【作者】 高丽；

【导师】 周然；

【作者基本信息】 湖北中医药大学 ， 方剂学， 2011， 博士

【摘要】 雷公藤有大毒,对消化、生殖、泌尿、心血管、骨髓及血液等系统有明确的病理损害。自1991年有文献报道雷公藤对骨髓及血液系统的毒副作用以来,有关雷公藤致骨髓及血液系统毒性的实验研究较少,其机制研究也未见报道。本课题选择体外培养的大鼠股骨骨髓细胞为研究对象,观察雷公藤醇提物对骨髓细胞增殖和凋亡的影响,以丰富对雷公藤骨髓及血液系统毒性的认识。同时,采用血清药理学方法,观察中药复方含药血清对雷公藤醇提物作用后骨髓细胞的保护作用,以期从中筛选出能有效对抗雷公藤骨髓及血液系统毒性的最佳方剂,并对其机制进行初步探讨。鉴于雷公藤药材尚未收入国家药品标准,本课题首先对本实验中所用的雷公藤药材进行质量分析研究。同时,为了进一步了解雷公藤的毒性作用,我们对雷公藤进行了急性毒性试验,测定LD50,以初步判断可能的主要毒性靶器官。第一部分雷公藤主要毒性成分分析及其急性毒性考察方法：选择雷公藤总二萜内酯和雷公藤甲素作为检测指标,分别采用比色法和HPLC法,从有效成分总量和单一有效成分两个水平,对本课题所用雷公藤进行质量分析研究。雷公藤醇提物口服灌胃一次给药,连续观察14d,记录小鼠毒性反应情况。结果：本课题所用雷公藤中总二萜内酯的含量为0.0839‰～0.0859‰,雷公藤甲素的含量为0.0507‰～0.0525‰。雷公藤醇提物的LD5o为5.6 g/kg,其95%的可信区间为4.8-6.4 g/kg。第二部分雷公藤醇提物对骨髓细胞增殖和凋亡的影响方法：体外培养大鼠股骨骨髓细胞,经不同浓度的雷公藤醇提物作用后,采用MTT比色法检测对骨髓细胞增殖的影响；采用流式细胞仪PI单标法检测对骨髓细胞周期的影响；采用流式细胞仪Annexin V-FITC/PI双标法检测对骨髓细胞凋亡的影响；采用紫外-可见分光光度法检测对骨髓细胞caspase3、caspase8和caspase9酶活性的影响；采用ELISA法检测对骨髓细胞培养液上清中GM-CSF、EPO、TPO三种细胞因子的影响。结果：雷公藤醇提物可明显抑制正常骨髓细胞增殖,并且此抑制作用呈明显的时间和剂量依赖性,在作用24,48,72 h后,IC50分别为8.742,5.106,4.684μg/mL。细胞周期进程的结果显示,与阴性对照组比较,雷公藤组Go/G1期细胞比例显著增加,S期和G2/M期细胞比例显著减少,细胞增殖指数显著降低,表明雷公藤醇提物阻滞骨髓细胞于Go/G,期。骨髓细胞凋亡的结果显示,与阴性对照组比较,雷公藤醇提物可引起骨髓细胞产生明显的凋亡,且随着雷公藤浓度的增加,早期凋亡细胞(Annexin V+/PI-)占细胞总数的比例也不断增加,说明雷公藤醇提物具有诱导骨髓细胞凋亡的作用。对骨髓细胞caspase3、caspase8和caspase9酶活性的影响结果显示,与阴性对照组比较,雷公藤醇提物组caspase3和caspase9的活性显著升高(P<0.01),caspase8的活性无显著性差异,说明雷公藤醇提物可能通过线粒体途径诱导骨髓细胞凋亡。对骨髓细胞培养液上清中GM-CSF、EPO、TPO三种细胞因子的影响结果显示,与阴性对照组比较,雷公藤醇提物组GM-CSF、EPO、TPO含量显著降低(P<0.01)。第三部分中药复方含药血清对雷公藤醇提物致骨髓细胞凋亡的保护作用方法：体外培养大鼠股骨骨髓细胞,经雷公藤醇提物(8μg/mL)作用后,加入5%、10%、15%、20%的六味地黄丸、金匮肾气丸和归脾丸含药血清,采用MTT比色法检测对骨髓细胞增殖的影响。对实验中显示促增殖作用明显的金匮肾气丸进行进一步研究,采用流式细胞仪PI单标法检测对骨髓细胞周期的影响；采用流式细胞仪Annexin V-FITC/PI双标法检测对骨髓细胞凋亡率的影响；采用紫外-可见分光光度法检测对骨髓细胞caspase3、caspase8和caspase9酶活性的影响；采用ELISA法检测对骨髓细胞培养液上清中GM-CSF、EPO、TPO三种细胞因子的影响。结果：对骨髓细胞存活率的影响结果显示,与模型对照组比较,六味地黄丸、金匮肾气丸、归脾丸含药血清均能显著升高骨髓细胞的存活率,以金匮肾气丸含药血清的效果最佳。对细胞周期进程影响的结果显示,当血清浓度为5%、10%、15%、20%时,与模型对照组比较,金匮肾气丸含药血清能显著升高骨髓细胞增殖指数(P<0.05或P<0.01),说明一定浓度的金匮肾气丸含药血清可促使骨髓细胞解除Go/G,期阻滞,由Go/G,期进入S期以及由S期向G2/M期转化,从而有利于骨髓细胞的增殖。对骨髓细胞凋亡影响的结果显示,与模型对照组比较,当血清浓度为10%、15%、20%时,金匮肾气丸组凋亡指数显著降低(P<0.05或P<0.01),说明金匮肾气丸含药血清对雷公藤醇提物所致的骨髓细胞凋亡有保护作用。对骨髓细胞caspase3、caspase8和caspase9酶活性的影响结果显示,与模型对照组比较,当血清浓度为15%、20%时,金匮肾气丸组caspase3、caspase9活性显著降低(P<0.05或P<0.01),caspase8活性未见明显改变,说明金匮肾气丸含药血清可能通过抑制线粒体途径而减少骨髓细胞凋亡。对骨髓细胞培养液上清中GM-CSF、EPO、TPO三种细胞因子的影响结果显示,与模型对照组比较,金匮肾气丸组GM-CSF、EPO、TPO的含量显著升高(P<0.05或P<0.01)。结论：1.本课题所用雷公藤药材中总二萜内酯的含量为0.0839‰～0.0859‰,雷公藤甲素的含量为0.0507%o-0.0525%o。2.雷公藤醇提物的LD50为5.6 g/kg,95%的可信区间为4.8～6.4 g/kg,肝脏为其急性毒性作用的主要靶器官之一3.雷公藤醇提物可明显抑制正常骨髓细胞增殖,具有时间和浓度依赖性,同时,使骨髓细胞阻滞于Go/G1期,并能诱导细胞凋亡,其诱导凋亡的作用可能是通过激活线粒体途径实现的；雷公藤醇提物对骨髓细胞的抑制作用还可能与骨髓造血微环境中细胞因子的含量下降有关。4.中药复方含药血清能显著增加雷公藤醇提物作用后骨髓细胞的存活率,金匮肾气丸、六味地黄丸、归脾丸三个复方中,金匮肾气丸效果最佳,六味地黄丸其次,再次是归脾丸。5.金匮肾气丸含药血清能诱导骨髓细胞由相对静止期进入增殖活跃期,并减少细胞凋亡,其机制可能是通过抑制caspase3和caspase9的表达而减少骨髓细胞凋亡,或是通过促进骨髓造血微环境中细胞因子的分泌而促进造血,从而发挥“补肾填精生血”的功效。更多还原

【Abstract】 Tripterygium wilfordii Hook. F. (TWH) is toxic to digestive system, reproductive system, urinary system, cardiovascular system, haematopoietic system, which relatively more incidents of human poisoning have been reported in recent years. Experiment on toxicity to haematopoietic system of TWH was few, and the mechanism is not very clear. So BMCs in vitro were chosen as research object, and effect and mechanism of TWH on cell growth and apoptosis were observed. And effect and mechanism of serum containing Chinese medical formulas on BMCs co-cultured with ethanol extraction of TWH were observed, using serum pharmacological test. Thinking of there was no national standard for TWH, contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were tested. And in order to study the toxicity, LD50 was tested.Part One Study on Contents of Tripterygium Total Lactones and Triptolide in TWH and Acute Toxicity of Ethanol Extraction of TWHMethods:A colorimetric method was applied for the assay of Tripterygium total lactones, and HPLC applied for triptolide. According to the results of preliminary experiments, mice were divided into six groups, administered ethanol extraction of TWH once by oral gavage. Toxic reactions were observed in continuous 14 days. Results:The contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were 0.0839%o-0.0859%o and 0.0507%o-0.0525%o in the experiment. The LD50 (95%confidence limit) of ethanol extraction of TWH in mice was 5.6 g/kg(4.8-6.4 g/kg).Part Two Study on Effects of Ethanol Extraction of TWH on Cell Growth and Apoptosis in Bone Marrow CellsMethods:BMCs were co-cultured in vitro with different concentrations of ethanol extraction of TWH. MTT assay was used to evaluate the growth inhibition of BMCs. The cell cycle and apoptosis rate were detertmined by FCM with PI staining and AnnexinⅤ/PI staining. The activities of caspase3, caspase8 and caspase9 were detected by spectrophotometry. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were measured by ELISA. Results:After applied with different concentrations of ethanol extraction of TWH, viability of BMCs were significantly reduced in a time-and dose-dependent manner. The IC50 were 8.742μg/mL for 24 h,5.106μg/mL for 48 h and 4.684μg/mL for 72 h. Cell cycle flow cytometry demonstrated that BMCs treated with different concentrations of ethanol extraction of TWH resulted in G1 phase arrest. Apoptosis rate were increased in BMCs treated with different concentrations of ethanol extraction of TWH tested by FCM with AnnexinⅤ/PI staining. The activities of caspase3 and caspase9, but not caspase8, were up-regulated in the experimental group compared with the control group (P<0.01), indicating ethanol extraction of TWH may induce apoptosis of BMCs through mitochondrion pathway. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were decreased in the experimental group compared with the control group (P<0.01).Part Three Study on Protective Effects of Serum Containing Chinese Medical Formulas on BMCs Co-cultured with Ethanol Extraction of TWHMethods:BMCs were co-cultured in vitro with ethanol extraction of TWH and different concentrations of serum containing LWDH, JGSQ and GP. MTT assay was used to evaluate the growth proliferation of BMCs. The cell cycle and apoptosis rate were detertmined by FCM with PI staining and AnnexinⅤ/PI staining. The activities of caspase3, caspase8 and caspase9 were detected by spectrophotometry. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were measured by ELISA. Results:After applied with different concentrations of serum containing LWDH, JGSQ and GP, viability of BMCs were significantly increased. The best was serum containing JGSQ. Cell cycle flow cytometry demonstrated that BMCs treated with different concentrations of serum containing JGSQ resulted in BMCs of Go/G1 phase decreased and BMCs of S and G2/M phase increased. Apoptosis rate were decreased in BMCs treated with different concentrations of serum containing JGSQ. The activities of caspase3 and caspase9, but not caspase8, were decreased in JGSQ group, indicating serum containing JGSQ might inhibit apoptosis of BMCs through mitochondrion pathway. The contents of GM-CSF, EPO and TPO in supernatant of BMCs were increased in LWDH group.Conclusion:1. The contents of Tripterygium total lactones and triptolide in ethanol extraction of TWH were 0.0839‰-0.0859‰and 0.0507‰-0.0525‰in the experiment.2. The LD50 (95%confidence limit) of ethanol extraction of TWH in mice was 5.6 g/kg(4.8-6.4 g/kg). Hepatotoxic effect of ethanol extraction of TWH is a major cause of mobility in mice.3. After applied with different concentrations of ethanol extraction of TWH, viability of BMCs were significantly reduced in a time-and dose-dependent manner, resulted in G1 phase arrest. Ethanol extraction of TWH could induce the apoptosis of BMCs. It is probably mediated by activating mitochondrion pathway. Decrease of contents of cytokines in bone marrow micro-enviroment might be relevant to inhibition.4. After applied with different concentrations of serum containing LWDH, JGSQ and GP, viability of BMCs were significantly increased. The best one was serum containing JGSQ, the second serum containing LWDH, the third serum containing GP.5. Serum containing JGSQ could significantly increase viability of BMCs, promoted BMCs of G0/G1 phase entering proliferation cycle. It might inhibit apoptosis of BMCs through mitochondrion pathway. It also could increase the contents of cytokines in bone marrow micro-enviroment.更多还原