本文選題：幾丁寡糖 + 順鉑　； 參考：《遼寧中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：目的:1.通過(guò)體外細(xì)胞實(shí)驗(yàn)及體內(nèi)動(dòng)物實(shí)驗(yàn),分析中藥降解產(chǎn)物幾丁寡糖單獨(dú)應(yīng)用及與順鉑聯(lián)合應(yīng)用對(duì)肺腺癌A549細(xì)胞增殖、遷移、凋亡能力的影響以及對(duì)裸鼠移植瘤的作用,探討兩藥聯(lián)合在抗腫瘤作用方面是否具有良好的協(xié)同效果,為未來(lái)幾丁寡糖在抗腫瘤治療領(lǐng)域中的開(kāi)發(fā)應(yīng)用及臨床轉(zhuǎn)化,提供實(shí)驗(yàn)基礎(chǔ)及理論依據(jù)。材料與方法:本研究采用三組藥物(幾丁寡糖組、順鉑組、幾丁寡糖聯(lián)合順鉑組)作用于人肺腺癌A549細(xì)胞系,通過(guò)MTS實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)、transwell小室遷移實(shí)驗(yàn)及流式細(xì)胞凋亡實(shí)驗(yàn),對(duì)比觀察各藥物組對(duì)肺腺癌A549細(xì)胞增殖、遷移及凋亡等生物學(xué)行為的影響;同時(shí)應(yīng)用肺腺癌A549細(xì)胞對(duì)裸鼠進(jìn)行皮下移植瘤造模,待造模成功后,按照給藥的不同將裸鼠隨機(jī)分為四組,分別為空白對(duì)照組、幾丁寡糖組、順鉑組和幾丁寡糖聯(lián)合順鉑組,先利用HE染色的方法鏡下觀察各組腫瘤組織的形態(tài)學(xué)變化,再應(yīng)用免疫組織化學(xué)的方法,觀察各組腫瘤組織中Ki67、P53及TTF-1的表達(dá)水平,通過(guò)對(duì)比各組藥物對(duì)Ki67、P53及TTF-1表達(dá)水平的影響,分析其對(duì)裸鼠移植瘤的作用并探討腫瘤的預(yù)后。結(jié)果:1.中藥降解產(chǎn)物幾丁寡糖具有抑制肺腺癌A549細(xì)胞增殖和遷移的作用,其效果隨著濃度的增加而增大。幾丁寡糖對(duì)肺腺癌A549細(xì)胞作用24h的IC50為6.840mg/mL,后續(xù)實(shí)驗(yàn)均以7mg/mL為濃度參數(shù)。2.幾丁寡糖作用于A549細(xì)胞24h、48h、72h及96h后的增殖抑制率分別為49.80%、55.90%、71.30%和73.50%,順鉑對(duì)肺腺癌細(xì)胞的增殖抑制率分別為25%、47.4%、61.7%和71.2%,而兩藥聯(lián)合對(duì)A549細(xì)胞的增殖抑制率分別為52.4%、73.7%、86.8%和87.4%。藥物作用24h后,幾丁寡糖聯(lián)合順鉑組對(duì)腫瘤細(xì)胞的抑制率與幾丁寡糖組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),但與順鉑組相比抑制率明顯增高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);藥物作用48h、72h和96h后幾丁寡糖聯(lián)合順鉑組對(duì)腫瘤細(xì)胞的抑制率與幾丁寡糖組和順鉑組相比均明顯增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。幾丁寡糖聯(lián)合順鉑能夠明顯抑制肺腺癌A549細(xì)胞的增殖能力,其作用大于單獨(dú)應(yīng)用幾丁寡糖或順鉑。3.細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,當(dāng)幾丁寡糖藥物濃度為1mg/mL時(shí),24h、48h相對(duì)劃痕寬度分別為82.33%、42.68%,當(dāng)幾丁寡糖藥物濃度為3mg/mL時(shí),24h、48h相對(duì)劃痕寬度分別為83.46%、60.52%,而空白對(duì)照組分別為:84.78%、21.79%,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。幾丁寡糖可以抑制肺腺癌A549細(xì)胞的遷移能力,且抑制作用隨幾丁寡糖藥物濃度的增加而增大。當(dāng)幾丁寡糖藥物濃度為7mg/mL時(shí),24h、48h相對(duì)劃痕寬度分別為81.79%、62.21%,當(dāng)順鉑藥物濃度為3μg/mL時(shí),24h、48h相對(duì)劃痕寬度分別為83.23%、55.91%,幾丁寡糖和順鉑聯(lián)合應(yīng)用時(shí),24h、48h相對(duì)劃痕寬度分別為83.91%、69.67%,而空白對(duì)照組則分別為:84.11%、23.09%,各組間比較差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。幾丁寡糖聯(lián)合順鉑組與幾丁寡糖組及順鉑組相比,可以更加顯著的降低肺腺癌A549細(xì)胞的遷移能力。4.Transwell小室遷移實(shí)驗(yàn)計(jì)算A549細(xì)胞的遷移數(shù)量,空白對(duì)照組578±26、幾丁寡糖組462±21、順鉑組423±28、幾丁寡糖聯(lián)合順鉑組236±23,幾丁寡糖聯(lián)合順鉑組與順鉑組、幾丁寡糖組和空白對(duì)照組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。幾丁寡糖聯(lián)合順鉑可以明顯抑制肺腺癌A549細(xì)胞的遷移能力,其作用大于單獨(dú)應(yīng)用幾丁寡糖或順鉑。5.細(xì)胞凋亡實(shí)驗(yàn)顯示幾丁寡糖組作用于肺腺癌A549細(xì)胞株24h后,早期凋亡率和晚期凋亡率分別為11.8%和5.3%(總凋亡率17.1%),順鉑組早期凋亡率和晚期凋亡率分別為5.3%和4.5%(總凋亡率9.8%),幾丁寡糖聯(lián)合順鉑組早期凋亡率和晚期凋亡率分別為6.7%和8.0%(總凋亡率14.7%),而空白對(duì)照組則分別為3.4%和2.8%(總凋亡率6.2%)。兩藥聯(lián)合組與幾丁寡糖組相比,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),但分別與順鉑組和空白對(duì)照組相比,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。中藥降解產(chǎn)物幾丁寡糖及與順鉑聯(lián)合應(yīng)用均能夠誘導(dǎo)肺腺癌A549細(xì)胞凋亡。6.對(duì)裸鼠移植瘤組織進(jìn)行HE染色鏡下觀察發(fā)現(xiàn),空白對(duì)照組、幾丁寡糖組及順鉑組腫瘤組織的形態(tài)學(xué)表現(xiàn)基本相同,即癌細(xì)胞呈彌漫性分布,且異型性明顯,可見(jiàn)病理性核分裂像,腫瘤組織內(nèi)部可見(jiàn)點(diǎn)狀壞死;而幾丁寡糖聯(lián)合順鉑組鏡下觀察腫瘤組織,除了有上述表現(xiàn)以外,還可見(jiàn)局灶性壞死,并有細(xì)胞核破碎、固縮及染色質(zhì)邊集。7.對(duì)裸鼠移植瘤組織進(jìn)行免疫組織化學(xué)染色結(jié)果顯示,空白對(duì)照組、幾丁寡糖組、順鉑組、幾丁寡糖聯(lián)合順鉑組各組之間Ki67免疫積分中位數(shù)分別為9、5、5、1,即Ki67的表達(dá)水平分別為強(qiáng)陽(yáng)性(+++)、陽(yáng)性(++)、陽(yáng)性(++)和弱陽(yáng)性(+),各用藥組與空白對(duì)照組相比,表達(dá)量均顯著降低,差異有統(tǒng)計(jì)學(xué)差異(P0.05);其中,幾丁寡糖聯(lián)合順鉑組與其他兩單藥組及空白對(duì)照組相比,表達(dá)量降低更為顯著,差異有統(tǒng)計(jì)學(xué)差異(P0.05)。P53免疫積分中位數(shù)分別為2、1、1、1,即P53的表達(dá)水平均為弱陽(yáng)性(+),各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。TTF-1免疫積分中位數(shù)分別為3、1、1、0,即TTF-1的表達(dá)水平分別為弱陽(yáng)性(+)、弱陽(yáng)性(+)、弱陽(yáng)性(+)和陰性(-),聯(lián)合用藥組與空白對(duì)照組及兩單藥組之間比較差異有統(tǒng)計(jì)學(xué)意義(P0.05),兩單藥組與空白對(duì)照組比較,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。中藥降解產(chǎn)物幾丁寡糖聯(lián)合順鉑能夠明顯下調(diào)裸鼠肺腺癌A549細(xì)胞移植瘤中Ki67、TTF-1的表達(dá)水平,其作用大于單獨(dú)應(yīng)用幾丁寡糖或順鉑。結(jié)論:1.本研究從體外細(xì)胞學(xué)水平及體內(nèi)動(dòng)物模型上證明中藥降解產(chǎn)物幾丁寡糖聯(lián)合順鉑在抑制肺腺癌A549細(xì)胞增殖、遷移以及誘導(dǎo)細(xì)胞凋亡方面具有良好的協(xié)同效果,其抑制增殖和遷移的作用優(yōu)于單獨(dú)應(yīng)用幾丁寡糖或順鉑。2.中藥降解產(chǎn)物幾丁寡糖聯(lián)合順鉑可能是通過(guò)下調(diào)Ki67、TTF-1的表達(dá)水平,尤其是Ki67的表達(dá)水平,來(lái)促進(jìn)腫瘤細(xì)胞的壞死,改善預(yù)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.

【學(xué)位授予單位】：遼寧中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R734.2

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