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[1]張瑞萍,邱相君,蘇兵,等.頭蛋白shRNA和骨形態(tài)發(fā)生蛋白2的協(xié)同作用對骨髓間充質(zhì)干細(xì)胞成骨能力的影響[J].中醫(yī)正骨,2019,31(09):1-6.
　ZHANG Ruiping,QIU Xiangjun,SU Bing,et al.The synergistic effects of Noggin shRNA and bone morphogenetic protein 2 on osteogenic ability of bone marrow mesenchymal stem cells[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(09):1-6.
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頭蛋白shRNA和骨形態(tài)發(fā)生蛋白2的協(xié)同作用對骨髓間充質(zhì)干細(xì)胞成骨能力的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期數(shù):
2019年09期
頁碼:
1-6
欄目:
基礎(chǔ)研究
出版日期:
2019-09-20

文章信息/Info

Title:
The synergistic effects of Noggin shRNA and bone morphogenetic protein 2 on osteogenic ability of bone marrow mesenchymal stem cells
作者:
張瑞萍1; 邱相君2; 蘇兵3; 鄭路路1
(1.洛陽市第一人民醫(yī)院,河南 洛陽 471002; 2.河南科技大學(xué),河南 洛陽 471003; 3.Quater診斷實驗室,紐約 布法羅 14200)
Author(s):
ZHANG Ruiping1; QIU Xiangjun2; SU Bing3; ZHENG Lulu1
1.The First People's Hospital of Luoyang,Luoyang 471002,Henan,China 2.Henan University of Science and Technology,Luoyang 471003,Henan,China 3.Quater diagnosis laboratory,Buffalo 14200,New York,America
關(guān)鍵詞:
間質(zhì)干細(xì)胞;  骨髓;  骨形態(tài)發(fā)生蛋白質(zhì)2;  轉(zhuǎn)染;  頭蛋白
Keywords:
mesenchymal stem cells;  bone marrow;  bone morphogenetic protein 2;  transfection;  Noggin
摘要:
目的:觀察頭蛋白shRNA和骨形態(tài)發(fā)生蛋白2(bone morphogenetic protein 2,BMP-2)的協(xié)同作用對骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)成骨能力的影響。方法:分離、培養(yǎng)兔BMSCs,分為頭蛋白shRNA基因干擾組、BMP-2過表達(dá)組、雙基因轉(zhuǎn)染組。構(gòu)建慢病毒頭蛋白shRNA干擾載體和BMP-2過表達(dá)載體。頭蛋白shRNA基因干擾組、BMP-2過表達(dá)組分別加入頭蛋白shRNA基因干擾載體和BMP-2過表達(dá)載體,雙基因轉(zhuǎn)染組同時加入頭蛋白shRNA基因干擾載體和BMP-2過表達(dá)載體,進行基因轉(zhuǎn)染。基因轉(zhuǎn)染成功后,采用熒光定量PCR檢測3組BMSCs中頭蛋白和BMP-2的mRNA相對表達(dá)量,采用茜素紅染色和堿性磷酸酶染色檢測3組BMSCs的誘導(dǎo)成骨能力。結(jié)果:①細(xì)胞培養(yǎng)和基因轉(zhuǎn)染鑒定結(jié)果。流式細(xì)胞儀檢測第2代培養(yǎng)細(xì)胞,CD44和CD29表達(dá)分別為61.3%和57.2%,CD34和CD45表達(dá)分別為3.6%和2.4%,符合BMSCs表型特征。基因轉(zhuǎn)染72 h后,3組BMSCs的轉(zhuǎn)染率為(92.5±2.5)%,基因轉(zhuǎn)染成功。②頭蛋白和BMP-2 mRNA表達(dá)檢測結(jié)果。基因轉(zhuǎn)染成功后,3組細(xì)胞中頭蛋白mRNA相對表達(dá)量的組間總體比較,差異有統(tǒng)計學(xué)意義(0.343±0.124,1.040±0.269,0.123±0.057; F=22.390,P=0.002); BMP-2過表達(dá)組較頭蛋白shRNA基因干擾組和雙基因轉(zhuǎn)染組高(P=0.016,P=0.005),頭蛋白shRNA基因干擾組和雙基因轉(zhuǎn)染組的組間差異無統(tǒng)計學(xué)意義(P=0.054)。3組細(xì)胞中BMP-2 mRNA相對表達(dá)量的組間總體比較,差異有統(tǒng)計學(xué)意義(1.120±0.363,3.667±0.431,4.833±0.475; F=59.680,P=0.000)。雙基因轉(zhuǎn)染組較頭蛋白shRNA基因干擾組和BMP-2過表達(dá)組高(P=0.000,P=0.035),BMP-2過表達(dá)組高于頭蛋白shRNA基因干擾組(P=0.001)。③茜素紅和堿性磷酸酶染色結(jié)果。茜素紅染色可見胞漿邊緣呈現(xiàn)不透光的礦化結(jié)節(jié)。3組細(xì)胞茜素紅染色陽性率的組間總體比較,差異有統(tǒng)計學(xué)意義[(62.317±3.969)%,(39.267±2.170)%,(72.320±4.133)%,F=68.870,P=0.000]; 雙基因轉(zhuǎn)染組高于頭蛋白shRNA基因干擾組和BMP-2過表達(dá)組(P=0.039,P=0.000),頭蛋白shRNA基因干擾組高于BMP-2過表達(dá)組(P=0.001)。堿性磷酸酶染色細(xì)胞內(nèi)呈現(xiàn)灰黑色顆粒或塊狀沉淀。3組細(xì)胞堿性磷酸酶染色陽性率組間總體比較,差異有統(tǒng)計學(xué)意義[(55.157±9.959)%,(35.353±6.494)%,(82.337±10.594)%,F=19.750,P=0.002]; 雙基因轉(zhuǎn)染組高于頭蛋白shRNA基因干擾組和BMP-2過表達(dá)組(P=0.032,P=0.003),頭蛋白shRNA基因干擾組高于BMP-2過表達(dá)組(P=0.045)。結(jié)論:頭蛋白shRNA和BMP-2的協(xié)同作用可以強化BMSCs的成骨能力。
Abstract:
Objective:To observe the synergistic effects of Noggin shRNA and bone morphogenetic protein 2(BMP-2)on osteogenic ability of bone marrow mesenchymal stem cells(BMSCs).Methods:The BMSCs were isolated from the femoral bone marrow of rabbits and were cultured in vitro.The second-generation BMSCs were randomly divided into Noggin shRNA gene interference group,BMP-2 overexpression group and double-gene transfection group.The lentivirus-mediated Noggin shRNA interference vectors and BMP-2 overexpression vectors were constructed.The BMSCs in Noggin shRNA gene interference group and BMP-2 overexpression group were transfected with Noggin shRNA gene interference vectors and BMP-2 overexpression vectors respectively,and the BMSCs in double-gene transfection group were co-transfected with Noggin shRNA gene interference vectors and BMP-2 overexpression vectors.After successful gene transfection,the relative expression levels of Noggin mRNA and BMP-2 mRNA in BMSCs were detected by using fluorescent quantitative PCR method,and the ability of BMSCs to induce osteogenesis was detected by using alizarin red staining(ARS)and alkaline phosphatase(ALP)staining.Results:The second-generation BMSCs were detected by using flow cytometry,and the results showed that the expressions of CD44,CD29,CD34 and CD45 were 61.3%,57.2%,3.6% and 2.4% respectively,which were consistent with the phenotypic characteristics of BMSCs.After 72-hour gene transfection,the transfection rate of BMSCs was(92.5+/-2.5)% in the 3 groups.After successful gene transfection,there was statistical difference in the relative expression level of Noggin mRNA in BMSCs between the 3 groups in general(0.343+/-0.124,1.040+/-0.269,0.123+/-0.057; F=22.390,P=0.002).The relative expression level of Noggin mRNA in BMSCs was higher in BMP-2 overexpression group compared to Noggin shRNA gene interference group and double-gene transfection group(P=0.016,P=0.005),and there was no statistical difference in the relative expression level of Noggin mRNA in BMSCs between Noggin shRNA gene interference group and double-gene transfection group(P=0.054).There was statistical difference in the relative expression level of BMP-2 mRNA in BMSCs between the 3 groups in general(1.120+/-0.363,3.667+/-0.431,4.833+/-0.475; F=59.680,P=0.000).The relative expression level of BMP-2 mRNA in BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.000,P=0.035),and was higher in BMP-2 overexpression group compared to Noggin shRNA gene interference group(P=0.001).The opaque mineralized nodules along the cytoplasmic margin were found through ARS.There was statistical difference in the positive rate of ARS for BMSCs between the 3 groups in general(62.317+/-3.969,39.267+/-2.170,72.320+/-4.133%,F=68.870,P=0.000).The positive rate of ARS for BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.039,P=0.000),and was higher in Noggin shRNA gene interference group compared to BMP-2 overexpression group(P=0.001).The gray-black granules or block-shaped sediments were found within BMSCs through ALP staining.There was statistical difference in the positive rate of ALP staining for BMSCs between the 3 groups in general(55.157+/-9.959,35.353+/-6.494,82.337+/-10.594%,F=19.750,P=0.002).The positive rate of ALP staining for BMSCs was higher in double-gene transfection group compared to Noggin shRNA gene interference group and BMP-2 overexpression group(P=0.032,P=0.003),and was higher in Noggin shRNA gene interference group compared to BMP-2 overexpression group(P=0.045).Conclusion:The synergistic effects of Noggin shRNA and BMP-2 can enhance the osteogenic ability of BMSCs.

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通訊作者:張瑞萍 E-mail:[email protected](收稿日期:2019-08-01 本文編輯:楊雅)
更新日期/Last Update: 2019-09-15