[1]李大伟,孙一,姜翠萍,等.重组人骨形态发生蛋白-2质粒转染诱导人脐血间充质干细胞软骨分化研究[J].中华老年骨科与康复电子杂志,2019,(02):75-81.[doi:10.3877/cma.j.issn.2096-0263.2019.02.003]
　Li Dawei,Sun Yi,Jiang Cuiping,et al.Research on human umbilical cord bloodmesenchymal stem cells transfected with the gene of human bone morphogenetic protein-2[J].Chin J Geriatr Orthop Rehabil(Electronic Edition),2019,(02):75-81.[doi:10.3877/cma.j.issn.2096-0263.2019.02.003]
点击复制

重组人骨形态发生蛋白-2质粒转染诱导人脐血间充质干细胞软骨分化研究()
分享到：

中华老年骨科与康复电子杂志[ISSN:1674-3911/CN:11-9292/R]

卷:
期数:
2019年02期
页码:
75-81
栏目:
基础研究
出版日期:
2019-04-05

文章信息/Info

Title:
Research on human umbilical cord bloodmesenchymal stem cells transfected with the gene of human bone morphogenetic protein-2
作者:
李大伟1; 孙一1; 姜翠萍2; 丛文斌3; 张海宁1
266000 青岛大学附属医院关节外科1，手术室2，放射科3
Author(s):
Li Dawei1;  Sun Yi1;  Jiang Cuiping2;  Cong Wenbin3;  Zhang Haining1
2Operating Room, 3Radiology, the Affiliated Hospital of Qingdao University, QingDao 266000, China
关键词:
重组人骨形态发生蛋白-2;  间充质干细胞;  基因编辑;  脂质体
Keywords:
Recombinant ; human ; bone morphogenetic protein-2;  Mesenchymal stem cells;  Gene editing;  Liposomes
DOI:
10.3877/cma.j.issn.2096-0263.2019.02.003
文献标志码:
A
摘要:
目的 探究从人脐血中提取间充质干细胞，重组质粒pIRES2-EGFP-hBMP-2转染干细胞并诱导其成软骨化的可能性。方法 采用密度梯度离心方法获取脐带血中细胞，依据贴壁时间不同获得间充质干细胞，流式细胞仪检测表面抗原表达鉴定细胞；然后把重组有pIRES2-EGFP-hBMP-2的质粒导入间充质干细胞，观察EGFP的表达；ELISA方法检测在不同时间收集的培养基上清中hBMP-2蛋白含量，采用免疫荧光和RT-PCR方法检测目的蛋白和基因表达。转染成功后继续培养细胞2 w，免疫组化检测细胞Ⅱ型胶原的表达和RT-PCR检测软骨特异性标志物软骨连接蛋白（CRLT1）的表达。结果 两种方法可以获取人脐血间充质干细胞，流式细胞术鉴定发现CD90、CD105、CD146高表达，CD34、CD45、Anti-HLA-DR不表达。非脂质载体包裹重组质粒pIRES2-EGFP-hBMP-2可成功导入脐血间充质干细胞，转染率为（27.7±7.6）%。ELISA检测实验组和对照组hBMP-2的表达结果有统计学差异（t=3.355，P＜0.01）。RT-PCR结果表明hBMP-2基因稳定转录，免疫荧光标记hBMP-2蛋白呈红色荧光。Ⅱ型胶原免疫组化染色示部分细胞被染成棕黄色，RT-PCR结果表明加入转染后的干细胞组CRLT1的表达量比未转染的干细胞组高（t=59.700，P＜0.05）。结论 重组hBMP-2基因可以成功转染人脐血间充质干细胞并在胞内稳定表达，且有hBMP-2分泌，并可促进其表达Ⅱ型胶原蛋白及软骨连接蛋白，可向软骨细胞化诱导。
Abstract:
Objective To gain mesenchymal stem cells from humanumbilical cord blood and transfect the recombinant plasmid pIRES2-EGFP-hBMP-2 into human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) with liposome and to induce hUCB-MSCs osteoblastization. Methods Separated cells from human umbilical cord blood with density gradient centrifugation, and hUCB-MSCs were gained with adherent method, using flow cytometry to identify the surface markers of hUCB-MSCs. Transfected recombinate plasmid pIRES2-EGFP-hBMP-2 into the third generation hUCB-MSCs with X-treme GENE HP DNA Transfection reagent, then detected the intensity of EGFP. Collected the medium at different time after transfection and the hBMP-2 content in the medium was measured by ELISA. Use immunofluorescent to locate the existence of hBMP-2 within the cells and RT-PCR techniques to measure the transcription of the hBMP-2 gene. Two weeks after transfection, immunohistochemistry were used to detect the type Ⅱ collagen for discovering the possible changes of hUCB-MSCs. Results HUCB-MSCs could be isolated from blood in umbilical cord by density gradient centrifugation and the different ability of adherence. Flow cytometry showed that the hUCB-MSCs positively expressed CD90, CD105 and CD146, and did not express CD34, CD45 and Anti-HLA-DR. The recombinant plasmid pIRES2-EGFP-hBMP-2 was successfully fused into umbilical cord blood mesenchymal stem cells, and the fusion rate was (27.7±7.6)%. The ELISA test compared the expression of hBMP-2 between the experimental group and the control group, P<0.01 showed a statistically significant difference. RT-PCR results showed that the hBMP-2 gene was stably transcribed and the immunofluorescently labeled hBMP-2 protein showed red fluorescence. Immunohistochemical staining of type Ⅱ collagen showed that some cells were browned. RT-PCR results showed that the expression of CRLT1 in umbilical cord blood mesenchymal stem cells transfected with BMP-2 was higher than that in untransfected umbilical cord blood mesenchymal stem cells and human synovial fibrosis group (P<0.05). Conclusions Recombinat plasmid pIRES2-EGFP-BMP-2 coated with X-treme CENE can be successfully transfected into hUCB-MSCs. Both the marker gene and objectivegene can be transcribed and expressed in hUCB-MSCs, and cells can synthesis hBMP-2, which stimulates the hUCB-MSCs’differentiation towards to chondrocytes.

参考文献/References:

1 Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets [J]. Arthritis Rheum, 2001, 44(6): 1237-1247.
2 胥少汀, 葛宝丰, 徐印坎, 等. 实用骨科学 [M] . 北京: 人民军医出版社, 2015: 1671-1677.
3 Bhardwaj N, Devi D, Mandal BB. Tissue-Engineered cartilage: the crossroads of biomaterials, cells and stimulating factors [J]. Macromol Biosci, 2015, 15(2): 153-182.
4 Yamasaki S, Mera H, Itokazu M, et al. Cartilage repair with autologous bone marrow mesenchymal stem cell transplantation: review of preclinical and clinical studies [J]. Cartilage, 2014, 5(4): 196-202.
5 Odabas S, Elçin AE, Elçin YM. Isolation and characterization of mesenchymal stem cells [J]. Methods Mol Biol, 2014, 1109: 47-63.
6 Hassan G, Kasem I, Soukkarieh C, et al. A simple method to isolate and expand human umbilical cord derived mesenchymal stem cells: using explant method and umbilical cord blood serum [J]. Int J Stem Cells, 2017, 10(2): 184-192.
7 Dufrane D. Impact of age on human adipose stem cells for bone tissue engineering [J]. Cell Transplant, 2017, 26(9, SI): 1496-1504.
8 Singh J, Mann A, Kumar D, et al. Cultured Buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells [J]. In Vitro Cell Dev Biol Anim, 2013, 49(6): 408-416.
9 Jaing TH. Umbilical cord blood: a trustworthy source of multipotent stem cells for regenerative medicine [J]. Cell Transplant, 2014, 23(4/5): 493-496.
10 Kim JS, Lee HK, Kim MR, et al. Differentially expressed proteins of mesenchymal stem cells derived from human cord blood (hUCB) during osteogenic differentiation [J]. Biosci Biotechnol Biochem, 2008, 72(9): 2309-2317.
11 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.
12 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.
13 Hua J, Gong J, Meng HB, et al. Comparison of different methods for the isolation of mesenchymal stem cells from umbilical cord matrix: Proliferation and multilineage differentiation as compared to mesenchymal stem cells from umbilical cord blood and bone marrow [J]. Cell Biol Int, 2014, 38(2): 198-210.
14 Lee DH, Ahn Y, Kim SU, et al. Targeting rat brainstem glioma using human neural stem cells and human messenchymal stem cells [J]. Clin Cancer Res, 2009, 15(15): 4925-4934
15 Jin HJ, Park SK, Oh W, et al. Down-regulation of CD105 is associated with multi-lineage differentiation in human umbilical cord blood-derived mesenchymal stem cells [J]. Biochem Biophys Res Commun, 2009, 381(4): 676-681.
16 Mohyeddin Bonab MA, Alimoghaddam KA, Goliaei ZA, et al. Which factors can affect cord blood variables [J]. Transfusion, 2004, 44(5): 690-693.
17 Romanov YA, Svintsitskaya VA, Smimov VN. Searching for alternative sources of postnatal human mesenehymal stem cells: candidate MSC-like cells from umbilical cord [J]. Stem Cells, 2003, 21(1): 105-110.
18 黄宏宇, 刘国平, 段莉, 等. 人脐血间充质干细胞培养方法的比较 [J]. 中国组织工程研究, 2014, 18(37): 5961-5966.
19 Feldmann RE, Bieback K, Maurer MH, et al. Stem cell proteomes: A profile of human mesenchymal stem cells derived from umbilical cord blood [J]. Electrophoresis, 2005, 26(14): 2749-2758.
20 郑德宇, 刘建生, 杨依勇, 等. 成骨诱导后的脐血间充质干细胞与β-磷酸三钙复合植骨材料对兔颅骨缺损的修复作用 [J]. 西安交通大学学报:医学版, 2011, 32(4): 441-444, 476.
21 Wang FS, Yang KD, Wang CJ, et al. Shockwave stimualates Oxygen radical-mediated osteogenesis of the mesenehymal cells from human umbilical cord blood [J]. J Bone Miner Res, 2004, 19(6): 973-982.
22 杨依勇, 朱慧敏, 秦书俭, 等. 脐带血间充质干细胞的成骨诱导及结果检测 [J]. 解剖学杂志, 2010, 33(5): 692-694.
23 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, 8(6): 32786.
24 Li H, Nie B, Du Z, et al. Bacitracin promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by stimulating the bone morphogenetic protein-2/Smad axis [J]. Biomed Pharmacother, 2018, 103: 588-597.
25 Bougioukli S, Sugiyama O, Pannell W, et al. Gene therapy for bone repair using human cells: superior osteogenic potential of bone morphogenetic protein 2-Transduced mesenchymal stem cells derived from adipose tissue compared to bone marrow [J]. Hum Gene Ther, 2018, 29(4): 507-519.
26 Nasrabadi D, Rezaeiani S, Eslaminejad MB, et al. Improved protocol for chondrogenic differentiation of bone marrow derived mesenchymal stem cells -Effect of PTHrP and FGF-2 on TGF beta 1/BMP2-Induced chondrocytes hypertrophy [J]. Stem Cell Rev, 2018, 14(5): 755-766.
27 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.
28 Deshmukh SN, Dive AM, Moharil R, et al. Enigmatic insight into collagen [J]. J Oral Maxillofac Pathol, 2016, 20(2): 276-283.
29 Lindenmair A, Nürnberger S, Stadler G, et.al. Intact human amniotic membrane differentiated towards the chondrogenic lineage [J]. Cell Tissue Bank, 2014, 15(2): 213-225.

相似文献/References:

[1]孙一,李大伟,张海宁.hIGF-1基因改良修饰人脐血间充质细胞的研究[J].中华老年骨科与康复电子杂志,2018,(06):346.[doi:10.3877/cma.j.issn.2096-0263.2018.06.006]
　Sun Yi,Li Dawei,Zhang Haining.The study on modification of human umbilical cord blood mesenchymal cells by hIGF-1 gene[J].Chin J Geriatr Orthop Rehabil(Electronic Edition),2018,(02):346.[doi:10.3877/cma.j.issn.2096-0263.2018.06.006]
[2]张洁,黄辉,王俊国,等.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[J].中华老年骨科与康复电子杂志,2019,(02):82.[doi:10.3877/cma.j.issn.2096-0263.2019.02.004]
　Zhang Jie,Huang Hui,Wang Junguo,et al.Research on bone marrow derived-mesenchymal stem cells and human umbilical cord blood derived-mesenchymal stem cells modified with the gene of human bone morphogenetic protein-2[J].Chin J Geriatr Orthop Rehabil(Electronic Edition),2019,(02):82.[doi:10.3877/cma.j.issn.2096-0263.2019.02.004]
[3]邬波,柳椰,马旭,等.3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用研究[J].中华老年骨科与康复电子杂志,2020,(03):123.[doi:10.3877/cma.j.issn.2096-0263.2020.03.001]
　Wu Bo,Liu Ye,et al.Effect of 3D printed collagen / hydroxyapatite scaffold on osteogenic differentiation of bone marrow mesenchymal stem cells[J].Chin J Geriatr Orthop Rehabil(Electronic Edition),2020,(02):123.[doi:10.3877/cma.j.issn.2096-0263.2020.03.001]

备注/Memo

备注/Memo:
基金项目：国家自然科学基金（81672197）
更新日期/Last Update: 2019-04-12