【作者】 张勇；

【导师】 徐永清； 王非；

【作者基本信息】 昆明医学院 ， 外科学， 2009， 硕士

【摘要】 目的尝试分离培养兔软骨终板细胞,观察其生物学特性,了解携带人胰岛素样生长因子-1(hIGF-1,Human insulin-like growth factor-1)的重组腺病毒载体转染不同代兔软骨终板细胞的效率及目的基因对细胞的生物学影响,探讨应用Ad-hIGF-1治疗椎间盘退变的可行性。方法体外分离软骨终板组织,应用胰蛋白酶消化30min后,Ⅱ型胶原酶消化不同时间后收集软骨终板细胞,分别进行计数和台盘蓝染色计算细胞存活率。传代培养软骨终板细胞,对1、3、6代细胞行甲苯胺蓝染色,观察不同代软骨终板细胞形态。检测Ad-hIGF-1的序列及应用50%组织感染计量法检测重组腺病毒载体的滴度,Ad-hIGF-1转染软骨终板细胞24h后提取细胞基因组DNA,PCR检测Ad-hIGF-1能否转染进细胞,RT-PCR检测转染后hIGF1的表达情况。将Ad-LacZ分别以20、50、100、200转染复数(MOI)转染软骨终板细胞后,利用标志基因β半乳糖苷酶基因(LacZ)观察最佳MOI。分别将Ad-hIGF-1转染第1、3、6代软骨终板细胞,RT-PCR半定量目的基因hIGF-1及细胞产物聚集蛋白聚糖、Ⅱ型胶原在基因水平的表达,MTT法检测软骨终板细胞的增殖能力,ELISA试剂盒检测软骨终板细胞对hIGF-1的分泌量,番红O染色检测软骨终板细胞对蛋白多糖的表达。结果Ⅱ型胶原酶消化3h后获取细胞数量最多,且细胞存活率高,4h后获取细胞数量明显减少,细胞存活率降低。第1代软骨终板细胞呈三角形、多角形,立体感强,第3代细胞生长变缓,形状出现长梭形,当细胞传至第6代,细胞核仁不清,细胞形状以长梭形为主,折光性弱,呈成纤维化改变。第1代软骨终板细胞甲苯胺蓝染色后胞核染色较深,胞质略淡,第3代细胞染色较第1代细胞变淡,第6代细胞染色较第3代明显变淡。由测序结果可判断Ad-hIGF-1的序列完整且方向正确,Ad-hIGF-1的滴度为2×10~9PFU/ml。Ad-hIGF-1能够成功转染进软骨终板细胞,且细胞能够表达目的基因hIGF-1。Ad-LacZ转染兔软骨终板细胞后,在100MOI范围内转染率与转染复数呈正比,200MOI转染率与100MOI转染率基本相等,均为95%左右。Ad-hIGF-1转染第1代软骨终板细胞后,hIGF-1和Ⅱ型胶原的mRNA表达量较对照组明显增加(P＜0.05),聚集蛋白聚糖表达量无明显变化(P＞0.05);当细胞传至第3代,第1代即转染Ad-hIGF-1后传代的细胞与传代后转染Ad-hIGF-1的细胞对hIGF-1、聚集蛋白聚糖与Ⅱ型胶原的表达无明显差异(P＞0.05),较对照组明显增加(P＜0.05);当软骨终板细胞传至第6代时,第1代即转染Ad-hIGF-1后传代的细胞与传代后转染Ad-hIGF-1的细胞对hIGF-1、聚集蛋白聚糖与Ⅱ型胶原的表达无明显差异(P＞0.05),较对照组明显增加(P＜0.05)。同时,转染Ad-hIGF-1后软骨终板细胞的增殖能力增强(P＜0.05),hIGF-1蛋白的表达量增加,且能够维持软骨终板细胞的表型。结论通过体外分离、培养和初步鉴定,成功进行兔软骨终板细胞培养,通过单层传代培养软骨终板细胞,细胞逐渐发生退变,可作为研究软骨终板的体外模型。携带hIGF1的腺病毒载体可转染发生退变的和正常的软骨终板细胞,在100MOI内转染率与转染复数呈正相关,转染复数为100MOI时转染效率达最高,且对细胞无特殊毒副作用,由腺病毒载体介导的hIGF1可在兔软骨终板细胞内表达,并能促进细胞表达和分泌蛋白多糖、Ⅱ型胶原,增强细胞增殖能力,维持软骨终板细胞表型,且作用持续较长时间。在软骨终板细胞退变早期应用携带hIGF1腺病毒载体上调其生物学活性较退变后期应用效果要好。更多还原

【Abstract】 Object To try isolating and culturing rabbit endplate cells,and to observe the biological characteristics of the endplate cells.To learn the efficiency of the adenovirus vector carrying human insulin-like growth factor(hIGF1) transfecfing different generation rabbit cartilage endplate cells and the biologic effect of purpose gene on cell.To discuss the feasibility of applying Ad-hIGF-1 treating disc degeneration.Methods Cartilage endplate was isolated in vitro,and then was digested 30 minute with trypsin.We collected endplate cells after tissue being digested for different time with typeⅡcollagenase,and counted cell number and calculated cell survival rate by typan blue staining. Endplate cells were cultured monolayer through passage.The p1、p3、p6 cells were stained with ammonium methylbenzene blue,and we observed the different passage cells’ shape.The gene sequence of Ad-hIGF-1 was tested and titre was measured using 50%tissue culture infectious dose.In order to study weather Ad-hIGF-1 could transfect into endplate cell,genomic DNA was extracted after cells being transfected 24 hours with Ad-hIGF-1,and then was tested using PCR, and the expression of hIGF-1 was measured by RT-PCR.After endplate cells being transfected with Ad-LacZ with 20,50,100,200 MOI respectively,we invested the optimum MOI applying marker gene Galacitiol nucleoside enzymes(LacZ).The p1,p3,p6 cells were transfected with Ad-hIGF-1,and then the gene expression of purpose gene hIGF1,aggrecan,collagenⅡwas semi-quantitatived by RT-PCR,and the capacity to proliferate was tested by MTT,and the secretion of hIGF-1 protein was tested by ELISA,and the secretion of aggrecan was tested by safranine O staining.Results We could harvest the largest cells number after tissue being digested 3 hours with typeⅡcollagenase,and the cells’ survival rate was high.After 4 hours,the cell number decreased,and cells survival rate reduced.The first generation cartilage endplate cells performed triangular,polygonal,and stereo sense is strong.For the third cells,capacity for proliferation weakened,and appeared spindle shape.When cells were passaged to sixth passages,nucleus became vague,and cells appeared long spindle shape,and the refractive was weak,and the cells were changed into fibrosis.Being stained by ammonium methylbenzene blue,for the first generation cells,nucleus is dark blue,and the cytoplasm is light blue;for the third generation cells, cellular color is lighter than first generation cells,and for the p6 cells,staining color became apparently light compared with the p3 cells.The gene sequence of Ad-hIGF-1 is complete and the orientation is correct,judging from sequencing result.The titre of Ad-hIGF-1 was 2×10~9PFU/ml. Ad-hIGF-1 could transfect into endplate cells successfully,and cells could express purpose gene hIGF-1.When endplate cells were transfected with Ad-LacZ,transfection rate varied following multiplicity of infection within 100MOI range,and the transfection rate was both about 95%when multiplicity of infection were 100MOI and 200MOI respectively.When first generation cells were transfected with Ad-hIGF1,the gene expression of both hIGF1 and collage typeⅡincreased significantly compared with control group,and the expression of aggrecan didn’t take place change(P＞0.05).When cells were passaged to third passage,the gene expression of hIGF1, aggrecan,collage typeⅡdidn’t exist difference between cells which were passaged after being transfected at first generation and cells which were transfected after being passaged(P＞0.05). However,the gene expression increased compared with control group(P＜0.05).When cells were passaged to sixth passage,the gene expression of hIGF1,aggrecan,collage typeⅡdidn’t exist difference between cells which were passaged after being transfected at first generation and cells which were transfected after being passaged(P＞0.05).However,the gene expression increased compared with control group(P＜0.05).At the same time,the capacity for proliferation of cells was enhanced while cells being transfected with Ad-hIGF-1,and the secretion of hIGF1 increased, and endplate cells could maintain its phenotype.Conclusion We cultured rabbit endplate cells in vitro successfully through isolation,culture, preliminary appraisal.The endplate cells degenerated gradually by monolayer culture.Therefore, the ceils could be studied as cartilage endplate models in vitro.Adenovirus vector carrying hIGF1 could transfect degenerated and normal endplate cells,and the transfection rate correlated positively with multiplicity of infection,and the transfection efficiency is the highest when multiplicity of infection is 100 MOI,and the adenovirus vector had not side-effect on cells.The rabbit endplate cells could express hIGF1 mediated by adenovirus vector,hIGF1 can promote cell expressing and secreting aggrecan,collage typeⅡ,and enhance cellular capability to proliferate,and maintain endplate cells phenotype,and the bioactivity could last long time.The biological effect of hIGF1 which was mediated by adenovirus vector was better when it was applied early disc degeneration than when it was applied latter disc degeneration.更多还原