Human telomerase reverse transcriptase gene-transfected effects on biological characteristics of Schwann cells

doi:10.3969/j.issn.2095-4344.2015.14.020

Abstract

Abstract:

BACKGROUND: Studies have shown that genetically modified Schwann cells can survive for a longer time in vivo, and promote nerve regeneration and functional recovery.

OBJECTIVE: To transfect human telomerase reverse transcriptase (hTERT) gene into rat Schwann cells cultured in vitro via PLXSN vector, and to detect the telomerase activity and biological characteristics of Schwann cells.

METHODS: Schwann cells from Wistar rats were cultured in vitro and transfected by PLXSN vector with (hTERT group) or without hTERT (empty vector group). Normal Schwann cells were selected as control group. RT-PCR and western blot methods were used to detect the hTERT protein and mRNA levels in Schwann cells, and flow cytometry was used to measure the cell cycle distribution. Cell growth was observed by cell growth curve and MTT colorimetric method.

RESULTS AND CONCLUSION: At 48 hours after transfection, the mRNA and protein expressions of hTERT were remarkably seen in Schwann cells. Compared with the control and empty vector groups, the cells grew faster, the number of cells at G₀/G₁ phase was reduced, but the number of S phase cells was increased in the hTERT group (P < 0.05). These findings indicate that PLXSN vector-mediated hTERT transfection of Schwann cells can significantly improve the activity of telomerase in Schwann cells as well as promote the proliferation of Schwann cells cultured in vitro.

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

全文链接：

Key words: Telomerase, Schwann Cells, Genes, Transfection

CLC Number:

R394.2

Liu Lin-lin, Deng Wei-min. Human telomerase reverse transcriptase gene-transfected effects on biological characteristics of Schwann cells [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(14): 2250-2254.

Figures/Tables 3

References

[1] 孙晓宇.雪旺细胞对于周围神经再生的功能与作用[J].内蒙古民族大学学报:自然科学版,2010,25(1):105-107.
[2] 褚庭纲,黄金龙.雪旺细胞在周围神经损伤及人工神经研制中的进展[J].中国临床康复,2004,8(19):3872-3873.
[3] 杨宝林,刘德明.异种雪旺细胞移植在中枢神经系统损伤修复中的作用[J].江西医学院学报,2005,45(5):185-187.
[4] 陈礼明,朴永旭,毛伯镛,等.pSVPoMCAT微基因修饰雪旺氏细胞移植治疗实验性脊髓损伤的酶及免疫组化评价[J].中国临床康复, 2002,6(6):906-907．
[5] 李锦,谢超,裴雪涛.端粒酶在干细胞研究中的主要进展[J].生理科学进展,2004,35(2):149-151.
[6] Assouline JG, Bosch EP, Lim R. Purification of rat Schwann cells from cultures of peripheral nerve: an immunoselective method using surfaces coated with anti-immunoglobulin antibodies. Brain Res. 1983;277(2):389-392.
[7] 劳杰,顾玉东,熊良俭,等. 改良成年SD大鼠雪旺细胞培养的实验研究[J].中华手外科杂志,1999,15(2):106-110.
[8] 张军,许百男,周定标.乳鼠雪旺细胞的培养和纯化[J].解放医学杂志,2002,27(7):610- 611.
[9] Scarpini E, Kreider BQ, Lisak RP, et al. Establishment of Schwann cell cultures from adult rat peripheral nerves. Exp Neurol. 1988;102(2):167-176.
[10] 韩岩,汤朝武,王剑波,等.利用Geneticin纯化雪旺细胞的实验研究[J].中华显微外科杂志,1997,20(4):227-230.
[11] 楚玉荣,宫凌涛,楚智慧,等.端粒、端粒酶与细胞衰老及肿瘤的关系[J].医学综述,2004,10(5):269-271.
[12] Abdallah BM, Haack-Sørensen M, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation. Biochem Biophys Res Commun. 2005; 326(3):527-538.
[13] Kawano Y, Kobune M, Yamaguchi M, et al. Ex vivo expansion of human umbilical cord hematopoietic progenitor cells using a coculture system with human telomerase catalytic subunit (hTERT)-transfected human stromal cells. Blood. 2003;101(2): 532-540.
[14] Pittenger MF, Mosca JD, McIntosh KR. Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma. Curr Top Microbiol Immunol. 2000;251:3-11.
[15] 黄文,张成,陈松林,等. 脑源性神经营养因子体外诱导骨髓间充质干细胞分化为神经元样细胞[J].第一军医大学学报,2004, 24(8):854-858.
[16] 邢莹,秦洁,曹孟德,等.细胞因子诱导人脐血间充质干细胞分化过程中细胞巢蛋白、神经丝亚单位和端粒酶逆转录酶mRNA的表达[J].郑州大学学报:医学版,2005,40(2):250-253.
[17] Mihara K, Imai C, Coustan-Smith E, et al. Development and functional characterization of human bone marrow mesenchymal cells immortalized by enforced expression of telomerase. Br J Haematol. 2003;120(5):846-849.
[18] Kobune M, Kawano Y, Ito Y, et al. Telomerized human multipotent mesenchymal cells can differentiate into hematopoietic and cobblestone area-supporting cells.Exp Hematol. 2003;31(8):715-722.
[19] 房佰俊,史明霞,廖联明,等.重建端粒酶活性延长胎儿肌肉源间充质干细胞寿命并维持其成神经潜能[J].中国生物工程杂志,2004, 24(8):48-53.
[20] 刘云燕,裘秀春,张殿忠,等.hTERT反义寡核苷酸对脊索瘤细胞周期及增殖的影响[J].现代肿瘤医学, 2011,19(10):1922-1924.
[21] 张晓辉,张建宁,康春生,等.hTERT正、反义表达载体转染大鼠骨髓间充质干细胞的实验研究[J].中华神经医学杂志,2006, 5(3): 217-221.
[22] 张志宏,曾永秋,税青林,等. hTERT-siRNA表达载体的构建及对MCF-7细胞生长、端粒酶活性的抑制作用[J].解放军医学杂志, 2007,32(6):561-564.
[23] 张洁,廖亚平,吴灵芝,等. hTERT基因转染对人胚胎大脑皮质神经元生长的影响[J].解剖学研究,2008,30(4):251-256.
[24] 吴灵芝,李水彬,程刚卫,等.hTERT基因转染对人神经元活力的影响[J].赣南医学院学报,2014,34(2):170-173.
[25] 靳斌,王伟,刘泽阳,等.外源hTERT基因转染对老年大鼠供肝缺血再灌注损伤的防护作用[J].山东大学学报:医学版, 2013,51(8): 13-16.
[26] 李宏良,曾荣香,陈鹏,等.清毒片和hTERT反义核酸对HL-60细胞增殖及凋亡的协同作用[J].辽宁中医杂志,2010,7(3):395-398.
[27] 孙来保,李成荣,文建明,等.反义hTERT基因对白血病细胞端粒酶表达及活性的抑制作用[J].中国病理学杂志,2004,33(5): 454-457.
[28] 王开利,迟淑萍,孙杰,等.外源hTERT基因对人肝细胞增殖及CYP450 19A1表达的影响[J].军医进修学院学报, 2011,32(11): 1137-1138.
[29] Abdallah BM, Haack-Sørensen M, Burns JS, et al. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation. Biochem Biophys Res Commun. 2005; 326(3):527-538.
[30] Kang SK, Putnam L, Dufour J, et al. Expression of telomerase extends the lifespan and enhances osteogenic differentiation of adipose tissue-derived stromal cells. Stem Cells. 2004; 22(7): 1356-1372.