【作者】 陈传好；

【导师】 汤锦波；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 目的:(1)检测结缔组织生长因子(CTGF)、转化生长因子β(TGF-β)、类胰岛素生长因子1 (IGF-1)、血管内皮生长因子(VEGF)、血小板源性生长因子(PDGF-B)和碱性成纤维细胞生长因子(bFGF)在肌腱损伤愈合早期的mRNA水平变化规律和TGF-β, bFGF和IGF-1在蛋白水平的表达变化。(2)探索微量注射法以不同转基因载体转基因至损伤肌腱的转基因效率、分布和组织反应。(3)比较腺病毒(adenoviral, Ad)、腺相关病毒(adeno-associated viral, AAV)和脂质体-质粒(liposome-plasmid)三种转基因载体在肌腱中的基因转染效率并观测腺相关病毒载体介导碱性成纤维细因子(basic fibroblast growth factor, bFGF)基因在肌腱愈合过程中表达。(4)探讨微小RNA导入肌腱细胞和损伤肌腱沉默转化生长因子(TGFβ)基因的作用及对I型胶原、Ш型胶原和CTGF基因表达的影响。方法:(1)在鸡趾的掌面掌趾关节与近节趾间关节间行Bruner切口,游离出趾深屈肌腱(FDP),并在趾半屈曲位时用锐刀将其从掌侧横形做完全切断,然后用5-0无创缝线做改良Kessler法缝合修复肌腱。在术后第3、5、7、9、14和21天分别取肌腱,做TGF-β, bFGF和IGF-1免疫组化染色,观测其表达变化和分布。通过实时PCR方法检测CTGF、TGF-β、VEGF、IGF-1、PDGF-B和bFGF基因的mRNA水平在术后第3、9、14和21天四个时间点表达变化。(2)用微量注射器将10微升携带增强绿色荧光蛋白(Enhanced Green Fluorescent Protein, EGFP)报告基因的pCMV-EGFP、pCAGGS-EGFP、AAV2-EGFP和Ad5-EGFP载体直接注射入18只鸡的48根横断伤的趾屈肌腱近侧断端截面上取两点注射。在术后第3、7、14、21天分别取肌腱,进行冰冻切片后在荧光显微镜下观察肌腱内的EGFP表达量及分布,并将肌腱切片作HE染色后观察各不同载体在肌腱中引起的炎症反应。另24根肌腱不作手术作为对照。(3)用微量注射器将携带增强绿色荧光蛋白报告基因的pCMV-EGFP、AAV2-EGFP和Ad5-EGFP注射入鸡的正常趾深屈肌腱中,术后第3、7、14和21天分别取肌腱进行冰冻切片,荧光显微镜观测肌腱内EGFP的表达。将携带有bFGF基因的AAV2-bFGF病毒颗粒注入完全切断的鸡的趾深屈肌腱,术后的第2、3和4周获取注射AAV2-bFGF实验组的肌腱及对照组肌腱做免疫组化染色。(4)针对鸡TGFβ1基因的mRNA序列,设计并合成4对miRNA TGFβ1 DNA序列和一对不编码序列,分别插入至miRNA质粒载体(pcDNA6.2)中,获得5个miRNA TGFβ1质粒载体,其分别命名为miRNA #1、#2、#3、#4和阴性对照。将不同的miRNA载体转染培养的肌腱细胞,用荧光显微镜观测载体携带的增强绿色荧光蛋白报告基因在肌腱细胞的表达,并使用流式细胞仪评价转基因的效率。采用实时PCR技术检测并分析miRNA导入肌腱细胞后TGFβ1、I、Ш型胶原和CTGF基因表达变化。选用抑制TGFβ1表达最明显的miRNA #1载体转染活体损伤肌腱。在转基因治疗肌腱一周和六周后,检测TGF-β1、I、Ш型胶原和CTGF基因在体内损伤肌腱中的表达。结果:(1)在肌腱损伤愈合早期CTGF、TGF-β基因mRNA表达水平较高,其中TGF-βmRNA在肌腱损伤后呈增高的趋势;VEGF和IGF-1基因mRNA表达水平次之;bFGF和PDGF-B基因mRNA在肌腱损伤后表达水平较低,且bFGF基因mRNA呈逐渐下降的趋势。TGF-β, bFGF和IGF-1免疫组化也显示相同结果。(2)荧光显微镜下观测发现,注射4种转基因载体的肌腱,转染3天后即可见肌腱内有增强绿色荧光蛋白表达;在第7天时表达最明显;第14天时,各组表达增强绿色荧光蛋白表达量下降;21天时很少有表达增强绿色荧光蛋白的细胞。用微量注射器在腱段两点注射能保证近损伤肌腱内转染细胞均匀分布。注射AAV2-EGFP和Ad5-EGFP的肌腱的增强绿色荧光蛋白明显多于质粒载体的增强绿色荧光蛋白,而AAV2-EGFP和Ad5-EGFP两组间无明显差异。HE染色发现质粒载体和Ad5-EGFP致肌腱的组织反应较重,可见较多炎症细胞浸润,以淋巴细胞和中性粒细胞为主。注射AAV2-EGFP的肌腱炎症反应较轻。(3)荧光显微镜下可见三种不同的载体在肌腱内均表达EGFP:转染3天后即可见肌腱内有EGFP表达;在第7天时表达最明显;第14天时,各组EGFP的表达下降;21天时很少有表达EGFP细胞。在同期的时间点,注射AAV2-EGFP和Ad5-EGFP的肌腱内EGFP表达明显强于质粒载体,而AAV2-EGFP和Ad5-EGFP两组之间无较大区别。术后的第2、3和4周免疫组化染色结果发现:相比于正常和对照组肌腱,注射AAV2-bFGF的肌腱表达很强的bFGF。(4)体外细胞培养中荧光显微镜观察到肌腱细胞有EGFP表达,流式细胞仪检测基因转染效率为20~25%。与阴性对照细胞组相比,实时PCR结果分析显示:miRNA #1和#2质粒载体治疗的细胞组TGFβ1基因表达分别下降了68%和43%;在miRNA #1治疗的细胞组,III型胶原和CTGF基因表达分别也下降70%和68%,这些变化在统计学上有显著性。使用miRNA #1干扰质粒转基因至体内损伤肌腱结果显示:术后一周,TGFβ1基因表达下降了67%,而I、Ш型胶原和CTGF基因表达没有变化;术后六周,TGFβ1和Ш型胶原基因表达分别下降了56%和58%,变化在统计学上有显著性。结论:(1)在肌腱损伤愈合的早期(3周内)TGF-β基因表达水平较高,呈逐渐升高趋势;CTGF, VEGF, and IGF-1表达次之;bFGF基因表达水平较低且逐渐降低;PDGF-B表达较低。(2)用微量注射器在腱段两点注射转基因载体能转染近损伤处的整个肌腱段的细胞。在研究的四种基因治疗方法中,AAV2和Ad5在在损伤肌腱中的转基因效率最强,在转基因后第7天表达最明显,而且AAV2引起的肌腱组织反应最轻。提示AAV2载体比Ad5和质粒载体有利于作为转基因治疗肌腱损伤的载体,微量注射载体是转基因至损伤肌腱的合适方法。(3)Ad5和AAV2载体的基因转染效率高于脂质体-质粒载体,AAV2携带的外源性bFGF基因肌腱细胞内较强表达,提示我们将来在体内转基因到肌腱的研究中能够选择腺病毒和腺相关病毒载体作为运载基因的工具。(4)miRNA导入肌腱细胞后TGFβ1、Ш型胶原和CTGF基因表达显著下降。导入miRNA至活体损伤肌腱后一周,TGFβ1基因表达显著下降;术后六周,TGFβ1和Ш型胶原基因表达显著下降。本研究提示miRNA能有效沉默肌腱TGFβ基因表达,可望成为减少损伤肌腱粘连的方法。更多还原

【Abstract】 Objective:(1) To observe the discipline of mRNA experssion level of CTGF, TGF-β, IGF-1, VEGF, PDGF-B and bFGF during healing early periods of injured flexor tendons, and the protein production expression action of TGF-β, bFGFand IGF-1 at multiple time-points during the early healing period in a chicken model.(2) To investigate efficiency and distribution of gene delivery to the injured tendons and tissue reactions caused by different vectors(3) To investigate efficiency of gene delivery of the adenoviral, adeno-associated viral and liposome-plasmid vectors to the uninjured tendons, and to explore the bFGF gene expression by AAV2 vectors during tendon healing.(4) To investigate the in vitro and in vivo effects of delivery of microRNA (miRNA) to silence expression of the transforming growth factorβ1 (TGFβ1) genes and its influence on expression of the typeⅠ,Ⅲcollagen and connective tissue growth factor (CTGF) genes in cultured tenocytes and injured tendons.Method:(1) Seventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis on days 3, 5, 7, 9, 14, or 21 post-surgery. Expression of 6 growth factors at 4 post-surgical time-points was studied with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time-points were studied by immunohistochemical staining with antibodies. Fourteen tendons that underwent no surgery served as day 0 controls. Tendon healing status was also assessed histologically.(2) Using a microinjection technique, 10μl of pCMV-EGFP, pCAGGS-EGFP, AAV2-EGFP, and Ad5-EGFP, harboring enhanced green fluorescence protein (EGFP) gene, respectively, were injected to two sites of the proximal stump of 48 transected digital flexor tendons in 18 chickens. At 3, 7, 14, and 21 days, the tendons were harvested. Under a fluorescence microscope, distribution and expression of EGFP was examined using frozen tissue sections. The tendon sections were also stained with hematoxylin and eosin to examine inflammation caused by these vectors. 24 flexor tendons were not injured, serving as the controls.(3) pCMV-EGFP, AAV2-EGFP, and Ad5-EGFP were injected to the uninjured digital flexor tendons of the chickens. At 3, 7, 14, and 21 days, the tendons were harvested. Under a fluorescence microscope, the expression of EGFP was examined. The AAV2-bFGF was injected to the injured digital flexor tendons of the chickens. Immunohistochemical staining of the tendons treated with AAV2-bFGF and non-treated with AAV2-bFGF was carried out at 2, 3 and 4 weeks after surgery.(4) Four miRNAs TGFβ1 DNA and one sham sequence vector control were designed and synthesized according to chicken TGFβ1 mRNA sequence. They were used to produce 5 plasmid expression vectors (miRNA #1、#2、#3、#4 and negative control). Cultured chicken tenocytes were transfected with these vectors. Under a fluorescence microscope and flow cytometry, the expression of EGFP was examined, to evaluate the transfection efficiency of the vectors. Expression of TGFβ1, collagen I, III, and CTGF genes were measured using real-time PCR. miRNA #1 plasmid, which had the highest inhibitory effects to the TGFβ1 gene was injected into injured digital flexor tendons of chickens. At week 1 and 6 postsurgery, expression of TGFβ1, collagen I, III, and CTGF gene were measured using real-time PCR.Results:(1) Throughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-β) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-βgene was up-regulated after injury, while basic fibroblast growth factor (bFGF) gene was down-regulated at all post-surgical time-points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks post-surgery. Platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of Immunohistochemistry corresponded to TGF-β, bFGF, and IGF-1 gene expression. (2) Compared with normal tendon tissues, the EGFP expression was observed in tendons at 3, 7, 14 and 21 day post-injection. The EGFP expression was observed at 3 days, and was the highest at 7 days for all vectors. At 14 days, we observed a decrease in the EGFP expression. EGFP was distributed even in the injected tendon segment adjacent to the cut level. The EGFP expression in the tendons injected with AAV2-EGFP and Ad5-EGFP was higher than that with pCMV-EGFP and pCAGGS-EGFP injection. We did not find remarkable differences in the EGFP expression between AAV2-EGFP and Ad5-EGFP vectors. Tissue reactions of the tendons caused by the liposome–plasmid vector (including pCMV-EGFP and pCAGGS-EGFP) were the most prominent among all vectors. Infiltration of Inflammatory cells, chiefly lymphocytes and neutrophilic granulocytes, were observed. Inflammatory reactions in the tendons injected with AAV2 vectors were the least severe.(3) Compared with normal tendon tissues, the EGFP expression was observed in tendons at 3, 7, 14 and 21 day post-injection. The EGFP expression was observed at 3 days, and was the highest at 7 days for all vectors. At 14 days, we observed a decrease in the EGFP expression. The EGFP expression in the tendons injected with AAV2-EGFP and Ad5-EGFP was higher than those of pCMV-EGFP injection. We did not find remarkable differences in the EGFP expression between AAV2-EGFP and Ad5-EGFP vectors. The AAV2-bFGF treatment remarkably increased the expression of bFGF at 2, 3 and 4 weeks after surgery.(4) EGFP expression was confirmed in tendon cell using fluorescence microscope and flow cytometry which showed that 20~25% of the tenocytes were transfected by the miRNA vectors. Compared with the negative control, expression of the TGFβ1gene in the tenocytes treated with miRNA #1 and #2 was decreased by 68% and 43%, respectively. In the cells treated with miRNA #1, expression of the typeⅢcollagen and CTGF genes was decreased by 70% and 68%, respectively. These changes were statistically significant. At week 1 postsurgery, expression of the TGFβ1 gene in the injured digital flexor tendons decreased by 67%, but expression of the collagen I, III, and CTGF genes were not changed. At week 6, expression of the TGFβ1 and typeⅢcollagen genes was decreased by 56% and 58%, respectively; the changes were statistically significantConclusion: (1) In this model, up to post-surgical 3 weeks, gene expression and production of TGF-βare high and are up-regulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.(2) Microinjection to two sites of each tendon stump delivers the transgene to the entire tendon segment adjacent to the cut. Efficiency of gene delivery by the AAV2 and Ad5 vectors is the highest among 4 vectors tested. Expression levels peak at 7 days post-injection. AAV2 vector causes the slightest tissue reactions in the tendons. The study suggests that the AAV2 vector is a promising gene delivery vector and microinjection is practical for tendon gene therapy.(3) Efficiency of gene delivery by the AAV2 and Ad5 vectors is the higher than the liposome-plasmid vectors. The tendons with AAV2-bFGF treating express the bFGF strongly, which can be maintained at a high level up to 4 weeks. The study suggests that the AAV2 and adenoviral vectors are promising gene delivery vectors for tendon gene therapy.(4) Expression of the TGFβ1, collagen III, and CTGF genes decreased substantially in tendon cells treated with miRNAs. Expression of the TGFβ1 gene was reduced in injured tendons at week 1 postsurgery; expression of both the TGFβ1 and collagen III genes was decreased at week 6. The study indicates that delivery of miRNA to silence expression of the TGFβ1 gene may be a promising in reducing tendon adhesion formations.更多还原