几丁寡糖联合顺铂对肺癌A549细胞及其移植瘤的影响

发布时间：2018-04-28 08:29

本文选题：几丁寡糖 + 顺铂　；参考：《辽宁中医药大学》2017年硕士论文

【摘要】：目的:1.通过体外细胞实验及体内动物实验,分析中药降解产物几丁寡糖单独应用及与顺铂联合应用对肺腺癌A549细胞增殖、迁移、凋亡能力的影响以及对裸鼠移植瘤的作用,探讨两药联合在抗肿瘤作用方面是否具有良好的协同效果,为未来几丁寡糖在抗肿瘤治疗领域中的开发应用及临床转化,提供实验基础及理论依据。材料与方法:本研究采用三组药物(几丁寡糖组、顺铂组、几丁寡糖联合顺铂组)作用于人肺腺癌A549细胞系,通过MTS实验、细胞划痕实验、transwell小室迁移实验及流式细胞凋亡实验,对比观察各药物组对肺腺癌A549细胞增殖、迁移及凋亡等生物学行为的影响;同时应用肺腺癌A549细胞对裸鼠进行皮下移植瘤造模,待造模成功后,按照给药的不同将裸鼠随机分为四组,分别为空白对照组、几丁寡糖组、顺铂组和几丁寡糖联合顺铂组,先利用HE染色的方法镜下观察各组肿瘤组织的形态学变化,再应用免疫组织化学的方法,观察各组肿瘤组织中Ki67、P53及TTF-1的表达水平,通过对比各组药物对Ki67、P53及TTF-1表达水平的影响,分析其对裸鼠移植瘤的作用并探讨肿瘤的预后。结果:1.中药降解产物几丁寡糖具有抑制肺腺癌A549细胞增殖和迁移的作用,其效果随着浓度的增加而增大。几丁寡糖对肺腺癌A549细胞作用24h的IC50为6.840mg/mL,后续实验均以7mg/mL为浓度参数。2.几丁寡糖作用于A549细胞24h、48h、72h及96h后的增殖抑制率分别为49.80%、55.90%、71.30%和73.50%,顺铂对肺腺癌细胞的增殖抑制率分别为25%、47.4%、61.7%和71.2%,而两药联合对A549细胞的增殖抑制率分别为52.4%、73.7%、86.8%和87.4%。药物作用24h后,几丁寡糖联合顺铂组对肿瘤细胞的抑制率与几丁寡糖组相比,差异无统计学意义(P0.05),但与顺铂组相比抑制率明显增高,差异具有统计学意义(P0.05);药物作用48h、72h和96h后几丁寡糖联合顺铂组对肿瘤细胞的抑制率与几丁寡糖组和顺铂组相比均明显增加,差异具有统计学意义(P0.05)。几丁寡糖联合顺铂能够明显抑制肺腺癌A549细胞的增殖能力,其作用大于单独应用几丁寡糖或顺铂。3.细胞划痕实验结果显示,当几丁寡糖药物浓度为1mg/mL时,24h、48h相对划痕宽度分别为82.33%、42.68%,当几丁寡糖药物浓度为3mg/mL时,24h、48h相对划痕宽度分别为83.46%、60.52%,而空白对照组分别为:84.78%、21.79%,差异具有统计学意义(P0.05)。几丁寡糖可以抑制肺腺癌A549细胞的迁移能力,且抑制作用随几丁寡糖药物浓度的增加而增大。当几丁寡糖药物浓度为7mg/mL时,24h、48h相对划痕宽度分别为81.79%、62.21%,当顺铂药物浓度为3μg/mL时,24h、48h相对划痕宽度分别为83.23%、55.91%,几丁寡糖和顺铂联合应用时,24h、48h相对划痕宽度分别为83.91%、69.67%,而空白对照组则分别为:84.11%、23.09%,各组间比较差异均具有统计学意义(P0.05)。几丁寡糖联合顺铂组与几丁寡糖组及顺铂组相比,可以更加显著的降低肺腺癌A549细胞的迁移能力。4.Transwell小室迁移实验计算A549细胞的迁移数量,空白对照组578±26、几丁寡糖组462±21、顺铂组423±28、几丁寡糖联合顺铂组236±23,几丁寡糖联合顺铂组与顺铂组、几丁寡糖组和空白对照组比较,差异有统计学意义(P0.05)。几丁寡糖联合顺铂可以明显抑制肺腺癌A549细胞的迁移能力,其作用大于单独应用几丁寡糖或顺铂。5.细胞凋亡实验显示几丁寡糖组作用于肺腺癌A549细胞株24h后,早期凋亡率和晚期凋亡率分别为11.8%和5.3%(总凋亡率17.1%),顺铂组早期凋亡率和晚期凋亡率分别为5.3%和4.5%(总凋亡率9.8%),几丁寡糖联合顺铂组早期凋亡率和晚期凋亡率分别为6.7%和8.0%(总凋亡率14.7%),而空白对照组则分别为3.4%和2.8%(总凋亡率6.2%)。两药联合组与几丁寡糖组相比,差异无统计学意义(P0.05),但分别与顺铂组和空白对照组相比,差异均具有统计学意义(P0.05)。中药降解产物几丁寡糖及与顺铂联合应用均能够诱导肺腺癌A549细胞凋亡。6.对裸鼠移植瘤组织进行HE染色镜下观察发现,空白对照组、几丁寡糖组及顺铂组肿瘤组织的形态学表现基本相同,即癌细胞呈弥漫性分布,且异型性明显,可见病理性核分裂像,肿瘤组织内部可见点状坏死;而几丁寡糖联合顺铂组镜下观察肿瘤组织,除了有上述表现以外,还可见局灶性坏死,并有细胞核破碎、固缩及染色质边集。7.对裸鼠移植瘤组织进行免疫组织化学染色结果显示,空白对照组、几丁寡糖组、顺铂组、几丁寡糖联合顺铂组各组之间Ki67免疫积分中位数分别为9、5、5、1,即Ki67的表达水平分别为强阳性(+++)、阳性(++)、阳性(++)和弱阳性(+),各用药组与空白对照组相比,表达量均显著降低,差异有统计学差异(P0.05);其中,几丁寡糖联合顺铂组与其他两单药组及空白对照组相比,表达量降低更为显著,差异有统计学差异(P0.05)。P53免疫积分中位数分别为2、1、1、1,即P53的表达水平均为弱阳性(+),各组间比较差异无统计学意义(P0.05)。TTF-1免疫积分中位数分别为3、1、1、0,即TTF-1的表达水平分别为弱阳性(+)、弱阳性(+)、弱阳性(+)和阴性(-),联合用药组与空白对照组及两单药组之间比较差异有统计学意义(P0.05),两单药组与空白对照组比较,差异无统计学意义(P0.05)。中药降解产物几丁寡糖联合顺铂能够明显下调裸鼠肺腺癌A549细胞移植瘤中Ki67、TTF-1的表达水平,其作用大于单独应用几丁寡糖或顺铂。结论:1.本研究从体外细胞学水平及体内动物模型上证明中药降解产物几丁寡糖联合顺铂在抑制肺腺癌A549细胞增殖、迁移以及诱导细胞凋亡方面具有良好的协同效果,其抑制增殖和迁移的作用优于单独应用几丁寡糖或顺铂。2.中药降解产物几丁寡糖联合顺铂可能是通过下调Ki67、TTF-1的表达水平,尤其是Ki67的表达水平,来促进肿瘤细胞的坏死,改善预后。
[Abstract]:Objective: 1. through in vitro cell experiments and in vivo animal experiments, the effects of the combination of chitosan oligosaccharides and cisplatin on the proliferation, migration and apoptosis of lung adenocarcinoma A549 cells and the effect of cisplatin combined with cisplatin on the xenografts in nude mice were analyzed, and the synergistic effect of the combination of the two drugs in the anti swelling tumor was discussed. In this study, three groups of drugs (oligosaccharides, cisplatin group, oligosaccharide combined cisplatin group) were used in the A549 cell line of human lung adenocarcinoma, and the MTS experiment, the cell scratch test, and the Transwell cell migration were used in this study. Experiments and flow cytometry were used to observe the effects of various drug groups on the biological behavior of A549 cells, such as proliferation, migration and apoptosis. At the same time, lung adenocarcinoma A549 cells were used to make subcutaneous transplantation tumor in nude mice. After the model was successful, the mice were randomly divided into four groups according to the different dosage, which were blank control group and several diced groups. The oligosaccharide group, the cisplatin group and the oligosaccharide combined with cisplatin group, the morphological changes of the tumor tissues were observed by HE staining method first, and then the expression of Ki67, P53 and TTF-1 in the tumor tissues was observed by immunohistochemistry. The effects of each group on the expression of Ki67, P53 and TTF-1 were compared and analyzed. The effect of xenograft in nude mice and the prognosis of tumor were discussed. Results: 1. the degradation product of chitosan oligosaccharides can inhibit the proliferation and migration of A549 cells in lung adenocarcinoma cells, and the effect increases with the increase of concentration. The IC50 of 24h in A549 cells of lung adenocarcinoma by chitosan oligosaccharides is 6.840mg/mL, followed by 7mg/mL as the concentration parameter.2. chitosan oligosaccharide oligosaccharide oligosaccharides The inhibitory rates of glucose on A549 cells 24h, 48h, 72h and 96h were 49.80%, 55.90%, 71.30% and 73.50% respectively. The proliferation inhibition rate of cisplatin to lung adenocarcinoma cells was 25%, 47.4%, 61.7% and 71.2% respectively, while the inhibition rate of the two drugs combined with A549 cells was 52.4%, 73.7%, 86.8% and 87.4%. drugs 24h, respectively, and the oligosaccharide combined with cisplatin. The inhibition rate of tumor cells was not statistically significant compared with that of the oligosaccharide group (P0.05), but the inhibition rate was significantly higher than that in the cisplatin group (P0.05). The inhibitory rate of the drug action 48h, 72h and 96h in the oligosaccharide combined with cisplatin group was significantly higher than that of the oligosaccharide group and cisplatin group. The difference was statistically significant (P0.05). A few oligosaccharides combined with cisplatin could significantly inhibit the proliferation of A549 cells in lung adenocarcinoma. The effect was greater than the results of the single use of chitosan oligosaccharides or cisplatin.3. cells. When the concentration of oligosaccharide drugs was 1mg/mL, the relative scratch width of 24h and 48h was 82.33%, 42.68%, respectively, when the oligosaccharides were oligosaccharides. When the drug concentration was 3mg/mL, the relative scratching width of 24h and 48h was 83.46% and 60.52% respectively, while the blank control group was 84.78%, 21.79%, and the difference was statistically significant (P0.05). The inhibition of chitosan oligosaccharides could inhibit the migration of A549 cells in lung adenocarcinoma, and the inhibitory effect was increased with the increase of the concentration of oligosaccharides. The relative scratch width of 24h and 48h was 81.79%, 62.21%, respectively, when the concentration of cisplatin was 3 mu g/mL, 24h, 48h relative scratch width was 83.23%, 55.91% respectively. The relative scratch width of 24h and 48h was 83.91% and 69.67% respectively when combined with oligosaccharide and cisplatin, while those in the blank control group were 84.11%, 23.09%, and the differences were all the differences in all groups. There were statistical significance (P0.05). Compared with the group of oligosaccharides and cisplatin, the group of oligosaccharide oligosaccharide combined with cisplatin could significantly reduce the migration ability of A549 cells in the lung adenocarcinoma cell.4.Transwell cell migration test to calculate the migration number of A549 cells, the blank control group was 578 + 26, the oligosaccharide group was 462 + 21, the cisplatin group was 423 + 28, and the oligosaccharide combined with the oligosaccharide group. The cisplatin group was 236 + 23, and the oligosaccharide oligosaccharide combined with cisplatin group and cisplatin group, the difference between the oligosaccharide group and the blank control group was statistically significant (P0.05). A few oligosaccharides combined with cisplatin could obviously inhibit the migration ability of A549 cells in lung adenocarcinoma, and the effect was greater than the single oligosaccharide or cisplatin.5. cell apoptosis experiment showed the oligosaccharide group. The early apoptosis rate and the advanced apoptosis rate were 11.8% and 5.3% (total apoptosis rate 17.1%), respectively. The early apoptosis rate and the late apoptosis rate of cisplatin group were 5.3% and 4.5% (total apoptosis rate 9.8%) respectively. The early apoptosis rate and late apoptosis rate of cisplatin combined with cisplatin group were 6.7% and 8%, respectively, and the total apoptosis rate was 14.7%, respectively, and the total apoptosis rate was 14.7%, respectively, and the rate of apoptosis was 14.7% in the early stage of cisplatin group. The control group was 3.4% and 2.8% (total apoptosis rate 6.2%) respectively. There was no significant difference between the combination group of two drugs and the oligosaccharide group (P0.05), but compared with the cisplatin group and the blank control group, the difference was statistically significant (P0.05). The combination of dicaropide oligosaccharides and cisplatin can induce A549 cell death in lung adenocarcinoma. The tumor tissues of nude mice were observed under the HE staining microscope. The morphological features of the tumor tissues of the blank control group, the oligosaccharide group and the cisplatin group were basically the same, that is, the diffuse distribution of the cancer cells and the obvious heteromorphosis, the pathological mitosis, and the spot necrosis in the inner part of the tumor tissue, and the combination of oligosaccharide and cisplatin in the group of.6.. In addition to the observation of tumor tissue, focal necrosis was observed in addition to the above findings, and nuclear fragmentation, condensation and chromatin.7. were used to stain the transplanted tumor tissue in nude mice. The median number of Ki67 immuno integral in the blank control group, the oligosaccharide group, the cisplatin group and the oligosaccharide combined cisplatin group The expression level of 9,5,5,1, namely Ki67, was strong positive (+ + +), positive (+ +), positive (+ +) and weak positive (+). Compared with blank control group, the expression level of each drug group was significantly lower than that in the blank control group (P0.05), and the expression of oligosaccharide combined with cisplatin group was significantly lower than that of the other two single drug groups and the blank control group. The difference was statistically significant (P0.05) the median of.P53 immune integral was 2,1,1,1, that is, the expression level of P53 was weak positive (+), and there was no significant difference between each group (P0.05), the median of.TTF-1 immune integral was 3,1,1,0, that is, the expression level of TTF-1 was divided into weak positive (+), weak positive (+), weak positive (+) and negative (-), combined use There was a significant difference between the drug group and the blank control group and the two single drug group (P0.05). The difference between the two single drug group and the blank control group was not statistically significant (P0.05). The degradation product of chitosan oligosaccharide combined with cisplatin could obviously reduce the expression level of Ki67 and TTF-1 in the A549 cell xenografts of nude mice, and the effect was greater than that of the individual. A few oligosaccharides or cisplatin were used. Conclusion: 1. this study showed that the biodegradable products of chitosan oligosaccharide combined with cisplatin have good synergistic effect on inhibiting the proliferation, migration and inducing apoptosis of A549 cells in lung adenocarcinoma cells, and the inhibition of proliferation and migration is better than that of several oligosaccharide oligosaccharides alone. Some oligosaccharides and cisplatin, the degradation product of sugar or cisplatin.2., may be by downregulating the expression level of Ki67 and TTF-1, especially the expression level of Ki67, to promote the necrosis of tumor cells and improve the prognosis.

【学位授予单位】：辽宁中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

【参考文献】