摘要
随着对PPARs基因的深入了解,PPARγ在肿瘤中的作用日益成为学者们研究的焦点,有不少研究证明,其激动剂在乳腺癌、结肠癌、胃癌、前列腺癌研究中能够促进肿瘤细胞的凋亡,淋巴结转移是非小细胞肺癌患者术后复发和死亡等预后不良的重要因素。其中PPARγ的特异性配体能诱导多种肿瘤的终末分化,抑制肿瘤细胞的增殖,在脂肪肉瘤和前列腺癌患者的临床治疗中,PPARγ配体已经显示了良好的抑制肿瘤效果。随着淋巴管内皮特异标记物和淋巴管生长因子的发现,NSCLC诱发淋巴管生成的分子机制和以淋巴管生成(lymphangiogenesis)作为抗肿瘤转移靶点的研究已成为目前研究的热点之一,抑制肿瘤新生淋巴管可能为控制非小细胞肺癌淋巴道转移提供新的思路,为此,本研究选择跟PPARγ表达有关的非小细胞肺癌肿瘤细胞系A549细胞,来证实罗格列酮通过下调IGF-1的表达来抑制非小细胞肺癌的淋巴转移及诱导凋亡,意在寻找治疗非小细胞肺癌的一种更好的方法和药物。
胰岛素样生长因子(Insulin-like growth factor, IGF)特别是IGF1不仅能够促进非小细胞肺癌的无限增殖,而且与淋巴结转移的关系密切,IGF-1可作为非小细胞肺癌生物学行为尤其是淋巴结转移的判断指标。罗格列酮是过氧化物酶体增殖激活受体γ(PPARγ)激动剂,它能够诱导非小细胞肺癌凋亡并抑制非小细胞肺癌细胞侵袭及转移。并且,罗格列酮已被证明能够通过PI3K／Akt途径抑制乳腺癌细胞中IGF-Ⅰ的表达。据此我们推测罗格列酮可能通过抑制IGF-Ⅰ的来抑制肺癌细胞的淋巴转移。为此,本试验观察罗格列酮作用与A549细胞后IGF-Ⅰ的表达,研究并探讨罗格列酮抑制非小细胞肺癌增值及转移的机制。
【研究目的】
探讨罗格列酮通过下调IGF-1的表达来抑制非小细胞肺癌的淋巴转移及诱导凋亡。
【研究方法】
1. A549细胞的培养及罗格列酮对A549细胞的处理；
2.采用Hoechst3222染色法检测罗格列酮对肺癌A549细胞凋亡的影响：转染后48小时,Hoechst3222染色,于荧光倒置显微镜下观测胞核变化。分别取5个高倍镜视野,计数凋亡变化的细胞个数所占比例为凋亡率,取平均值,并与空白对照组细胞做比较。重复研究3次；
3.透射电镜观察细胞超微结构的改变；
4. Real time PCR检测罗格列酮对A549细胞VEGF-C表达的影响；
5.采用免疫荧光化学法观察罗格列酮处理A549细胞后VEGF-C的表达；
6. Western blot对下调后的IGF-1进行半定量分析：罗格列酮处理细胞24小时后,提取细胞的总蛋白,Bicinchoninic acid assay (BCA)蛋白测定试剂盒测定蛋白浓度。进行SDS-PAGE电泳,目的条带用AlphaEaseFC软件分析系统进行灰度值分析。PPARγ条带与actin条带吸光度的比值表示蛋白表达量；
7.对结果进行统计学分析,采用均数±标准差的方式对数据进行描述,凋亡率采用χ2分析,VEGF-C及IGF-1的表达采用方差分析。
【研究结果】
1.罗格列酮诱导A549细胞凋亡呈浓度依赖性,浓度分别为0,10,20,30μM的罗格列酮,处理48小时后,凋亡率分别为0.012±0.033、0.105±0.177、0.388±0.201、0.437±0.189；A549细胞在在处理后出现早期核凋亡改变,核膜皱缩,成波浪状,染色体凝集成块状。48小时后,凋亡细胞明显增加,染色质致密浓染呈粗大的颗粒状,边集,或整个核呈辐轮状；有些细胞已有凋亡小体突出；或整个细胞核致密浓染,或分裂成凋亡小体。并用透射电镜观察细胞超微结构的改变
2.罗格列酮下调VEGF-C的表达,罗格列酮作用48小时后,可见处理组的荧光比空白组明显减弱,而阴性对照组未见特异性荧光表达。
3.罗格列酮下调IGF-1的表达成浓度依赖性,罗格列酮处理A549细胞24小时后,IGF-1的表达量分别为0.761±0.006、0.4101±0.0142、0.361355±0.006、0.209±0.018。
【研究结论】
罗格列酮能够通过下调IGF-1的表达实现其对非小细胞肺癌淋巴转移及增殖的抑制作用,IGF-1可能是存在于诱导细胞凋亡及抑制其转移的关键位点。而罗格列酮能够降低IGF-1的原位表达,为非小细胞肺癌的治疗供新的试验基础及方向。
With the deeper understanding of PPARs gene, there was an increasing interest in the roles of PPARγ's in cancer. Several studies have indicated that PPARγ's agonist could induce apoptosis of tumor cells in breast cancer colorectal carcinoma、gastric cancer、Prostate Cancer, The metastasis of Lymph node in non-small cell lung cancer is an important factor in acromegalic poor prognosis such as recurrence and death, In which the corresponding ligands of PPARγcan induce physical terminal differentiation of a variety of tumors, inhibit tumor cell proliferation, in clinical treatments of liposarcoma and prostate cancer, PPARγligands have shown good anti-tumor effect. With the discovery of the lymphatic endothelium-specific markers and lymphatic growth factor, The molecular mechanisms of lymphangiogenesis by NSCLC-induced and with lymphangiogenesis as a target of anti-tumor metastasis has become a hot spot in the current study, The inhibition of tumor lymphangiogenesis may be provide new ideas for the controlling of non-small cell lung cancer lymph node metastasis. In this test, we use the cancer cells A549, who have been improved to be with expression of PPARγ. To study the effects of rosiglitazone on inhibiting lymph node metastasis and the induction of apoptosis of non-small cell lung cancer by down-regulating the expression of IGF-1. We observe the changes of these cells to investigate the roles of PPARγin non-small cell lung cancer and to find a better way to cure this disease.
Insulin-like growth factor (Insulin-like growth factor, IGF) in particular, IGF1 not only promote the infinite proliferation of non-small cell lung cancer, but also make a close relationship with lymph node metastasis, IGF-1 can be used as biological behavior of non-small cell lung cancer, especially can be the indicators to judge the transfer of lymph node. Rosiglitazone is a peroxisome proliferator-activated receptorγ(PPARγ) agonists, which can induce apoptosis in non-small cell lung cancer and inhibit non-small cell lung cancer cell invasion and metastasis. Moreover, rosiglitazone has been shown to inhibit breast cancer cells through the PI3K/Akt channels in IGF-I expression. Accordingly, we speculated that rosiglitazone may be by inhibiting IGF-I to inhibit lung cancer cells in lymph node metastasis. Therefore, this experiment observed the role of rosiglitazone on A549 cells with the expression of IGF-1 to study and to explore mechanism of rosiglitazone inhibit non-small cell lung cancer metastasis and Numerical increasing.
【Objective】
To rosiglitazone on inhibiting lymph node metastasis and the induction study the effects of of apoptosis of non-small cell lung cancer by down-regulating the expression of IGF-1.
【Methods】
1. The culture of A549 cells and the treatment of rosiglitazone on A549 cells;
2. Hoechst3222 staining was adopted to detect apoptosis of the A549 cell treated by rosiglitazone with different concentration. At 48 hours after transfection, Hoechst3222 staining was performed, fluorescence microscope was used to observing changes in nuclei. Choose five high-power vision, count the number of apoptotic nuclei, and compare with blank control group cells;
3. TEM was used to observe the ultrastructural changes of cell;
4. Real time PCR was used to detect the local expression of VEGF-C in lung cancer A549 cells dealed by rosiglitazone;
5. Immunofluorescence technique was used to detect the local expression of VEGF-C in lung cancer A549 cells dealed by rosiglitazone.And make the bar graph of the cells;
6. Western blot was used to do the semi- quantitative analysis of the IGF-1 expression. Westen blotting detect expression of PPARγprotein Cells were harvested at 24 hours after transfection for SDS-PAGE electrophoresis. AlphaEaseFC software analysis system was used to analysis of gray value of the strip;
7. Data was described in the mode of x±s.Apoptosis ratio was analyzed by the way of chi square test analosis, and the expression of VEGF-C and IGFI-1 was analyed by the way of ANOVA.
【Results】
1. Apoptosis of A549 cells induced by rosiglitazone is a dose-dependent. Apoptosis ratio induced by Rosiglitazone with different concentration of 0,10,20,30μM were 0.012±0.033.0.105±0.177、0.388±0.201、0.437±0.189. After transfection, Early signs of apoptosis took place in small part of nuclear in interfering group. Nuclear membrane shrinked and turned into a wavy; After 48 hours, a marked increase of apoptotic cells appeared. Chromatin was concentrated into large granular and was of margination. Apoptotic bodies had appeared in a few cells or the entire nucleus turned into dense concentration or apoptotic bodies. TEM was used to observe the ultrastructural changes of cell.
2. Rosiglitazone down-regulated local expression of VEGF-C in a dose-dependent manner. After 48h treated by Rosiglitazone, fluorescence was weaken compared with blank control group, while there was no specific fluorescence in the negative control group.
3. The expression of IGF-1 were 0.761±0.006、0.410±0.0142、0.361355±0.006、0.209±0.018 after treated by different concentration of Rosiglitazone treating 24h later.
【Conclusions】
IGF-1 pathway may play a key role in the mechanism in which Rosiglitazone significantly inhibit metastasis and induce apoptosis of non-small cell lung cancer cells. IGF-1 may play a key role in inducing apoptosis and inhibiting lymph node metastasis.
引文
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