【作者】 张纯；

【导师】 陈燕；

【作者基本信息】 华中科技大学 ， 血液病学， 2006， 博士

【摘要】 第一部分雷公藤内酯醇抗淋巴瘤细胞增殖效应的实验研究【目的】研究雷公藤内酯醇在体外抗淋巴瘤细胞增殖的效应。【方法】选择Burkitt’s淋巴瘤细胞系Raji细胞,与浓度为0、6.25 nmol/L、12.5 nmol/L、25 nmol/L、50 nmol/L、100 nmol/L的雷公藤内酯醇分别培养0h、24 h、36 h、48 h、60 h和72 h。采用MTT法检测对Raji细胞增殖的作用。【结果】雷公藤内酯醇以时间和剂量依赖方式抑制Raji细胞的增殖,加药组与未加药的对照组相比,OD值明显减少。24 h、36 h、48 h、60 h和72h的半数抑制浓度IC50值分别为43.06 nmol/L、25.08 nmol/L、7.32 nmol/L、2.66nmol/L和0.58 nmol/L。【结论】雷公藤内酯醇具有抗淋巴瘤细胞体外增殖的效应。第二部分雷公藤内酯醇影响非霍奇金淋巴瘤SDF-1/CXCR4生物学轴效应的研究【目的】研究雷公藤内酯醇对非霍奇金淋巴瘤(NHL)SDF-1/CXCR4生物学轴的作用。【方法】采用RT-PCR方法检测雷公藤内酯醇对NHL患者淋巴结基质细胞SDF-1α表达的影响;采用流式细胞术检测雷公藤内酯醇对NHL淋巴结瘤细胞CXCR4表达的作用;采用Transwell微孔隔离室迁移实验观察雷公藤内酯醇对NHL淋巴结瘤细胞体外迁移的影响。【结果】①雷公藤内酯醇能抑制NHL淋巴结基质细胞SDF-1α的表达,其中较低剂量组(12.5nmol/L)与对照组相比有一定的降低,但差异无显著性(1.15±0.33 vs 1.22±0.30 P>0.05),而较高剂量组(25、50nmol/L)则可明显抑制SDF-1α的表达(0.88±0.35;0.74±0.32,P<0.01),并具有量效关系;②NHL淋巴结瘤细胞经不同浓度雷公藤内酯醇(12.5、25、50nmol/L)处理后,CXCR4表达率与对照组(17.37%±7.21%)相比,逐渐降至(11.13%±5.66%,P<0.05)、(8.30%±4.61%,P<0.01)和(3.31%±2.35%,P<0.01),此抑制效应呈剂量依赖性;③雷公藤内酯醇能抑制重组人SDF-1α对NHL淋巴结瘤细胞的趋化作用,不同浓度雷公藤内酯醇(12.5、25、50nmol/L)处理后细胞迁移率由对照组(75.4%±15.8%)降至(59.4%±17.3%)、(30.3%±13.8%)和(21.4%±9.9%);此外雷公藤内酯醇还能抑制NHL淋巴结基质细胞对瘤细胞的体外迁移,细胞迁移率由(61.1%±15.5%)降至(53.4%±17.3%)、(20.8%±7.4%)、(15.4%±6.3%),雷公藤内酯醇对趋化作用的抑制也具有剂量依赖性。【结论】雷公藤内酯醇能抑制NHL淋巴结基质细胞SDF-1和瘤细胞表面CXCR4受体的表达,阻断NHL淋巴结瘤细胞向重组人SDF-1或接种的淋巴结基质细胞的迁移,因此雷公藤内酯醇能阻断NHL淋巴结瘤细胞通过淋巴结的转移,具有抗NHL肿瘤侵袭的作用,其机制与雷公藤内酯醇对NHL SDF-1/CXCR4生物学轴的抑制效应有关。第三部分雷公藤内酯醇对Raji细胞黏着斑激酶表达的影响【目的】研究雷公藤内酯醇对Raji细胞黏着斑激酶表达的影响。【方法】选择Burkitt’s淋巴瘤细胞系Raji细胞,用浓度为25nmol/L的雷公藤内酯醇分别培养4 h和24 h。采用Western Blot、免疫沉淀的测定方法研究雷公藤内酯醇对Burkitt淋巴瘤细胞系Raji细胞粘着斑激酶蛋白表达和活力的影响。【结果】①雷公藤内酯醇分别作用于Raji细胞4h和24h,加入雷公藤内酯醇4h后,FAK的蛋白质含量变化不明显;而作用24h后黏着斑激酶蛋白的含量较对照组明显降低(P<0.05);②雷公藤内酯醇作用Raji细胞4h和24h,4h及24h作用后黏着斑激酶的活力较对照组均明显降低(4h P<0.05,24h P<0.01)。【结论】雷公藤内酯醇可降低Burkitt淋巴瘤细胞系Raji细胞粘着斑激酶的含量和活力,提示雷公藤内酯醇抗肿瘤机制可能与抑制粘着斑激酶介导细胞信号转导有关。更多还原

【Abstract】 Part I Antiproliferative effects of triptolide on Burldtt’slymphoma cell line Raji cells in vitro[Objiective] To investigate the antiproliferative effect of triptolide on Burkitt’slymphoma cell line Raji cells in vitro.[Method] Raji cells were grown in RPMI-1640 culture medium supplementedwith 10% fetal calf serum（FCS） and 100 u/ml penicillin and 100 u/ml streptomycinat 37°C in a 5% CO₂ incubator. Raji cells were treated with different concentrationsof triptolide (（0、 6.25 nmol/L、 12.5 nmol/L、 25 nmol/L、 50 nmol/L、 100nmol/L）for 0, 24 h, 36 h, 48 h, 60 h, and 72 h. The effects of triptolide on the growth of Rajicells were studied by 3-（4,5-Dimethyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazolium（MTT） assay.[Results] Triptolide inhibited the proliferation of Raji cells in a dose- andtime-dependent way with a 24-h , 36-h , 48-h, 60-h, and 72-h IC₅₀ value of 43.06 ,25.08 nmol/L, 7.32 nmol/L, 2.66 nmol/L and 0.58 nmol/L.[Conclusion] Triptolide could inhibit the proliferation of Burkitt’s lymphoma cellline Raji cells in vitro. Part II Inhibitary effect of triptolide on lymph node metastasis in patients with non-Hodgkin lymphoma by regulating SDF-1/CXCR4axis in vitro[Objective] To investigate the effect of triptolide on lymph node metastasis in patients with non-Hodgkin’s lymphoma （NHL） in vitro, and to explore the underlying mechanism regulating SDF-1/CXCR4 axis.[Methods] The effects of triptolide on SDF-1 mRNA expression in lymph node stromal cells from patients with NHL were determined by reverse transcriptase-polymerase chain reaction （RT-PCR）. The effects of triptolide on CXCR4 expression on lymphoma cells freshly isolated from lymph node of these patients were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of primary lymphoma cells towards recombinant human SDF-1α （rhSDF-1α） or cultured lymph node stromal cells in vitro.[Results] The expression of SDF-1α mRNA in lymph node stromal cells obtained from patients with NHL was decreased treated by triptolide at concentrations of 25 and 50 nmol/L for 24 h. Flow cytometry analysis showed that the CXCR4 expression on primary lymphoma cells were downregulated gradually in a dose-dependent manner following triptolide treatment. Chemotaxis assays revealed that the migration of freshly isolated lymphoma cells towards either rhSDF-1 or cultured lymph node stromal cells was markedly inhibited by the addition of triptolide in vitro, and the inhibition was dose-dependent.[Conclusions] Triptolide was able to inhibit the migration of lymphoma cells via lymph node in vitro. The potential antitumor mechanisms of triptolide are related to the blockage of SDF-1/CXCR4 biological axis. Part III Effects of triptolide on the expression and activity of focal adhesion kinase in Burkitt’s lymphoma cell line Raji cells[Objective] To observe the effects of triptolide on the expression and activity offocal adhesion kinase in Burkitt’s lymphoma cell line Raji cells.[Methods] Raji cells were grown in RPMI-1640 culture mediumsupplemented with 10% fetal calf serum and 100 u/ml penicillin and100 u/ml streptomycin at a concentration of 25 nmol/L for 4 h and 24 h.Western blot and immunoprecipitation-tyrosine kinase assay were usedto study the expression and activity of focal adhesion kinase inBurkitt’s lymphoma cell line Raji cells.[Results] The expression of focal adhesion kinase on Burkitt’slymphoma cell line Raji cells was decreased treated by 25 nmol/Ltriptolide for 24h （P<0.05） as compared with the control; moreover, there wasa decrease in the activity of the focal adhesion kinase on cultured Rajicells after treated with 25 nmol/L triptolide for 4 h and 24 h （4hP<0.05,24hP<0.01） .[Conclusion] The expression and activity of focal adhesion kinaseon cultured Raji cells were regulated by triptolide, and the regulationmight be important in the tumor-pathogenesis.更多还原