川芎嗪联合顺铂影响Lewis肺癌小鼠移植瘤生长及血管生成的研究

发布时间：2018-06-10 06:05

本文选题：血管生成 + 含凝血酶敏感素基序I型的解聚素样金属蛋白酶　；参考：《河北北方学院》2016年硕士论文

【摘要】：原发性支气管肺癌(primary bronchogenic carcinoma)的发病率和死亡率位于各种恶性肿瘤之首,大约85%的肺癌是非小细胞肺癌,侵袭和转移是肺癌治疗失败的重要原因之一。肿瘤血管生成与恶性肿瘤的侵袭、转移以及治疗后复发密切相关,血管生成是一个多基因参与,多步骤循序渐进的复杂过程,研究血管生成相关因子及具体的分子机制为肿瘤的治疗提供更多基因位点。因此阻断血管生成已经成为研究肿瘤治疗和预防的热点。含凝血酶敏感素基序I型的解聚素样金属蛋白酶(A disintegrin and metallopeptidase with thrombospondin motif type1,ADAMTS1)是最近发现的具有抗肿瘤活性的金属蛋白酶,能够抑制血管生成,但在肺癌中的功能尚不明确。众所周知,血管内皮生长因子是在促进血管生成方面作用最强的因子,能够很好的反应血管内皮的生长状态,降低血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达,可以破坏血管生成网络进而抑制肿瘤生长。CD105(endoslin,EDG)在恶性肿瘤血管生成、发生、发展及生物学行为中发挥重要作用。川芎嗪是我国传统中医药川芎的有效成分,临床上普遍用于心脑血管疾病的治疗。研究发现川芎嗪能够诱导肿瘤细胞向凋亡方向发展,提高机体免疫功抑制血管内皮细胞中促血管生成因子的表达来抑制肿瘤的生长。顺铂为目前临床上最常用的肿瘤化疗药物之一,能够作用于多种肿瘤、药效强、与其他化疗药物联用时能够增加疗效,但长期使用易产生耐药。有研究发现,川芎嗪与具有细胞毒性化疗药物联用,可提高抗肿瘤的治疗效果。本试验通过应用川芎嗪与顺铂对接种Lewis肺腺癌细胞株小鼠进行药物作用后,通过测量小鼠肿瘤体积及肿瘤重量的变化,观察上述药物对小鼠移植瘤生长的影响。同时通过应用川芎嗪及顺铂对肺癌移植瘤小鼠进行一定的药物影响,检测ADAMTS1、VEGF、MVD-CD105三种蛋白表达水平的变化,观察肺癌组织中瘤细胞及微血管形态的变化,证实川芎嗪是否具有抗肿瘤作用,并阐述川芎嗪抗肿瘤血管生成的可能机制,从而为研究川芎嗪抗肿瘤血管生成可能的发生机制以及今后用于临床治疗提供实验依据。具体内容如下:健康雄性C57BL/6小鼠购买后适应性喂养一周后,将培养好的Lewis肺腺癌细胞接种于C57BL/6小鼠右腋下,细胞液体总量为0.2ml,建立移植瘤模型,2周后接种处皮下可触及肿块大小约8mm。按数字表法将实验动物随机分为四组:生理盐水组,川芎嗪组,顺铂组,联合治疗组。各组小鼠接种细胞后在动物饲养室内常规饲养,接种后2周开始按计划给药。生理盐水组:腹腔注射0.9%氯化钠0.2 m L,每天1次,连续14天;顺铂组,腹腔注射2 mgkg-1,顺铂组0.2 m L。每天1次,连续14天;川芎嗪组,腹腔注射,川芎嗪0.2 m L。每天1次,连续14天。联合组,腹腔注射川芎嗪100 mgkg-1+顺铂2 mgkg-1,共0.2 m L。每天1次,连续14天。每次给药前,称量体重后调整用药剂量。实验小鼠在治疗结束后第5天脱颈法处死,解剖瘤组织,固定液固定。所有瘤组织用电子天平测量瘤重量,并用游标卡尺测量瘤组织长短径,计算瘤体积,抑瘤率及肿瘤坏死率,透射电镜观察瘤组织超微结构,免疫组织化学及蛋白印迹方法检测药物作用前后ADAMTS1、VEGF、微血管密度的变化,微血管密度的变化由CD105染色来确定。1.治疗后小鼠生存质量和毒副反应的评估治疗方案结束时,以对照组小鼠的一般情况为标准,通过精神状态、活动状况、饮食情况、体重及肿瘤体积变化等指标的观察,评估小鼠生存质量和对化疗药物的毒副反应。与生理盐水组比较,川芎嗪组小鼠饮食及活动良好,小鼠体重和肿瘤体积增长幅度相当。顺铂组小鼠精神萎靡,饮食量少,活动量明显减少,皮毛缺乏光泽,并出现脱毛现象,喜蜷缩成堆,体重下降最明显,肿瘤体积比川芎嗪组增长慢。联合治疗组小鼠的饮食及活动量,皮毛色泽及脱毛程度较顺铂组为好,而差于川芎嗪组。结果提示:川芎嗪组小鼠用药后,生存质量较好,未产生严重药物毒副反应;联合治疗组小鼠的毒副反应轻于顺铂治疗组,顺铂组毒副反应表现最重,肿瘤体积动态变化情况依次为:生理盐水组、川芎嗪组、顺铂组、联合组,结果提示川芎嗪与顺铂联用能够降低单用顺铂治疗的毒副作用。2.移植瘤小鼠抑瘤率及肿瘤坏死率的观察药物治疗后观察移植瘤小鼠肿瘤的抑瘤率:川芎嗪组(35.58%)、顺铂组(40.41%)、联合治疗组(62.48%),与生理盐水组比较,各用药组肿瘤抑瘤率明显增高,差异有显著性(P0.05)。移植瘤小鼠肿瘤的坏死率:川芎嗪组(15.58±3.19)、顺铂组(24.64±11.96)以及联合治疗组(31.76±15.20)肿瘤的坏死高于生理盐水组(9.37±1.36),差异有显著性(P0.05)。与顺铂组相比,联合治疗组移植瘤小鼠抑瘤率、肿瘤坏死率明显增高,差异有显著性(P0.05),而顺铂组优于川芎嗪组,提示川芎嗪及顺铂联合应用其抗肿瘤作用优于顺铂。3.含凝血酶敏感素基序I型的解聚素样金属蛋白酶,血管内皮生长因子,微血管密度在Lewis肺癌组织中的表达待肿瘤组织全部取出后,免疫组织化学方法检测VEGF、ADAMTS1结果显示:VEGF在四组中呈阳性表达,与生理盐水组(56367.82±7441.12)比较,川芎嗪组(28674.58±2331.40)、顺铂组(23932.76±3151.59)以及联合治疗组(19183.99±1453.64)表达明显减少,联合治疗组表达最低,差异显著(P0.05)。ADAMTS1在四组中呈阳性表达,川芎嗪组(15475.27±2064.88)、顺铂组(18382.79±2162.08)以及联合治疗组(26403.85±3293.36)表达明显高于对照组(1646.74±212.96),联合治疗组表达最高,生理盐水组表达最低,差异显著(P0.05)。蛋白印迹方法结果显示:川芎嗪组(0.57±0.02)、顺铂组(0.79±0.01)、联合治疗组(0.98±0.02)ADAMTS1的表达明显高于生理盐水组(0.48±0.01),差异显著(P0.05),而VEGF的表达生理盐水组(0.87±0.03)表达最高,联合治疗组(0.28±0.01)表达最低。提示免疫组织化学方法与蛋白印迹方法结果基本一致,川芎嗪、顺铂均可降低肺腺癌VEGF的表达,升高其ADAMTS1的表达,川芎嗪联合顺铂治疗能够进一步增强其抑制VEGF表达能力,以及促进ADAMTS1的表达能力。4.微血管密度计数的检测免疫组织化学方法测定微血管密度指标CD105,光学显微镜下观察肿瘤血管,参照Weidner法进行微血管密度(microvessel density,MVD)的计数,请病理科专家3名,独立观察5个高倍视野,最终计算平均值,结果表明:生理盐水组表达最高(4.9×104±7.3×103),联合治疗组表达最低(1.4×104±6.2×103)。5.透射电镜下观察肿瘤细胞形态及血管结构透射电镜下结果显示生理盐水组肿瘤细胞分布密集,细胞核仁大而明显,核型不规则,线粒体及粗面内质网等细胞器丰富,偶见凋亡细胞。川芎嗪组可见部分肿瘤细胞脂肪变性,胞浆内脂滴增多,形成不完整细胞膜,线粒体空泡化,可见坏死细胞及碎片,细胞器减少。顺铂组及联合组瘤细胞分布稀少,可见部分细胞胞浆线粒体肿胀,可见较多细胞碎片,常见凋亡肿瘤细胞,偶见凋亡小体形成。综上所述,Lewis肺腺癌细胞接种于C57BL/6小鼠后8~10天,可造模成功。通过实验荷瘤小鼠的一般生存状态、体重、瘤体积、瘤重、血管超微结构、肿瘤细胞、血管生成因子的变化,抑瘤率的比较通过实验观察移植瘤小鼠生存状态、体重、肿瘤体积、微血管超微结构、血管生成蛋白表达水平变化,以及肿瘤坏死率及抑瘤率的比较,说明川芎嗪与化疗药物顺铂同时应用可提高小鼠生存质量,降低毒副反应;川芎嗪能够抑制Lewis肺癌小鼠移植瘤生长,与顺铂联合具有协同作用。川芎嗪联合顺铂能通过抑制VEGF表达,促进ADAMTS1的表达,减少微血管生成,进而抑制肿瘤生长。
[Abstract]:The incidence and mortality of primary bronchogenic carcinoma (primary bronchogenic carcinoma) are the first of all kinds of malignant tumors. About 85% of the lung cancer is non small cell lung cancer. Invasion and metastasis are one of the important reasons for the failure of lung cancer treatment. Generation is a complex process of multi gene participation and step by step process. The study of angiogenesis related factors and specific molecular mechanisms provides more gene loci for the treatment of tumor. Therefore, blocking angiogenesis has become a hot spot in the study of tumor treatment and prevention. The depolymerization hormone like protease containing thrombin sensitive preorder I (A disintegrin and metallopeptidase with thrombospondin motif Type1, ADAMTS1) is a recently discovered metalloproteinase with antitumor activity, which inhibits angiogenesis, but the function in lung cancer is not clear. It is well known that vascular endothelial growth factor is the strongest factor in promoting angiogenesis and can be very good. The expression of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) can destroy the angiogenesis network and inhibit the growth of.CD105 (endoslin, EDG) and play an important role in the angiogenesis, development, development and biological behavior of malignant tumor. Ligustrazine is a traditional Chinese tradition. The effective components of Ligusticum chuanxiong, a traditional Chinese medicine, are widely used in the treatment of cardiovascular and cerebrovascular diseases. It is found that Ligustrazine can induce tumor cells to develop to the direction of apoptosis and improve the immune function to inhibit the expression of angiogenic factors in vascular endothelial cells to inhibit the growth of tumor. One of the drugs, which can act on a variety of tumors, is potent and can increase the efficacy when combined with other chemotherapeutic drugs, but it is easy to produce drug resistance in the long term. It is found that Ligustrazine is combined with cytotoxic chemotherapeutic drugs to improve the effect of antitumor treatment. This experiment has been conducted with Ligustrazine and cisplatin on Lewis lung adenocarcinoma cells. The effect of the above drugs on the growth of transplanted tumor in mice was observed by measuring the changes of tumor volume and weight of tumor in mice, and the effects of Ligustrazine and cisplatin on the lung cancer transplanted mice were affected by the use of Ligustrazine and cisplatin. The changes of the expression level of the three proteins of ADAMTS1, VEGF and MVD-CD105 were detected and the lung was observed. The changes of tumor cells and microvascular morphology in cancer tissues confirm whether tetramethylpyrazine has anti-tumor effect, and expounds the possible mechanism of Ligustrazine antitumor angiogenesis, thus providing experimental basis for the study of the possible mechanism of Ligustrazine antitumor angiogenesis and clinical treatment in the future. The specific contents are as follows: healthy male C57BL/ After 6 mice were fed for one week after adaptive feeding, the cultured Lewis lung adenocarcinoma cells were inoculated into the right axillary C57BL/6 mice. The total amount of cell liquid was 0.2ml, and the transplanted tumor model was established. After 2 weeks, the size of the palpable mass in the subcutaneous area of the inoculation was divided into four groups randomly: saline group, ligustrazine group and cisplatin group. The mice were inoculated in the treatment group. After inoculation, the mice were fed in the animal feeding room routinely. 2 weeks after inoculation, the saline group was given 0.9% sodium chloride 0.2 m L, 1 times a day for 14 days; cisplatin group, intraperitoneal injection of 2 mg? Kg-1, 0.2 m L. per day for 14 days; Ligustrazine group, intraperitoneal injection of Ligustrazine 0.2 m L. each. 1 times a day for 14 days. The combined group was intraperitoneally injected with Ligustrazine 100 mg? Kg-1+ cisplatin 2 mg? Kg-1, a total of 0.2 m L., 1 times a day for 14 days. The dose of the drug was adjusted after each administration, after weighing the body weight. The experimental mice were sacrificed at fifth days after the treatment, and the tumor tissue and fixed solution were fixed. All tumor tissues measured the weight of the tumor by electronic balance. The length and diameter of tumor tissue were measured with vernier caliper, the tumor volume, tumor suppressor rate and tumor necrosis rate were calculated. The ultrastructure of the tumor tissue was observed by transmission electron microscope. The changes of ADAMTS1, VEGF, microvessel density before and after the action of the drug were detected by immunohistochemistry and Western blot. The changes of microvascular density were determined by CD105 staining to determine the survival quality of the mice after.1. treatment. At the end of the evaluation of the dose and side effects, the general condition of the mice in the control group was taken as the standard. The quality of life and the side effects on the chemotherapeutic drugs were evaluated through the observation of mental state, activity condition, diet, weight and tumor volume, and the diet and activity of the Ligustrazine group in the group of Ligustrazine, compared with the saline group. The body weight and tumor volume increased fairly well. The cisplatin group was depressed, the diet was less, the amount of activity was reduced, the hair lacked luster, and the hair was dehairing, and the body weight decreased most obviously. The volume of tumor was slower than that of the Ligustrazine group. The diet and activity of the mice in the combined treatment group, the color of fur and the hair removal process in the combined treatment group were more than that of the Ligustrazine group. Compared with the cisplatin group, it was better than the Ligustrazine group. The results suggested that the qustrazine group had better quality of survival and no serious drug side effects after the use of the tetramethylpyrazine group. The side effects of the combined treatment group were less than the cisplatin treatment group. The side effects of the cisplatin group were the heaviest, and the dynamic changes of the tumor volume were in the normal saline group, the Ligustrazine group, and the group of tetramethylpyrazine. The combination of cisplatin and cisplatin, the results suggest that the combination of Ligustrazine and cisplatin can reduce the tumor suppressor rate and tumor necrosis rate of.2. transplanted tumor mice with cisplatin alone. After the treatment, the tumor suppressor rate of the transplanted tumor mice was observed: Ligustrazine group (35.58%), cisplatin group (40.41%), combined treatment group (62.48%), compared with the saline group. The tumor tumor inhibition rate in each group was significantly higher (P0.05). The necrosis rate of the tumor in the transplanted mice was (15.58 + 3.19), the tumor necrosis in the cisplatin group (24.64 + 11.96) and the combined treatment group (31.76 + 15.20) were higher than that of the normal saline group (9.37 + 1.36), and the difference was significant (P0.05). The combined treatment group was compared with the cisplatin group. The tumor necrosis rate and tumor necrosis rate of tumor mice increased significantly (P0.05), while cisplatin group was superior to the Ligustrazine group. It suggested that the combination of Ligustrazine and cisplatin was better than cisplatin.3. containing thrombin sensitive I type depolymer like metalloproteinase, vascular endothelial growth factor and microvascular density in Lewis lung cancer tissue. After all the tumor tissues were removed, VEGF was detected by immunohistochemistry. The results of ADAMTS1 showed that VEGF was positive in the four groups, compared with that of the normal saline group (56367.82 + 7441.12), the Ligustrazine group (28674.58 + 2331.40), the cisplatin group (23932.76 + 3151.59) and the combined treatment group (19183.99 + 1453.64), and the combined treatment of the group (19183.99 + 1453.64). The expression of the treatment group was the lowest, the difference was significant (P0.05).ADAMTS1 positive expression in the four groups, the Ligustrazine group (15475.27 + 2064.88), the cisplatin group (18382.79 + 2162.08) and the combined treatment group (26403.85 + 3293.36) was significantly higher than the control group (1646.74 + 212.96), the combined treatment group was the highest expression, the physiological saline group was the lowest, the difference was significant (P0.05). Eggs (P0.05). The results of white blot showed that the group of tetramethylpyrazine (0.57 + 0.02) and cisplatin group (0.79 + 0.01), the expression of (0.98 + 0.02) ADAMTS1 in the combined treatment group was significantly higher than that of the saline group (0.48 + 0.01), and the difference was significant (P0.05), while the expression of VEGF in the saline group (0.87 + 0.03) was the highest, and the expression of the combined treatment group (0.28 + 0.01) was the lowest. The results are basically consistent with the results of Western blot. Tetramethylpyrazine and cisplatin can reduce the expression of VEGF in lung adenocarcinoma and increase the expression of ADAMTS1. Ligustrazine Combined with cisplatin can further enhance its inhibition of VEGF expression, and promote the expression of ADAMTS1 in.4. microvessel density count by immunohistochemical method. The vascular density index (CD105), the tumor vessels were observed under the optical microscope, the count of microvessel density (MVD) was counted by the Weidner method. 3 experts in the pathology department were asked to observe 5 high times of visual field independently, and the average value was calculated. The results showed that the expression of the normal saline group was the highest (4.9 * 104 + 7.3 x 103), and the expression of the combined treatment group was the lowest (1.4). The morphology and vascular structure of the tumor cells were observed under transmission electron microscope (104 + 6.2 x 103). The results of transmission electron microscopy showed that the tumor cells in the saline group were densely distributed, the nucleolus were large and obvious, the karyotype was irregular, the mitochondria and the rough endoplasmic reticulum were abundant, and the apoptotic cells were found. The Ligustrazine group showed that some tumor cells were fatty degeneration and cell. The plasma lipid droplets increased, formed incomplete cell membrane, mitochondria vacuolation, necrotic cells and fragments, and organelles decreased. The distribution of tumor cells in cisplatin group and combined group was rare, the mitochondria swelling in the cytoplasm of some cells, more cell fragments, common apoptotic tumor cells, and occasional apoptotic bodies formed. To sum up, Lewis lung adenocarcinoma cells The survival state, body weight, tumor volume, tumor weight, vascular ultrastructure, changes of tumor cells, angiogenic factors and tumor suppressor rate were compared through experiments to observe the survival state, body weight, tumor volume, microvascular ultrastructure and angiogenesis of transplanted mice through experiment. The change of protein expression level and the comparison of tumor necrosis rate and tumor suppressor rate showed that Ligustrazine and cisplatin can improve the quality of survival and reduce the side effects. Ligustrazine can inhibit the growth of transplanted tumor in Lewis lung cancer mice, and have synergistic effect with cisplatin. Ligustrazine Combined with cisplatin can inhibit the expression of VEGF by inhibiting the combination of cisplatin and cisplatin. It can promote the expression of ADAMTS1, reduce angiogenesis and inhibit tumor growth.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R734.2

【参考文献】