多肽Epithalon的合成及其對(duì)人胎肝細(xì)胞L-02端粒酶和端粒的作用研究
發(fā)布時(shí)間：2019-01-12 14:51
【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(chǔ),人工合成的多肽“Epithalon”其結(jié)構(gòu)為:Ala-Glu-Asp-Gly。研究其作用于人肝細(xì)胞L-02后對(duì)細(xì)胞的生長(zhǎng)情況、細(xì)胞端粒長(zhǎng)度以及細(xì)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(fù)(縮合—洗滌—去保護(hù)—中和—洗滌)操作,達(dá)到所要合成的肽鏈長(zhǎng)度;最后將肽鏈從樹脂上裂解下來(lái),經(jīng)過(guò)高效液相法(簡(jiǎn)稱HPLC純化法)純化處理,運(yùn)用核磁技術(shù)檢測(cè)H譜、C譜對(duì)其進(jìn)行進(jìn)一步分析鑒定。 (2)通過(guò)MTT法檢測(cè)Epithalon多肽對(duì)人肝細(xì)胞株L-02增殖與活力的影響;用細(xì)胞形態(tài)學(xué)觀察用藥后細(xì)胞的生長(zhǎng)情況。 (3)用藥一段時(shí)間后,運(yùn)用端粒酶重復(fù)序列擴(kuò)增——焦磷酸根酶聯(lián)發(fā)光技術(shù)(TRAP-ELIDA)檢測(cè)人肝細(xì)胞株L-02端粒酶活性,流式熒光原位雜交法(FLOW-FISH)檢測(cè)端粒長(zhǎng)度。 結(jié)果:(1)核磁與質(zhì)譜結(jié)合分析鑒定的結(jié)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(cè)Epithalon多肽對(duì)人肝細(xì)胞株L-02增殖與活力的影響顯示:細(xì)胞在2.5umol/l至25umol/l這段隨藥物濃度增大而增大。對(duì)L-20細(xì)胞增殖的促進(jìn)作用逐漸增加,且對(duì)濃度的限制性不大。 (3)運(yùn)用端粒酶重復(fù)序列擴(kuò)增——焦磷酸根酶聯(lián)發(fā)光技術(shù)(TRAP-ELIDA)檢測(cè)人肝細(xì)胞株L-02端粒酶活性,流式熒光原位雜交法(FLOW-FISH)檢測(cè)端粒長(zhǎng)度的變化,發(fā)現(xiàn)Epithalon多肽能有效的激活細(xì)胞端粒酶,雖然,L-20細(xì)胞是永生細(xì)胞,但端粒酶活性表達(dá)并不強(qiáng),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(zhǎng)度的檢測(cè)結(jié)果顯示,加入Epithalon多肽后細(xì)胞端粒的長(zhǎng)度得到延長(zhǎng)。 結(jié)論:研究顯示Epithalon多肽具有激活端粒酶,延長(zhǎng)端粒的作用。從目前主要的衰老理論之一的基因程控學(xué)說(shuō)(端粒理論)角度來(lái)說(shuō),Epithalon多肽能以激活端粒酶活性延長(zhǎng)端粒進(jìn)而達(dá)到延長(zhǎng)細(xì)胞壽命、生物體壽命。且由于端粒縮短的減緩,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(dǎo)致的癌癥。衰老、癌癥是多因素積累的結(jié)果,但從預(yù)防角度上,Epithalon多肽也可能會(huì)是一種可行的較安全的預(yù)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(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R341
本文編號(hào)：2407900
[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(xué)位授予單位】：重慶大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R341
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 彭喜平;褪黑素的生物學(xué)功能研究進(jìn)展[J];青海大學(xué)學(xué)報(bào)(自然科學(xué)版);2004年02期
2 劉瓊,王紅,胡德聰,丁朝建,肖恒,徐輝碧,舒柏華,徐順清;亞硒酸鈉對(duì)肝細(xì)胞L-02端粒酶活性和端粒長(zhǎng)度的作用[J];生物化學(xué)與生物物理學(xué)報(bào);2003年12期
3 胡建,覃文新,萬(wàn)大方,顧健人;端粒及端粒酶研究的最新進(jìn)展[J];生命科學(xué);2001年03期
4 梁錚錚,胡劍;端粒、端粒酶結(jié)構(gòu)功能研究進(jìn)展[J];生物技術(shù)通訊;2003年04期
5 趙朝暉,陳曉春,朱元貴,黃春,師廣斌,曾育琦,李永坤,彭小松,金建生,彭旭;人參皂苷Rg1延緩細(xì)胞衰老過(guò)程中端粒長(zhǎng)度和端粒酶活性的變化[J];中國(guó)藥理學(xué)通報(bào);2005年01期
6 崔巍,陳健萌;流式原位雜交法檢測(cè)白血病和骨髓異常增生綜合征患者端粒長(zhǎng)度的研究[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2002年01期
，本文編號(hào)：2407900
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2407900.html
最近更新
教材專著
