【作者】 潘海涛；

【导师】 郑启新；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分兔骨髓基质干细胞的获取及生物学鉴定目的体外获取兔骨髓基质干细胞为骨组织工程研究提供种子细胞。方法取4周龄新西兰大白兔骨髓,密度梯度离心法获取BMSC,通过倒置相差显微镜、BrdU标记染色观察其形态学特征和生长状况,流式细胞仪分析其表面标志物抗原表达情况。结果分离培养的细胞在第10～12代以前生长性状稳定,增殖能力强。70%以上的BMSC细胞核被BrdU标记。流式细胞仪检测显示兔BMSC表面抗原CD44阳性细胞百分率为93.85%,CD90阳性细胞百分率为75.28%,CD34阳性细胞百分率为2.98%,CD45阳性细胞百分率为1.70%,CD11b阳性细胞百分率为0.45%,Lamine阳性细胞百分率为0.24%。结论分离培养的细胞成分较为单一,具有干细胞特性,适用于骨组织工程学研究。第二部分双功能多肽(K)16GRGDSPC的设计、合成及介导细胞特异性粘附目的制备双功能多肽(K)16GRGDSPC并评价其能否介导BMSC特异性粘附。方法设计RGD肽(K)16GRGDSPC。固相合成法合成(K)16GRGDSPC,用质谱仪和高压液相色谱仪鉴定。通过细胞粘附和粘附竞争性抑制实验评价(K)16GRGDSPC介导细胞粘附的可行性和特异性。结果质谱仪显示合成肽的平均分子量为2741.54 m/z,与理论计算分子量一致。高压液相色谱仪显示合成肽的纯度为99.2796%,完全满足实验要求。RGD肽GRGDSPC、(K)16GRGDSPC及BSA、(K)16均能促进BMSC粘附,以(K)16GRGDSPC作用最为显著;非RGD肽GRGESPC对BMSC粘附无促进作用。仅GRGDSPC、(K)16GRGDSPC可显著抑制BMSC在(K)16GRGDSPC预处理培养板的粘附,而非RGD肽GRGESPC无明显影响,BSA、(K)16甚至进一步促进BMSC在(K)16GRGDSPC预处理培养板的粘附。结论(K)16GRGDSPC易于合成,可以通过RGD结构域介导BMSCs特异性粘附。第三部分多肽(K)16GRGDSPC介导基因转染及影响因素目的评价多肽(K)16GRGDSPC作为一种非病毒载体介导基因转染的可行性及其介导基因转染的靶向特异性;探讨优化(K)16GRGDSPC介导基因转染的各种影响因素。方法用(K)16GRGDSPC介导报告基因虫荧光素酶质粒和绿色荧光蛋白质粒转染BMSC,通过液闪计数仪和荧光显微镜观察基因表达。流式细胞仪检测基因转染效率,并与商业化脂质体Lipofetamine 2000的转染效率进行比较。通过转染竞争性抑制实验检测(K)16GRGDSPC介导基因转染的靶向特异性。在保持其它因素一致的条件下,通过变化单一因子来评估其影响并优化该参数。结果虫荧光素酶pGL3质粒和EGFP质粒均能在BMSC内表达。FCM显示(K)16GRGDSPC的转染效率为19.47%,与Lipofectamine 2000的转染效率19.98%无显著性差异(P>0.05)。加入含RGD肽GRGDSPC和(K)16GRGDSPC使虫荧光素酶的表达活性明显下降;而非RGD肽GRGESPC对转染无明显影响。在(K)16GRGDSPC介导基因转染的过程中,氯喹和PEI均可以改善其基因转染效率。尤其是氯喹,对多肽载体介导基因转染起着不可或缺的作用,BMSCs在氯喹溶液中的暴露时间至少应维持在8h以上;在载体/质粒复合物中暴露时间过短(<4h)或过长(>24h)对转染都有不利影响;质粒浓度在2～4μg时转染效果最佳;载体/质粒质量比为3:1时有最高的转染效率;血清对质粒/载体复合物与细胞的结合有明显抑制作用;膜融合肽P20对基因转染效率也有促进作用,但仅约为氯喹的75%;脂质体Lipofectamine和多肽对基因转染有明显的协同作用;加入转铁蛋白也能显著提高基因的转染和表达,且在25μg/ml时转染效率达到最高。结论多肽(K)16GRGDSPC不仅可以作为一种RGD肽介导细胞特异性粘附,还可以作为一种非病毒载体介导靶向特异性基因转染并获得理想的转染效率。优化转染条件可提供改善多肽载体转染效率的多种策略,为多肽载体在基因转染领域的开发应用和本实验下一步进行基质材料表面基因修饰研究奠定了理论基础。第四部分载基因仿生基质材料的制备及介导基因转染目的制备载基因仿生基质材料并检测其能否介导外源基因转染。方法通过交连剂Sulfo-LC-SPDP将非病毒载体(K)16GRGDSPC共价接枝到骨基质材料PLGA-[ASP-PEG]支架材料上构建非病毒基因转染体系,XPS图谱检测接枝反应的发生。将BMSC与其复合培养,扫描电镜观察细胞生长情况,通过沉淀法、微管吮吸法检测细胞粘附性能的变化。激光共聚焦显微镜下观察细胞骨架主要成分肌动蛋白(F-actin)的形态。该非病毒基因转染体系与TGF-β1基因复合制备载基因仿生基质材料,将兔BMSC与其复合培养,通过RT-PCR、Western Blot、ELISA及免疫组化染色检测TGF-β1基因的表达。结果XPS图谱证实含RGD肽(K)16GRGDSPC被成功地接枝到PLGA- [ASP-PEG]表面。细胞粘附性能和粘附强度显著改善。RT-PCR、Western Blot、ELISA以及免疫组化检测均显示TGF-β1基因在BMSC内表达。结论这种载基因基质材料作为一种基因活化基质,可以作为一种非病毒载体介导外源基因转染。第五部分载基因仿生基质材料调控BMSC成骨定向分化目的评价载基因仿生基质材料能否调控骨髓基质干细胞(BMSC)向成骨细胞定向分化。方法制备载基因基质材料并与BMSC复合培养,采用四甲基偶氮唑蓝微量酶反应比色法(MTT)、考马斯亮蓝法和流式细胞仪(FCM)检测细胞体外增殖活性,并进行成骨诱导扩增,检测转基因细胞成骨标志物碱性磷酸酶、骨钙蛋白、骨桥蛋白、Ⅰ型胶原、核心结合因子a1(Cbfa1)的表达及矿化情况。FCM分析DNA含量和细胞骨架染色评估载基因基质材料的安全性。结果载基因基质材料复合培养细胞的体外增殖活性明显高于对照组(P<0.01);经成骨诱导后其成骨特征性表型(ALP、OCN、OPN和CollⅠ、Cbfa1)的表达及矿化与对照组有显著的差异(P<0.01);细胞与载基因基质材料长期复合培养后无异倍体,细胞骨架形态正常。结论载基因基质材料可以调控BMSC向成骨细胞方向定向分化;TGF-β1可以作为理想的调控因子之一。更多还原

【Abstract】 PartⅠIsolation and Identification of Rabbit-derived Bone Marrow Stromal CellsObjective To obtain rabbit autogenous bone marrow stromal cells (BMSC) and provide seed cells for our researches on bone defect restoration in bone tissue engineering. Methods The bone marrow tissues were fetched from rabbit and BMSCs were isolated by the method of density centrifuge .Inverted phase contrast microscope and BrdU label were employed to observe the cell morphologic features and growth statuses. Flow cytometry was also used to analyze the CD markers on cell surfaces. Results The cultured BMSC grew well and displayed strong capabilities of proliferation until 10th to 12th generations. Above 70 percentage of the cells were labelled by Brdu. FCM analyses showed the positive rates of CD44, CD90, CD34, CD45, CD11b and Lamine on the cell membranes were 93.85%, 75.28%, 2.98%, 1.70%, 0.45% and 0.24% respectively.Conclusion The cultured cells were mostly composed of undifferentiated BMSCs, being of the property of stem cells. The cells were suitable for tissue engineering.PartⅡDesign and synthesis of a bifunctional oligopeptide and specific adhesion by the synthetic peptide Objective To design and sythesize a bifunctional oligopeptide and explore the specifility of cell adhesion mediated by the synthetic peptide.Methods Design a RGD-containing peptide consisting of 23 amino residues. The peptide was synthesized by solid-phase synthesis method and characterized by mass spectrometry and high pressure liquid chromatography. The feasibility and speicifility of cell adhesion mediated by the synthetic peptide was examined by cell attachment inhibition assay.Results MS showed the average molecular weight of the synthetic peptide was 2741.54 m/z, which was accorded with the theoretic molecular weight. HPLC showed the purity of the synthetic peptide was 99.2796%, which was pure enough for our experiments. RGD-containing peptides of GRGDSPC and (K)16GRGDSPC and BSA, (K)16 all promoted the attachments of BMSCs, especially (K)16GRGDSPC. Only the RGD-containing peptides of GRGDSPC and (K)16GRGDSPC could significantly inhibit the cell attachments to pretreated flusk by (K)16GRGDSPC. Non-RGD-containing peptide of GRGESPC displayed no effects and (K)16 and BSA showed not inhibitory effects but further promoting the attachments.Conclusion Our designed peptide (K)16GRGDSPC was easy to synthesize and could mediate BMSC cell adhesion specifically via RGD tripeptide motif.PartⅢGene delivery by the synthetic peptide and analysis of influence factorsobjective To evaluate the feasibilty and the specifility of gene delivery mediated by the synthetic peptide and to analyze the related influence factors on gene delivery. Methods The synthetic peptide was used as vector and two kinds of reporter genes of luciferase and EGFP were individually transfected into BMSCs. The gene expressions were observed by Liquid Scintillation Counting detector and fluorescent microscope. The transfection efficiency was compared with another commercial liposome Lipofectamine 2000. The targeted specifility of gene delivery by the peptide was examined by the gene delivery inhibition tests. On the conditions of keeping other factors constant, one factors was changed and its influences on the gene delivery was also evaluated and optimized.Results Both the two reporter genes of luciferase and EGFP were expressed in BMSCs. The transfection efficiency of synthetic peptide by FCM was 19.47%, which had no sigficant difference with that of Lipofectamine 2000(19.98%). RGD- containing peptides GRGDSPC and (K)16GRGDSPC decreased the transfection efficiency significantly, but non-RGD contaiing peptide GRGESPC failed. Chloroquine and PEI could facilitate the gene delivery by the synthetic peptide, especially Chloroquine. Endosomal buffer chloroquine was essential for the peptide-mediated gene delivery and optimal exposure time to chloroquine is more than 8 hours and its optimal final concentration was 100μM. When the concerntration of DNA is 2～4μg and the weight to weight ratio of vector to DNA was 3 to 1 the optimal transfection effects were received. The time exposure to vector/DNA complex shorter than 4 hours or longer than 24 hours was adverse to the targeted gene expression. Serum significantly inhibited the combination of vector/DNA complexes with cells. Fusion peptide based on hemagglutinin N terminum 20 residues and cationic liposome Lipofectamine 2000 facilitated the gene delievery, especially the fusion peptide had significant synergistic effects with the synthetic RGD-containing peptide.Transferrin increased TGFβ1 expression greatly as well and the optimal effects were obtained when 25μg/ml.Conclusion The synthetic peptide (K)16GRGDSPC not only mediated specific cell adhesion but also mediated gene delivery targetedly and specifically obtaining ideal transfection efficiency. That optimizing kinds of influence fators could provide tactics for improving peptide-mediated gene delivery efficiency and provide foundations for the wide application of peptide vector in gene delivery fields and next experiments on the gene modification of bone matrix materials.PartⅣFibrication of gene-loaded biomimetic matrices and gene delivery mediated by the novel gene activated matricesObjective To fibricate transforming growth factor beta 1 gene-loaded biomimetic matrices and evaluate whether the modified matrices could mediate gene delivery into BMSCs.Methods The synthetic peptide vector (K)16GRGDSPC was conjugated with PLGA-[ASP-PEG] matrices by cross linker Sulfo-LC-SPDP and examined by XPS. Then BMSCs obtained from rabbit were mixed cultured onto the scaffolds and observed by scanning electonic microscope. Cell adhension behaviors of the cells were analyzed by conventional precipitation method and micropipette aspiration technique. The feagures of cytoskeleton F-actin were observed under laser scanning confocal microscope. Then the modified mtrices were mixtured with transforming growth factor beta 1 gene and BMSCs were cultured on them. The expressions of transduced gene were examined by RT-PCR, western blot, ELISA and immunohistochemistry.Results XPS patterns showed that the synthetic peptide were successfully conjugated into PLGA-[ASP-PEG] mitrices and significantly improved their adhesion capabilities. RT-PCR, western blot, ELISA and immunohistochemistry all showed TGF-β1 gene expressed in BMSCs.Conclusion TGF-β1 gene-loaded matrices was in fact a gene-activated matrix and could mediate exogenous genes into BMSCs as a novel nonviral vector.PartⅤgene-loaded matrices regulated the osteoblastic differentiation of bone marrow stromal cellsObjective To evaluate the osteoblastic differentiation of bone marrow stromal cells regulated by TGF-β1 gene-loaded matrices.Methods TGF-β1 gene-loaded matrices were fabricated and BMSCs were mixed cultured onto them. The prolification behaviors were examined by MTT assay, Coomassie Brilliant Blue dyes and flow cytometry analysis. With osteoinductive medium culture, the osteoblast-associated markers ALP, OCN, OPN, CollagenⅠand Cbfa1 mRNAs and mineralization were assayed. The safety of the gene-loaded matrices were evaluated by DNA contents and cytoskeleton staining.Results The proliferation activities of BMSCs cultured on the gene-loaded matrices were significantly higher than the control groups. The osteoblastic phenotype expressions of ALP, OCN, OPN, CollagenⅠand Cbfa1 and minerization of experimental group were also significantly higher than the control groups.The cells mixed cultured with gene-loaded matrices were found no heteroploids and normal cytoskeletons.Conclusion TGF-β1 gene-loaded matrices were capable of regulating the osteoblastic differentiation of bone marrow stromal cells and TGF-β1 could be an ideal inducer for BMSCs.更多还原