人脐带间充质干细胞促进创面愈合及体外诱导分化为表皮样细胞的实验研究

发布时间：2018-05-08 04:26

本文选题：脐带间充质细胞 + 创面愈合　；参考：《中国人民解放军军医进修学院》2011年博士论文

【摘要】：目的探讨从新生儿脐带华通氏胶中分离、培养人脐带间充质干细胞(umbilical cord mesenchymal stem cells, UCMSCs),并对其表型鉴定以及生物学特性进行检测。将UCMSCs与兔异体皮+自体微粒皮复合移植于兔皮肤全层缺损创面,观察UCMSCs促进微粒皮修复创面的效果,为其临床应用提供实验支持。观察UCMSCs在不同孔径的聚碳酸酯膜上生长和迁移情况,体外快速分离、培养表皮干细胞(epidermal stem cells, ESCs),将UCMSCs与ESCs分别接种在聚碳酸酯膜正反两面进行共培养,诱导UCMSCs分化为表皮样细胞,为以UCMSCs做为组织工程皮肤种子细胞提供实验依据。方法1取正常足月产健康新生儿脐带,采用酶消化法及组织块法的方法分离培养UCMSCs。以相差显微镜、HE染色及透射电镜观察UCMSCs形态及超微结构；以免疫荧光及流式细胞术鉴定细胞表面标志物；通过MTT法检测第1、3、5代细胞的增殖状况,并绘制生长曲线；流式细胞术检测第2、4代细胞周期；通过成脂以及成骨实验检测人脐带间充质干细胞多向分化的潜能。 2以腺病毒介导GFP基因体外转染UCMSCs 48h,于兔耳皮下注射移植,于3d、7d、14d、21d取材冰冻切片荧光显微镜下观察GFP表达情况。取成年日本大耳兔8只,随机抽取2只分为一组,同时构建兔背部急性皮肤全层缺损创面模型(每只兔2个创面),交换移植同等大小面积的反削断层皮,其真皮面均匀粘附兔自体微粒皮。所有兔头侧创面为UCMSCs移植组(A组)、尾侧为PBS空白自身对照组(B组)。于术后14d、21d观察创面情况,计算21d创面愈合率,进行统计学分析。于术后21d切取创面愈合区域组织,对标本行HE染色观察。 3取健康包皮环切术患者包皮皮片,采用中性蛋白酶和胰酶消化结合Ⅳ型胶原黏附法分离、培养hESCs,以相差显微镜及HE染色观察ESCs形态及生长特点;以免疫荧光染色鉴定细胞标志CK19、P63和β1-integrin。 4UCMSCs以丝裂霉素C处理后接种于常用的0.4、3.0和8.0μm三种膜孔径的6孔板Transwell插件中,培养7d,观察、计数三种孔径插件底部贴壁细胞,计算迁移率,并在扫描电镜下观察细胞在多孔膜上的生长、迁移情况。 5在聚碳酸酯膜底面预培养ESCs的Transwell插件内接种UCMSCs,使UCMSCs与ESCs在聚碳酸酯膜正反两面生长形成非直接接触式共培养,诱导UCMSCs向表皮细胞分化。共培养10d后,消化收集细胞,爬片后进行形态学观察；并进行免疫荧光染色检测细胞标志物CK19、P63和β1-integrin的变化；收集诱导后细胞通过Western blotting及qRT-PCR的方法,在mRNA和蛋白水平检测CK19、P63和β1-integrin表达情况；通过流式细胞术检测Transwell新型共培养与传统共培养诱导后细胞CK19表达率。结果1采用复合酶消化法和组织块法培养均从脐带华通氏胶中分离出成纤维样细胞,细胞呈梭形或多角形,呈旋涡状生长。透射电镜观察,细胞核大且不规则,核仁明显,细胞浆较少,细胞器以粗面内质网和线粒体为主。MTT法绘制生长曲线显示,各代细胞经过1d的潜伏期后,进入对数增殖期,第7d之后开始出现不同的接触抑制。细胞周期检测80%以上的细胞处于静止期。流式细胞仪检测显示分离培养的细胞表达CD44 (98.07%)、CD105 (95.29%)、CD73 (98.58%)、CD29 (99.50%)和HLA-Ⅰ(99.80%),不表达CD34 (0.59%)、CD31 (0.24%)、CD45 (1.91%)和HLA-DR (1.18%)。免疫荧光染色显示,细胞CD90和CD44阳性表达, CD31和CD45阴性表达。在体外特殊诱导条件下,这些细胞能够向脂肪细胞和成骨细胞分化,显示它们具有多向分化潜能。 2腺病毒介导的GFP基因转染UCMSCs后48h,荧光显微镜观察显示转染UCMSCs可有效表达GFP。于兔耳皮下注射移植后3d、7d时取材冰冻切片观察,UCMSCs较强表达GFP；14d、21d取材观察仍有GFP减弱表达。术后21d创面愈合率A组高于B组(P0.05)。愈合后创面取材病理学观察可见表皮层下大量不规则胶原纤维,成纤维细胞以及小血管,缺少皮肤附件；移植组表皮层可发现表皮钉突样结构,而对照组表皮层底部平坦,与基底结合较为疏松。 3中性蛋白酶和胰酶消化结合Ⅳ型胶原黏附法分离、培养ESCs,形态学观察可见hESCs呈扁圆状多角形,融合后呈铺路石样生长；免疫荧光染色鉴定细胞标志CK19、P63和β1-integrin均呈阳性表达。 4培养7d后,0.4μm、3.0μm和8.0μm三种Transwell插件内贴壁UCMSCs的迁移率分别为0、1.8%、8.0%,0.4μm孔径聚碳酸酯膜膜细胞迁移率为0。扫描电镜下观察到细胞在3.0μm和8.0gm孔径聚碳酸酯膜底面生长以及穿越微孔的现象,而在0.4μm孔径膜则未发现。 5UCMSCs与ESCs在聚碳酸酯膜正反两面非直接接触式共培养10d后,诱导组UCMSCs形态呈扁圆状多角形,而对照组细胞形态未变化；免疫荧光染色、Western blotting检测诱导组细胞CK19、P63阳性表达,对照组则为阴性；实时定量PCR检测CK19、P63 mRNA表达,诱导组表达水平显著高于对照组,差异具有统计学意义(P0.01)。而β1-integrin的各种表达诱导组细胞与对照组相比没有明显减弱。流式细胞术检测CK19阳性率,新型共培养诱导组(54.3%)与传统Transwell共培养诱导组(11.4%)相比,差异具有统计学意义(P0.01)。结论通过两种方法能够从脐带分离、培养出MSCs, UCMSCs增殖能力强,经细胞表面标志物、生物学特性以及多向分化能力检测,符合MSCs评价标准。UCMSCs可促进创面微粒皮生长,缩短创面愈合时间。Ⅳ型胶原黏附法能够快速、有效分离表皮干细胞；而0.4μm孔径的聚碳酸酯膜能够阻止细胞迁移通过,在该孔径聚碳酸酯膜正反两面近距离共培养UCMSCs和ESCs,能够较为有效地诱导UCMSCs向表皮样细胞分化。UCMSCs能够成为组织工程皮肤所需理想的种子细胞。
[Abstract]:Objective to study the isolation of umbilical cord mesenchymal stem cells (UCMSCs) from Huatong's gum in newborn umbilical cord, and to detect the phenotypic identification and biological characteristics of human umbilical cord mesenchymal stem cells (UCMSCs). The transplantation of UCMSCs with rabbit skin and autologous particle skin to the full-thickness skin defect of rabbit skin was carried out, and UCMSCs promoted the particle skin. To repair the effect of the wound, we provide experimental support for its clinical application. Observe the growth and migration of UCMSCs on polycarbonate membranes with different pore sizes, rapid separation in vitro, culture of epidermal stem cells (epidermal stem cells, ESCs), and co culture UCMSCs and ESCs in the positive and negative sides of polycarbonate membrane, and induce UCMSCs to differentiate into epidermis. The like cells provide an experimental basis for using UCMSCs as a seed cell for tissue-engineered skin.
Methods 1 healthy newborn umbilical cord was obtained from normal term, and UCMSCs. was isolated and cultured by enzyme digestion method and tissue block method. The morphology and ultrastructure of UCMSCs were observed by phase contrast microscope, HE staining and transmission electron microscopy. The cell surface markers were identified by immunofluorescence and flow cytometry, and the proliferation of 1,3,5 cells was detected by MTT method. The growth curve was plotted and the cell cycle of the 2,4 generation was detected by flow cytometry; the multidirectional differentiation potential of human umbilical cord mesenchymal stem cells was detected by lipid formation and osteogenesis.
2 transfection of GFP gene with adenovirus mediated UCMSCs 48h in vitro and subcutaneous injection in rabbit ear. The expression of GFP in 3D, 7d, 14d, 21d was observed under frozen section fluorescence microscope. 8 adult Japanese big ear rabbits were randomly selected and 2 were randomly divided into one group. At the same time, the rabbit model of the full layer defect on the skin of the rabbit back (2 wounds of each rabbit) was constructed, and the exchange shift was carried out. The true skin surface adhered to the rabbit autogenous skin with the same size and area. All the rabbit head side wounds were UCMSCs transplantation group (group A), and the tail side was PBS blank control group (group B). After 14d, 21d, the wound healing rate was observed after 14d and 21d, and statistical analysis was made to calculate the healing rate of the 21d wound. After 21d, the wound healing regional tissue was cut by 21d, right after operation. The mark was observed by HE staining.
3 the patients with circumcision of healthy circumcision were separated by neutral protease and trypsin digestion combined with type IV collagen adhesion, and hESCs was cultured. The morphology and growth characteristics of ESCs were observed by phase contrast microscope and HE staining. Immunofluorescence staining was used to identify cell markers CK19, P63 and beta 1-integrin..
4UCMSCs was treated with mitomycin C and inoculated into 6 pore Transwell plug-ins with three kinds of pore diameter of 0.4,3.0 and 8 mu m, and cultured 7d. Observe and count the wall cells at the bottom of three kinds of aperture plug-ins, calculate the mobility, and observe the growth and migration of the cells on the porous membrane under scanning electron microscope.
5 inoculating UCMSCs in the Transwell plug-in of pre culture ESCs on the bottom of polycarbonate membrane, which makes UCMSCs and ESCs grow in the positive and negative sides of the polycarbonate membrane to form a non direct contact co culture and induce the differentiation of UCMSCs into the epidermal cells. After co culture 10d, the cells are collected and the cells are collected to observe the morphologic observation after climbing the tablet, and the immunofluorescence staining is used to detect the cells. The changes in the markers CK19, P63 and beta 1-integrin; the cells were collected and induced by Western blotting and qRT-PCR to detect CK19, P63 and beta 1-integrin expressions at mRNA and protein levels, and the cell CK19 expression rate was detected by the flow cytometry for the Transwell new co culture and the traditional co culture.
Results 1 the fibroblast like cells were isolated from the Huatong's gum with compound enzyme digestion method and tissue block method. The cells were spindle shaped or polygonal, and the cells were vortexed. The cell nuclei were large and irregular, the nucleolus was obvious, and the cytoplasm was less. The growth curves were plotted by the.MTT method of rough surface endoplasmic reticulum and mitochondria. After the incubation period of 1D, each cell entered the logarithmic proliferation period. After 7d, different contact inhibition began to appear. Cell cycle detection more than 80% cells were at rest. Flow cytometry showed that the cultured cells expressed CD44 (98.07%), CD105 (95.29%), CD73 (98.58%), CD29 (99.50%) and HLA- I (99.80%). CD34 (0.59%), CD31 (0.24%), CD45 (1.91%) and HLA-DR (1.18%). Immunofluorescence staining showed that the positive expression of CD90 and CD44 and negative expression of CD31 and CD45 were expressed. Under special induction conditions, these cells could differentiate into adipocytes and osteoblasts, showing their multipotential differentiation potential.
2 adenovirus mediated GFP gene was transfected to 48h after UCMSCs, and the fluorescence microscope showed that transfection of UCMSCs could effectively express GFP. in 3D after subcutaneous injection of rabbit ear, and the frozen section was observed in 7d, and UCMSCs strongly expressed GFP; 14d, 21d observation still had the attenuation of GFP. Pathological observation showed a large number of irregular collagen fibers under the epidermis, fibroblasts and small vessels and lack of skin appendages. The epidermis of the transplantation group could find the peg like structure of epidermis, while the epidermis of the control group was flat and loosely combined with the base.
3 neutral protease and trypsin digestion combined with type IV collagen adhesion method were isolated and cultured for ESCs. Morphological observation showed that hESCs showed flat polygonal polygon. After fusion, it was paved stone like growth. Immunofluorescence staining was used to identify cell markers CK19, P63 and beta 1-integrin were all positive.
4 after culture of 7D, the migration rates of UCMSCs in the three Transwell plug-ins of 0.4, 3, and 8 m were 0,1.8%, 8%, and 0.4 mu m membrane cell membrane cell migration rate was 0. scanning electron microscopy to observe the growth of the cells at the bottom of the 3 and 8.0gm aperture polycarbonate membrane and through the micropores, while the 0.4 mu m aperture membrane was not found.
After the 5UCMSCs and ESCs were co cultured on the positive and negative two sides of the polycarbonate membrane, the UCMSCs morphology of the induced group was flat round polygon, but the cell morphology of the control group was not changed, and the immunofluorescence staining, Western blotting detected the cells CK19, P63 positive and negative in the control group, and the real-time quantitative PCR detection CK19, P63 mRNA expression, The expression level of the induced group was significantly higher than that in the control group (P0.01), but the cells in the induction group of beta 1-integrin were not significantly decreased compared with the control group. The positive rate of CK19 in the flow cytometry, the new co culture induction group (54.3%) and the traditional Transwell Co culture induction group (11.4%) were statistically significant. Meaning (P0.01).
Conclusion by two methods, MSCs can be isolated from the umbilical cord, and the proliferation ability of UCMSCs is strong. The cell surface markers, biological characteristics and multidirectional differentiation ability are detected. The MSCs evaluation standard.UCMSCs can promote the growth of the wound particle skin and shorten the healing time of the wound. The polycarbonate membrane with 0.4 micron m aperture can prevent cell migration from passing through, and co culture UCMSCs and ESCs in the near and opposite sides of the porous polycarbonate membrane. It can effectively induce UCMSCs to differentiate into epidermal like cells and become ideal seed cells for tissue engineering skin.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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