【摘要】： 目的:本課題以松果體分泌物,一種名為“Epithalamin”的縮氨酸為基礎,人工合成的多肽“Epithalon”其結構為:Ala-Glu-Asp-Gly。研究其作用于人肝細胞L-02后對細胞的生長情況、細胞端粒長度以及細胞端粒酶活性所產(chǎn)生的影響。 方法:(1)采用SPPS固相多肽合成法線性合成制備Epithalon多肽,重復(縮合—洗滌—去保護—中和—洗滌)操作,達到所要合成的肽鏈長度;最后將肽鏈從樹脂上裂解下來,經(jīng)過高效液相法(簡稱HPLC純化法)純化處理,運用核磁技術檢測H譜、C譜對其進行進一步分析鑒定。 (2)通過MTT法檢測Epithalon多肽對人肝細胞株L-02增殖與活力的影響;用細胞形態(tài)學觀察用藥后細胞的生長情況。 (3)用藥一段時間后,運用端粒酶重復序列擴增——焦磷酸根酶聯(lián)發(fā)光技術(TRAP-ELIDA)檢測人肝細胞株L-02端粒酶活性,流式熒光原位雜交法(FLOW-FISH)檢測端粒長度。 結果:(1)核磁與質(zhì)譜結合分析鑒定的結果是合成產(chǎn)物正是Epithalon多肽,只是還含少許雜質(zhì)。 (2)MTT法檢測Epithalon多肽對人肝細胞株L-02增殖與活力的影響顯示:細胞在2.5umol/l至25umol/l這段隨藥物濃度增大而增大。對L-20細胞增殖的促進作用逐漸增加,且對濃度的限制性不大。 (3)運用端粒酶重復序列擴增——焦磷酸根酶聯(lián)發(fā)光技術(TRAP-ELIDA)檢測人肝細胞株L-02端粒酶活性,流式熒光原位雜交法(FLOW-FISH)檢測端粒長度的變化,發(fā)現(xiàn)Epithalon多肽能有效的激活細胞端粒酶,雖然,L-20細胞是永生細胞,但端粒酶活性表達并不強,幾乎沒有,但加入Epithalon多肽后卻有效提高了端粒酶的活性。端粒長度的檢測結果顯示,加入Epithalon多肽后細胞端粒的長度得到延長。 結論:研究顯示Epithalon多肽具有激活端粒酶,延長端粒的作用。從目前主要的衰老理論之一的基因程控學說(端粒理論)角度來說,Epithalon多肽能以激活端粒酶活性延長端粒進而達到延長細胞壽命、生物體壽命。且由于端�？s短的減緩,則可能更好的起到穩(wěn)定染色體的作用,減少染色體因不穩(wěn)定而引起的畸變等,從而減少染色體畸變導致的癌癥。衰老、癌癥是多因素積累的結果,但從預防角度上,Epithalon多肽也可能會是一種可行的較安全的預防治療性藥物。
[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.
【學位授予單位】：重慶大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R341

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