【作者】 罗衍波；

【导师】 陈斌；

【作者基本信息】 南方医科大学 ， 外科学， 2009， 硕士

【摘要】 骨髓间充质干细胞(Bone mesenchymal stem cell,BMSCs)具有极强的自我复制和多向分化潜能,来源广泛,易于获取并且具有低免疫源性,可将BMSCs用于细胞移植治疗,具有非常重要的理论研究意义和临床价值。近年来的研究提示,骨髓间充质干细胞能转分化为肝细胞,这一现象为临床上应用BMSCs移植治疗肝损伤和急慢性肝功能衰竭展示了一个可喜的前景。现已证实,在体外培养条件下BMSCs可由肝细胞生长因子(hepatocyte growth factor,HGF)等细胞因子诱导向肝细胞分化,并可释放多种肝细胞因子,但对BMSCs体内移植后能否向具肝细胞功能的细胞分化并对受损肝脏起修复作用的研究则鲜见报道。因此,本课题将大鼠骨髓中分离获得的BMSCs在体外纯化、扩增后通过门静脉移植入肝损伤大鼠体内,观察BMSCs移植后在肝脏内的分布及受损肝脏修复情况,为临床应用提供理论依据。第一部分大鼠骨髓间充质干细胞的分离、培养与签定目的建立大鼠骨髓间充质干细胞分离和培养扩增的方法,同时对其生物学特性进行研究,为BMSCs作为种子细胞治疗临床疾病提供理论依据。方法联合应用密度梯度离心法和贴壁细胞培养法进行分离获取大鼠BMSCs,应用流式细胞仪测定细胞表面标记物来鉴定所培养的细胞是否为BMSCs;在倒置显微镜下观察BMSCs的形态学特点,细胞计数测定BMSCs接种贴壁率,并绘制出生长曲线。结果通过联合密度梯度离心法与贴壁细胞培养方法分离培养出的BMSCs纯度高。BMSCs体外培养生长旺盛,具有活跃的增殖能力。培养的BMSCs阳性表达CD29、CD44,阴性表达CD34、CD45。结论通过联合密度梯度离心与贴壁细胞培养方法能从骨髓中获得骨髓间充质干细胞;获得的BMSCs在体外生长稳定,且增殖较快,可作为组织工程的种子细胞。第二部分慢病毒转染大鼠骨髓间充质干细胞研究目的对分离、培养到第三代的大鼠骨髓间充质干细胞进行慢病毒载体介导的基因转染,观察其转染效率及对骨髓间充质干细胞生物活性的影响,为骨髓间充质干细胞基因治疗研究提供实验基础。方法利用密度梯度离心、贴壁筛选法分离培养大鼠骨髓间充质干细胞。利用脂质体法进行慢病毒感染大鼠BMSCs。结果体外分离培养的骨髓间充质干细胞,经携带绿色荧光蛋白(greenfluorescence protein,GFP)的慢病毒转染72h后可见绿色荧光蛋白的表达,96h后达到高峰。转染后的骨髓间充质干细胞体外继续培养,1月后仍可见绿色荧光蛋白的表达,但表达强度较前减弱。在一定病毒复数范围内,慢病毒对骨髓间充质干细胞的转染率随病毒复数的增加而增高,当病毒复数为5时,转染率达到80%以上;但病毒复数超出一定范围后,转染率反而下降。慢病毒转染后的骨髓间充质干细胞生长曲线和未转染的骨髓间充质干细胞生长曲线未见明显变化。结论慢病毒对大鼠BMSCs有较高的转染率,且携带的GFP能在BMSCs内有效表达,可以作为骨髓间充质干细胞基因治疗的载体和体内示踪。第三部分骨髓间充质干细胞移植对肝功能影响的实验研究目的观察同种异体骨髓间充质干细胞移植在肝脏特定微环境下是否向具肝细胞功能的细胞分化并探讨其对大鼠受损肝脏修复作用的可行性。方法利用密度梯度离心、贴壁筛选法分离培养大鼠骨髓间充质干细胞。经慢病毒转染后的第3代BMSCs从门静脉植入肝大部分切除大鼠体内,分别于移植后7、14、28d行血清肝功能检测,分析BMSCs对肝功能的作用。移植后的第28d取出肝脏,通过冰冻切片荧光检测,观察BMSCs在大鼠肝脏内定居分布情况。结果大鼠连续用2-乙酰氨基芴(2-acetylaminofluorene,2-AAF)灌喂6d后出现精神萎靡、厌食、少活动、尿深黄;肉眼见肝脏明显充血肿大、边缘圆钝、表面可见针尖样细小颗粒、呈局灶性,偶见有小出血点;HE见部分肝细胞肿胀、胞质疏松。术后BMSCs移植组和DMEM/F12注射组各肝功能检测指标均随着时间的延长逐渐恢复;BMSCs移植组与DMEM/F12注射组比较,ALB、ALT和AST均有显著性差异(P＜0.05)。术后28d,BMSCs移植组与正常组比较,ALB、ALT、AST、ALP均无显著性差异(P＞0.05);DMEM/F12注射与正常组比较,ALB、ALT均有显著性差异(P＜0.05)。这些表明BMSCs移植对肝功能的恢复有促进作用。术后冰冻切片,BMSCs移植组可见BMSCs定居于肝脏内,主要在汇管区周围,肝实质里也可见散在分布,而心、肺、脾、肾未见BMSCs。HE染色BMSCs移植组肝小叶结构正常,肝索排列规则;DMEM/F12注射组仍见部分肝索排列紊乱,肝窦增宽或变窄、消失。结论经门静脉移植的BMSCs能在受损肝脏内定居、增殖,BMSCs移植可以在一定程度上修复受损肝脏。通过以上实验研究,掌握了BMSCs的分离与培养的方法,证实了BMSCs具有干细胞极强的自我复制和多向分化潜能的生物学特性;成功应用携带GFP的慢病毒进行转染BMSCs,且GFP高效、稳定表达,为进一步携带目的基因的慢病毒转染BMSCs用于基因治疗提供理论依据;慢病毒转染后的BMSCs经门静脉移植后能定居于肝脏内,并可以在一定程度上修复受损肝脏,证实了BMSCs在肝脏特定微环境下对受损肝脏起修复作用。更多还原

【Abstract】 Bone mesenchymal stem cells are thought to be pluripotent cells that can be differentiated into a variety of cell or tissue types and can be ideal resources of transplantation therapy.Recently accumulating evidence indicates that bone mesenchymal cells that can diferentiate into hepatocyte.It is a very good prospects that BMSCs transplantation be used in the treatment of acute and chronic liver failure. However,little is known about the mechanism of bone mesenchymal stem cells diferentiation into hepatocytes.It is important to determine suitable culture conditions in which bone mesenchymal stem cells will be differentiated into hepatocytes not only for understanding diferentiation mechanisms but also for eficient amplification of hepatocyte-progenitor cells of bone marrow origin,this being a prerequisite for potential therapeutic use.Recently accumulating evidence indicates that bone mesenchymal cells that can diferentiate into hepatocyte under the condition of hepatocyte growth factor in vitro,and can release the various hepatocyte factors.But whether transplanted bone mesenchymal stem cells can repopulate hepatic and biliary duct epithelia in vivo is rarely reported.For this reason,in the present,we study to observe whether bone mesenchymal stem cells have the potential to transdifferentiate into functional hepatocyte-like cells in the special niche as well as the therapeutic feasibility to repair damaged liver after transplantation in rats. PartⅠApproaches to isolate,culture and identify the bone marrowderived mesenchymal stem cells of ratObjective To establish a method for isolation and cultivation of bone mesenchymal stem cells from rat in vitro and explore their biological properties in order to provide an experimental basis for applying BMSCs to achieve the therapeutic benefit as seed cells in clinical diseases.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.Cell surface antigens were detected through flow cytometer.The configuration was observed under phase - contrast microscope consecutively and the growth curve of cells was drew.Results BMSCs were very purified by density gradient centrifugation and adherent culture.The subcultured cells showed active proliferative ability.These cultured cells showed immunoreactivity to CD29 and CD44 but not CD34 and CD45.Conclusion BMSCs can be cultured and purified in vitro by density gradient centrifugation and adherent culture.BMSCs showed stable and rapid growth in vitro. All the characteristics make it possible to BMSCs to be the seed cells for tissue engineering.PartⅡLentiviral transfection of mesenchymal stem cells derived from rat’s bone marrow and its biological characteristicsObjective To isolate and culture bone mesenchymal stern cells derived from rat’s bone marrow and transfected the cells by lentiviral vectors.To provide an experimental basis which will play a role in researching on the function of BMSCs in gene engineering.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.The BMSCs were transfected by lentiviral vectors using polybrene. Results 72h after transfection,the fluorescent intensity of GFP was found in the third generation bone mesenchymal stem cells under confocal microscopy,96h after transfection,the higher fluorescent intensity of GFP was found.One month after transfection,we can still see fluorescent intensity of GFP,but it is weaken.In certain viral plural number scope,the lentiviral virus advances to the bone mesenchymal stem cell transfection efficiency along with the viral plural number increase.When the multiply of infection is five,the transfection efficiency will reach to 80%.when the viral plural number surpasses certain scope,the transfection efficiency decreases.The growth curve have not obvious changed between transfected and not transfected bone mesenchymal stem cells.Conclusion Lentivirus/GFP was transfected into bone mesenchymal stem cells of rats,the expression of GFP was efficiency and stable,which will play a role in researching on the function of BMSCs in gene engineering.PartⅢTransplanted bone mesenchymal stem cells influence on hepatic functionObjective To observe whether bone mesenchymal stem cells have the potential to transdifferentiate into functional hepatocyte-like cells in the special niche as well as the therapeutic feasibility to repair damaged liver after transplantation in rats.Methods BMSCs were isolated from femur in the rat by the density gradient centrifugation with Percoll solution and purified by the characteristic of the tissue culture plastic.The third generation of BMSCs,after transfected by lentiviral vectors, were injected into portal vein of liver resection of rats.At the 7th,14th and 28th day after transplantation,liver function indexes were inspected by automated biochemical analyzer.At the 28th day after transplantation,liver samples were collected.The distribution of BMSCs in the liver were observed by fluorescence.Results After continuously feeded with 2-AAF 6 day,rats appeared to the energetic dispirited,lose of appetite,few activitied,deep yellow urine.The liver was hyperemia and enlarger,the edge of liver was obtuse,small particles was seen on the surface of liver,seen the small bleeding point in the partner.The hepatocyte was swelling,the cytoplasm was osteoporosis.All experimental animals survived after hepatectomy.The hepatic function gradually restored with the time.After 28 days,the BMSCs transplanted group’s liver function restored normally,but the DMEM/F 12 transplanted group did not.The BMSCs were more effective to promote the restoration of hepatic function than the DMEM/F12.BMSCs can successfully reside and multiplied in the damage of liver.BMSCs mainly collected around the district deparment of the surrouding,also distributed in the parenchyma of liver.The hepatic lobule structure is normal,hepatic cord arrangemented rule in the BMSCs transplanted group with HE stain.The hepatic cord arrangemented disorder,hepatic sinusoid expanded,narrowed or disappesr in the DMEM/F12 transplanted group with HE stain.Conclusion BMSCs transplanted through portal vein can successfully reside and multiplied in the damage of liver.Transplantation of BMSCs might amend the damaged tissue of host liver to a certain extent.Through above the experimental study,we’ve grasped experimental skills,such as cell culture,abstraction and separation stem cell.BMSCs have the biological characteristics of extremely self-renew and multiplex differentiation potential;Lentivirus/GFP was transfected into bone mesenchymal stem cells,the expression of GFP was efficiency and stable,which will play a role in researching on the function of BMSCs in gene engineering.BMSCs transplanted through portal vein can successfully reside and multiplied in the damage of liver,transplantation of BMSCs might amend the damaged tissue of host liver to a certain extent.更多还原