Optimization of culture and secretion of parathyroid cells via increasing the parathyroid hormone level in cell supernatant

doi:10.3969/j.issn.2095-4344.1780

Abstract

Abstract:

BACKGROUND: Telomerase reverse transcriptase is an important enzyme that regulates cell differentiation and proliferation, and it has various biological activities. hTERT gene modification may improve the viability and proliferation of parathyroid cells, and can optimize the in vitro culture of parathyroid cells.
OBJECTIVE: To transfect rat parathyroid cells with retroviral PLXSN carrying hTERT gene, and to observe the biological characteristics and secretory function of transfected cells in order to optimize the culture of parathyroid cells.
METHODS: Twenty-eight male Wistar rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. The study protocol was approved by the Animal Experiment Ethics Committee of Tianjin Medical University with the approval No. 2017-0652. Under anesthesia, the parathyroid glands of rats were surgically removed and pathologically confirmed. The parathyroid cells were digested by collagenase II. The protein expression level of calcium-sensing receptor, parathyroid hormone and glial cells missing-2 were detected by western blot. The parathyroid cells were divided into parathyroid cell group, empty virus group and hTERT transfection group. The expression levels of hTERT gene and protein in parathyroid cells were detected by RT-PCR and western blot after 3 days of transfection, respectively. Cell growth was observed by cell growth curve and cell counting kit-8. The cell cycle distribution was measured by flow cytometry. Enzyme-linked immunosorbent assay was used to detect the level of parathyroid hormone in the cell supernatant.
RESULTS AND CONCLUSION: Western blot results showed that the parathyroid cells were positive for calcium-sensing receptor, parathyroid hormone and glial cells missing-2, indicating the cultured cells were parathyroid cells. After transfection of hTERT, hTERT gene and protein levels were significantly increased in the hTERT transfection group as compared with parathyroid cell group and empty virus group (P < 0.05). The cell growth rate in the hTERT transfection group noticeably increased (P < 0.05). The cell number in G₀/G₁ phase was significantly decreased and the number of cells in the S phase was increased (P < 0.05). The level of parathyroid hormone in the culture supernatant was significantly increased (P < 0.05). To conclude, transfection of the PLXSN carrying the hTERT gene can promote the proliferation of rat parathyroid cells in vitro and increase the level of parathyroid hormone (PTH) in the supernatant of parathyroid cells.

Key words: parathyroid cells, cell culture, telomerase reverse transcriptase, gene transfection, parathyroid hormone, hTERT gene

CLC Number:

R446
R318

Wang Hui . Optimization of culture and secretion of parathyroid cells via increasing the parathyroid hormone level in cell supernatant[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(25): 4025-4030.

Figures/Tables 6

References

[1]陈佳,霍景山.甲状旁腺功能减退症的非手术治疗进展[J].中华临床医师杂志(电子版),2014,8(19):81-85.

[2]全婷婷,王鸥,聂敏,等.甲状旁腺功能减退症发病机制的遗传学进展[J].中华骨质疏松和骨矿盐疾病杂志,2017,10(2):186-193.

[3]张凯凯,刘晋闽.甲状腺功能减退致骨质疏松症的机制及治疗进展[J].中国骨质疏松杂志,2017,23(6):837-840.

[4]刘洋.甲状腺癌全切术后甲状旁腺功能减退及血钙变化规律[D].吉林大学, 2015.

[5]Lourida I,ThompsoncoonJ,Dickens CM,etal. ParathyroidHormone, Cognitive Function and Dementia:A Systematic Review.Plos One. 2015;10(5):e0127574.

[6]Russo R,Ruospo M,Cozzolino M,et al.Effects of vitamin D on parathyroid hormone and clinical outcomes in peritoneal dialysis:a narrative review. J Nephrol. 2014 Oct; 27(5):483-494.

[7]Kakava K,Tournis S,Papadakis G,et al. Postsurgical Hypoparathyroidism: A Systematic Review. Vivo. 2016;30(3):171.

[8]徐德全,代文杰.甲状旁腺激素替代疗法治疗甲状旁腺功能减退研究进展[J].中国实用外科杂志,2014,34(5):459-461.

[9]孙海晨,刘爽,崔叶青,等.成人甲状旁腺细胞的培养方法及其分泌功能研究[J].中国普外基础与临床杂志,2014,21(9):1120-1123.

[10]赵越,王晓辉,刘爽,等.成人甲状旁腺细胞的培养和鉴定[J].中国普外基础与临床杂志,2017,24(1):32-36.

[11]赵越.甲状旁腺功能减退症的治疗现状[J].医学研究生学报, 2016, 29(1):104-108.

[12]Choi MJ,Cho KH,Lee S,et al.hTERT mediates norepinephrine- induced Slug expression and ovarian cancer aggressiveness. Oncogene.2015; 34(26):3402-3412.

[13]Guo W,Lu J,Dai M,etal.Transcriptionalcoactivator CBP upregulateshTERT expression and tumor growth and predicts poor prognosis in human lung cancers.Oncotarget. 2014;5(19): 9349-9361.

[14]Schneiderstock R,Emrich T,Peters B,etal.Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoidliposarcomas using Light Cycler real-time quantitative reverse transcriptase- polymerase chain reaction. Electrophoresis. 2015;22(6):1098-1101.

[15]Zhao Q,Zhai YX,LiuH Q,et al.MicroRNA-491-5p suppresses cervical cancer cell growth by targeting hTERT.Oncology Reports. 2015;34(2):979.

[16]于宏建,赵杨,赵世铭,等.hTERT基因沉默对乳腺癌细胞生物学特性及COX-2表达的影响[J].中国实验诊断学, 2016,20(11):1817-1821.

[17]秦玲.hTERT基因修饰神经干细胞移植修复大鼠脊髓损伤[J].医学研究杂志,2016,45(2):99-104.

[18]Mitsui T,Narumi S,Inokuchi M,et al.Comprehensive next-generation sequencing analyses of hypoparathyroidism: identification of novel GCM2 mutations. J Clin Endocrinol Metab. 2014 Nov;99(11): E2421-2428.

[19]阮莹,侯佳宁,刘仁斌,等.条件重编辑共培养技术体外培养人甲状旁腺细胞的研究[J].中华实验外科杂志,2018,35(9):1641-1643.

[20]李佳丽,赵瑞力.干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退中的研究进展[J].中华细胞与干细胞杂志(电子版), 2018,8(3): 168-171.

[21]陈宇嘉.补脾肾活血方对慢性肾衰鼠甲状旁腺细胞增生及凋亡影响[D].广州:广州中医药大学,2015:1-41.

[22]赵越,罗斌.水凝胶化成人甲状旁腺细胞形态和功能的观察[J].国际外科学杂志,2016,43(2):85-87,封3.

[23]Li SH,Wang DD,Xu YJ,et al.ExogenoushTERT gene transfected endothelial progenitor cells from bone marrow promoted angiogenesis in ischemic myocardium of rats.Int J ClinExp Med. 2015;8(8):14447-14453.

[24]Liu AQ,GeL Y,Lu XL,et al.Silencing of the hTERT Gene by shRNA Inhibits Colon Cancer SW480 Cell Growth In Vitro and In Vivo. Plos One.2014;9(9):e107019.

[25]刘志平,沙翔垠,王智崇,等.转染hTERT基因对人角膜内皮细胞增殖及功能维持的影响[J].广东医学,2016,37(7):965-969.

[26]尹博文.慢病毒介导转染hTERT基因建立永生化树鼩小肠上皮细胞系[D].北京协和医学院中国医学科学院,2017:1-71.

[27]莫霄.hTERT基因转染大鼠雪旺细胞的实验研究[D].大连:大连医科大学, 2013.

[28]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.

[29]Ling-Zhao MO,De-Quan LI,Yu X,et al.Effect of hTERT Gene Expression on Proliferation and Apoptosis of Cervical Cancer Caski Cells by Lentivirus-Mediated Small Interfering RNA.J Int Transl Med 2016;4(2):114-120.

[30]李佳丽,赵瑞力.干细胞向甲状旁腺细胞的分化及在甲状旁腺功能减退中的研究进展[J].中华细胞与干细胞杂志(电子版), 2018,8(3): 168-171.