Human preproenkephalin gene-modified bone marrow mesenchymal stem cell line: its biological characteristics

doi:10.3969/j.issn.2095-4344.2016.19.001

Abstract

Abstract:

BACKGROUND: Previous studies have found that the transplantation of modified cell lines exhibit analgesic effect. But little is reported on the biological characteristics of human preproenkephalin gene-modified bone marrow mesenchymal stem cell lines.
OBJECTIVE: To observe the biological characteristics of human preproenkephalin gene-modified bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells were isolated and cultured to establish human preproenkephalin gene-modified bone marrow mesenchymal stem cell lines.
RESULTS AND CONCLUSION: After freezing-thawing, passage 4 human bone marrow mesenchymal stem cells, human bone marrow mesenchymal stem cells-pBABE and human bone marrow mesenchymal stem cells-human preproenkephalin exhibited no significant changes in the cell viability (P > 0.05), as well as in the fat cell proportion after adipogenic induction (P > 0.05). Moreover, red calcium deposition was presented in all these cells by alizarin red staining after osteogenic induction. Flow cytometry results showed that passage 4 human bone marrow mesenchymal stem cells, human bone marrow mesenchymal stem cells-pBABE and human bone marrow mesenchymal stem cells-human preproenkephalin could express CD29 and CD44, but not express CD34 and CD45. Human preproenkephalin genes were highly expressed in human bone marrow mesenchymal stem cells-pBABE, but lowly expressed in passage 4 human bone marrow mesenchymal stem cells. Additionally, recombinant plasmid pBABE-preproenkephalin-modified human bone marrow mesenchymal stem cells could express enkephalin protein. In conclusion, human preproenkephalin gene-modified human bone marrow mesenchymal stem cell lines can maintain the pluripotent differentiation or proliferation capacity of bone marrow mesenchymal stem cells, and secrete enkephalin protein.

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: Stem Cells, Tissue Engineering, Enkephalins

CLC Number:

R394.2

Li Xin-mao. Human preproenkephalin gene-modified bone marrow mesenchymal stem cell line: its biological characteristics[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(19): 2741-2747.

Figures/Tables 1

2.1 培养的人骨髓间充质干细胞的形态骨髓间充质干细胞原代培养24 h时开始出现贴壁细胞，贴壁细胞多呈多角形或者长梭形，贴壁细胞增殖比较快，培养至第10天细胞达到90%以上融合。传代培养24 h内出现完全贴壁，约第5天完全融合(图1A)。 2.2 反转录病毒包装和克隆筛选情况经过转染后的PHOENIX-293T细胞筛选后3周，实验组细胞一部分死亡，一部分存活(图1B)，对照组细胞均死亡。存活细胞继续生长为抗性细胞克隆，克隆的周围细胞呈多边形，呈放射状，中心细胞呈圆形，出现融合。单个细胞继续扩大培养，细胞呈现多边形，继续扩大培养，得到P293T-pBABE和P293T-人前脑啡肽单克隆细胞。 2.3 病毒感染后的人骨髓间充质干细胞抗性细胞形态病毒感染后的人骨髓间充质干细胞筛选3周后，实验组细胞一部分死亡，一部分出现抗性细胞克隆，对照组的细胞均死亡。克隆细胞四周呈长梭形，出现放射性分布，中心细胞呈多角形，有融合现象，单细胞克隆扩大培养后，细胞呈多边形(图1C)，继续扩大培养，得到人骨髓间充质干细胞-pBABE和-人前脑啡肽单克隆细胞。 2.4 细胞的生物学特性经过转染后人骨髓间充质干细胞-pBABE和-人前脑啡肽细胞系为多边形或者梭形，旋涡状分布。传代至15代，细胞的冻存和复苏对细胞的形态和增殖没有影响。锥虫蓝检查冻存复苏后的P4代人骨髓间充质干细胞、人骨髓间充质干细胞-pBABE和-人前脑啡肽细胞活细胞的比例分别为(91.45±4.78)%，(89.87±4.15)%，(90.65±3.21)%，组间差异无显著性意义(P > 0.05)。表明冻存和复苏不影响转染后细胞的活力。 2.5 人骨髓间充质干细胞的鉴定结果流式细胞仪检测显示：P4代人骨髓间充质干细胞、人骨髓间充质干细胞-pBABE和-人前脑啡肽细胞的CD29和CD44的表达均呈阳性，CD34和CD45的表达均呈阴性，且各组之间比较差异无显著性意义(P > 0.05；表1)。 2.6 转染前后各种细胞的成脂诱导和成骨诱导情况培养第3天可以看草细胞内出现脂滴，呈圆形或者椭圆形，油红染色后出现橙红色，P4代人骨髓间充质干细胞、人骨髓间充质干细胞-pBABE和人骨髓间充质干细胞-人前脑啡肽细胞之间脂肪细胞占总细胞的比例为(79.33± 4.43)%，(81.24±7.22)%，(80.87±4.23)%，差异无显著性意义(P > 0.05)。成骨诱导分化后，茜素红染色可见红色钙沉积，对照组未见红色钙沉积(图2)。表明3种细胞在体外均可诱导分化为脂肪细胞和成骨细胞。 2.7 人骨髓间充质干细胞中人前脑啡肽基因的表达PCR结果显示，P4代人骨髓间充质干细胞、人骨髓间充质干细胞-pBABE和人骨髓间充质干细胞-人前脑啡肽细胞均有Homo-GAPDH mRNA表达，3种细胞均可反转录出特异性161 bp基因条带(图3)，其中人前脑啡肽基因在人骨髓间充质干细胞-pBABE细胞中高表达，在P4代人骨髓间充质干细胞中低表达。 2.8 人骨髓间充质干细胞-人前脑啡肽细胞系脑啡肽表达免疫荧光染色结果显示：转染重组质粒pBABE-人前脑啡肽的人骨髓间充质干细胞中脑啡肽蛋白表达比较明显，而空载体组人骨髓间充质干细胞中脑啡肽蛋白呈低表达(图4)。"

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