二硫鍵紫杉醇聚合物膠束抑制乳腺腫瘤的體內(nèi)外評(píng)價(jià)
發(fā)布時(shí)間：2019-04-22 20:30
【摘要】：紫杉醇(PTX)被廣泛用于乳腺癌、非小細(xì)胞肺癌等實(shí)體腫瘤的一線治療。但紫杉醇水溶性差,須將無水乙醇和聚氧乙基代蓖麻油作為溶媒,但易引發(fā)嚴(yán)重的過敏反應(yīng),限制了其臨床使用。為克服紫杉醇的水溶性差及實(shí)現(xiàn)腫瘤靶向控釋,諸多紫杉醇的新型制劑應(yīng)運(yùn)而生。其中,氧化還原型納米紫杉醇膠束制劑利用腫瘤細(xì)胞內(nèi)外及腫瘤細(xì)胞與正常細(xì)胞之間的氧化還原電位差,可實(shí)現(xiàn)高效的腫瘤細(xì)胞內(nèi)藥物觸發(fā)釋放。減少血液循環(huán)或正常細(xì)胞內(nèi)的藥物泄漏,降低相關(guān)的藥物毒副作用。目的:本研究以一種選擇性氧化還原型紫杉醇納米制劑[P(PEGMEA)-co-P(PDPHEMA)-g-PTX]作為研究對(duì)象,通過體內(nèi)外實(shí)驗(yàn)評(píng)價(jià)其作用于乳腺癌細(xì)胞株和動(dòng)物模型的效果,為該新型紫杉醇的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)。方法:(1)以生理鹽水稀釋成不同濃度梯度的二硫鍵紫杉醇處理乳腺癌MCF-7細(xì)胞株,24h后,MTT法測(cè)定MCF-7細(xì)胞生長抑制情況,并繪制濃度抑制率曲線,摸索其IC50。將MCF-7、MDA-MB-231,MCF-10A及U937共4種細(xì)胞系分別按照空白對(duì)照組:共聚物,陽性對(duì)照組:紫杉醇(PTX),給藥組:二硫鍵紫杉醇納米制劑。24h后用MTT法檢測(cè)細(xì)胞生長的抑制情況。另用受試藥物培養(yǎng)一組MDA-MB-231細(xì)胞,48h后,流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。(2)制作MCF-7裸鼠乳腺癌動(dòng)物模型。選擇荷瘤裸鼠18只,隨機(jī)分為3組(空白基質(zhì)對(duì)照組:共聚物,16mg/kg。陽性對(duì)照組:PTX,5mg/kg。給藥組:二硫鍵紫杉醇納米制劑,16mg/kg),并為裸鼠稱重、測(cè)量瘤體大小。給藥結(jié)束后檢測(cè)血常規(guī),計(jì)算腫瘤抑制率;同時(shí),留取裸鼠心、肝、脾、瘤等臟器進(jìn)行HE染色。另用同樣方法培養(yǎng)三組裸鼠,但投藥濃度為(共聚物,5mg/kg,PTX,5mg/kg;二硫鍵紫杉醇,5mg/kg)。(3)實(shí)驗(yàn)結(jié)果以均數(shù)±標(biāo)準(zhǔn)差(x±SD)表示,組間比較采用ANOVA方差分析,兩組間比較用t-檢驗(yàn),用SPSS 18.0對(duì)組間進(jìn)行統(tǒng)計(jì)分析,以P0.05為統(tǒng)計(jì)學(xué)差異具有顯著性。結(jié)果:(1)MTT試驗(yàn)結(jié)果顯示二硫鍵紫杉醇納米制劑隨著濃度增加,對(duì)MCF-7細(xì)胞活力抑制作用逐漸加強(qiáng),存在非常顯著的劑量與效應(yīng)關(guān)系,IC50=6.13μg/ml。以10μg/ml進(jìn)行投藥,與共聚物相比,對(duì)MCF-7細(xì)胞、MDA-MB-231細(xì)胞以及人正常乳腺細(xì)胞MCF-10A的抑制率分別為:52.4%,56.0%,22.4%(p0.05),對(duì)U937細(xì)胞活力具有促進(jìn)作用(24.3%),與10μg/ml紫杉醇組相比,對(duì)不同細(xì)胞的抑制率差異無統(tǒng)計(jì)學(xué)意義。二硫鍵紫杉醇與紫杉醇具有誘導(dǎo)MDA-MB-231細(xì)胞凋亡的作用。(2)體內(nèi)試驗(yàn)中,共聚物對(duì)照組和二硫鍵紫杉醇組裸鼠狀態(tài)較好。紫杉醇組裸鼠在給藥2周后精神狀態(tài)開始萎靡,活動(dòng)量減少并出現(xiàn)2只裸鼠死亡。(1)表觀藥物濃度相同時(shí),紫杉醇對(duì)照組和二硫鍵紫杉醇給藥組腫瘤生長被抑制,分別為:3590±877、4125±792mm3;瘤重分別為:3.62±1.0、4.58±0.9g。(2)實(shí)際紫杉醇含量相同,PTX組為:1936±399mm3,二硫鍵紫杉醇組為:1637±371 mm3。紫杉醇及二硫鍵紫杉醇組瘤體重量均為2.00±0.18g,抑瘤率為33.7%。二硫鍵紫杉醇無明顯骨髓抑制。HE染色組織切片顯示:腫瘤組織可見明顯壞死區(qū)域,心肺腎無明顯改變,肝、脾見少量炎細(xì)胞浸潤,二硫鍵紫杉醇組與紫杉醇組對(duì)腫瘤組織殺傷方面在病理學(xué)上未見明顯差別。結(jié)論:二硫鍵紫杉醇為水溶性藥物,無需聚氧乙基代蓖麻油和乙醇作為溶媒,消除了過敏反應(yīng)。二硫鍵紫杉醇納米制劑對(duì)乳腺癌MCF-7細(xì)胞活力具有非常顯著的抑制作用,其抑制作用具有濃度依賴性。二硫鍵紫杉醇與PTX均明顯抑制乳腺癌細(xì)胞增殖,對(duì)巨噬細(xì)胞U937具有免疫促進(jìn)作用,可誘導(dǎo)MDA-MB-231細(xì)胞凋亡。在體內(nèi)實(shí)驗(yàn)中,在取得與紫杉醇相同的抑瘤效果時(shí),二硫鍵紫杉醇組的紫杉醇含量更低。在達(dá)到最佳有效濃度后,再加大二硫鍵紫杉醇的濃度并不能明顯提高抑瘤效果。其抑制性作用主要集中于腫瘤組織,毒副作用低。
[Abstract]:Paclitaxel (PTX) is widely used for the first-line treatment of solid tumors such as breast cancer and non-small cell lung cancer. However, the water-solubility of paclitaxel is poor, and absolute ethyl alcohol and polyoxyethylene ether can be used as the solvent, but it is easy to cause serious allergic reaction and limit its clinical use. In order to overcome the water-solubility difference of the paclitaxel and to realize the targeted controlled release of the tumor, the novel preparation of the taxol has emerged. In which, the oxidation-reduction type nano-taxol micelle preparation can realize the drug-triggered release of the high-efficiency tumor cell by utilizing the redox potential difference between the inside of the tumor cells and the tumor cells and the normal cells. And the drug leakage in the blood circulation or normal cells is reduced, and the related medicine toxic and side effect is reduced. Objective: In this study, a selective oxidation-reduction type paclitaxel nano-preparation[P (PEGMEA)-co-P (PDPHEMA)-g-PTX] was used as the research object, and the effect of it on the breast cancer cell line and animal model was evaluated by out-of-vivo experiment, and the experimental basis for clinical application of the novel paclitaxel was provided. Methods: (1) The MCF-7 cell line of breast cancer was treated with the disulfide-bond paclitaxel which was diluted with normal saline to different concentration gradient. After 24 h, the growth inhibition of MCF-7 cells was determined by MTT method, and the concentration-inhibition curve was plotted and the IC50 of the MCF-7 cell line was determined. MCF-7, MDA-MB-231, MCF-10A and U937 cell lines were divided into two groups according to the control group: the copolymer, the positive control group, the paclitaxel (PTX) and the treated group: the disulfide-bond paclitaxel nano-preparation. The inhibition of cell growth was detected by the MTT method after 24 h. After a group of MDA-MB-231 cells were cultured with the test drug, the cell apoptosis was detected by flow cytometry after 48 h. And (2) preparing the MCF-7 nude mouse mammary cancer animal model. 18 tumor-bearing nude mice were randomly divided into 3 groups (blank matrix control group: copolymer,16 mg/ kg). Positive control group: PTX,5 mg/ kg. Treated group: disulfide-bond paclitaxel nano-preparation,16 mg/ kg) and weighed in nude mice to measure the size of the tumor. After the administration is over, the blood routine is detected and the tumor inhibition rate is calculated; at the same time, the viscera of the naked mouse heart, the liver, the spleen, the tumor and the like are left to carry out HE staining. Three groups of nude mice were cultured in the same manner, but the administration concentration was (copolymer,5 mg/ kg, PTX,5 mg/ kg; disulfide bond paclitaxel,5 mg/ kg). (3) The results of the experiment were represented by mean square standard deviation (x-SD). The ANOVA and variance analysis were used for the comparison between the two groups. The statistical analysis between the two groups was performed with SPSS 18.0, and the difference was significant between the two groups. Results: (1) The results of the MTT assay showed that the concentration of the disulfide-linked paclitaxel nano-preparation increased with the increase of the concentration, and the inhibitory effect on the activity of the MCF-7 cells was gradually enhanced, and there was a very significant dose-and-effect relationship, with the IC50 of 6.13. m The inhibition rate of MCF-7 cells, MDA-MB-231 cells and human normal breast cells (MCF-10A) was 52.4%, 56.0% and 22.4% (p0.05), respectively. The inhibition rate of different cells was not statistically significant. Disulfide and paclitaxel play a role in inducing the apoptosis of MDA-MB-231 cells. (2) In vivo test, the state of the nude mice of the copolymer control group and the disulfide bond paclitaxel group was better. After 2 weeks of administration, the state of mental state of the nude mice of the Paclitaxel group began to fall, and the amount of activity decreased and two nude mice died. (1) When the apparent drug concentration was the same, the tumor growth of the paclitaxel-treated group and the paclitaxel-treated group was inhibited, namely,3590-877,4125-792 mm3, and the tumor weight: 3.62-1.0, 4.58-0.9g, respectively. (2) The actual taxol content was the same, the PTX group was:1936-399 mm3, and the disulfide-bond paclitaxel group was 1637-371mm3. The weight of the paclitaxel and the disulfide-bonded paclitaxel group was 2.00-0.18 g, and the tumor-inhibiting rate was 33.7%. Disulfide-bound paclitaxel had no significant bone marrow inhibition. The histological sections of HE staining showed that the tumor tissue had obvious necrotic area, no obvious change in the heart and lung, and small amount of inflammatory cell infiltration in the liver and spleen, and no significant difference was seen in the pathology of the anti-killing of the tumor by the combination of the paclitaxel group and the paclitaxel group. Conclusion: The disulfide-bond paclitaxel is a water-soluble drug, and it is not necessary to use the polyoxyethylene ether and the ethanol as the solvent to eliminate the allergic reaction. The disulfide-linked taxol nano-preparation has a very significant inhibitory effect on the activity of the breast cancer MCF-7 cell, and the inhibitory effect of the disulfide-linked taxol nano-preparation has the concentration dependence. Both the disulfide bond and PTX inhibit the proliferation of breast cancer cells, and it has an immune function to the macrophage U937 and can induce the apoptosis of the MDA-MB-231 cells. In vivo experiments, the paclitaxel content of the disulfide-bond paclitaxel group was lower when the same tumor-inhibiting effect as paclitaxel was obtained. After the optimal effective concentration is reached, the concentration of the disulfide-bound paclitaxel can not be increased obviously, and the tumor-inhibiting effect can not be obviously improved. The inhibitory effect is mainly concentrated on the tumor tissues, and the toxic and side effects are low.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
本文編號(hào)：2463175
[Abstract]:Paclitaxel (PTX) is widely used for the first-line treatment of solid tumors such as breast cancer and non-small cell lung cancer. However, the water-solubility of paclitaxel is poor, and absolute ethyl alcohol and polyoxyethylene ether can be used as the solvent, but it is easy to cause serious allergic reaction and limit its clinical use. In order to overcome the water-solubility difference of the paclitaxel and to realize the targeted controlled release of the tumor, the novel preparation of the taxol has emerged. In which, the oxidation-reduction type nano-taxol micelle preparation can realize the drug-triggered release of the high-efficiency tumor cell by utilizing the redox potential difference between the inside of the tumor cells and the tumor cells and the normal cells. And the drug leakage in the blood circulation or normal cells is reduced, and the related medicine toxic and side effect is reduced. Objective: In this study, a selective oxidation-reduction type paclitaxel nano-preparation[P (PEGMEA)-co-P (PDPHEMA)-g-PTX] was used as the research object, and the effect of it on the breast cancer cell line and animal model was evaluated by out-of-vivo experiment, and the experimental basis for clinical application of the novel paclitaxel was provided. Methods: (1) The MCF-7 cell line of breast cancer was treated with the disulfide-bond paclitaxel which was diluted with normal saline to different concentration gradient. After 24 h, the growth inhibition of MCF-7 cells was determined by MTT method, and the concentration-inhibition curve was plotted and the IC50 of the MCF-7 cell line was determined. MCF-7, MDA-MB-231, MCF-10A and U937 cell lines were divided into two groups according to the control group: the copolymer, the positive control group, the paclitaxel (PTX) and the treated group: the disulfide-bond paclitaxel nano-preparation. The inhibition of cell growth was detected by the MTT method after 24 h. After a group of MDA-MB-231 cells were cultured with the test drug, the cell apoptosis was detected by flow cytometry after 48 h. And (2) preparing the MCF-7 nude mouse mammary cancer animal model. 18 tumor-bearing nude mice were randomly divided into 3 groups (blank matrix control group: copolymer,16 mg/ kg). Positive control group: PTX,5 mg/ kg. Treated group: disulfide-bond paclitaxel nano-preparation,16 mg/ kg) and weighed in nude mice to measure the size of the tumor. After the administration is over, the blood routine is detected and the tumor inhibition rate is calculated; at the same time, the viscera of the naked mouse heart, the liver, the spleen, the tumor and the like are left to carry out HE staining. Three groups of nude mice were cultured in the same manner, but the administration concentration was (copolymer,5 mg/ kg, PTX,5 mg/ kg; disulfide bond paclitaxel,5 mg/ kg). (3) The results of the experiment were represented by mean square standard deviation (x-SD). The ANOVA and variance analysis were used for the comparison between the two groups. The statistical analysis between the two groups was performed with SPSS 18.0, and the difference was significant between the two groups. Results: (1) The results of the MTT assay showed that the concentration of the disulfide-linked paclitaxel nano-preparation increased with the increase of the concentration, and the inhibitory effect on the activity of the MCF-7 cells was gradually enhanced, and there was a very significant dose-and-effect relationship, with the IC50 of 6.13. m The inhibition rate of MCF-7 cells, MDA-MB-231 cells and human normal breast cells (MCF-10A) was 52.4%, 56.0% and 22.4% (p0.05), respectively. The inhibition rate of different cells was not statistically significant. Disulfide and paclitaxel play a role in inducing the apoptosis of MDA-MB-231 cells. (2) In vivo test, the state of the nude mice of the copolymer control group and the disulfide bond paclitaxel group was better. After 2 weeks of administration, the state of mental state of the nude mice of the Paclitaxel group began to fall, and the amount of activity decreased and two nude mice died. (1) When the apparent drug concentration was the same, the tumor growth of the paclitaxel-treated group and the paclitaxel-treated group was inhibited, namely,3590-877,4125-792 mm3, and the tumor weight: 3.62-1.0, 4.58-0.9g, respectively. (2) The actual taxol content was the same, the PTX group was:1936-399 mm3, and the disulfide-bond paclitaxel group was 1637-371mm3. The weight of the paclitaxel and the disulfide-bonded paclitaxel group was 2.00-0.18 g, and the tumor-inhibiting rate was 33.7%. Disulfide-bound paclitaxel had no significant bone marrow inhibition. The histological sections of HE staining showed that the tumor tissue had obvious necrotic area, no obvious change in the heart and lung, and small amount of inflammatory cell infiltration in the liver and spleen, and no significant difference was seen in the pathology of the anti-killing of the tumor by the combination of the paclitaxel group and the paclitaxel group. Conclusion: The disulfide-bond paclitaxel is a water-soluble drug, and it is not necessary to use the polyoxyethylene ether and the ethanol as the solvent to eliminate the allergic reaction. The disulfide-linked taxol nano-preparation has a very significant inhibitory effect on the activity of the breast cancer MCF-7 cell, and the inhibitory effect of the disulfide-linked taxol nano-preparation has the concentration dependence. Both the disulfide bond and PTX inhibit the proliferation of breast cancer cells, and it has an immune function to the macrophage U937 and can induce the apoptosis of the MDA-MB-231 cells. In vivo experiments, the paclitaxel content of the disulfide-bond paclitaxel group was lower when the same tumor-inhibiting effect as paclitaxel was obtained. After the optimal effective concentration is reached, the concentration of the disulfide-bound paclitaxel can not be increased obviously, and the tumor-inhibiting effect can not be obviously improved. The inhibitory effect is mainly concentrated on the tumor tissues, and the toxic and side effects are low.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9
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