侵袭性真菌性鼻—鼻窦炎IKKα调控maspin蛋白表达的实验研究
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摘要
随着真菌感染疾病的增多，人类对真菌病的认识不断提高，研究逐步深入，真菌性鼻-鼻窦炎日益受到关注。真菌性鼻-鼻窦炎是指真菌在鼻腔、鼻窦内引起的一种感染性或(和)变应性疾病。根据组织受侵犯的临床表现和病理表现不同，分为非侵袭性和侵袭性两大类型。所谓侵袭性真菌性鼻窦炎（Invasive FungalRhinosinusitis, IFRS）属于侵袭性真菌感染，是指真菌菌丝侵入黏膜、黏膜下、血管、骨质等组织结构中，而非侵袭性真菌性鼻窦炎真菌菌丝聚集在窦腔，未侵入黏膜及深部组织，两者在临床表现和预后方面差异很大，特别是急性侵袭性真菌性鼻窦炎病情凶险，病人可在数日内由于真菌侵袭性损害而死亡。
各种环境因素及宿主因素与真菌性鼻窦炎的发病有关，其中机体免疫功能状态与真菌性鼻窦炎的病理分型有关，当免疫功能不足时，易患侵袭性真菌性鼻窦炎。骨髓移植和器官移植的接受者、未经控制血糖的糖尿病患者和艾滋病感染者都是侵袭性鼻窦炎的高危人群。烟曲霉菌是真菌性鼻窦炎最常见条件致病菌，烟曲霉菌可分泌许多胞外蛋白酶，如碱性蛋白酶、金属蛋白酶、丝氨酸蛋白酶等，这些酶之间协同作用，造成组织的破坏和侵袭。在烟曲霉分泌的许多蛋白酶中，丝氨酸蛋白酶分泌量高，发挥重要的毒力作用。
近年的突破性研究表明，乳腺丝氨酸蛋白酶抑制（mammary serine proteinaseinhibitor,Maspin），在抑制肿瘤的侵袭和转移中发挥重要的作用。在前列腺癌晚期时局部炎症活化IκB激酶-α（IκB kinase-α，IKKα)，继而下调maspin蛋白的表达，促进肿瘤的侵袭和转移。侵袭性真菌性鼻-鼻窦炎病变向周围组织扩散的进程与恶性肿瘤的浸润和转移过程具有相似性，在侵袭性真菌性鼻窦炎时maspin是否也参与了病变的侵袭和扩散，其炎症组织中是否有IKKα的活化及其介导的maspin下调呢？而且真菌侵袭的主要致病毒素之一即为丝氨酸蛋白酶，而maspin恰恰是丝氨酸蛋白酶的抑制剂，探讨maspin与真菌侵袭的关系有重要的临床意义。
本课题共分3部分，将应用体外细胞学的方法，观察真菌诱导呼吸道上皮IKKα、maspin蛋白表达下调；建立侵袭性真菌性鼻窦炎动物模型；在活体动物实验的条件下，侵袭性真菌性鼻窦炎发病中maspin与IKKα的表达情况。
第一部分
烟曲霉菌诱导大鼠呼吸道上皮细胞IKKα调控maspin表达的初步研究
目的研究烟曲霉诱导呼吸道上皮细胞时是否存在IκB激酶-α（IκB kinase-α，IKKα）调控maspin蛋白表达下调。
方法体外培养大鼠呼吸道上皮细胞(REC)，制备灭活的烟曲霉丝(AFH)悬液诱导REC细胞，以PBS缓冲液为对照，采用RT-PCR技术检测REC中maspin基因的表达，并通过免疫细胞化学技术观察maspin蛋白及IKKα在REC的表达情况。进一步制备浓度依次增高的AFH1～3悬液诱导REC细胞，Western Blot技术检测REC的maspin和IKKα蛋白表达，应用方差分析和两变量的相关性分析统计实验数据。
结果RT-PCR检测显示AFH组与对照组的maspin蛋白的mRNA相对表达量分别为0.128±0.059和2.972±0.353，差异具有统计学意义（t=15.883P＜0.05）。免疫细胞化学结果显示，maspin蛋白在两组中均表达，其中AFH组呈弱阳性，而对照组呈中度阳性，综合计分两组差异有统计学意义（t=3.721P＜0.05）；IKKα显色，AFH组中度阳性，对照组弱阳性，综合计分差异有统计学意义（t=6.825P＜0.05）。Western Blot结果maspin与β-actin的灰度比值对照组为0.912±0.023，AFH1～3组分别为0.607±0.030、0.476±0.019、0.416±0.017，组间差异有统计学意义（F=281.91，P＜0.05），且任两组比较差异均有统计学意义；IKKα与β-actin灰度比值对照组为0.624±0.012，AFH1～3组分别为0.739±0.020、0.778±0.010、0.927±0.017，组间差异有统计学意义（F=200.91，P＜0.05），且任两组比较差异均有统计学意义。两变量相关性分析结果，maspin蛋白表达与IKKα蛋白表达呈负相关关系（r=－0.911，P＜0.05）。
结论烟曲霉诱导大鼠呼吸上皮细胞IKKα表达上调并伴有maspin蛋白表达下调。应用体外细胞学的方法，观察真菌诱导呼吸道上皮IKKα、maspin mRNA及其蛋白表达下调,以便探寻侵袭性真菌性鼻窦炎的侵袭机制。
第二部分
建立免疫抑制大鼠侵袭性真菌性鼻窦炎动物模型的实验研究
目的鉴于国内外侵袭性真菌性鼻-鼻窦炎动物模型报道极少的情况，本实验旨探索建立侵袭性真菌性鼻-鼻窦炎的动物模型的方法。
方法本实验按照临床侵袭性真菌性鼻窦炎的发病特点，以50只健康Wistar大鼠为实验动物，利用Merocel棉鼻腔填塞导致上颌窦窦口阻塞，应用环磷酰胺作为免疫抑制，烟曲霉孢子悬液鼻腔导入的方法建立侵袭性真菌性鼻窦炎的模型。实验分5组，分别是空白对照组、单纯鼻塞组、鼻塞加真菌组、鼻塞加免疫抑制组、侵袭性真菌鼻窦炎组。通过大体观察、检查白细胞数及血清半乳甘露聚糖、组织真菌培养和病理学检查来确定各组的发病情况。
结果大体观察结果表明，本实验中大鼠鼻塞后出现打喷嚏及挠鼻增多，体重增加速度减缓。鼻塞加免疫抑制组和侵袭性真菌鼻窦炎组第7天大鼠注射环磷酰胺后开始，大鼠开始出现毛发不荣，食量及饮水量减少，体重下降明显。侵袭性真菌鼻窦炎组第11天真菌导入后有血涕现象，结膜充血，2只大鼠气喘紫绀，1只偏瘫；而鼻塞加真菌组第11天真菌导入后体重未见下降，无结膜充血，无气喘紫绀现象。第18天前侵袭性真菌鼻窦炎组死亡2只，而其余各组在18天未见死亡。
实验检测结果表明，鼻塞对大鼠中性粒细胞计数无影响，鼻塞加真菌促使大鼠中性粒细胞计数显著增加，而鼻塞加腹腔注射环磷酰胺可导致大鼠中性粒细胞显著下降（低于0.5×109/L）。侵袭性真菌鼻窦炎组血清半乳甘露聚糖较其他组升高（P＜0.05）。侵袭性真菌鼻窦炎组的组织匀浆真菌培养阳性率鼻窦和肺组织分别为62.5%和25%，而其他组组织匀浆真菌培养为阴性结果。病理学检查见侵袭性真菌性鼻窦炎组病变可见大量菌丝，真菌侵犯血管，引起血管炎、血管栓塞、骨质破坏和组织坏死等，而其他组大鼠未见符合侵袭性真菌性鼻窦炎的病理改变的特征。
结论本实验探索了建模的程序、鼻塞及用免疫抑制药的时机，真菌侵入的条件，成功建立大鼠侵袭性真菌性鼻窦炎的动物模型。
第三部分
大鼠侵袭性真菌性鼻窦炎IKKα调控maspin蛋白表达的实验研究
目的本课题前期体外细胞学实验研究结果提示，呼吸道上皮细胞存在烟曲霉刺激下的IKKα诱导maspin mRNA及其蛋白表达下调。本实验旨在探讨在活体动物实验的条件下，侵袭性真菌性鼻窦炎发病中maspin与IKKα的表达情况。
方法建立大鼠侵袭性真菌性鼻窦炎动物模型，以空白对照组、单纯鼻塞组、鼻塞加真菌组、鼻塞加免疫抑制组大鼠为对照，通过组织真菌培养和病理学检查来检测各组的发病情况，应用ELISA法检测大鼠血清TNFα浓度，采用免疫组化染色技术、RT-PCR技术、Western Blot杂交技术，检测大鼠鼻窦粘膜组织maspin及IKKα表达的情况。
结果ELISA法检测大鼠血清TNFα浓度，组间比较方差分析F=26.78P<0.05，各组TNFα浓度不同。其中鼻塞加真菌组大鼠血清TNFα浓度最高，与其他组比较，差异具有统计学意义(P<0.05)。鼻塞加免疫抑制组和侵袭性真菌性鼻窦炎组血清TNFα浓度最低，与其他各组比较，差异具有统计学意义(P<0.05)。
免疫组化染色技术以染色强度结合阳性细胞数百分比综合计分，各组的maspin染色评分结果组间比较方差分析F=14.58(P<0.05)，组间差异具有统计学意义。其中侵袭性真菌鼻窦炎组评分最低，与其他各组比较，差异具有统计学意义(P<0.05)。IKKα染色评分结果组间比较方差分析F=18.80(P<0.05)，组间差异具有统计学意义。其中鼻塞加真菌组、侵袭性真菌性鼻窦炎组评分较高与分别与其他各组相比差异具有统计学意义(P<0.05）。
RT-PCR技术检测结果， maspin的mRNA相对表达量组间比较方差分析F=13.678(Ｐ<0.05)，可见各组maspin的mRNA相对表达量不同。其中侵袭性真菌鼻窦炎组最低，且与其他各组比较，差异显著具有统计学意义(P<0.05)。
Western Blot杂交技术结果maspin／β-actin灰度比值组间比较方差分析F=6.976(P<0.05)，组间差异具有统计学意义。其中侵袭性真菌鼻窦炎组maspin相对表达量最低，且与其他各组比较，差异显著具有统计学意义(P<0.05)。各组IKKα／β-actin灰度比值组间比较方差分析F=18.156(P<0.05)，组间差异具有统计学意义，鼻塞加真菌组和侵袭性真菌性鼻窦炎组IKKα蛋白相对表达量较高，和其他各组两两比较，差异均具有统计学意义(P<0.05)。
结论在体实验研究情况下，不存在IKKα活化--maspin下调的简单的线性关系。免疫功能正常的情况下，真菌侵袭IKKα活化但未见maspin下调；而在免疫功能缺陷的情况下真菌侵袭IKKα活化促进了maspin下调。
结论
1、体外细胞培养的条件下，大鼠呼吸道上皮存在烟曲霉介导的IKKα活化和maspin的表达下调；
2、按照临床侵袭性真菌性鼻窦炎的发病特点，借鉴细菌性鼻窦炎的成功经验，应用环磷酰胺为免疫抑制剂，在鼻塞的基础上真菌侵入，成功探索构建大鼠侵袭性真菌性鼻窦炎的动物模型的方法。
3、在体动物实验研究的条件下，免疫功能正常，烟曲霉刺激IKKα活化，未见maspin下调，而侵袭性真菌性鼻窦炎在免疫功能缺陷的情况下烟曲霉刺激IKKα活化促进了maspin下调。
4、本课题的创新之处在于首次探讨maspin蛋白及IKKα与侵袭真菌病发病的关系,揭示了IKKα活化介导的maspin蛋白下调是侵袭性真菌性鼻窦炎侵袭的重要的分子机制之一。
Invasive fungal rhinosinusitis (IFRS) is a serious disease with a high mortalityrate that has increased over the last decade due to the growing number ofimmunocompromised patients.To explore the pathogenesis of IFRS, there sections ofexperiments were constructed.
Section1A preliminary study on aspergillus fumigates-induced IKKαcontroled maspin protein expression in respiratory epithelial cells
Objective To study if the expression of maspin protein in respiratory epithelialcells was downregulated through IκB kinase-α (IKKα)-controlled mechanism in anaspergillus fumigates-induced model in rat.
Method Inactivated Aspergillus fumigatus hyphae (AFH) suspension was usedto induce respiratory epithelial cells (REC) cultured in vitro in rat, with PBS buffer ascontrol. By RT-PCR, the mRNA expression of maspin was quantified, and byimmunocytochemistry the expressions of maspin and IKKα in REC were observed.Furthermore AFH (from level1to level3) suspension was prepared to induce REC.Then Western blot hybridization techniques were used to detect the expression ofmaspin and IKKα protein. All data were processed by analysis of variance andtwo-variable correlation analysis.
Result By RT-PCR, a statistically significant (t=2.463, P <0.05) expression ofmaspin mRNA was found (0.128±0.059in AFH group vs2.972±0.353in controlgroup). By Immunocytochemistry, the difference of maspin protein color in differentgoups was shown statistically significant in integrated scoring (t=3.721, P <0.05)(weak positive in AFH group, moderately positive in control group). While in IKKαcolor study, the difference between the two groups was also statistically significant (t =6.825, P <0.05) in integrated scoring, with a moderate positive in AFH group andweak positive in control group. By Western Blot hybridization, Grayscale ratio ofmaspin and β-actin was0.912±0.023,in control group，0.607±0.030,0.476±0.019,0.416±0.017in AFH1~3groups，respectively, with a statistically significantdifference within the four groups (F=281.91, P <0.05); While Grayscale ratio ofIKKα and β-actin was0.624±0.012in control group,0.739±0.020,0.778±0.010,0.927±0.017, respectively, in AFH1~3groups; with a statistically significantdifference within the four groups (F=200.91，P＜0.05). Moreover, the differencebetween any two groups from both AFH group (including supgroup1,2and3) andcontrol group was statistically significant. Two-variable correlation analysis indicateda negative correlation between expression of maspin and IKKα (r=-0.911, P <0.05).
Conclusion Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.
Section2A novel model of invasive fungal rhinosinusitis in rats
Objective Invasive fungal rhinosinusitis (IFRS) is a life-threateninginflammatory disease that affects immunocompromised patients, but animal modelsof the disease are scarce. This study aimed to develop an IFRS model in neutropenicrats.
Method The model was established in three consecutive steps: unilateral nasalobstruction with a Merocel sponge, followed by the administration ofcyclophosphamide (CPA), and finally nasal inoculation with Aspergillus fumigatus.Fifty healthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. Hematology, histology, and mycology investigations wereperformed.
Result The changes in the rat absolute neutrophil counts (ANCs) werestatistically different across the groups. The administration of CPA decreased theANCs, whereas nasal obstruction with fungal inoculation increased the ANCs, andnasal obstruction did not change them. Histological examination of the rats in groupV revealed the hyphal invasion of sinus mucosa and bone, thrombosis, and tissueinfarction. No pathology indicative of IFRS was observed in the remaining groups.Positive rates of fungal culture in tissue homogenates from the maxillary sinus(62.5%) and lung (25%) were found in group V, whereas groups I, II, III and IVdemonstrated no fungal culture in the homogenates.
Conclusion A rat IFRS model was successfully developed through nasalobstruction, CPA-induced neutropenia, and fungal inoculation. The disease modelclosely mimics the pathophysiology of anthropic IFRS.
Section3Experimental Study on IKKα regulation of Maspin Expression inRats Invasive Fungal Rhinosinusitis
Objective The early study showed that induced by aspergillus fumigates, the ratrespiratory epithelia might upregulate the expression of IKKα with a downregulatedexpression of maspin protein.This study was aimed to explore the expression ofmaspin and IKKα in rats model of invasive fungal rhinosinusitis.
Method The rats model of invasive fungal rhinosinusitis were developed. Fiftyhealthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. By RT-PCR, the mRNA expression of maspin was quantified,and by immunohistochemical study the expressions of maspin and IKKα in thegroups were observed. Then Western blot hybridization techniques were used todetect the expression of maspin and IKKα protein. All data were processed byanalysis of variance.
Result By immunohistochemical study, the difference of maspin protein color indifferent goups was shown statistically significant in integrated scoring (F=14.58 P<0.05)(weak positive in group V, moderately positive in the remaining groups).While in IKKα color study, the difference between the five groups was alsostatistically significant (F=18.80P<0.05) in integrated scoring, with a moderatepositive in groups III and V, weak positive in the remaining groups.
By RT-PCR, a statistically significant (F=13.678P<0.05) expression of maspinmRNA was found and the expression in group V was lower than that in the remaininggroups (P<0.05).
By Western Blot hybridization, there was a statistically significant differencewithin the five groups in the grayscale ratio of maspin and β-actin (F=6.976P<0.05).The ratio in group V was lower than that in the remaining groups (P<0.05); WhileGrayscale ratio of IKKα and β-actin was a statistically significant difference in thefive groups (F=18.156P<0.05).The ratio in groups III and V were higher than that inthe remaining groups (P<0.05).
The results of invasive fungal sinusitis IKKα protein up expression, anddownregulation of maspin; stuffy nose with fungal group, IKKα protein proteinenhanced expression without maspin downregulated; the blank control group, simplystuffy nose group, stuffy nose with immunosuppression group did not see IKKαprotein enhanced expression and downregulation of maspin.
Conclusion IKKα activation does not cause maspin down in normal immunefunction, while in the case of the immunodeficiency, the activation of IKKα promoteddown of the maspin.
Conclusion
The innovation of this study was the discussion on relationship between IFRSand IKKα regulation of maspin expression.
1. Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.
2. A rat IFRS model was successfully developed through nasal obstruction,CPA-induced neutropenia, and fungal inoculation. The disease model closely mimics the pathophysiology of anthropic IFRS.
3. There is not simple linear relationship between upregulated IKKα anddownregulated maspin in vivo,which is different from it in vitro study. IKKαactivation does not cause maspin down in normal immune function, while in the caseof the immunodeficiency, the activation of IKKα promoted down of the maspin. Itmight be one of the molecular mechanisms of invasion mechanism in IFRS.
各种环境因素及宿主因素与真菌性鼻窦炎的发病有关，其中机体免疫功能状态与真菌性鼻窦炎的病理分型有关，当免疫功能不足时，易患侵袭性真菌性鼻窦炎。骨髓移植和器官移植的接受者、未经控制血糖的糖尿病患者和艾滋病感染者都是侵袭性鼻窦炎的高危人群。烟曲霉菌是真菌性鼻窦炎最常见条件致病菌，烟曲霉菌可分泌许多胞外蛋白酶，如碱性蛋白酶、金属蛋白酶、丝氨酸蛋白酶等，这些酶之间协同作用，造成组织的破坏和侵袭。在烟曲霉分泌的许多蛋白酶中，丝氨酸蛋白酶分泌量高，发挥重要的毒力作用。
近年的突破性研究表明，乳腺丝氨酸蛋白酶抑制（mammary serine proteinaseinhibitor,Maspin），在抑制肿瘤的侵袭和转移中发挥重要的作用。在前列腺癌晚期时局部炎症活化IκB激酶-α（IκB kinase-α，IKKα)，继而下调maspin蛋白的表达，促进肿瘤的侵袭和转移。侵袭性真菌性鼻-鼻窦炎病变向周围组织扩散的进程与恶性肿瘤的浸润和转移过程具有相似性，在侵袭性真菌性鼻窦炎时maspin是否也参与了病变的侵袭和扩散，其炎症组织中是否有IKKα的活化及其介导的maspin下调呢？而且真菌侵袭的主要致病毒素之一即为丝氨酸蛋白酶，而maspin恰恰是丝氨酸蛋白酶的抑制剂，探讨maspin与真菌侵袭的关系有重要的临床意义。
本课题共分3部分，将应用体外细胞学的方法，观察真菌诱导呼吸道上皮IKKα、maspin蛋白表达下调；建立侵袭性真菌性鼻窦炎动物模型；在活体动物实验的条件下，侵袭性真菌性鼻窦炎发病中maspin与IKKα的表达情况。
第一部分
烟曲霉菌诱导大鼠呼吸道上皮细胞IKKα调控maspin表达的初步研究
目的研究烟曲霉诱导呼吸道上皮细胞时是否存在IκB激酶-α（IκB kinase-α，IKKα）调控maspin蛋白表达下调。
方法体外培养大鼠呼吸道上皮细胞(REC)，制备灭活的烟曲霉丝(AFH)悬液诱导REC细胞，以PBS缓冲液为对照，采用RT-PCR技术检测REC中maspin基因的表达，并通过免疫细胞化学技术观察maspin蛋白及IKKα在REC的表达情况。进一步制备浓度依次增高的AFH1～3悬液诱导REC细胞，Western Blot技术检测REC的maspin和IKKα蛋白表达，应用方差分析和两变量的相关性分析统计实验数据。
结果RT-PCR检测显示AFH组与对照组的maspin蛋白的mRNA相对表达量分别为0.128±0.059和2.972±0.353，差异具有统计学意义（t=15.883P＜0.05）。免疫细胞化学结果显示，maspin蛋白在两组中均表达，其中AFH组呈弱阳性，而对照组呈中度阳性，综合计分两组差异有统计学意义（t=3.721P＜0.05）；IKKα显色，AFH组中度阳性，对照组弱阳性，综合计分差异有统计学意义（t=6.825P＜0.05）。Western Blot结果maspin与β-actin的灰度比值对照组为0.912±0.023，AFH1～3组分别为0.607±0.030、0.476±0.019、0.416±0.017，组间差异有统计学意义（F=281.91，P＜0.05），且任两组比较差异均有统计学意义；IKKα与β-actin灰度比值对照组为0.624±0.012，AFH1～3组分别为0.739±0.020、0.778±0.010、0.927±0.017，组间差异有统计学意义（F=200.91，P＜0.05），且任两组比较差异均有统计学意义。两变量相关性分析结果，maspin蛋白表达与IKKα蛋白表达呈负相关关系（r=－0.911，P＜0.05）。
结论烟曲霉诱导大鼠呼吸上皮细胞IKKα表达上调并伴有maspin蛋白表达下调。应用体外细胞学的方法，观察真菌诱导呼吸道上皮IKKα、maspin mRNA及其蛋白表达下调,以便探寻侵袭性真菌性鼻窦炎的侵袭机制。
第二部分
建立免疫抑制大鼠侵袭性真菌性鼻窦炎动物模型的实验研究
目的鉴于国内外侵袭性真菌性鼻-鼻窦炎动物模型报道极少的情况，本实验旨探索建立侵袭性真菌性鼻-鼻窦炎的动物模型的方法。
方法本实验按照临床侵袭性真菌性鼻窦炎的发病特点，以50只健康Wistar大鼠为实验动物，利用Merocel棉鼻腔填塞导致上颌窦窦口阻塞，应用环磷酰胺作为免疫抑制，烟曲霉孢子悬液鼻腔导入的方法建立侵袭性真菌性鼻窦炎的模型。实验分5组，分别是空白对照组、单纯鼻塞组、鼻塞加真菌组、鼻塞加免疫抑制组、侵袭性真菌鼻窦炎组。通过大体观察、检查白细胞数及血清半乳甘露聚糖、组织真菌培养和病理学检查来确定各组的发病情况。
结果大体观察结果表明，本实验中大鼠鼻塞后出现打喷嚏及挠鼻增多，体重增加速度减缓。鼻塞加免疫抑制组和侵袭性真菌鼻窦炎组第7天大鼠注射环磷酰胺后开始，大鼠开始出现毛发不荣，食量及饮水量减少，体重下降明显。侵袭性真菌鼻窦炎组第11天真菌导入后有血涕现象，结膜充血，2只大鼠气喘紫绀，1只偏瘫；而鼻塞加真菌组第11天真菌导入后体重未见下降，无结膜充血，无气喘紫绀现象。第18天前侵袭性真菌鼻窦炎组死亡2只，而其余各组在18天未见死亡。
实验检测结果表明，鼻塞对大鼠中性粒细胞计数无影响，鼻塞加真菌促使大鼠中性粒细胞计数显著增加，而鼻塞加腹腔注射环磷酰胺可导致大鼠中性粒细胞显著下降（低于0.5×109/L）。侵袭性真菌鼻窦炎组血清半乳甘露聚糖较其他组升高（P＜0.05）。侵袭性真菌鼻窦炎组的组织匀浆真菌培养阳性率鼻窦和肺组织分别为62.5%和25%，而其他组组织匀浆真菌培养为阴性结果。病理学检查见侵袭性真菌性鼻窦炎组病变可见大量菌丝，真菌侵犯血管，引起血管炎、血管栓塞、骨质破坏和组织坏死等，而其他组大鼠未见符合侵袭性真菌性鼻窦炎的病理改变的特征。
结论本实验探索了建模的程序、鼻塞及用免疫抑制药的时机，真菌侵入的条件，成功建立大鼠侵袭性真菌性鼻窦炎的动物模型。
第三部分
大鼠侵袭性真菌性鼻窦炎IKKα调控maspin蛋白表达的实验研究
目的本课题前期体外细胞学实验研究结果提示，呼吸道上皮细胞存在烟曲霉刺激下的IKKα诱导maspin mRNA及其蛋白表达下调。本实验旨在探讨在活体动物实验的条件下，侵袭性真菌性鼻窦炎发病中maspin与IKKα的表达情况。
方法建立大鼠侵袭性真菌性鼻窦炎动物模型，以空白对照组、单纯鼻塞组、鼻塞加真菌组、鼻塞加免疫抑制组大鼠为对照，通过组织真菌培养和病理学检查来检测各组的发病情况，应用ELISA法检测大鼠血清TNFα浓度，采用免疫组化染色技术、RT-PCR技术、Western Blot杂交技术，检测大鼠鼻窦粘膜组织maspin及IKKα表达的情况。
结果ELISA法检测大鼠血清TNFα浓度，组间比较方差分析F=26.78P<0.05，各组TNFα浓度不同。其中鼻塞加真菌组大鼠血清TNFα浓度最高，与其他组比较，差异具有统计学意义(P<0.05)。鼻塞加免疫抑制组和侵袭性真菌性鼻窦炎组血清TNFα浓度最低，与其他各组比较，差异具有统计学意义(P<0.05)。
免疫组化染色技术以染色强度结合阳性细胞数百分比综合计分，各组的maspin染色评分结果组间比较方差分析F=14.58(P<0.05)，组间差异具有统计学意义。其中侵袭性真菌鼻窦炎组评分最低，与其他各组比较，差异具有统计学意义(P<0.05)。IKKα染色评分结果组间比较方差分析F=18.80(P<0.05)，组间差异具有统计学意义。其中鼻塞加真菌组、侵袭性真菌性鼻窦炎组评分较高与分别与其他各组相比差异具有统计学意义(P<0.05）。
RT-PCR技术检测结果， maspin的mRNA相对表达量组间比较方差分析F=13.678(Ｐ<0.05)，可见各组maspin的mRNA相对表达量不同。其中侵袭性真菌鼻窦炎组最低，且与其他各组比较，差异显著具有统计学意义(P<0.05)。
Western Blot杂交技术结果maspin／β-actin灰度比值组间比较方差分析F=6.976(P<0.05)，组间差异具有统计学意义。其中侵袭性真菌鼻窦炎组maspin相对表达量最低，且与其他各组比较，差异显著具有统计学意义(P<0.05)。各组IKKα／β-actin灰度比值组间比较方差分析F=18.156(P<0.05)，组间差异具有统计学意义，鼻塞加真菌组和侵袭性真菌性鼻窦炎组IKKα蛋白相对表达量较高，和其他各组两两比较，差异均具有统计学意义(P<0.05)。
结论在体实验研究情况下，不存在IKKα活化--maspin下调的简单的线性关系。免疫功能正常的情况下，真菌侵袭IKKα活化但未见maspin下调；而在免疫功能缺陷的情况下真菌侵袭IKKα活化促进了maspin下调。
结论
1、体外细胞培养的条件下，大鼠呼吸道上皮存在烟曲霉介导的IKKα活化和maspin的表达下调；
2、按照临床侵袭性真菌性鼻窦炎的发病特点，借鉴细菌性鼻窦炎的成功经验，应用环磷酰胺为免疫抑制剂，在鼻塞的基础上真菌侵入，成功探索构建大鼠侵袭性真菌性鼻窦炎的动物模型的方法。
3、在体动物实验研究的条件下，免疫功能正常，烟曲霉刺激IKKα活化，未见maspin下调，而侵袭性真菌性鼻窦炎在免疫功能缺陷的情况下烟曲霉刺激IKKα活化促进了maspin下调。
4、本课题的创新之处在于首次探讨maspin蛋白及IKKα与侵袭真菌病发病的关系,揭示了IKKα活化介导的maspin蛋白下调是侵袭性真菌性鼻窦炎侵袭的重要的分子机制之一。
Invasive fungal rhinosinusitis (IFRS) is a serious disease with a high mortalityrate that has increased over the last decade due to the growing number ofimmunocompromised patients.To explore the pathogenesis of IFRS, there sections ofexperiments were constructed.
Section1A preliminary study on aspergillus fumigates-induced IKKαcontroled maspin protein expression in respiratory epithelial cells
Objective To study if the expression of maspin protein in respiratory epithelialcells was downregulated through IκB kinase-α (IKKα)-controlled mechanism in anaspergillus fumigates-induced model in rat.
Method Inactivated Aspergillus fumigatus hyphae (AFH) suspension was usedto induce respiratory epithelial cells (REC) cultured in vitro in rat, with PBS buffer ascontrol. By RT-PCR, the mRNA expression of maspin was quantified, and byimmunocytochemistry the expressions of maspin and IKKα in REC were observed.Furthermore AFH (from level1to level3) suspension was prepared to induce REC.Then Western blot hybridization techniques were used to detect the expression ofmaspin and IKKα protein. All data were processed by analysis of variance andtwo-variable correlation analysis.
Result By RT-PCR, a statistically significant (t=2.463, P <0.05) expression ofmaspin mRNA was found (0.128±0.059in AFH group vs2.972±0.353in controlgroup). By Immunocytochemistry, the difference of maspin protein color in differentgoups was shown statistically significant in integrated scoring (t=3.721, P <0.05)(weak positive in AFH group, moderately positive in control group). While in IKKαcolor study, the difference between the two groups was also statistically significant (t =6.825, P <0.05) in integrated scoring, with a moderate positive in AFH group andweak positive in control group. By Western Blot hybridization, Grayscale ratio ofmaspin and β-actin was0.912±0.023,in control group，0.607±0.030,0.476±0.019,0.416±0.017in AFH1~3groups，respectively, with a statistically significantdifference within the four groups (F=281.91, P <0.05); While Grayscale ratio ofIKKα and β-actin was0.624±0.012in control group,0.739±0.020,0.778±0.010,0.927±0.017, respectively, in AFH1~3groups; with a statistically significantdifference within the four groups (F=200.91，P＜0.05). Moreover, the differencebetween any two groups from both AFH group (including supgroup1,2and3) andcontrol group was statistically significant. Two-variable correlation analysis indicateda negative correlation between expression of maspin and IKKα (r=-0.911, P <0.05).
Conclusion Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.
Section2A novel model of invasive fungal rhinosinusitis in rats
Objective Invasive fungal rhinosinusitis (IFRS) is a life-threateninginflammatory disease that affects immunocompromised patients, but animal modelsof the disease are scarce. This study aimed to develop an IFRS model in neutropenicrats.
Method The model was established in three consecutive steps: unilateral nasalobstruction with a Merocel sponge, followed by the administration ofcyclophosphamide (CPA), and finally nasal inoculation with Aspergillus fumigatus.Fifty healthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. Hematology, histology, and mycology investigations wereperformed.
Result The changes in the rat absolute neutrophil counts (ANCs) werestatistically different across the groups. The administration of CPA decreased theANCs, whereas nasal obstruction with fungal inoculation increased the ANCs, andnasal obstruction did not change them. Histological examination of the rats in groupV revealed the hyphal invasion of sinus mucosa and bone, thrombosis, and tissueinfarction. No pathology indicative of IFRS was observed in the remaining groups.Positive rates of fungal culture in tissue homogenates from the maxillary sinus(62.5%) and lung (25%) were found in group V, whereas groups I, II, III and IVdemonstrated no fungal culture in the homogenates.
Conclusion A rat IFRS model was successfully developed through nasalobstruction, CPA-induced neutropenia, and fungal inoculation. The disease modelclosely mimics the pathophysiology of anthropic IFRS.
Section3Experimental Study on IKKα regulation of Maspin Expression inRats Invasive Fungal Rhinosinusitis
Objective The early study showed that induced by aspergillus fumigates, the ratrespiratory epithelia might upregulate the expression of IKKα with a downregulatedexpression of maspin protein.This study was aimed to explore the expression ofmaspin and IKKα in rats model of invasive fungal rhinosinusitis.
Method The rats model of invasive fungal rhinosinusitis were developed. Fiftyhealthy Wistar rats were randomly divided into five groups, with group I as thecontrols, group II undergoing unilateral nasal obstruction alone, group III nasalobstruction with fungal inoculation, group IV nasal obstruction with theadministration of CPA, and group V nasal obstruction with the administration of CPAand fungal inoculation. By RT-PCR, the mRNA expression of maspin was quantified,and by immunohistochemical study the expressions of maspin and IKKα in thegroups were observed. Then Western blot hybridization techniques were used todetect the expression of maspin and IKKα protein. All data were processed byanalysis of variance.
Result By immunohistochemical study, the difference of maspin protein color indifferent goups was shown statistically significant in integrated scoring (F=14.58 P<0.05)(weak positive in group V, moderately positive in the remaining groups).While in IKKα color study, the difference between the five groups was alsostatistically significant (F=18.80P<0.05) in integrated scoring, with a moderatepositive in groups III and V, weak positive in the remaining groups.
By RT-PCR, a statistically significant (F=13.678P<0.05) expression of maspinmRNA was found and the expression in group V was lower than that in the remaininggroups (P<0.05).
By Western Blot hybridization, there was a statistically significant differencewithin the five groups in the grayscale ratio of maspin and β-actin (F=6.976P<0.05).The ratio in group V was lower than that in the remaining groups (P<0.05); WhileGrayscale ratio of IKKα and β-actin was a statistically significant difference in thefive groups (F=18.156P<0.05).The ratio in groups III and V were higher than that inthe remaining groups (P<0.05).
The results of invasive fungal sinusitis IKKα protein up expression, anddownregulation of maspin; stuffy nose with fungal group, IKKα protein proteinenhanced expression without maspin downregulated; the blank control group, simplystuffy nose group, stuffy nose with immunosuppression group did not see IKKαprotein enhanced expression and downregulation of maspin.
Conclusion IKKα activation does not cause maspin down in normal immunefunction, while in the case of the immunodeficiency, the activation of IKKα promoteddown of the maspin.
Conclusion
The innovation of this study was the discussion on relationship between IFRSand IKKα regulation of maspin expression.
1. Induced by aspergillus fumigates, the rat respiratory epithelia mightupregulate the expression of IKKα with a downregulated expression of maspinprotein.
2. A rat IFRS model was successfully developed through nasal obstruction,CPA-induced neutropenia, and fungal inoculation. The disease model closely mimics the pathophysiology of anthropic IFRS.
3. There is not simple linear relationship between upregulated IKKα anddownregulated maspin in vivo,which is different from it in vitro study. IKKαactivation does not cause maspin down in normal immune function, while in the caseof the immunodeficiency, the activation of IKKα promoted down of the maspin. Itmight be one of the molecular mechanisms of invasion mechanism in IFRS.
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