人脂肪间充质干细胞的分离培养及生物学特性研究

目的：探索高效分离和扩增人脂肪间充质干细胞（adipose derived mesenchymal stem cells,ADSCs）的方法,观察其生物学特性,并通过形态学、免疫表型及多向分化能力进行鉴定。方法：以手术中剩余的人皮下脂肪组织为材料来源,利用胶原酶消化法分离ADSCs,体外扩增后传代,倒置显微镜观察,MTT比色法测定细胞生长曲线,流式细胞仪检测细胞表面标记CD29、CD31、CD34、CD45、CD90、CD105的表达。在DMEM及胎牛血清培养基、地塞米松、IBMX、吲哚美辛的诱导下向脂肪细胞定向分化;在DMEM及胎牛血清培养基、地塞米松、抗坏血酸、β-磷酸甘油、胰岛素的诱导下向成骨细胞定向分化。结果：原代和传代细胞呈梭形外观,生长增殖能力良好。细胞传代后2天内处于潜伏期,第3天进入生长期,5天后进入平台期。流式细胞仪检测CD29、CD31、CD34、CD45、CD90、CD105阳性率分别为73.4%、3.6%、4.5%、2.0%、97.3%、80.4%。经定向诱导分化后,细胞分别呈现脂肪细胞、骨细胞的表型特征。结论：酶消化法能有效分离纯化人ADSCs,细胞生长稳定,增殖能力活跃,具有ADSCs的一般生物学特性,为其成为组织工程理想的种子细胞提供了进一步的支持。     

持续负压灌洗引流在糖尿病足合并足底脓肿清创术中的应用

目的 了解早期清创结合持续负压灌洗引流在糖尿病足合并足底脓肿患者保肢手术中应用的可行性.方法 糖尿病足合并足底脓肿患者10例共10只患足,实施手术清创后留置胃管进行持续负压灌洗引流7～14 d,停止灌洗后维持负压吸引3～5 d,拔除引流管,术后3周左右拆线.观察创面愈合情况. 结果经上述处理,患者局部炎性反应明显减轻,创面愈合,足部功能恢复良好,外形不受影响. 结论清创术结合局部持续负压灌洗引流,有利于糖尿病足合并足底脓肿的治疗,可保留患肢原始长度.     

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.     

延期皮肤移植术在面颈部畸形修复中的应用

目的:探讨并比较面颈部大范围自体皮移植手术中,延期手术与一次手术的成功率及愈后效果。方法:针对面颈部瘢痕、巨痣等在实施大面积自体皮移植手术时,手术分两种术式进行:一期术式即切除病变组织后立即行自体皮移植;延期术式则先行常规病变组织切除,包扎创面后48h再进行自体皮移植。术后交换敷料时间均为术后第7天,观察比较两种术式在皮片成活率、创面感染发生率、皮下血肿发生率、瘢痕增生发生率等方面的差异,以及患者治疗周期及治疗费用上的差异。结果:本组病例共49例,行一期术式者13人,行延期术式者36人,在皮片成活率(91.9%±4.5%vs99.5%±1.2%)、皮下血肿发生率(92.3%vs22.2%)、感染发生率(7.7%vs2.8%)方面,二者有显著性差异(P0.05)。两组在治疗周期上无显著性差异。治疗费用方面,延期手术组略高。随访结果显示,两组远期效果基本类似,但一期术式组的瘢痕形成及增生情况多于延期术式组。结论:面颈部实施大范围自体皮移植手术时采取延期植皮的方式,对保证手术的成功及愈后效果有重要作用。     

转化生长因子-β1对体外培养的人毛囊干细胞生物学特性的影响

目的 观察转化生长因子-β1(TGF-β1)对体外培养的毛囊干细胞(HFSCs)生物学特性的影响.方法 体外分离培养人HFSCs,观察其在10-μg/L TGF-β1条件培养基下的细胞形态变化,噻唑蓝(MTT)比色法于24、48、72 h分别检测加入条件培养基前后细胞增殖能力,细胞接近铺满时划痕,并于0、24、48、72 h后用目镜测微尺测量划痕宽度检测迁移能力,将细胞接种于Transwell小室上层,下层加入含10 μg/L TGF-β1条件培养基,48 h后计数穿透细胞数,免疫荧光化学方法检测加入条件培养基前后角蛋白19、β-整合素、E钙黏蛋白、N钙黏蛋白、波形蛋白的表达.结果在TGF-β1作用下,HFSCs之间的紧密连接消失,细胞由圆形或多角形向梭形细胞转变,MTT结果显示,在TGF-β1作用下HFSCs各时间点A值与对照组比较,差异无统计学意义(P>0.05),细胞划痕实验结果显示,TGF-β1能够促进细胞迁移,对照组差异有统计学意义(P<0.01).Transwell穿透实验结果表明,加入TGF-β1后,HFSCs细胞定向迁移穿透基底膜数量与对照组比较显著增多(47.9±8.8比35.8±6.7,P<0.01),免疫荧光结果显示TGF-β1能够降低角蛋白19、β-整合素和E钙黏蛋白表达,而N钙黏蛋白、波形蛋白的表达增加.结论 TGF-β1通过诱导体外培养的HFSCs上皮间质转化而促进其迁移.     

热损伤角质形成细胞培养上清对真皮成纤维细胞增殖与胶原mRNA表达的影响

目的 观察热损(创)伤后角质形成细胞(KC)培养上清对真皮成纤维细胞(Fb)增殖与胶原mRNA表达的影响.方法 建立KC热损伤模型,收集正常及热损伤12 h后培养上清配制不同浓度的细胞条件培养液;分离培养人正常Fb,实验组分别加入含不同浓度的细胞条件培养液,以单纯DMEM组为对照,采用噻唑蓝(MTT)比色法测定24 h后真皮成纤维细胞增殖;分别以流式细胞仪、实时荧光定量聚合酶链反应(Real-time PCR)测定DMEM组、50%浓度条件培养液组相同时间Fb生长周期及Ⅰ型胶原mRNA表达.结果 细胞增殖测定:热损伤上清(10%、30%、50%、70%)条件培养液组A值(0.151±0.004、0.165±0.009、0.195±0.006、0.202±0.008)均高于正常上清(10%、30%、50%、70%)条件培养液组(0.144±0.004,0.159±0.002,0.180±0.005,0.181±0.006)及对照组(0.144±0.007),除10%浓度组外各实验组A值与对照组比较差异均有统计学意义(t值分别为:4.01、11.42、11.41;P＜0.05),热损伤上清与正常上清条件培养液组比较:50%、70%浓度组间差异有统计学意义(t值分别为:2.03、1.94;P＜0.05);热损伤上清条件培养液50%与70%浓度组间差异无统计学意义(t值为1.34;P＞0.05).细胞周期测定见50%浓度热损伤上清组可明显促进Fb通过G1/S及S/G2限制点,S期及G2/M期细胞与对照组比较增多,G0/G1期细胞与对照组比较明显减少(t值分别为:5.87、11.3、4.86;P＜0.05).Ⅰ型胶原mRNA表达测定中,50%浓度热损伤上清组与对照组比较表达明显上调,差异亦有统计学意义(t=1.72;P＜0.05).结论 热损(创)伤后KC上清液可促进Fb的增殖,同时可上调Ⅰ型胶原mRNA的表达.

Abstract：

Objective To observe the effects of heat injured keratinocyte (KC) supematant on proliferation activity and collagen mRNA expression of the normal fibroblast (Fb).Methods A model of heat injured KC in vitro was reproduced,the supernatants of heat injured KC were collected and used as conditioned medium,and DMEM was used as control medium.Normal Fb were treated with different concentrations of conditioned medium.After treatment for 24 h,the proliferation activity,cell cycle and collagen Ⅰ mRNA levels in treated Fb were measured by using methylthiazol tetrazolium ( MTT),flow cytometry (FCM) and real-time polymerase chain reaction (PCR) techniques.Results As compared with the control group,the absorbance (A) values of MTT in treated groups were significantly increased (t = 4.01,11.42,11.41 ;P ＜0.05),except in 10% group (t = 1.34,P ＞0.05).As compared with normal supernatant treated group (0.159 ± 0.002,0.180 ± 0.005,0.181 ± 0.006),the A values in 50% and 70% conditioned medium group (0.195 ± 0.006,0.202 ± 0.008 ) were significantly increased ( t = 2.03,1.94;P ＜0.05).The 50% conditioned medium increased the percentage of G1 to S and S to G2 phase transit.As compared with control group,the number of cells in S and G2/M phase was increased significantly (t =5.87,11.3;P＜0.05 ) and that in G0/G1 phase decreased significantly ( t = 4.86;P＜0.05 ).Real-time PCR revealed that the collagen Ⅰ mRNA level in 50% conditioned medium group was significantly up-regulated (t= 1.72;P＜0.05 ) as compared with control group.Conclusion Heat injured KC supernatant may promote the proliferation of dermal Fb,and up-regulate the mRNA expression of collagen.     

烫伤大鼠肠道P物质含量和P物质能神经的观察分析

以３０％ＴＢＳＡⅢ度烫伤大鼠为模型，采用ＲＩＡ和ＩＨＣ的方法，动态观察了伤后７２小时大鼠空肠免疫活性Ｐ物质（ｉＳＰ）的含量及绒毛内Ｐ物质－免疫反应（ＳＰ－ＩＲ）阳性神经纤维的变化。结果发现：①烫伤大鼠空肠内ｉＳＰ伤后１小时出现显著升高，４小时仍处于高水平状态；８小时后明显下降，伤后７２小时一直处于低水平状态。②绒毛内ＳＰ－ＩＲ阳性神经纤维在形态、分布密度和纤维内ＳＰ－ＩＲ物质的含量都有明显的改变。图像分析定量结果表明：绒毛内ＳＰ－ＩＲ阳性神经纤维分布密度，伤后１小时明显增加，随后减少，４８小时再次出现增加；神经纤维内ＳＰ－ＩＲ阳性物质伤后１小时及４小时明显升高，１２小时明显下降，２４小时后再次升高。实验结果说明：烫伤后大鼠空肠内Ｐ物质出现应激性释放与消耗，Ｐ物质可能参与了烫伤后大鼠的肠道损伤；肠绒毛内的Ｐ物质能神经纤维是肠粘膜屏障损伤的直接参与因素。     

高压液—固相微型催化反应器—色谱仪联合装置

对于催化剂快速初评及催化动力学的研究,微型催化反应器装置是很有效的工具。可是目前一般均限于气—固相的反应过程。为了适应某些液—固相反应过程的开发,研制了本装置。操作压力最高可达6MPa(可调),催化剂装量仅为0.5—2克。在连续流动的反应过程中,还能进行在线取样分析,并用微处理机自动打印结果。     

硝酸镧块染法显示肠粘膜屏障通透性的电镜技术

硝酸镧块染法显示肠粘膜屏障通透性的电镜技术史颖辉，李学荣，胡大海，谷法有，陈壁，刘健（西安第四军医大学电镜室，西京医院烧伤科．西安７１００３２）我们将镧示踪法用于肠粘膜屏障的研究．取得了良好的效果，现介绍如下．１材料和方法１．１动物及模型用纯系ＳＤ大...