【作者】 刘晓强；

【导师】 陈宗雄；

【作者基本信息】 福建医科大学 ， 外科学， 2010， 硕士

【摘要】 目的:(1)分离、培养及鉴定骨髓源性间充质干细胞( Bone Marrow Mesenchymal Stem Cells,BMSCs),并获取高纯度的BMSCs;(2)分离、培养及鉴定关节软骨细胞(Articular Cartilage Cells,ACs),并获取高纯度的ACs;(3)BMSCs与ACs共同培养诱导,并检测其诱导结果;(4)对共培养生物诱导与TGF-β1化学诱导的结果进行比较。方法:(1)获取并培养、增殖高纯度骨髓间充质干细胞:从SD大鼠(4周龄)的股骨及胫骨干中取得骨髓,联合利用密度梯度离心法及差异贴壁法分离、纯化BMSCs,体外扩增并传代后,相差显微镜下观察细胞的形态及流式细胞仪检测细胞表面标记:CD29、CD90、CD34及CD45,再通过成脂肪样、成骨样及成神经样细胞多向分化诱导,检测其诱导结果,确定所得细胞为BMSCs,为下一步实验打下基础。(2)获取并培养、增殖高纯度的关节软骨细胞:从SD大鼠(4周龄)的正常股骨头表面取得关节软骨片,先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化软骨片,当相差显微镜下见有大量软骨细胞游离后,离心、弃上清液,加入含15%FBS的软骨细胞培养液进行培养、扩增传代,并取样行台盼蓝染色计数。当细胞传至第三代时,运用甲苯胺蓝染色及HE染色检查、鉴别该细胞,确定所得细胞为关节软骨细胞,为下一步实验打下基础。(3)BMSCs与ACs在Transwell共培养:分别吸取第三代BMSCs和ACs,按1:1的细胞比例分别植于Transwell共培养系统中,下室植入BMSCs,上室植入ACs,同时设置相同浓度单独BMSCs空白对照组,分别在用含10%FBS的高糖DMEM培养基培养,相差显微镜下观察细胞的增殖和基质合成情况,并对各组细胞爬片进行Ⅱ型胶原免疫组化染色检查。(4)共培养生物诱导与TGF-β1化学诱导的比较:将BMSCs用于TGF-β1(10 ng/m1)诱导,同时与1:2,1:1,2:1(关节软骨细胞:BMSCs)浓度的细胞共培养诱导组比较对照。并运用MTT比色法检查、氨基聚糖(GAG)含量检测及WESTERN BLOT检测各组细胞Ⅱ型胶原基因表达量,确定其诱导情况。结果:(1)获取并培养、增殖高纯度BMSCs:相差显微镜下观察结果:原代细胞接种12h后可见部分细胞贴壁,24h后细胞大量贴壁,外观呈现纺锤形或多角形。72h后细胞数量开始增多,大部分细胞形态呈长梭形、似成纤维细胞。培养10d左右细胞铺满培养瓶底达80%左右,细胞呈极性排列,部分集落可呈漩涡状。细胞表面标记物经流式细胞仪检测结果:BMSCs均一地表达CD29及CD90,阳性率分别为92.5%及94.4%;而CD34和CD45为阴性,阴性率分别为99.3%和99.7%。成脂肪样、成骨样及成神经样细胞诱导均获得成功。(2)获取并培养高纯度的ACs:运用上述方法可获得一定纯度的ACs。在倒置相差显微镜下观察发现:软骨细胞接种12h后开始贴壁,48h完成贴壁。原代培养的ACs呈圆形及多角形,胞浆丰富,胞核圆形,居中,细胞长成一片可呈明显的铺路石样外观,与长梭形的成纤维细胞较易区分。试验证明:经本方法消化培养后仅见单一的软骨细胞生长。(3)BMSCs与ACs共培养:共培养组BMSCs数量增多,细胞外基质合成丰富,其基质能被Ⅱ型胶原免疫组化染色,细胞染色呈现黄色。(4)共培养生物诱导与TGF-β1化学诱导的比较:共培养细胞生物诱导的MTT比色法检查、氨基聚糖(GAG)含量检测及Ⅱ型胶原WESTERN BLOT检测结果显示:各组细胞共培养诱导结果均不差于TGF-β1 10ng/m的l化学诱导结果,但1:1、2:1共培养组诱导统计结果差异不明显。结论:(1)密度梯度离心和差异贴壁法可以获取纯度较高的BMSCs;(2)高纯度的ACs可以经胰蛋白酶、Ⅱ型胶原酶联合消化后,过滤、离心、培养获得;(3)BMSCs与ACs共培养可作为修复关节软骨的种子细胞。更多还原

【Abstract】 Objectives: (1) To isolate, culture and identificate Bone marrow Mesenchymal stem cells, (BMSCs),obtaining high- concentration and high -purity BMSCs;(2)To isolate, culture and identificate articular cartilage cells (ACs),obtaining high- concentration and high -purity ACs;(3)To Induce BMSCs co-cultured with the ACs and detect the induction ;(4)To compare co-cultured biological induction and chemical induction。Methods:(1)Obtain, culture and proliferate high-purity BMSCs: Bone marrow were obtained from femoral shaft of SD rats (4 weeks) and BMSCs were isolated by gradient centrifugation and adherent separation and passaged after Amplification in vitro. The cell shape was observed under phase contrast microscope and the expression of CD 29、CD90、CD34 and CD 45 of the cells were analyzed by flow cytometry. Make sure the cells obtained were BMSCs by inducing of fat-like, osteoblast-like and adult neural-like cellstolay the foundation for the next step experiment. (2)Obtain, culture and passage high- concentration and high–purity ACs:Articular cartilage were obtained from the surface of normal femoral head of SD rats(4-week),digested by trypsin(0.25%)and CollageaseⅡ(0.2%).After observing numerous ACs free under phase contrast microscope,centrifuge, deposit Supernatant and place the cells into cartilage cell culture medium to Cultivate, proliferate, and passage; sample, count with trypan blue staining and detect and identify ACs with Toluidine blue and HE staining methods to lay the foundation for the next step experiment.。(3)Co-culture BMSCs and ACs in Transwell:Aspire the third passage BMSCs and ACs and place them respectively into Transwell co-culture system by1:1,BMSCs were placed in the bottom and in the upper ACs。Meanwhile set the same concentration of BMSCs control group separately, cultured in culture medium of NMEM containing 10% FBS respectly. Then Cell proliferation and matrix synthesis would be observed under phase contrast microscopy and Envision immunohistochemical stain typeⅡcollagen was used to analyze each group of cell-seeded cover slips (.4)Comparison of Co-culture bio-induced and TGF-β1 Comparison of chemically induced:Use TGF-β1(10 ng/m1)induction and compare with co-culture induced group of BMSCs of 1:2,1:1,2:1 . MTT assay, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test were adopted to check the induction of each group cells.Result:(1)Obtain, culture and proliferate high-purity BMSCs:Some cells anchored were observed under Inverted phase contrast , after 24 hours,most wall-adhering cells were seen , which appeared as a fusiform polygonal appearance ,and cell nucleus were observed too。72h later the number of cells increased and their shapes were fibroblast-like, as with fibroblastoid cells. Meanwhile pairs of nucleolar could be seen in most nucleus and Cells were colony-like growth. Till 10d the cells covered the bottom of culture bottle and up to 90%,Polar cells were arranged, and the colony was swirling. Cell surface markers were tested by flow cytometry: BMSCs uniformly express CD29 and CD90, the positive rates were 92.5%and 94.4%,; while CD34 and CD45 were negative, the negative rates were 99.3%and 99.7%。Fat-like, osteoblast-like and adult neural-like cells induction succeeded. (2) acquire and develop high-grade ACs: the use of the above method can get a certain purity of ACs. Observation under the inverted phase contrast microscope showed: ACs vaccinated after 12h began to adhere, 48h completed adherence. Primary cultured chondrocytes was round and polygonal with abundant cytoplasm, round nucleus which exists in the center; the cells grew into a clear cobblestone appearance, distinguished with long spindle-shaped fibroblasts easily.Only single cartilage cell could be seen growing after digestion of this method.(3)BMSCs were co-cultured with ACs:The number of co-cultured BMSCs increased ,synthesis of extracellular matrix was rich ,which can be stained by typeⅡcollagen immunohistochemical staining and cells staining took on yellow.(4)Comparison of Co-culture bio-induction and TGF-β1 chemical induction: MTT colorimetric examination of Co-cultured cell bio-induction, glucose amino glycan (GAG) content detection and typeⅡcollagen WESTERN BLOT test results showed that:The induction of cell co-culture results were better than the results of TGF-β1 chemical induction,but no significant differences appeared in statistical results of1:1,1:2 co-culture induction.Conclusion:(1) High-purity BMSCs can be obtained with density gradient centrifugation and differential adhesion method ;(2)High-purity ACs can be obtained by filtrating, centrifugating and culturing after digestion in trypsin and CollagenⅡ. (3)BMSCs co-cultured with ACs can act as seed cells repairing cartilago articularis.更多还原