【作者】 陈伟；

【导师】 白晓东；

【作者基本信息】 河北医科大学 ， 外科学， 2009， 硕士

【摘要】 目的:骨肉瘤是一种常见于青少年的恶性肿瘤,易复发和转移,病死率及致残率极高,严重危害了人类的健康。骨肉瘤的发生、发展涉及多种癌基因、抑癌基因。其中,细胞凋亡与肿瘤的形成密切相关,其中抗凋亡基因Bcl-2和Bag-1在骨肉瘤的发生、发展过程及预测、预防方面有可能起着十分重要的作用。文献中只有检测Bcl-2单一指标与骨肉瘤关系的的研究,关于Bag-l与骨肉瘤间的关系及Bcl-2和ag-l两基因在骨肉瘤中的相互作用关系目前国内尚无报道。本研究采用免疫组织化学法(SP法)对骨肉瘤中Bcl-2和Bag-1表达进行检测,分析二者在骨肉瘤组织中的单独及联合表达情况.探讨基因Bcl-2和Bag-1在骨肉瘤中的表达及两者的相互关系,以期找到指示骨肉瘤临床病理特征的可靠分子指标,为开展骨肉瘤基因治疗研究提供理论依据。方法:研究对象随机选取30例中国医科大学病理学教研室2001—2007年截肢、肿瘤切除或手术切取活体检查的骨肉瘤标本,术前未行化疗、放疗,术后所有标本均作病理检查,根据骨肉瘤的诊断标准并有三位病理专家证实以确定诊断。其中男性19例,女性11例,年龄12岁～46岁,平均22.4岁。骨肉瘤组织分型,其中骨母细胞型l3例,软骨母细胞型9例,纤维母细胞型8例。另有15例正常对照骨标本取自秦皇岛市第一医院骨科非肿瘤非炎症病人因治疗需要而术中切除的正常骨作为对照组,其中男性9例,女性6例,年龄18～65岁,平均33.2岁。采用S-P免疫组织化学法检测Bcl-2和Bag-1在骨肉瘤及正常骨组织中的表达情况。实验数据用SPSS 12.0软件进行统计学处理,Bcl-2抗原和Bag-l抗原阳性率比较采用x2检验,变量间的相关分析应用Spearman等级相关分析,以α=0.05为检验水准。结果:1 Bcl-2在正常骨组织低表达,阳性表达率为0.00%(3/15),而在骨肉瘤组织中高表达,阳性表达率为63.33%(19/30),Bcl-2在骨肉瘤组织阳性表达率明显高于正常骨组织,两组之间差异有统计学意义(x~2=5.3804,P<0.05);Bcl-2在骨母细胞型的骨肉瘤组织中阳性表达率为61.54%(8/13),在软骨母细胞型的骨肉瘤组织中的阳性表达率为66.67%(6/9),而在纤维母细胞型骨肉瘤组织中的阳性表达率为62.50%(5/8),三组之间差异无明显统计学意义(x2=0.0635,P=O.9688>O.05);Bcl-2在骨肉瘤中的表达与患者年龄、性别在统计学上均无意义(P>0.05)。2 Bag-1在骨肉瘤组织中呈高表达,阳性表达率为76.67%(23/30),而在正常骨组织组中无表达或呈微弱表达,其阳性表达率6.67%(1/1 5),两组之间差异有明显统计学意义(x~2=19.6875,P<O.05)。Bag-1表达与骨肉瘤组织类型无明显关联,Bag-l在骨母细胞型的骨肉瘤组织中的阳性表达率为76.92%(10/13),在软骨母细胞型的骨肉瘤组织中的阳性表达率为66.67%(6/9),在纤维母细胞型骨肉瘤组织中的阳性表达率为87.50%(7/8),三组之间差异无明显统计学意义(x2 =1.0284,P>0.05)。Bag-1表达与患者的年龄、性别在统计学上均无意义(P>O.05)。3 Bcl-2和Bag-1的表达关系23例Bag-1阳性表达的病例中,Bcl-2的表达阳性者为15例,阴性者为8例;在13例Bcl-2阴性表达的病例中,Bag-1的表达阴性者为5例,表达阳性者为8例。二者在骨肉瘤组织中的阳性表达成正相关(r=0.4462,P=0.0134<0.05)。结论:1 Bcl-2在骨肉瘤组织中高表达,而在正常骨组织中低表达,Bcl-2在骨肉瘤组织的阳性表达率明显高于正常骨组织;提示Bcl一2基因的高度表达与骨肉瘤的发生发展有关。2 Bag-1在骨肉瘤组织中高表达,而在正常骨组织中微弱表达或不表达,Bag-l在骨肉瘤组织中阳性表达率远远高于正常骨组织;提示Bag-1高表达在骨肉瘤的发生、发展中可能发挥了重要的作用。3骨肉瘤细胞凋亡受到了多种基因的调控,Bcl-2和Bag-1基因可能均与骨肉瘤的细胞凋亡有关,在骨肉瘤中Bag-1基因的更高表达,提示两基因中Bag-1的作用可能更加明显。4 Bcl-2阳性表达的骨肉瘤组织中,Bag-1亦出现较高阳性表达,两者呈正相关。提示抗凋亡基因Bcl-2与Bag-1在骨肉瘤的发生、发展中抗凋亡作用密切关联。更多还原

【Abstract】 Objective: The osteosarcoma is one of the most common malignant tumors among young people.lt is easy to palindromia and metastasize. The fatality rate and mutilation rate are exceeding high. It is severely detrimental to people’s health. The occurrence and development of osteosarcoma is closely concerned with multiple oncogenes as well as anti-oncogenes. Apoptosis closely affect the occurrence of osteosarcoma, and The anti- apoptosis gene Bcl-2 and Bag-1 may are most important action in the occurrence, development, anticipation and prophylaxis of osteosarcoma. Most studies only detect the relationship of the expression of the single indicator Bcl-2 with the development of osteosarcoma ,but local authority has not been reported about the relationship of bag-1 in osteosarcoma and the interreaction relationship of Bcl-2 and Bag-1 in osteosarcoma . The research was to detect the expression of Bcl-2 and Bag-1 in osteosarcoma tissues using immunohistochemical method (SP), and to analyze the separate and associated expression of Bcl-2 and Bag-1 in osteosarcoma. Research the expression and the interreaction relationship to find a safety molecular indicatrix for patients to demonstrate the clinical Pathology characteristic, to provid theory evidence for Part I :The Culture in Vitro of Fetal Mesenchymal Stem Cells Derived from Amniotic Fluid and the induction into Epidermic like CellsObjective:To observe the possibility of the induction of fetal mesenchymal stem cells (MSCs) derived from amniotic fluid into epidermic cells during mid-pregnancy.Method: Chose pregnant women who accepted prenatal diagnosis and induction, obtained ten Amniotic fluid of pregnant women whose pregnant weeks were 16~22 by ultrasound -guided amniocentesis.Mechanical dissociate the cells and then passage culture in vitro. Generated, amplificated and amplificated to the 1st~3rd generation after culture and proliferation.Used a flow cytometry to detect surface antigen expression.Chose the 3rd generation MSCs from fetus which grew well.Used culture solution B(70% culture solution A+30% supernatant fluid from fibroblast medium+10 ng/LEGF) to induce the fetal MSCs differentiate into epidermic cells。Chose fetal MSCs which were continuous passage cultured by solution A as control roup.Observed the change of cells morphology structure after induction through inverted phase contrast microscope.After 20d, detect the express of CK19 by immunohistochemistry.Result: fetal MSCs into epidermic cells during mid-pregnancy generate fast in the culture in vitro. After 3d, we could see a single attached cell or a few colony through inverted phase contrast microscope,most of them were almost shuttle-shaped or needle tip-shaped. A part of them were round or polygon, the cell nucleus were in the middle,we could also see the nucleoli. After 8d, these were sporadic MSCs colony of fibroblas, the main of which were spindle cells,and epithelioid cell colony, the main were polygonal cells, every colony didn’t contact each other. Mechanical chose fetal MSCs colony and inoculation culture, the cells grew homogeneous distribution, the shape of which were Fusiform, apiciform or Stellate.After 7~8d, the number and size of colony increase obviously, the cells ranked directivity, which were turbulent, reticulate or reticulate, and the fusion rate up to 90%, the were no obviously limit between each other. Fetal mesenchymal stem cells derived from amniotic fluid are: CD44 (+), CD34(-).After 20d Inducing cultured with culture solution B(70% culture solution A+30% supernatant fluid from fibroblast medium+10 ng/LEGF), the cells which were fusiform fibroblas became short gradually, and they ranked tight .Identify the structure by cytochemistry, there were the express of CK19、β1.Conclusion: 1 Fetal MSCs can separate in vitro, purify culture, grow stable, generate fast and serial passage.2 Fetal MSCs have the tendency to differentiate into epidermic cells under the culture with supernatant fluid from fibroblast mediumand EGF in vitro.Part II: The effect of hydrochloric Fasudil to differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid.Objective:To observe the effect of different concentrations of hydrochloric Fasudil to differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid. Hydrochloric Fasudil is blocking agent of Rho molecular signal path.Method: According to the method in the first experiment,we got amniotic fluid at mid-pregnancy and then cultured and passaged the cells in vitro. We chose the 3 rd generation of MSCs from fetus which grew well and divided them into 5 groups. Group 1 was continuously cultured for passsaging by culture solution A(90%L-DMEM+10%FBS+p enicillin100U/ml+phytomycin 100μg/ ml). As to group 2、3、4、5,we washouted solution A with sterile PBS and respectively used substitute as follows: culture solution B (70%culture solution A +30%clear supernatant liquid from fibroblast nutritive medium +10 ng/LEGF) ; culture solution C: (HA1077 culture solution B with a concentration of 10μmol/L); culture solution D (HA1077 culture solution B with a concentration of 20umol/L); culture solution E: (HA1077 culture solution B with a concentration of 40μmol/L).Puted them into incubator for culture and changed their culture solution every 2~3 days until the fusion rate rase up to 90%,and then we digested them with 0.25%parenzyme -lmmol EDTA to passage them by 1: 2.We cultured 3 generations.Group 1 was blank group, group 2 was single induced group. We observed the cell morphous and groweing status in every group regularly.20 days after inducted,we detected the expression of antibody of CK19 with flow cytometry in group 1~5. Group 2 and 4 were detected for RT-PCR.Result:The positive rate of CK19 in group 1 detected with flow cytometry was 0.37% on 20d,and group 2 was 3.08%.We observed the 3 rd fetus MSCs on 20d,which was induced respectively with solution C and E.The cells which are fusiform fibroblas become short gradually, and they rank tight . Identify the structure by cytochemistry, the positive expression of CK19 was 2.93%and 3.02%.There were changes in cell morphous 18d induced dy solution D,which occurred earlier than the blank group. Identified the structure by cytochemistry on 20d, the positive expression of CK19 was 3.40%. As to group 2 and group 4 detected with RT-PCR,we observed the tendency to differentiate into epidermic cells in Group 4 was trengthened. Conclusion: Different concentrations of hydrochloric Fasudil (HA1077 ) can influence the differentiation of epidermic cells from fetal mesenchymal stem cells derived from amniotic fluid.更多还原