摘要
1863年Virchow提出，肿瘤的形成源自慢性炎症，从此展开了对肿瘤与炎症之间关系的研究。目前较为明确的观点认为，相当一部分肿瘤与慢性炎症密切相关，但当肿瘤和炎症共存时，二者之间的相互作用和影响则少见报道。炎症的过程涉及了肉芽组织的形成，即成纤维细胞的增殖和间质细胞的构架、血管生成以及炎症细胞的浸润，这些都与肿瘤的形成过程颇为相似。炎症的过程是局限且受机体控制的生物学过程，而肿瘤则不然，炎症的受控性与肿瘤的失控性形成了鲜明的对比。本研究利用黑色素瘤这一常见的恶性肿瘤为模型，建立了同时存在手术切口的荷瘤小鼠，通过观察急性炎症对肿瘤体积、微血管密度(Microvessel density，MVD)、血管内皮生长因子(Vascular endothelial growthfactor，VEGF)-a、基质金属蛋白酶(Matrix metalloproteinase，MMPs)家族中的MMP-2和MMP-9的表达及其活性，及体内外实验动态观察转化生长因子(Transforming growth factor，TGF)-β和干扰素(Interferon，IFN)-γ对B16F10细胞的影响，来评价当机体同时存在急性炎症(手术伤口)和肿瘤时，急性炎症对黑色素瘤的影响，并对其相关分子机制进行初步探讨。主要研究内容如下：
第一部分：小鼠黑色素瘤急性炎症模型的建立
本研究选择了黑色素瘤，这一高度恶性且早期血行转移的肿瘤作为研究模型，依靠C57BL小鼠和B16F10黑色素瘤细胞来构建急性炎症-肿瘤的动物模型，待黑色素瘤长至500 mm~3左右时，建立不同类型的急性炎症-黑色素瘤模型：A组为单纯创伤-黑色素瘤模型，B组为重复扩创-黑色素瘤模型，C组为创伤/化脓菌-黑色素瘤模型，D组为创伤/冰乙酸-黑色素瘤模型，E组为单纯肿瘤模型(对照组)。每日观察肿瘤生长及伤口愈合情况、绘制肿瘤生长曲线，比较不同急性炎症-肿瘤模型的效果。
实验结果表明：在建立急性炎症模型以前，对照组与实验组肿瘤体积集中在450-550 mm~3之间，在伤口处施加重复扩创、金葡菌和冰乙酸三种不同干扰后，肿瘤生长出现了不同趋势，A组与E组肿瘤生长比较接近，其他实验组肿瘤生长速度趋缓，体积均小于对照组，这说明急性炎症没达到一定的强度和时间，对肿瘤生长无影响，当达到一定的强度并持续一定时间，可以抑制肿瘤的生长。统计分析证实，B、C、D三种手段均可抑制肿瘤的生长(P＜0．05)，且三者对肿瘤生长抑制的程度没有区别(P＞0．05)。
三种建模的方式均可达到持续炎症刺激的效果，但创面和其他情况各不相同：B组小鼠创面干净，结痂和渗出均不明显，组织成分新老交替，但需每日麻醉小鼠后扩创，操作复杂；C组小鼠创面有脓痂覆盖，建模操作简便，但小鼠有罹患败血症的危险，且由于金葡菌感染力强，要求单独饲养；D组小鼠的创面较C组干净，创口处组织成分较B组均一，但冰乙酸具有腐蚀能力，建模过程中易损伤大中型血管及其他敏感部位。根据上述实验结果，重复扩创模型可以尽量避免外源因素的干扰，并且结果稳定可靠，本研究选用重复扩创-黑色素瘤模型作为下一步实验的动物模型。
第二部分：急性炎症对黑色素瘤体积及血管生成的影响
肿瘤血管生成是肿瘤细胞、血管内皮细胞与其微环境相互影响的复杂的过程，多种活性物质可调节肿瘤血管的生成。炎症过程中产生的多种细胞因子也可调控内皮细胞的激活、增殖、迁移、塑形和凋亡，从而在血管生成过程发挥重要作用。根据第一部分实验结果，选择重复扩创-黑色素瘤模型为本研究的急性炎症荷瘤鼠模型。HE染色检测实验组和对照组肿瘤MVD，免疫组织化学方法检测肿瘤组织VEGF表达，探讨急性炎症对黑色素瘤组织血管生成的影响。
实验将小鼠分为对照组与实验组。建模后隔日分别测量对照组和实验组的肿瘤体积，绘制肿瘤生长曲线，内眦取血。建立急性炎症模型后第7和11天处死动物，分离肿瘤组织，福尔马林固定并制备蜡块。
实验结果表明：1．急性炎症对肿瘤的生长呈现了先抑制后失抑制的表象，即在造模后第7天之内表现为急性炎症抑制肿瘤的生长(早期抑制时相)，而第7天之后，这种抑制作用减弱，至第11天，实验组的肿瘤体积恢复到与对照组相同水平，差异无统计学意义(P＞0．05，晚期失抑制时相)。2．抑制时相期肿瘤组织的MVD计数对照组高于实验组，差异有统计学意义(P＜0．05)。3．抑制时相期肿瘤免疫组织化学染色VEGF阳性表达率，对照组显著高于实验组，差异有统计学意义(P＜0．05)。
本研究所建立的模型是通过模拟急性炎症的病理生理过程来观察其直接和间接对肿瘤组织的影响作用，特别关注急性炎症或伤口修复过程中的“调控性”因素对肿瘤组织生长和血管生成的影响。这种影响作用主要体现为先抑制后失抑制的过程，其抑制作用不但与炎症因子的直接刺激有关，也可能与某些炎症灶释放入血的抑制因子有关，失抑制则可能与炎症晚期分泌某种因子的拮抗作用有关。
第三部分：急性炎症对黑色素瘤基质金属蛋白酶表达及活性的影响
研究发现，肿瘤组织决不是癌细胞简单相加的总和，肿瘤细胞与间质细胞有着密切的协作，这些细胞包括成纤维细胞、炎性免疫细胞比如巨噬细胞、平滑肌细胞以及血管内皮细胞，这些细胞通过细胞外基质(Extracellular matrix，ECM)相互联结共同构成肿瘤间质成分。肿瘤间质成分对肿瘤细胞本身的生长和迁移都产生重要的影响。基质金属蛋白酶家族在肿瘤及其微环境中参与降解和重塑ECM，对肿瘤的生长、侵袭、转移具有重要的作用，同时也与急性炎症创伤愈合肉芽组织形成密切相关。MMP-2与MMP-9是MMPs的重要成员，在肿瘤微循环和创伤愈合过程中具有重要作用。
本部分研究利用免疫组织化学染色观察肿瘤组织MMP-2和MMP-9的表达情况，利用明胶酶谱法定量分析肿瘤组织MMP-2和MMP-9的活性来评价当机体同时存在急性炎症和肿瘤时，急性炎症对肿瘤的MMPs表达与活性的影响。
实验结果表明：在第7天时，MMP-2在实验组的表达明显小于对照组，平均阳性细胞率约为对照组的20％(P＜0．05)，而到第11天后该差别减小而无统计学意义(P＞0．05)。实验组与对照组中MMP-9的表达在第7天和11天差异均具有统计学意义(P＜0．05)。急性炎症对MMP-2，MMP-9的活性的影响，在第7天时对照组肿瘤组织内MMP-2和MMP-9酶活性均高于实验组(P＜0．05)；而在第11天时该差别消失(P＞0．05)，其MMPs活性变化趋势与其表达变化趋势相符。
由此可见，急性炎症可能通过某种途径影响了肿瘤细胞的MMP-2和MMP-9的表达量和活性，这种影响作用也呈现为早期时相的抑制和晚期时相的失抑制，与本研究第二部分对于肿瘤体积的观察结果相吻合，但为何晚期出现失抑制现象有待于进一步研究。
第四部分：IFN-γ和TGF-β对黑色素瘤细胞增殖、迁移和侵袭作用影响的研究
急性炎症的过程涉及到早期的炎症因子释放和晚期的肉芽组织修复两个阶段，而本实验所制造的伤口模型由于重复扩创，充当了早期炎症因子释放这个阶段。该阶段涉及了多种炎症因子和炎细胞浸润，炎症组织局部表现为炎细胞浸润，炎细胞浸润主要为粒细胞系统，而影响肿瘤的则可能是炎症灶释放的炎症因子通过血液循环间接干扰肿瘤本身。
为了进一步证实炎症局部释放的炎症因子对肿瘤细胞增殖、迁移和侵袭能力的影响，本研究通过第二部分研究所获得的血清对细胞因子进行了分析和筛选，发现IFN-γ和TGF-β具有一定的动态变化意义，进而选择C57BL小鼠来源B16F10细胞系进行体外培养，通过施加细胞因子IFN-γ和TGF-β，观察两者对B16F10细胞增殖、迁移和侵袭能力的影响，自小鼠尾静脉注入IFN-γ，观察其对肿瘤生长的影响。
磺酰罗丹明(Sulforhodamine B，SRB)实验结果表明：TGF-β干预组肿瘤细胞存活率高于对照组，差异具有统计学意义(P＜0．05)；IFN-γ干预组肿瘤细胞存活率低于对照组，差异具有统计学意义(P＜0．05)；同时给予IFN-γ和TGF-β干预时，肿瘤细胞存活率明显低于对照组，差异具有统计学意义(P＜0．05)。给予不同细胞因子处理时，随细胞存活率的变化，肿瘤细胞表现为不同的形态，说明IFN-γ可抑制肿瘤细胞的增殖、迁移和侵袭能力，而TGF-β则可拮抗IFN-γ的这种作用，使肿瘤细胞重获并增强其增殖、迁移和侵袭的能力。自小鼠尾静脉注入IFN-γ(注射组)，生长曲线表现为早期抑制时相和晚期失抑制时相。明胶酶谱分析表明IFN-γ降低黑色素瘤MMPs活性，TGF-β升高MMPs活性，二者存在拮抗作用。
该实验揭示了肿瘤细胞分泌TGF-β对抗IFN-γ这一生物学现象，解释了急性炎症对肿瘤影响作用呈现为早期抑制和晚期失抑制的可能机制，提出IFN-γ/TGF-β的动态变化可能在急性炎症对肿瘤生长影响的过程中发挥重要作用。
综上所述，本研究通过建立急性炎症荷瘤鼠动物模型，应用免疫组化染色、明胶酶谱分析及体外细胞培养等技术，观察急性炎症对肿瘤体积、MVD、VEGF-a表达、MMP-2和MMP-9表达及活性的影响，并通过体内外实验观察TGF-β和IFN-γ的动态变化对肿瘤的影响。主要结论如下：
1．建立单纯创伤、创伤+化脓菌、创伤+冰乙酸和重复扩创与荷瘤鼠四种急性炎症-肿瘤复合动物模型。结果表明：单纯创伤-黑色素瘤模型对肿瘤生长无影响，后三者均可抑制肿瘤生长、且对肿瘤生长抑制程度相同，说明急性炎症未达到一定的强度和时间对肿瘤生长无影响，当达到一定的强度并持续一定时间时，可以抑制肿瘤的生长。
2．三种有效抑制肿瘤生长的动物模型各有所长，应结合实际条件，遵循科学合理、方便可行的原则进行选择。本研究选择重复扩创-黑色素瘤模型作为本研究动物模型。
3．急性炎症时实验组肿瘤体积小于对照组，肿瘤组织中VEGF-a表达减少，MVD降低，说明急性炎症对肿瘤组织的影响主要体现为抑制，包括抑制肿瘤生长和抑制血管生成。
4．通过对肿瘤组织的免疫组织化学染色观察和明胶酶谱分析表明，急性炎症抑制肿瘤组织中MMP-2、MMP-9的表达与活性，从而抑制肿瘤血管生成和肿瘤生长。
5．观察细胞因子IFN-γ、TGF-β对炎症-肿瘤模型的动态影响和对体外培养B16F10细胞影响的结果表明：IFN-γ可抑制肿瘤细胞的增殖、迁移和侵袭能力，而TGF-β则可对抗IFN-γ的作用，使肿瘤细胞重获生长和侵袭能力，增强肿瘤细胞的增殖、迁移和侵袭能力，这种炎性因子对肿瘤细胞生物学特性的正性与负性影响的结果，可解释前期实验观察到的急性炎症对肿瘤生长前期抑制和后期失抑制的现象。
As early as 1863,Virchow first postulated that cancer originates at sites ofchronic inflammation,in part based on his hypothesis that some classes of irritantsthat cause inflammation also enhance cell proliferation.In the past decades,scientistshave made considerable progress in the research on the relationship between cancerand inflammation.This process of inflammation involves the classic model ofinflammatory response,including the formation of granulation tissue,leukocyteinfiltration,angiogenesis factor,and cytokines network.All of those are very similarto tumor growth and progression,but since there are two different consequences,theformer is limited and controlled;the latter is unlimited and irregular.In this study,amouse tumor model undergoes manufactured surgical wound,building a model ofacute inflammation,to evaluate the relationship between acute inflammation orwound healing and the process of tumor growth.HE staining was performed tocompare the microvessel density (MVD) among these groups.Immunhistochemistrywas employed to detect the expression of VEGF,metalloproteinases-2 (MMP-2) andMMP-9.Gelatin zymography was used to examine the levels of MMP-2 andMMP-9 activity.In vitro experiments,the role of TGF-β/IFN-γin the inflammationwas evaluated.This study provides a better understanding of the relationship betweentumor and inflammation,and contributes to a deeper understanding of tumor cells andattacks on immunity.The main contents are as following:
1.The Construct of melanoma mouse model of acute inflammation
Melonoma,metastasis occurs in the early phase by blood,is selected toconstructed the model.When the volum of melanoma is up to about 500 mm~3,B16F10 melanoma cells suspension was used to establish different types ofmelanoma - acute inflammation model.
Group A:Melanoma- a simple model of trauma,Group B:Melanoma-therepeated debridement model,Group C:Melanoma - traumatic suppurative bacteriamodel,Group D:Melanoma - the trauma model of glacial acetic acid,E:controlgroup--imple tumor.Information of tumor from daily observation was used to mapthe curves of growth to compare the different effects of inflammation in the tumor model.
The experimental results show that:In this experiment,the tumor volume of thecontrol group and experimental group focused on among 450-550 mm~3 before theestablishment of acute inflammation model.After treantments with the repeatedimposition of the wound debridement,Staphylococcus aureus and glacial acetic acidinterference,different trends of the tumor growth of emerged.Tumor growth ofGroup A is similar to that of Group E,tumor growth rate slowed down in otherexperimental groups,the volume is less than the control group gradually.On the fifthday of modeling,tumor volume of Group B,C and D focused on between 350-450mm~3,which suggest that the acute inflammation had no effect on tumor growth untilto a certain intensity and duration.When reaches a certain intensity and duration,inflammation can inhibit tumor growth.
All differences of three groups had statistical significancein in inhibition of tumorgrowth (P＜0.05),t here was no difference between them (P＞0.05) .All three waysof modeling can achieve the effect of sustained inflammatory stimulus,but thewounds and other conditions vary:
Mice in Group B has clean wound,cruste and exudative are not clear,thetransition of tissue from the old to the new,suitable for higher sterile experiments.But it needs daily debridement and it's complicated to operate.Mice in Group B havepus covered eggplant,modeling method is simple,but there is risk of suffering fromsepsis,Staphylococcus aureus infection and as a result of strong demands for keepingseparate;Wound of mice in Group D is cleaner than that of Group C;Composition ofwound in Group D is more homogeneous than that of Group B.But acetic acid hasthe capability to cause corrosion and is strict with the experimental site.In the processof modeling,it is easy to damage large and medium-sized blood vessels and othersensitive parts.Also there are less interfere effect on B models.Based on the aboveexperimental results,this study selected melanoma - the repeating debridement as ouranimal experimental model of the next step.
2.The effect of acute inflammation on angiogenesis in melanoma.
Tumor angiogenesis is a complex process of the interaction between tumor cells,vascular endothelial cells with their micro-environment.A variety of active substances can regulate tumor angiogenesis.A variety of cytokines resulting from theprocess of inflammation may also control the activation,migration,proliferation,remodeling and apoptosis of endothelial cells,which play a critical role in promotingtumor angiogenesis.According to the first part of the experimental results,we selectmelanoma - the continuing debridement as our animal experimental model.In orderto investigate the effect of acute inflammation on angiogenesis in melanoma,HEstaining was performed to compare MVD among these groups andimmunhistochemistry was employed to detect the expression of VEGF.Mice will bedivided into two groups:The control group:tumor without wound;the experimentgroup:tumor with wound on the opposite side of bodies.After modeling,tumorvolume of the control group and the experiment group were measured on alternatedays and calculated according to the method reported in the literature.Tumor growthcurve were mapped.On the 7th day of modeling,the animals were sacrificed,andthen tumor tissues were isolated,formalin fixed for paraffin block preparation.
The experimental results show that:1.The experimental model present two phasein the process of tumor-inflammation interaction:shown as inhibition phase (before7d) and inhibition-missing phase (after 11 d).2.The MVD in the in two groups weresignificantly different in inhibition phase:Control＞Experiment (P＜0.05) 3.Thepositive expression ratio of VEGF in the experiment group statistically differentsfrom the control group in inhibition phase (P＜0.05);
Model established in this study was used to observe the direct and indirect impactof inflammation on tumor tissue through the simulation of the pathophysiology ofacute inflammatory process,with particular attention to effect of acute inflammationon angiogenesis in melanoma.The effect characterized by inhibition,including theinhibition of tumor growth and angiogenesis.
3.The effect of acute inflammation on expression of MMPs
Study found that cancer cells in tumor tissue are not simply the sum.There isclose collaboration between tumor cells and stromal cells.These cells includefibroblasts,inflammatory cells such as macrophages and endothelial cells.These cellsrely on extracellular matrix (ECM) and link with each other constitute thecomponents of tumor stroma.Stromal tumor components have an important impact on the growth of tumor cells themselves and migration.Matrix metalloproteinasefamily participates in the degradation and remodeling of ECM in the tumor and itsmicro-environment.Acute inflammation plays an important role in tumor growth,invasion and metastasis,as well as wound healing and the formation of granulationtissue.MMP-2 and MMP-9 is two key members of MMPs,which closely effecttumor microcirculation and wound healing.
Study in this part is to investigate the effect of acute inflammation on expressionof MMP-2 and MMP-9.The expression of MMP-9 and MMP-2 were examined byimmunohistochemistry staining and Gelatin Zymography.Evaluate the effect of acuteinflammation on the expression of MMPs under the condition of the existence ofacute inflammation and tumors at the same time.
The experimental results show that:immunohistochemical staining was used todetect positive cell count,in 7d,MMP-2 expression was 2-fold down-regulationcompared to the control group,same as MMP-9.both statistically significantdifference (P = 0.000);but at lld,MMP-2 has missing statistically significantdifference,MMP-9 also restored.Gelatin zymography analysis showed that theactivity of MMP-2 and MMP-9 were down-regulated compared with the controlgroup within the tumor tissue,both statistically significant (P＜0.05) .At 11 d thisdifference were whole missing.The results suggest that the expression and theactivity of MMP-2 and MMP-9 are both subject to the effects of acute inflammation.Acute inflammation may effect expression and activity of MMP-2 and MMP-9 bysome way.This effect is also shown to the early inhibition phase and loss of inhibitionin late-phase,with the second part of the experimental results anastomosis.This isidentical with the result in the second part.
4.A pilot study of IFN-γand TGF-βon melanoma cell proliferation,migrationand invasion
The process of acute inflammation may involve two stages:the initial stage ofrelease of inflammatory factors and the late stage of repairing granulation tissue.As aresult of continued wound debridement in this experimental,it mainly served as theinitial stage of release of inflammatory factors.This stage involved a wide range ofinflammatory cytokines and infiltration of inflammatory cell.Local areas of inflammation characterized by inflammatory cell infiltration,mainly myeloid cells,and the effect on the tumor may be caused by the release of inflammatory factors,directly or indirectly.
In order to further confirm the effect on tumor cell proliferation,migration andinvasion capacity caused by the local release of inflammatory factors,serum from thesecond part was used to analysis and screening cytokine.We found that dynamicchanges of IFN-γand TGF-βhave certain significance.So we choose B16F10 celllines in vitro cell culture in our experiment.Effects on on the B16F10 cellproliferation,migration and invasion capacity caused by cytokines IFN-γandTGF-βweer observed.The SRB show that the survival rate of cells in TGF-βgroup ishigher than that of control group,with statistically significant differences (P＜0.05) ;but the survival rate of cells in IFN-γgroup is lower than that of control group,withstatistically significant differences (P＜0.05) ;The survival rate of cells in experimentgroup is lower than that of control group when IFN-γand TGF-βwere administeredat the same time.Cell morphology varied with the changes of cell survival result fromdifferent administration of cytokines.All of that suggest that IFN-γinhibited cellsproliferation,migration,and invasion,and TGF-βcan confront the role of IFN-γ,sothat tumor cells can restore proliferation,migration,as well as the ability to resumeinvasion and the activity of MMPs.Growth curve of IFN-γinjection group (injectionthrough mouse tail vein) showed inhibition in early phase and loss of inhibition inlate-phase.Gelatin zymography analysis showed that IFN-γcan reduce the activity ofMMP-2 and MMP-9.On the other hand,TGF-βconfronted IFN-γto recover theactivity of MMPs,and increased the activity of MMP-2 and MMP-9.
The experiment revealed that the tumor cells can secrete TGF-βand IFN-γ,whichexplains the possible mechanisms of inhibition in early phase and loss of inhibition inlate-phase.This suggested that the balance of IFN-γ/TGF-βplay an important role inthe interaction between acute inflammation and tumor.
In conclusion,we established animal models of acute inflammation.Immunohistochemistry,gelatin zymography and in vitro cell culture techniques werecarried out to detect the effect of inflammation on tumor volume,microvessel density,expression of VEGF-a,MMP-2 and MMP-9 ,activity of MMP-2 and MMP-9 and role of balance of IFN-γ/TGF-βin tumor.The main conclusions are as follows:
1.The Construct of melanoma mouse model of acute inflammation.This studyselected melanoma - the repeated debridement as our animal experimental model ofthe next step.
2.The effect of acute inflammation on angiogenesis in melanoma.Theinhibitory effect not only results from the direct stimulation of inflammatory factors,but also may from the release of certain inflammatory cytokines into the blood.
3.The effect of acute inflammation on expression of MMPs.The results suggestthat the expression and the activity of MMP-2 and MMP-9 are both subject to theeffects of acute inflammation.
4.A pilot study of IFN-γand TGF-βon melanoma cell proliferation,migrationand invasion.The experiment revealed that the tumor cells can secrete TGF-βandIFN-γ,which explains the possible mechanisms of inhibition in early phase and lossof inhibition in late-phase.This suggested that the balance of IFN-γ/TGF-βplay animportant role in the interaction between acute inflammation and tumor.
引文
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