發(fā)布時(shí)間：2018-06-09 06:50

  本文選題：面神經(jīng)損傷 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《昆明醫(yī)科大學(xué)》2016年博士論文

【摘要】：[背景與目的]面神經(jīng)軸索損傷(facial nerve axotomy, FNA)后若面部表情肌功能無(wú)法恢復(fù),嚴(yán)重影響患者生活質(zhì)量。目前臨床上以面神經(jīng)原位修復(fù)為主,雖術(shù)后面肌功能臨床恢復(fù)滿意,但仍有部分患者感到神經(jīng)功能同前有明顯差異,因此面神經(jīng)修復(fù)與重建是整形及神經(jīng)領(lǐng)域的一大難題。面神經(jīng)軸索損傷后,面神經(jīng)核內(nèi)面神經(jīng)元(facial motoneuron, FMN)會(huì)出現(xiàn)凋亡,維持神經(jīng)元存活狀態(tài)是損傷后神經(jīng)功能恢復(fù)的基礎(chǔ)。軸索損傷后面神經(jīng)核內(nèi)有40%的FMN存活依賴有功能的外周免疫系統(tǒng)和／或神經(jīng)營(yíng)養(yǎng)因子的參與。因此探討軸索損傷后神經(jīng)元凋亡發(fā)生的機(jī)制、采取方法改變面神經(jīng)核內(nèi)神經(jīng)免疫炎癥微環(huán)境,阻止FMN的凋亡是目前研究的熱點(diǎn)之一。骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)移植后可在中樞神經(jīng)系統(tǒng)微環(huán)境下轉(zhuǎn)化為類(lèi)神經(jīng)元樣細(xì)胞或神經(jīng)干細(xì)胞,替代凋亡或壞死的神經(jīng)元,還可分泌多種有利于神經(jīng)元生長(zhǎng)存活的神經(jīng)營(yíng)養(yǎng)因子,因此可以治療神經(jīng)系統(tǒng)疾病或損傷。然而大量研究表明移植的BMSCs由于局部損傷性炎癥微環(huán)境、缺氧或血流不足等因素,活率較低。面神經(jīng)軸索損傷后,斷端損傷信號(hào)沿近端軸突進(jìn)入FMN,導(dǎo)致面神經(jīng)核內(nèi)神經(jīng)免疫-炎癥微環(huán)境失去生理性平衡變?yōu)榇傺谞顟B(tài),該促炎微環(huán)境趨化外周T淋巴細(xì)胞及自體干細(xì)胞向面神經(jīng)核內(nèi)募集。其中T淋巴細(xì)胞亞群中CD4+ CD25+調(diào)節(jié)性T細(xì)胞(Regulatory T cell,Treg)在維持面神經(jīng)核內(nèi)免疫炎性微環(huán)境平衡中起主要作用,且對(duì)神經(jīng)元凋亡具有保護(hù)作用。自體干細(xì)胞可分化為神經(jīng)細(xì)胞替代或營(yíng)養(yǎng)損傷FMN,但損傷初期面神經(jīng)核內(nèi)的促炎微環(huán)境不利于干細(xì)胞存活,趨化的自體干細(xì)胞也不足以彌補(bǔ)損失的FMN。研究認(rèn)為BMSCs具有免疫抑制作用,可以抑制T淋巴細(xì)胞細(xì)胞增殖,調(diào)節(jié)機(jī)體內(nèi)免疫反應(yīng),減輕免疫反應(yīng)的程度,提高體外混合淋巴細(xì)胞培養(yǎng)中Treg細(xì)胞比例�；谝陨侠碚摶A(chǔ),本課題組提出假設(shè),尋找SD大鼠BMSCs與脾臟單核細(xì)胞最佳共培養(yǎng)比例,進(jìn)行腦干立體定位面神經(jīng)核層面共培養(yǎng)細(xì)胞移植,一方面可提高面神經(jīng)核內(nèi)具有神經(jīng)保護(hù)做用的Treg細(xì)胞比例,同時(shí)改善面神經(jīng)核內(nèi)神經(jīng)-免疫炎癥微環(huán)境,提供有利于移植BMSCs趨化、存活、分泌神經(jīng)營(yíng)養(yǎng)因子的微環(huán)境,挽救微依賴環(huán)境存在的面神經(jīng)核內(nèi)40%的FMN,從而為臨床進(jìn)行面神經(jīng)損傷的修復(fù)與重建提供基礎(chǔ)性研究依據(jù)。研究干細(xì)胞移植后在體內(nèi)分布、遷移、分化及歸巢等現(xiàn)象,有助于了解干細(xì)胞的修復(fù)機(jī)制,因此本研究采用MR分子成像標(biāo)記物超順磁性氧化鐵納米顆粒(superparamagnetic iron oxide, SPIO)與綠色熒光蛋白(green fluorescent protein,GFP)共同標(biāo)記BMSCs,同時(shí)采用組織學(xué)與活體MR示蹤方法顯示BMSCs的歸巢現(xiàn)象。[方法]1.體外將從SD大鼠脛腓骨骨髓分離培養(yǎng)鑒定后的BMSCs及轉(zhuǎn)染GFP的BMSC (GFP-BMSC)標(biāo)記不同濃度的SPIO后,檢測(cè)BMSC/GFP-BMSCs的增殖活性、細(xì)胞周期,明確不同濃度SPIO對(duì)細(xì)胞的活性的影響；試管內(nèi)MR成像,了解不同濃度SPIO標(biāo)記細(xì)胞體外MR成像信號(hào)強(qiáng)度變化；將標(biāo)記后的BMSC/ GFP-BMSC進(jìn)行成骨、成脂、成類(lèi)神經(jīng)元樣細(xì)胞誘導(dǎo)分化,明確SPIO對(duì)BMSCs分化的影響。2.將BMSCs與單核細(xì)胞按不同比例共培養(yǎng)后檢測(cè)BMSCs對(duì)T細(xì)胞增殖的抑制作用及對(duì)Treg細(xì)胞(Regulatory T cell,)比例影響,抑制最明顯及Treg比例最高組設(shè)為共培養(yǎng)1組；采用蛋白芯片篩選共培養(yǎng)細(xì)胞及單獨(dú)培養(yǎng)細(xì)胞裂解液及上清液內(nèi)差異蛋白；檢測(cè)單核細(xì)胞對(duì)BMSCs向面神經(jīng)元分化的影響,將向神經(jīng)元分化最好的一組設(shè)為共培養(yǎng)2組；3.建立右側(cè)面神經(jīng)損傷模型,4天后將最佳配比共培養(yǎng)細(xì)胞及單獨(dú)培養(yǎng)4天的細(xì)胞行腦干立體定位面神經(jīng)核層面細(xì)胞移植。4.根據(jù)移植細(xì)胞成分不同,將動(dòng)物分為6組(陰性對(duì)照組,陽(yáng)性對(duì)照組,單獨(dú)單核淋細(xì)胞組,共培養(yǎng)1組,單獨(dú)BMSCs組,共培養(yǎng)2組)移植后當(dāng)天及第3d、7d、14d、21d、28d MR掃描活體示蹤及面神經(jīng)核層面組織學(xué)示蹤BMSCs歸巢情況；各時(shí)間點(diǎn)檢測(cè)面神經(jīng)核內(nèi)神經(jīng)-免疫炎癥微環(huán)境相關(guān)細(xì)胞因子(蛋白芯片篩選出的差異蛋白)趨化因子,神經(jīng)營(yíng)養(yǎng)因子表達(dá)量變化及信號(hào)通路變化,比較免疫炎癥微環(huán)境相關(guān)因子變化與面神經(jīng)核內(nèi)神經(jīng)元凋亡數(shù)量的相關(guān)性。[結(jié)果]1.成功從SD大鼠脛腓骨分離BMSCs細(xì)胞,第三代培養(yǎng)4天后檢測(cè)CD29,CD90陽(yáng)性,CD34陰性；成功將BMSC/GFP-BMSCs標(biāo)記不同濃度的SPIO (25μg/ml, 50μg/ml,75μg/ml,100μg/ml, 0μg/ml, A-E組)；檢測(cè)BMSC/GFP-BMSCs的增殖活性D濃度組最低,細(xì)胞周期A～E組無(wú)差異,試管內(nèi)MR成像A-D組信號(hào)均減低,D組最低,C、D濃度組無(wú)統(tǒng)計(jì)學(xué)差異。MR成像SWI較T2*WI敏感；成功將標(biāo)記后的BMSCs/GFP-BMSCs誘導(dǎo)成骨、成脂、成類(lèi)神經(jīng)元樣細(xì)胞分化,結(jié)果顯示75μg/ml的SPIO信號(hào)減低明顯且對(duì)BMSCs分化無(wú)影響。2.將單核細(xì)胞與BMSCs按不同比例(單獨(dú)單核細(xì)胞,單獨(dú)單核細(xì)胞與PHA,1：1,1：10,1：30,1：50,單獨(dú)BMSCs,1：1,10：1,30：1,50：1,即A～K組)共培養(yǎng)后,單核細(xì)胞與BMSCs比例為1：30時(shí)(共培養(yǎng)1組),CD4+T/CD8+T比值及Treg細(xì)胞比例最高；比例為30：1時(shí)(共培養(yǎng)2組),BMSCs向類(lèi)神經(jīng)元細(xì)胞誘導(dǎo)分化最好。蛋白芯片篩選結(jié)果顯示,共培養(yǎng)1、2組較單獨(dú)培養(yǎng)細(xì)胞細(xì)胞裂解液上調(diào)的蛋白為：VEGF、BDNF、CXCR4、CD34,上清液上調(diào)蛋白為VEGF、TGF-β、 IL-4、EL-10,下調(diào)的蛋白為IL-2,INF-γ; PCR檢測(cè)Tub 3, Nestin, NSE, Syt 1 mRNA表達(dá)量,誘導(dǎo)前較誘導(dǎo)后明顯升高。3.成功建立單側(cè)面神經(jīng)高位損傷模型125只,建立陰性對(duì)照組(耳后切口找到面神經(jīng),不切斷)模型25只,建模后第4天進(jìn)行腦干立體定位面神經(jīng)核層面細(xì)胞移植均成功,均未出現(xiàn)死亡或其他并發(fā)癥。4.150只SD大鼠,移植當(dāng)天MR掃描均顯示,移植處SWI小點(diǎn)狀低信號(hào)。移植后3d,7d,14d,21d,28d MR顯示小點(diǎn)狀低信號(hào)逐漸向面神經(jīng)核區(qū)域移動(dòng),其中共培養(yǎng)2組移動(dòng)最快,其次為共培養(yǎng)1組,與此同時(shí)病理切片顯示GFP陽(yáng)性細(xì)胞向面神經(jīng)核歸巢;各組各時(shí)間點(diǎn)Tunel染色及Weston blot檢測(cè)Caspase-3及Bcl-2表達(dá)量變化顯示,3-21d細(xì)胞凋亡量增加,21d后開(kāi)始下降,共培養(yǎng)2組細(xì)胞凋亡數(shù)量最少；Weston blot及Q-PCR檢測(cè)面神經(jīng)核內(nèi)各移植組各因子均在3-14d表達(dá)量增加,一般14d或21d達(dá)高峰,21d后開(kāi)始下降：抗炎細(xì)胞因子IL-4,IL-10,TGF-β1及JAK/STAT6信號(hào)通路,趨化因子SDF-1/CXCR 4軸,BDNF及trkB/ERK信號(hào)通路,共培養(yǎng)2組表達(dá)量均最高,其次為共培養(yǎng)1組,再次為單獨(dú)BMSCs組,陽(yáng)性對(duì)照組表達(dá)量最低；促炎細(xì)胞因子INF-γ,IL-2及IL-6共培養(yǎng)2組表達(dá)量最低,共培養(yǎng)1組稍高,陽(yáng)性對(duì)照組表達(dá)量最高。[結(jié)論]1.GFP與75μg/mL的SPIO雙標(biāo)不影響B(tài)MSCs的增殖及分化能力；SWI序列較T2*WI更敏感的顯示75μg/mLSPIO標(biāo)記的GFP-BMSCs引起的MR信號(hào)強(qiáng)度變化。2. BMSCs在體外可以抑制PHA引起的淋巴細(xì)胞增殖,提高CD4+T/CD8+T比值及CD4+CD25+T (Treg)細(xì)胞比例,使Thl/Th2向Th2抗炎方向“漂移”,促進(jìn)TGF-γ、IL-4,IL-10分泌,減少I(mǎi)L-2、INF-γ分泌,從而改變BMSCs生存微環(huán)境,最佳單核細(xì)胞與BMSCs細(xì)胞數(shù)比例為1：30。3. BMSCs與單核細(xì)胞共培養(yǎng),可促進(jìn)BMSCs向類(lèi)神經(jīng)元方向分化,向面神經(jīng)核趨化,提高BMSCs分泌BDNF. VEGF,最佳單核細(xì)胞與BMSC細(xì)胞數(shù)比例為30：1。4.莖乳孔處高位切斷面神經(jīng)可建立穩(wěn)定可重復(fù)的單側(cè)面神經(jīng)損傷模型。5.腦干立體定位面神經(jīng)核層面細(xì)胞移植方法可靠,采用建模后第4天移植,細(xì)胞量為1×106/5μL PBS.6.面神經(jīng)軸索損傷致面神經(jīng)核內(nèi)抗炎／促炎微環(huán)境發(fā)生“漂移”,神經(jīng)-免疫炎癥微環(huán)境發(fā)生變化使FMN凋亡增加。7. BMSCs及單核細(xì)胞聯(lián)合移植,改善面神經(jīng)核內(nèi)神經(jīng)-免疫炎癥微環(huán)境,IL-4通過(guò)JAK/STST6信號(hào)通路抑制炎癥發(fā)生,促進(jìn)移植BMSCs趨化、分化、分泌BDNF, BDNF通過(guò)trkB/erk信號(hào)通路抗神經(jīng)元凋亡。
[Abstract]:Bone marrow mesenchymal stem cells ( FMN ) can be transformed into neuron - like cells or neural stem cells in the central nervous system . In vitro , the effects of SPIO on proliferation , cell cycle and cell cycle of BMSC / GFP - BMSC were detected by means of histological and living MR tracer method .
MR imaging in vitro was performed to understand the signal intensity changes of different concentrations of SPIO labeled cells in vitro ;
The BMSC / GFP - BMSC after labeling was divided into bone , fat - forming , adult - like neuron - like cells to induce differentiation , and the influence of SPIO on the differentiation of bone marrow cells was determined .
the protein chip is used for screening the co - cultured cells and separately culturing the cell lysate and the supernatant ;
To detect the effect of monocytes on the differentiation of bone marrow cells into neurons , the best group of differentiating neurons was co - cultured with 2 groups .
3 . The right facial nerve injury model was established . Four days later , the cells were co - cultured and cultured for 4 days . 4 . The animals were divided into 6 groups ( negative control group , positive control group , single mononuclear cell group , co - cultured group 1 , group of positive control group , co - cultured group 2 ) on the same day and 3d , 7d , 14d , 21d and 28d after transplantation .
The changes of the expression quantity of neurotrophic factors and the changes of signal pathway were detected in the peripheral nerve - immune inflammatory microenvironment related cytokines ( protein chips ) in various time points , and the correlation between the changes of the related factors of immune inflammatory microenvironment and the number of apoptotic neurons in facial nerve nuclei was compared .
The different concentrations of SPIO ( 25 渭g / ml , 50 渭g / ml , 75 渭g / ml , 100 渭g / ml , 0 渭g / ml , A - E ) were successfully labeled with BMSC / GFP - BMSC .
The proliferation activity of BMSC / GFP - BMSC was lowest , there was no difference in cell cycle A - E group , the signal of MR imaging group A - D in test tube was reduced , there was no statistical difference in group D , C and D . MR imaging SWI was sensitive to T2 * WI .
The results showed that the ratio of monocytes to bone marrow cells was 1 鈭，

本文編號(hào)：1999436