Recombinant adenovirus-mediated hypoxia inducible factor 1alpha mutant promotes proliferation of adipose-derived mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.23.006

Abstract

Abstract:

BACKGROUND: To improve the survival rate of transplanted tissue, most scholars focus on cell therapy, particularly cell-assisted fat grafting.
OBJECTIVE: To analyze the effect of recombinant adenovirus-mediated hypoxia inducible factor 1alpha (HIF1α) mutant on survival rate of transplanted fat particles through transfection of adipose-derived mesenchymal stem cells.
METHODS: Recombinant adenovirus-mediated triple-mutant HIF1α was inserted into an adenovirus pAdEasy-1 system, followed by viral packaging and titer determination. Human adipose-derived mesenchymal stem cells were cultured, passaged and identified, and subsequently transfected with three kinds of viruses and blank vector (experimental group with transfection of the triple mutant of HIF1α; positive control group; negative control group; blank control group). Transfection efficiency was determined using enhanced green fluorescent protein labeling. Additionally, MTT assay was used to detect cell proliferation.
RESULTS AND CONCLUSION: The recombinant adenovirus was successfully constructed and packaged in line with transfection requirements. Moreover, adipose-derived mesenchymal stem cells were successfully identified by adipogenic, osteogenic and chondrogenic induction and could be used as seed cells for subsequent experiments. RT-PCR results showed that HIF1α mRNA expression in the experimental group and positive control group was significantly higher than that in the other two groups(P < 0.05). Western blot analysis showed that the relative absorbance value in the experimental group was significantly higher than that in the other three groups (P < 0.05). A significant increase in the cell proliferation was found in the experimental group, significantly different from the other three groups (P < 0.05). Therefore, our findings indicate that transfection of adenovirus-mediated triple-mutant HIF1α not only can sustain the expression of target protein in transfected adipose-derived mesenchymal stem cells under normoxic conditions, but also can promote the proliferation of transfected adipose-derived mesenchymal stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Mesenchymal Stem Cells, Hypoxia-Inducible Factor 1, Tissue Engineering

CLC Number:

R394.2

Zhang Xin, Zhou Lin, Zhang Xiao-lei. Recombinant adenovirus-mediated hypoxia inducible factor 1alpha mutant promotes proliferation of adipose-derived mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(23): 3386-3393.

Figures/Tables 2

2.1 低氧诱导因子1α突变鉴定结果测序结果显示供体质粒中所携带的低氧诱导因子1α基因编码区402位氨基酸由CCA(脯氨酸)突变成GCA(丙氨酸)，564位氨基酸由CCC(脯氨酸)突变成GCC(丙氨酸)，803位氨基酸由ATT(天冬酰胺)突变成AGC丙氨酸(图1)。 2.2 腺病毒包装结果 HEK293A细胞经腺病毒转染后均观察到有大量绿色荧光蛋白表达并出现相似的细胞病变效应(Cytopathic effect，CPE)(图2，3)。滴度检测结果依次为实验组病毒液1.76×108 pfu/mL，阳性对照组病毒液2.09×108 pfu/mL，阴性对照组病毒液1.92×108 pfu/mL，符合转染要求。 2.3 脂肪间充质干细胞鉴定结果在成脂诱导培养2周后，细胞内开始出现部分脂滴，同时进行油红O染色鉴定，结果显示：成脂诱导组脂肪间充质干细胞油红O染色阳性，对照组阴性(图4)。在成骨诱导的第14天，通过茜素红染色鉴定可以观察到红色典型钙化结节形态，而对照组的细胞形态仍为成纤维细胞样，大小基本没有变化，茜素红染色均不着色或着色极为虚弱(图5)。在成软骨诱导培养后，脂肪间充质干细胞的形态逐渐由梭形向多角形或多边形转变。诱导14 d后发现软骨细胞结构层清晰，核被染成蓝色，胞浆几乎不着色，基质呈淡蓝紫色，对照组不着色或着色极为虚弱(图6)。 2.4 脂肪间充质干细胞增殖检测结果各组细胞分别在培养24，48，72，96，120 h后利用MTT法检测细胞的吸光度值，同时绘制增殖曲线。结果提示，阳性对照组及空白组的脂肪间充质干细胞生长速度较慢，两组相比较差异无显著性意义(P > 0.05)；实验组细胞增殖程度显著增加，相比于另外两组差异有着显著性意义(P < 0.05)，说明三点突变型低氧诱导因子1α可以显著提高脂肪间充质干细胞的增殖(图7)。 2.5 RT-PCR检测结果实验组和阳性对照组细胞内低氧诱导因子1α mRNA表达两组之间差异无显著性意义(P > 0.05)；阴性对照组和空白组两组低氧诱导因子1α mRNA的表达量非常低，几乎可以忽略，差异也无显著性意义(P > 0.05)；而前两组细胞内低氧诱导因子1α mRNA表达量均明显高于后两组，差异有显著性意义 (P < 0.05，图8)。 2.6 Western blot检测结果实验组细胞中低氧诱导因子1α蛋白表达明显高于阳性对照组、阴性对照组和空白组(P < 0.05)；而阳性对照组、阴性对照组和空白组3组间差异无显著性意义(P > 0.05，图9)。结果显示，三点突变型低氧诱导因子1α可以在常氧条件下高效表达，有利于促进移植种子细胞的增殖及新血管生成。"

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