腺病毒介导TGF-β1基因转染兔骺板软骨细胞的实验探究
一、中文摘要 (2)
二、英文摘要 (4)
三．论文正文
1、前言 (7)
2、材料和方法 (9)
3、实验结果 (17)
4、讨论 (20)
四、结论 (24)
五、附图 (29)
六、论文参考文献 (25)
七、中英文缩略词表 (39)
八、综述
1、综述正文 (42)
2、参考文献 (47)
腺病毒介导TGF-β1基因转染兔骺板软骨细胞
的实验研究
中文摘要
目的：通过腺病毒介导TGF-β1基因转染兔骺板软骨细胞(Epiphyal Chondrocytes，ECs)，观察TGF-β1的表达及ECs合成和分泌软骨基质的情况，为构建新型的组织工程骺板软骨提供实验依据。
方法：1.利用3周龄新西兰大白兔股骨远端骺板软骨，分离出ECs并传代培养，观察ECs生
长情况并鉴定。选用第3代ECs进入下一步基因转染。2. 构建携带TGF-β1基因的腺病毒载体。3.分3组进行转染：分别为TGF-β1载体组、空载体组和空白组。转染后在倒置显微镜下观察各组细胞形态以及增殖情况；并在荧光显微镜下观察各组病毒转染情况，同时计算转染效率。利用MTT法检测重组腺病毒转染后的ECs增殖能力是否有明显变化。应用ELISA法检测转染后收集到的各组培养液中的TGF-β1浓度。应用Ⅱ型胶原免疫荧光评价转染后细胞的成软骨能力。采用RT-PCR检测各组细胞TGF-β1 mRNA的表达情况，并用Western-blot检测各组细胞Ⅱ型胶原和TGF-β1蛋白表达情况。
结果：1、成功的从兔骺板软骨中分离出ECs，并进行了传代培养及鉴定。2、在荧光显微镜下观察各组转染后细胞，发现TGF-β1载体组和空载体组有较多荧光细胞出现，而空白组始终没有出现荧光细胞，细胞转染成功。通过MTT法检测证明，腺病毒介导TGF-β1基因转染后的ECs的增殖能力短期内没有影响。3、转染14d后，TGF-β1载体组免疫荧光观察到Ⅱ型胶原较对照组明显增强。4、RT-PCR检测发现转染组标本中TGF-β1 mRNA的表达较对照组明显增强。4、Western-blot结果显示转染组中CollagenⅡ和TGF-β1表达也明显增强。
结论：1、本实验成功的从兔骺板软骨中分离出ECs，并进行了传代培养及鉴定，证实了第
3代ECs表型稳定，骺板软骨细胞的生物学及功能学特性表达良好。2、本实验成功的构建了携带TGF-β1基因的重组腺病毒载体，并运用重组腺病毒载体成
功转染ECs，且转染率高，对细胞毒性小。3、利用基因转染技术将TGF-β1基因转染至ECs，可增强ECs合成和分泌软骨基质。
关键词：TGF-β1 腺病毒基因转染骺板软骨细胞
The experimental study of recombinant adenovirus-mediated TGF - beta 1 Gene transfecting Epiphyal Chondrocytes
Abstract
Objective: According to the gene transfection technology, u the adenovirus to mediate TGF - beta 1 gene transfecting Epiphyal plate cartilage cells (Epiphyal Chondrocytes, ECs), and obrve the expression of TGF - beta 1 and ECs synthesis and cretion of cartilage matrix , to provide a experimental basis for building a new type of Epiphyal plate cartilage tissue engineering.
Methods: 1. ECs were isolated from 3-week-old New Zealand white rabbits’femoral distal epiphyal plate cartilage. Then ECs were cultured and amplified in vitro. ECs at passage 3 were ud for the transfect ion.2. Build a TGF - beta 1 gene adenovirus vector. 3. The experiment was divided into three groups, respectively named TGF - beta 1vector group, empty vector group, and control group. After transfection, the cell morphology was obrved under fluorescence microscope. Meanwhile, the number and fluorescence intensity of fluorescent cells were obrved under fluorescence microscope. The transfection efficiency was estimated by counting fluorescent cells in each field of vision.And the cell Proliferation activity was assayed by using MTT. The TGF - beta 1 conceniration in the culture media was detected by ELISA /doc/e82055467.html
e Collagen Ⅱimmunofluorescence to evaluate cartilage capability of transfect cells. U RT-PCR to detect TGF - beta 1 mRNA expression in each cell, and Western- blot to detect each group of cells Ⅱ collagen type and TGF - beta 1 protein expression.
Results: 1. ECs was successfully isolated from rabbit epiphyal plate cartilage and subcultured and identified .2. Each group of transfected cells was obrved under a fluorescence microscope, TGF-β1 carrier group and empty vector group have more fluorescent cells, while the control group did not have fluorescent cells. It has been proved that the proliferative capacity of adenovirus-mediated TGF-β1 gene transfection
of ECs has no effect in the short term by MTT assay . 3.14 days after transfection, compared with the control group the obrved collagen type II significantly incread through TGF-β1 carrier group immunofluorescence. 4.RT-PCR detected that in the transfection group specimens TGF-β1 mRNA expression significantly incread compared with the control group 5.Western-blot results showed that the transfected group Collagen Ⅱ and TGF-β1 expression also significantly enhanced . Conclusion: 1. ECs was successfully isolated from rabbit epiphyal plate cartilage and subcultured and identified, rabbits in vitro epiphyal plate 3th reprents the type of stable cells, the expression of biology and functional characteristics of a good school.2. The recombinant adenovirus carrying hTGF-β1 gene had been constructed successfully ,u the recombin
ant adenovirus vector to transfect ECs successfully, and the transfection rate is very high, meanwhile , small toxicity to cells. 3. Using the gene transfection technology to make TGF - beta 1 gene transfect ECs, and strengthen the ability of ECs synthesis and cretion of cartilage matrix.
Keywords：Transforming growth factor beta 1, adenovirus, gene transfection, Epiphyal Chondrocytes
前言
骺板损伤修复的临床治疗和实验研究是小儿骨科中一个难点，也是一个热点，因为骺板损伤常在损伤部位形成骨桥，导致骺板生长缺陷，从而造成肢体短缩或成角畸形，严重者影响肢体功能[1] 。目前对于小儿骺板损伤的治疗效果不佳。治疗骺板损伤的方法在人们不断的探究中进步，最初的治疗方法是将骺板损伤部位形成的骨桥切除，填充明胶海绵、骨腊、自体脂肪组织和骨水泥等材料，这些材料只能防止损伤部位骨桥形成，缺乏再生修复能力，疗效不佳[2]；研究发现将有血供的骺板作为移植修复材料，解决了骺板修复的供血问题，但容易导致供区缺损和畸形，而且取材受限[3] 。目前，对于骺板损伤的治疗，疗效
欠佳。但软骨组织工程技术的发展，为骺板损伤的治疗提供了新的研究方向。
组织工程学是运用生命科学和工程学的一般原理及技术，通过研究生物替代物来改善或修复受损器官功能的一门新兴学科。组织工程构建的生物材料较传统材料更具优越性，研究表明通过构建新型组织工程材料修复骺板软骨损伤[4-6]，取得了可喜的实验成果。Planka等[7]使用异体骨髓间充质干细胞复合由Ⅰ型胶原蛋白和壳聚糖纳米纤维构建的支架材料，修复猪股骨远端骺板缺损，4w后观察到修复材料在缺损处形成了类似骺板结构的透明软骨样组织，肢体畸形程度较对照组明显减轻。Yoshida等[8]体外培养滑膜间充质干细胞构建成无支架的组织工程材料，用以修复6w龄新西兰白兔的胫骨近端的骺板损伤模型，4w后X线摄片显示胫骨出现畸形愈合，但畸形程度低于对照组；而且细胞有向软骨样细胞分化趋势。研究发现软骨细胞在体外培养过程中，随着传代次数的增加，软骨细胞的细胞活力及生物学功能明显下降。
组织工程包含三要素：种子细胞、支架材料和细胞因子。种子细胞作为组织工程学的研究基础，在现阶段的骨组织工程研究中得到重视[9]。在构建组织工程化骺板的研究中，人们应用不同的种子细胞来构建各种新型组织工程化骺板，但是ECs 因其具有其他种子细胞所
不具备的特点，成为国内外研究的热点。ECs合成和分泌胶原蛋白及蛋白聚糖等软骨基质能力优于其他种子细胞，是修复骺板损伤的最佳种子细胞[10]。周强等[11]运用离心管内软骨细胞聚集生成软骨技术，按原代ECs离心收集于离心管内后，加入含10％胎牛血清的IMDM培养液，4w后得到了直径为4.5
