蜂毒穴位注射抑制肺癌生長(zhǎng)轉(zhuǎn)移及其抗血管生成作用機(jī)制研究
本文選題:肺癌 + 蜂毒��; 參考:《重慶醫(yī)科大學(xué)》2017年碩士論文
【摘要】:背景:肺癌是最常見(jiàn)的一種惡性腫瘤,并且是癌癥相關(guān)死亡的重要原因之一,大約90%的肺癌患者死于肺癌轉(zhuǎn)移而不是肺癌,而新生血管生成是腫瘤生長(zhǎng)和轉(zhuǎn)移的關(guān)鍵環(huán)節(jié)。腫瘤的無(wú)限制生長(zhǎng)、組織浸潤(rùn)及周?chē)M織與全身器官轉(zhuǎn)移均依賴(lài)于血管生成,原位癌在無(wú)血管期的生長(zhǎng)直徑不超過(guò)2mm,并且該原位癌沒(méi)有轉(zhuǎn)移能力。當(dāng)原位癌生長(zhǎng)至直徑達(dá)到2mm時(shí),就需要新生血管的生成以提供足夠的氧及營(yíng)養(yǎng)物質(zhì)使其繼續(xù)生長(zhǎng)。沒(méi)有血管提供的氧和營(yíng)養(yǎng)物質(zhì),原位癌就無(wú)法繼續(xù)生長(zhǎng),而依然處于對(duì)宿主無(wú)害的狀態(tài),血管的生成使腫瘤的生長(zhǎng)從沒(méi)有血管的緩慢生長(zhǎng)階段變?yōu)橛醒艿目焖偕L(zhǎng)階段。因此,抑制原位癌的腫瘤血管生成就可顯著的抑制腫瘤的生長(zhǎng)和組織及器官轉(zhuǎn)移。所以,以新生腫瘤血管為靶點(diǎn),抑制腫瘤血管生成,阻斷腫瘤細(xì)胞及腫瘤組織的氧和營(yíng)養(yǎng)物質(zhì)的來(lái)源及擴(kuò)散通道,是抑制腫瘤組織生長(zhǎng)以及組織器官轉(zhuǎn)移的有效手段。蜂毒(Bee venom)是工蜂副腺和毒腺分泌出的一種透明液體,具有強(qiáng)烈刺激性氣味、苦味和芳香氣味,平時(shí)貯存于工蜂的毒囊中,于蟄刺時(shí)由蟄針排出。多方面研究表明蜂毒及針灸均對(duì)腫瘤有抑制作用。目的:本研究通過(guò)應(yīng)用蜂毒干預(yù),以lewis肺癌細(xì)胞為靶細(xì)胞,結(jié)合c57bl/6j小鼠實(shí)驗(yàn),測(cè)量并計(jì)算lewis肺癌小鼠移植瘤體積、瘤重、抑瘤率、腫瘤血管數(shù)目,解剖顯微鏡下觀察肺癌小鼠肺臟轉(zhuǎn)移結(jié)節(jié)數(shù),檢測(cè)酪氨酸2(januskinase2,jak2)、磷酸化的酪氨酸2(phosphorylation-januskinase2,p-jak2)、轉(zhuǎn)錄因子3(signaltransducerandactivatoroftranscription,stat3)、磷酸化的轉(zhuǎn)錄因子3(phosphorylation-signaltransducerandactivatoroftranscription,p-stat3)、細(xì)胞因子信號(hào)抑制物3(suppressorofcytokinesignaling,socs3)、缺氧誘導(dǎo)因子-1α(hypoxiainduciblefactor-1,hif1α)、血管內(nèi)皮生長(zhǎng)因子(vascularendothelialgrowthfactor,vegf)的表達(dá)情況,從jak2/stat3信號(hào)通路的角度探討蜂毒穴位注射抑制肺癌腫瘤血管生成及轉(zhuǎn)移的具體作用機(jī)制。方法:建立lewis肺癌荷瘤小鼠模型,第7天,當(dāng)c57bl/6j小鼠瘤體生長(zhǎng)至最大徑為0.5~0.7cm時(shí),將其隨機(jī)分5組,每組10只,其中模型組經(jīng)腹腔注射0.1ml生理鹽水,貝伐單抗組經(jīng)腹腔注射0.1ml(0.22mg)貝伐單抗,蜂毒非經(jīng)穴注射組于小鼠前肢上非經(jīng)非穴處注射蜂毒0.1ml(0.04mg),蜂毒穴位注射組分別于小鼠合谷、尺澤穴位注射蜂毒0.025ml(每穴0.01mg,共0.04mg),每日1次,共17天。治療期間,每3天測(cè)量小鼠體重和瘤體大小,第24天麻醉取材,稱(chēng)量瘤重、計(jì)算肺表面轉(zhuǎn)移結(jié)節(jié)數(shù),he染色后在光學(xué)顯微鏡下觀察肺組織的組織學(xué)改變,采用免疫組化法檢測(cè)腫瘤組織中cd34陽(yáng)性的表達(dá)而后計(jì)算腫瘤血管密度,westernblot法、rt-qpcr法檢測(cè)腫瘤組織中jak2、p-jak2、stat3、p-stat3、socs3、hif-1α及vegf的表達(dá)。結(jié)果:腫瘤體積及瘤質(zhì)量,貝伐單抗組、蜂毒非經(jīng)穴組,蜂毒穴位注射組明顯低于模型組(p0.05),腫瘤體積及瘤質(zhì)量蜂毒穴位注射低于蜂毒非經(jīng)穴組(p0.05)。解剖顯微鏡下計(jì)數(shù)肺上轉(zhuǎn)移結(jié)節(jié),貝伐單抗組、蜂毒非經(jīng)穴組及蜂毒穴位注射組明顯低于模型組(p0.05),肺上轉(zhuǎn)移結(jié)節(jié)數(shù)蜂毒穴位注射低于蜂毒非經(jīng)穴組(p0.05)。免疫組化法檢測(cè)腫瘤微血管密度,貝伐單抗組、蜂毒非經(jīng)穴組及蜂毒穴位注射組明顯低于模型組(p0.05),腫瘤微血管密度蜂毒穴位注射低于蜂毒非經(jīng)穴組(p0.05)。rt-qpcr法檢測(cè)p-jak2、p-stat3、hif-1α、vegf蛋白表達(dá),貝伐單抗組、蜂毒非經(jīng)穴組及蜂毒穴位注射組明顯低于模型組(p0.05),蜂毒穴位注射組明顯低于貝伐單抗組(p0.05),蜂毒穴位注射組明顯低于蜂毒非經(jīng)穴組(p0.05),p-jak2、p-stat3、hif-1α、vegf蛋白表達(dá)蜂毒穴位注射組與貝伐單抗組無(wú)明顯差異(p0.05);socs3蛋白表達(dá),貝伐單抗組及蜂毒穴位注射組明顯高于蜂毒非經(jīng)穴組及模型組(p0.05),蜂毒穴位注射組明顯高于貝伐單抗組(p0.05),蜂毒穴位注射組明顯高于貝伐單抗組(p0.05),socs3蛋白表達(dá)蜂毒非經(jīng)穴組與模型組無(wú)明顯差異(p0.05)。實(shí)時(shí)熒光定量pcr檢測(cè)hif-1α、vegfmrna的表達(dá),貝伐單抗組、蜂毒非經(jīng)穴組及蜂毒穴位注射組明顯低于生理鹽水組(p0.05),hif-1α、vegfmrna的表達(dá)蜂毒穴位注射組明顯低于蜂毒非經(jīng)穴組(p0.05),hif-1α、vegfmrna的表達(dá)蜂毒穴位注射組與貝伐單抗組無(wú)明顯差異(p0.05);SOCS3mRNA的表達(dá),貝伐單抗組及蜂毒穴位注射組明顯高于蜂毒非經(jīng)穴組及生理鹽水組(P0.05),SOCS3mRNA的表達(dá)蜂毒穴位注射組明顯高于貝伐單抗組(P0.05)。結(jié)論:蜂毒穴位注射能抑制Lewis肺癌荷瘤小鼠腫瘤血管生成及轉(zhuǎn)移,其作用機(jī)制與抑制JAK2/STAT3信號(hào)傳導(dǎo)有關(guān)。
[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.
【學(xué)位授予單位】:重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2017
【分類(lèi)號(hào)】:R245
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