脂质体介导TGF-β1基因转染脂肪干细胞分化为软骨细胞构建组织工程软骨的实验研究

论文标题:脂质体介导TGF-β1基因转染脂肪干细胞分化为软骨细胞构建组织工程软骨的实验研究
Adipose Tissue Derived Stem Cells Transferred with TGF-β1 Gene, Differentiated into Chondrocytes, and Used to Constructed Tissue-engineering Cartilage
论文作者
论文导师吕刚,论文学位博士,论文专业外科学
论文单位中国医科大学,点击次数 28,论文页数 105页File Size7286K
2007-04-01论文网 http://www.lw23.com/lunwen_673071852/
transforming growth factor-β1 (TGF-β1) ; platelet-rich plasma (PRP) ; adipose tissue derived stem cells (ATSCs); fetal bovine serum (FBS) ; Cell immunofluorescence; immunohistochemistry; gene transfection; reverse transcription polymerase chain reaction (RT-PCR); Western-blot
目的关节软骨缺损和退变是骨科常见病、多发病，创伤、骨软骨炎、骨性关节炎、髌骨软化等均可引起软骨的病损。但关节软骨的病变难以自身修复，目前治疗除人工关节置换外尚无其他良策，人工关节置换的高昂费用和并发症不容忽视。组织工程方法修复关节软骨缺损已取得初步成功，但移植后组织工程软骨时间延长出现老化退变现象，从而制约了其进一步临床应用，老化和退变的主要原因是软骨细胞本身增殖、传代能力差，软骨细胞所具有特异性细胞外基质(extracellular matrix，ECM)合成减少，从而影响软骨细胞的表型和活性，虽然有报道在细胞培养过程中应用藻酸盐支架和添加细胞因子进行改善，但受到作用时间短、易于流失需反复添加、价格昂贵等因素制约。转基因技术(gene transfer)是指将克隆化的外源基因通过特定的手段导入真核细胞的方法，具有以下优点：①受体细胞可持续高效地表达目的基因，以调控其自身和其他效应细胞生长并获得所需特定功能。②转基因细胞合成分泌的内源性蛋白经过适当的翻译后修饰过程，能够更有效地同细胞表面受体结合，表达产物的生物活性更高。③价格远低于纯化的重组蛋白。因此，若结合应用转基因技术，将能够促进软骨细胞ECM合成的目的基因导入其中，以此为种子细胞构建一种基因修饰的组织工程软骨(gene modified ti ssue engineering cartilage，GMTEC)无疑非常具有意义。本课题旨在进行脂肪干细胞分离、培养及富血小板血浆PRP诱导脂肪干细胞成骨作用进行鉴定；并利用转基因技术，以TGF-β1目的基因修饰脂肪干细胞，使其向软骨方向分化；将TGF-β1目的基因修饰脂肪干细胞在藻酸盐载体中进行立体培养：进行TGF-β1目的基因修饰脂肪干细胞复合藻酸盐在裸鼠体内异位软骨生成的实验，为基因修饰的组织工程软骨的构建和其实际应用奠定实验基础。材料与方法一、实验材料成年雄性Wistar大鼠，SPF级裸鼠4只由中国医科大学实验动物部提供。二、实验方法 1、脂肪干细胞的分离、培养取成年雄性Wistar大鼠腹股沟处脂肪组织，仔细清除包裹脂肪组织的假包膜以及深入脂肪组织的血管、结缔组织，在超净工作台中，将组织剪碎，加入0.25％的Ⅰ型胶原酶，37℃、搅拌3h，加入两倍体积的D-Hank’s，1200rpm，离心5mi．n，含15％FBS的高糖DMEM培养基稀释，纱巾过滤，离心，将细胞收集到1-2瓶50ml培养瓶中培养。细胞长满80％用0.125％/EDTA胰酶消化传代培养。 2、CD44免疫细胞化学染色取第2代脂肪干细胞爬片行CD44的免疫细胞化学染色，Ⅰ抗：兔抗CD44抗体(1：100)，FITC标记的Ⅱ抗(1；40)，荧光显微镜下观察。对照组不加Ⅰ抗用PBS代替，其余步骤同实验组。 3、PRP的制备经同一大鼠腹主动脉抽取全血10ml(预先加入10％枸橼酸钠抗凝剂)，以1500r/min速度离心10min，吸取上层血浆及血小板，再以3000rpm速度离心10min，其沉淀即为PRP。对全血及PRP进行血小板计数，确保PRP中血小板的数量是全血的4倍以上。将1ml PRP加入98ml DMEM培养基中，同时加入1ml含5000 U牛凝血酶的100g/L CaCl_2溶液，即为含10ml/L PRP的DMEM条件培养基。 4、MTT检测PRP对脂肪干细胞增殖的影响 5、碱性磷酸酶(ALP)组织化学染色取10ml/L PRP的DMEM条件培养基培养14d的细胞爬片，10％中性福尔马林固定10 min，碱性磷酸酶(ALP)改良Kaplow氏法染色． 6、ALP活性测定将第2代脂肪干细胞以5×10~4/孔的密度接种96孔板中，含10ml/L PRP的DMEM条件培养基培养7，14，21，28d后，ALP检测试剂盒检测ALP活性。第2代脂肪干细胞培养7，14，21，28d分别测ALP活性作对照。 7、茜素红染色钙结节取成骨诱导14d的细胞爬片，PBS漂洗，95％酒精固定5分钟，2％茜素红染液染色5分钟，PBS冲洗两遍。 8、TGF-β1基因转染脂肪干细胞及阳性细胞克隆株的筛选选取第2代脂肪干细胞，5×10~5细胞传至35mm单皿中，37℃，5％CO_2培养至细胞密度为90％时进行转染，按照Lipofectamine2000使用说明进行转染，6h后更换完全生长培养液。转染一天后，将细胞按1：10的稀释比例传代至新鲜生长培养液中，第二天加入G418 400μg/ml进行筛选。筛选21d后克隆形成，将其挑至24孔板，生长至融合转移至6孔板，最后转至50ml培养瓶中。 9、RT-PCR检测TGF-β1、FN、ColⅡ、Aggrecan、MMP-1、MMP-2、MMP-3 mRNA TGF-β1稳定转染的细胞弃培养液，每50ml培养瓶中加1 ml Trizol，按Trizol试剂盒用一步法提取总RNA，用DNA/RNA测定仪测定RNA浓度和纯度。逆转录反应体系中加入Mgcl_2 2μl、10×RT Buffer 1μl、RNase Free dH_2O 3.75μl、dNTPMixture(各10mM)1μl、RNase inhibitor 0.25μl、Reverse Transcriptase 0.5μl、Random 9 mers 0.5μl、样品RNA 1μl。反应条件：30℃10min，42℃30min，99℃5min，5℃5min。 PCR反应条件：94℃2min 94℃30sec 50-65℃30sec 72℃1min 32个循环72℃延伸7min。用内参照β-肌动蛋白(β-actin)检测逆转录效率。PCR产物用2％琼脂糖凝胶电泳，溴化乙啶(EB)显色。用GDS8000凝胶自动成像仪进行摄像分析。用DL2000 Marker为分子大小对照确定阳性电泳条带。 10、Western blot检测TGF-β1 RIPA buffer 200μl裂解标本；采用Folin-酚试剂法进行蛋白定量；聚丙烯酰胺凝胶电泳；硝酸纤维素膜(Millipore，USA)转膜2h，加入TGF-β1兔多克隆抗体(1：400，Santa Cruz)和β-actin兔多克隆抗体(1：400，Santa Cruz)，4℃过夜。加入碱性磷酸酶标记的羊抗兔单克隆二抗，室温孵育2h，碱性磷酸酶显色15 min，于自动电泳凝胶成像分析系统下成像。 11、扫描电镜观察TGF-β1稳定转染的细胞在藻酸盐凝胶中的生长情况将TGF-β1基因转染后的脂肪干细胞细胞悬液以1×10~6个/ml的细胞浓度与藻酸钠混匀后滴入Cacl_2溶液，边滴入边搅拌直到形成均匀的凝胶，将凝胶在15％FBS高糖-DMEM培养一周。无细胞的藻酸盐凝胶做为对照。S-450日立扫描电镜观察。 12、细胞藻酸钠复合体甲苯胺兰染色观察 TGF-β1基因转染后的脂肪干细胞复合藻酸钠培养一周后，进行压片，甲苯胺兰染色观察 13、构建组织工程化软骨将准备好TGF-β1基因转染后的脂肪干细胞悬液以5×10~6个/ml的细胞浓度与藻酸钠混匀后滴入Cacl_2溶液，边滴入边搅拌直到形成均匀的凝胶，之后将凝胶注入裸鼠(4只裸鼠由中国医科大学动物部提供)背部皮下一侧，另一侧注入无细胞的藻酸盐凝胶作为对照。分别于4周、8周取材进行HE、Masson染色、Ⅱ型胶原免疫组化检测 14、统计学方法实验结果以(?)±s表示，应用SPSS医用软件统计软件包进行统计。结果原代培养细胞第二天贴壁，7-10d长满，倒置显微镜下见细胞呈纤维样，各代细胞形态无明显变化，传30代后细胞形态及生长速度仍无明显改变。脂肪干细胞的CD44免疫荧光染色呈阳性。MTT检测结果：相对于对照组，实验组有显著的统计学意义(P<0.01)。PRP诱导14天，ALP染色明显阳性。ALP活性检测显示诱导组与对照组有显著性差异(P<0.01)。茜素红染色显示诱导14天可见钙结节形成。 RT-PCR结果显示：实验组(TGFβ1稳定转染组)的TGFβ1、FN、ColⅡ、Aggrecan、MMP-2mRNA的表达较空染组和正常对照组均明显增多，而MMP-1mRNA的表达较空染组和正常对照组均明显减少(P<0.01)，MMP-3mRNA的表达较空染组和正常对照组亦明显减少(P<0.05) Western blot检测实验组(TGFβ1稳定转染组)TGFβ1蛋白的表达较空染组和正常对照组均明显增多(P<0.01)。扫描电镜观察可见藻酸盐凝胶呈网格状，由许多孔隙，TGF-β1基因修饰的ATSCs在凝胶的孔隙中生长良好，并有基质分泌。细胞藻酸钠复合体甲苯胺兰染色观察：TGF-β1基因修饰的ATSCs甲苯胺兰染色阳性，可见细胞分裂相。组织学观察：实验组4周后切片观察，植入物内含有大量的萎缩成团的细胞，细胞形态并不完全一致。细胞间基质较多且松散，有部分凝胶未吸收。Masson染色显示较大区域蓝色的软骨基质，包裹软骨样细胞。术后8周，实验组在植入区内新生软骨组织逐渐融合成片，支架基本完全吸收，Masson染色发现位于陷窝内的软骨样细胞，可见细胞分裂相，以及细胞周围大片蓝色基质，为典型的软骨样结构。术后4周和8周，组织Ⅱ型胶原免疫组化呈阳性表达。结论 1、成功地分离大鼠的脂肪干细胞(ATSCs)，细胞表面标志CD44呈现阳性。 2、富血小板血浆(PRP)能够促使ATSCs向成骨细胞分化。 3、以脂质体介导法将TGF-β1目的基因成功转染ATSCs，转染后成软骨分化的特异性细胞外基质增多，表明TGF-β1可以促使ATSCs向软骨方向分化。 4、TGF-β1目的基因上调MMP-2，下调MMP-1，3。MMP-1，3分泌量减少是导致其成软骨分化特异性细胞外基质增多的原因之一。 5、TGF-β1基因修饰的ATSCs复合藻酸盐凝胶在裸鼠体内成功地构建组织工程软骨。
Introduction The defect and degeneration of articular cartilage are common in orthopedic field. Although artificial prothesis is the main and effective choice, high cost and complication could not be neglected. Repairing the defect of articular cartilage with tissue engineering cartilage succeeded preliminarily, but clinical application was suspended because the transplanted tissue engineering cartilage degenerated as time going. The main reason of the degeneration is that the proliferation and passage ability of chondrocyte is limited. The phenotype and activity of chondrocyte are affected, as the specific extracellular matrix (ECM) components are synthesized decreased during passage. There are some reports about application of alginate and growth factors to improve the synthesis of ECM, but there are some shortages, such as short biological half-lives, high price and repeatedly addition. Gene transfer, which means to induce aim gene into eukaryotic cells with special method, has following advantages: 1) the transferred cells are able to express aim gene continuously and effectively that can modulate the growth of themselves and other effective cells and to obtain specific functions. 2) The endogenous aim proteins produced by transferred cells have undergone authentic post-translational modification and therefore have improved potencies to combine with receptors on cells" surface as well as more biological activities. 3) The price of gene transfer is lower than that of purified recombinant proteins. Therefore, it is of great value to induce aim gene which could increase ECM synthesis into cells by employing gene transfer techniques. With these gene modified seed cells, the new gene modified tissue engineering cartilage could be constructed in order to repair the defect and degeneration of articular cartilage. The purposes of present study were to isolate and culture adipose tissue derived stem cells of rat"s inguinal region, and to investigate the feasibility of oriented differentiation into osteoblast by PRP.The adipose tissue derived stem cells are transferred with TGF-β1 gene and differentiated into chondrocytes. The gene modified stem cells from adipose tissue are cultured in the three-dimensioned system of alginate gel and observed through transmission electron microscope(TEM).Heterotopic chondrogenesis of TGF-β1 gene modified stem cells loading alginate gel was observed in athymic mice. Materials and methods Materials Ripe male Wistar rats and 4 athymic mice (SPF) were supplied by laboratory animal department of china medical university. Methods 1. ATSCs were isolated and cultured The inguinal adipose tissue of the rats was dissected under sterile conditions, and digested by 0.25 % typeⅠcollagenase at 37℃for 3h.The resulting cell suspension was diluted with D-Hank"s solution and centrifuged at 1200rpm for 5 minutes. The pellet was resuspended with HG-DMEM including 15% fetal bovine serum and cultured at 37℃in a humidified atmosphere and 5% CO_2. 2. CD44 immunofluorescence staining The second passage ATSCs grown in monolayer were fixed in acetone before histologic examination. Immunofluorescence staining procedures were performed using standard protocols. 3. PRP DMEM preparation The rat"s blood was taken suction from its abdominal aorta and centrifuged at 1500 rpm for 10min.The upper hematoplasma and platelet were taken suction,then were centrifuged at 3000 rpm for 10min. The pellet was PRP.1ml PRP was added into 98ml DMEM and the same time 1ml 100g/L CaCl_2 solution including 5000 U bovine thrombin was added. 4. The effect of PRP on ATSCs proliferation was detected through MTT 5. Histochemical staining of ALP The twice-passaged ATSCs were cultured with PRP DMEM for 14 days. The cells were fixed with 4% paraformaldehyde for 10 min and stained according to Kaplow"s method 6. Detection of ALP activity The twice-passaged ATSCs were seeded in 96-well plates at 5×10~4/well and cultured in PRP DMEM for 7, 14, 21, 28 days respectively. After the respective cultivation, ALP activity was detected with ALP detective reageant.Simultaneously the twice-passaged ATSCs were seeded in 96-well plates at 5×10~4/well and cultured in DMEM as controls 7. Calcium nodule staining with alizarin red The cells cultured in PRP DMEM for 14 days were washed with PBS, fixed with 95% alcohol for 5 min, stained with 2% alizarin red staining solution and washed twice with PBS. 8. TGF-β1 gene transferring ATSCs for stable cells The twice-passaged ATSCs were plated at 5×10~5 cells in the 35mm plate so that they will be 90% confluent at the time of transfection. ATSCs were transferred according to Lipofectamine2000 direction for use. After 6h, growth medium was replaced. Passage the cells at a 1:10 dilution into fresh growth medium after 24h.Add selective medium the following day. The cell clones formed and was plated into 24-well plate after 21 days. When the cells were confluent they were seeded into 6-well plate. Finally they were seeded in culture flask. 9.RT-PCR for TGF-β1、FN、ColⅡ、Aggrecan、MMP-1、MMP-2、MMP-3 mRNA Total RNA of each sample were isolated by using Trizol Solution, then the concentration and purity of RNA were detected through DNA/RNA detective machine. Reverse transcription reaction tube included 10×PCR Buffer 1μl, dNTP 1μl、MgCl_2 2μl、Random 9mers 0.5μl、RNAlμl、RNase inhibitor 0.25μl、Reverse Transcriptase 0.5μl、RNase Free dH_2O 3.75μl。Followed by the conditions: 10min at 30℃, 30min at 42℃,5min at 99℃and 5min at 5℃. PCR amplification was performed on a PCR thermal cycler. PCR reaction conditions included initial denaturation for 2min at 95℃, followed by 30 cycles of denaturation of 95℃for 1min and annealing at 72℃for 1min, then extension at 72℃for 7min. The efficiency were detected byβ-actin. PCR products were loaded onto a 8% agarose gels electrophoresis containing ethidium bromide and the bands were visualized under ultraviolet light, then ascertained with the comparison to Makers (TaKaRa Co). 10. Western-blot analysis The samples were lysed in 200μ1 RIPA buffer. The protein concentration was measured according to the Lowry method;The protein was loaded onto polyacrylamide gel eletrophoresis;After 2h the product was transferred to PVDF membrane,the blot was probed with rabbit multiclonal anti-TGF-β1 antibody (1: 400, Santa Cruz,USA) and rabbit multiclonal anti-β-actin antibody (1:400, Santa Cruz,USA),incubated at 4℃for 12h,incubated with the secondary goat anti-rabbit monoclonal antibody conjugated with alkaline phosphatase for 2h at room temperature, stained with alkaline phosphatase coloration solution for 15min.The positive blot were scanned and then measured for intensity by using the UPV gel imaging-analyzing system (Chemi Imager 5500, USA). 11. Observe the stable cells growing in alginate gel through SEM The stable cells at 1×10~6个/ml was blended with 1.25 % algin solution, dropped into 102M Cacl_2 solution and stirred so that alginate gel formed. The complex was cultured in 15% FBS DMEM for a week. The alginate gel without stable cells was made control. Observe the stable cells growing in alginate gel through SEM 12.Observe complex of the stable cells and alginate gel through toluidine blue staining The complex was cultured in 15% FBS DMEM for a week, made stressed plate and stained with blue toluidine solution. 13. Construction of tissue engineering cartilage The cells of TGF-β1 gene stable transfection at 5×10~6个/ml was blended with 1.25 % algin solution, dropped into 102M Cacl_2 solution and stirred at the same time so that alginate gel formed. Then the complex was injected into athymic mice subcutaneouly. The complexes were dissected from the athymic mice respectively after 4, 8 weeks, made paraffin sections and stained with respect to HE,Masson. and immunohistochemistry. 14. The data were analyzed by the SPSS12.0 software. Results The ATSCs of primary culture adhered to the plate the second day and were confluent between 7 to 10 days. They exhibited fibroblast-like phenotype, without obvious variation among passages. To confirm ATSCs, expression of CD44 was determined by immunofluorescence. MTT demonstrated experimental groups were different from control groups significantly (P＜0.01).The PRP inducing ATSCs expressed positive after 14 days by ALP staining. Detection of ALP activity demonstrated experimental groups differentiated with control groups significantly (P＜0.01). Alizarin red staining expressed that the PRP inducing ATSCs formed calcium nodules after 14 days. RT-PCR demonstrated that TGFβ1、FN、ColⅡ、Aggrecan、MMP-2 mRNA of experimental groups increased more evidently than control groups (P＜0.01). MMP-1mRNA of experimental groups decreased more evidently than control groups (P＜0.01), at the same time MMP-3 mRNA of experimental groups also declined more evidently than control groups(P＜0. 05). Western blot demonstrated that TGFβ1 protein of experimental groups increase more evidently than control groups significantly (P＜0.01) Alginate gel was observed high porosity, and that TGFβ1 modified ATSCs grew up well and secrete extracellular matrix by scanning electronic microscope. TGF-β1 modified ATSCs cultured in alginate gel expressed positive toluidine blue staining and splitting phase of the nucleus was observed. Histologic findings demonstrated that the constructs included many cartilage-like cells and extracellular matrix, were similar to cartilage tissue. Many cells located in lacunes. Extracellular matrix was loose and minority of alginate gel was not absorbed at 4 weeks after injection. Extracellular matrix demonstrated blue and enclosed cartilage-like cells by Masson staining. At 8 weeks after injection, alginate gel was completely absorbed, simultaneously cartilage-like cells located in lacunes and extracellular matrix was stained blue by Masson staining. The conducts at 4 and 8 weeks after injection expressed collagenⅡthrough immunohistochemistry. Conclusions 1. ATSCs of Wistar rats could be separated and cultured successfully and their expression of CD44 was positive 2. Platelet-rich plasma (PRP) induced ATSCs to differentiate into osteoblast 3. TGF-β1 gene was transferred into ATSCs at the first time in our country. The specific extracellular matrix of chondrocyte differentiation increased evidently. The results demonstrated TGF-β1 gene could induce ATSCs into chondrocyte. 4. TGF-β1 gene promoted MMP-2 to increase and promoted MMP-1,3 to decrease. The decrease of MMP-1,3 was one of reasons that the specific extracellular matrix of chondrocyte differentiation increased. 5. TGF-β1 gene modified ATSCs-alginate gel complex successfully constructed tissue-engineering cartilage。