論文標題:脂質體介導TGF-β1基因轉染脂肪干細胞分化為軟骨細胞構建組織工程軟骨的實驗研究
Adipose Tissue Derived Stem Cells Transferred with TGF-β1 Gene, Differentiated into Chondrocytes, and Used to Constructed Tissue-engineering Cartilage
論文作者
論文導師 呂剛,論文學位 博士,論文專業 外科學
論文單位 中國醫科大學,點擊次數 28,論文頁數 105頁File Size7286K
2007-04-01論文網 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 關節軟骨缺損和退變是骨科常見病、多發病，創傷、骨軟骨炎、骨性關節炎、髕骨軟化等均可引起軟骨的病損。但關節軟骨的病變難以自身修復，目前治療除人工關節置換外尚無其他良策，人工關節置換的高昂費用和并發癥不容忽視。 組織工程方法修復關節軟骨缺損已取得初步成功，但移植后組織工程軟骨時間延長出現老化退變現象，從而制約了其進一步臨床應用，老化和退變的主要原因是軟骨細胞本身增殖、傳代能力差，軟骨細胞所具有特異性細胞外基質(extracellular matrix，ECM)合成減少，從而影響軟骨細胞的表型和活性，雖然有報道在細胞培養過程中應用藻酸鹽支架和添加細胞因子進行改善，但受到作用時間短、易于流失需反復添加、價格昂貴等因素制約。 轉基因技術(gene transfer)是指將克隆化的外源基因通過特定的手段導入真核細胞的方法，具有以下優點：①受體細胞可持續高效地表達目的基因，以調控其自身和其他效應細胞生長并獲得所需特定功能。②轉基因細胞合成分泌的內源性蛋白經過適當的翻譯后修飾過程，能夠更有效地同細胞表面受體結合，表達產物的生物活性更高。③價格遠低于純化的重組蛋白。因此，若結合應用轉基因技術，將能夠促進軟骨細胞ECM合成的目的基因導入其中，以此為種子細胞構建一種基因修飾的組織工程軟骨(gene modified ti ssue engineering cartilage，GMTEC)無疑非常具有意義。 本課題旨在進行脂肪干細胞分離、培養及富血小板血漿PRP誘導脂肪干細胞成骨作用進行鑒定；并利用轉基因技術，以TGF-β1目的基因修飾脂肪干細胞，使其向軟骨方向分化；將TGF-β1目的基因修飾脂肪干細胞在藻酸鹽載體中進行立體培養：進行TGF-β1目的基因修飾脂肪干細胞復合藻酸鹽在裸鼠體內異位軟骨生成的實驗，為基因修飾的組織工程軟骨的構建和其實際應用奠定實驗基礎。 材料與方法 一、實驗材料 成年雄性Wistar大鼠，SPF級裸鼠4只由中國醫科大學實驗動物部提供。 二、實驗方法 1、脂肪干細胞的分離、培養 取成年雄性Wistar大鼠腹股溝處脂肪組織，仔細清除包裹脂肪組織的假包膜以及深入脂肪組織的血管、結締組織，在超凈工作臺中，將組織剪碎，加入0.25％的Ⅰ型膠原酶，37℃、攪拌3h，加入兩倍體積的D-Hank’s，1200rpm，離心5mi．n，含15％FBS的高糖DMEM培養基稀釋，紗巾過濾，離心，將細胞收集到1-2瓶50ml培養瓶中培養。細胞長滿80％用0.125％/EDTA胰酶消化傳代培養。 2、CD44免疫細胞化學染色 取第2代脂肪干細胞爬片行CD44的免疫細胞化學染色，Ⅰ抗：兔抗CD44抗體(1：100)，FITC標記的Ⅱ抗(1；40)，熒光顯微鏡下觀察。對照組不加Ⅰ抗用PBS代替，其余步驟同實驗組。 3、PRP的制備 經同一大鼠腹主動脈抽取全血10ml(預先加入10％枸櫞酸鈉抗凝劑)，以1500r/min速度離心10min，吸取上層血漿及血小板，再以3000rpm速度離心10min，其沉淀即為PRP。對全血及PRP進行血小板計數，確保PRP中血小板的數量是全血的4倍以上。將1ml PRP加入98ml DMEM培養基中，同時加入1ml含5000 U牛凝血酶的100g/L CaCl_2溶液，即為含10ml/L PRP的DMEM條件培養基。 4、MTT檢測PRP對脂肪干細胞增殖的影響 5、堿性磷酸酶(ALP)組織化學染色 取10ml/L PRP的DMEM條件培養基培養14d的細胞爬片，10％中性福爾馬林固定10 min，堿性磷酸酶(ALP)改良Kaplow氏法染色． 6、ALP活性測定 將第2代脂肪干細胞以5×10~4/孔的密度接種96孔板中，含10ml/L PRP的DMEM條件培養基培養7，14，21，28d后，ALP檢測試劑盒檢測ALP活性。第2代脂肪干細胞培養7，14，21，28d分別測ALP活性作對照。 7、茜素紅染色鈣結節 取成骨誘導14d的細胞爬片，PBS漂洗，95％酒精固定5分鐘，2％茜素紅染液染色5分鐘，PBS沖洗兩遍。 8、TGF-β1基因轉染脂肪干細胞及陽性細胞克隆株的篩選 選取第2代脂肪干細胞，5×10~5細胞傳至35mm單皿中，37℃，5％CO_2培養至細胞密度為90％時進行轉染，按照Lipofectamine2000使用說明進行轉染，6h后更換完全生長培養液。轉染一天后，將細胞按1：10的稀釋比例傳代至新鮮生長培養液中，第二天加入G418 400μg/ml進行篩選。篩選21d后克隆形成，將其挑至24孔板，生長至融合轉移至6孔板，最后轉至50ml培養瓶中。 9、RT-PCR檢測TGF-β1、FN、ColⅡ、Aggrecan、MMP-1、MMP-2、MMP-3 mRNA TGF-β1穩定轉染的細胞棄培養液，每50ml培養瓶中加1 ml Trizol，按Trizol試劑盒用一步法提取總RNA，用DNA/RNA測定儀測定RNA濃度和純度。逆轉錄反應體系中加入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、樣品RNA 1μl。反應條件：30℃10min，42℃30min，99℃5min，5℃5min。 PCR反應條件：94℃2min 94℃30sec 50-65℃30sec 72℃1min 32個循環72℃延伸7min。用內參照β-肌動蛋白(β-actin)檢測逆轉錄效率。PCR產物用2％瓊脂糖凝膠電泳，溴化乙啶(EB)顯色。用GDS8000凝膠自動成像儀進行攝像分析。用DL2000 Marker為分子大小對照確定陽性電泳條帶。 10、Western blot檢測TGF-β1 RIPA buffer 200μl裂解標本；采用Folin-酚試劑法進行蛋白定量；聚丙烯酰胺凝膠電泳；硝酸纖維素膜(Millipore，USA)轉膜2h，加入TGF-β1兔多克隆抗體(1：400，Santa Cruz)和β-actin兔多克隆抗體(1：400，Santa Cruz)，4℃過夜。加入堿性磷酸酶標記的羊抗兔單克隆二抗，室溫孵育2h，堿性磷酸酶顯色15 min，于自動電泳凝膠成像分析系統下成像。 11、掃描電鏡觀察TGF-β1穩定轉染的細胞在藻酸鹽凝膠中的生長情況 將TGF-β1基因轉染后的脂肪干細胞細胞懸液以1×10~6個/ml的細胞濃度與藻酸鈉混勻后滴入Cacl_2溶液，邊滴入邊攪拌直到形成均勻的凝膠，將凝膠在15％FBS高糖-DMEM培養一周。無細胞的藻酸鹽凝膠做為對照。S-450日立掃描電鏡觀察。 12、細胞藻酸鈉復合體甲苯胺蘭染色觀察 TGF-β1基因轉染后的脂肪干細胞復合藻酸鈉培養一周后，進行壓片，甲苯胺蘭染色觀察 13、構建組織工程化軟骨 將準備好TGF-β1基因轉染后的脂肪干細胞懸液以5×10~6個/ml的細胞濃度與藻酸鈉混勻后滴入Cacl_2溶液，邊滴入邊攪拌直到形成均勻的凝膠，之后將凝膠注入裸鼠(4只裸鼠由中國醫科大學動物部提供)背部皮下一側，另一側注入無細胞的藻酸鹽凝膠作為對照。分別于4周、8周取材進行HE、Masson染色、Ⅱ型膠原免疫組化檢測 14、統計學方法 實驗結果以(?)±s表示，應用SPSS醫用軟件統計軟件包進行統計。 結果 原代培養細胞第二天貼壁，7-10d長滿，倒置顯微鏡下見細胞呈纖維樣，各代細胞形態無明顯變化，傳30代后細胞形態及生長速度仍無明顯改變。脂肪干細胞的CD44免疫熒光染色呈陽性。MTT檢測結果：相對于對照組，實驗組有顯著的統計學意義(P<0.01)。PRP誘導14天，ALP染色明顯陽性。ALP活性檢測顯示誘導組與對照組有顯著性差異(P<0.01)。茜素紅染色顯示誘導14天可見鈣結節形成。 RT-PCR結果顯示：實驗組(TGFβ1穩定轉染組)的TGFβ1、FN、ColⅡ、Aggrecan、MMP-2mRNA的表達較空染組和正常對照組均明顯增多，而MMP-1mRNA的表達較空染組和正常對照組均明顯減少(P<0.01)，MMP-3mRNA的表達較空染組和正常對照組亦明顯減少(P<0.05) Western blot檢測實驗組(TGFβ1穩定轉染組)TGFβ1蛋白的表達較空染組和正常對照組均明顯增多(P<0.01)。 掃描電鏡觀察可見藻酸鹽凝膠呈網格狀，由許多孔隙，TGF-β1基因修飾的ATSCs在凝膠的孔隙中生長良好，并有基質分泌。細胞藻酸鈉復合體甲苯胺蘭染色觀察：TGF-β1基因修飾的ATSCs甲苯胺蘭染色陽性，可見細胞分裂相。 組織學觀察：實驗組4周后切片觀察，植入物內含有大量的萎縮成團的細胞，細胞形態并不完全一致。細胞間基質較多且松散，有部分凝膠未吸收。Masson染色顯示較大區域藍色的軟骨基質，包裹軟骨樣細胞。術后8周，實驗組在植入區內新生軟骨組織逐漸融合成片，支架基本完全吸收，Masson染色發現位于陷窩內的軟骨樣細胞，可見細胞分裂相，以及細胞周圍大片藍色基質，為典型的軟骨樣結構。術后4周和8周，組織Ⅱ型膠原免疫組化呈陽性表達。 結論 1、成功地分離大鼠的脂肪干細胞(ATSCs)，細胞表面標志CD44呈現陽性。 2、富血小板血漿(PRP)能夠促使ATSCs向成骨細胞分化。 3、以脂質體介導法將TGF-β1目的基因成功轉染ATSCs，轉染后成軟骨分化的特異性細胞外基質增多，表明TGF-β1可以促使ATSCs向軟骨方向分化。 4、TGF-β1目的基因上調MMP-2，下調MMP-1，3。MMP-1，3分泌量減少是導致其成軟骨分化特異性細胞外基質增多的原因之一。 5、TGF-β1基因修飾的ATSCs復合藻酸鹽凝膠在裸鼠體內成功地構建組織工程軟骨。
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個/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個/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。
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