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

doi:10.3877/cma.j.issn.2096-0263.2019.02.003

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。

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

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.

Key words: Recombinant human bone morphogenetic protein-2, Mesenchymal stem cells, Gene editing, Liposomes

图1 细胞贴壁7 d后，生长状态良好，呈长梭状（100X）

图2 流式细胞仪鉴定：CD34（0.13%），CD45（0.14%），CD90（99.88%），CD105（99.95%），CD146（73.91%），Anti-HLA-DR（1.57%）

图3~5 荧光显微镜下绿荧光蛋白（EGFP）的表达（200X）。图3 换液后EGFP表达情况，亮度较强；图4 48 h EGFP表达达到最大数目；图5 72 hEGFP强度降低

图11 ELISA检验结果，转染组BMP-2含量明显高于未转染组，转染组在转染后BMP-2蛋白量持续增高，峰值出现在转染后48 h，为3 006 pg/ml，其后开始下降，但仍高于对照组其他时段

表1 加入重组基因后不同时间点对照组与实验组hBMP-2蛋白浓度（pg/ml，±s）

组别	12 h	24 h	36 h	48 h	60 h	72 h	84 h	96 h
对照组	128.5±1.9	212.4±2.1	321.6±2.0	448.5±3.1	400.1±2.9	384.3±1.4	375.8±1.4	366.2±3.0
实验组	787.1±1.2	1215.7±4.9	1948.3±1.6	3006.0±4.6	2664.9±7.6	2367.3±8.4	2196.0±4.6	1995.3±5.2
t值	662.562	418.266	1429.045	1027.186	622.344	522.479	849.123	609.180
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

表1 加入重组基因后不同时间点对照组与实验组hBMP-2蛋白浓度（pg/ml，±s）

组别	12 h	24 h	36 h	48 h	60 h	72 h	84 h	96 h
对照组	128.5±1.9	212.4±2.1	321.6±2.0	448.5±3.1	400.1±2.9	384.3±1.4	375.8±1.4	366.2±3.0
实验组	787.1±1.2	1215.7±4.9	1948.3±1.6	3006.0±4.6	2664.9±7.6	2367.3±8.4	2196.0±4.6	1995.3±5.2
t值	662.562	418.266	1429.045	1027.186	622.344	522.479	849.123	609.180
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

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Research on human umbilical cord bloodmesenchymal stem cells transfected with the gene of human bone morphogenetic protein-2

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