细胞因子基因转染的骨骺干细胞复合明胶-硫酸软骨素-透明质酸钠支架修复大鼠骨骺生长板缺损的实验研究

A combination of epiphysis stem cell transfected with cytokine genes and bio-scaffold consists of gelatin,chondroitin sulfate and sodium hyaluronate for repairing epiphyseal growth plate defect in rats: an experimental study

目的:观察细胞因子基因转染的骨骺干细胞复合明胶-硫酸软骨素-透明质酸钠支架修复大鼠骨骺生长板缺损的效果。方法:取SD大鼠肋软骨透明带细胞,用Percoll密度梯度离心法获取骨骺干细胞。从低氧诱导因子(hypoxia-inducible factor,HIF)-1α、甲状旁腺激素相关肽(parathyroid hormone-related peptide,PTHr P)、重组人源核因子-κB(recombinant human nuclear factor-κB,rh NF-κB)、HIF-1α+PTHr P、rh NF-κB+PTHr P、HIF-1α+rh NF-κB、rh NF-κB+HIF-1α+PTHr P共7种细胞因子及细胞因子组合中筛选出对骨骺干细胞增殖作用最佳的细胞因子或细胞因子组合。利用筛选出的细胞因子或细胞因子组合,构建能稳定表达重组细胞因子的骨骺干细胞。取20只SD大鼠,制作右胫骨上端骨骺生长板缺损模型。造模后将大鼠随机分为2组,每组10只,分别采用从自体肋软骨透明带细胞获取的骨骺干细胞移植(自体移植组)和重组骨骺干细胞植入明胶-硫酸软骨素-透明质酸钠载体(组织工程组)两种方法修复骨骺生长板缺损。分别于修复术后4周、8周时,拍摄2组大鼠双下肢X线片,计算双侧胫骨长度差值和双侧胫骨角差值。X线检查结束后从每组随机选取5只大鼠,取肋软骨获取骨骺干细胞,以Western Blot法测定CollagenⅡ及CollagenⅩ蛋白水平;同时切取大鼠右胫骨上端,制作切片,HE染色后光镜下观察骨骺生长板损伤修复情况。结果:最终筛选出的细胞因子组合为HIF-1α+PTHr P,将其构建入p IRES2-EGFP真核表达载体,转染获取的骨骺干细胞,获得稳定表达重组细胞因子的骨骺干细胞。骨骺生长板缺损修复术后4周、8周时,组织工程组大鼠的双侧胫骨长度差值、双侧胫骨角差值均小于自体移植组[(0.07±0.01)cm,(0.35±0.05)cm,t=17.360,P=0.000;(0.83±0.45)cm,(1.51±0.13)cm,t=4.590,P=0.001;1.50°±0.69°,2.55°±0.40°,t=4.160,P=0.001;14.26°±1.87°,33.98°±2.17°,t=21.770,P=0.000],组织工程组大鼠的CollagenⅡ、CollagenⅩ蛋白水平均高于自体移植组[0.21±0.02,0.10±0.01,t=15.560,P=0.000;0.37±0.03,0.14±0.01,t=16.260,P=0.000;0.22±0.03,0.08±0.01,t=14.010,P=0.000;0.33±0.02,0.11±0.01,t=21.000,P=0.000]。HE染色结果显示,修复术后4周时,组织工程组胫骨上端骨骺生长板缺损由新生软骨部分填充,自体移植组尚未形成骨骺生长板组织结构;修复术后8周时,组织工程组胫骨上端骨骺生长板部分接近闭合,呈薄层柱状结构,自体移植组有新骨骺形成。结论:以骨骺干细胞作为种子细胞,以HIF-1α+PTHr P为细胞因子,以明胶-硫酸软骨素-透明质酸钠载体作为生物支架,能有效修复大鼠骨骺生长板缺损。

Objective:To observe the role of a combination of epiphysis stem cell transfected with cytokine genes and bio-scaffold consists of gelatin,chondroitin sulfate and sodium hyaluronate in repairing epiphyseal growth plate defect in rats.Methods:The zona-pellucida cells were fetched out from costicartilage of SD rats and the epiphysis stem cells were obtained by using Percoll density gradient centrifugation method.Cytokine or cytokine combinations which had the best promoting effect on proliferation of epiphysis stem cells were selected out of seven kinds of cytokines and cytokine combinations,including hypoxia-inducible factor(HIF)-1α,parathyroid hormonerelated peptide(PTHrP),recombinant human nuclear factor-κB(rhNF-κB),HIF-1α+PTHrP,rhNF-κB+PTHrP,HIF-1α+rhNF-κB and rhNF-κB+HIF-1α+PTHrP.Then the epiphysis stem cells which could steadily express recombinant cytokines were constructed by using selected cytokines or cytokine combinations.Twenty SD rats were selected to make models of defect of epiphyseal growth plate of superior extremity of right tibias.After modeling,the rats were randomly divided into 2 groups,10 cases in each group.The epiphyseal growth plate defects were repaired through transplantation of epiphysis stem cells which were obtained from zona-pellucida cells of autologous costicartilage(autotransplantation group)and using a combination of recombinant epiphyseal stem cells and bio-scaffold consists of gelatin,chondroitin sulfate and sodium hyaluronate(tissue engineering group)respectively.The X-ray films of double lower limbs were taken in rats of the 2 groups and the differences between bilateral tibias in length and angle were measured at 4 and 8 weeks after repairing surgery respectively.After the X-ray examination,five rats were randomly selected from each group and the epiphysis stem cells were obtained from their costicartilage,and the levels of CollagenⅡprotein and CollagenⅩprotein were measured by using Western Blot assays.The superior extremities of right tibias of rats were sectioned for HE staining and the repair of epiphyseal growth plate defect was observed under light microscope.Results:HIF-1α+PTHrP was selected out of the cytokine combination in the end and was imported into pIRES2-EGFP eukaryotic expression vector.The obtained epiphysis stem cells were transfected and then they could steadily express recombinant cytokines.The differences between bilateral tibias in length and angle were smaller in tissue engineering group compared to autotransplantation group at 4 and 8 weeks after epiphyseal growth plate defect repairing surgery(0.07+/-0.01 vs 0.35+/-0.05 cm,t=17.360,P=0.000;0.83+/-0.45 vs 1.51+/-0.13 cm,t=4.590,P=0.001;1.50+/-0.69 vs 2.55+/-0.40 degrees,t=4.160,P=0.001;14.26+/-1.87 vs 33.98+/-2.17 degrees,t=21.770,P=0.000),and the levels of CollagenⅡprotein and CollagenⅩprotein were higher in tissue engineering group compared to autotransplantation group(0.21+/-0.02 vs 0.10+/-0.01,t=15.560,P=0.000;0.37+/-0.03 vs 0.14+/-0.01,t=16.260,P=0.000;0.22+/-0.03 vs 0.08+/-0.01,t=14.010,P=0.000;0.33+/-0.02 vs 0.11+/-0.01,t=21.000,P=0.0000).The HE staining results showed that epiphyseal growth plate defect of tibial superior extremity was partially filled with newborn cartilage in tissue engineering group and epiphyseal growth plate structure was not found in autotransplantation group at 4 weeks after repairing surgery.Partial epiphyseal growth plate of tibial superior extremity was approximately closed and presented with lamellar columnar organization in tissue engineering group,and newborn epiphysis was found in autotransplantation group at 8 weeks after repairing surgery.Conclusion:Epiphyseal growth plate defect in rats can be effectively repaired by choosing epiphyseal stem cells as seed cells,HIF-1α+PTHrP as cytokine,and carrier consists of gelatin,chondroitin sulfate and sodium hyaluronate as bio-scaffold.