人表皮细胞的培养与移植

用细胞悬液法,原代培养人表皮细胞,在373瓶原代培养中,3T3细胞作滋养层的74瓶,生长成细胞单层的占64.8％;以鼠尾腱胶原作支持物的299瓶生长成细胞单层的占83.6％。用扫描电镜观察,见细胞表面有微绒毛和突起,细胞间桥,有丝分裂,培养后10d形成细胞单层。将培养的细胞单层,初步试用在14例次烧伤病人的切(削)痂创面及感染肉芽创面,结果5例次成活,4例次部分成活,5例次因感染失败。     

早期烧伤患者血清介导培养血管内皮细胞损伤

目的　探讨严重烧伤早期血管内皮细胞 (EC)损伤机制 .方法　以培养脐静脉 EC为模型 ,应用扫描电镜技术 ,3H-Td R特异性释放实验 ,观察了终浓度为 2 0 0 m L· L- 1 的早期烧伤患者血清 (EBPS)介导 PMN与 EC相互作用后 ,EC及PMN的表面结构 ,PMN与 EC粘附特性及 EC3H- Td R特异释放率的变化 .结果　正常人血清 (NHS)调理的 PMN为园形、规则 ,EBPS调理的 PMN形态不规则 .NHS对照组 PMN与 EC连接松散 ,EC与 EC连接紧密 ,EBPS刺激 EC与 NHS调理的 PMN相互作用 ,EC与 EC连接出现微小间隙 ,EC表面突起增多 ,PMN形态不规则 ,主要粘附于 EC间隙 .NHS刺激的 EC与 EBPS调理的 PMN相互作用后 ,PMN偏平状锚于 EC表面 .EBPS刺激的 EC与 EBPS调理的 PMN相互作用 ,PMN形态不规则 ,表面微绒毛显著减少 ,EC从膜上分离脱落 ,EC3H- Td R特异性释放率与对照组比较有显著差异(P<0 .0 1) .结论　 EBPS可激活 EC和 PMN,激活的 EC与PMN相互作用 ,可引起 EC损伤 ,EC损伤依赖 PMN的参与 ,EBPS可能不是作为一种直接的损伤因素而是作为一种细胞激活剂参与了烧伤早期 EC的损伤过程     

血小板源生长因子-AB对成纤维细胞周期及DNA合成的影响

目的：探讨血小板源生长因子（ＰＤＧＦ）－ＡＢ在创面愈合及瘢痕增生过程中的作用机制;方法：取第３代～６代体外培养的人正常皮肤成纤维细胞（ＮｓＦｂ）及增生性瘢痕成纤维细胞（ＨＴｓＦｂ）,经ＰＤＧＦ－ＡＢ作用后,用＾３Ｈ－ＴｄＲ参入法测定ＤＮＡ合成的变化,用汉式细胞术测定细胞周期的变化。结果：ＰＤＧＦ－ＡＢ作用后,两种细胞ＤＮＡ民均明显增加,Ｓ期细胞数百分比均明显增高,Ｇ１期细胞不断进入Ｓ期,但作用有划     

脂质体介导质粒转染角朊细胞的影响因素

探讨利用脂质体介导真核表达质粒转染体外培养的人角朊细胞的最佳转染条件。体外分离培养正常人角朊细胞 ,培养至 6 0 %、70 %、80 %、90 %及 10 0 %融合时 ,应用不同浓度的LipofectAMINE包被真核表达质粒pCMV·SPORT β gal,分别转染 6、8、10、12及 2 4h。细胞经转染后再培养 48h ,行 β 半乳糖苷酶原位染色 ,镜下观察并计算阳性转染率。被转染的阳性细胞 ,可见质粒 β 半乳糖苷酶基因的良好表达 ;当细胞融合率为 80 %、90 % ,以12 5 μL/ 10 0 μL的LipofectAMINE包被 1 5 μg/ 10 0 μL的pCMV·SPORT β gal,转染时间为 8h的转染率最高 ,可分别达到 (31 35± 1 35 ) %、(32 32± 2 4) %。该实验说明LipofectAMINE可有效地介导真核表达质粒转染培养的人角朊细胞 ;转染率与细胞生长状态、脂质体包被质粒的浓度比例、及转染时间直接相关。     

人胎儿毛囊隆突细胞的分离、培养与鉴定

目的:建立一种简单高效的毛囊隆突细胞的培养方法. 方法: 将5～7 mo终止妊娠胎儿头皮的毛干中上部剪下后Ⅰ型胶原酶消化,采用成纤维细胞和毛囊隆突细胞对胰酶的不同敏感性进行纯化,流式细胞仪进行细胞周期分析,MTT法检测细胞克隆率,免疫组化检测细胞K19的表达情况. 结果: 消化法原代培养可以每小时获得100个左右毛囊隆突,经姨酶消化不同时间可以纯化毛囊隆突细胞,流式细胞仪检测第二代细胞G1期占88.20%,细胞克隆形成率为18.2%,K19表达呈阳性. 结论: 建立了一种简单高效的毛囊隆突细胞的分离、培养方法,并证明其具有某些干细胞的特征性表现.     

复合皮真皮-表皮连接区结构再生与功能重建

目的：为了解复合皮（ＣＳ）真皮－表皮连接（ＤＥＪ）区结构再生及其对ＣＳ功能重建的作用机制。方法：用经胰蛋白酶处理的异体乳猪真皮与自体表皮重组ＣＳ,并以自体表皮为对照（Ｃ组）,对移植术后２～１２４周的活检标本作光镜和透射电镜观察。结果：①ＣＳ组表皮和真皮生长快、成熟早,乳头层组织疏松有序,毛细血管增生迅速,２０～２４周时,乳头和皮钉及以致密板为中心的半桥粒、锚丝、锚纤维等ＤＥＪ区结构形成,以靠近毛细     

甲壳胺对人角朊细胞培养DNA合成速率的影响

０引言甲壳胺是从甲壳类动物如虾、蟹等外壳中经化学方法提取的一种高分子化合物氨基多糖,有关文献报道了用甲壳胺制作的人工皮肤,手术缝合线对创面愈合具有促进作用［１］．我们采用角朊细胞悬液培养法检测了不同浓度的甲壳胺对角朊细胞ＤＮＡ合成速率的影响．１材料和...     

烧伤早期及丹参对中性粒细胞化学发光的影响

目的：探讨烧伤早期患者血清（ＥＢＰＳ）调理的中性普细胞（ＰＭＮ）对血管内皮细胞的损伤机制及丹能的保护作用,并对ＥＢＰＳ和丹参注射液（ＳＭ）调理ＰＭＮ的氧化活性及吞噬活性进行了研究。方法：以１００ｍＬ／ＬＥＢＰＳ,１００ｍＬ｜ＬＥＢＰＳ加１０ｍＬＳＭ及１００ｍＬ／Ｌ正常血清（ＮＨＳ）分别丐健康人全血ＰＭＮ孵育不同时间,检测ＰＭＮ生物化学发光（ＰＭＮ－ＣＬ）。结果：１００ｍＬＥＢＰＳ调理ＰＭＮ０￣１５     

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.     

胰岛素对脂肪干细胞生长因子分泌功能的影响

Objective To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). Methods ADSCs were isolated from human ab-dominal adipose tissue and cultured. The immunophenotype and adipose induced-differenciation were identi-fied, and the third generation cells were collected. The collected cells were assigned to 1 × 10-8, 1 × 10-7, 1 × 10-6 mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. Results The secretion amounts of VEGF and HGF of ADSCs in 1 × 10-8 and 1 × 10-7 mol/L insulin groups [ (471±41, 762±66 ng/L), (643±64, 930±67 ng/L) , respectively ] were significantly higher as compared with those in control group (286±47, 577±84 ng/L) ( P <0.05 orP <0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1±10-6 mol/L insulin group ( P >0.05 ). The supernatant fluid of ADSCs' nutrient medium of 1 ± 10-8 ,1 ± 10-7 mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 ± 10-7 mol/L group ( P < 0.05 or P < 0. 01 ). Conclusions Insulin in the concentrations of 1 ± 10-8 and 1 ± 10-7 mol/L can notably promote ADSCs' function of secreting VEGF and HGF.