【作者】 张庚；

【导师】 单保恩； 刘运江；

【作者基本信息】 河北医科大学 ， 免疫学， 2014， 博士

【摘要】 肿瘤的发病率和死亡率呈逐年上升的趋势，严重危胁人类健康。目前，药物抗肿瘤治疗疗效仍不理想，主要原因在于传统的抗肿瘤药物在杀伤患者体内肿瘤细胞的同时，其巨大的毒副作用不可避免。因此，寻找新型高效且低毒的抗肿瘤药物是生物医药领域的重大课题。天然产物化学研究一直是制药业新药研制的重要途径和主要来源，天然产物成分具有结构新颖、疗效高、毒副作用小等优点，所以目前天然产物化学及其研究成果已广泛应用于医药业。Sansalvamide A是1999年被报道的从海洋真菌中分离出的一种天然环五肽酯，具有很高的亲脂性及显著的抗肿瘤能力。Sansalvamide A环肽是由Sansalvamide A改造所得到的一类化合物。经过多年的研究，新报道的Sansalvamide A环肽具有200余种，其中很多衍生物的活性明显提高。在本课题中，由河北科技大学药用分子化学重点实验室提供了9种新合成Sansalvamide A环肽衍生物，选取小鼠恶性黑色素瘤B16细胞，人乳腺癌MCF-7细胞和人乳腺癌MDA-MB-231细胞三种细胞模型做体外抗肿瘤活性研究，观察这9种新化合物的抗肿瘤活性。同时，应用细胞培养技术原代培养人外周血淋巴细胞和SD大鼠主动脉血管平滑肌细胞，用于研究9种新化合物对这两种正常细胞模型生长的抑制作用。通过抗肿瘤活性筛选，选择活性较好的化合物5（H-10）和化合物9（H-15）为研究重点，用小鼠恶性黑色素瘤B16细胞为模型，对化合物H-10和化合物H-15抑制肿瘤细胞增殖的机制做进一步研究。具体研究内容和结果如下：第一部分Sansalvamide A环肽衍生物抗肿瘤活性及对正常细胞毒性的研究目的：9种新型SansalvamideA环肽衍生物抗肿瘤活性筛选及其对正常细胞毒性的研究。方法：应用细胞培养技术培养小鼠恶性黑色素瘤B16细胞，人乳腺癌MCF-7细胞和人乳腺癌MDA-MB-231细胞，做为肿瘤细胞模型用于9种新型SansalvamideA环肽衍生物抗肿瘤活性筛选。原代培养人外周血淋巴细胞和SD大鼠主动脉血管平滑肌细胞，用于研究9种新化合物对这两种正常细胞模型生长的抑制作用。磺酰罗丹明B（SRB）比色分析法分析结果。到置相差显微镜观察化合物作用不同时间后B16细胞，淋巴细胞和大鼠平滑肌细胞（VSMC）形态，数量及生长状态的变化情况。结果：19种Sansalvamide A环肽衍生物对小鼠恶性黑色素瘤B16细胞，人乳腺癌MCF-7细胞和人乳腺癌MDA-MB-231细胞的增殖具有抑制作用，用SRB比色法测定空白组以及1%DMSO组48h后吸光度（OD）值，通过比较发现1%DMSO（溶解化合物溶剂的最大量）对B16，MCF-7和MDA-MB-231三种细胞的增殖无影响，P>0.05。除化合物6，8和9在50μM对MDA-MB-231增殖无抑制作用外，9种化合物对这三种细胞都显示出了抗肿瘤活性。对于B16细胞，化合物2，5和6抑制率最强，分别为82.43%，81.79%和83.19%；对于MCF-7细胞，抑制率最强的三种化合物分别为化合物5，64.19%，化合物9，51.08%和化合物4，50.89%；对于MDA-MB-231细胞，化合物4和5作用最强，抑制率分别为68.04%和66.56%。29种化合物对人外周血淋巴细胞和SD大鼠主动脉血管平滑肌细胞的增殖无明显抑制作用。3化合物H-10可使B16细胞出现凋亡和分化的形态学改变；化合物H-15可使B16细胞出现明显的分化形态改变。结论：9种SansalvamideA环肽衍生物具有抗肿瘤活性且对正常细胞毒性较小；化合物H-10对多种肿瘤细胞具有活性，考虑具有诱导B16细胞凋亡的作用；化合物H-15可能通过诱导B16细胞分化抑制其增殖。第二部分新型环五肽H-10抑制B16细胞增殖机制的研究目的：研究化合物H-10抑制B16细胞增殖的机制。方法：磺酰罗丹明B比色分析法研究化合物H-10抑制B16细胞增殖的浓度依赖性，细胞计数法绘制生长曲线研究化合物H-10抑制B16增殖的时间依赖性。流式细胞学技术分析B16细胞经化合物H-10作用后凋亡细胞DNA含量。Western Blot检测凋亡相关蛋白caspase-3，caspase-8和caspase-9表达的情况。结果：1化合物H-10抑制B16细胞生长具有浓度依赖性：1%DMSO组与空白组相比，增殖率无明显变化，P>0.05。经不同浓度化合物H-10（0.1、1、10、50和100μM）处理48h后，B16细胞增殖率逐渐降低。与对照组相比，100μM组P<0.01，50μM组P<0.01，10μM组P<0.05，其增值率均明显降低。同时在光学显微镜下可以明显观察到B16经化合物H-10作用后呈现的凋亡形态。经公式计算得到化合物H-10作用B16细胞48h的IC50（半抑制浓度）为39.68μM。2化合物H-10抑制B16细胞生长具有时间依赖性：经浓度为50μM的化合物H-10作用B16细胞24h、48h、72h、96h、120h和144h后计数细胞数，与对照组相比，细胞生长受到明显抑制。3化合物H-10具有诱导B16细胞凋亡的作用：流式细胞学结果显示，经浓度为50μM的化合物H-10作用B16细胞24h后，出现明显DNA亚二倍体峰，凋亡细胞比例为(9.21±4.62)%，与对照组凋亡比例(0.96±0.22)%相比，P<0.05，结果具有统计学意义。Western Blot结果显示，经10、30和50μM化合物H-10作用后，caspase-3随药物浓度升高其表达量上升。4化合物H-10诱导B16细胞凋亡通过线粒体途径：经10、30和50μM化合物H-10作用后，通过线粒体途径诱导细胞凋亡的caspase-9随药物浓度升高其表达量上升，而与外源性凋亡相关的caspase-8没有明显变化。结论：化合物H-10对B16细胞的增殖具有很强的抑制作用，且作用效应具有显著的浓度依赖性和时间依赖性。化合物H-10作用B16细胞48h的IC50为39.68μM。化合物H-10具有诱导B16细胞凋亡的作用，化合物H-10诱导B16细胞凋亡通过线粒体途径。第三部分新型环五肽H-15抑制B16细胞增殖机制的研究目的：研究化合物H-15抑制B16细胞增殖的机制。方法：磺酰罗丹明B比色分析法研究化合物H-15抑制B16细胞增殖的浓度依赖性，细胞计数法绘制生长曲线研究化合物H-15抑制B16增殖的时间依赖性。检测B16细胞经化合物H-15作用后黑色素分泌量的变化。RT-PCR检测B16细胞经化合物H-15作用后干细胞基因Foxd3，Klf4，Sox2和Nanog的表达。Western Blot检测酪氨酸酶，Sox2和Foxd3表达的情况。结果：1化合物H-15抑制B16细胞生长具有浓度依赖性：1%DMSO组与空白组相比，增殖率无明显变化，P>0.05。经不同浓度化合物H-15（0.1、1、10、50和100μM）处理48h后，B16细胞增殖率逐渐降低。化合物H-15作用B16细胞与对照组相比，50μM组和10μM组P<0.01，其增值率均明显降低。经公式计算得到化合物H-15作用B16细胞48h的IC50为69.44μM。2化合物H-15抑制B16细胞生长具有时间依赖性：经浓度为50μM的化合物H-15作用B16细胞24h、48h、72h、96h、120h和144h后计数细胞数，与对照组相比，细胞生长受到明显抑制。350μM化合物H-15作用B16细胞48h后细胞黑色素含量较对照组增高：经酶标仪测定50μM化合物H-15作用B16细胞48h后吸光度值为0.1743±0.0227，对照组吸光度值为0.0788±0.0039，两组间比较P<0.01，差异具有统计学意义，说明经化合物H-15作用后的B16细胞分泌黑色素含量明显增加。450μM化合物H-15作用B16细胞48h后酪氨酸酶（TYR）的表达增加：经浓度为50μM的化合物H-15作用B16细胞48后，酪氨酸酶（TYR）的表达较空白组增高。5化合物H-15具有抑制Sox2, Foxd3基因表达的作用：RT-PCR结果显示，经10、30和50μM化合物H-15作用24h后，Sox2和Foxd3的表达呈下降趋势，Klf4的表达没有明显变化，Nanog的表达没有规律性。Western-blot结果显示，经10、30和50μM化合物H-15作用24h后，Sox2和Foxd3的表达呈下降趋势。说明化合物H-15具有抑制B16细胞干细胞特性的作用。结论：化合物H-15对B16细胞的增殖具抑制作用，且作用效应具有显著的浓度依赖性和时间依赖性。化合物H-15作用B16细胞48h的IC50为69.44μM。化合物H-15具有诱导B16细胞分化的作用，经化合物H-15作用过的B16细胞分泌黑色素的能力增强。化合物H-15抑制了B16细胞Sox2和Foxd3的表达，使其肿瘤干细胞的特性受到抑制。更多还原

【Abstract】 As there is an upward trend in the incidence and mortality, tumorbecomes a serious threat to human health. At present, the efficacy ofchemotherapy is not ideal. The main reason is the side effects of traditionalantitumor drugs in the clinical treatment. Therefore, looking for new highefficiency and low toxicity of antitumor drugs is a major subject in the field ofbiomedicine. Natural product chemistry has been the important way indevelopment of new pharmaceutical industry. Natural products have theadvantages of novel structure, high curative effect and side effects, so thenatural products chemistry and its research results have been widely used inpharmaceutical.Sansalvamide A is a cyclic depsipeptide produced by a marine fungus,and it is first reported in1999. It is a natural part of five peptides has the veryhigh lipotropy and significant anti-tumor ability. Sansalvamide A cyclicpeptides are compounds by the transformation of Sansalvamide A. After yearsof research, more than200new derivatives of Sansalvamide A cyclic peptideshave been reported. The activity of any derivatives has been improvedsignificantly. In this study,9new synthetic Sansalvamide A cyclic peptidederivatives were provided by the State Key Laboratory Breeding Base-HebeiProvince Key Laboratory of Molecular Chemistry for Drug (Hebei Universityof Science and Technology). Malignant melanoma B16cells in mice, humanbreast cancer MCF-7cells and human breast cancer MDA-MB-231cells wereselected to observe the antitumor activity of these new compounds. At thesame time, peripheral blood mononuclear cells and SD rat aortic vascularsmooth muscle cells were prepared by primitive culture for experiment. B16cell was selected as the tumor model and the compound H-10and H-15werein further research on the mechanism of inhibition of cell proliferation.The research contents and results are as follows: Part I The study of antitumor activity and the toxicity of normal cell onSansalvamide A cyclic peptide derivativesObjective: to observe the activity and toxicity of the nine new cyclicpeptide derivatives of Sansalvamide A.Methods: malignant melanoma B16cells in mice, human breast cancerMCF-7cells and human breast cancer MDA-MB-231cells were cultured astumor cell models for antitumor activity screening. Peripheral bloodmononuclear cells and SD rat aortic vascular smooth muscle cells wereprepared by primitive culture for observing the toxicity of these compounds.The results were measured by sulforhodamine B (SRB) colorimetric assay.The changes and the growth states of B16cells, lymphocytes, and smoothmuscle cells (VSMC) of rats were observed by inverted phase contrastmicroscope.Results:1Nine Sansalvamide A cyclic peptide derivatives have inhibiting effecton malignant melanoma B16cells in mice, human breast cancer MCF7cellsand human breast cancer MDA-MB-231cells. After treated48h, blank groupand1%DMSO group didn’t reach statistical significance, P>0.05. Except thecompound6,8and9in50μM for MDA-MB-231, all compounds showedantitumor activity on the three kinds of tumor cells. For B16cells, compound2,5, and6have the strongest inhibition rate,82.43%,81.79%and83.19%, forMCF-7cell, the strongest inhibition rate of three kinds of compounds werecompound5,64.19%, compound9,51.08%and compound4,50.89%, forMDA-MB-231, strongest were compound4and5, and the inhibition rate were68.04%and66.56%.29compounds on human peripheral blood mononuclear cells and SD rataortic vascular smooth muscle cell proliferation did have inhibitory effect.3Compound H-10have the function to make B16cells apoptosis anddifferentiation of morphology change, compound H-15can make B16cellsappear obvious differentiation changes. Conclusion:9Sansalvamide A cyclic peptide derivatives have antitumoractivity and less toxicity to normal cells, Compound H-10have a wide varietyof tumor cells with activity, and have the function to induce apoptosis of B16cells, compound H-15could induce differentiation of B16cells and inhibit theproliferation.Part II The mechanism of H-10inhibit proliferation of B16cellsObjective: to study the mechanism of H-10on inhibiting proliferation ofB16cells.Methods: sulforhodamine B (SRB) colorimetric method was used tocalculate Percentage Growth (PG) of different concentration of H-10treatedB16cells for48h and cell counting method to draw growth curve study H-10inhibit the proliferation of B16dependencies. Flow cytometric analysis todetect the apoptotic cell death. The expression of caspase-8, caspase-9andcaspase-3were detected by western-blot.Results:1H-10has concentration-dependent effect on B16cell growth:Compared with blank group, the proliferation rate of1%DMSO group has nosignificant change, P>0.05. After the treatment of different concentrations ofH-10(0.1,1,10,50and100μM) for48h, proliferation rate of B16cell weregradually decreased. Compared with control group, the proliferation rate of100μM,50μM and10μM were significantly decreased (100μM P<0.01,50μMP<0.01and10μM P<0.05). The morphological changes could be seen throughlight microscopy.2H-10has time-dependent effect on B-16cell growth: Time-dependenteffect of H-10on cell proliferation was measured by cell number. After treatedby50μM H-10for24h,48h,72h,96h,120h and144h, cell numbers werecounted and compared with control group. H-10caused time-dependentinhibition of B-16cell lines.3H-10induced apoptosis of B16cells: Flow cytometric analysis of cellsamples has demonstrated the ability of H-10induce apoptosis. Apoptoticcells were defined as those with subdiploid DNA content and presented as the percentage of all counted cells per sample. The proportion of apoptotic cells ofuntreated B-16cells was (0.96±0.22)%. Treated with50uM H-10for24h, thepercentage of apoptotic cells was (9.21±4.62)%and the difference wasstatistically significant. Caspase-3plays a key role in cell apoptosis and is theimportant molecular in apoptosis initiation. In this study, the expression ofcaspase-3was determined by Western Blot and this further confirmed thatH-10has the function of inducing apoptosis. After treated by10,25and50μMH-10, the expression of caspase-3increased with the concentration.4H-10induced apoptosis of B16cells via mitochondrial pathway: Theresults of western blot showed ascendant trend of the expression of caspase-3and capsase-9. And the expression of caspase-8didn’t change significantlyamong the control and the test group. These results suggest H-10inducedapoptosis of B16cells via mitochondrial pathway.Conclusion: H-10has strong inhibition on the proliferation of B16cells,and the effect has significant concentration dependence and time dependence.The IC50of H-10on B16cells in48hours is39.68μM. H-10has the functionof induce apoptosis of B16cells, and H-10induces B16cells apoptosis bymitochondrial pathway.Part III The mechanism of H-15inhibit proliferation of B16cellsObjective: to study the mechanism of H-15on inhibiting proliferation ofB16cells.Methods: sulforhodamine B (SRB) colorimetric method was used tocalculate Percentage Growth (PG) of different concentration of H-15treatedB16cells for48h and cell counting method to draw growth curve study H-15inhibit the proliferation of B16dependencies. Detect melanin production ofB16cells after treated by H-15. The expression of stem cell gene Foxd3, Klf4,Sox2and Nanog were detected by RT-PCR. The expression of TYR, Sox2and Foxd3were detected by Western Blot.Results:1H-15has concentration-dependent effect on B16cell growth:Compared with blank group, the proliferation rate of1%DMSO group has nosignificant change, P>0.05. After the treatment of different concentrations of H-15(0.1,1,10,50and100μM) for48h, proliferation rate of B16cell weregradually decreased. Compared with control group, the proliferation rate of50uM and10uM were significantly decreased, P<0.05. The morphologicalchanges could be seen through light microscopy. The IC50of H-15on B16cells in48hours is69.44μM.2H-15has time-dependent effect on B-16cell growth: Time-dependenteffect of H-15on cell proliferation was measured by cell number. After treatedby50μM H-15for24h,48h,72h,96h,120h and144h, cell numbers werecounted and compared with control group. H-15caused time-dependentinhibition of B16cell lines.3The melanin content is higher than control after treated by50μM H-15for48hours: the OD value of B16cells after treated by50μM H-15for48hours was0.1743±0.0227compared with the control0.0788±0.0039, therewas statistically significant between the two groups, P<0.01.4The expression of tyrosine (TYR) enzyme was increased after treatedby50μM H-15for48hours: detected by Western Blot, the expression of TYRwas increased.5H-15has the function of inhibit the expression of Sox2and Foxd3:according to the results of RT-PCR, after treated by H-15for24h, theexpression of Sox2and Foxd3had a downward trend, Klf4expression was notsignificantly change, and Nanog expression was not regularity. It suggestedthat H-15has the function of inhibit the stem cell properties of B16cells.Conclusion: H-15has strong inhibition on the proliferation of B16cells,and the effect has significant concentration dependence and time dependence.The IC50of H-15on B16cells in48hours is69.44μM. H-15may have thefunction of the induced differentiation of B16cells, and the ability of secretionmelanin in B16cells treated by H-15was stronger than control. H-15couldinhibit the expression of Sox2and Foxd3, and inhibit the stem cell propertiesof B16cells.更多还原