纤调蛋白在小细胞肺癌肿瘤血管生成中的作用及机制研究

发布时间：2018-05-08 00:40

本文选题：纤调蛋白 + 肿瘤血管生成　；参考：《第三军医大学》2016年博士论文

【摘要】：背景肿瘤新生血管为肿瘤细胞的生长和转移提供了重要的物质来源和结构基础,因此抑制肿瘤血管生成不仅可以切断肿瘤细胞的营养供给,使肿瘤萎缩退化、缺血坏死,还可以阻断肿瘤细胞经血管途径向远处转移,抑制转移瘤的形成[10]。小细胞肺癌(small cell lung cancer,SCLC)是侵袭性极高的恶性肿瘤,SCLC细胞具有分化程度低、恶性程度高的生物学特性,易早期侵犯血管发生远处转移。据统计,广泛期小细胞肺癌的平均生存期仅7-12个月,5年生存率低于2%,疾病预后极差[3]。另外,小细胞肺癌也是血管依赖性肿瘤,其丰富的新生血管网为肿瘤细胞提供氧气和营养物质,清除局部代谢产物,也为肿瘤细胞早期局部扩散和远处转移建立了血性通道。因此,探索引起小细胞肺癌肿瘤血管异常增生的促血管生成因子及其作用机制有望为研发有效的小细胞肺癌血管生成抑制剂提供研究基础。纤调蛋白(fibromodulin,FMOD)是富含亮氨酸重复序列蛋白聚糖(small leucine-rich proteoglycans,SLRPs)家族成员[11]。近年来诸多研究报道,FMOD是一种新发现的内源性促血管生成因子,在生理性和病理性血管增生活动中具有重要的调控作用[4-6]。本课题组前期研究比较SCLC亲代细胞株H446及其耐药细胞株H446/DDP的基因表达谱,发现FMOD在两种细胞株中均高表达,两者mRNA表达水平没有显著差异。采用RT-PCR和Western Blot进一步验证获得一致结果。由此说明FMOD的异常表达与SCLC的发生发展存在潜在的联系,但与SCLC的获得性耐药没有显著相关性。采用免疫组织化学法(immunohistochemistry,IHC)检测FMOD在SCLC患者肿瘤组织标本中的表达情况,发现FMOD在近一半肿瘤组织标本中表达阳性,而在正常肺组织标本中无一例阳性,这进一步表明FMOD在SCLC中的异常表达可能与其发生发展密切相关。基于FMOD的促血管生成作用,我们推测FMOD可能参与了SCLC的血管生成。目前FMOD在肿瘤血管生成中的作用尚不清楚,其作用机制也有待深入研究。因此,本课题将在SCLC的疾病研究背景下探讨FMOD对肿瘤血管生成的作用及其分子机制。首先采用基因沉默技术下调FMOD基因表达,接着从临床水平、动物水平、细胞和分子水平多个层面研究FMOD对SCLC肿瘤血管生成的影响,阐明FMOD促SCLC肿瘤血管生成的作用及其相关机制。旨在为以FMOD作为靶点开发新的SCLC抗血管生成药物提供实验研究的基础,并最终达到抑制SCLC发生发展,改善疾病预后的目的。方法1.收集SCLC临床组织标本,以及正常肺泡上皮组织标本作为对照,经甲醛固定、石蜡包埋和切片处理后采用免疫组织化学法检测FMOD在组织标本中的表达情况。采集标本所对应临床患者的病历资料,包括年龄、性别、吸烟、肿瘤分期和预后等临床特征参数,采用?2检验法分析FMOD的表达水平和各项临床指针的相关性。免疫组化法检测组织标本中血管内皮标记物CD31的表达,计数微血管密度,初步验证FMOD异常表达与SCLC血管生成的相关性。2.将靶向FMOD的shRNA序列5’-CCGCATGAAGTACGTCTACTT-3’[7]克隆到慢病毒载体pGLV3/H1/GFP+Puro Vector中构建重组慢病毒穿梭质粒LV3-shFMOD。同时构建包含无序片段5’-TTCTCCGAACGTGTCACGT-3’的重组慢病毒LV3-shCON。用重组慢病毒转染H446细胞,嘌呤霉素筛选转染细胞,Western Blot方法检测经转染的H446细胞内合成的FMOD和培养液中分泌的FMOD表达水平,目的是验证H446细胞是否能合成和分泌FMOD蛋白,LV3-shFMOD是否能有效抑制FMOD的表达和分泌。3.将重组慢病毒转染后的H446细胞种植在免疫缺陷小鼠(severe combined immunodeficiency mice,SCID mice)背部皮下组织,建立SCLC移植瘤动物模型。定期称量SCID鼠体重,监测体重随时间的变化规律;定期测量移植瘤径长,评估瘤体的生长情况;经尾静脉注入对比增强剂进行血管造影B超检查[9],检测肿瘤区域的血流动力学指标:血流峰灰阶值和峰值时间,评估肿瘤的血流灌注情况;摘取移植瘤,用免疫组化法检测FMOD和CD31的表达,进行微血管计数,从在体水平进一步验证FMOD在肿瘤血管生成中的作用。4.用CCK8试剂盒测定H446细胞的增殖活性,transwell小室检测H446细胞的侵袭和迁移能力,分析FMOD对H446细胞生物学活性的影响。收集H446培养上清作为条件培养基(conditioned medium,CM)。将人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)与CM共培养,用CCK8试剂盒测定不同表达水平的分泌型FMOD对HUVECs增殖活性的影响。将HUVECs和H446细胞分别培养于transwell共培养系统的上室和下室,测定分泌型FMOD对HUVECs侵袭和迁移能力的影响。Western Blot检测HUVECs与CM共培养后,促血管生成生长因子的表达情况。结果1.共收集27例SCLC肺穿针吸组织标本,其中4例临床病历资料不全,3例石蜡切片标本未检测出癌性组织,剩余20例标本纳入研究,进行免疫组化检测和统计学分析。(1)实验结果显示,FMOD在SCLC临床组织中的阳性表达率为55%,阳性表达定位于细胞核和细胞质,FMOD在正常肺组织中均表达阴性。采用Fisher’s确切概率法分析得出,FMOD的表达与患者年龄、性别、吸烟和预后无相关性,与SCLC肿瘤分期显著相关(P=0.017)。(2)FMOD表达阳性的组织标本微血管数量为43.89?10.45,FMOD表达阴性的组织微血管数量为23.84?10.54,采用t检验法分析得出,微血管数量在FMOD阳性表达的肿瘤组织中显著高于FMOD阴性组织(P=0.013)。2.重组质粒的测序结果表明慢病毒重组载体LV3-shFMOD构建正确,包含靶向FMOD的shRNA目的序列。慢病毒滴度测定结果为1?108 TU/ml,慢病毒感染H446细胞72h,荧光强度最强。用嘌呤霉素筛选转染成功的细胞,Western Blot检测细胞内和培养上清液中的FMOD表达水平,结果显示转染shFMOD的H446细胞内和培养液中的FMOD含量均显著性低于对照组(分别为P0.001,P0.05)。结果一方面表明H446细胞能合成和分泌FMOD蛋白;另一方面表明重组质粒构建正确,shRNA-FMOD能有效抑制FMOD表达和分泌。3.实验组(接种H446-shFMOD细胞)SCID鼠和对照组(接种H446-shCON细胞)SCID鼠均在细胞种植后第4天形成新生瘤体,实验组平均瘤体体积小于对照组(P=0.001)。随后在第7天、第14天、第21天和第28天测量得出实验组瘤体体积均小于对照组(P=0.003,0.003,0.004,0.002)。实验组SCID鼠的体重在观察期间持续增长,对照组SCID鼠体重在第14天后开始下降(P0.05),恶病质表现明显,出现SCID鼠死亡。血管B超造影检查发现实验组肿瘤区域的平均血流峰值(10.08?3.49)显著低于对照组(26.27?4.93,P0.001),实验组肿瘤的血流峰值时间(9.28?4.88s)较对照组(2.80?0.77s)显著延长(P=0.022)。采用免疫组化法对CD31标记的肿瘤微血管染色,计数结果显示实验组瘤体组织微血管数量为16.03?2.84,对照组相应值为35.87?13.41,t检验分析发现实验组微血管数量明显减少(P=0.006)。4.H446细胞转染LV3-shFMOD后,细胞增殖、侵袭和迁移活性较对照组均无显著差异(P0.05)。将HUVECs分别培养于培养上清CM-shFMOD和CM-shCON中,CCK-8检测发现HUVEC在CM-shFMOD中的增殖活性较对照显著下降(P0.05);将HUVECs分别与H446-shFMOD和H446-shCON共培养,检测结果显示与H446-shFMOD共培养的HUVECs侵袭和迁移能力均下降,具有统计学差异(P0.001)。5.将HUVECs分别培养于条件培养基CM-shFMOD和CM-shCON中,用Western Blot检测HUVECs的促血管生成生长因子的表达水平。结果显示,与对照组相比,HUVECs与CM-shFMOD共培养后,血管生长因子VEGF、FGF-2、PDGF-B、TGF-?1表达均显著下调(P0.05)。结论1.FMOD在SCLC临床组织中的异常表达与肿瘤分期相关,提示FMOD可能成为预测SCLC肿瘤分期的潜在标志物。2.FMOD在SCLC临床组织中的异常表达与微血管密度相关,提示FMOD可能参与了小细胞肺癌的肿瘤血管生成。3.成功构建靶向FMOD基因的shRNA重组慢病毒载体,重组载体能显著抑制SCLC细胞的FMOD表达和分泌。4.移植瘤动物模型充分证明FMOD能诱导SCLC的肿瘤血管生成,并能增加肿瘤组织的血流灌注,从而促进肿瘤的生长。5.SCLC细胞合成的内源性FMOD对其自身的增殖活性、侵袭和迁移能力无显著影响。6.分泌型FMOD促使静息型内皮细胞的血管生成活性被激活,表现为增殖、侵袭和迁移能力的增强。7.FMOD促肿瘤血管生成的分子机制是:SCLC细胞合成并分泌FMOD至细胞外,经旁分泌途径作用于肿瘤基质中的血管内皮细胞。FMOD是多种血管生长因子的上游调控子,刺激血管内皮细胞上调表达促血管生长因子VEGF,FGF-2,TGF-?1和PDGF-B,开启“血管生成开关”(angiogenic switch),形成有利肿瘤血管生成的局部分子微环境,从而激活血管内皮细胞的生物学功能,促进肿瘤血管新生。
[Abstract]:Background neovascularization of tumor provides important material source and structural basis for tumor cell growth and metastasis. Therefore, inhibition of tumor angiogenesis can not only cut off the nutritional supply of tumor cells, make tumor atrophy and degeneration, ischemic necrosis, but also block the metastasis of tumor cells through the blood tube pathway to inhibit the formation of metastatic tumor [10 Small cell lung cancer (SCLC) is an aggressive malignant tumor. SCLC cells have low differentiation and high malignant biological characteristics, and early invasion of the distant metastasis of blood vessels. According to the statistics, the average survival time of the extensive stage small cell lung cancer is only 7-12 months, the 5 year survival rate is less than 2%, and the prognosis of the disease is extremely poor [3].. In addition, small cell lung cancer is also a vascular dependent tumor. Its rich neovascularization network provides oxygen and nutrients for tumor cells, scavenging local metabolites, and establishing bloody channels for early local diffusion and distant metastasis of tumor cells. Therefore, it is necessary to explore the angiogenic factors that cause abnormal proliferation of tumor vessels in small cell lung cancer. And its mechanism is expected to provide a basis for research and development of effective angiogenesis inhibitors for small cell lung cancer. Fibromodulin (FMOD) is a member of the small leucine-rich proteoglycans (SLRPs) family, a member of the family [11]., and FMOD is a newly discovered endogenous blood vessel. Generation factors have important regulatory roles in physiological and pathological vascular proliferation activities [4-6].. Earlier studies compared the gene expression profiles of SCLC parent cell line H446 and its drug-resistant cell line H446/DDP. It was found that FMOD was highly expressed in two cell lines, and there was no significant difference in the level of mRNA tables. RT-PCR and Western were used. Blot further verified the consistent results. This shows that the abnormal expression of FMOD is associated with the occurrence and development of SCLC, but has no significant correlation with the acquired resistance of SCLC. The expression of FMOD in the tumor tissue specimens of SCLC patients is detected by immunohistochemistry (immunohistochemistry, IHC), and it is found that FMOD is nearly half of the expression of FMOD. There is no positive expression in the normal lung tissue specimens, which further indicates that the abnormal expression of FMOD in SCLC may be closely related to its development. Based on the angiogenesis effect of FMOD, we speculate that FMOD may be involved in the angiogenesis of SCLC. The role of FMOD in tumor angiogenesis is not yet. It is clear that its mechanism of action needs to be further studied. Therefore, this topic will explore the role and molecular mechanism of FMOD on tumor angiogenesis under the background of SCLC disease research. Firstly, gene silencing technique is used to reduce the expression of FMOD gene, and then the study of FMOD on SCLC tumor from the clinical level, animal level, cell and molecular level. The effect of angiogenesis and the effect of FMOD on the angiogenesis of SCLC tumor and its related mechanism. The aim is to provide the basis for the development of new SCLC anti angiogenesis drugs with FMOD as the target, and to achieve the goal of inhibiting the development of SCLC and improving the prognosis of the disease. The method 1. collects the specimens of SCLC clinical tissue and normal alveolus. As control, the expression of FMOD in tissue specimens was detected by immunohistochemical staining by formaldehyde fixation, paraffin embedding and slice treatment. The medical records of the corresponding clinical patients were collected, including age, sex, smoking, tumor staging and preclinical characteristics, and the analysis of FMOD by the 2 test method. The correlation between expression level and clinical pointer. Immunohistochemical method was used to detect the expression of vascular endothelial marker CD31 in tissue specimens, count microvascular density, and preliminarily verify the correlation between abnormal expression of FMOD and SCLC angiogenesis, and the shRNA sequence 5 '-CCGCATGAAGTACGTCTACTT-3' [7] targeted to FMOD was cloned to the lentivirus vector pGLV3/H1/GFP+. The recombinant lentivirus shuttle plasmid LV3-shFMOD. was constructed in Puro Vector and the recombinant lentivirus containing disordered fragment 5 '-TTCTCCGAACGTGTCACGT-3 was constructed with recombinant lentivirus transfected H446 cells, purinamycin was used to screen transfected cells. Western Blot method was used to detect FMOD in the transfected H446 cells and FM secretion in the culture medium The expression level of OD is to verify whether the H446 cells can synthesize and secrete the FMOD protein, and whether LV3-shFMOD can effectively inhibit the expression of FMOD and secrete.3. to implant the recombinant lentivirus transfected H446 cells into the subcutaneous tissue of the immune deficient mice (severe combined immunodeficiency mice, SCID), and establish a transplanted tumor animal model. The weight of SCID rats was weighed regularly to monitor the change of body weight with time; the length of the tumor size was measured regularly and the growth of the tumor was evaluated; the contrast enhancement agent was injected into the tail vein to perform the angiography B ultrasound examination [9], and the hemodynamic indexes of the tumor area were measured, the peak time and the peak time of the blood flow peak were measured, and the blood flow perfusion of the tumor was evaluated; The expression of FMOD and CD31 was detected by immunohistochemical method, the microvessel count was detected, and the role of FMOD in the angiogenesis of the tumor was further verified from the body level..4. was used to determine the proliferation activity of H446 cells by CCK8 kit, and the ability to detect the invasion and migration of H446 cells by Transwell chamber, and to analyze the effect of FMOD on the biological activity of H446 cells. H446 culture supernatant was collected as a conditioned medium (conditioned medium, CM). Human umbilical vein endothelial cells (human umbilical vein endothelial cells, HUVECs) were co cultured with CM, and the effects of secretory cells with different levels of expression on the proliferation activity were measured with CCK8 kit. The effects of secretory FMOD on the invasion and migration of HUVECs,.Western Blot was used to detect the expression of angiogenic growth factor after co culture of HUVECs and CM. Results 1. a total of 27 specimens of SCLC lung needle aspiration were collected, of which 4 cases of clinical records were incomplete and 3 paraffin sections did not detect cancer. Tissue, the remaining 20 specimens were included in the study to perform immunohistochemical detection and statistical analysis. (1) the results showed that the positive expression rate of FMOD in SCLC clinical tissues was 55%, the positive expression was located in the nucleus and cytoplasm, FMOD was negative in normal lung tissue. The expression of Fisher 's and the expression of FMOD were obtained. There was no correlation between age, sex, smoking and prognosis, and significant correlation with SCLC tumor staging (P=0.017). (2) the number of microvasculature in tissue specimens with positive FMOD expression was 43.89? 10.45, and the number of microvascular tissues with negative FMOD expression was 23.84? 10.54. The number of microvessels was significantly higher in the FMOD positive expression of tumor tissues by t test. The sequencing of FMOD negative tissue (P=0.013).2. recombinant plasmid showed that lentivirus recombinant vector LV3-shFMOD was constructed correctly, including shRNA target sequence targeting FMOD. The result of lentivirus titer was 1? 108 TU/ml, lentivirus infected H446 cells 72h, and the fluorescence intensity was the strongest. The successful transfection of cells was screened with purinamycin, Western Blot detection was fine. The expression level of FMOD in the intracellular and culture supernatant showed that the content of FMOD in H446 cells transfected with shFMOD was significantly lower than that of the control group (P0.001, P0.05). The results showed that H446 cells could synthesize and secrete FMOD protein on the one hand; on the other hand, the construction of recombinant plasmid was correct and shRNA-FMOD could effectively inhibit FMOD. The expression and secretion of.3. experimental group (inoculated H446-shFMOD cells) SCID mice and control group (inoculated H446-shCON cell) SCID mice were formed fourth days after cell cultivation, the average volume of tumor body in the experimental group was less than the control group (P=0.001). Then the volume of the tumor body in the experimental group was less than that of the control group on the seventh day, fourteenth days, twenty-first days and twenty-eighth days (P =0.003,0.003,0.004,0.002). The weight of SCID rats in the experimental group continued to increase during the observation period. The weight of SCID mice in the control group began to decline after fourteenth days (P0.05), and the cachexia was obvious and the SCID mice died. The mean blood flow peak of the tumor area in the experimental group (10.08? 3.49) was significantly lower than that of the control group (26.27? 4.93, P0.001). The peak time of blood flow (9.28? 4.88s) in the experimental group was significantly longer than that of the control group (2.80? 0.77s) (P=0.022). The microvascular staining of CD31 labeled tumor by immunohistochemistry was used in the experimental group. The count results showed that the number of microvessels in the tumor tissue of the experimental group was 16.03? 2.84, the corresponding value of the control group was 35.87? 13.41, and the number of microvessels in the experimental group was detected by t test. After transfecting (P=0.006).4.H446 cells to LV3-shFMOD, the proliferation, invasion and mobility of cells were not significantly different than those of the control group (P0.05). HUVECs was cultured in culture supernatant CM-shFMOD and CM-shCON respectively. CCK-8 detection found that the proliferation activity of HUVEC in CM-shFMOD was significantly lower than that of control (P0.05). H446-shCON co culture, the detection results showed that the invasion and migration ability of HUVECs co cultured with H446-shFMOD decreased, with statistical difference (P0.001).5. HUVECs was cultured in the conditioned medium CM-shFMOD and CM-shCON respectively, and Western Blot was used to detect the expression level of HUVECs vascular generation growth factor. The results showed that the.5. was compared with the control group. After co culture of HUVECs and CM-shFMOD, the expression of vascular growth factor VEGF, FGF-2, PDGF-B, TGF-? 1 was significantly down (P0.05). Conclusion the abnormal expression of 1.FMOD in the SCLC clinical tissue is related to the tumor staging, suggesting that FMOD may be a potential marker for predicting SCLC tumor staging. The abnormal expression of.2.FMOD in clinical tissue and microvascular density may be the potential marker of SCLC tumor staging. Degree related, suggesting that FMOD may be involved in the angiogenesis of small cell lung cancer.3. to successfully construct the recombinant lentivirus vector of the target FMOD gene. The recombinant vector can significantly inhibit the FMOD expression of SCLC cells and secrete the.4. transplanted tumor animal model, which fully proves that FMOD can induce SCLC tumor angiogenesis and can increase the blood flow of the tumor tissue. Perfusion, thus promoting the growth of endogenous FMOD synthesized by.5.SCLC cells, has no significant effect on its own proliferative activity, invasion and migration ability, and the.6. secretory FMOD stimulates the activation of the angiogenesis activity of resting endothelial cells, which shows the molecular mechanism of proliferation, invasion and migration of.7.FMOD to promote tumor angiogenesis. SCLC cells synthesize and secrete FMOD to extracellular, the vascular endothelial cell.FMOD, which is mediated by paracrine pathway in the tumor matrix, is an upstream regulator of a variety of vascular growth factors, stimulating vascular endothelial cells to increase expression of vascular growth factor VEGF, FGF-2, TGF-? 1 and PDGF-B, and to open the "vasculogenic switch" (angiogenic switch). The local molecular microenvironment, which is favorable for tumor angiogenesis, activates the biological functions of vascular endothelial cells and promotes tumor angiogenesis.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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