体外诱导人胎盘间充质干细胞分化为软骨细胞的实验研究

发布时间：2018-05-21 08:22

本文选题：胎盘 + 间充质干细胞　；参考：《中国医科大学》2008年硕士论文

【摘要】： 目的间充质干细胞(Mesenchymal stem cells,MSCs)是属于中胚层的一类多能干细胞,主要存在于结缔组织和器官间质中,以骨髓组织中含量最为丰富。近年来,随着人们对间充质干细胞生物学特性及功能的深入研究,已成功地从人骨髓、外周血、肌肉、脂肪、脐血、羊水及胎儿组织中分离并鉴定出MSCs。胎盘作为胚胎发育中维系母体和胎儿氧气及营养物质交换的重要暂时性器官,由起源于细胞滋养层和胚外中胚层的胎儿丛密绒毛膜和母体子宫基蜕膜共同组成,无论从解剖结构还是在发育行为上,都包含了较为幼稚的胚胎及趋于成熟的成体干细胞成分;胎盘在胎儿娩出后即完成使命,成为“废弃”物,对其研究不会涉及伦理道德问题,因此目前已成为寻找人类间充质干细胞新来源及提高临床应用效果的研究热点。本文就人胎盘来源的间充质干细胞(Placenta-derived Mesenchymal Stem Cells,PMSCs)体外分离和培养的方法及PMSCs在单层培养中向软骨细胞定向分化的条件作以论述。方法 1、人胎盘间充质干细胞的提取取足月剖宫产胎儿的胎盘,剥离子体蜕膜,剪取蜕膜下组织,PBS冲洗两次,室温静置10min,将组织块剪碎约1mm~3,均匀置于培养器底部,应用10%DMEM培养基培养于37℃5%CO_2培养箱,每3d换液一次,待细胞沿培养器底部生长面积达80%～90%时,按常规方法传代。 2、胎盘细胞表型测定取第3代细胞,以0.25%胰蛋白酶消化3min后,调整细胞数为1×10~6/L,加入5μL小鼠血清封闭15min后,加入鼠抗人PE标记的单克隆抗体,冰上避光反应20min,PBS洗2次后,4℃1000r/min离心5min,弃上清后加入200μL冷PBS吹打混匀后,流式细胞仪检测。 3、诱导分化将第3代胎盘来源的间充质干细胞胰酶消化后制成细胞悬液,离心弃上清后PBS洗3遍,分别以2×10~4/cm~2密度接种于预先以0.1%明胶涂抹的六孔细胞培养液中,待细胞贴壁后改换软骨诱导剂,置于37℃5%CO_2培养箱中培养,每3d换液一次。 4、甲苯胺蓝染色取培养21d的两组细胞,PBS换2遍后,4%多聚甲醛固定15～30min,PBS洗3遍后,1%甲苯胺蓝染色2～4h,95%酒精洗3遍后光镜下观察。 5、免疫细胞化学检测Ⅱ型胶原表达具体步骤按SABC染色试剂盒说明书操作,并弃孔板中培养液,PBS(0.02mol/LpH7.2～7.6)液洗3遍,4%多聚甲醛固定30min,0.3%H_2O_2灭活内源性酶,BSA封闭后分别加入1:200稀释的兔抗人-抗Ⅱ型胶原,4℃孵育过夜,用山羊抗兔-小鼠IgG孵育1h,DAB显色剂显色,苏木精复染后光镜下观察。结果组织块培养11d左右,可见少量细胞爬出,呈圆形或多角形,随细胞数目增多,细胞呈旋涡状生长,胞体变细长,形态类似成纤维细胞,培养3w左右,细胞可达80%融合,细胞传代贴壁后,重新长成梭形细胞,该细胞传至20代仍可稳定生长。流式细胞仪检测显示,来源于人胎盘组织的第3代细胞表达CD13、CD44、CD73、CD90、CD166、HLA-AB,不表达CD34、CD45、HLA-DR,与骨髓来源的间充质干细胞具有相似的细胞表面标志。细胞在诱导开始时,细胞增殖速度较慢,诱导一周时即已停止增殖,细胞形态发生改变,细胞体积变大,部分细胞由原来的梭形逐渐变成多边形,细胞形态不一,整个诱导分化过程细胞未传代。细胞在诱导21d时甲苯胺蓝染色可见细胞间基质呈蓝紫色。Ⅱ型胶原免疫细胞化学检测均发现细胞胞浆内有棕黄色颗粒的阳性结果。结论 1、人胎盘来源的间充质干细胞可成功在体外培养并大量扩增,生物学性状稳定,是组织工程优良的种子细胞来源之一。 2、本实验在单层培养条件下,成功将人胎盘间充质干细胞诱导分化成软骨细胞。经诱导培养后形成的软骨细胞具有软骨细胞的形态特征,并能分泌软骨细胞特异性Ⅱ型胶原。
[Abstract]:objective
Mesenchymal stem cells (MSCs) is a kind of pluripotent stem cell belonging to the mesoderm. It mainly exists in connective tissue and organ interstitium, and is the most abundant in bone marrow. In recent years, human bone marrow, peripheral blood and muscle have been successfully developed with the in-depth study of the biological characteristics and functions of mesenchymal stem cells. The isolation and identification of fat, umbilical blood, amniotic fluid and fetal tissues and the identification of MSCs. placenta as an important temporary organ for the exchange of oxygen and nutrients in the embryonic development of the embryo and the fetus, is composed of the fetal plexus and the mother's uterine decidua, which originate in the cell trophoblast and the mesoderm of the embryo, and from the mother's uterus decidua. The development behavior includes the more immature embryos and mature adult stem cells. The placenta completes its mission and becomes "abandoned" after the birth of the fetus. The research does not involve the ethical and moral problems. Therefore, it has become a hot spot for finding new sources of human mesenchymal stem cells and improving the effect of clinical application. In this paper, the methods of isolation and culture of Placenta-derived Mesenchymal Stem Cells (PMSCs) from human placenta and the conditions for directing differentiation of PMSCs into chondrocytes in monolayer culture are discussed.
Method
1, extraction of human placental mesenchymal stem cells
Take the placenta of the term cesarean section, peel the decidua, cut the decidua, cut the tissue under the decidua, rinse PBS for two times. Shi Wenjing set up 10min, cut the tissue block up to about 1mm~3, and put it at the bottom of the incubator evenly. The 10%DMEM medium is used to cultivate the 5%CO_2 culture box at 37 degrees C, each 3D is replaced once, and the cell line along the bottom of the incubator reaches 80% ~ 90%, according to the routine Method of passage.
2, phenotypic determination of placental cells
After taking third generations of cells and digesting 3min with 0.25% trypsin, the number of cells was adjusted to 1 x 10~6/L. After adding the 5 mu L mouse serum to close the 15min, the mouse anti human PE labeled monoclonal antibody was added to the mouse, the ice on the light reaction was 20min, and the PBS was washed 2 times, and the 5min was centrifuged at 4 1000r/min, after the supernatant was added to 200 mu L cold PBS and blended, the flow cytometer detected.
3, induced differentiation
After digestion of the third generation placental derived mesenchymal stem cells, the cell suspensions were digested. After the centrifuge was abandoned, PBS was washed 3 times, and 2 x 10~4/cm~2 density was inoculated in the six hole cell culture medium with 0.1% gelatin. After the cells were adhered to the wall, the chondrocytes were changed to the 37 C 5%CO_2 culture incubator, and each 3D was replaced once.
4, toluidine blue staining
Two groups of cells were cultured for 21d. After PBS was changed for 2 times, 4% paraformaldehyde was fixed 15 to 30min. After PBS washing for 3 times, 1% toluidine blue was stained 2 to 4h, 95% alcohol was washed for 3 times, and then observed under light microscope.
5, immunocytochemical detection of type II collagen expression
The concrete steps are operated according to the instructions of the SABC staining kit, and the culture liquid in the orifice plate is abandoned, PBS (0.02mol/LpH7.2 ~ 7.6) is washed 3 times, 4% polyformaldehyde is fixed to 30min, and 0.3%H_2O_2 is used to inactivate the endogenous enzymes. After the BSA is closed, the Rabbit anti human anti type II collagen is added to the 1:200 diluted rabbit, 4 degrees centigrade for the night, the goat is incubated with the rabbit against the rabbit mouse IgG, and the DAB chromogenic agent is coloured. The hematoxylin was observed under the light microscope after redyeing.
Result
In the tissue mass of 11d, a small number of cells were found to climb out, round or polygonal. With the number of cells increasing, the cells were vortexed, the cell body became elongated, the morphology was similar to fibroblasts, the cells were cultured about 3W, and the cells could reach 80% fusion. After the cells were adhered to the cells, the cells could grow into spindle cells again. The cells could still grow steadily to the 20 generation. Flow cells still can still grow steadily. Flow cells still can still grow. Flow cells still can still grow. Flow cells can still grow steadily. The third generation cells from human placenta tissue express CD13, CD44, CD73, CD90, CD166, HLA-AB, which do not express CD34, CD45, HLA-DR, and have similar cell surface markers with mesenchymal stem cells derived from bone marrow. Cell proliferation at the beginning of induction is slow, and the cell morphology changes at one week. The cell volume became larger and some cells were gradually transformed from the original spindle shape to polygon, and the cell morphology was different. The cells were not passable throughout the induction process. The cells were blue purple when the cells were induced by toluidine blue staining, and the positive results of the brown yellow granules in the cytoplasm of the cells were detected by the type II collagen immunocytochemical test.
conclusion
1, human placenta derived mesenchymal stem cells can be successfully cultured in vitro and amplified in large quantities, with stable biological properties.
2, in this experiment, human placental mesenchymal stem cells were successfully induced and differentiated into chondrocytes under single layer culture. The chondrocytes formed after induction were characterized by chondrocytes and could secrete the specific type II collagen of chondrocytes.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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