雷公藤甲素及其氨基葡萄糖结合物的表观溶解度、表观油水分配系数和解离常数的测定

目的 测定雷公藤甲素及其氨基糖结合物的表观溶解度、表观油水分配系数和解离常数(pKTa).方法 采用平衡溶解度法、摇瓶法测定雷公藤甲素及其氨基糖结合物的表观溶解度和油水分配系数;采用分光光度指示剂法测定雷公藤甲素-氨基糖结合物的解离常数.结果 雷公藤甲素的表观溶解度随pH的增加而增加,其表观油水分配系数随pH的增加而降低,但变化并不显著.雷公藤甲素-氨基葡萄糖结合物的表观油水分布系数随pH的增加而增加,在纯水中的表观溶解度为1.9325±0.1373 g,pKT =5.938 ±0.191.结论 雷公藤甲素是一个难溶性的亲脂性化合物,而雷公藤甲素-氨基葡萄糖结合物是一个弱碱性的亲水性化合物.     

Anti-tumour and immuno-modulation effects of triptolide-loaded polymeric micelles

Triptolide (TP) possesses both anti-tumour and immuno-suppressive activities. Its immuno-suppressive activity may be disadvantageous for the therapy of cancers. A novel polymeric micelle system containing TP (TP-PM) was constructed by the solvent evaporation method using methoxypolyethylene glycol–poly(d,l-lactic acid)-block copolymer as the carrier, and was characterised using photon correlation spectroscopy, transmission electron microscopy and high performance liquid chromatography. The anti-tumour and immuno-modulation effects of TP-PM were evaluated in sarcoma 180-bearing mice and A2780 cells. Results demonstrated that TP-PM had an average diameter of 78.9 nm, encapsulation efficiency of 66.7%, core–shell morphology and a long-term stability. TP-PM could significantly inhibit tumour growth via intravenous injections at the dose levels of 0.0375, 0.075 and 0.15 mg/kg, and their inhibition rates were 42.5%, 46.0% and 49.9%, respectively; they showed similar cytotoxicity against A2780 cells compared to that of TP. Simultaneously, TP-PM had no effect on the thymus index, spleen index, spleen lymphocyte proliferation and the TNF-α and IL-2 levels in serum as compared with TP. Therefore, TP encapsulated in polymeric micelles does not demonstrate immuno-suppressive activity but does not lose its anti-tumour effect. These results show that polymeric micelles are a promising carrier for cancer therapy using TP.     

雷公藤甲素通过诱导细胞自噬促进结肠癌CT26细胞死亡

目的探讨雷公藤甲素诱导结肠癌CT26细胞自噬与细胞死亡之间的关系。方法体外培养小鼠结肠癌CT26细胞,应用免疫荧光、荧光双标自噬腺病毒载体(Ad-mRFP-GFP-LC3)技术,在激光扫描共焦显微镜下观察LC3的表达轮廓及水平,并结合免疫印迹法对LC3蛋白、P62蛋白做半定量分析;进而用单溶液细胞增殖检测(MTS)试剂测定CT26细胞增殖抑制率;用Annexin V-FITC/PI双染、流式细胞术检测细胞凋亡/死亡率。结果雷公藤甲素能诱导CT26细胞出现明确的自噬流;激活细胞自噬可提高CT26细胞的晚期凋亡率。与雷公藤甲素组比较,雷帕霉素与其联用能显著增强CT26细胞死亡。结论自噬可能介导了雷公藤甲素诱导CT26细胞死亡的过程。     

雷公藤甲素对U251细胞自噬活性的影响及机制研究

目的 探讨雷公藤甲素对U251细胞自噬活性的影响及机制.方法 采用四甲基偶氮唑蓝(MTT)法检测雷公藤甲素对U251细胞的增殖抑制率、半数抑制浓度(IC50);免疫荧光化学法测定细胞中LC3-Ⅱ表达,Western blot法测定LC3-Ⅱ/LC3-Ⅰ、Beclin-1、磷酸化P70、磷酸化Erk、磷酸化AKT蛋白的表达.结果 雷公藤甲素作用于U251细胞后,细胞增殖受抑制,24 h的IC50为0.73 μmol/L,48 h的IC50为0.27μmol/L.免疫荧光法测定6、12、24 h后LC3-Ⅱ水平增高;Western blot法测定LC3-Ⅱ水平、LC3-Ⅱ/LC3-Ⅰ比值随时间增高;Beclin-1水平增高,磷酸化P70、磷酸化Erk表达降低、磷酸化AKT无明显变化.结论 雷公藤甲素可抑制U251细胞增殖并促进自噬,并可通过上调Beclin-1表达和抑制mTOR的活性来促进自噬.     

Enhancement of triptolide-loaded micelles on tumorigenicity inhibition of human ovarian cancer

Triptolide (TP), a diterpenoid obtained from Tripterygium wilfordii Hook.f, has shown its antitumor activities against a variety of cancers in vitro in recent years. Unfortunately, TP has a small margin between the therapeutic and toxic doses and shows serious toxicity which limits its uses in antitumor treatment. In our previous study, Triptolide-loaded micelles (TP micelles), a TP drug delivery system with a sustained release behavior, had been reported to decrease TP uptake in the liver to relieve its toxicity, and increase TP distribution in the ovary to enhance its effects. This work therefore aimed at evaluating the inhibitory ability of TP micelles in the proliferation, apoptosis, invasion, and migration, and compared with free TP in SKOV3 cells. Our results showed that TP micelles inhibited the proliferation of SKOV3 in a time- and dose-dependent manner, and exhibited enhanced inhibition following 48 and 72 h treatment compared to TP. Cell cycle analysis revealed that TP and TP micelles inhibited cell proliferation by blocking their progression from the G2/M phase to the S phase. Although TP induced a significant increase in cell apoptosis, TP micelles showed a superior effect following 48 and 72 h treatment. Induction of caspase-dependent way and inhibition of NF-κB activation were found to be involved in the mechanism of TP micelles-induced apoptosis. Furthermore, the wound healing assay and transwell assay showed that both TP and TP micelles could obviously inhibit SKOV3 cells migration and invasion. Overall, TP micelles exhibited enhanced therapeutic efficacy in ovarian cancer in vitro due to its prolonged release and redistribution compared with the free TP. TP micelles might lead to an increase in tumorigenicity inhibition and a decrease in resistance and incidence simultaneously, indicating that it offers a new strategy with promising characteristics for TP chemotherapy application for ovarian cancer.     

雷公藤内酯醇诱导人淋巴细胞凋亡的作用与细胞周期的关系

目的：通过观察雷公藤仙酯醇诱导淋巴细胞发生凋亡与细胞周期的关系。探讨该化合物导致细胞凋亡作用的机制。方法;以人外周血Ｔ细胞为研究对象,选择流式细胞分析术,ＤＮＡ凝胶电泳及ＤＮＡ片段测定等作为检测细胞凋亡的方法。结果;雷公藤内酯醇只能造成活化的Ｔ细胞发生细胞凋亡,且与雷公藤内酯醇的浓度正相关;雷公藤内酯醇诱导活化Ｔ细胞发生细胞凋亡的作用与细胞活化的程度密切相关;     

环孢素A及雷公藤内酯醇对CD4＋T细胞、角质形成细胞和HeLa细胞增殖的影响

目的比较环孢素A(CyA)和雷公藤提取物T0 对CD4 T细胞、角质形成细胞和HeLa细胞增殖的影响。方法采用人外周血单一核细胞(PBMC)、人表皮角质形成细胞和HeLa细胞培养 ,3H TdR掺入法测定药物对细胞DNA合成的影响。结果T0 和CyA都能抑制ConA诱导的人外周血T淋巴细胞增殖。T0 在体外可抑制生长于低钙无血清角质形成细胞培养基中的正常人角质形成细胞和HeLa细胞株的生长 ,而CyA在药理浓度下无抑制作用。结论T0 与CyA均能抑制T细胞增殖 ,但对上皮细胞的作用不同。     

白鹤冲剂对TNF-α刺激的血管内皮细胞与嗜中性粒细胞黏附的影响

目的:观察白鹤冲剂对人脐静脉内皮细胞(HUVEC)与人外周血嗜中性粒细胞黏附以及细胞间黏附分子-1(ICAM-1)和CD18表达及核转录因子NF-κB(NF-κB)的影响,探讨白鹤冲剂治疗血管炎的作用机制。方法:将细胞分为正常对照组、TNF-α组、TNF-α+白鹤冲剂大剂量组、TNF-α+白鹤冲剂小剂量组和TNF-α+雷公藤内酯醇组,共五组。分别进行细胞黏附实验;以细胞ELISA法观察HVVEC的ICAM-1表达;以免疫细胞化学间接法检测CD18表达;间接免疫荧光法观察白鹤冲剂对HUVEC中NF-κB活化。结果:白鹤冲剂能抑制肿瘤坏死因子-α(TNF-α)刺激的HUVEC中ICAM-1表达和人外周血嗜中性粒细胞CD18表达(P<0.05),同时呈剂量依赖性抑制人外周血嗜中性粒细胞与HUVEC的黏附。结论:通过影响HUVEC中NF-κB活化从而抑制ICAM-1表达和嗜中性粒细胞CD18的表达,最终抑制嗜中性粒细胞与HUVEC的黏附可能是白鹤冲剂治疗血管炎的作用机制。     

卵巢癌中雷公藤内脂醇的研究进展

卵巢癌是严重威胁女性生殖健康的肿瘤之一,死亡率居各类妇科肿瘤的首位。多药耐药是治疗后复发、转移甚至死亡的主要原因。雷公藤内酯醇（triptolide,TP）具有很强的抗肿瘤活性,其在卵巢癌治疗方面的作用也引起了众多学者的重视,尤其是在多药耐药卵巢癌治疗中的应用。本文从抑制卵巢癌细胞增殖、诱导癌细胞凋亡两大方面综述雷公藤内酯醇在卵巢癌治疗中的研究进展。TP的毒性反应、半衰期短等缺点限制了其临床应用,纳米给药技术的出现为TP用于卵巢癌治疗提供了更广阔的应用前景。     

雷公藤甲素对TM4细胞氧化应激及PI3K/AKT通路的影响

目的：探讨雷公藤甲素（triptolide ,TP）对TM4小鼠睾丸支持细胞氧化应激、凋亡的影响及相关分子信号调控机制。方法：不同浓度雷公藤甲素对TM4细胞染毒24 h;细胞增殖实验检测雷公藤甲素对TM4细胞增殖的抑制作用;通过2′,7′ 二氯荧光黄双乙酸盐（6-carboxy-2′,7′ dichlorofluorescein diacetate,DCFH-DA）探针检测TM4细胞内活性氧水平的变化;Annexin V/PI双染检测雷公藤甲素诱导TM4细胞凋亡变化; Western blot免疫印迹法分别检测凋亡标志蛋白cleaved PARP和PI3K/Akt通路相关蛋白表达情况。结果：细胞增殖实验提示雷公藤甲素对TM4细胞有显著抑制作用,呈浓度梯度上升［10 nmol/L：（73.77±20.95）%, 100 nmol/L：（51.60±10.43）%, 500 nmol/L：（44.34±5.78）%］;雷公藤甲素处理使细胞内活性氧水平上调（P<0.01）,呈浓度依赖性;给药组TM4细胞早期凋亡和晚期凋亡比例均上调［对照组：（3.84±1.50）%, 100 nmol/L：（13.04±2.03）%, 200 nmol/L：（16.24±1.34）%, 400 nmol/L：（18.76±3.45）%］,各给药组cleaved PARP表达上调（P<0.01）;给药组TM4细胞中Akt、p70S6K磷酸化水平上调（P<0.01）,mTOR磷酸化水平未见显著变化（P>0.05）。结论：在体外培养条件下,雷公藤甲素可抑制TM4睾丸支持细胞增殖,并诱导TM4细胞凋亡增加,呈浓度依赖性;雷公藤甲素同时可引起TM4细胞中氧化应激水平上调,这可能是其细胞毒性机制之一;雷公藤甲素可导致Akt和p70S6K的激活,磷酸化水平升高,提示PI3K/Akt信号通路可能参与雷公藤甲素诱导的TM4细胞氧化应激反应,激活PI3K/Akt信号通路可能是其分子调控机制之一。