本文選題：顱腦損傷 + 肝臟�。� 參考：《天津醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：觀察顱腦損傷后經(jīng)腸系膜靜脈注射特定抗原免疫耐受建立的情況,初步探討肝臟非實(shí)質(zhì)細(xì)胞誘導(dǎo)免疫耐受的機(jī)制。 方法：1.選取新西蘭兔32只,隨機(jī)分成4組,每組8只,依次命名為A-D組,A組為單純顱腦損傷組,B組為注射1ml生理鹽水組,C組為注射1mlMBP(2mg/ml)組,D組為注射1ml腦組織單細(xì)胞懸液組(細(xì)胞密度為106/ml)。1d四組均開顱造模,取0.8*0.4*0.5cm腦組織制備單細(xì)胞懸液；制備好腦組織單細(xì)胞懸液后在30min內(nèi)開腹尋找腸系膜靜脈,B組注射lml生理鹽水。C、D組分別經(jīng)腸系膜靜脈分別注射1mlMBP和1ml單細(xì)胞懸液。7d全部處死,取損傷灶周圍腦組織做Fas/FasL的定量PCR。 2.選取20只SD大鼠,隨機(jī)分為3組,對照組5只、假手術(shù)組5只和損傷組10只,損傷組按傷后時(shí)間分為2、4、8、16h和24h5個(gè)采血點(diǎn),在傷后不同時(shí)間點(diǎn)采用眼眶取血法,取靜脈血離心后得血清刺激原代培養(yǎng)的大鼠小膠質(zhì)細(xì)胞,培養(yǎng)24h后流式細(xì)胞儀測定小膠質(zhì)細(xì)胞MHC-Ⅱ的表達(dá)�！� 3.另選取新西蘭兔24只,隨機(jī)分成3組,每組8只,依次命名為E-G組,E組為注射1ml生理鹽水組,F組為注射1mlMBP(2mg/ml)組,G組為注射lml單細(xì)胞懸液組(細(xì)胞密度為106/ml)。手術(shù)腦損傷造模、注射抗原同第一部分。分別于術(shù)后1d、3d、5d、7d、14d和21d抽取靜脈血,做Elisa測血清中TGF-β,IL-2和IL-10的含量,21d處死,取肝臟組織做免疫熒光測定Foxp+3表達(dá)情況。 結(jié)果：1.實(shí)時(shí)定量PCR顯示：損傷后7d單純顱腦損傷組和各處理組Fas均高表達(dá),各組之間未見明顯差異(p＞0.05)；A、B兩組FasL未見表達(dá)增高,C組FasL表達(dá)顯著升高,與A、B兩組比較有統(tǒng)計(jì)學(xué)差異(p0.05),D組表達(dá)略有升高,與A、B兩組比較未見統(tǒng)計(jì)學(xué)差異(p＞0.05)。 2.流式細(xì)胞結(jié)果顯示：空白對照組MHC-Ⅱ微量表達(dá);假手術(shù)組有較低表達(dá)；損傷組各個(gè)時(shí)間點(diǎn)均有表達(dá),且隨著時(shí)間的推移,MHC-Ⅱ表達(dá)增高。損傷組與對照組兩兩比較,差異有統(tǒng)計(jì)學(xué)意義(p＜0.05)。 3.Elisa實(shí)驗(yàn)顯示：與E組相比,F組TGF-β、IL-10顯著升高,于14d左右到達(dá)高峰,隨后緩慢下降,差異有統(tǒng)計(jì)學(xué)意義(p0.05),IL-2相應(yīng)減低,差異有統(tǒng)計(jì)學(xué)意義(p0.05)；G組TGF-β、IL-10于10-14d可見略有升高,差異無統(tǒng)計(jì)學(xué)意義(p0.05),IL-2未見明顯減低,差異無統(tǒng)計(jì)學(xué)意義(p＞0.05)。 4.免疫熒光化學(xué)顯示：在注射生理鹽水組和注射腦組織單細(xì)胞懸液組中,肝臟石蠟切片中T淋巴細(xì)胞未看到明顯橙紅色激發(fā)光。在注射MBP組中可看到靜脈竇中和血管周圍T淋巴細(xì)胞中可見橙紅色熒光,說明注射MBP可使肝臟T淋巴細(xì)胞表達(dá)Foxp+3,與其他兩組相比,有統(tǒng)計(jì)學(xué)差異(p0.05)。 結(jié)論：1.各處理組中,Fas在損傷后均高表達(dá),說明損傷灶周圍有大量淋巴細(xì)胞聚集,炎癥反應(yīng)強(qiáng)烈；經(jīng)腸系膜靜脈注射特定抗原并不能減少Fas的表達(dá)。腸系膜靜脈注射MBP可以誘導(dǎo)免疫耐受；注射腦組織單細(xì)胞懸液的抗原特異性不高,不能完全誘導(dǎo)免疫耐受。 2.大鼠腦損傷后24h內(nèi)的血清可使小膠質(zhì)細(xì)胞MHC-Ⅱ的表達(dá)增高,小膠質(zhì)細(xì)胞MHC-Ⅱ表達(dá)隨著時(shí)間的推移不斷升高。從細(xì)胞水平支持了顱腦損傷早期炎癥反應(yīng)的劇烈程度。 3.經(jīng)腸系膜靜脈注射MBP后,可見與E組相比,顯著下降；而注射腦組織單細(xì)胞懸液組僅在14d才有下降,表明注射MBP可以誘導(dǎo)免疫耐受,而后者抗原特異性較差。 4. TGF-β、IL-10均是強(qiáng)效免疫抑制因子,它們可以抑制細(xì)胞毒性T淋巴細(xì)胞的增殖和活化,抑制T,B淋巴細(xì)胞的增殖及免疫球蛋白的分泌。注射MBP比注射腦組織單細(xì)胞懸液更容易可以誘導(dǎo)免疫耐受。 5.經(jīng)腸系膜靜脈注射MBP后,21d后,在熒光顯微鏡下顯示,靜脈竇及周圍肝組織的T淋巴細(xì)胞呈橙紅色熒光,說明其表達(dá)了Foxp+3,,側(cè)面提示免疫耐受的成功建立。而在另外兩組中,并未發(fā)現(xiàn)T細(xì)胞有橙紅色熒光物質(zhì)表達(dá)。說明腦細(xì)胞尚不能誘導(dǎo)Foxp+3的表達(dá)。
[Abstract]:Objective : To investigate the mechanism of immune tolerance induced by non - essential cells in liver by observing the establishment of immune tolerance of specific antigen in mesenteric vein after craniocerebral injury .

Methods : 1 . Thirty - two New Zealand rabbits were randomly divided into four groups : group A - D group , group A as group A - D , group A as group A - D , group B was injected with 1 ml of MBP ( 2 mg / ml ) group , group D was injected 1ml of brain tissue single cell suspension group ( cell density 106 / ml ) .
In group B , 1 ml of MBP and 1 ml of single cell suspension were injected respectively through mesentery vein in group B . All the rats were sacrificed at 7d , and the brain tissue around the lesion was taken as the quantitative PCR of Fas / FasL .

2 . Twenty SD rats were randomly divided into three groups : control group ( 5 ) , sham operation group ( 5 ) and injury group ( n = 10 ) . The injured group was divided into 2 , 4 , 8 , 16 h and 24 h after injury . After injury , the rats were divided into 2 , 4 , 8 , 16 h and 24 h5 blood sampling points . After injury , the rats were divided into 2 , 4 , 8 , 16 h and 24 h5 blood sampling points . After the injury , the rat microglial cells were cultured in primary culture . After 24 h , the expression of MHC - 鈪，

本文編號：1749422