正常人皮肤深Ⅱ度烫伤动物模型的建立

利用正常人皮肤裸鼠移植模型,建立一种深Ⅱ度烫伤动物模型.首先将正常人全厚皮肤移植于裸鼠背部,待皮片完全成活后,用高压蒸气消毒锅及自制蒸气烫伤装置,以压力0.12 MPa、直径1.5 cm 在裸鼠背部植皮区作用8 s,分别于伤后24 h、2、4、6周取标本行组织学观察,观察毛囊、汗腺等皮肤附件的损伤程度、创面愈合时间及被毛生长等情况.结果表明,正常人全厚皮肤于移植后2周完全成活,具有正常的组织结构;高压蒸气作用8 s,可造成所移植皮肤组织的深Ⅱ度烫伤,创面于伤后3周愈合,6周后有少量被毛生长.该模型可成功复制正常人皮肤深Ⅱ度烫伤创面,为研究创面愈合过程中皮肤附件的病理变化提供了实验工具.     

肿瘤患者自体CIK细胞治疗及其对患者免疫功能的影响

目的　从恶性肿瘤患者外周血中高效诱导扩增CIK细胞并观察自体CIK过继免疫治疗对患者免疫功能的影响.方法体外利用抗CD3单抗、IL 2、γ IFN等细胞因子从12例肿瘤患者外周血单个核细胞(PBMC)中诱导扩增CIK细胞,分3次自体回输;并用流式细胞仪检测回输前后患者T细胞亚群和NK细胞变化.结果　CIK细胞经13d培养后细胞总数和CD3+CD56+T效应细胞均获得大量增殖,分别平均增殖123(46～301)倍和1656(177～5130)倍.肿瘤患者经自体CIK细胞免疫治疗后,CD3、CD4T细胞和NK细胞比例均显著提高(P<0.01).结论　可从不同恶性肿瘤患者PBMC中高效诱导扩增CIK细胞;自体CIK细胞过继免疫治疗可以显著提高患者免疫功能,且副作用小,具有良好的应用前景.     

组织工程真皮替代物的构建及在创伤修复中的作用

以双层胶原-壳聚糖海绵为支架构建组织工程真皮替代物并研究其在大鼠创伤愈合中的作用.将双层胶原-壳聚糖海绵支架埋植于SD大鼠皮下以考察其组织相容性;将SD大鼠皮肤中分离的成纤维细胞种植在支架上体外构建人工真皮替代物,并将该替代物移植到SD大鼠皮肤缺损处以修复创面.皮下埋植实验显示支架植入早期有炎症反应,但一周后炎症反应逐渐减轻,而且有成纤维细胞和毛细血管的侵入.皮肤移植实验结果显示:术后第7天和第14天实验组创面表皮再生和肉芽组织增生比对照组更快更明显;第14天和第21天实验组创面面积明显小于对照组(p<0.05).因此以双层胶原-壳聚糖海绵为支架构建的组织工程真皮替代物具有良好的生物相容性,并能促进皮肤创面的愈合,可以作为人工真皮替代物用于皮肤缺损的修复.     

无细胞猪真皮的生物相容性评价

采用猪皮制备的无细胞真皮基质,在基质上接种血管内皮细胞(VEC),鼠成纤维细胞(3T3),在细胞水平检测其生物相容性.采用MTT法检测VEC、3T3在基质上增殖活力;将复合培养1周的VEC-真皮复合物作组织切片,光镜下观察细胞生长状况.结果发现VEC在无细胞猪真皮基质上增殖迅速,并已经形成单层细胞膜片,某些局部区域已形成2～3层细胞膜片.结果说明采用的无细胞猪真皮基质具有良好的生物相容性,该材料可能有广泛的应用前景.     

筛状全厚皮片原位回植治疗皮肤大面积撕脱伤

目的:探索大面积皮肤撕脱伤急诊创面覆盖方法.方法:将撕脱皮肤"反取皮"制成筛状全厚皮片原位移植.结果:156例患者中皮片平均成活面积达95%,且创面具有良好的感觉,外形及功能正常.结论:"反取皮"制成筛状全厚皮片原位回植是早期治疗大面积皮肤撕脱伤的一种很实用的方法,且操作简便,皮片成活质量高,对患者损伤小.     

脐带源间充质干细胞的生物学性状及其成脂肪细胞诱导分化

以健康产妇脐带为材料分离脐带间充质干细胞,体外培养扩增传代,做细胞形态观察,计数细胞并绘制细胞生长曲线,用流式细胞仪测定细胞周期以及免疫表型,测定细胞定向诱导分化成脂肪细胞的能力.发现人脐带源间充质干细胞能在体外培养扩增,可定向诱导分化为脂肪细胞并且具有和骨髓来源的间充质干细胞相似的生物学形态和抗原表型.成功建立了脐带源间充质干细胞系,可以作为干细胞研究和应用的新的材料来源.     

DiI荧光标记示踪兔皮肤成纤维细胞修复前交叉韧带损伤的研究

体内采用组织工程方法构建前交叉韧带种子细胞筛选研究,提供了一种理想的细胞标记的方法.体外分离培养兔皮肤成纤维细胞(SF),比较皮肤成纤维细胞使用荧光染料CM-DiI体外标记前后细胞的生长状态、增殖活性以及检测SF复合纤维生物蛋白胶植入前交叉韧带上12周后的荧光表达情况.结果表明CM-Dil标记SF效果良好,标记阳性率高于95%.而标记前后SF增殖活性无明显变化,荧光标记在体内实验12周后仍有表达.从而证实CM-DiI是筛选前交叉韧带组织工程种子细胞筛选研究中细胞标记、示踪的良好标记物.     

细胞因子诱导的NK细胞过继免疫治疗恶性肿瘤的初步应用

目的　采集不同类型恶性肿瘤患者外周血,扩增CINK细胞,并观察自体CINK细胞过继免疫治疗的疗效和安全性.方法　利用抗CD3 单抗、IL-2、PHA从9 例肿瘤患者PBMC中诱导扩增CINK细胞,2 周左右开始自体回输.结果　CINK细胞经培养后细胞总数和CD3- CD56+NK细胞均获大量增殖.肿瘤患者经自体CINK 细胞免疫治疗后, CD3、CD4 T 细胞、NK 细胞、CD4/CD8的比例均显著提高(P<0.01),睡眠、食欲等指标也有相应改善,治疗过程中未见副作用. 结论　自体CINK细胞过继免疫治疗可以显著提高患者免疫功能,改善患者生活质量,且副作用小.     

以组织工程技术构建活性双层人工皮肤

用冷冻干燥胶原凝胶方法制备胶原海绵,经戊二醛交联处理,作为活性双层人工皮肤的支架;以幼儿包皮为细胞来源获得真皮成纤维细胞和皮肤角质细胞,体外扩增后作为种子细胞;在胶原海绵上接种成纤维细胞和角质细胞,体外构建活性双层人工皮肤。研究结果表明,人工真皮内的成纤维细胞生长良好,并且有人纤粘连蛋白类生物活性物质存在,活性双层人工皮肤表皮层有类似干天然皮肤表皮层的组织结构。     

小鼠体细胞核移植及ES细胞样集落分离

利用小鼠皮肤成纤维细胞为核供体进行体细胞核移植并从重构胚中分离胚胎干细胞(ES)样集落,以便对体细胞核移植重构胚来源的ES细胞样集落进行研究.结果显示,小鼠皮肤成纤维细胞作为核供体,核移植重构胚激活率为60.48%(254/420),囊胚发育率为6.90%(29/420),6个囊胚中分离出ES细胞样集落,分离率为1.43%(6/420),3个ES细胞样集落能够稳定传代,至第5代时核型正常率分别为77.84%,75.18%,77.20%.分离出的ES细胞样集落具有岛屿状团状隆起结构,碱性磷酸酶染色呈阳性,体外可自发分化成上皮样或梭形细胞.实验证实小鼠唇部皮肤成纤维细胞能够支持体细胞核移植重构胚发育至囊胚,并能分离出可以稳定传代的ES细胞样集落.     

Analysis of fibroblast proteins from patients with Duchenne muscular dystrophy by two-dimensional gel electrophoresis.

Duchenne muscular dystrophy (DMD), the most common and severe form of the muscular dystrophies, is an X-linked inborn error of metabolism with multiple tissue involvement. Although the major pathological changes are observed in skeletal muscle, abnormalities have also been detected in the heart, nervous system, red blood cells, lymphocytes and cultured skin fibroblasts. For many reasons, such as readily available tissue material, fewer secondary changes and the potential for prenatal diagnosis, cultured skin fibroblasts should be the tissue of choice to search for the primary defect. Several abnormalities have been reported in DMD fibroblasts, suggesting that the genetic abnormality is expressed in these cells. To search for potentially mutant protein(s) we have compared the protein composition of normal and DMD fibroblasts by two-dimensional gel electrophoresis and have now found one protein spot consistently missing in DMD cells. The nature of this protein and its relation to the DMD gene are unknown.     

Advances in tissue engineering

Tissue engineering is a newly developed specialty involved in the construction of tissues and organs either in vitro or in vivo. Tremendous progress has been achieved over the past decade in tisse construction as well as in other related areas, such as bone marrow stromal cells, embryonic stem cells and tissue progenitor cells. In our laboratory, tissues of full-thickness skin, bone, cartilage and tendon have been successfully engineered, and the engineered tissues have repaired full-thickness skin wound, cranial bone defects, articular cartilage defects and tendon defects in animals. In basic research areas, bone marrow stromal cells have been induced and transformed into osteoblasts and chondrocytes in vitro. Mouse embryo stem cell lines we established have differentiated into neuron precursor, cardiac muscle cells and epithelial cells. Genetic modifications of seed cells for promoting cell proliferation, delaying cell aging and inducing immune tolerance have also been investigated.     

Stimulation of connective tissue cell growth by substance P and substance K

Connective tissue cells proliferate actively when cultured in the presence of serum. Platelet-derived growth factor (PDGF), a basic protein of relative molecular mass approximately 30,000, has been identified as the major serum mitogen for these cells; its main physiological/pathophysiological role may be to initiate wound healing in connection with tissue injury. However, growth of cultured cells is also influenced by several other factors, including epidermal growth factor, fibroblast growth factor, insulin and somatomedins. Furthermore, Rozengurt and Sinnett-Smith recently showed that bombesin, a neuroendocrine peptide isolated from frog skin, stimulates DNA synthesis and cell division in cultures of a specific subtype of 3T3 cells. Substance P and substance K (also known as neurokinin A or neuromedin L) are mammalian peptides belonging to the tachykinin family. Substance P has been studied extensively; it is distributed widely throughout the central and peripheral nervous system, including primary sensory neurones, and can be released in the periphery from axon collaterals of stimulated pain fibres and contribute to the inflammatory response. Substance K is a member of the tachykinin family isolated from mammalian spinal cord; Nawa et al. determined the primary structure of two types of substance P precursors, one of which contained a sequence homologous to substance K, as well as the sequence of substance P. We report here that substance P and substance K stimulate DNA synthesis in cultured arterial smooth muscle cells and human skin fibroblasts, and that this stimulation is inhibited by the substance P-antagonist spantide.     

伤口收缩及瘢痕攣缩的机制

本文通过复习伤口收缩及瘢痕收缩的有关文献和通过有关病理标本的观察,指出伤口收缩与瘢痕收缩是两个过程,并认为两者收缩的机制不同。目前伤口收缩机制的假说主要有肌纤维母细胞说,伤口内容物说和肌肉收缩说。通过比较,作者认为肌肉收缩说比较合理。关于瘢痕挛缩机制的学说,目前文献已否定胶原纤维老化收缩学说,只提肌纤维母细胞学说。通过观察有广泛瘢痕病变的标本(如门脉性肝硬化,原发及继发性肾固缩等),未发现有病变器官的主质细胞和血管由于瘢痕组织收缩而导致的萎缩和受压现象。作者认为,这些病变器官及瘢痕缩小的主要机制是由于病灶内主质细胞的丢失,以及肉芽组织及瘢痕组织内充血血管不再充血、数量减少,以及其间质水肿液消退所引起的。     

新型前交叉韧带支架材料的构建及其生物相容性的实验研究

探讨以聚羟基丁酸己酯/聚左旋乳酸(PHB/PLLA1∶1)胶原杂化支架作为前交叉韧带组织工程载体材料的可行性。制备"三明治"样结构PHB/PLLA共聚物并测量其孔隙率等指标。以I型胶原对制备的PHB/PLLA支架进行杂化,获得PHB/PLLA胶原杂化支架。扫描电镜观察其表面结构。将兔皮肤成纤维细胞(SF)接种于PHB/PLLA胶原杂化支架,共培养5d后,扫描电镜下观察其在材料上生长情况。PHB/PLLA支架杂化后胶原填充于纤维空隙,分布比较均匀。体外培养的皮肤成纤维细胞成功种植在支架材料上,在材料上粘附、生长良好。说明构建的支架材料具有良好的三维构型和生物相容性,有望为前交叉韧带损伤的修复提供了一种新型的支架材料。     

Induction of hair growth by implantation of cultured dermal papilla cells

Mammalian hairs are formed by differentiation and keratinization of cells produced in the epidermal matrix (Figs 3, 4). Using the rodent vibrissa follicle as a model, transplantation studies have shown that the dermal papilla, a discrete population of specialized fibroblasts, is of prime importance in the growth of hair. Papillae induce hair growth when implanted into follicles and can interact with skin epidermis to form new hair follicles. When grown in culture, papilla cells display singular morphological and behavioural characteristics compared with connective tissue cells from other skin sources. We report here that serially cultured adult papilla cells can induce the growth of hair when implanted into follicles which otherwise would not grow hairs. This finding presents an opportunity to characterize properties distinguishing the papilla cell population from other skin fibroblasts, and, more specifically, those which control hair growth. The eventual application of this work to human hair replacement techniques can also be envisaged.     

鹿茸的组织结构及离体细胞的显微结构观察

本文对鹿茸的组织结构、鹿茸皮肤及顶端肉质部分的显微结构进行了较详尽的观察,同时对从鹿茸分离的细胞及由鹿茸组培养出的细胞形态结构也作了初步研究。