野生型p16基因体外诱导人瘢痕疙瘩成纤维细胞衰老的研究

目的：观察野生型p16基因对人瘢痕疙瘩成纤维细胞的生物学效应，探讨对瘢痕疙瘩进行基因治疗的可行性。方法：通过基因转染将含p16 cDNA的真核表达载体pcDNA3-p16导入人瘢痕疙瘩成纤维细胞中，筛选出阳性克隆后用测定细胞生长曲线、细胞周期及细胞超微结构等方法观察细胞的变化，用衰老相关-β-半乳糖苷酶染色法鉴定细胞形态。结果：细胞在转染野生型p16基因后自第3代起形态发生明显变化，细胞增殖明显减慢，细胞周期分析显示有96．7％的细胞处于G1期；电镜分析表明，细胞出现明显的溶酶体超载现象。衰老相关-β-半乳糖苷酶染色证实细胞进入衰老期。结论：野生型p16基因可诱导入瘢痕疙瘩成纤维细胞提前进入衰老阶段，这为瘢痕疙瘩基因治疗提供了实验依据。     

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.     

ATP－MgCl_2对烫伤大鼠肠粘膜保护作用的实验观察

为研究ＡＴＰ－ＭｇＣｌ２对创伤应激动物肠粘膜的保护作用，采用３０％ＴＢＳＡⅢ度烫伤大鼠模型，观察了腹腔注射ＡＴＰ－ＭｇＣｌ２复合液对回肠粘膜丙二醛（ＭＤＡ）含量及回肠组织形态学改变的影响。结果显示：烫伤后６～２４小时，ＭＤＡ含量明显升高，并伴有形态学病理改变。经ＡＴＰ－ＭｇＣｌ２注射治疗的大鼠伤后２４小时内ＭＤＡ含量维持于正常对照水平，且回肠粘膜病理改变减轻。提示：ＡＴＰ－ＭｇＣｌ２能够保护烫伤大鼠肠粘膜，其作用机理可能与降低肠粘膜的脂质过氧化反应有关。     

人视网膜色素上皮细胞的冻存和复苏培养

目的：建立人视网膜色素上皮(retinal pigment epithelium，RPE)细胞的冻存和复苏方法。方法：根据慢冻速融的细胞冻存原则，将原代或传代1～2次培养的人RPE细胞，加入10%二甲基亚砜作为保护剂，液氮中冻存。复苏时将冻存细胞在60℃、2分钟内融化，用台盼蓝染色测定细胞存活率，用抗人角蛋白抗体免疫细胞化学染色作细胞鉴定，每日计算细胞数以确定细胞生长曲线。 结果：经冻存复苏后的RPE细胞的存活率达90%，生长状态与对照组无差异，抗人角蛋白染色阳性，细胞对数生长期在1～4天，群体细胞倍增期约为1.55天。 结论：人RPE细胞液氮冻存可保持生物活性，复苏后生长良好，保持了细胞特征。这可提供细胞系，方便了体外实验，并可能作为RPE细胞移植治疗一些眼底病的细胞库。 （中华眼底病杂志，1997，13：157-159）     

P物质上调正常人真皮成纤维细胞TGF-β1的mRNA表达

目的 探讨神经肽P物质（SubstanceP,SP）与瘢痕形成间的调控关系。方法 体外分离培养正常人真皮成纤维细胞，分别加入SP及其受体特异性拮抗剂L－703，606乙酸盐，MTT法测定细胞生长增殖量；采用细胞爬片法培养细胞，加入SP对细胞作用后，应用TGF－β1mRNA特异性探针行细胞原位杂交，计算细胞的TGF－β1mRNA阳性表达率，并经图像分析测定阳怀细胞TGF－β1mRNA的表达强度，分析SP与真皮纤维细胞TGF－β1mRNA表达间的关系。结果 SP可促进体外培养的正常人真皮成纤维细胞的生长，其作用与SP浓度呈现依赖关系，当浓度达到25ng/ml时，刺激细胞生长的速度达最大值，可使细胞较对照组提前4d融合；正常人真皮纤维细胞受25ng/ml时时，刺激细胞生长的速率达最大值，可命名细胞较对照组提前4d后即可见细胞表达TGF－β1mRNA的阳性率及强度均显著增加；SP对正常人真皮纤维细胞的上述效应，均可被其受体拮抗剂L－703，606乙酸盐所抑制。结论 SP可促进正常人真皮纤维细胞的生长增殖，同时上调细胞的TGF－β1mRNA表达；提示SP可能为皮肤损伤后痕形成过程中启动成纤维细胞表型转化的重要调节因子。     

活化的人淋巴细胞与不同猪真皮共同培养的免疫反应特征

目的探讨临床应用异种无细胞真皮基质(Xeno-ADM)的免疫反应。方法用网状Xeno-ADM与超薄自体皮片复合移植于3例大面积Ⅲ度烧伤患者,成活后4～8周分离外周血单个核细胞(PBMC),以同期单纯自体皮移植的大面积烧伤患者PBMC为对照,用新鲜小猪整张真皮片(wholexenodermis)、微粒真皮(mincedxenodermis)和MincedXeno-ADM作抗原刺激物,通过不同的猪真皮→人淋巴细胞共同培养(DLCC)、3H-TdR掺入、免疫组织化学技术,检测反应细胞增殖水平和细胞类型。结果DLCC后,新鲜小猪微粒真皮组对活化和未被活化人淋巴细胞增殖刺激作用均明显强于猪整张真皮组和Xeno-ADM组(P<0.05～0.01),增殖和浸润到真皮内的细胞以CD3+CD4+/CD45RO+T淋巴细胞为主、单核细胞和B细胞次之。结论以CD3+-CD4+/CD45RO+为主的T淋巴细胞参与可能是异种真皮移植后的免疫反应特征;真皮微血管内皮细胞ICAM-1的表达可能与诱导淋巴细胞浸润和增殖有关,与未活化的淋巴细胞相比,采用活化的淋巴细胞进行DLCC,可更真实地反映宿主对Xeno-ADM的免疫反应状况。     

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