高代次人皮肤成纤维细胞致肿瘤性的研究

目的 研究高代次人皮肤成纤维细胞的染色体变异性和致肿瘤性,为验证其作为组织工程肌腱种子细胞的安全性提供相应依据.方法 采用常规染色体G带处理方法分别对8个样本的第2、第10代人皮肤成纤维细胞进行核型分析,并通过裸鼠皮下致肿瘤实验对其致肿瘤性进行考察.结果 各样本、各代次的人皮肤成纤维细胞均未发现染色体核型异常改变,裸鼠皮下致肿瘤实验在观察期内均未见结节或可疑病灶产生,符合国家标准.结论 高代次(第10代以内)人皮肤成纤维细胞具有正常的染色体核型,且无致肿瘤性,作为组织工程肌腱种子细胞具有较高的安全性.     

人端粒酶逆转录酶真核表达质粒转染人成纤维细胞的体外培养及生物学特性研究

目的　　分析人端粒酶逆转录酶 (h TERT)真核表达质粒 p GRN14 5转染人成纤维细胞的生物学特性。方法　体外分离培养小儿包皮成纤维细胞 ,脂质体法将构建的 p GRN14 5转染人成纤维细胞 ,经潮霉素 B筛选 ,扩增培养阳性克隆。 RT- PCR法、TRAP- PCR法分析细胞端粒酶活性。流式细胞术检测细胞增殖周期及细胞凋亡率 ,对转染后细胞行染色体核型分析及免疫组织化学法检测胶原分泌能力。结果　转染后培养的人成纤维细胞形态与转染前相比无明显改变 ,转染细胞稳定地表达端粒酶活性 ,流式细胞术分析转染前后成纤维细胞的增殖周期无明显改变 ,细胞凋亡率较转染前降低。经 p GRN14 5转染的人成纤维细胞保持正常的二倍体核型 ,具有正常分泌 、 型胶原的能力。结论　 p GRN14 5转染的人成纤维细胞稳定地表达端粒酶活性 ,细胞寿命明显延长。初步证实了用这种方法建立组织工程标准化种子细胞系的可行性     

悬滴培养法维持瘢痕疙瘩成纤维细胞表面标志CD105的表达及其功能

目的 探讨悬滴培养法对维持体外培养的瘢痕疙瘩成纤维细胞表面标志的作用,并初步研究CD105调节细胞功能的作用。方法 以耳部瘢痕疙瘩标本3例培养成纤维细胞,将第5代细胞分别常规贴壁培养和悬滴培养1周,对同一患者来源常规培养的第4代(P4)、第5代(P5)以及悬滴培养的第5代细胞(P5HD),采用多色流式细胞仪检测表面标志CD105、CD90、CD73和CD44的表达;并采用Real-time PCR的方法,检测上述表面标志,以及瘢痕疙瘩成纤维细胞相关功能基因CTGF、Col IA1和Col IA2的m RNA表达。结果 流式检测显示CD105+和CD73+CD90+CD105+细胞比例在P4和P5HD组均高于P5组,统计学分析显示有显著性差异,但P4和P5HD组之间无差异;而CD73+、CD90+和CD44+各组细胞比例无差异。Real-time PCR结果显示,各组细胞CD105的m RNA表达与流式结果一致;且各组CTGF和Col IA1表达差异与CD105一致。结论 悬滴培养法有助于维持体外瘢痕疙瘩成纤维细胞CD105,及其与纤维化和胶原合成相应功能基因的表达,从而保持细胞的生物学功能,但机制有待于进一步的研究。     

高代次人皮肤成纤维细胞的热原检测及致敏性研究

目的对多个样本的高代次(第2～10代)人皮肤成纤维细胞进行热原检测及致敏性研究,为其作为组织工程肌腱种子细胞的安全性提供相关依据。方法采用胶原酶消化法获取人皮肤成纤维细胞,在体外扩增培养、传代至第10代并收集培养细胞的上清液作为实验组,同时收集第2代细胞培养上清液作为对照,根据《医疗器械生物学评价》等国家规定的相关标准实验方法,分别对所收集的不同样本上清液进行热原检查和致敏性分析。结果各样本实验组和对照组动物均未发生明显的体温异常变化和皮肤过敏反应,根据国家相应标准判断为阴性结果。结论高代次(第10代以内)人皮肤成纤维细胞无热原,且不会产生致敏反应,用于组织工程肌腱构建具有较高的安全性。     

成骨诱导的人骨髓基质干细胞生物安全性评价

目的分析人骨髓基质干细胞在体外成骨诱导培养条件下，其染色体核型及端粒酶活性是否发生异常的改变，细胞是否产生致肿瘤性，为验证人骨髓基质干细胞作为骨组织工程种子细胞的安全性提供实验依据。方法采用常规细胞染色体G显带处理方法对3例成骨诱导培养的人骨髓基质干细胞样本进行核型分析，采用端粒酶活性PCRELISA检测法分析细胞的端粒酶活性，并通过裸鼠皮下致肿瘤试验对细胞的致肿瘤性进行研究。结果人骨髓基质于细胞体外成骨诱导培养至第7代，未发现染色体核型异常改变，相应的端粒酶活性也未出现异常增高。致肿瘤试验，在观察期内均未见结节形成或可疑病灶产生，符合围家医疗产品生物学评价标准的要求。结论人骨髓基质干细胞在体外长期成骨诱导培养条件下，染色体核型及端粒酶活性均未出现异常改变，且无致肿瘤性，符合作为骨组织工程种子细胞的生物安全性应用要求。     

巢蛋白在人真皮成纤维细胞中的表达

目的探讨巢蛋白(nestin)在人皮肤真皮组织成纤维细胞(adu lt hum an derm is fibrob lasts,HDF)中的表达。方法采用体外培养、免疫组织/细胞化学技术检测巢蛋白在6例成人皮肤真皮组织及体外培养3、5、7、10、12代龄皮肤真皮成纤维细胞中的表达,计数分析人皮肤真皮组织成纤维细胞中巢蛋白+成纤维细胞数量,权重法分析体外培养成纤维细胞各代间巢蛋白的表达差异。结果人皮肤真皮组织中,巢蛋白+成纤维细胞表达数量占成纤维细胞总数(9.5±3.0)%,其数量为(103.3±67.4)个/mm2。与人皮肤真皮组织巢蛋白+成纤维细胞的表达数量相比,体外培养的真皮成纤维细胞中巢蛋白+成纤维细胞数量明显升高,并出现代龄之间的差异(P<0.05),其中第7、10代细胞表达量高于第5、12代细胞,第3代细胞最少(P<0.05)。结论在人皮肤真皮组织成纤维细胞中存在着巢蛋白+成纤维细胞。体外培养条件下,人皮肤真皮巢蛋白+成纤维细胞数量增加,提示体外培养作为刺激因子可能使成纤维细胞反分化为其前体细胞,作为干细胞标志物的巢蛋白有可能成为用于成纤维前体细胞鉴定的标志物之一。     

四配子异源嵌合体导致的真两性畸形机制研究

目的 :报告 1例四配子异源嵌合体导致的真两性畸形并讨论其发病机制。　方法 :对 1例外生殖器模糊的患者外周血的淋巴细胞、经培养的皮肤成纤维细胞、两种不同性腺组织的成纤维细胞进行染色体核型分析 ,同时用X和Y染色体探针进行双色荧光原位杂交 (FISH) ;对患者红细胞血型、人类白细胞抗原 (HLA)和 77个短重复序列 (STR)微卫星标记进行检测 ;对患者性腺的 2种不同组织进行组织病理学检查 ;同时对患者的父母进行红细胞血型、HLA和STR检测。　结果 :患者外周血淋巴细胞、皮肤成纤维细胞、呈白色和黄色性腺组织的成纤维细胞染色体核型均为 4 6 ,XX/ 4 6 ,XY ;FISH检测所有细胞都显示了XX或XY的杂交信号。 4 6 ,XY的核型在外周血淋巴细胞、皮肤成纤维细胞和白色性腺组织的成纤维细胞中占优势 ;4 6 ,XX的核型在黄色性腺组织的成纤维细胞中占优势。外周血淋巴细胞及 3种不同组织培养物的STR位点检测、ABO血型分析和HLA检测都显示有 2个不同的单倍体来自父亲 ,1个单倍体来自母亲。组织病理学检查患者同一性腺上有两种不同组织 ,呈白色的组织是睾丸 ,呈黄色的组织是卵巢。　结论 :性腺组织病理学检查、染色体核型分析、FISH是鉴定真两性畸形患者的有效方法 ,红细胞血型、HLA和STR可为鉴定四配子异源嵌合体提供?     

连续传代人胚骨骼肌成肌细胞生物学特性研究

目的 探讨成肌细胞连续传代能力,选择适宜肌组织工程研究的成肌细胞。方法 常规传代培养人胚骨骼肌细胞,以生长曲线、融合率分别观察细胞增列、分化能力,探讨成纤维细胞沾染对传代细胞的影响。结果 第６代以内细胞成纤维细胞沾染轻,主要表现出成肌细胞的生长特性,增殖较旺盛,分化能力高。第８代￣第１６代细胞成纤维细胞沾染重,表现出成纤维细胞的生长特性,增殖速度明显加快但分化能力低。第２０代细胞退变明显,细胞增殖     

碱性成纤维细胞因子对猪弹性软骨细胞增殖和成软骨能力的影响

目的　研究碱性成纤维细胞因子 (b FGF)对体外培养的弹性软骨细胞增殖和成软骨能力的影响 ,探讨b FGF对软骨组织工程的意义。方法　细胞取自猪耳软骨 ,用含 1 0、2 5、50 μg/L3种浓度b FGF的培养液 ,体外培养至第 3代 ,从细胞形态、数量和基质分泌等方面进行研究。结果　含b FGF组细胞贴壁呈类成纤维细胞样 ,撤去b FGF ,细胞形态可恢复正常 ;含b FGF各组在第 3代 ,细胞总数是无b FGF组的 60～ 70倍 (P <0 .0 1 ) ,但 3种不同浓度b FGF对细胞增殖差异无显著性 ;培养液氨基糖氨多糖 (GAG)含量测定各组差异无显著性 ;免疫组织化学和原位杂交示各组第 3代软骨细胞仍有Ⅱ型胶原表达。结论　应用b FGF能在 3周内从少量软骨组织获取大量软骨细胞 ,且未改变细胞表型和功能 ,对软骨组织工程有重要意义 ;在 1 0、2 5、50 μg/L 3种浓度中 ,1 0μg/L为b FGF在软骨组织工程应用中的最佳浓度     

雷公藤提取物抑制增生性瘢痕成纤维细胞的实验研究

目的　为雷公藤提取物 (LLZ)治疗烧伤后增生性瘢痕提供实验依据。　方法　体外培养增生性瘢痕成纤维细胞 ,培养液中加入不同浓度的LLZ(5× 10 -3 、5× 10 -4、5× 10 -5、5× 10 -6g/L) ,2 4h后观察细胞形态、增殖活性及药物雷公藤提取物的细胞毒性。　结果　不同浓度的LLZ均能改变成纤维细胞形态 ,减少细胞数量 ,同时可明显降低细胞增殖活性。　结论　LLZ对烧伤后增生性瘢痕成纤维细胞形态和增殖均有明显的抑制作用 ,此作用并非毒性所至。     

Autologous and Allogeneic Skin Cell Grafts in the Treatment of Severely Burned Patients: Retrospective Clinical Study

Background

Transplantation of skin cells (keratinocytes and fibroblasts) cultured in vitro is a method of choice for the treatment of severe and extensive burns in patients with a deficit of donor sites for free split-thickness skin grafts, and when the grave medical condition of the patient excludes the possibility of an operation under general anesthetic. Appropriate amounts of keratinocytes and/or fibroblasts cultured in vitro are grafted as a suspension in platelet-leukocyte-rich gel directly on the prepared acceptor site. Approximately 3 weeks are needed for autologous cell culture to grow. Allogeneic cells are obtained from patients who died before their own autologous cell transplantation. Therefore allogeneic cells are considered as ready to use product. The aim of the study was to evaluate the efficiency of in vitro cultured autologous/allogeneic skin cell grafts in the treatment of burns.

Percutaneous transplantation of genetically-modified autologous fibroblasts in the rabbit femoral artery: A novel approach for cardiovascular gene therapy

OBJECTIVE: Arterial cell and gene therapies are promising strategies for the treatment of cardiovascular diseases; however, the optimal cell type and delivery technique for such treatment remain to be determined. The aim of the present study was to design a new approach for arterial cell and gene therapy in which genetically modified autologous skin fibroblasts are percutaneously delivered in stented rabbit femoral arteries in vivo. METHODS: Autologous skin fibroblasts underwent in vitro transfection with the cationic lipid FuGene and plasmids expressing the human form of the tissue inhibitor of metalloproteinase (hTIMP-1) or nls-LacZ reporter genes. RESULT: Transfection efficiency was about 50% and there were high levels of hTIMP-1 secretion up to 14 days after gene transfer. We demonstrated the feasibility of in vivo percutaneous transplantation of fluorescent fibroblasts in the rabbit femoral artery. Results were confirmed by scanning electron microscopy. In vivo local delivery of hTIMP-1-expressing fibroblasts in stented femoral arteries also resulted in high-levels of hTIMP-1 secretion ex vivo for 7 days. Fibroblast transplantation resulted in a modest increase in intimal hyperplasia at the target site, which was reversed with hTIMP-1-transfected fibroblasts. CONCLUSION: Percutaneous transplantation of genetically modified autologous fibroblasts could be used as a cellular platform for locoregional secretion of therapeutic proteins to treat either specific arterial diseases or the diseased organ (eg, the heart) supplied by the target artery. CLINICAL RELEVANCE: Cell and gene therapies are potential new treatments for cardiovascular diseases. We demonstrated that autologous fibroblasts could be easily harvested from a skin biopsy specimen, genetically modified in vitro with nonviral vectors, and percutaneously seeded in vivo in rabbit femoral arteries, leading to locoregional secretion of abundant amounts of recombinant proteins. This new approach has important advantages over alternative approaches that use endothelial cells, viral vectors, and intraoperative cell delivery. Clinical applications may include local treatment of atherosclerotic plaques or aneurysms and also treatment of the diseased organs supplied by the target artery (eg, ischemic or failing heart).     

Development of a reconstructed human skin model for angiogenesis

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.     

Skin cell isolation and expansion for cell transplantation is limited in patients using tobacco, alcohol, or are exhibiting diabetes mellitus

The aim of this exploratory study was to investigate the isolation and expansion of keratinocytes and fibroblasts from donors with certain medical histories. Biopsies were taken from donors (N=32) falling into one or more of the following categories: a history of heavy smoking and/or alcohol abuse, drug abuse, diabetes mellitus or steroid treatment. Cells from donors who did not fall into any of the above-mentioned categories were used as controls. Proliferation and growth behaviour of cells were analyzed by measurement of passage duration, absorbance (MTT-assay) and light microscopy. Donors with a specific medical history required larger biopsy areas than the control group for isolating a sufficient number of fibroblasts and keratinocytes. Times to confluence were significantly prolonged and absorbances (MTT) were significantly reduced in several donor groups when compared to control cultures. Biopsies from donors with steroid treatment, drug abuse and combined nicotine and alcohol abuse could not be established beyond passage 0 degrees or 1 degree, respectively. We conclude that isolation and expansion of skin cells from donors with certain medical histories may require larger biopsies, prolonged expansion times or may even result in failure. These findings may therefore be of clinical importance in the field of autologous skin cell transplantation.     

Transplantation of autologous cells and porous gelatin microcarriers to promote wound healing

Definitive treatment to achieve wound healing in major burns frequently include skin transplantation, where split-thickness skin grafts is considered gold standard. This method is associated with several drawbacks. To overcome these hurdles, efforts have been made to develop tissue engineered skin substitutes, often comprised of a combination of cells and biomaterials. In the present study, we aimed to investigate transplantation of autologous keratinocytes and fibroblasts seeded on porous gelatin microcarriers using a porcine wound model. Pre-seeded microcarriers were transplanted to a total of 168 surgical full-thickness wounds (2 cm diameter) on eight adult female pigs and covered with occlusive dressings. The experimental groups included wounds transplanted with microcarriers seeded with the combination of keratinocytes and fibroblasts, microcarriers seeded with each cell type individually, microcarriers without cells, each cell type in suspension, and NaCl control. Wounds were allowed to heal for one, two, four or eight weeks before being excised and fixated for subsequent histological and immunohistochemical analysis. In vitro, we confirmed that viable cells populate the surface and the pores of the microcarriers. In vivo, the microcarriers were to a large extent degraded after two weeks. After one week, all treatment groups, with the exception of microcarriers alone, displayed significantly thicker neo-epidermis compared to controls. After two weeks, wounds transplanted with microcarriers seeded with cells displayed significantly thicker neo-epidermis compared to controls. After four weeks there was no difference in the thickness of neo-epidermis. In conclusion, the experiments performed illustrate that autologous cells seeded on porous gelatin microcarriers stimulates the re-epithelialization of wounds. This method could be a promising candidate for skin transplantation. Future studies will focus on additional outcome parameters to evaluate long-term quality of healing following transplantation.     

体外培养的不同个体人肌腱细胞与皮肤成纤维细胞中胶原表达水平的比较研究

目的 对几种胶原在体外培养的不同个体的人第二代肌腱细胞和皮肤成纤维细胞中的表达水平进行比较研究,以探讨利用皮肤成纤维细胞作为组织工程肌腱构建种子细胞的可行性.方法 在无菌条件下取外伤病人术中修剪的废弃的肌腱和皮肤组织,经胶原酶和胰蛋白酶消化获取两种细胞,体外培养至第二代后,取两种细胞进行细胞学检测.采用基因芯片和RT-PCR技术对两种细胞几种胶原进行检测及比较.结果 体外培养的第二代人肌腱细胞和皮肤成纤维细胞两种细胞贴壁后形态相似.从基因水平可以看出体外培养的第二代人肌腱细胞和皮肤成纤维细胞在几种胶原的合成方面差异不大.结论 第二代人肌腱细胞与皮肤成纤维细胞的胶原表达基本相似,皮肤成纤维细胞有可能替代肌腱细胞,作为肌腱组织工程新的种子细胞来构建组织工程化肌腱.     

皮肤来源的前体细胞培养和向神经元分化的观察

目的 研究皮肤来源的前体细胞(SKPs)的体外培养方法，为神经移植提供一种新的细胞来源。方法 分离培养小鼠皮肤组织的细胞，在无血清的培养基中培养，用机械方法对细胞进行传代，免疫细胞化学方法对细胞进行鉴定。结果 从成年和幼年的小鼠皮肤组织中分离培养出SKPs,这种细胞在体外可以长期增殖和传代，体外培养可超过8代，这种细胞大部分表达纤维粘连蛋白，约50％的细胞表达巢蛋白。在有血清的培养基中培养，约5％的细胞分化为神经元样细胞，表达神经元特异性烯醇化酶和神经微丝，部分细胞分化为脂肪细胞，其余的细胞分化为成纤维细胞样细胞。结论 皮肤组织中存在的前体细胞可在体外稳定增殖，能够分化为神经细胞、脂肪细胞和成纤维细胞样细胞。这种前体细胞有潜力成为神经移植的一种细胞来源。     

自体组织工程化皮肤修复全层皮肤缺损的实验研究

目的为制成含表皮细胞与成纤维细胞的双层皮肤替代物,直接用于修复全层皮肤缺损.方法选用长枫杂交仔猪10只,酶消化法获取皮肤表皮细胞与成纤维细胞,将原代培养处于对数生长期的表皮细胞、成纤维细胞分别与30%氧化异丙烯F-127 混匀成细胞悬液后,种植聚羟基乙酸(polyglycolic acid,PGA)形成细胞-生物材料复合物,用于修复自体动物背部直径4 cm全层皮肤缺损,以单纯生物材料(PGA+氧化异丙烯)充填的创面作为对照组.修复术后1,2,4,8周取材,通过组织学和基底膜特殊染色等方法对新生组织进行评价.结果第1周新生组织即出现表皮与真皮2层结构,特殊染色观察到连续的基底膜.第2周表皮与真皮均较前增厚.修复后第8周组织工程化皮肤的形态结构均与正常皮肤相似.对照组则无皮肤形成,仅见大量肉芽组织.结论应用组织工程技术,以PGA+氧化异丙烯为表皮细胞、成纤维细胞载体构建的组织工程化皮肤可修复全层皮肤缺损.     

组织工程技术修复皮肤缺损的动物实验

目的　探索应用组织工程技术修复包括皮下组织的皮肤缺损的方法。方法　长枫杂交仔猪 2 0只 ,取腹部 2cm× 2cm全厚皮肤 ,酶消化法获取表皮细胞与成纤维细胞。经原代培养 ,将处于对数生长期的表皮细胞、成纤维细胞分别与 30 %氧化异丙烯F 1 2 7(pluronicF 1 2 7)混匀成细胞悬液后 ,种植于聚羟基乙酸 (polyglycolicacid ,PGA)形成细胞 生物材料复合物 ,用于修复自体背部直径 4cm皮肤缺损 ,以单纯生物材料 (PGA 30 %氧化异丙烯F 1 2 7)修复作为对照组。术后 1、2、4、8周取材 ,通过组织学方法评价新生组织。结果　实验组 :第 1周 ,形成含表皮与真皮两层结构的组织工程化皮肤 ;第 2周 ,表皮与真皮均较前增厚 ;第 8周 ,组织工程化皮肤的结构与正常皮肤相似 ,仅缺乏毛发、毛囊和汗腺等附属器。对照组 :则无皮肤形成 ,仅见大量肉芽组织。结论　以原代培养的表皮细胞与成纤维细胞作为种子细胞 ,PGA 30 %氧化异丙烯F 1 2 7为细胞载体的方法可修复皮肤缺损。     

Autologous fibroblast transplantation at the vesico-ureteral junction as potential reconstructive cell replacement in an animal model

Purpose

To evaluate the cellular survival of donor fibroblasts after transplantation at the vesico-ureteral junction (VUJ) and to analyse their potential for reconstructive cell replacement in an animal model as autologous fibroblasts have been used as soft tissue augmentation material for scared and damaged tissue.

Methods

Muscles biopsies were procured from the lower limb muscles of 4 pigs; cytoplasm of fibroblasts was labelled with nano-sized iron oxide particles. Six weeks after taking of the muscle biopsies, fibroblast transplantation was performed, 3 × 10⁶ cells suspended in transplantation medium (in 1-ml syringes) were injected at the VUJ using the modified STING technique. Animals were killed 8 weeks later; seeded fibroblasts were identified using prussian blue staining protocol; histological evaluation and morphological analysis were performed by light microscopy (Mayer’s haematoxylin-eosin staining); and bladders were scanned by MRI for visualization and localization of the iron-labelled donor cells.

Results

Donor fibroblast cell colonization and cellular viability at the VUJ was demonstrated by MRI and histochemically indicating cellular uptake of iron particles at the VUJ. It was also evident that transplanted fibroblasts integrate into the extracellular matrix of the distal ureter augmenting ureteral host tissue.

Conclusions

Labelled implanted autologous fibroblasts were visualized by staining procedure as well as MRI scan demonstrating persistence at the VUJ, suggesting that in vitro expanded fibroblasts survived in vivo after transplantation.