【作者】 李琰；

【导师】 张金巧； 宋晓宁；

【作者基本信息】 河北医科大学 ， 内科学（专业学位）， 2014， 硕士

【摘要】 目的：多发性骨髓瘤（Multiple Myeloma，MM）是以骨髓中浆细胞恶性克隆性增生为主要特征的血液系统肿瘤，化疗为主要治疗手段。硼替佐米（Bortezomib，BTZ）作为MM患者一线治疗中的基础药物，其疗效已经得到广泛认可。然而，仍有近30%的患者对硼替佐米治疗无效，37~44%的患者在治疗过程中因出现严重不良反应被迫停药。因此，迫切需要高效能、低毒性或能够增强硼替佐米抗骨髓瘤作用的化学药物出现。去甲斑蝥素（Norcantharidine，NCTD）是斑蝥素（一种传统抗肿瘤中药）的去甲基衍生物，不仅保留了斑蝥素原有的抗肿瘤活性，同时使其对肾脏的毒性刺激作用大大降低，得到了许多研究者的认可。我们的前期工作已经证实，NCTD在体外可通过调节NF-κB通路及下游信号分子的表达来诱导U266细胞凋亡，并起到MM细胞对硼替佐米的化疗增敏作用。本实验通过建立人骨髓瘤动物模型，进一步验证NCTD在体内的抗骨髓瘤效应及增强硼替佐米抗骨髓瘤效应的机制，为临床应用NCTD治疗或辅助治疗MM提供更确切的理论依据。方法：1NCTD急性毒性试验：50只健康昆明小鼠随机分成5组。首先通过进行预试验找出NCTD的最大及最小致死剂量，分别为250mg/kg及102.4mg/kg，之后各组剂量用0.9%氯化钠以1:0.8比例依次稀释，按0.2ml/10g体重分别给各组小鼠一次性腹腔注射给药。观察并记录不同NCTD给药剂量下各组小鼠的死亡分布情况，按改良寇氏法计算NCTD的半数致死剂量LD50。2建立MM移植瘤裸鼠模型：32只BALB/C裸鼠，每只肩胛部皮下注射2×106U266细胞，待瘤体体积生长到约100mm3左右，随机分成4组。分别腹腔注射下列药物：（1）对照组：生理盐水100μL/10g，3次/周，连续4周；（2）NCTD组：以接近或略低于1/5半数致死量的NCTD（20mg/kg），3次/周，连续4周；（3）BTZ组：BTZ0.25mg/kg，1次/周，连续4周；（4）联合用药组：NCTD，20mg/kg，3次/周+BTZ，0.25mg/kg，1次/周，连续4周。给药后每周2次对裸鼠瘤体体积进行跟踪观测。于末次给药4小时后以颈椎脱位法将裸鼠处死，剥离移植瘤标本，进行免疫组化及其他后续实验。3病理检查及凋亡、免疫组化检测：移植瘤标本以石蜡包埋并切片至5μm厚，HE染色后光镜下观察移植瘤组织细胞结构，并分别应用原位末端标记（TUNEL）技术及免疫组化方法检测移植瘤组织的细胞凋亡率及PCNA、NF-κB P65的表达量。结果：1NCTD半数致死剂量LD50：根据急性毒性试验结果，各组小鼠死亡率之和Σp=1.0+0.9+0.7+0.6+0.2=3.4，相邻两组剂量对数值之差i=lg250/200=lg1.25=0.1，最大剂量对数值Xm=lg250=2.4，代入寇氏法公式LD50=log-1[Xm–i (Σp–0.5)]=128mg/kg。2各组裸鼠瘤体生长情况：各组小鼠移植瘤体积在用药前无差异(P>0.05)。给药后观察4周，对照组小鼠瘤体生长迅速，肿瘤表面血管丰富，部分出现中心坏死，NCTD及BTZ组肿瘤体积明显小于对照组（P＜0.05），两组的抑瘤率分别为31.83%、36.32%，而以两药联合组的瘤体最小，抑瘤率达64.49%，与BTZ单药组对比，存在显著性差异（P＜0.05）。3各组裸鼠一般状况及生化指标比较：整个治疗过程中，各组小鼠均没有明显的惊恐、不安或拒食等现象，而由于瘤体较大，对照组小鼠出现活动受限。4周后对小鼠的生化指标进行检测，各组小鼠均未出现明显的肾功能损害，血肌酐平均水平均＜0.5mg/dL，比较各组谷丙转氨酶水平，P>0.05，无显著性差异。4TUNEL检测各组移植瘤组织细胞凋亡：各治疗组均可见明显的凋亡细胞，其中以两药联合组最为显著，凋亡细胞核多呈固缩状态、经DAB显色后为深棕色，而对照组中的凋亡细胞并不明显；高倍镜下计数，BTZ、NCTD组的细胞凋亡率分别为29.91%、42.06%，而对照组仅为14.95%，存在显著性差异（P <0.05）；联合用药组的细胞凋亡率为81.80%，与BTZ单药组比较，有统计学意义（P <0.05）。5细胞增殖核抗原PCNA在各组移植瘤组织中的表达情况：作为评价细胞增殖状态的一项常用指标，其阳性表达位于细胞核，棕黄色为阳性反应细胞。本试验中免疫组化结果显示，对照组骨髓瘤细胞增殖极度活跃，其PCNA的阳性表达率高达（89.91±13.59）%，BTZ组与NCTD组的阳性表达率则分别为（71.85±8.93）%和（58.39±9.43）%，明显低于对照组（P<0.05）；而联合用药组PCNA的表达率仅为（11.61±2.94）%，其瘤细胞增殖活性更显著低于BTZ单药组，差异有统计学意义（P <0.05）。6NF-κB P65在各组移植瘤组织中的表达：免疫组化结果显示，棕黄色为NF-κB P65的阳性表达反应，多位于细胞浆。本试验中对照组NF-κBP65的阳性表达率高达（85.23±16.62）%，而BTZ与NCTD的阳性表达率分别为（71.51±9.86）%和（64.80±11.73）%，明显低于对照组（P <0.05）；而联合用药组的阳性表达率仅为（11.95±3.13）%，与BTZ单药组比较，差异具有显著性（P <0.05）。结论：1NCTD能够在体内抑制骨髓瘤细胞的增殖，诱导其凋亡；2NCTD在体内能够增强硼替佐米的抗骨髓瘤细胞增殖及促进凋亡作用；3与体外作用机制相同，NCTD在体内通过抑制NF-κB/IκBα途径，来增强硼替佐米的抗骨髓瘤效应。更多还原

【Abstract】 Objective: As the second most common hematological malignancy,multiple myeloma (MM) is characterized by the accumulation of monoclonalplasma cells in patient’s bone marrow, and chemotherapy is the mainlymedical treatment. Bortezomib(BTZ) is the basic medication in the first-linetreatment of patients with multiple myeloma, and its curative effect has beenwidely recognised. However, nearly30%of patients have no response to BTZ,and37–44%of patients are compelled to discontinue their treatment due tothe serious adverse reaction. Therefore, there is an imperative need for somenew chemical entities, which have high performance but low toxicity, or couldenhance the anti-myeloma activity of BTZ.Norcantharidin (NCTD), the demethylated analog of cantharidin, retainsthe antitumor activity with reduced nephrotoxicity and recognised by manyresearchers. In previous studies, we have demonstrated that NCTD inducedapoptosis in U266cells and enhance the anti-myeloma activity of BTZthrough down-regulate NF-kB and NF-kB downstream molecules. Therefore,in this study, we investigated the anti-myeloma activity of NCTD in vivo byestablishing the animal models of multiple myeloma, and then provide moreexact theoretical basis for clinical application of NCTD against MM.Methods:1The toxicity test of NCTD: The maximum and minimum lethaldose(250and102.4mg/kg) of NCTD were defected by trial test. Then50micewere randomized into5groups(n=10/group) and treated with differentconcentrations of NCTD(250,200,160,128and102.4mg/kg, IP). Record thedeath distribution of each mice group for7days, and compute the LD50concentration of NCTD with the modified Karber formula.2Nude mouse xenograft assay:2×106U266cells were subcutaneously injected into the subscapularis of each6-week-old male nu/nu nude mice. Thetumor volume (V) was calculated with the following formula: V=[tumorlength in mm×(tumor width2in mm)]/2. When the tumor volume reached100mm3in size, the mice were randomly assigned into four groups (n=8/group).The control group was intraperitoneally (IP) treated with100mL normalsaline3/week for4weeks. The NCTD group was administered NCTD (20mg/kg, IP)3/week for4weeks. The BTZ group received BTZ (0.25mg/kg, IP)once per week for4weeks. The combination group was treated with acombination of NCTD (20mg/kg, IP) and BTZ (0.25mg/kg, IP). Tumorgrowth and animal body weights were monitored twice a week. The mice wereeuthanized4h after the last dose, and the tumor xenografts were removed forimmunohistochemical (IHC) analysis.3Pathology and apoptosis, immunohistochemistry analysis: The tumorxenografts were parrffin-embedded and sliced into5μm per section. Thesections were stained by HaematoxylinEosin and then observed with lightmicroscope. Apoptosis in tumors was determined by TUNEL assay and theexpression of NF-kB p65, PCNA was detected through Immunohistochemicalanalysis.Results：1LD50concentration of NCTD:According to the results of the toxicitytest, Σp=1.0+0.9+0.7+0.6+0.2=3.4, i=lg250/200=lg1.25=0.1, Xm=lg250=2.4, the LD50of NCTD was determined with the modified Karber formula:LD50=log-1[Xm–i (Σp–0.5)]=128mg/kg。2The tumor growth observation: There was no difference betweenaverage volumes of nude mice xenograft in both4groups before treatment(P>0.05). However, tumor volumes in3treatment groups were much smaller thanthe untreated control group after4weeks(P<0.05). The tumor volumereduction rates were respectively31.83,36.32%in NCTD, BTZ groups, whilein the combination group, the reduction rate reached64.49%, significantlygreater than BTZ-alone group(P<0.05).3The general condition and biochemical indexes comparison: There was no apparently panic, anxiety, or antifeeding and other phenomena throughoutthe whole intervention process but only limited mobility in the control group.Biochemical indexes detection after4weeks showed that in the3treatmentgroups, there was no significant renal impairment(average serum creatininelevels<0.5mg/dL) neither difference in alanine aminotransferase levels(P<0.05).4Apoptosis detection by TUNEL assay: Obviously apoptosis cells wereobserved in both the four treatment groups, characterized by karyopyknosisand brown nucleus after stained with diaminobenzidine. The apoptosis rateswere respectively29.91%,42.06%in NCTD, BTZ groups, with respect to14.95%in the control group. While in the NCTD-BTZ combination group, theapoptosis rate reached81.80%, much greater than BTZ-alone group(P<0.05).5The protein expression of PCNA in nude mice xenografts: As acommon index for evaluation of cell proliferation, the positive expression ofPCNA is located in nucleus and appears as a diffuse brown particles. OurImmunohistochemical tests showed that the PCNA positive expression rate upto (89.91±13.59)%, presented much lower in BTZ and NCTD group,respectively (71.85±8.93)%and (58.39±9.43)%. While in the NCTD-BTZcombination group, the positive expression rate was only (11.61±2.94)%,there was a significant difference compared with BTZ-alone group(P<0.05).6The protein expression of NF-κB P65in xenografts: The positiveexpression is located mostly in cytoplasm and appears as a diffuse brownparticles. Our Immunohistochemical tests showed that the NF-κB P65positiveexpression rate was up to (85.23±16.62)%, presented much lower in BTZ andNCTD group, respectively (71.51±9.86)%and (64.80±11.73)%. While in theNCTD-BTZ combination group, the positive expression rate was only(11.95±3.13)%, there was a significant difference compared with BTZ-alonegroup(P<0.05).Conclusions：1NCTD could significantly inhibit the proliferation and induce theapoptosis of myeloma cells in vivo. 2NCTD could potentiate the proliferate-inhibitor and apoptosis-induction effects of BTZ, both in vitro and in vivo.3With the same mechanism in vitro, NCTD could potentiate theanti-tumor effect of BTZ in vivo through down-regulating the NF-κB/IκBαsignaling pathway and can be administered in clinic.更多还原