【作者】 王世知；

【导师】 袁庆文；

【作者基本信息】 南昌大学 ， 外科学， 2010， 硕士

【摘要】 目的：探讨成人外周血来源的平滑肌祖细胞(smooth muscle progenitor cells,SPCs)与成熟血管平滑肌细胞(smooth muscle cells, SMCs)细胞形态、增殖潜能等方面的异同点,为血管组织工程提供可靠的种子细胞来源。方法：1、密度梯度离心法分离成人外周血中单个核细胞,于用纤粘连蛋白包被的含血小板衍生生长因子BB (Platelet derived growth factor BB, PDGF-BB)和EGM-2培养基的培养瓶中诱导培养,观察细胞形态变化,常规换液、传代、冻存及复苏。2、通过手术取得实验用大隐静脉3cm,采用胶原蛋白酶消化、差异贴壁法原代培养,含PDGF-BB的EGM-2培养基培养,观察细胞形态,获得平滑肌细胞。3、平滑肌祖细胞及平滑肌细胞分别进行细胞爬片,应用细胞免疫荧光检测平滑肌肌动蛋白(a-SMA)和调宁蛋白(calponinl)的表达。4、用RT-PCR检测平滑肌肌动蛋白.(alpha-smooth muscle actin, a-sma)和调宁蛋白(calponin 1, cap)的mRNA表达。5、MTT(四唑盐比色实验)分别检测第6代平滑肌祖细胞与平滑肌细胞的增殖能力。结果：1、SPCs传代细胞培养达80-90%融合时,呈螺旋状排列,并可呈典型“峰、谷”样形态,符合平滑肌细胞形态学特征。2、平滑肌祖细胞可表达平滑肌细胞特异性标记α-SMA和calponinl。3、第6代的SPCs(n=10, A=0.43±0.02)增殖能力明显强于第6代的SMCs(n=10, A=0.21±0.02, P<0.01)。结论：1、外周血中单个核细胞在PDGF-BB的刺激下可以分化为具有平滑肌细胞特性的平滑肌祖细胞。2、平滑肌祖细胞较平滑肌细胞增殖旺盛,培养3个月未见老化,具有发展为-种理想的血管组织工程种子细胞来源的可能性。更多还原

【Abstract】 Objective:Compared the cell morphology and proliferation potential of smooth muscle progenitor cells(SPCs) derived from human peripheral blood with mature vessel wall smooth muscle cells(SMCs) to provide a reliable source of seed cells.Methods:1. Used Ficoll density gradient centrifugation to separate the mononuclear cells from adult peripheral blood. Cultured in the fibronectin-coated culture flask,and add EGM-2 medium which contails platelet-derived growth factor(PDGF-BB).Observed the morphological changes, liquid passage, cryopreservation and resuscitation by conventional methods.2. Obtained experimental vena saphena magna 3cm through the surgery, uses the collagen proteinase digestion, the difference to paste the wall law original generation of raise, raise in the EGM-2 culture medium including PDGF-BB. observation the change of cellular form, obtains the smooth muscle cell.3. Smooth muscle progenitor cells and smooth muscle cells were cell-climb filmed to detect the expression of a-SMA and calponinl by immunofluorescence.4. Reverse transcription-polymerase chain reaction detected the mRNA expression of smooth muscle actin (a-SMA) and calponinl.5. MTT detected the sixth generation proliferation of the smooth muscle progenitor cells and smooth muscle cell.Results:1. SPCs culture passage up to 80-90% confluence, appearing spiral arrangement,and presenting typical "peak, valley" form, sign smooth muscle cell morphology.2. Smooth muscle progenitor cells can express smooth muscle cell specific marker a-SMA and calponinl.3. The sixth generation of SPCs (n=10, A=0.43±0.02) proliferation was significantly faster than the same generations of SMCs (n=10, A=0.21±0.02, P <0 .01). Conclusion:1.Peripheral blood mononuclear cells in the stimulus of platelet-derived growth factor can differentiate into smooth muscle progenitor cells.2.Compared to smooth muscle cells, smooth muscle progenitor cells proliferate better, and not to see the aging in 3 months cultivation. Smooth muscle progenitor cells has the development of the possibility which one kind of ideal blood vessel organization project seed cell originates.更多还原