应用人自体血清培养人口腔黏膜上皮的实验研究

目的 研究人自体血清培养人口腔黏膜移植生长的生物学特性，为组织工程化尿道提供新材料。方法 将人自体血清培养黏膜移植于裸鼠体内，分别于移植后2、3、4、6周观察培养黏膜生长与转归，应用anti—HLA免疫荧光鉴定成活黏膜组织属性，应用抗人Ⅳ型胶原及抗人层黏蛋白为基底膜形成指标。结果 裸鼠体内移植培养黏膜成活生长分化良好，anti—HLA免疫荧光证实为移植的培养人黏膜组织；免疫组化发现移植后3周开始形成基底膜，4周形成完整的基底膜。结论 自体血清培养的人口腔黏膜可形成功能完整的上皮组织。     

纤维蛋白凝胶移植培养口腔黏膜细胞的研究

目的 为培养上皮细胞寻找良好的移植载体.方法 用含自体血清培养基培养人口腔黏膜角质细胞,实验组细胞置于纤维蛋白凝胶中,对照组细胞置于DMEM:F12细胞培养液中,将悬液细胞以1×106个/ml浓度分别移植于裸鼠皮下和创面,于移植后1、2、3、4、6周取材,分别行苏木素-伊红染色及冰冻切片抗人HLA-Ⅰ免疫荧光染色及抗人Ⅳ型胶原及抗人层黏蛋白免疫组织化学检查.结果 皮下移植时,凝胶组角质细胞逐步增殖分化形成较大上皮管腔(4.62×103个/cm2),组织有多量血管形成(5.72×103个/cm2),对照组中上皮管腔(1.76×103 个/cm2)及血管数量少(0.88×103 个/cm2),两者差异有统计学意义(P＜0.01),管腔上皮抗人-HLA免疫荧光阳性;实验组创面愈合平均时间为11 d,愈合处皮肤粗糙、角化,抗人-HLA免疫荧光强阳性,免疫组织化学染色示基底膜形成完整,对照组愈合时间为18.5 d,愈合创面凹陷、光滑无角化,抗人-HLA免疫荧光弱阳性,两组创面愈合时间差异有统计学意义(P＜0.05).结论 纤维蛋白凝胶可促进移植黏膜上皮细胞的增殖分化并形成功能完备的上皮组织,是上皮细胞移植的良好载体.     

不同来源血清体外培养骨髓间充质干细胞的研究

目的 适当的血清浓度可维持骨髓问充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的体外培养,而不同来源的血清对BMSCs的培养效果是否小同.本研究探讨自体血清、脐带血清、AB血清、胎牛血清在体外培养人BMSCs的可行性.方法 Ficoll密度梯度离心法结合贴壁法分离纯化人BMSCs,在培养过程中分别加入白体血清、脐带血清、AB血清、胎牛血清,观察比较细胞形态、表面抗原、生长曲线及各组在诱导剂作用下表面抗原和诱导分化的相关性.结果 从BMSCs的形态学、生长曲线、表面抗原、诱导分化上看各组无明显差异,生长状况良好.结论 自体血清、脐带血清、AB血清、胎牛血清均可维持BMSCs的体外培养,而自体血清解决异种和异体血清带来的一些潜在的风险,更为安全.     

不同血清浓度对淋巴管内皮细胞培养的影响

目的探讨不同浓度的血清在人淋巴管内皮细胞培养中的作用,得出最优血清浓度,优化淋巴管内皮细胞(Lymphatic endothelial cells,LECs)的培养方案。方法通过免疫磁珠法获得人LECs,将不同浓度(0%、2.5%、5%、7.5%、10%)的胎牛血清加入EGM-2培养液中,培养LECs,并通过免疫荧光、RT-PCR、流式分析、成管试验、吞脂试验等方法,鉴定LECs特异性标记及功能。结果通过免疫磁珠法获得的人LECs表达特异性指征和功能。不同浓度的血清培养液对人LECs的吞脂功能无明显影响,无血清组丧失淋巴管成管能力,7.5%血清组成管能力和增殖能力最强。结论人LECs培养过程中,血清在细胞增殖和功能保持方面具有重要作用,最适宜的血清浓度是7.5%,此浓度血清培养的细胞其增殖能力以及功能都是最优的。     

烧伤前后大鼠血清对骨髓基质细胞生物学行为的影响

目的:观察烧伤前后大鼠血清对体外培养的大鼠骨髓基质细胞的生物学行为的影响,为骨髓基质细胞在创伤修复中的应用奠定实验基础.方法:取Wistar大鼠,处死分离骨髓,原代培养骨髓基质细胞,传代后分别用含有10%胎牛血清、正常大鼠血清、烧伤后3d大鼠血清和烧伤后14d大鼠血清的F-12培养基培养,用MTT法测定其生长曲线,PI染色,流式细胞仪分析细胞周期、DNA含量和细胞凋亡率.结果:用含有10%大鼠血清的F-12培养基传代培养的细胞生长曲线相似,但与含有FCS的F-12培养基传代培养的明显不同.前者倍增时间缩短,N组、B1组、B2组细胞群体倍增时间分别为23.7h、18.6h、20.2h.平台期所能达到的细胞总数明显增加.烧伤后大鼠血清处理过的细胞中G0/G1期细胞比例明显低于其它两组,而S期细胞的比例则相反.正常大鼠血清处理过的细胞中G2+M期细胞的比例则比其它组明显升高.大鼠血清处理过的细胞中DNA含量明显高于胎牛血清培养的细胞,而烧伤后的大鼠血清处理过的细胞中DNA含量则明显高于正常大鼠血清处理过的细胞.而烧伤后3d的大鼠血清处理过的细胞的凋亡率比其它组细胞明显升高.结论:大鼠血清比胎牛血清能更好地促进大鼠体外培养的骨髓基质细胞生长;烧伤大鼠血清内可能含有多种能影响骨髓基质细胞生长的物质,且其含量随烧伤的时间改变而变化,对骨髓基质细胞生长的影响是复杂的.     

同种异体血清在体外培养骨髓间充质干细胞的作用研究

[目的]适当的血清浓度可维持骨髓间充质干细胞(MSCs)的体外培养,血清浓度过低不利于细胞的生长,而高浓度血清则易引起细胞分化。而血清的不同来源对MSCs的培养同样产生影响。传统的培养方法用胎牛血清作为营养支持,临床应用时存在潜在的风险。本研究探讨同种异体血清在体外培养人骨髓间充质干细胞(hBM-SCs)的可行性。[方法]无菌条件下收集人骨髓悬液,分离hBMSCs,分别用含胎牛血清和人血清的培养基培养。分别测定2种方法培养的hBMSCs细胞生长曲线;采用流式细胞仪检测分析培养的hBMSCs表面抗原类型;并将培养的hBMSCs诱导分化为软骨细胞及神经细胞,诱导后的软骨细胞及神经细胞采用免疫组化染色分析。[结果]2种方法培养的间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性,表达强度无显著性差异;人血清培养的hBMSCs细胞生长速度快于胎牛血清培养组,但分化效率低于后者。[结论]通过生长特性、表面抗原表达以及分化潜能等方面的对比研究,人血清培养hBMSCs与胎牛血清培养差别不大,可以作为一种适合临床的安全培养方法。     

组织工程人口腔黏膜的制备及异体移植的临床应用

目的观察自行制备的复层组织工程人口腔黏膜移植后生长情况.方法取3月龄患儿唇裂术中切取的多余口腔黏膜组织,分离成纤维细胞与上皮细胞,分别接种于聚乳酸/聚羟基乙酸共聚物(polylactic/glycolic acid copolymer,PLGA)胶原复合膜上培养,后将其移至气液面进行复合,制成复层组织工程口腔黏膜.7例行口腔内良性肿瘤切除后遗留黏膜缺损的患者,缺损范围为1.8 cm×1.6 cm～3.2 cm×2.6 cm.采用组织工程口腔黏膜修复,术后观察其生长情况.1例志愿者在移植术后18和30 d分别取移植部位黏膜行组织学观察.结果制备的组织工程口腔黏膜生长良好,具有上皮层和上皮下层双层结构,之间为PLGA膜;上皮层5～6层细胞,角蛋白染色阳性,上皮下层3～7层细胞,角蛋白染色阴性.7例患者移植修复术后10 d,组织工程口腔黏膜与创面生长良好,颜色较正常黏膜深;18 d后仍可辨出移植区与正常黏膜;30 d后无法区别界限,创面均Ⅰ期愈合,无明显瘢痕组织.组织学观察:18 d创面上皮层及下方肉芽组织生长良好,毛细血管增生有上皮钉突形成,成纤维细胞卵圆形;30 d胶原化更明显,移植区结构与周边正常组织相似.结论应用组织工程口腔黏膜异体移植修复后,黏膜生长良好,但愈合组织上皮的来源还需进一步研究.     

转化生长因子-β1和血清对人骨髓基质干细胞增殖及向软骨细胞诱导分化的影响

目的探讨转化生长因子-β1(TGF-β1)和不同浓度胎牛血清对体外培养人骨髓基质干细胞(BMSCs)增殖及向软骨细胞诱导分化的作用,为软骨组织工程提供有用的种子细胞。方法分别采集3例志愿者骨髓各6mL,用Percoll细胞分离液分离,收集单个核细胞,在含10%胎牛血清的低糖DMEM培养液中培养扩增。将第3代细胞分为3组:A组为对照组,B组为含5%胎牛血清的诱导培养基组,C组为含10%胎牛血清的诱导培养基组。倒置显微镜下观察细胞生长状况,免疫组化检测Ⅱ型胶原分泌,原位杂交检测Ⅱ型胶原mRNA表达,3H标记的胸腺嘧啶脱氧核苷(3H-TdR)掺入实验检测细胞的增殖情况。结果经密度梯度离心后,活细胞比例在96%以上。贴壁后的细胞形态均一,由梭形向多角形转变。B组细胞在诱导培养7d后Ⅱ型胶原免疫组化均为阳性,原位杂交可检测到Ⅱ型胶原mRNA的表达,而A、C两组分别为阴性和弱阳性。三组细胞3H-TdR摄入量的大小顺序为C>B>A。结论密度梯度离心法是一种高效、可大量获取人BMSCs的方法,细胞在体外生长稳定;低浓度的胎牛血清和10ng/mL的TGF-β1联合作用既可促进人BMSCs的增殖,又可诱导其向软骨细胞方向分化,而高浓度胎牛血清仅对细胞的增殖有促进作用。     

血清对体外培养成年人关节软骨细胞生长的影响

目的观察不同血清及血清浓度对成人关节软骨的细胞（AHAC）生长的影响，为软骨细胞移植的临床应用提供血清选择。方法用无血清、胎牛血清（FBS）和人AB血清及不同浓度血清在加或不加生长因子条件下培养成年人关节软骨细胞，比较细胞增殖和生长情况。结果对于AHAC，人AB血清培养优于FBS；合适的人AB血为10％；合适的FBS浓度为20％；无血清培养细胞增殖非常缓慢；不加因子用人AB血清培养细胞梭形化明显，加用生长因子后与使用FBS在形态上一致。结论10％浓度的人血清是AHAC体外培养的合适血清和浓度选择。     

应用组织工程口腔黏膜重建尿道的初步实验研究

目的 探讨兔口腔黏膜细胞的体外培养方法,并以其为种子细胞与异体膀胱黏膜下脱细胞基质(bladder acellular matrix graft,BAMG)复合,构建组织工程尿道. 方法 18只10周龄雄性新西兰大耳白兔,体重0.3～0.5 kg.取其中12只兔口腔黏膜组织,分离获得口腔黏膜细胞.将口腔黏膜细胞分别接种于有3T3细胞及无3T3细胞的培养皿进行培养,接种后第2天于倒置相差显微镜下观察细胞形态变化,绘制细胞生长曲线,观察生长增殖情况,并行免疫荧光组织化学染色鉴定.取余6只兔膀胱,经脱细胞处理制备BAMG,随机取1 cm×1 cm组织行HE染色,观察脱细胞效果.将接种于有3T3细胞培养皿培养获得的第2代口腔黏膜细胞种植于BAMG上,培养1周后,行HE染色及扫描电镜观察口腔黏膜细胞与BAMG的复合状况. 结果 倒置相差显微镜下观察,接种于有3T3细胞培养皿的口腔黏膜细胞可传至7～8代,其细胞形态均一,功能良好;无3T3细胞培养皿的口腔黏膜细胞形态多样,传至第2代时,即开始出现老化.细胞生长曲线显示,两种方法培养的口腔黏膜细胞均于第8天开始呈对数增长,第14天达峰值.接种于有3T3细胞培养皿的口腔黏膜细胞,培养至融合时所获得细胞数量明显多于接种于无3T3细胞培养皿的u腔黏膜细胞.两种方法培养的口腔黏膜细胞行免疫荧光染色,均呈绿色荧光.HE染色观察,BAMG经脱细胞处理后未见细胞,脱细胞效果良好.复合培养1周后,HE染色及扫描电镜观察口腔黏膜细胞与BAMG复合良好. 结论 兔口腔黏膜细胞可在体外成功培养、扩增,与同种异体BAMG复合后生长良好,为构建组织工程尿道提供了一种新的选择.     

不同血清对成人骨髓基质干细胞成骨诱导分化的影响

[目的]探讨三种不同来源血清对体外培养成人骨髓基质干细胞(hBMSCs)向成骨诱导分化的作用.[方法]将体外第3代hBMSCs向成骨诱导分化培养,分为胎牛血清组(对照组)、AB 血清组和自体血清组.对比观察细胞碱性磷酸酶(ALP)染色、钙结节染色.诱导培养后4、7、14 d 和 21 d,各血清组分别进行钙黄绿素法荧光显微镜动态观察矿盐沉积,检测 ALP 活性,实时荧光定量 PCR(RT-qPCR)法检测成骨基因(ALP)、骨桥蛋白(OPN)和骨钙素(OCN)的表达.[结果]ALP 染色和钙结节染色结果显示,与自体血清(AS)组、胎牛血清(FBS)组相比,AB 血清(ABS)组明显提高了 hBMSCs 的染色阳性率.荧光显微镜下观察诱导 21 d 的 hBMSCs,ABS 组较 FBS 组、AS 组呈现更多的钙盐沉积.ALP 活性结果显示,同一时间点 ABS 组的 ALP 活性均明显高于 FBS 组和 AS 组,差异有统计意义(P<0.05).RT-qPCR 结果显示,在7、14 d 和 21 d,ABS 组 ALP、OPN 和 OCN 成骨基因表达均明显高于 FBS 组、AS 组,其中,ALP 基因表达在 7 d 出现峰值,OPN 基因表达在 14 d 出现峰值,OCN 基因表达在 21 d 出现峰值,差异均有统计学意义(P<0.05).[结论]ABS 对 hBMSCs 成骨分化作用较 FBS、AS 明显增强.ABS 有望替代 FBS 建立符合骨组织工程临床应用要求的体外 hBMSCs 培养体系.     

Motility and growth of human bone-marrow mesenchymal stem cells during ex vivo expansion in autologous serum

Human bone-marrow mesenchymal stem cells have an important role in the repair of musculoskeletal tissues by migrating from the bone marrow into the injured site and undergoing differentiation. We investigated the use of autologous human serum as a substitute for fetal bovine serum in the ex vivo expansion medium to avoid the transmission of dangerous transfectants during clinical reconstruction procedures. Autologous human serum was as effective in stimulating growth of bone-marrow stem cells as fetal bovine serum. Furthermore, medium supplemented with autologous human serum was more effective in promoting motility than medium with fetal bovine serum in all cases. Addition of B-fibroblast growth factor to medium with human serum stimulated growth, but not motility. Our results suggest that autologous human serum may provide sufficient ex vivo expansion of human bone-marrow mesenchymal stem cells possessing multidifferentiation potential and may be better than fetal bovine serum in preserving high motility.     

Effect of media and growth promoters on in vitro cultivation of normal murine urinary bladder epithelium

Growth potential of normal murine epithelium was investigated by means of 3 culture media: RPMI 1640 with fetal calf serum, Dulbecco's with fetal calf serum and McCoy's with horse serum. The effect of 3 growth promoters was tested separately with each of the 3 media. A total of 886 explants were cultured from 29 murine bladders and epithelial outgrowth was obtained in 35.2 per cent. The growth medium 1640 with 20 per cent fetal calf serum and epithelial growth factor produced the most abundant outgrowth of explants. McCoy's medium containing 15 per cent horse serum produced significantly lower outgrowth compared to the medium containing fetal calf serum (p less than 0.001). Epidermal growth factor has a stimulating effect and horse serum has an inhibitory effect on growth of normal murine epithelial cells.     

Characterization of wound drainage fluids as a source of soluble factors associated with wound healing: comparison with platelet-rich plasma and potential use in cell culture

Wound fluids, human serum from platelet-poor and platelet-rich plasma (SPPP and SPRP), contain various soluble factors involved in cell growth and proliferation. Levels of cytokines, chemokines, and matrix metalloproteinases (MMPs) in drainage fluids (DFs) harvested from subcutaneous wounds, punctured fluids (PF) from seroma, and SPPP were measured. SPPP and SPRP from four healthy volunteers were also subjected to the analysis. Biochemical profiles of DF reflected the sequential stages of wound healing. Early-phase DF contained high concentrations of basic fibroblast growth factor and platelet-derived growth factor and EGF. The levels of keratinocyte growth factor, interleukin-6, and MMP-8 in DF peaked on days 2-3, while vascular endothelial growth factor, hepatocyte growth factor, interleukin-8, and MMP-1 increased over time during days 0-6. Punctured fluids contained high levels of TGF-beta1, keratinocyte growth factor, vascular endothelial growth factor, hepatocyte growth factor, and MMP-1. Experiments using human adipose-derived stem cells and dermal fibroblasts cultured in media containing various concentrations of DF and fetal bovine serum suggested that for some cell types, DF-contained growth factors that are not obtained from SPRP could be used to supplement or substitute for serum in culture media. SPRP and DF are economical ready-made mixtures of serum and autologous soluble factors, and may be differentially useful for regenerative therapies.     

表皮黑素细胞专用培养基组分与细胞外信号调节激酶蛋白磷酸化关系初步观察

目的了解表皮黑素细胞专用培养基HU16中维持细胞生长的主要物质人重组碱性成纤维细胞生长因子(bFGF)、FBS和环腺苷酸激动剂与细胞外信号调节激酶(ERK1/2)蛋白磷酸化的关系。方法从环切包皮原代培养人表皮黑素细胞,用western blot检测去bFGF、去FBS以及去环腺苷酸激动剂培养基培养后ERK1/2蛋白磷酸化水平的改变。结果去bFGF和去FBS处理后,表皮黑素细胞ERK1/2蛋白的磷酸化水平明显下降(P0.05),恢复完全培养基培养后0.5h,ERK1/2蛋白的磷酸化水平即重新上升至处理前水平。去环腺苷酸激动剂处理后,ERK1/2蛋白的磷酸化水平无明显改变。结论 HU16培养基中bFGF和FBS的浓度可影响体外培养的人表皮黑素细胞中ERK1/2蛋白的磷酸化水平。     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.     

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

Traditionally, culture medium is supplemented with foetal bovine serum (FBS). However, in cultures of osteoblasts intended for human re-implantation, such serum presents potential risks of foreign protein contamination and transmission of viral or prionrelated material, if used. We cultured human osteoblasts from 16 patients in 10% autologous human serum, 10% pooled human serum, 10% FBS or 2% Ultroser G. Non-synthetic sera were tested in both heat-treated and non-heat-treated forms. We determined cell growth and osteoblast phenotype. Cell proliferation in all types of human serum was significantly greater than in FBS. This was most marked in heat-treated autologous human serum. Cells cultured in Ultroser G had less proliferation than all other groups. The phenotypic tests showed that cells cultured in human and foetal bovine serum displayed an osteoblast phenotype, with greater protein expression in cells cultured in human serum. We conclude that culture of human osteoblasts in autologous human serum enhances cell proliferation, while maintaining an osteoblast phenotype. These findings have implications for the use of cultured osteoblasts in self-cell therapy. Human osteoblast growth is supported by autologous human serum, which allows re-implantation of cultured cells, while avoiding the risk of foreign protein carry-over with enhancement of cell proliferation.