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目的:通过体内外实验观察雷公藤内酯醇(triptolide,TPL)对人胰腺癌PANC-1细胞生长和凋亡的抑制作用,并分析其对Toll样受体4(Toll-like receptor 4,TLR4)、血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)的表达和肿瘤血管生成的影响。方法:以0、20、40、80 ng/ml的TPL作用于PANC-1细胞,MTT法和流式细胞术分别检测TPL对PANC-1细胞增殖和凋亡的影响,Western blotting检测TPL作用后PANC-1细胞中TLR4和VEGF的表达。建立PANC-1细胞裸鼠荷瘤模型并随机分为TPL组、PBS组,测量移植瘤的体积变化,治疗35 d后摘取瘤块,免疫组织化学方法检测移植瘤组织内TLR4、VEGF和CD31的表达,并计算微血管密度(microvessel density,MVD)。结果:与0 ng/ml组相比,PANC-1细胞经20、40和80 ng/ml的TPL处理24 h后,细胞凋亡率均显著升高[(4.7±1.0)%、(10.5±2.0)%、(21.1±4.2)%vs(2.6±0.5)%,P<0.05或P<0.01];48 h后,细胞增殖率均显著下降[(68.0±5.3)%、(59.6±5.0)%、(51.6±4.2)%vs(99.6±5.2)%,均P<0.01],并较相同浓度TPL处理24 h时显著降低(P<0.05或P<0.01)。80 ng/ml TPL组处理后PANC-1细胞中TLR4蛋白[(20.2±4.7)%vs(57.5±6.3)%,P<0.01]和VEGF蛋白[(35.8±4.0)%vs(92.1±8.3)%,P<0.01]的表达量显著低于未处理组。TPL治疗组第34天的裸鼠移植瘤体积显著小于PBS对照组[(510.9±79.8)vs(1 220.6±127.2)mm3,P<0.01];TPL治疗组移植瘤组织内的TLR4、VEGF表达均显著低于PBS组[(3.2±0.6)vs(6.7±1.1),(3.7±0.7)vs(7.1±1.2);均P<0.01),其MVD也显著低于PBS组[(12.2±4.0)vs(22.7±5.6),P<0.01]。结论:TPL能够抑制人胰腺癌PANC-1细胞及其裸鼠移植瘤的生长,并促进PANC-1细胞凋亡,其机制可能与TPL抑制TLR4、VEGF表达及肿瘤血管生成有关。
OBJECTIVE: To observe the inhibitory effect of triptolide (TPL) on the growth and apoptosis of human pancreatic cancer cell line PANC-1 in vitro and in vivo, and to investigate its effect on the expression of Toll-like receptor 4 (TLR4) , Vascular endothelial cell growth factor (VEGF) expression and tumor angiogenesis. Methods: The effects of TPL on the proliferation and apoptosis of PANC-1 cells were detected by MTT and flow cytometry (TPL) with 0, 20, 40 and 80 ng / ml TPL respectively. After TPL treatment by Western blotting Expression of TLR4 and VEGF in PANC-1 cells. The tumor-bearing model of PANC-1 cells in nude mice was established and randomly divided into TPL group and PBS group. The volume changes of the transplanted tumors were measured. After 35 days of treatment, the tumor masses were removed and the expressions of TLR4, VEGF and CD31 were detected by immunohistochemistry The expression of microvessel density (MVD) was calculated. Results: Compared with 0 ng / ml group, the apoptotic rates of PANC-1 cells were significantly increased after treated with 20, 40 and 80 ng / ml TPL for 24 h [(4.7 ± 1.0)%, (10.5 ± (68.0 ± 5.3)%, (59.6 ± 5.0)%, (21.1 ± 4.2)% vs (2.6 ± 0.5)%, P <0.05 or P < %, (51.6 ± 4.2)% vs (99.6 ± 5.2)% respectively, all P <0.01], and were significantly lower than those at the same concentration of TPL for 24 h (P <0.05 or P <0.01). The expressions of TLR4 protein [(20.2 ± 4.7)% vs (57.5 ± 6.3)%, P <0.01] and VEGF protein [35.8 ± 4.0% vs 92.1 ± 8.3% %, P <0.01] was significantly lower than the untreated group. The tumor volume in nude mice on day 34 in TPL treatment group was significantly smaller than that in PBS control group [(510.9 ± 79.8) vs (220.6 ± 127.2) mm3, P <0.01]. The expression of TLR4 and VEGF in TPL treatment group (P <0.01), and the MVD in PBS group was significantly lower than that in PBS group [(3.2 ± 0.6) vs (6.7 ± 1.1) vs (3.7 ± 0.7) vs (7.1 ± 1.2) vs (22.7 ± 5.6), P <0.01]. CONCLUSION: TPL can inhibit the growth of human pancreatic cancer PANC-1 cells and xenografts in nude mice and promote the apoptosis of PANC-1 cells. The mechanism may be related to TPL inhibiting the expression of TLR4, VEGF and tumor angiogenesis.