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目的研究鬼臼毒素二元醇质体(POD-BE)体外抗宫颈人乳头瘤病毒(HPV)感染的效果。方法采用超声注入法制备POD-BE,采用透析法测定POD-BE包封率(EE%),观察POD-BE形态学、外观性状及稳定性,运用CCK-8法和流式细胞术(FCM)分别检测POD-BE和POD处理后永生化人宫颈上皮细胞(H8细胞)的增殖和凋亡,以透射电镜和激光共聚焦显微镜(LCSM)分别观察细胞的形态变化及药物在细胞的分布。结果制备的POD-BE混悬液电镜下为类球状小囊泡,平均粒径(84.6±9.2)nm,Zeta电位(-27.2±4.2)mV。保存在4℃冰箱内的POD-BE样本,6个月内仍保持半透明、质地均匀外观,未见沉淀及药物结晶析出。3000 r/min离心20 min未发现分层现象。而保存在室温下的样本,24 h未见任何变化,3~6个月时出现少许混浊,震荡后样本恢复均一外观,未见药物结晶析出。POD-BE组和POD组均呈剂量和时间依赖性地抑制H8细胞的增殖。在相同浓度同一时间条件下,POD-BE组对H8细胞的增殖抑制效果明显强于POD组(P<0.01)。POD-BE组12、24、48 h的半数抑制浓度(IC50)分别为782、595、97μg/L,而POD组的IC50则分别为1966、1570、758μg/L;空白BE组对细胞无明显毒性。与空白对照组相比,POD-BE组和POD组均能明显诱导H8细胞凋亡(P<0.01);然而POD-BE组诱导凋亡的效果则显著强于POD组(P<0.01)。10μg/L的POD-BE和POD分别处理H8细胞24 h后,透射电镜下显示:H8细胞被药物处理后其染色质高度凝聚、边缘化,形成凋亡小体。H8细胞分别被POD-BE和POD处理24 h后,两组的荧光均表现出聚集于细胞核的趋势,且POD-BE组较POD组荧光信号强度显著增强,而空白BE组和空白对照组未见有阳性荧光信号表达。结论 POD-BE具备理想的理化性质,POD-BE较POD对H8细胞具有更强的增殖抑制和凋亡诱导效果。
Objective To study the effect of podophyllotoxin dihydric alcohol (POD-BE) on human cervical papillomavirus (HPV) infection in vitro. Methods POD-BE was prepared by ultrasonic injection method. The encapsulation efficiency of EE-POD-BE was measured by dialysis method. Morphology, appearance and stability of POD-BE were observed. By using CCK-8 method and flow cytometry ) Were used to detect the proliferation and apoptosis of immortalized human cervical epithelial cells (H8) after treatment with POD-BE and POD respectively. The morphological changes of cells and the distribution of the drugs were observed by transmission electron microscopy and confocal laser scanning microscopy (LCSM). Results The POD-BE suspension was electron-microscopic under the electron microscope. The mean diameter was (84.6 ± 9.2) nm and the Zeta potential was (-27.2 ± 4.2) mV. POD-BE samples stored in a refrigerator at 4 ° C remained semi-transparent within 6 months with uniform appearance and no precipitation and drug crystallization. No delamination was observed after 3000 r / min centrifugation for 20 min. The samples stored at room temperature, 24 h without any change, 3 to 6 months when a little turbidity, the samples were recovered after the shock appearance, no drug crystallization. Both POD-BE group and POD group inhibited the proliferation of H8 cells in a dose-and time-dependent manner. Under the same concentration and same time, the proliferation of H8 cells in POD-BE group was significantly stronger than that in POD group (P <0.01). The IC50 of POD-BE group were 782, 595 and 97μg / L at 12, 24 and 48 h, respectively, while the IC50 of POD-BE group was 1966, 1570 and 758 μg / L, respectively. toxicity. Compared with the blank control group, POD-BE group and POD group could obviously induce the apoptosis of H8 cells (P <0.01). However, the apoptosis of POD-BE group was significantly stronger than that of POD group (P <0.01). HUVECs were treated with 10 μg / L POD-BE and POD for 24 h, respectively. Transmission electron microscopy revealed that the H8 cells were highly aggregated and marginalized to form apoptotic bodies after drug treatment. Fluorescence of H8 cells treated with POD-BE and POD for 24 h showed a trend of aggregation in the nucleus, and the fluorescence intensity of POD-BE group was significantly increased compared with that of POD-BE group and blank control group See positive fluorescent signal expression. Conclusion POD-BE possesses ideal physical and chemical properties. POD-BE has stronger inhibition of proliferation and apoptosis induction than POD on H8 cells.