大鼠深Ⅱ度烫伤合并创面感染模型的构建

目的建立一种稳定性高、重复性好的深Ⅱ度大鼠烫伤合并创面感染的动物模型,用于烫伤合并创面感染的修复及治疗的研究。方法选取40只SD大鼠,随机分成5组,采用自制烫伤仪(蒸汽温度94℃)对大鼠皮肤进行烫伤,致伤时间点分别为4、6、8、10和12 s(每只大鼠脊柱两侧分别致伤2个相同创面),烫伤24 h后观察创面组织学变化,分析形成深Ⅱ度烫伤创面的最佳致伤时间。选取30只SD大鼠,随机分为3组,每组10只,采用最佳致伤时间形成深Ⅱ度烫伤创面,3组大鼠一侧创面分别接种浓度为1×10~7、1×10~8和1×10~(9 )CFU/mL铜绿假单胞菌菌液0.3 mL,另一侧均接种等体积生理盐水作为对照,接种24 h后观察大鼠创面一般状况,采用HE染色对创面进行染色并观察炎症反应,分别于接种1、2、4、7和14 d后检测痂下细菌数量,并记录大鼠创面愈合时间。结果(1)组织病理学结果显示,致伤时间为8 s是形成深Ⅱ度创面的最佳致伤时间。(2)大鼠深Ⅱ度烫伤创面接种1×10~8、1×10~(9 )CFU/mL细菌后均有明显的炎性细胞浸润,后者痂下炎性浸润更明显。烫伤创面接种1×10~(9 )CFU/mL细菌14 d内,痂下细菌含量均高于1×10~(5 )CFU/g,且随着时间延长细菌含量呈持续上升趋势。大鼠创面愈合时间为(21.4±2.4)d,较生理盐水对照组创面愈合时间(18.4±1.7)d明显延长(t=2.72,P0.05)。结论温度94℃,烫伤时间8 s为形成深Ⅱ度烫伤的最佳致伤时间;创面接种浓度为1×10~(9 )CFU/mL铜绿假单胞菌可建立烫伤深度一致、重复性高、稳定性好的SD大鼠深Ⅱ度烫伤合并感染模型。     

激活Hacat细胞表面Toll样受体促进创面愈合的机制

目的：细胞表面Toll样受体对激发先天性免疫、抵抗微生物感染有重要作用。最近研究发现激活细胞表面Toll样受体不仅可以预防微生物感染，还可以促进组织再生。实验通过脂多糖激活体外培养的角质细胞株Hacat细胞表面Toll样受体，观察其促进创面愈合的可能机制。 方法：实验于2007-07在解放军第四军医大学西京医院烧伤与皮肤外科完成。①实验分组及方法：角质细胞株Hacat由解放军第四军医大学西京医院皮肤科馈赠；脂多糖为Sigma公司产品；小鼠抗入Toll样受体2单克隆抗体、兔抗人Toll样受体4多克隆抗体购于ebioscience公司。体外培养Hacat细胞，根据脂多糖（500ng/L）与Hacat细胞共培养24，48，72h将细胞随机分为脂多糖作用24h组、脂多糖作用48h组、脂多糖作用72h组，另设对照组，只加溶剂二甲基亚枫。②实验评估：蛋自免疫印迹技术检测脂多糖对Hacat细胞Toll样受体2、Toll样受体4及核因子KB表达的影响：荧光定量聚合酶链反应检测脂多糖作用后Hacat细胞内基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子的表达。 结果：光学显微镜下正常入皮肤表皮及角质细胞Toll样受体2、Toll样受体4表达呈蓝黑色：脂多糖作用于Hacat细胞24，48，72h后Toll样受体2、Toll样受体4、核因子-κB表达均高于对照组（P〈0．01）。与对照组相比，其他3组基质金属蛋白酶3及血管内皮生长因子表达均增高（P〈0．01，P〈0．05）。脂多糖作用24h组基质金属蛋白酶9表达与对照组差异无显著性（P〉0．05），脂多糖作用48，72h组与对照组比较，差异显著（P〈0．05）。 结论：激活Hacat细胞表面Toll样受体有促进创面愈合作用，其机制可能与脂多糖作用了：Toll样受体从而激活核因子KB信号传导通路促进细胞释放基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子等因素有关?     

人脐静脉血管内皮细胞体外培养的方法研究

目的建立人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)体外高效稳定培养的方法,为组织工程及相关医学基础研究提供稳定的细胞来源。方法取自愿捐赠足月妊娠分娩的新生儿脐带,用自制空针软管静脉注入0.1%Ⅱ型胶原酶,置于37℃培养箱中,消化收集,采用含5%FBS及1%内皮细胞生长因子(endothelial cell growth factor,ECGS)的内皮细胞专用培养基进行培养。将消化前后的脐带标本行HE染色,观察HUVECs脱壁情况;流式细胞仪检测原代细胞纯度;细胞培养期间于倒置相差显微镜下观察细胞形态;取第3代HUVECs行免疫细胞化学染色观察、MTT检测细胞增殖情况,并将细胞接种于细胞外基质胶Matrigel上培养24 h,观察细胞管腔形成情况。结果经Ⅱ型胶原酶消化后的脐带内HUVECs大量脱落,细胞消化完全。经Ⅱ型胶原酶37℃培养箱中消化15 min后,可获得纯度为99.56%的HUVECs;原代HUVECs培养后2～3 d生长最快,呈典型的铺路石或鹅卵石样排列,4～6 d融合成片。MTT法检测显示第3代HUVECs培养后3～4 d细胞生长最快,5 d左右融合;免疫细胞化学染色显示内皮细胞Ⅷ因子相关抗原表达阳性,培养24 h后在细胞外基质胶Matrigel上可见类似于毛细血管的完整闭合管腔形成。结论经自制空针软管静脉注入0.1%Ⅱ型胶原酶,使静脉充分充盈,内皮消化完全,采用含5%FBS和1%ECGS的内皮细胞专用培养基,可迅速获取大量高纯度、高存活率的HUVECs。     

离体角质形成细胞热损伤模型建立及细胞凋亡观察

Objective To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature. Methods Human KCs were cultured in vitro, and they were incubated at 37, 41, 43,45, 48, and 51 ℃ respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The prolifera-tion activity of KC after heat injury was detected by MTT test. Results The results of trypan blue stai-ning, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necro-sis of KCwererespectively(12.3±3.2)% and (14.1±1.6)% at 45℃, (27.7±5.1)% and (58.0± 4.2)% at 48 ℃. Rate of KC necrosis reached up to (83.0±5.3)% at 51℃. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 ℃ as observed with MTT test. Con-clusions Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 ℃ for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.     

离体角质形成细胞热损伤模型建立及细胞凋亡观察

Objective To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature. Methods Human KCs were cultured in vitro, and they were incubated at 37, 41, 43,45, 48, and 51 ℃ respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The prolifera-tion activity of KC after heat injury was detected by MTT test. Results The results of trypan blue stai-ning, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necro-sis of KCwererespectively(12.3±3.2)% and (14.1±1.6)% at 45℃, (27.7±5.1)% and (58.0± 4.2)% at 48 ℃. Rate of KC necrosis reached up to (83.0±5.3)% at 51℃. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 ℃ as observed with MTT test. Con-clusions Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 ℃ for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.     

激活Hacat细胞表面Toll样受体促进创面愈合的机制★

目的：细胞表面Toll样受体对激发先天性免疫、抵抗微生物感染有重要作用。最近研究发现激活细胞表面Toll样受体不仅可以预防微生物感染，还可以促进组织再生。实验通过脂多糖激活体外培养的角质细胞株Hacat细胞表面Toll样受体，观察其促进创面愈合的可能机制。 方法：实验于2007-07在解放军第四军医大学西京医院烧伤与皮肤外科完成。①实验分组及方法：角质细胞株Hacat由解放军第四军医大学西京医院皮肤科馈赠；脂多糖为Sigma公司产品；小鼠抗人Toll样受体2单克隆抗体、兔抗人Toll样受体4多克隆抗体购于ebioscience公司。体外培养Hacat细胞，根据脂多糖(500 ng/L)与Hacat细胞共培养24，48，72 h将细胞随机分为脂多糖作用24 h 组、脂多糖作用48 h 组、脂多糖作用72 h 组，另设对照组，只加溶剂二甲基亚枫。②实验评估：蛋白免疫印迹技术检测脂多糖对Hacat细胞Toll样受体2、Toll样受体4及核因子-κB表达的影响；荧光定量聚合酶链反应检测脂多糖作用后Hacat细胞内基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子的表达。 结果：光学显微镜下正常人皮肤表皮及角质细胞Toll样受体2、Toll样受体4表达呈蓝黑色；脂多糖作用于Hacat细胞24，48，72 h 后Toll样受体2、Toll样受体4、核因子-κB表达均高于对照组（P < 0.01）。与对照组相比，其他3组基质金属蛋白酶3及血管内皮生长因子表达均增高（P < 0.01，P < 0.05）。脂多糖作用24 h 组基质金属蛋白酶9表达与对照组差异无显著性（P > 0.05），脂多糖作用48，72 h 组与对照组比较，差异显著（P < 0.05）。 结论：激活Hacat细胞表面Toll样受体有促进创面愈合作用，其机制可能与脂多糖作用于Toll样受体从而激活核因子-κB信号传导通路促进细胞释放基质金属蛋白酶3、基质金属蛋白酶9及血管内皮生长因子等因素有关。     

成年大鼠许旺细胞快速扩增的改良方法

目的对三种来源不同的神经组织进行许旺细胞（SCs）培养，比较其纯度、增殖能力，以找到一个快速获取大量SCs的方法。方法取成年大鼠预变性坐骨神经、成年大鼠新鲜坐骨神经、新生大鼠坐骨神经，使用低浓度胰酶快速消化法结合差速贴壁法分别进行SCs培养，进行形态学观察、S-100免疫细胞化学鉴定，检测并比较各组SCs纯度及增殖能力。结果各组细胞生长状态良好，S-100染色阳性，SCs纯度分别为：成年预变性组98％，成年新鲜组92％，新生大鼠组93％。各组SCs生长曲线表明预变性组细胞较新鲜组及新生组SCs增殖旺盛。结论成年大鼠预变性坐骨神经制作取材方便、细胞量充足，纯度高、细胞增殖旺盛，可以被用作细胞生物学研究及组织工程等研究。     

离体角质形成细胞热损伤模型建立及细胞凋亡观察

Objective To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature. Methods Human KCs were cultured in vitro, and they were incubated at 37, 41, 43,45, 48, and 51 ℃ respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The prolifera-tion activity of KC after heat injury was detected by MTT test. Results The results of trypan blue stai-ning, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necro-sis of KCwererespectively(12.3±3.2)% and (14.1±1.6)% at 45℃, (27.7±5.1)% and (58.0± 4.2)% at 48 ℃. Rate of KC necrosis reached up to (83.0±5.3)% at 51℃. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 ℃ as observed with MTT test. Con-clusions Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 ℃ for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.     

离体角质形成细胞热损伤模型建立及细胞凋亡观察

目的 建立离体KC热损伤模型,了解不同温度热损伤后KC凋亡情况.方法 体外培养人KC,分别以37、41、43、45、48、51℃孵育KC 10 min.用锥虫蓝染色初步观察KC死亡情况,Hoechst 33258荧光染色检测细胞凋亡情况,应用流式细胞仪对细胞凋亡和死亡情况进行定量分析.采用噻唑蓝法检测热损伤对KC增殖活性的影响.结果 锥虫蓝染色、Hoechst 33258荧光染色、流式细胞仪检测结果 显示,随着热损伤温度的升高,KC中凋亡和死亡细胞逐渐增加;45℃时KC凋亡率和死亡率分别为(12.3±3.2)%、(14.1±1.6)%,48℃时各为(27.7±5.1)%、(58.0±4.2)%,51℃时KC死亡率高达(83.0±5.3)%.噻唑蓝法检测结果 显示,45℃热损伤条件下KC生长出现平台期.结论 热损伤能造成离体KC凋亡、生长受抑制,45℃孵育10 min为构建KC热损伤模型的较好条件.该模型可用于研究KC热损伤后的凋亡情况及其生物学特性.     

离体角质形成细胞热损伤模型建立及细胞凋亡观察

Objective To reproduce a model of heat injured KC in vitro and explore its apoptosis rate of KC due to heat injury at different temperature. Methods Human KCs were cultured in vitro, and they were incubated at 37, 41, 43,45, 48, and 51 ℃ respectively for 10 mins in water bath. Trypan blue staining and Hoechst 33258 fluorescence staining were used respectively to determine necrosis and apoptosis of KC. Rates of apoptosis and necrosis of KC were analyzed quantitatively by flow cytometer. The prolifera-tion activity of KC after heat injury was detected by MTT test. Results The results of trypan blue stai-ning, Hoechst 33258 fluorescence staining, and flow cytometer demonstrated that number of apoptotic and necrotic KC increased gradually along with a rise of water bath temperature. The rates of apoptosis and necro-sis of KCwererespectively(12.3±3.2)% and (14.1±1.6)% at 45℃, (27.7±5.1)% and (58.0± 4.2)% at 48 ℃. Rate of KC necrosis reached up to (83.0±5.3)% at 51℃. Inhibition of KC growth reached a stationary phase when the injurious temperature reached 45 ℃ as observed with MTT test. Con-clusions Heat injury can induce apoptosis and growth inhibition of KC in vitro. Incubating KC at 45 ℃ for 10 mins is a good condition to reproduce a model of heat injured KC in vitro. This model may be used to study the biological character and apoptosis of KC after burn injury.