hIGF-1基因改良修饰人脐血间充质细胞的研究

doi:10.3877/cma.j.issn.2096-0263.2018.06.006

目的

研究hIGF-1基因改良修饰人脐血间充质干细胞的方法，为关节软骨组织工程修复提供良好的种子细胞。

方法

综合应用密度梯度离心法和细胞贴壁法分离培养人脐血间充质干细胞，流式细胞仪进行细胞鉴定。通过X-tremen HP介导将含hIGF-1基因全长的真核表达载体pIRES2-EGFP-hIGF-I转染人脐血间充质干细胞，倒置荧光显微镜检测转染后荧光蛋白的表达，计算转染效率，ELISA检测转染后细胞上清液中hIGF-1的含量变化，免疫荧光与RT-PCR检测hIGF-1在细胞中的表达，免疫组化检测Ⅱ型胶原表达。

结果

人脐血间充质干细胞表面表达CD105（99.93%）、CD90（99.85%）、CD146（73.63%），不表达Anti-HLA-DR（1.38%）、CD45（0.13%）、CD34（0.11%）。hIGF-1基因可在X-tremen HP介导下转染人脐血间充质干细胞，48 h转染效率为（29±8）%。转染后48 h分泌的hIGF-1最多，为（34.89±0.38）ng/ml，且与对照组相比，差异具有统计学意义（P<0.01）。转染后细胞可检测到hIGF-1 mRNA、蛋白以及Ⅱ型胶原表达。

结论

hIGF-1基因可通过X-tremen HP介导改良修饰人脐血间充质干细胞，促进hIGF-1和Ⅱ型胶原的表达与分泌。

关键词: 胰岛素样生长因子1, 脂质体, 间充质干细胞, 基因转染

Objective

To study the modification of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) by hIGF-1 gene, and provide good seed cells for tissue engineering repair of articular cartilage.

Methods

hUCB-MSCs were isolated and cultured by density gradient centrifugation and cell adherence method, and cell identification was performed by flow cytometry. The eukaryotic expression vector, pIRES2-EGFP-hIGF-I, containing the full length of hIGF-1 gene was transfected into hUCB-MSCs via X-tremen HP. After transfection, the expression of fluorescent protein was detected by reverse fluorescence microscope and the transfection efficiency was calculated. ELISA was used to detect the content of hIGF-1 in the cell supernatant after transfection, and the immunofluorescence and RT-PCR were used to detect hIGF-1 in the hUCB-MSCs. The expression of the typeⅡ collagen was detected by immunohistochemistry.

Results

Flow cytometry showed that the hUCB-MSCs expressed CD90, CD105 and CD146 positively, and CD34, CD45, Anti-HLA-DR negatively. hIGF-1 gene was introduced into hUCB-MSCs with X-treme GENE HP DNA transfection reagent, and the transfection efficiency is (29+8)%. The hIGF-1 protein concentration in the supernatants determined by ELISA had significant difference between transfection group and control group (P<0.01). ELISA result showed that the hIGF-1 protein concentration in the supernatants determined after transfection at high level [(35±0.4)ng/ml] at 48 h point. The expression of hIGF-1 was detected by immunofluorescence and RT-PCR identification. Immunohistochemistry showed positive expression of type Ⅱcollagen after transfection.

Conclusions

hIGF-1 gene can modify human umbilical cord blood mesenchymal stem cells through X-tremen HP mediated modification, and promote the expression and secretion of hIGF-1 and type Ⅱ collagen.

Key words: Insulin-like growth factor 1, Liposomes, Gene transfection, Mesenchymal stem cells

图3 细胞生长曲线，3~6 d对数生长期，7 d后进入平台期

图11 RT-PCR结果（M为Marker；A、C为内参照；B为实验组pIRES2-EGFP-hIGF-I；D为空质粒对照组）

图14 细胞免疫组化显示未转染后细胞无Ⅱ型胶原的表达（×200）

表1 两组人脐血干细胞基因转染后不同时间点hIGF-1蛋白浓度的比较（ng/ml，±s）

组别	6 h	12 h	18 h	24 h	36 h	48 h	72 h	96 h	7d
实验组	4.13±0.16	7.46±0.40	11.97±0.39	17.56±0.80	21.86±0.19	34.89±0.38	33.23±0.38	26.77±0.36	10.20±0.23
对照组	3.10±0.12	3.24±0.27	3.02±0.21	3.28±0.06	3.34±0.19	3.36±0.25	3.36±0.25	3.20±0.14	3.19±0.12
F值	4.769	4.962	5.931	6.834	9.885	15.267	13.814	11.652	5.598
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

表1 两组人脐血干细胞基因转染后不同时间点hIGF-1蛋白浓度的比较（ng/ml，±s）

组别	6 h	12 h	18 h	24 h	36 h	48 h	72 h	96 h	7d
实验组	4.13±0.16	7.46±0.40	11.97±0.39	17.56±0.80	21.86±0.19	34.89±0.38	33.23±0.38	26.77±0.36	10.20±0.23
对照组	3.10±0.12	3.24±0.27	3.02±0.21	3.28±0.06	3.34±0.19	3.36±0.25	3.36±0.25	3.20±0.14	3.19±0.12
F值	4.769	4.962	5.931	6.834	9.885	15.267	13.814	11.652	5.598
P值	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01	<0.01

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