In vitro construction of tissue engineered adipose using vascular endothelial growth factor 165 gene-modified human adipose derived stem cells with chitosan-surface modified silk fibroin scaffolds

doi:10.3969/j.issn.2095-4344.2014.52.016

Abstract

Abstract:

BACKGROUND: To construct tissue-engineered adipose tissues, suitable seed cells and excellent performance of scaffold materials are indispensable.

OBJECTIVE: To investigate the feasibility of constructing tissue-engineered adipose in vitro utilizing vascular endothelial growth factor 165 (VEGF165) gene-modified human adipose derived stem cells with chitosan-modified silk fibroin scaffolds.

METHODS: Passage 3 human adipose derived stem cells growing well were selected, and transfected with or without lentivirus-VEGF165-EGFP. To test the impact of lentivirus infection on adipogenic capability of human adipose derived stem cells, oil red O staining was performed after adipogenic induction for 14 days. To evaluate the proliferation and adipogenic capability of lentivirus infected human adipose derived stem cells on the chitosan-modified silk fibroin scaffolds, lentivirus infected and uninfected passage 3 human adipose derived stem cells were seeded on modified silk fibroin scaffolds respectively, and MTT tests were used to evaluate cell proliferation on the scaffolds. After adipogenic induction for 14 days, RT-PCR was performed to detect the expression of peroxisome proliferator-activated receptor γ-2. Besides, oil red O staining was adopted to illustrate the mature adipocytes.

RESULTS AND CONCLUSION: After adipogenic induction for 14 days, oil red O staining results showed mature adipocytes existed in lentivirus infected and uninfected passage 3 human adipose derived stem cells, and there was no statistical difference between lentivirus infected and uninfected cells. The proliferation curves of lentivirus infected and uninfected cells on the scaffolds showed no significant difference. After adipogenic induction for 14 days, oil red O staining and RT-PCR results could demonstrate that human adipose derived stem cells could be induced to mature adipocytes. These findings indicate that after infected by lentivirus-VEGF165-EGFP, the adipogenic differentiation and growth of human adipose derived stem cells cannot be affected. Lentivirus-infected human adipose derived stem cells can be combined with the chitosan-modified silk fibroin scaffolds to construct tissue-engineered adipose tissues successfully.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: tissue engineering, mesenchymal stem cells, entivirus infections, vascular endothelial growth factors

CLC Number:

R318

Kang Ting, Wang Gang, Liu Yi, Liu Gang-qiang. In vitro construction of tissue engineered adipose using vascular endothelial growth factor 165 gene-modified human adipose derived stem cells with chitosan-surface modified silk fibroin scaffolds[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(52): 8450-8455.

Figures/Tables 3

2.1 人脂肪间充质干细胞的体外分离培养及形态学特点原代细胞提取后培养24 h换液，利用不同细胞贴壁性的差异去除杂细胞，可见原代细胞呈短梭形(图1A)，培养至7-9 d时，细胞呈长梭形，形态规则；细胞达到80%融合时，可呈“漩涡”状排列(图1B)。 2.2 人脂肪间充质干细胞的体外成脂、成骨诱导成脂诱导第3天，光镜下可见胞质内有少量折光性强的小脂滴形成，细胞形态由长梭形逐渐变圆；当成脂诱导14 d后，小脂滴融合形成大的脂滴，油红O染色呈阳性(图2A)。成骨诱导3 d后，细胞变为多角形，并开始聚集叠层样生长；诱导14 d后，细胞密集处经茜素红染色呈现红色的结节状沉淀物，表明有钙化结节形成(图2B)。细胞在体外具有多向分化能力证明了所提取细胞为人脂肪间充质干细胞。 2.3 壳聚糖/丝素蛋白支架的扫描电镜观察壳聚糖/丝素蛋白支架材料呈海绵状，内部布满三维网状结构，孔隙彼此相连，孔隙率为90%，孔径大小为70-140 μm(图3)。 2.4 慢病毒感染人脂肪间充质干细胞用携带重组人血管内皮细胞生长因子165基因的慢病毒感染人脂肪间充质干细胞(MOI=50)，感染后的最初48 h内荧光显微镜下未见绿色荧光蛋白表达，感染72 h后可见微弱的荧光(图4A)，96 h后荧光明显增强，阳性率可达80%(图4B)。 2.5 慢病毒感染人脂肪间充质干细胞后的成脂诱导成脂诱导14 d后，光镜下两组细胞胞质内均可见大小不等的脂滴，细胞形态由长梭形变为椭圆型。荧光显微镜下可见实验组被感染人脂肪间充质干细胞内大小不等的脂滴(图5A)。行油红O染色后，实验组(图5B)与对照组(图5C)均可见红色脂滴。使用Imge-Pro Plus图像分析软件计算每200倍视野油红O染色阳性面积和吸光度值(表1)并进行t 检验，结果显示两组比较差异无显著性意义(P > 0.05)。 2.6 慢病毒感染人脂肪间充质干细胞在壳聚糖/丝素蛋白支架材料上的生长增殖 MTT结果显示实验组与对照组细胞在壳聚糖/丝素蛋白支架材料上的生长无统计学差异(P > 0.05)，表明慢病毒感染后对人脂肪间充质干细胞在壳聚糖/丝素蛋白支架材料上的增殖能力无明显影响(图6)。 2.7 慢病毒感染人脂肪间充质干细胞在改性丝素蛋白上的成脂诱导慢病毒感染的P3代人脂肪间充质干细胞和未感染的P3代人脂肪间充质干细胞分别接种于支架材料后，荧光显微镜下可以观察实验组呈现绿色荧光(图7A)。成脂诱导14 d后，油红O染色，光镜下实验组(图7B)和对照组(图7C)中均可见椭圆形的成熟脂肪细胞生成黏附于壳聚糖/丝素蛋白支架材料上。 RT-PCR扩增产物琼脂糖凝胶电泳可见两组中成脂特异性基因过氧化物酶增殖物活化受体γ2条带(图8)，进一步表明慢病毒感染的人脂肪间充质干细胞在壳聚糖/丝素蛋白支架材料上经成脂诱导后可分化为成熟脂肪细胞。"

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