蜂毒穴位注射抑制肺癌生长转移及其抗血管生成作用机制研究

发布时间：2018-06-12 07:08

本文选题：肺癌 + 蜂毒　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景:肺癌是最常见的一种恶性肿瘤,并且是癌症相关死亡的重要原因之一,大约90%的肺癌患者死于肺癌转移而不是肺癌,而新生血管生成是肿瘤生长和转移的关键环节。肿瘤的无限制生长、组织浸润及周围组织与全身器官转移均依赖于血管生成,原位癌在无血管期的生长直径不超过2mm,并且该原位癌没有转移能力。当原位癌生长至直径达到2mm时,就需要新生血管的生成以提供足够的氧及营养物质使其继续生长。没有血管提供的氧和营养物质,原位癌就无法继续生长,而依然处于对宿主无害的状态,血管的生成使肿瘤的生长从没有血管的缓慢生长阶段变为有血管的快速生长阶段。因此,抑制原位癌的肿瘤血管生成就可显著的抑制肿瘤的生长和组织及器官转移。所以,以新生肿瘤血管为靶点,抑制肿瘤血管生成,阻断肿瘤细胞及肿瘤组织的氧和营养物质的来源及扩散通道,是抑制肿瘤组织生长以及组织器官转移的有效手段。蜂毒(Bee venom)是工蜂副腺和毒腺分泌出的一种透明液体,具有强烈刺激性气味、苦味和芳香气味,平时贮存于工蜂的毒囊中,于蛰刺时由蛰针排出。多方面研究表明蜂毒及针灸均对肿瘤有抑制作用。目的:本研究通过应用蜂毒干预,以lewis肺癌细胞为靶细胞,结合c57bl/6j小鼠实验,测量并计算lewis肺癌小鼠移植瘤体积、瘤重、抑瘤率、肿瘤血管数目,解剖显微镜下观察肺癌小鼠肺脏转移结节数,检测酪氨酸2(januskinase2,jak2)、磷酸化的酪氨酸2(phosphorylation-januskinase2,p-jak2)、转录因子3(signaltransducerandactivatoroftranscription,stat3)、磷酸化的转录因子3(phosphorylation-signaltransducerandactivatoroftranscription,p-stat3)、细胞因子信号抑制物3(suppressorofcytokinesignaling,socs3)、缺氧诱导因子-1α(hypoxiainduciblefactor-1,hif1α)、血管内皮生长因子(vascularendothelialgrowthfactor,vegf)的表达情况,从jak2/stat3信号通路的角度探讨蜂毒穴位注射抑制肺癌肿瘤血管生成及转移的具体作用机制。方法:建立lewis肺癌荷瘤小鼠模型,第7天,当c57bl/6j小鼠瘤体生长至最大径为0.5~0.7cm时,将其随机分5组,每组10只,其中模型组经腹腔注射0.1ml生理盐水,贝伐单抗组经腹腔注射0.1ml(0.22mg)贝伐单抗,蜂毒非经穴注射组于小鼠前肢上非经非穴处注射蜂毒0.1ml(0.04mg),蜂毒穴位注射组分别于小鼠合谷、尺泽穴位注射蜂毒0.025ml(每穴0.01mg,共0.04mg),每日1次,共17天。治疗期间,每3天测量小鼠体重和瘤体大小,第24天麻醉取材,称量瘤重、计算肺表面转移结节数,he染色后在光学显微镜下观察肺组织的组织学改变,采用免疫组化法检测肿瘤组织中cd34阳性的表达而后计算肿瘤血管密度,westernblot法、rt-qpcr法检测肿瘤组织中jak2、p-jak2、stat3、p-stat3、socs3、hif-1α及vegf的表达。结果:肿瘤体积及瘤质量,贝伐单抗组、蜂毒非经穴组,蜂毒穴位注射组明显低于模型组(p0.05),肿瘤体积及瘤质量蜂毒穴位注射低于蜂毒非经穴组(p0.05)。解剖显微镜下计数肺上转移结节,贝伐单抗组、蜂毒非经穴组及蜂毒穴位注射组明显低于模型组(p0.05),肺上转移结节数蜂毒穴位注射低于蜂毒非经穴组(p0.05)。免疫组化法检测肿瘤微血管密度,贝伐单抗组、蜂毒非经穴组及蜂毒穴位注射组明显低于模型组(p0.05),肿瘤微血管密度蜂毒穴位注射低于蜂毒非经穴组(p0.05)。rt-qpcr法检测p-jak2、p-stat3、hif-1α、vegf蛋白表达,贝伐单抗组、蜂毒非经穴组及蜂毒穴位注射组明显低于模型组(p0.05),蜂毒穴位注射组明显低于贝伐单抗组(p0.05),蜂毒穴位注射组明显低于蜂毒非经穴组(p0.05),p-jak2、p-stat3、hif-1α、vegf蛋白表达蜂毒穴位注射组与贝伐单抗组无明显差异(p0.05);socs3蛋白表达,贝伐单抗组及蜂毒穴位注射组明显高于蜂毒非经穴组及模型组(p0.05),蜂毒穴位注射组明显高于贝伐单抗组(p0.05),蜂毒穴位注射组明显高于贝伐单抗组(p0.05),socs3蛋白表达蜂毒非经穴组与模型组无明显差异(p0.05)。实时荧光定量pcr检测hif-1α、vegfmrna的表达,贝伐单抗组、蜂毒非经穴组及蜂毒穴位注射组明显低于生理盐水组(p0.05),hif-1α、vegfmrna的表达蜂毒穴位注射组明显低于蜂毒非经穴组(p0.05),hif-1α、vegfmrna的表达蜂毒穴位注射组与贝伐单抗组无明显差异(p0.05);SOCS3mRNA的表达,贝伐单抗组及蜂毒穴位注射组明显高于蜂毒非经穴组及生理盐水组(P0.05),SOCS3mRNA的表达蜂毒穴位注射组明显高于贝伐单抗组(P0.05)。结论:蜂毒穴位注射能抑制Lewis肺癌荷瘤小鼠肿瘤血管生成及转移,其作用机制与抑制JAK2/STAT3信号传导有关。
[Abstract]:Background: lung cancer is one of the most common malignant tumors, and is one of the important causes of cancer related death. About 90% of lung cancer patients die from lung cancer metastasis rather than lung cancer. Neovascularization is the key link of tumor growth and metastasis. Unrestricted growth of tumor, tissue infiltration, surrounding tissue and systemic organ metastasis are all dependent In angiogenesis, the growth of carcinoma in situ is not more than 2mm in the vascular stage, and the carcinoma in situ does not have the ability to metastasate. When in situ cancer grows to a diameter of 2mm, it requires the formation of new blood vessels to provide sufficient oxygen and nutrients to continue to grow. Without the oxygen and nutrients supplied from the blood vessels, cancer in situ can not continue to grow. Long, but still in a harmless state to the host, the formation of blood vessels makes the tumor grow from the slow growth stage without blood vessels into the rapid growth stage of the blood vessel. Therefore, the inhibition of the angiogenesis of the tumor in situ can significantly inhibit the growth of the tumor and the tissue and organ transfer. Therefore, the tumor angiogenesis is the target of the tumor. Tumor angiogenesis, blocking the source and diffusion channel of oxygen and nutrients in tumor cells and tumor tissues, is an effective means to inhibit the growth of tumor tissue and tissue and organs. Bee venom is a transparent liquid secreted by the accessory glands and poison glands of the worker bee. It has a strong irritation smell, bitter taste and aromatic smell, and is stored at ordinary times. In the venom of the worker bee, the stinging needles are discharged when stings. Many studies have shown that bee venom and acupuncture have inhibitory effects on the tumor. Objective: to measure and calculate the volume, tumor weight, tumor suppressor rate and number of tumor vessels in Lewis lung cancer rats by using the intervention of bee venom and using Lewis lung cancer cells as the target cells and c57bl/6j mice experiments. The number of lung metastatic nodules in lung cancer mice was observed under anatomic microscope, and tyrosine 2 (januskinase2, JAK2), phosphorylated tyrosine 2 (phosphorylation-januskinase2, p-jak2), transcription factor 3 (signaltransducerandactivatoroftranscription, STAT3), phosphorylated transcription factor 3 (phosphorylation-signaltransducerandactivatoroftranscri) were detected. Ption, p-STAT3), cytokine signal inhibitor 3 (suppressorofcytokinesignaling, SOCS3), hypoxia inducible factor -1 alpha (hypoxiainduciblefactor-1, HIF1 alpha), vascular endothelial growth factor (vascularendothelialgrowthfactor, VEGF) expression, from the angle of jak2/ STAT3 signal pathway to investigate the suppression of lung cancer tumor vessels by acupoint injection of bee venom The specific mechanism of formation and transfer. Methods: to establish a Lewis tumor bearing mouse model of lung cancer, seventh days, when the c57bl/6j mice grew to the maximum diameter of 0.5~0.7cm, they were randomly divided into 5 groups, 10 rats in each group. The model group was intraperitoneally injected with 0.1ml saline, bevacizumab group was intraperitoneally injected with 0.1ml (0.22mg) bevacizumab, and the bee venom was not injected into the meridian. Injection of bee venom 0.1ml (0.04mg) on the forelimb of mice on the forelimb of mice. The acupoint injection group of the bee venom was used in mice Hegu respectively. The acupoint of the injection of bee venom was injected with bee venom 0.025ml (0.01mg, 0.04mg), 1 times a day for 17 days. During the treatment, the weight of the mice and the mass of the tumor were measured every 3 days, the twenty-fourth days of intoxication were taken, the tumor was weighed and the metastatic nodules of the lung surface were calculated. After he staining, the histological changes of lung tissue were observed under the optical microscope. The expression of CD34 positive in tumor tissues was detected by immunohistochemical method and then the tumor vascular density was calculated, and the expression of JAK2, p-jak2, STAT3, p-STAT3, SOCS3, HIF-1 A and VEGF in tumor tissues was detected by Westernblot. Results: the volume and quality of the tumor, the mass of the tumor, and the mass of the tumor. FV group, bee venom non acupoint group, acupoint injection group of bee venom was significantly lower than model group (P0.05), tumor volume and injection of bee venom were lower than non meridian acupoint group (P0.05). Anatomic microscope was used to count pulmonary metastasis nodules, bevacizumab group, bee venom non meridian group and bee venom acupoint injection group were significantly lower than model group (P0.05), pulmonary metastasis. Point injection of nodule number of bee venom was lower than that of non acupoint acupoint group (P0.05). Immuno histochemical method was used to detect tumor microvascular density. Bevacizumab group, non acupoint group of bee venom and acupoint injection group of bee venom were significantly lower than that of model group (P0.05). The acupoint injection of tumor microvascular density was lower than that of non meridian acupoint group (P0.05).Rt-qpcr method for detecting p-jak2, p-STAT3, HIF-1 a, VEGF protein expression, bevacizumab group, bee venom non acupoint group and acupoint injection group were significantly lower than model group (P0.05), bee venom acupoint injection group was significantly lower than bevacizumab group (P0.05), bee venom acupoint injection group was significantly lower than the non meridian acupoint group (P0.05), p-jak2, p-STAT3, HIF-1 a, VEGF protein expression of bee venom acupoint injection group and bevacizumab group There was no significant difference (P0.05), the expression of SOCS3 protein, bevacizumab group and acupoint injection group of bee venom were significantly higher than that of the non meridian acupoint group and model group (P0.05), the acupoint injection group of the bee venom was obviously higher than the bevacizumab group (P0.05), the acupoint injection group of the bee venom was obviously higher than the bevacizumab group (P0.05), and the SOCS3 protein expressed no obvious point in the acupoint group and the model group of the bee venom. Difference (P0.05). Real time fluorescence quantitative PCR detection of HIF-1 a, VEGFmRNA expression, bevacizumab group, bevacizumab group, non meridian acupoint group and acupoint injection group were significantly lower than normal saline group (P0.05), HIF-1 alpha, the expression of bee venom in acupoint injection group was obviously lower than that of non acupoint group (P0.05), HIF-1 alpha, VEGFmRNA expression of bee venom and beg The expression of SOCS3mRNA, bevacizumab group and acupoint injection group of bevacizumab group were significantly higher than that of non meridian acupoint group and saline group (P0.05). The expression of bee venom in acupoint injection group (SOCS3mRNA) was significantly higher than that of bevacizumab group (P0.05). Conclusion: acupoint injection of bee venom can inhibit the angiogenesis of tumor bearing mice of Lewis lung cancer and P0.05. The mechanism of metastasis is related to the inhibition of JAK2/STAT3 signaling.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245

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