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多肽Epithalon的合成及其对人胎肝细胞L-02端粒酶和端粒的作用研究

发布时间:2019-01-12 14:51  文章来源:笔耕文化传播
【摘要】: 目的:本课题以松果体分泌物,一种名为“Epithalamin”的缩氨酸为基础,人工合成的多肽“Epithalon”其结构为:Ala-Glu-Asp-Gly。研究其作用于人肝细胞L-02后对细胞的生长情况、细胞端粒长度以及细胞端粒酶活性所产生的影响。 方法:(1)采用SPPS固相多肽合成法线性合成制备Epithalon多肽,重复(缩合—洗涤—去保护—中和—洗涤)操作,达到所要合成的肽链长度;最后将肽链从树脂上裂解下来,经过高效液相法(简称HPLC纯化法)纯化处理,运用核磁技术检测H谱、C谱对其进行进一步分析鉴定。 (2)通过MTT法检测Epithalon多肽对人肝细胞株L-02增殖与活力的影响;用细胞形态学观察用药后细胞的生长情况。 (3)用药一段时间后,运用端粒酶重复序列扩增——焦磷酸根酶联发光技术(TRAP-ELIDA)检测人肝细胞株L-02端粒酶活性,流式荧光原位杂交法(FLOW-FISH)检测端粒长度。 结果:(1)核磁与质谱结合分析鉴定的结果是合成产物正是Epithalon多肽,只是还含少许杂质。 (2)MTT法检测Epithalon多肽对人肝细胞株L-02增殖与活力的影响显示:细胞在2.5umol/l至25umol/l这段随药物浓度增大而增大。对L-20细胞增殖的促进作用逐渐增加,且对浓度的限制性不大。 (3)运用端粒酶重复序列扩增——焦磷酸根酶联发光技术(TRAP-ELIDA)检测人肝细胞株L-02端粒酶活性,流式荧光原位杂交法(FLOW-FISH)检测端粒长度的变化,发现Epithalon多肽能有效的激活细胞端粒酶,虽然,L-20细胞是永生细胞,但端粒酶活性表达并不强,几乎没有,但加入Epithalon多肽后却有效提高了端粒酶的活性。端粒长度的检测结果显示,加入Epithalon多肽后细胞端粒的长度得到延长。 结论:研究显示Epithalon多肽具有激活端粒酶,延长端粒的作用。从目前主要的衰老理论之一的基因程控学说(端粒理论)角度来说,Epithalon多肽能以激活端粒酶活性延长端粒进而达到延长细胞寿命、生物体寿命。且由于端粒缩短的减缓,则可能更好的起到稳定染色体的作用,减少染色体因不稳定而引起的畸变等,从而减少染色体畸变导致的癌症。衰老、癌症是多因素积累的结果,但从预防角度上,Epithalon多肽也可能会是一种可行的较安全的预防治疗性药物。
[Abstract]:Objective: based on the pineal secretion, a peptide named "Epithalamin", the synthetic polypeptide "Epithalon" is composed of Ala-Glu-Asp-Gly.. The effects of L-02 on cell growth, telomere length and telomerase activity were studied. Methods: (1) the normal synthesis of SPPS peptide was used to prepare Epithalon peptide. The peptide chain length was obtained by repeated operation (condensation-washing-deprotection-neutralization-washing). Finally, the peptide chain was dissociated from the resin and purified by high performance liquid phase (HPLC) method. The H spectrum was detected by nuclear magnetic technique, and the C spectrum was further analyzed and identified. (2) the effect of Epithalon polypeptide on the proliferation and activity of L-02 cell line was detected by MTT, and the growth of L-02 cells was observed by cell morphology. (3) telomerase activity was detected by telomerase repeat amplification (TRAP-ELIDA) and telomere length by flow in situ hybridization (FLOW-FISH). Results: (1) the results of nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis showed that the synthetic product was Epithalon polypeptide with a few impurities. (2) the effect of Epithalon polypeptide on the proliferation and activity of L-02 cell line was detected by MTT assay. The results showed that the cell increased from 2.5umol/l to 25umol/l with the increase of drug concentration. The proliferation of L-20 cells increased gradually, and the concentration was not limited. (3) telomerase activity was detected by telomerase repeat amplification (TRAP-ELIDA) and telomere length was detected by flow in situ hybridization (FLOW-FISH). It was found that Epithalon peptide could activate telomerase effectively. Although L-20 cells were immortalized cells, the expression of telomerase activity was not strong, almost none, but the activity of telomerase was increased after adding Epithalon peptide. The results of telomere length test showed that the length of telomere was prolonged with the addition of Epithalon peptide. Conclusion: Epithalon peptide can activate telomerase and prolong telomere. From the point of view of gene programming theory (telomere theory) which is one of the main aging theories at present, Epithalon peptide can prolong telomere activity by activating telomerase activity and then prolong cell life and organism life. As telomere shortening slows down, it may play a better role in stabilizing chromosomes, reducing chromosomal aberrations caused by instability, and thus reducing cancer caused by chromosomal aberrations. Aging and cancer are the result of multifactorial accumulation, but from a preventive point of view, Epithalon peptide may also be a feasible and safe preventive therapeutic drug.
【学位授予单位】:重庆大学
【学位级别】:硕士
【学位授予年份】:2007
【分类号】:R341

【参考文献】

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