中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
5期
412-418
,共7页
抗真菌药物/治疗%伏立康唑%纳米药物%银/药理学%烟曲霉菌%角膜炎%近交系C57BL小鼠
抗真菌藥物/治療%伏立康唑%納米藥物%銀/藥理學%煙麯黴菌%角膜炎%近交繫C57BL小鼠
항진균약물/치료%복립강서%납미약물%은/약이학%연곡매균%각막염%근교계C57BL소서
Antifungal agents/therapeutic use%Voriconazole%Nanomedicine%Silver/pharmacology%Aspergillus fumigatus%Keratitis%Mice,inbred C57BL
背景 伏立康唑具有良好的抗真菌活性,但目前临床上所用剂型的生物利用度并不理想.改良伏立康唑眼用剂型并提高药物的生物利用度对于改善真菌性角膜炎的预后,减少药物不良反应具有重要意义. 目的 研究伏立康唑纳米银/聚合物复合材料在治疗真菌性角膜炎过程中的缓释作用、药物疗效和安全性.方法 选择6～8周龄雌性SPF级C57BL/6小鼠210只,其中30只用于药物的生物相容性验证,180 只小鼠用于疗效观察,均以左眼作为实验眼.药物的生物相容性研究中将30只小鼠按照随机数字表法分为正常对照组、纳米银/羧基石墨烯/壳聚糖季铵盐复合物(CS-ETA/Ag/GO)组和包被伏立康唑的纳米银/羧基石墨烯/壳聚糖季铵盐复合物(CS-ETA/Ag/GO/Vor)组,分别将自行构建的CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜贴敷于小鼠左眼角膜,分别于贴敷后1d、7d行角膜组织的常规组织病理学检查.药物疗效观察实验中,应用随机数字表法将180只小鼠随机分为模型对照组、CS-ETA/Ag/GO组和CS-ETA/Ag/GO/Vor组.3个组小鼠均用33G注射器针头于左眼角膜基质内注射5×107 CFU/ml烟曲霉菌悬浮液2.0μl建立烟曲霉菌性角膜炎小鼠模型,CS-ETA/Ag/GO组和CS-ETA/Ag/GO/Vor组小鼠分别在模型眼角膜贴敷相应的药膜.分别于造模后第1、3、5、7天用裂隙灯显微镜对术眼角膜炎症进行评分及组织病理学检查,采用真菌平板并计算角膜载菌量;采用real-time PCR法检测角膜组织中炎性因子TNF-α mRNA和IL-1β mRNA的相对表达量.结果 CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜贴敷于正常小鼠角膜后1～7d,经组织病理学检查均未见异常.CS-ETA/Ag/GO/Vor组各时间点小鼠的角膜炎症评分明显低于模型对照组和CS-ETA/Ag/GO组,3个组间和不同时间点角膜炎症评分的总体差异均有统计学意义(F分组=237.29,P=0.00;F时间=260.33,P=0.00).角膜组织病理学检查显示,模型对照组小鼠角膜水肿,部分角膜基质溶解坏死,第7天部分小鼠角膜穿孔.CS-ETA/Ag/GO组于造模后角膜炎症表现轻于模型对照组,随造模后时间的延长,角膜炎症逐渐减轻,而各个时间点CS-ETA/Ag/GO/Vor组小鼠角膜炎症反应最为轻微,造模后第7天角膜炎症接近痊愈.造模后CS-ETA/Ag/GO/Vor组小鼠角膜载菌量最低,且随着时间延长,载菌量逐渐减少,各组不同时间点角膜载菌量的总体差异比较差异均有统计学意义(F分组=113.15,P=0.00;F时间=126.52,P=0.00).CS-ETA/Ag/GO/Vor组小鼠角膜中IL-1β mRNA和TNF-α mRNA相对表达量均明显低于模型对照组和CS-ETA/Ag/GO组,其相对表达量均于造模后第5天达峰,第7天明显下降.3个组间小鼠在造模后不同时间点角膜中相对表达量的差异均有统计学意义(IL-1β:F分组=189.90,P=0.00;F时间=108.56,P=0.00;TNF-α:F分组=82.55,P=0.00;F时间=44.36,P=0.00). 结论 CS-ETA/Ag/GO/Vor药物缓释膜通过伏立康唑和银离子的协同作用共同抑制真菌活性,减轻真菌诱导的角膜炎症反应.CS-ETA/Ag/GO/Vor药物缓释膜贴敷于角膜后生物相容性好,未发现角膜组织的毒性反应.
揹景 伏立康唑具有良好的抗真菌活性,但目前臨床上所用劑型的生物利用度併不理想.改良伏立康唑眼用劑型併提高藥物的生物利用度對于改善真菌性角膜炎的預後,減少藥物不良反應具有重要意義. 目的 研究伏立康唑納米銀/聚閤物複閤材料在治療真菌性角膜炎過程中的緩釋作用、藥物療效和安全性.方法 選擇6～8週齡雌性SPF級C57BL/6小鼠210隻,其中30隻用于藥物的生物相容性驗證,180 隻小鼠用于療效觀察,均以左眼作為實驗眼.藥物的生物相容性研究中將30隻小鼠按照隨機數字錶法分為正常對照組、納米銀/羧基石墨烯/殼聚糖季銨鹽複閤物(CS-ETA/Ag/GO)組和包被伏立康唑的納米銀/羧基石墨烯/殼聚糖季銨鹽複閤物(CS-ETA/Ag/GO/Vor)組,分彆將自行構建的CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜貼敷于小鼠左眼角膜,分彆于貼敷後1d、7d行角膜組織的常規組織病理學檢查.藥物療效觀察實驗中,應用隨機數字錶法將180隻小鼠隨機分為模型對照組、CS-ETA/Ag/GO組和CS-ETA/Ag/GO/Vor組.3箇組小鼠均用33G註射器針頭于左眼角膜基質內註射5×107 CFU/ml煙麯黴菌懸浮液2.0μl建立煙麯黴菌性角膜炎小鼠模型,CS-ETA/Ag/GO組和CS-ETA/Ag/GO/Vor組小鼠分彆在模型眼角膜貼敷相應的藥膜.分彆于造模後第1、3、5、7天用裂隙燈顯微鏡對術眼角膜炎癥進行評分及組織病理學檢查,採用真菌平闆併計算角膜載菌量;採用real-time PCR法檢測角膜組織中炎性因子TNF-α mRNA和IL-1β mRNA的相對錶達量.結果 CS-ETA/Ag/GO膜和CS-ETA/Ag/GO/Vor膜貼敷于正常小鼠角膜後1～7d,經組織病理學檢查均未見異常.CS-ETA/Ag/GO/Vor組各時間點小鼠的角膜炎癥評分明顯低于模型對照組和CS-ETA/Ag/GO組,3箇組間和不同時間點角膜炎癥評分的總體差異均有統計學意義(F分組=237.29,P=0.00;F時間=260.33,P=0.00).角膜組織病理學檢查顯示,模型對照組小鼠角膜水腫,部分角膜基質溶解壞死,第7天部分小鼠角膜穿孔.CS-ETA/Ag/GO組于造模後角膜炎癥錶現輕于模型對照組,隨造模後時間的延長,角膜炎癥逐漸減輕,而各箇時間點CS-ETA/Ag/GO/Vor組小鼠角膜炎癥反應最為輕微,造模後第7天角膜炎癥接近痊愈.造模後CS-ETA/Ag/GO/Vor組小鼠角膜載菌量最低,且隨著時間延長,載菌量逐漸減少,各組不同時間點角膜載菌量的總體差異比較差異均有統計學意義(F分組=113.15,P=0.00;F時間=126.52,P=0.00).CS-ETA/Ag/GO/Vor組小鼠角膜中IL-1β mRNA和TNF-α mRNA相對錶達量均明顯低于模型對照組和CS-ETA/Ag/GO組,其相對錶達量均于造模後第5天達峰,第7天明顯下降.3箇組間小鼠在造模後不同時間點角膜中相對錶達量的差異均有統計學意義(IL-1β:F分組=189.90,P=0.00;F時間=108.56,P=0.00;TNF-α:F分組=82.55,P=0.00;F時間=44.36,P=0.00). 結論 CS-ETA/Ag/GO/Vor藥物緩釋膜通過伏立康唑和銀離子的協同作用共同抑製真菌活性,減輕真菌誘導的角膜炎癥反應.CS-ETA/Ag/GO/Vor藥物緩釋膜貼敷于角膜後生物相容性好,未髮現角膜組織的毒性反應.
배경 복립강서구유량호적항진균활성,단목전림상상소용제형적생물이용도병불이상.개량복립강서안용제형병제고약물적생물이용도대우개선진균성각막염적예후,감소약물불량반응구유중요의의. 목적 연구복립강서납미은/취합물복합재료재치료진균성각막염과정중적완석작용、약물료효화안전성.방법 선택6～8주령자성SPF급C57BL/6소서210지,기중30지용우약물적생물상용성험증,180 지소서용우료효관찰,균이좌안작위실험안.약물적생물상용성연구중장30지소서안조수궤수자표법분위정상대조조、납미은/최기석묵희/각취당계안염복합물(CS-ETA/Ag/GO)조화포피복립강서적납미은/최기석묵희/각취당계안염복합물(CS-ETA/Ag/GO/Vor)조,분별장자행구건적CS-ETA/Ag/GO막화CS-ETA/Ag/GO/Vor막첩부우소서좌안각막,분별우첩부후1d、7d행각막조직적상규조직병이학검사.약물료효관찰실험중,응용수궤수자표법장180지소서수궤분위모형대조조、CS-ETA/Ag/GO조화CS-ETA/Ag/GO/Vor조.3개조소서균용33G주사기침두우좌안각막기질내주사5×107 CFU/ml연곡매균현부액2.0μl건립연곡매균성각막염소서모형,CS-ETA/Ag/GO조화CS-ETA/Ag/GO/Vor조소서분별재모형안각막첩부상응적약막.분별우조모후제1、3、5、7천용렬극등현미경대술안각막염증진행평분급조직병이학검사,채용진균평판병계산각막재균량;채용real-time PCR법검측각막조직중염성인자TNF-α mRNA화IL-1β mRNA적상대표체량.결과 CS-ETA/Ag/GO막화CS-ETA/Ag/GO/Vor막첩부우정상소서각막후1～7d,경조직병이학검사균미견이상.CS-ETA/Ag/GO/Vor조각시간점소서적각막염증평분명현저우모형대조조화CS-ETA/Ag/GO조,3개조간화불동시간점각막염증평분적총체차이균유통계학의의(F분조=237.29,P=0.00;F시간=260.33,P=0.00).각막조직병이학검사현시,모형대조조소서각막수종,부분각막기질용해배사,제7천부분소서각막천공.CS-ETA/Ag/GO조우조모후각막염증표현경우모형대조조,수조모후시간적연장,각막염증축점감경,이각개시간점CS-ETA/Ag/GO/Vor조소서각막염증반응최위경미,조모후제7천각막염증접근전유.조모후CS-ETA/Ag/GO/Vor조소서각막재균량최저,차수착시간연장,재균량축점감소,각조불동시간점각막재균량적총체차이비교차이균유통계학의의(F분조=113.15,P=0.00;F시간=126.52,P=0.00).CS-ETA/Ag/GO/Vor조소서각막중IL-1β mRNA화TNF-α mRNA상대표체량균명현저우모형대조조화CS-ETA/Ag/GO조,기상대표체량균우조모후제5천체봉,제7천명현하강.3개조간소서재조모후불동시간점각막중상대표체량적차이균유통계학의의(IL-1β:F분조=189.90,P=0.00;F시간=108.56,P=0.00;TNF-α:F분조=82.55,P=0.00;F시간=44.36,P=0.00). 결론 CS-ETA/Ag/GO/Vor약물완석막통과복립강서화은리자적협동작용공동억제진균활성,감경진균유도적각막염증반응.CS-ETA/Ag/GO/Vor약물완석막첩부우각막후생물상용성호,미발현각막조직적독성반응.
Background Voriconazole is the traditionally used antifungal agent,but its ophthalmic form is unsatisfactory.A novel ophthalmic drug delivery system with biomedical devices may be of promising for the prognosis of fungal keratitis.Objective This study was to investigate the sustained release,therapeutic effect and biocompatibility of effect and quaternized chitosan functionalized with carboxylated graphene and nano-silver and voriconazole (CS-ETA/Ag/GO/Vor) for fungal keratitis.Methods This study complied with the Regulations for the Administration of Affair Concerning Experimental Animals of State Science and Technology Commission.Two hundred and ten SPF female C57BL/6 mice were selected with the age 6-8 weeks for the biocompatibility experiment (30 mice) and therapeutic observation of CS-ETA/Ag/GO/Vor (180 mice).CS-ETA/Ag/GO and CS-ETA/Ag/GO/Vor were attached on the normal corneas of mice and compared with the normal mice to assess the histopathological changes.Aspergillus fumigatus-infected mouse models were established in the left eyes of 180 mice by intrastromally injection of 2.0 μl Aspergillus fumigatus suspension with the density of 5 × 107 CFU/ml,then the mice were randomized into the model control group,CS-ETA/Ag/GO group and CS-ETA/Ag/GO/Vor group,and the corresponding membrane were attached the central corneas in different groups.In 1 day,3,5,7 days after modeling,the corneas were examined under the slit lamp microscope and scored,and corneal sections were prepared for the histopathological examination.Fungal activity was confirmed by plate counts,and real-time PCR was employed to assay the relative expressions of interleukin-1β (IL-1β) mRNA and tumor necrosis factor-α (TNF-α) mRNA in the corneas.Results No morphological abnormality was seen in the corneas in the normal control group,CS-ETA/Ag/GO group and CSETA/Ag/GO/Vor group.Corneal inflammatory score was significantly lower in the CS-ETA/Ag/GO/Vor group in various time points,with a significant differences among the groups and time points (Fgroup =237.29,P=0.00;Ftime =260.33,P=0.00).The edema,necrosis or perforation of cornea were seen in the model control group,and slighter inflammatory response in the CS-ETA/Ag/GO group,and corneal edema was gradually disappear in the CS-ETA/Ag/GO/Vor group.The corneal fungal loads were highest in the model control group and lowest in the CS-ETA/Ag/GO/V or group,with significant differences among the three groups and various time points (Fgroup =113.15,P =0.00;Ftime =126.52,P=0.00).The relative expressions of IL-1β mRNA and TNF-α mRNA in the corneas peaked in the fifth day after modeling in all of the three groups,and the expression levels of IL-1β mRNA and TNF-α mRNA in the corneas were lowest in the CS-ETA/Ag/GO/Vor group,showing significant differences among the groups and time points (IL-1β:Fgroup =189.90,P =0.00;Ftime =108.56,P =0.00;TNF-α:Fgroup =82.55,P =0.00;Ftime =44.36,P =0.00).Conclusions CS-ETA/Ag/GO/Vor delivery system plays an anti-fungal activity in fungal keratitis by the synergistic effect of voriconazole and Ag+.In addition,CS-ETA/Ag/GO/Vor appears to have a good safety after topical application.
