香加皮提取物抗肿瘤及免疫调节作用的实验研究
河北医科大学第四医院科研中心 赵连梅
【摘要】目的：研究中药香加皮两种提取物的抗肿瘤和免疫调节作用以及它们的作用机制，为治疗食管癌寻找有效药物，同时为中药香加皮的二次开发利用提供实验依据。材料与方法：采用MTT法分析不同浓度香加皮杠柳苷(CPP)作用不同时间后，对四种恶性程度不同的食管癌细胞株TE-13、Eca-109、TE-1、TE-10增殖反应的影响。2. 用不同浓度CPP分别处理TE-13细胞48h后，采用Gimsa染色技术和流式细胞技术检测细胞凋亡和细胞周期分布情况。3. 采用Western blot技术分析细胞周期蛋白依赖性激酶CDK2和CDK4的表达变化，以探讨CPP诱导细胞凋亡及细胞生长周期阻滞的可能机制。4. 采用RT-PCR法分析四种食管癌细胞株TE-13、 Eca- 109、TE-1和TE-10 syk mRNA的表达状态，以及经CPP 处理后syk mRNA表达的改变情况，同时采用流式细胞技术分析CPP处理细胞前后细胞中syk蛋白表达的变化。5. 无菌分离健康人外周血淋巴细胞，MTT法分析不浓度香加皮羽扇豆烷乙酸酯(CPLA)对人外周血淋巴细胞增殖和对巨噬细胞吞噬功能的影响，6. 用ELISA和RT-PCR法分析CPLA对人外周血淋巴细胞产生细胞因子功能的影响。结果：1.香加皮提取物杠柳苷(CPP)体外对恶性程度不同的食管癌细胞TE-13、Eca-109、TE-1、TE-10的增殖反应均有显著抑制作用(P<0.01); 2. CPP作用于肿瘤细胞后，可诱导典型的细胞凋亡形态变化; 流式细胞分析结果显示，CPP能使TE-13细胞发生凋亡和周期阻滞，处理48h时，凋亡率明显增加；G0/G1细胞明显增加，S期细胞明显减少，与对照组相比，差异有显著性(P<0.01)。3.CPP能够使TE-13细胞CDK4蛋白表达水平明显下调，与对照组相比，差异有显著性(P<0.01)。CPP对CDK2的蛋白表达没有明显影响(P>0.05); 4.RT-PCR法分析结果显示高度恶性的细胞株TE-13、Eca-109 syk mRNA(514bp)表达弱阳性，恶性程度比较低的食管癌细胞株TE-1syk mRNA表达水平明显高于TE-13、Eca-109细胞，而在恶性程度比较低的食管癌细胞株TE-10中却未检测到syk mRNA的表达。经CPP处理后，TE-13、Eca-109、TE-1 syk mRNA表达增强, 对TE-10细胞syk mRNA表达没有影响(P>0.05)。流式细胞技术显示上述细胞syk蛋白表达水平结果与mRNA表达情况相符合。5.香加皮提取物羽扇豆烷乙酸酯(CPLA)能明显促进PHA活化的人外周血淋巴细胞的增殖以及吞噬细胞的吞噬作用; 6. ELISA检测结果显示，经CPLA作用后，人外周血淋巴细胞产生TNF-α和IL-2水平显著升高(P<0.01)。RT-PCR分析结果显示，20μg/ml和40μg/ml CPLA处理后，能明显增强 PHA的活化作用，使淋巴细胞IFN-γ mRNA的表达水平明显增强(P<0.01)。结论：香加皮提取物杠柳苷(CPP)体外能明显抑制食管癌细胞的增殖; CPP对食管癌细胞TE-13的抑制作用可能与其诱导细胞凋亡和细胞周期阻滞有关，CPP能使TE-13细胞的生长周期阻滞在G0/G1期，其机制可能与CPP下调细胞周期蛋白依赖性激酶CDK4的表达有关; CPP的抑瘤作用可能还与其恢复候选抑癌基因syk mRNA和蛋白的表达有关，表明CPP发挥抑瘤作用是多基因、多靶点的; 香加皮羽扇豆烷乙酸酯(CPLA)成份能增强人外周血淋巴细胞的免疫功能，能促进PHA活化的淋巴细胞增殖；CPLA能增强吞噬细胞对肿瘤细胞的杀伤功能，其机制可能与CPLA能促进吞噬细胞产生TNF-α有关; CPLA能促进外周血淋巴细胞产生TNF-α和IL-2等细胞因子，并能增强外周血淋巴细胞IFN-γ mRNA的表达。
Exprimental study on the Antineoplastic Activity and Immunoregulatory Activity of cortex periplocae extracts
[Abstruct] Objective: In order to study antitumor and Immuno -regulation mechanism of both different extracts of cortex periplocae, which are helpful for finding the effective therapeutic measures for curing of human esophageal carcinoma. Meanwhile, which provide us with some experimental bases to develop and exploite Chinese medicine herb of cortex periplocae. Methods: 1. MTT assays was used to detect the suppressive effect of CPP on different human esophageal carcinoma cells such as TE-13, Eca-109, TE-1 and TE-10. 2. Affter treatment with CPP for 48h, The cycle and apoptosis of human esophageal carcinma cells TE-13 was studied by flow-cyto -metry (FCM); 3. Expression of CDK2 and CDK4 were detected with western blot. 4. The expression of syk mRNA in four kinds of human esophageal carcinma cells line and the changes of syk mRNA induced by CPP were analyzed by RT-PCR, and the changes of syk protein was measured by FCM; 5. Lymphocytes from human peripheral blood were isolataed by density gradient centrifugation with lymphocyte isolation. Effect of CPP on proliferation of lymphocytes and phogocitoxity to macrophages on carcinoma cells were studied wit MTT; 6. The influence of CPLA on cytokines production by lymphocytes was analyzed with ELISA and RT-PCR. Results: CPP inhibited the proliferation of human esophageal carcinoma cells such as TE-13, Eca-109, TE-1 and TE-10 in a time and dose-dependent manner(P<0.01); After treatment with CPP, TE-13 cells shown some typical morphologic features; The result of FCM showed that CPP can induce apoptosis and cell cycle of TE-13 cells; TE-13 cells cycle arresting induced by CPP may be related with CDK4 protein decreaing, but it have no relate with CDK2 protein expression; Expression level of syk mRNA in TE-13 and Eca-109 are very lowly, while the expression level of syk mRNA was high in TE-1 cell and was negative in TE-10 cell; After treatment with CPP for 48h, the expression of syk mRNA(514bp) in TE-13、Eca-109 and TE-1 increase markly, but CPP have no effect on that in TE-10 cells.The result of syk protein measured by FCM are consistent with the FCM result; The proliferation of lymphocytes was enhanced by 5μg/ml-40μg/ml CPLA with acting of PHA, and in a dose dependent manner(p<0.01); CPLA(5-40μg/ml) can strengthen the phagocytosis effect of macrophage on esophageal carcinoma cells significantly(p< 0.01); The production of IL-2 and TNF-α by lymphocytes could be augmented by treatment with CPLA significantly, which is in a dose-dependment (P<0.01); The expression of IFN-γ mRNA in lymphocytes was also enhanced by 20μg/ml and 40μg/ml CPLA. Conclusions: 1. CPP showed very strong inhibitory effect on the proliferation of esophague carcinoma cells such as TE-13、Eca-109, TE-1 and TE-10 cells; Inhabitory effect of CPP on TE-13 cells can be related to induce apoptosis of cells and arrest of cell cycle at G0/G1, and it may be related with CDK4 protein expressin and have no related with CDK2 protein expression; Syk mRNA and protein expression in human esophageal carcinoma cells are low and it can be enhanced by CPP; The results showed that inhabitory effect of CPP on human esophageal carcinoma cells have relation with syk up-regulation; The proliferation of lymphocytes was enhanced by CPLA, which also can strengthen macrophage to inhabit the proliferation of esophageal carcinoma cells significantly. The production of several tumor immunity-related cytokines can be augmented by treatment with CPLA.
|
