携Herceptin包裹紫杉醇的高分子造影剂的制备及体外显影实验研究

目的 制备一种携Herceptin包裹紫杉醇的高分子造影剂,并进行体外显影实验.方法 通过双乳化法制备出包裹紫杉醇的高分子PLGA-COOH造影刺,然后通过碳二亚胺化学连接法将制备出的载药造影剂与Herceptin连接,检测其一般性质及其包封率与载药量,观察所制备的载药靶向高分子造影剂与乳腺癌细胞的结合能力,并进行体外显影实验.结果 载药靶向高分子造影剂的平均粒径大小为(733.4±30.7)nm,包封率为(65.08±2.31)%,载药量为(6.51±0.23)%,体外寻靶能力实验可观察到载药靶向高分子造影剂与乳腺癌细胞有较强的结合能力,体外显影实验显示载药靶向造影剂显影效果好.结论 成功制备出的携Herceptin包裹紫杉醇的高分子造影剂具有较高的包封率和与乳腺癌细胞结合的能力,在体外显影实验中显影效果好.     

携抗HER-2抗体PLGA高分子纳米超声造影剂的制备及其体外寻靶实验

目的以高分子聚合物聚乳酸-羟基乙酸（PLGA）为成膜材料制备携抗HER-2抗体空心纳米靶向超声造影剂,并考察其体外寻靶及显像效果。方法以樟脑为致孔剂,通过改进的双乳化溶剂挥发法制备PLGA纳米超声造影剂,利用扫描电子显微镜、透射电子显微镜及激光粒度仪对其一般特性进行表征;并用碳二亚胺法将造影剂与抗HER-2抗体耦联制备携抗HER-2抗体的PLGA靶向纳米超声造影剂,用激光共聚焦扫描显微镜对其体外寻靶能力进行初步评估,考察其体外成像效果。结果 PLGA纳米超声造影剂的平均粒径为（152.00±58.08）nm,粒子呈规则球形,大小均一,分散性好。体外寻靶实验显示,携抗HER-2抗体的PLGA靶向造影剂较多牢固地聚集到乳腺癌细胞表面。体外成像实验显示,PLGA靶向纳米超声造影剂显像呈细腻均匀的点状高回声,后方回声未见明显衰减。结论本研究成功制备了携抗HER-2抗体的PLGA靶向纳米超声造影剂,其能与HER-2受体高表达的乳腺癌细胞体外特异性靶向结合,且体外显像效果较好。     

载紫杉醇靶向PLGA高分子造影剂对乳腺癌细胞(SKBR-3)增殖和凋亡的影响

目的成功制备靶向载药PLGA高分子造影剂并比较其对乳腺癌细胞增殖及凋亡的影响。方法通过双乳化法制得载药造影剂、碳二亚胺法制备出载紫杉醇及抗HER-2抗体造影剂,MTT法检测靶向载药组与非靶向载药组对乳腺癌细胞的增殖抑制作用的差异,用细胞流式术检测不同浓度靶向载药组、非靶向载药组对乳腺癌细胞凋亡的影响。结果 MTT法检测到靶向载药组不同时间段的抑制率均高于非靶向载药组,在诱导乳腺癌凋亡试验中,靶向载药组凋亡率高于非靶向组,且随着药物浓度增加,差异随之增大。结论载紫杉醇及抗HER-2抗体PLGA-COOH高分子造影剂比载紫杉醇PLGA-COOH造影剂对SBKR-3乳腺癌细胞的增殖抑制作用更高,且能够更有效地促进SKBR-3乳腺癌细胞的凋亡。     

靶向癌胚抗原载药纳米超声造影剂体外抑制卵巢癌细胞生长

目的 制备靶向癌胚抗原（CEA）载药相变型PLGA纳米粒（Ab-PTX-NPs）,探讨该纳米粒的体外寻靶及抑制肿瘤细胞生长的效能。方法 乳化溶剂挥发法联合碳二亚胺法制备靶向CEA载紫杉醇纳米粒（Ab-PTX-NPs）,采用马尔文粒径分析仪检测其粒径大小。采用高效液相色谱法检测紫杉醇包封率及载药量,恒温摇床透析法检测该纳米粒体外释药特征。激光共聚焦显微镜及流式细胞术观察该纳米粒体外寻靶情况,CCK-8试剂检测卵巢癌细胞存活率。结果 制备的Ab-PTX-NPs粒径为（397.70±99.95）nm,紫杉醇包封率及载药量分别为（67.26±4.15）%和（6.31±0.39）%。抗CEA单克隆抗体与纳米粒连接率为（92.74±5.75）%。共聚焦显微镜下观察到靶向组卵巢癌SKOV3细胞周围见较多造影剂黏附,流式细胞术测得靶向组细胞平均荧光强度明显高于其余各组（P<0.05）,CCK-8试剂法测得靶向组在24 h时,细胞存活率高于纯药组（P<0.05）,而低于无靶组（P<0.05）,当48 h时,靶向组与纯药组细胞存活率差异无统计学意义（P>0.05）。结论 成功制备靶向CEA载药相变型PLGA纳米粒,该纳米粒经低功率聚焦超声治疗仪（LIFU）致相变后可明显增强超声显影,且载药量高,释药快,靶向能力强。     

载10-羟基喜树碱靶向脂质微泡的制备及体外寻靶能力实验研究

目的 制备携人肝癌单抗Hab18载10-羟基喜树碱的脂质微泡,并观察其体外寻靶能力.方法 制备生物素化抗人肝癌Hab18单克隆抗体,检测其生物素化程度;用机械振荡法制备载药脂质微泡,以生物素-亲和素桥连方式构建携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声微泡,检测其一般特性、包封率和载药量,以免疫荧光法检测微泡与抗体的连接情况,在光镜下观察靶向载药微泡的寻靶能力,并与载药非靶向微泡进行比较.结果 每个单抗分子平均可与13个生物素分子结合.载药靶向脂质超声微泡分布均匀,平均粒径为1.52 μm,包封率为76.32%,载药量为21.81%.免疫荧光法显示微泡表面可见明亮的红色环状荧光,体外寻靶实验显示该载药靶向微泡可与人肝癌7721细胞牢固结合.结论 携人肝癌单抗Hab18载10-羟基喜树碱的脂质超声载药靶向微泡可成功制备,其包封率和载药量较高,具有较强的体外寻靶能力.     

携Herceptin靶向高分子造影剂的制备及体外寻靶能力研究

目的 制备一种携Herceptin的靶向高分子造影剂,并研究其体外寻靶能力.方法 通过双乳化法制备出高分子PLGA-COOH造影剂,采用碳二亚胺法将高分子PLGA-COOH造影剂与Herceptin相连制备出靶向高分子造影剂,检测其一般性质,然后通过免疫荧光法检测其与抗体的连接情况,用光镜及激光共聚焦显微镜观察其与细胞的结合能力,并与普通高分子造影剂做比较.结果 靶向高分子造影剂的粒径范围为466.8～857.6 nm,平均粒径为(662.2±69.7)nm,有85.9%的微球粒径落于该范围内,通过免疫荧光法可在荧光显微镜下观察到许多绿色点状荧光,体外寻靶能力实验显示靶向高分子造影剂能与乳腺癌细胞较好的结合.结论 成功制备出的携Herceptin的靶向高分子造影剂在体外与人乳腺癌细胞有较好的结合能力.     

载紫杉醇靶向超声脂质微泡对于卵巢癌细胞体外存活率的实验研究

目的制备一种载紫杉醇的靶向相变型纳米级超声造影剂,并评价其一般特性。方法薄膜水化法、乳化法制备载紫杉醇的非靶向相变型脂质纳米粒(PTX-LNP),通过生物素-亲和素法将生物素化的促黄体生成素释放激素(LHRH)连接于非靶向相变型脂质纳米粒制备载紫杉醇的LHRH靶向相变型脂质纳米粒(PTX-LNP-LHRH),于观察室温下PTX-LNP-LHRH的形态,载药量及包封率,致人卵巢癌OVCAR3细胞的凋亡率。结果 PTX-LNP-LHRH的平均粒径为(303±57.76)nm,载药量及包封率分别为(13.37±0.95)%和(80.72±5.19)%,OVCAR3细胞的存活率随各组培养时间延长而下降。空白微球组的细胞存活率均90%。提示该组对细胞活性影响较小。而靶向组联合低强度聚焦超声组(PTX-LNP-LHRH+LIFU)的细胞存活率最低,与其他实验组比较,差异具有统计学意义(P0.05)。结论成功制备外接LHRH的载药微泡,并且不影响紫杉醇的载药量及包封率,靶向造影剂组的卵巢癌细胞凋亡率显著增高,且差异均具有统计学意义(P0.05)。     

靶向包载经PEG修饰的重组腺病毒PLGA造影剂的制备及其体外寻靶实验

目的采用共价结合的方法制备靶向包载经PEG修饰的重组腺病毒PLGA-COOH造影剂,并考察其体外寻靶能力。方法采用碳二亚胺法将包载经PEG修饰的重组腺病毒PLGA-COOH造影剂与抗体共价结合,制备靶向高分子超声造影剂,检测该造影剂一般特性及体外寻靶能力。结果体外寻靶实验显示,该靶向超声造影剂与普通包载经PEG修饰的重组腺病毒PLGA COOH造影剂对照能聚集并牢固结合到人脐静脉血管内皮细胞表面。结论成功制备出靶向包载经PEG修饰的重组腺病毒PLGA-COOH造影剂,该靶向造影剂在体外具有较强的特异性寻靶结合能力。     

抗VEGF抗体介导靶向超声造影剂的体外实验研究

目的以抗血管内皮因子（VEGF）抗体为配体研制能与血管内皮细胞特异性结合的靶向脂质体超声造影剂并检测其体外寻靶能力。 方法以静电吸附法将抗VEGF抗体连接到脂质体造影剂微泡的表面；体外培养ECV304人血管内皮细胞，用免疫荧光法检测靶向造影剂与其体外结合能力，以普通造影剂为对照组。 结果所制备的靶向超声造影剂与普通微泡无显著差异；免疫荧光实验结果显示靶向造影剂能在体外与血管内皮细胞特异性结合。 结论携抗VEGF抗体的脂质体靶向造影剂能通过静电吸附法成功制备，且在体外能与血管内皮细胞特异性结合。     

载ACE2激动剂的胶原靶向纳米粒的制备及体外显像与靶向研究

目的制备一种以心肌胶原蛋白标记物CNA35为靶点的载ACE2激动剂三氮脒(DIZE)的液态氟碳脂质纳米粒(CNA35/PFP/DIZE@NPs),评价其体外显像和寻靶能力。方法采用薄膜旋蒸联合乳化法将PFP和DIZE装载于脂质体内,碳二亚胺法连接CNA35。检测其一般性质及载药情况,LIFU辐照后评估超声显像能力,共聚焦显微镜观察其与兔心肌纤维化冰冻切片中胶原蛋白的结合,评估体外寻靶能力。结果制备的靶向载药纳米粒,形态均一,平均粒径为(302.90±4.89)nm,载药量4.00%±0.78%。体外LIFU辐照可增强现象,共聚焦下见纳米粒可与较多胶原结合。结论成功制备了CNA35/PFP/DIZE@NPs,具有良好的体外显像和胶原靶向能力。     

The immunocytokine scFv23/TNF sensitizes HER-2/neu-overexpressing SKBR-3 cells to tumor necrosis factor (TNF) via up-regulation of TNF receptor-1

Overexpression of HER-2/neu confers cellular resistance to tumor necrosis factor (TNF)-mediated cytotoxicity to SKBR-3 breast cancer cell lines. To understand the correlation between HER-2/neu expression and TNF resistance, we examined the unique signaling pathways associated with the cytotoxic effects of the immunocytokine scFv23/TNF, recombinant single-chain antibody fusion constructs containing TNF and targeting HER-2/neu, in TNF-resistant SKBR-3-LP cells. We found that treatment of HER-2/neu-overexpressing SKBR-3-LP cells with scFv23/TNF resulted in a 5- to 7-fold higher level of TNF receptor-1 expression 48 hours after exposure. In addition, treatment of SKBR-3-LP cells with scFv23/TNF resulted in down-regulation of Akt phosphorylation and induced apoptosis through cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase. ScFv23/TNF-induced cytotoxicity was inhibited by blocking of the binding of the TNF component of scFv23/TNF to TNF receptor-1 and was dependent on activation of caspase-8 and caspase-3. These results indicate that the immunocytokine scFv23/TNF sensitizes TNF-resistant HER-2/neu-overexpressing SKBR-3-LP cells to TNF-induced apoptosis via the overexpression of TNF receptor-1 and suggest that the overexpression of TNF receptor-1 plays a crucial role in TNF sensitivity in HER-2/neu-overexpressing cancer cells. ScFv23/TNF targeting the HER-2/neu may be an effective cytotoxic agent against HER-2/neu-overexpressing cancer cells, which are inherently resistant to TNF.     

Epidermal growth factor receptor (EGFR)-related protein inhibits multiple members of the EGFR family in colon and breast cancer cells

Inactivation of epidermal growth factor receptor (EGFR) family members represents a promising strategy for the development of selective therapies against epithelial cancers. Current anti-EGFR therapies, such as cetuximab (Erbitux), gefitinib (Iressa), or trastuzumab (Herceptin), target EGFR or HER-2 but not both. Because solid tumors express different EGFRs, identification of inhibitor(s), targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. We have identified a natural inhibitor of EGFRs called EGFR-related protein (ERRP), a 53 to 55 kDa protein that is present in most, if not all, normal human epithelial cells. The growth of colon (HCT-116, Caco2, and HT-29) and breast (MDA-MB-468 and SKBR-3) cancer cells expressing varying levels of EGFR, HER-2, and/or HER-4 was inhibited by recombinant ERRP in a dose-dependent manner. In contrast, ERRP caused no inhibition of growth of normal mouse fibroblast cell lines (NIH-3T3, NIH-3T3/P67), and the growth of nontransformed rat small intestinal IEC-6 cells expressing relatively low levels of EGFRs was inhibited only at high doses of ERRP. Transforming growth factor-alpha or heparin-binding epidermal growth factor-induced activation of EGFR and HER-2 was inhibited by ERRP in colon and breast cancer cells expressing high levels of EGFR or HER-2. In contrast, cetuximab inhibited the growth- and ligand-induced activation of EGFR in cell lines expressing high levels of EGFR, whereas trastuzumab was effective only in HER-2-overexpressing cells. ERRP and trastuzumab, but not cetuximab, attenuated heregulin-alpha-induced activation of colon and breast cancer cells that expressed high levels of HER-2. Furthermore, ERRP, but not cetuximab or trastuzumab, significantly induced apoptosis of colon and breast cancer cells. None of these agents induced apoptosis of either NIH-3T3 mouse fibroblast or normal rat small intestinal IEC cells. Our results suggest that ERRP is an effective pan-erbB inhibitor and, thus, may be a potential therapeutic agent for a wide variety of epithelial cancers expressing different levels and subclasses of EGFRs.     

肽功能化载GOD智能响应型相变纳米粒用于乳腺癌超声诊疗的实验研究

目的制备一种肿瘤归巢穿膜肽tLyp-1介导的载葡萄糖氧化酶（GOD）和全氟正戊烷（PFP）纳米粒（tLyp-1-GOD@PFP NPs），观察其基本表征、体内外超声成像以及对人乳腺癌MDA-MB-231细胞的体外靶向及体内外抗肿瘤能力。 方法采用乳化法制备tLyp-1-GOD@PFP NPs，观察其形态特点，检测其理化性质（粒径、电位、包封率、释药率）；低强度聚焦超声（LIFU）辐照纳米粒后，光镜下观察其相变情况，并观察其体内外超声成像效果；激光共聚焦显微镜和流式细胞仪检测纳米粒对MDA-MB-231细胞的靶向能力；使用CCK-8法和流式法评估在LIFU辐照下该纳米粒对MDA-MB-231细胞的抗肿瘤能力；通过荷瘤裸鼠尾静脉注射纳米粒并观察肿瘤体积变化和裸鼠体重以探索其体内抑瘤效果。 结果制备的纳米粒呈球形壳核结构，大小均一，其平均粒径为（227.4±12.5）nm，平均电位为（-16.5±2.7）mV，GOD的包封率为（12.93±0.46）%，载药率为（1.62±0.06）%，24 h释药率可达（51.73±3.22）%。光镜下相变和体外超声成像具有辐照时间依赖性。CCK-8和流式细胞仪检测结果证明tLyp-1-GOD@PFP NPs具有良好的生物安全性，并且在LIFU辐照后其抑瘤效果较好。同时，体内实验证实该纳米粒具有良好的超声造影能力和靶向能力，体内治疗结果也表明该纳米粒可有效抑制肿瘤增殖。 结论本研究成功制备了tLyp-1介导的载GOD和PFP的相变型纳米粒，对MDA-MB-231细胞具有特异靶向能力，在LIFU辐照下，可实现体内外超声成像，同时产生良好的抗肿瘤效果。     

Bortezomib (PS-341, Velcade) increases the efficacy of trastuzumab (Herceptin) in HER-2-positive breast cancer cells in a synergistic manner

BACKGROUND: Preclinical and clinical studies have shown that the proteasome inhibitor bortezomib (PS341, Velcade) is highly effective when combined with chemotherapeutic agents. The value of trastuzumab (Herceptin) in HER-2-positive (3+ score by immunohistochemistry or fluorescence in situ hybridization positive) breast cancer is also known; however, the response rate is <40% for metastatic breast cancer. These two pharmacologic agents prevent nuclear factor-kappaB (NF-kappaB) activation and induce nuclear accumulation of the cyclin-dependent kinase inhibitor p27(kip1), suggesting that combining bortezomib with trastuzumab could increase trastuzumab efficacy. METHODS: Drug cytotoxicity, both individually and together, and drug effects on p27 localization and NF-kappaB activation were investigated on four breast cancer cell lines: SKBR-3 (HER-2+++), MDA-MB-453 (HER-2++), HER-2-transfected MCF-7 (HER-2+++), and MCF-7 (HER-2-). RESULTS: Bortezomib induced apoptosis in HER-2-positive and HER-2-negative breast cancer cells in a dose- and time-dependent manner. Together, these drugs induced apoptosis of HER-2++/+++ cells at low concentrations, which had no effect when used alone, indicating there was a synergistic effect. Sequential treatment (trastuzumab then bortezomib) induced either necrosis or apoptosis, depending on the trastuzumab preincubation time. Susceptibility to bortezomib alone and the drug combination correlated with NF-kappaB activity and p27 localization. CONCLUSIONS: The addition of bortezomib to trastuzumab increases the effect of trastuzumab in HER-2+++/++ cell lines in a synergistic way. This effect likely results from the ability of these two drugs to target the NF-kappaB and p27 pathways. The potential clinical application of this drug combination is under current evaluation by our group in a phase 1 clinical trial.     

HER-2/neu胞外配体第2结构域致敏的DC与CIK共培养对高表达HER-2/neu乳腺癌细胞杀伤作用的研究

目的探讨HER-2/neu胞外配体第2结构域(RLD2)作为肿瘤疫苗在抗乳腺癌免疫中的作用。方法纯化出目的蛋白HER-2/neu胞外配体第2结构域,同时选取10例经免疫组化证实为HER-2/neu阳性的乳腺癌,用RLD2分别负荷这10名患者外周血来源的树突状细胞(DC),再诱导抗原特异的细胞因子诱导的杀伤细胞(CIK),将DC与C IK共培养,研究其对HER-2/neu阳性、阴性肿瘤细胞株和自体乳腺癌细胞杀伤活性的作用。结果RLD2诱导的特异性CIK对HER-2/neu阳性肿瘤细胞SKBR-3以及自体乳腺癌细胞的杀伤活性,较之单独CIK组更高,差异有统计学意义(P<0.01);RLD2诱导的特异性C IK对HER-2/neu阴性细胞MDA-435的杀伤活性与单独C IK组之间差异无统计学意义(P>0.05)。结论负载RLD2的DCs诱导的C IK对HER-2/neu阳性肿瘤细胞以及自体乳腺癌肿瘤有特异性杀伤能力,RLD2可能成为新型肿瘤疫苗应用于临床乳腺癌免疫治疗。     

Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer

Clinical resistance to the HER-2 oncogene-targeting drug trastuzumab (Herceptin) exists, but studies of the resistance mechanisms are hampered by the lack of suitable experimental model systems. We established a carcinoma cell line (designated JIMT-1) from a pleural metastasis of a 62-year old patient with breast cancer who was clinically resistant to trastuzumab. JIMT-1 cells grow as an adherent monolayer and form xenograft tumors in nude mice. JIMT-1 cells have an amplified HER-2 oncogene, which showed no identifiable mutations in its coding sequence. JIMT-1 cells overexpress HER-2 mRNA and protein, and the levels of HER-1, HER-3, and HER-4 mRNA and protein were similar to the trastuzumab-sensitive cell line SKBR-3. The cell line lacks expression of hormone receptors (estrogen receptors and progesterone receptors) and is phenotypically of epithelial progenitor cell origin, as evidenced by immunohistochemical positivity for both cytokeratins 5/14 and 8/18. JIMT-1 cells were insensitive to trastuzumab and another HER-2-inhibiting drug, pertuzumab (2C4), in vitro and in xenograft tumors. Small molecule tyrosine kinase inhibitors Ci1033 and ZD1839 inhibited the JIMT-1 cell growth but to a lesser degree than in trastuzumab-sensitive BT-474 cells. The lack of growth inhibition was rationalized by the unaltered Akt phosphorylation in JIMT-1 cells. Erk1/2 phosphorylation was slightly reduced but still evident in JIMT-1 cells. We conclude that the JIMT-1 cell line provides a valuable experimental model for studies of new trastuzumab-resistance mechanisms.     

超声介导微气泡造影剂促反义寡核苷酸转染的有效性研究

目的探讨超声介导脂质体微气泡转染反义寡核苷酸HA-2741的有效性。方法12孔板培养人乳腺癌细胞,分组为:(1)单纯超声照射;(2)单纯造影剂;(3)单纯HA-2741;(4)超声照射 HA-2741;(5)造影剂 HA-2741;(6)造影剂 HA-2741 超声照射;(7)脂质体 HA-2741;(8)脂质体 HA-2741 超声照射。造影剂浓度2%,超声波发射频率1.3MHz,强度-3dB,照射时间30s,予与实验刺激后,流式细胞仪定量分析HA-2741的转染率,RT-PCR的检测乳腺癌细胞的HER-2mRNA水平,免疫细胞化学检测HER-2蛋白的表达。结果造影剂 HA-2741 超声照射组HA-2741的转染率显著高于其余各组,约94.6%,造影剂 HA-2741 超声照射组乳腺癌细胞的HER-2mRNA水平显著降低,HER-2蛋白表达明显减低,其程度与HA-2741转染率呈正相关。结论超声介导微气泡造影剂显著增加基因的转染效率,方法简便,安全,具有良好的靶向性。