各免疫組脾臟及生殖道T淋巴細(xì)胞表型的改變。 結(jié)果免疫之后,各實(shí)驗(yàn)組培養(yǎng)上清較對(duì)照組相比可以顯著的增加淋巴細(xì)胞的遷移,尤其是重組乳酸桿菌免疫組,并且生殖道的微環(huán)境更傾向于增加T細(xì)胞的比例,并促進(jìn)Tcell尤其是γδT細(xì)胞向下室的遷移。 結(jié)論經(jīng)陰道黏膜加強(qiáng)免疫后,局部的微環(huán)境能夠顯著的增加T淋巴細(xì)胞的比例,尤其是γδT細(xì)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 偽E尾7 , 偽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 偽E尾7 , 偽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 緯未T cells .

Conclusion The local microenvironment can significantly increase the proportion of T lymphocytes , especially 緯未T cells .
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R392

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本文編號(hào)：1971408