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目的探讨以重建细胞端粒酶活性,达到延长经ptsA58H质粒转化的人胚肌腱细胞系(THETC)寿命的可行性。方法构建含人端粒酶逆转录酶(hTERT)cDNA的质粒pGRN145,体外转染THETC;通过形态学观察、平板克隆形成率检测、软琼脂培养、不同培养条件下细胞生长曲线的测定,免疫组化法染色分析胶原分泌类型,hTERTmRNA表达及端粒重复序列扩增-PCR法检测细胞端粒酶活性等,对转染前后的细胞生物学特性进行比较。结果经pGRN145质粒体外转染的THETC(telT)有端粒酶活性表达,并继续保持旺盛的增殖能力,寿命延长。telT保持原有的温度依赖性及血清营养依赖性生长的特点,并正常分泌I型胶原,无恶性转化趋向。结论重建端粒酶活性可以延长THETC寿命,为建立组织工程标准细胞系提供了新的实验手段。
Objective To explore the feasibility of reconstructing telomerase activity and prolonging the lifespan of human embryonic tendon cell line (THETC) transformed with plasmid ptsA58H. Methods The plasmid pGRN145 containing human telomerase reverse transcriptase (hTERT) cDNA was constructed and transfected into THETC cells in vitro. Morphological observation, plate colony formation assay, soft agar culture, cell growth curve under different culture conditions, The method of histochemical staining for collagen secretion, hTERT mRNA expression and telomere repeat amplification-PCR assay of cell telomerase activity were used to compare the cell biological characteristics before and after transfection. RESULTS: THETC (telT) transfected with plasmid pGRN145 in vitro showed telomerase activity and maintained its robust proliferative capacity with prolonged life expectancy. TelT maintain the original temperature dependence and serum-dependent growth characteristics, and normal secretion of type I collagen, no tendency to malignant transformation. Conclusion The reconstruction of telomerase activity can prolong the life of THETC and provide a new experimental method for the establishment of tissue engineering standard cell line.