本文選題：新生隱球菌 + 慢病毒�。� 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景隱球菌(Cryptococcus)系環(huán)境腐生菌,也是重要的機(jī)會(huì)致病真菌,常在免疫功能低下人群引起感染。中樞神經(jīng)系統(tǒng)感染是隱球菌病最主要的臨床表現(xiàn),其中隱球菌性腦膜炎/腦膜腦炎(Cryptococca meningitis/meningoencephalitis,CME)是其高病死率的最主要病因。強(qiáng)烈的嗜中樞性是隱球菌病的一個(gè)顯著特點(diǎn),有超過90%的隱球菌病侵犯中樞神經(jīng)系統(tǒng),但目前其機(jī)制不明。血腦屏障便是隱球菌侵襲中樞神經(jīng)系統(tǒng)的必經(jīng)關(guān)卡,所以新生隱球菌侵襲血腦屏障(Blood-Brain Barrier,BBB)的途徑只能是:或穿過腦微血管內(nèi)皮細(xì)胞;或穿越細(xì)胞間的緊密連接結(jié)構(gòu)。目前有三個(gè)機(jī)制假說:1.細(xì)胞旁途徑;2.“特洛伊木馬”機(jī)制;3.跨細(xì)胞途徑(即內(nèi)皮細(xì)胞胞吞胞吐)。S100A10蛋白是鈣結(jié)合蛋白家族中S100蛋白家族中的一員,存在于很多細(xì)胞的胞漿和胞核中。S100A10蛋白可與膜聯(lián)蛋白A2(annexin A2)結(jié)合為異四聚體(S100A10)2-(annexinA2)2并作為膜蛋白存在于多種細(xì)胞的細(xì)胞膜上。繼而使纖溶酶原被tPA(組織型纖溶酶原激活劑)、uPA(尿激酶型纖溶酶原激活劑)激活為纖溶酶,同時(shí)激活基質(zhì)金屬蛋白酶,降解細(xì)胞基質(zhì)。它與細(xì)胞的胞吞胞吐、物質(zhì)轉(zhuǎn)運(yùn)等密切相關(guān)。MMP9蛋白是一類結(jié)構(gòu)高度同源的分泌型或膜相關(guān)性鋅內(nèi)肽酶MMPs(Matrix metalloproteinase,MMP)家族中的一員,包括分子量為92kDa的無活性形式ProMMP9和82kDa的活性形式MMP9。體內(nèi)絕大多數(shù)細(xì)胞并不儲(chǔ)備MMPs,只有當(dāng)需要MMPs的信號(hào)傳遞到細(xì)胞后才臨時(shí)合成,然后以無活性的酶原形式分泌到胞外。隨后被激活并呈現(xiàn)瀑布效應(yīng)。MMPs與血腦屏障開放有關(guān),其中MMP-9可分解IV、V、VII、X、XI、彈性蛋白、微纖維蛋白、層粘連蛋白、骨連蛋白。針對(duì)隱球菌感染血腦屏障內(nèi)皮細(xì)胞后上調(diào)S100A10是否影響MMP9的表達(dá),本研究進(jìn)行了深入研究,進(jìn)一步驗(yàn)證MMP9是否在新生隱球菌的嗜中樞性機(jī)制中起到一定作用,并探索抑制MMP9來保護(hù)血腦屏障的新方法。研究目的第一,觀察新生隱球菌通過小鼠腦微血管內(nèi)皮細(xì)胞(b.End3)S100A10影響基質(zhì)金屬蛋白酶9(MMP9)在mRNA水平和細(xì)胞內(nèi)外蛋白質(zhì)水平的表達(dá);第二,沉默小鼠腦微血管內(nèi)皮細(xì)胞MMP9基因后,驗(yàn)證基質(zhì)金屬蛋白酶9表達(dá)情況;構(gòu)建該細(xì)胞系體外血腦屏障模型,探究此時(shí)新生隱球菌侵襲血腦屏障的能力;第三,探索基質(zhì)金屬蛋白酶抑制劑GM6001對(duì)新生隱球菌穿過血腦屏障是否有一定的保護(hù)作用,為進(jìn)一步臨床應(yīng)用研究提供體外實(shí)驗(yàn)基礎(chǔ)。研究方法第一部分將慢病毒轉(zhuǎn)染小鼠腦微血管內(nèi)皮細(xì)胞(b.end3)細(xì)胞,分別篩選穩(wěn)定下調(diào)s100a10、mmp9以及空載的細(xì)胞系,分別用熒光顯微鏡和qpcr定性和定量驗(yàn)證其下調(diào)程度(該部分已發(fā)表)。以新生隱球菌b3501感染由lv-muss100a10-shrnab.end3和ncb.end3構(gòu)建的簡易體外血腦屏障模型,在mrna水平和蛋白水平檢測(cè)基質(zhì)金屬蛋白酶9(mmp9)表達(dá)量的差異。第二部分將lv-musmmp9-shrnab.end3和ncb.end3分別構(gòu)建transwell體外血腦屏障模型,新生隱球菌b3501感染該模型,檢測(cè)其穿透血腦屏障的活菌數(shù)和bbb的完整性。第三部分基質(zhì)金屬蛋白酶抑制劑在新生隱球菌性腦炎/腦膜炎防治中的作用初步探索。以小鼠腦微血管內(nèi)皮細(xì)胞(b.end3)細(xì)胞構(gòu)建體外血腦屏障模型,放置于不同濃度的gm6001環(huán)境中。檢測(cè)gm6001作用時(shí),穿過血腦屏障的活性隱球菌數(shù)量以及血腦屏障穩(wěn)定性的改變程度,以探討gm6001對(duì)血腦屏障的保護(hù)作用及最佳濃度。結(jié)果第一部分成功構(gòu)建了ncb.end3細(xì)胞系、lv-muss100a10-shrnab.end3細(xì)胞系和lv-musmmp9-shrnab.end3細(xì)胞系,后兩者mmp9基因表達(dá)下調(diào)分別在76%和78.8%。經(jīng)s-n-k檢驗(yàn),lv-muss100a10-shrnab.end3細(xì)胞系和lv-musmmp9-shrnab.end3細(xì)胞系與nc組、b.end3組差別有統(tǒng)計(jì)學(xué)意義,p0.05,而b.end3組與nc組的mmp9表達(dá)的差異無統(tǒng)計(jì)學(xué)意義(該部分已發(fā)表,見附錄)。ncb.end3組與lv-muss100a10-shrnab.end3設(shè)置對(duì)照實(shí)驗(yàn),兩組分別與新生隱球菌b3501構(gòu)建相應(yīng)體外血腦屏障感染模型。mrna水平和細(xì)胞內(nèi)外蛋白水平的檢測(cè)發(fā)現(xiàn),lv-muss100a10-shrnab.end3組在s100a10下調(diào)后,基質(zhì)金屬蛋白酶9(mmp9)表達(dá)量與ncb.end3組mmp9表達(dá)量的差異有統(tǒng)計(jì)學(xué)意義(p0.05),且均較nc組低。第二部分lv-musmmp9-shrnab.end3和空載ncb.end3分別構(gòu)建體外血腦屏障的新生隱球菌感染模型。驗(yàn)證表明,mmp9下調(diào)后,在mrna水平和細(xì)胞內(nèi)外蛋白水平分別比較兩組間基質(zhì)金屬蛋白酶9(mmp9)表達(dá)量的差異,經(jīng)檢驗(yàn)差異有統(tǒng)計(jì)學(xué)意義,p0.05。在此基礎(chǔ)上,用一定量的新生隱球菌b3501感染相應(yīng)分別構(gòu)建好的兩組transwell體外血腦屏障模型模型,檢測(cè)血腦屏障穩(wěn)定性的下降程度和穿透血腦屏障模型的活菌數(shù),以證明其在不同血腦屏障模型的侵襲能力。結(jié)果發(fā)現(xiàn):第一,LV-musMMP9-shRNA b.End3所構(gòu)建的血腦屏障模型,其跨內(nèi)皮細(xì)胞電阻(TEER)的變化幅度與NC-b.End3組相比有明顯差異,且有統(tǒng)計(jì)學(xué)意義(P0.05)。兩組BBB的TEER變化幅度有差異,而由LV-musMMP9-shRNA b.End3所構(gòu)建的血腦屏障模型在經(jīng)歷新生隱球菌侵襲時(shí)TEER變化幅度小,穩(wěn)定性更高,完整性更好。第二,體外血腦屏障模型中,NC-b.End3組與LV-musMMP9-shRNA b.End3組的transwell下室內(nèi)新生隱球菌數(shù)目隨時(shí)間延長而增多,且兩組各時(shí)點(diǎn)菌落負(fù)荷均出現(xiàn)差異,經(jīng)成組T檢驗(yàn),P0.05,各時(shí)點(diǎn)兩組差異均具有統(tǒng)計(jì)學(xué)意義。第三部分小鼠腦微血管內(nèi)皮細(xì)胞bEnd.3構(gòu)建血腦屏障模型,放置于不同濃度金屬蛋白酶抑制劑GM6001的微環(huán)境,統(tǒng)計(jì)B3501穿過血腦屏障的數(shù)目。結(jié)果提示,GM6001 10μM處理組,transwell下室的活性菌數(shù)量減少,各組間差異有統(tǒng)計(jì)學(xué)意義。結(jié)論MMP9在新生隱球菌通過細(xì)胞旁途徑穿透血腦屏障過程中起到重要作用,其表達(dá)水平依賴于S100A10的表達(dá)情況。隱球菌嗜中樞感染時(shí),金屬蛋白酶抑制劑可作為一種血腦屏障保護(hù)劑,緩解并減少感染的擴(kuò)散,為臨床輔助治療隱球菌性腦炎/腦膜炎提供一種新的可能性。
[Abstract]:Cryptococcus neoformans (Cryptococcus), an important opportunistic pathogenic fungus, is an important opportunistic pathogenic fungus and often causes infection in people with low immune function. Central nervous system infection is the most important clinical manifestation of cryptococcosis, and cryptococcal meningitis / meningoencephalitis (Cryptococca meningitis/meningoencephalitis, CME) is a high mortality. The most important cause of the rate is the strong centrism is a prominent feature of cryptococcosis, with more than 90% of cryptococcosis invading the central nervous system, but the mechanism is unclear. The blood brain barrier is a necessary barrier for Cryptococcus neoformans to attack the central nervous system, so Cryptococcus neoformans invades the blood brain barrier (Blood-Brain Barrier, BBB). Only: or through the cerebral microvascular endothelial cells; or through the close connections between cells. There are three mechanism hypotheses: 1. cell side pathways; 2. "Troy Trojan" mechanism; 3. cross cell pathway (i.e. endothelin) is a member of the S100 protein family in the family of calcium binding proteins and exists in many cells. The.S100A10 protein in the cytoplasm and nucleus can be combined with the membrane protein A2 (annexin A2) as the heterogeneous four polymer (S100A10) 2- (annexinA2) 2 and as membrane protein exists on the cell membrane of a variety of cells. Then the plasminogen activator of the fibrinolytic enzyme is activated by tPA (tissue type plasminogen activator) and uPA (urokinase type plasminogen activator) as a fibrinolytic enzyme, and activates the plasminogen activator. .MMP9 protein is a member of a family of highly homologous secretory or membrane related zinc endopeptidase MMPs (Matrix metalloproteinase, MMP), including the active form of 92kDa and the active form MMP9. of 92kDa and 82kDa. Most of the cells in the body do not reserve MMPs, and they are temporarily synthesized only when the signal of MMPs is passed to the cell, and then secreted to the extracellular in the form of inactive zymogen. Then it is activated and presents the waterfall effect.MMPs related to the opening of the blood brain barrier, in which MMP-9 can decompose IV, V, VII, X, XI, elastin, microfibrin, laminin. This study conducted in-depth studies to further verify whether MMP9 plays a role in the central mechanism of Cryptococcus neoformans, and explores a new method to inhibit the protection of the blood brain barrier by MMP9. The first of this study is to observe the new purpose of the study. Cryptococcus neoformans affects the expression of matrix metalloproteinase 9 (MMP9) at mRNA level and protein level inside and outside of the mouse brain microvascular endothelial cells (b.End3). Second, the expression of matrix metalloproteinase 9 is verified after the silence of the MMP9 gene of the mouse brain microvascular endothelial cells, and the blood brain barrier model in vitro is constructed. At this time, the ability of Cryptococcus neoformans to invade the blood brain barrier; third, to explore the protective effect of matrix metalloproteinase inhibitor GM6001 on Cryptococcus neoformans through the blood brain barrier, and to provide the experimental basis for further clinical application. The first part of the research method transfected the lentivirus into the mouse brain microvascular endothelial cells (b.end3 The cells were screened for the stable downregulation of S100A10, MMP9 and unloaded cell lines. The down-regulation of the cells was qualitatively and quantitatively verified by fluorescence microscopy and qPCR, respectively. The simple external blood brain barrier model constructed by lv-muss100a10-shrnab.end3 and ncb.end3 for Cryptococcus neoformans b3501 infection was detected at mRNA level and protein level. The difference in the expression of matrix metalloproteinase 9 (MMP9). The second part constructs a Transwell in vitro blood brain barrier model of Transwell and ncb.end3 respectively. Cryptococcus neoformans b3501 infects the model to detect the number of living bacteria that penetrate the blood brain barrier and the integrity of BBB. The third part of the matrix metalloproteinase inhibitor in Cryptococcus neoformans Preliminary exploration of the role of encephalitis / meningitis prevention and control. A mouse brain microvascular endothelial cell (b.end3) cell is used to construct an in vitro blood brain barrier model, which is placed in a different concentration of GM6001 environment. The number of active Cryptococcus through the blood brain barrier and the change of the stability of the blood brain barrier are detected by the detection of the effect of GM6001 on the blood brain screen, in order to explore the effect of GM6001 on the blood brain screen. Results the first part successfully constructed ncb.end3 cell line, lv-muss100a10-shrnab.end3 cell line and lv-musmmp9-shrnab.end3 cell line. The down regulation of MMP9 gene expression in the latter two was 76% and 78.8%. by s-n-k test, lv-muss100a10-shrnab.end3 fine cell line and lv-musmmp9-shrnab.end3 cell line and NC group, B, respectively, B. The difference in.End3 group was statistically significant, P0.05, but there was no significant difference in the expression of MMP9 in group b.end3 and NC group (this part has been published, see Appendix).Ncb.end3 group and lv-muss100a10-shrnab.end3 set a control experiment. The two groups were constructed with Cryptococcus neoformans b3501 to construct the corresponding.Mrna level and intracellular protein of the blood brain barrier infection model in vitro. It was found that the expression of matrix metalloproteinase 9 (MMP9) expression and MMP9 expression in group ncb.end3 were significantly different from that of group ncb.end3 (P0.05), and were lower than those in the NC group after the downregulation of S100A10 in the lv-muss100a10-shrnab.end3 group. The model of Cryptococcus neoformans infection in the blood brain barrier was constructed by second partial lv-musmmp9-shrnab.end3 and no load ncb.end3. The results showed that the difference in the expression of matrix metalloproteinase 9 (MMP9) between the two groups was compared between the two groups after the downregulation, and the difference was statistically significant. On the basis of p0.05., two groups of Transwell in vitro blood brain barrier model model established by a certain amount of Cryptococcus neoformans b3501 infection were constructed. Type, detection of the decline in the stability of blood brain barrier and the number of living bacteria that penetrated the blood brain barrier model to prove its invasion ability in different blood brain barrier models. The results were as follows: first, the blood brain barrier model constructed by LV-musMMP9-shRNA b.End3 was significantly different from that of the NC-b.End3 group. And there was statistical significance (P0.05). The TEER changes in the two group of BBB were different, and the blood brain barrier model constructed by LV-musMMP9-shRNA b.End3 had little change, higher stability and better integrity during the invasion of Cryptococcus neoformans. Second, in the model of blood brain barrier in vitro, NC-b.End3 group and LV-musMMP9-shRNA b.End3 group Transwell. The number of Cryptococcus neoformans increased with time, and the colony load of the two groups at each time point was different. The difference between the two groups was statistically significant after the group T test, P0.05 and each time point. The third part of the mouse brain microvascular endothelial cells (bEnd.3) constructed the blood brain barrier model, which was placed at different concentrations of the metalloproteinase inhibitor GM6001. Microenvironment, statistical B3501 passes through the number of blood brain barriers. The results suggest that the number of active bacteria in the Transwell lower chamber is reduced in the GM6001 10 M treatment group. Conclusion MMP9 plays an important role in the process of penetrating the blood brain barrier through the paracellular pathway of Cryptococcus neoformans. The expression level of Cryptococcus neoformans depends on the expression of S100A10. When Cryptococcus neoformans are central infection, the inhibitor of metalloproteinase can be used as a protective agent for blood brain barrier to alleviate and reduce the spread of infection. It provides a new possibility for clinical adjuvant treatment of cryptococcal encephalitis / meningitis.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R519

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 陳雪雯;朱紅梅;溫海;;MMP9、S100A10基因沉默穩(wěn)轉(zhuǎn)小鼠細(xì)胞系的構(gòu)建及驗(yàn)證[J];中國真菌學(xué)雜志;2017年01期

2 邱天文;朱紅梅;溫海;;新生隱球菌通過S100A10活化尿激酶-纖溶酶系統(tǒng)侵襲血腦屏障的機(jī)制研究[J];中國真菌學(xué)雜志;2016年03期

3 Tobias Ruck;Stefan Bittner;Sven G.Meuth;;Blood-brain barrier modeling: challenges and perspectives[J];Neural Regeneration Research;2015年06期

4 Sugreev Verma;Kousik Kesh;Nilanjan Ganguly;Sayantan Jana;Snehasikta Swarnakar;;Matrix metalloproteinases and gastrointestinal cancers: Impacts of dietary antioxidants[J];World Journal of Biological Chemistry;2014年03期

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本文編號(hào)：1996532