酮基类胡萝卜素的检测、代谢研究及对HaCaT细胞紫外线损伤的作用

【作者】 胡雅馨；

【导师】 惠伯棣；

【作者基本信息】 首都师范大学 ， 细胞生物学， 2008， 硕士

【摘要】 目的研究在装备了二极管阵列检测器（PDA）的高压液相色谱（HPLC）上,应用C18、C30柱子分离与鉴定酮基类胡萝卜素的方法;分析北京市场上的5种食用虾中酮基类胡萝卜素的含量与组成;探索酮基类胡萝卜素在大鼠体内器官以及血清中的积累情况;研究酮基类胡萝卜素对人角质形成细胞（HaCaT）增殖的影响;研究酮基类胡萝卜素在长波紫外线（UVA）和中波紫外线（UVB）照射后HaCaT细胞损伤中的防治作用。方法C18色谱柱为DiamonsilTM,流动相为酸性乙腈-水、乙酸乙酯,线性梯度洗脱,流速1.0 ml.min^-1,检测波长480nm,波长范围280-580nm,进样量20μl;C30色谱柱为Waters YMC Carotenoid S-5,流动相酸性乙腈-水、甲基叔丁基醚,线性梯度洗脱,流速1.0ml.min^-1,检测波长480nm,波长范围250nm-600nm,进样量20μl。酮基类胡萝卜素灌胃雄性SD大鼠后收集其血清、脏器及粪便,萃取其中的酮基类胡萝卜素组分,C30-HPLC-PDA对萃取物进行分离并测其几何异构体的组成。分别用一定剂量的UVA和UVB照射HaCaT细胞,立即加入含有酮基类胡萝卜素的培养基,继续培养24小时后检测细胞上清液中乳酸脱氢酶水平,通过MTT法检测细胞增殖情况,并与单照射UVA或UVB的对照组及未照UV也未加酮基类胡萝卜素的空白组进行比较。结果应用C18-HPLC-PDA和C30-HPLC-PDA酸性流动相中,酮基类胡萝卜素得到了良好的分离,可以利用此法鉴定酮基类胡萝卜素。虾青素单体在大鼠血清中及脾脏中均有积累。斑蝥黄质在大鼠体内的消化、吸收、血液运输和肝脏代谢中,未发现顺式异构体比例的上升。灌胃2小时后大鼠血清中斑蝥黄质的积累达到最高水平。酮基类胡萝卜素能显著提高HaCaT细胞的增殖率,能降低UV辐照后细胞上清液中的乳酸脱氢酶水平。结论通过比较色谱行为和光谱特征,酮基类胡萝卜素类化合物可被鉴定,这使C18-HPLC-PDA定量研究亦成为可能。应用C30-HPLC-PDA对酮基类胡萝卜素几何异构体进行检测具有可行性。酮基类胡萝卜素对紫外照射引起的HaCaT细胞的损伤具有保护作用。更多还原

【Abstract】 ObjectiveThe separation of keto-carotenoid by HPLC equipped with PDA on both C18 and C30 columns; the assessment of five species of shrimps and lobsters from the local market of Beijing for astaxanthin amount and composition;the investigation into accumulation in different organs and the configuration variation of keto-carotenoid molecule in the metabolism of SD rats;the influence of keto-carotenoid on activity of LDH and proliferation after irradiated by ultraviolet A and ultraviolet B in human HaCaT cells.MethodsBy HPLC equipped with PDA keto-carotenoid was readily separated on a C18 column under following conditions:a Diamonsil^TM column;mobile phase A=acidic acetonitrile-water;mobile phase B=ethyl acetate;linear gradient;monitoring wavelength=480nm;the wavelength range of PDA=280-580nm;sample injection volume=20μl.The conditions of C30-HPLC-PDA was a Waters YMC Carotenoid S-5 column;mobile phase A=acidic acetonitrile-water;mobile phase B=MTBE;linear gradient;monitoring wavelength=480nm;the wavelength range of PDA=250-600nm;sample injection volume=20μl.Keto-carotenoid fraction was extracted from serum,organs and dung after fed into SD rat and the geometrical isomer composition of keto-carotenoid was assessed by C30-HPLC-PDA.Human HaCaT cell irradiated by UVB/UVA was treated with keto-carotenoid and cultured for 24 hours.The LDH level was detected and the cell proliferation was observed by MTT essay.ResultBy HPLC-PDA,keto-carotenoid was readily separated on both C18 and C30 columns acidic acetonitrile mobile phase.This result is going to form the possibility of their quantization on C30-HPLC-PDA.Astaxanthin monomers accumulated in spleen and serum of SD rat.The geometrical isomer composition of canthaxanthin was assessed by C30-HPLC-PDA.No significant increase in the Z-isomer ratio of canthaxanthin in vivo was observed during its absorption,transportation and metabolism in liver.Keto-carotenoid increases the proliferation of HaCaT cells and relieves the LDH level of supernatant after UVB/UVA irradiation.ConclusionAccording to the comparison of its chromatographic behavior and spectral characters with those of reference samples,keto-carotenoid was identified.Its quantification was therefore able to be considered by C18-HPLC-PDA.It was very possible to assess the geometrical isomer composition of keto-carotenoid by C30-HPLC-PDA.The present results indicate that keto-carotenoid relieve damage of HaCaT cells irradiated by UVA or UVB.更多还原