谢谭芳 朱小勇 黄天衍

中圖分类号 R285.5;R943 文献标志码 A 文章编号 1001-0408（2021）13-1589-07

DOI 10.6039/j.issn.1001-0408.2021.13.09

摘 要 目的：研究壳聚糖氧化石墨烯载体（CS-GO）负载冬凌草甲素（CS-GO-oridonin）对人肺癌细胞A549增殖及凋亡的影响。方法：以A549细胞为对象，采用CCK-8法检测经不同质量浓度的CS-GO（3、6、12、24、48 μg/mL）和负载了等质量冬凌草甲素的CS-GO-oridonin（3、6、12、24、48 μg/mL，以冬凌草甲素质量计，下同）作用后的细胞存活率，并计算CS-GO-oridonin的半数抑制浓度（IC50）;采用显微镜观察CS-GO和CS-GO-oridonin（均为32 μg/mL）作用2、4、10、24 h后的细胞形态，并采用荧光标记法观察细胞对CS-GO、冬凌草甲素、CS-GO-oridonin（均为32 μg/mL）的摄取情况;采用流式细胞术观察不同质量浓度CS-GO（16、32、64 μg/mL）和CS-GO-oridonin（16、32、64 μg/mL）作用后细胞的凋亡情况及细胞中活性氧（ROS）的含量，采用Western blot法检测细胞中抗凋亡相关蛋白[髓样细胞白血病1（Mcl-1）、Bax、Bak]蛋白的表达情况。结果：经不同质量浓度CS-GO作用后，细胞的存活率仍不低于90%;经不同质量浓度CS-GO-oridonin作用后，细胞存活率呈下降趋势，且显著低于相同质量浓度的CS-GO组（P<0.01）;CS-GO-oridonin的IC50为32.61 μg/mL。经CS-GO作用后，细胞形态未见明显改变;经CS-GO-oridonin作用后，细胞出现皱缩、成团脱落、悬浮物增多等现象;当细胞摄取冬凌草甲素和CS-GO-oridonin后，其荧光均较摄取CS-GO的细胞有所增强。与空白组比较，CS-GO 16、32、64 μg/mL组细胞的凋亡率和细胞中ROS含量、凋亡相关蛋白的表达水平均无显著变化（P>0.05）;而CS-GO-oridonin 16、32、64 μg/mL组细胞的凋亡率、细胞中ROS含量和Bax、Bak蛋白的表达水平均显著升高，Mcl-1蛋白的表达水平均显著降低，且上述指标水平均显著高于或低于同质量浓度CS-GO组（P<0.05）。结论：CS-GO不会影响A549细胞的增殖及凋亡;CS-GO- oridonin对细胞有明显的抑制和促凋亡作用，这种作用可能与增加细胞中ROS的产生、调控凋亡相关蛋白的表达有关。

关键词 壳聚糖氧化石墨烯载体;冬凌草甲素;A549细胞;增殖;凋亡

Effects of Oridonin-loaded Chitosan Graphene Oxide on the Proliferation and Apoptosis of A549 Cells

XIE Tanfang，ZHU Xiaoyong，HUANG Tianyan（School of Pharmacy， Guangxi University of TCM/Key Laboratory of Common Technology of Traditional Chinese Medicine Preparation in Universities of Guangxi/Modern Chinese Medicine Preparation Engineering Technology Research Center， Nanning 530022， China）

ABSTRACT   OBJECTIVE： To study the effects of chitosan graphene oxide carrier （CS-GO） loaded with oridonin （CS-GO- oridonin） on the proliferation and apoptosis of human lung cancer A549 cells. METHODS： Taking A549 cells as objects， the survival rate of cells were detected by CCK-8 method after treated with different concentrations of CS-GO（3， 6， 12， 24， 48         μg/mL） and CS-GO-oridonin loaded with same mass of oridonin （3， 6， 12， 24， 48 μg/mL， by the weight of oridonin， the same below）. IC50 of CS-GO-oridonin was calculated. The cell morphology were observed by microscope after treated with CS-GO and CS-GO-oridonin （both 32 μg/mL） for 2， 4， 10， 24 h. The uptake of CS-GO， oridonin， CS-GO-orionin （all 32 μg/mL） by cells was observed with fluorescence labeling method. The apoptosis of cells and the content of ROS were observed by flow cytometry after treated with different concentrations of CS-GO （16， 32， 64 μg/mL） and CS-GO-oridonin（16， 32， 64 μg/mL）. The expression of anti-apoptosis related proteins （Mcl-1， Bax and Bak） were detected by Western blot. RESULTS： After treated with different concentrations of CS-GO， the survival rate of cells was still above 90%; after treated with different concentrations of CS-GO-oridonin， the survival rate of cells showed a downward trend， and was significantly lower than that of CS-GO group （P<0.01）; IC50 of CS-GO-oridonin was 32.61 μg/mL. After CS-GO treatment， the cell morphology did not change significantly; after CS-GO-oridonin treatment， the cells shrunk and fell off in clusters， and the suspended matter increased; the fluorescence of oridonin and CS-GO-orionin taken up by cells was enhanced than CS-GO. Compared with blank group， there was no significant change in the apoptosis rate， the content of ROS and the expression of apoptosis-related protein in 16， 32， 64 μg/mL CS-GO groups （P>0.05）; apoptosis rate， the content of ROS， the protein expression of Bax and Bak in 16， 32， 64 μg/mL CS- GO-oridonin groups were increased significantly， while the protein expression of Mcl-1 were decreased significantly. Above indexes were significantly higher or lower than the same concentration CS-GO group （P<0.05）. CONCLUSIONS： CS-GO dose not affect the proliferation and apoptosis of A549 cells; CS-GO-oridonin has obvious inhibition and apoptosis promoting effect on cells， which may be related to increasing ROS production and regulating the expression of apoptosis related proteins.

KEYWORDS   Chitosan graphene oxide carrier; Oridonin; A549 cells; Proliferation; Apoptosis

肺癌是全世界最常见的肿瘤之一，是多因素、多基因共同作用所导致的恶性肿瘤[1-2]。目前，对于肺癌患者的临床治疗，西医一般采用手术切除、放疗、化疗等方法。而对于大部分不适合手术切除的实体肿瘤患者，化疗常被作为其首选治疗方法，但化疗的毒副作用较大，导致患者常会出现恶心呕吐等毒副反应、生存质量明显下降[3-4]。……