hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究
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摘要
目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法,并比较修饰结果。方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞,通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞,倒置荧光显微镜检测荧光强度并计算转染效率,qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达,免疫组化检测细胞中Ⅱ型胶原的变化。结果成功分离、培养出骨髓和人脐血间充质干细胞,两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组h BMP-2质粒成功转染骨髓和人脐血间充质干细胞,转染骨髓间充质干细胞效率[(18.44±5.94)%]低于人脐血间充质干细胞[(27.74±7.59)%],且差异有统计学意义(t=3.027,P<0.05)。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。结论 hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞,且能促进其向软骨细胞方向分化。
Objective To study the modification of bone marrow and human umbilical cord blood mesenchymal stem cells with hBMP-2 gene and compare the modification results. Methods Bone marrow and human umbilical cord blood mesenchymal stem cells were separated and cultured by density gradient centrifugation and adherent culture. The recombinant hBMP-2 plasmid was transfected into bone marrow and human umbilical cord blood mesenchymal stem cells by high-performance non-liposome reagent Xtreme GENE. The fluorescence intensity was measured by inverted fluorescence microscopy and the transfection efficiency was calculated. The hBMP was detected by qPCR. The expression of-2 and chondronectin in two kinds of cells was detected by immunohistochemistry. Results Bone marrow and human umbilical cord blood mesenchymal stem cells were successfully isolated and cultured. The two cells had different growth characteristics. The recombinant hBMP-2 plasmid mediated by high-performance non-liposome reagent was successfully transfected into bone marrow and human umbilical cord blood mesenchymal stem cells. The efficiency of transfection of bone marrow mesenchymal stem cells(18.44±5.94)% was lower than that of human umbilical cord blood mesenchymal stem cells(27.74±7.59)%, and the difference was statistically significant(t=3.027, P<0.05). The expression of hBMP-2, cartilage connectin and collagen type II were detected in both transfected cells. Conclusion hBMP-2 gene can effectively modify bone marrow and human umbilical cord blood mesenchymal stem cells and promote chondrocyte differentiation.
Objective To study the modification of bone marrow and human umbilical cord blood mesenchymal stem cells with hBMP-2 gene and compare the modification results. Methods Bone marrow and human umbilical cord blood mesenchymal stem cells were separated and cultured by density gradient centrifugation and adherent culture. The recombinant hBMP-2 plasmid was transfected into bone marrow and human umbilical cord blood mesenchymal stem cells by high-performance non-liposome reagent Xtreme GENE. The fluorescence intensity was measured by inverted fluorescence microscopy and the transfection efficiency was calculated. The hBMP was detected by qPCR. The expression of-2 and chondronectin in two kinds of cells was detected by immunohistochemistry. Results Bone marrow and human umbilical cord blood mesenchymal stem cells were successfully isolated and cultured. The two cells had different growth characteristics. The recombinant hBMP-2 plasmid mediated by high-performance non-liposome reagent was successfully transfected into bone marrow and human umbilical cord blood mesenchymal stem cells. The efficiency of transfection of bone marrow mesenchymal stem cells(18.44±5.94)% was lower than that of human umbilical cord blood mesenchymal stem cells(27.74±7.59)%, and the difference was statistically significant(t=3.027, P<0.05). The expression of hBMP-2, cartilage connectin and collagen type II were detected in both transfected cells. Conclusion hBMP-2 gene can effectively modify bone marrow and human umbilical cord blood mesenchymal stem cells and promote chondrocyte differentiation.
引文
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16李兴福,段莉,梁宇杰,等.直接共培养诱导人脐带间充质干细胞向软骨细胞的分化[J].中国组织工程研究,2017,21(1):18-24.
17张军,徐辉,温绣蔺,等.Sox9基因转染的人脐带血干细胞复合骨基质明胶/生物蛋白胶构建组织工程软骨的实验研究[J].生物医学工程与临床,2018(1):13-19.
18刘英,王姣杰,张玉红,等.脐血干细胞移植治疗膝盖软骨损伤一例疗效观察[J].中华临床医师杂志:电子版,2011,5(15):4586-4587.
19徐建民,王兆朋,王恒孝.骨形态发生蛋白-2在兔骨折愈合过程中的作用[J].中国矫形外科杂志,2016,24(10):920-925.
20 Zhou J,He T,Bi Y,et al.Effect of BMP-2 and-3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertebral disc cells in vitro[J].Nan Fang Yi Ke Da Xue Xue Bao,2013,33(7):977-982.
21 Kwon SH,Lee TJ,Park J,et al.Modulation of BMP-2-Induced chondrogenic versus osteogenic differentiation of human mesenchymal stem cells by Cell-Specific extracellular matrices[J].Tissue Eng Part A,2013,19(1/2):49-58.
22 Qian XD,Zhang C,Chen GJ,et al.Effects of BMP-2 and FGF2 on the osteogenesis of bone Marrow-Derived mesenchymal stem cells in Hindlimb-Unloaded rats[J].Cell Biochem Biophys,2014,70(2):1127-1136.
23沈鹏飞,王斌,谢子康,等.软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响[J].中国生物工程杂志,2016,36(10):1-7.
24李小飞,张涛,程庆书,等.重组人骨形态发生蛋白诱导气管软骨再生的剂量效应[J].中华实验外科杂志,2004,21(3):96-97.
25张泉,邓飞龙.骨形态发生蛋白复合胶原释放系统的研究进展[J].国际口腔医学杂志,2012,39(2):248-251.
26殷力,娄超举,韩奇财,等.骨形成蛋白-2转染兔骨髓基质细胞修复软骨缺损的研究[J].中华实验外科杂志,2012,29(3):515-517.
27秦国斌,王秀利,肖鹏,等.骨形态发生蛋白-2对兔骨髓基质细胞向软骨细胞分化基因表达的影响[J].中华实验外科杂志,2011,28(8):1370-1372.
28尹承慧,邱俊钦,曾昭勋,等.腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞[J].中国组织工程研究,2013,17(14):2488-2494.
2张海宁,冷萍,王英振,等.自体骨软骨柱及骨膜骨柱混合嵌入修复大面积骨软骨复合缺损[J].中华创伤杂志,2009,25(6):543-544.
3 Yamagata K,Nakayamada S,Tanaka Y.Use of mesenchymal stem cells seeded on the scaffold in articular cartilage repair[J].Inflamm Regen,2018,12(38):4.
4 Redler LH,Caldwell JM,Schulz BM,et al.Management of articular cartilage defects of the knee[J].Phys Sportsmed,2012,40(1):20-35.
5 Koc ON,Lazarus HM.Mesenchymal stem cells:heading into the clinic[J].Bone Marrow Transplant,2001,27(3):235-239.
6周广东,王晓云,苗春雷,等.骨髓基质细胞修复猪膝关节非负重区软骨与骨复合缺损的实验研究[J].中华医学杂志,2004,84(11):925-931.
7周广东,王晓云,刘德莉,等.GFP基因转染对骨髓基质细胞体内向软骨细胞分化的示踪作用[J].细胞与分子免疫学杂志,2004,20(1):27-30.
8 Kim HS,Shin TH,Lee BC,et al.Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2[J].Gastroenterology,2013,145(6):1392-1403.
9 Erices A,Conget P,Mingudl JJ,et al.Meaenchymaal progenitor cells in human umbilical cord blood[J].Br J Heamatol,2000,109(1):235-242.
10 Cardenas C,Kwon JY,Maeng YS.Human cord Blood-Derived CD133+/C-Kit(+)/Lin(-)cells have bipotential ability to differentiate into mesenchymal stem cells and outgrowth endothelial cells[J].Stem Cells Int,2016:7162160.
11 Heo JS,Choi Y,Kim HS,et al.Comparison of molecular profiles of human mesenchymal stem cells derived from bone marrow,umbilical cord blood,placenta and adipose tissue[J].Int J Mol Med,2016,37(1):115-125.
12 Gomez-Leduc T,Hervieu M,Legendre FA,et al.Chondrogenic commitment of human umbilical cord blood-derived mesenchymal stem cells in collagen matrices for cartilage engineering[J].Sci Rep,2016,6:32786.
13 Shawki S,Gaafar T,Erfan H,et al.Immunomodulatory effects of umbilical cord-derived mesenchymal stem cells[J].Microbiol Immunol,2015,59(6):348-356.
14 Li XF,Duan L,Liang YJ,et al.Human umbilical cord Blood-Derived mesenchymal stem cells contribute to chondrogenesis in coculture with chondrocytes[J].Biomed Res Int,2016(1):1-9.
15 DesancéM,Contentin R,Bertoni L,et al.Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy[J].Int J Mol Sci,2018,19(2):pii:E537.
16李兴福,段莉,梁宇杰,等.直接共培养诱导人脐带间充质干细胞向软骨细胞的分化[J].中国组织工程研究,2017,21(1):18-24.
17张军,徐辉,温绣蔺,等.Sox9基因转染的人脐带血干细胞复合骨基质明胶/生物蛋白胶构建组织工程软骨的实验研究[J].生物医学工程与临床,2018(1):13-19.
18刘英,王姣杰,张玉红,等.脐血干细胞移植治疗膝盖软骨损伤一例疗效观察[J].中华临床医师杂志:电子版,2011,5(15):4586-4587.
19徐建民,王兆朋,王恒孝.骨形态发生蛋白-2在兔骨折愈合过程中的作用[J].中国矫形外科杂志,2016,24(10):920-925.
20 Zhou J,He T,Bi Y,et al.Effect of BMP-2 and-3 overexpression on osteogenic and chondrogenic differentiation of prenatal mouse intervertebral disc cells in vitro[J].Nan Fang Yi Ke Da Xue Xue Bao,2013,33(7):977-982.
21 Kwon SH,Lee TJ,Park J,et al.Modulation of BMP-2-Induced chondrogenic versus osteogenic differentiation of human mesenchymal stem cells by Cell-Specific extracellular matrices[J].Tissue Eng Part A,2013,19(1/2):49-58.
22 Qian XD,Zhang C,Chen GJ,et al.Effects of BMP-2 and FGF2 on the osteogenesis of bone Marrow-Derived mesenchymal stem cells in Hindlimb-Unloaded rats[J].Cell Biochem Biophys,2014,70(2):1127-1136.
23沈鹏飞,王斌,谢子康,等.软骨寡聚基质蛋白过表达对BMP-2诱导骨髓间充质干细胞分化的影响[J].中国生物工程杂志,2016,36(10):1-7.
24李小飞,张涛,程庆书,等.重组人骨形态发生蛋白诱导气管软骨再生的剂量效应[J].中华实验外科杂志,2004,21(3):96-97.
25张泉,邓飞龙.骨形态发生蛋白复合胶原释放系统的研究进展[J].国际口腔医学杂志,2012,39(2):248-251.
26殷力,娄超举,韩奇财,等.骨形成蛋白-2转染兔骨髓基质细胞修复软骨缺损的研究[J].中华实验外科杂志,2012,29(3):515-517.
27秦国斌,王秀利,肖鹏,等.骨形态发生蛋白-2对兔骨髓基质细胞向软骨细胞分化基因表达的影响[J].中华实验外科杂志,2011,28(8):1370-1372.
28尹承慧,邱俊钦,曾昭勋,等.腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞[J].中国组织工程研究,2013,17(14):2488-2494.