摘要
背景
目前普遍认为,肿瘤的生长存在两个明显不同的阶段,即无血管的缓慢生长阶段和有血管的快速生长阶段。前者主要依靠弥散供氧,氧的最大弥散距离为150μm,当肿瘤直径超过1-2mm时,其继续生长就需要血液供应。研究发现,绝大多数实体瘤在缺少血管的情况下,生长扩散受到了极大限制,在有血管的情况下,其瘤体增长迅速。正常人体的血管内皮细胞倍增时间为一年,而肿瘤的血管内皮细胞倍增时间仅四天。因此,阻断肿瘤血供,从而抑制肿瘤的生长是目前研究的热点之一。
恶性肿瘤肝转移的途径主要是通过局部扩散转移、淋巴道转移、血道转移和癌细胞脱落形成种植转移等。肝脏是人体最大的实质性脏器,门静脉及肝动脉双重血液供应的特点使其成为许多恶性肿瘤转移的主要部位。而结肠癌引起的肝转移又是肝脏最常见的血道转移方式。文献报道约10%～25%的结直肠癌患者在接受原发灶的手术治疗时已经出现了肝转移,另有约25%的患者在术后随访中发现肝转移,50%以上的结直肠癌最终发生肝转移,而肝转移是结直肠癌死亡的主要因素之一。因此,抑制结肠癌肝转移是提高结肠癌远期疗效的关键问题之一。
近几年,随着超声造影技术的发展和新型超声造影剂的面市,关于微泡造影剂生物学效应的问题也逐渐引起人们的重视,超声造影在肝脏疾病诊断中得到了较广泛的应用,并取得了长足的进步。超声的物理特性和生物学效应(热效应、空化效应、机械效应等)的运用已渗透到了人体保健、疾病预防、疾病治疗(包括体外碎石、止血、肿瘤切除等)、生物技术(如基因转染)等医学领域。超声造影微泡不仅作为一种人为的空化核,降低超声的空化阈值,增强空化效应,并减少产生空化效应所需的超声能量;超声空化效应本身对周围组织细胞可产生各种生物学效应,运用高机械指数超声造影对原发灶进行间歇辐照,增强超声空化效应,可能对结肠癌肝转移产生抑制效应。
目的
本实验在建立转移性肝癌动物模型的基础上,采用高机械指数超声造影对其原发灶进行间歇辐照,希望通过微泡增强的空化效应,引起肿瘤组织及其周围肿瘤微血管的破坏。并经体外实验观察对肿瘤细胞迁移运动、增殖、凋亡、以及细胞微丝、微管等细胞骨架的影响,进一步探讨高机械指数超声造影增强空化效应,是否能够导致肿瘤肝转移的数目及大小的改变,为抑制肿瘤肝脏转移探索新的手段。
方法
1.高机械指数超声造影对结肠癌肝转移动物体内的实验研究:
(1)转移性肝癌动物模型的制备:结肠癌Lovo细胞常规培养于含10%灭活的胎牛血清的DMEM培养液中,取对数生长期的细胞作为实验研究。研究对象为62只正常雄性SD大鼠(体重200～250g),其中10只在SD大鼠脾脏直接接种结肠癌Lovo细胞后第2天、第4天及第6天分别脱颈处死,解剖SD大鼠肝脏,观察转移灶的数目及大小,确定最佳辐照时间。
(2)实验分组:随机分为4组(对照组、微泡+超声组、单纯超声组和单纯微泡组),每组12只。其中微泡+超声辐照组的SD大鼠在接种结肠癌Lovo细胞第4天时,用8%的硫化钠给予SD大鼠腹部脱毛,1%戊巴比妥钠40mg/kg进行腹腔注射麻醉,将SD大鼠仰卧固定于木板上,经尾静脉注入SonoVue造影剂(1ml/kg)后,用高频探头寻找脾脏定位后,转换为相控阵探头,将探头置于SD大鼠脾脏处,固定频率为1.5MHz,机械指数为1.7,显像深度固定在4cm。经SD大鼠尾静脉快速推注SonoVue造影剂(1ml/kg),随后推注1ml生理盐水冲洗管道,辐照6s,间歇6s,共20次。
(3)将分组处理后的SD大鼠肝脏及脾脏采用HE染色,进行病理组织学检查及透射电镜观察其超微结构的变化。
2.高机械指数超声造影对恶性肿瘤影响的体外实验:
(1)结肠癌Lovo细胞常规培养于含10%灭活的新生小牛血清的DMEM培养液中,取对数生长期细胞,观察细胞折光性强,将其分为4组(对照组、微泡+超声组、单纯超声组和单纯微泡组),其中微泡+超声组在采用ViVid Five型彩色多普勒超声仪,调节超声探头频率为1.5MHz,机械指数为1.7,显像深度固定在4cm,用医用胶带将探头环形缠绕呈现一凹槽,将耦合剂置于槽内,使得培养板与耦合剂充分接触,超声辐照6s,间歇6s,共20次。
(2)采用细胞迁移运动试验(Transwell法)和激光共聚焦显微镜检测细胞迁移运动能力及对结肠癌Lovo细胞微丝、微管的影响。
(3)细胞增殖试验(MTT法)检测肿瘤细胞增殖活性的变化。
(4)流式细胞仪检测结肠癌细胞周期及凋亡,观察G1期、S期及G2/G1期细胞比例的变化。
结果
1.高机械指数超声造影对结肠癌肝转移影响的动物实验研究显示:
(1)将62只正常雄性SD大鼠,其中10只经脾脏直接接种结肠癌Lovo细胞后第2天、第4天及第6天分别脱颈处死,解剖SD大鼠肝脏,发现在接种结肠癌Lovo细胞第4天即可见少数白色结节转移灶,直径在1-2mm左右,取此时为最佳辐照时间。余52只SD大鼠除4只因麻醉意外死亡,其余48只纳入实验研究,而其中41只于接种结肠癌Lovo细胞14天后,解剖肝脏均发现直径1～5mm的白色结节状转移灶,成功率为85.4%。
(2)分组处理后10天,将SD大鼠脱颈处死,对照组肝脏可见多个局灶性灰白色的微小转移灶、病灶直径在2～5mm之间。微泡+超声辐照组肝脏可见肿瘤微小转移灶的数目明显减少,直径均在1～2mm之间。微泡+超声辐照组与对照组比较肿瘤数目及大小显著减少(P<0.01),单纯超声组及单纯微泡组与对照组比较肿瘤数目、肿瘤大小差异没有显著意义(P>0.05)。
(3)将对照组、微泡+超声组、单纯辐照组及单纯微泡组分组处理后10天,解剖SD大鼠肝脏及脾脏作病理学检查。各组大鼠肝脏内均有肿瘤细胞生长,瘤细胞多呈圆形或椭圆形,呈团状分布,但在微泡+辐照组肝脏肿瘤细胞明显减少,脾脏组织内出现大片出血坏死及明显血栓形成,对照组、单纯辐照组及单纯微泡组的血栓数量明显少于微泡+超声组。
(4)透射电镜观察脾脏组织超微结构的改变:对照组透射电镜可见肿瘤细胞核大,胞浆和胞核比例减小,线粒体较多,未见肿胀。内皮细胞形态规则,之间连接紧密,线粒体正常。微泡+超声组透射电镜可见明显的超微结构的破坏,肿瘤细胞除具有对照组特点之外,线粒体明显肿胀,呈球状。内皮细胞形态多样,紧密连接断开,线粒体肿大。单纯超声组、单纯微泡组与对照组之间无显著差别。
2.高机械指数超声造影对结肠癌Lovo细胞迁移运动、增殖及凋亡的影响:
(1)采用Transwell法发现,对照组Lovo细胞数发生明显迁移,而进行微泡+超声辐照后Lovo细胞迁移数明显减少(P<0.01),微泡+超声辐照组可见大量悬浮不透亮的死亡细胞;而单纯超声组细胞迁移数和单纯微泡组的细胞迁移数目与对照组比较,无显著差异(P>0.05)。
(2)用激光共聚焦显微镜检测各组,细胞内微丝蛋白F-actin的表达,结果显示:对照组微管染色表现为细胞致密的丝状网络结构,向细胞边缘呈放射性延伸;单纯超声组与单纯微泡组与对照组无明显差别;微泡+超声组微管表达较对照组减弱、稀疏,网络样结构主要沿细胞长轴排列。对照组细胞微丝染色表现为细胞致密网络状细丝样结构,向四周伸出许多细短的毛刺状突起,有明显的拉丝状和方向性;单纯超声组与单纯超声微泡组与对照组无明显差别;微泡+超声组Lovo细胞胞质中部的网络状细丝明显减少,荧光暗淡,细短的毛刺状突起明显减少。
(3) MTT法检测细胞增殖活性结果显示,对照组细胞PI增殖指数明显多于微泡+超声组(P<0.01),而单纯超声组细胞增殖数和单纯微泡组的细胞增殖指数与对照组比较,无显著差异(P>0.05)。
(4)流式细胞仪检测各组结肠癌Lovo细胞周期及凋亡的改变,结果显示:对照组G1期细胞比例显著降低(P<0.01),而S期细胞比例显著增高(P<0.01);单纯超声组和单纯微泡组与对照组各细胞周期的比例无显著差异(P>0.05);与对照组相比,微泡+超声辐照组G1期细胞比例显著增高(P<0.01),S期细胞比例显著降低,且凋亡峰与对照组相比较明显增高(P<0.01)。
结论
1.高机械指数超声造影能够引起转移性肝癌的数目、大小发生改变,抑制肿瘤肝转移;
2.高机械指数超声造影能够引起原发肿瘤组织坏死,促进肿瘤内部及周围组织微血栓形成,使肿瘤细胞超微结构发生变化;
3.高机械指数超声造影能够抑制结肠癌Lovo细胞迁移,引起细胞骨架的改变;
4.高机械指数超声造影能引起结肠癌Lovo细胞周期的改变,并可诱导结肠癌Lovo细胞凋亡、抑制增殖。
Background:
It is generally thought that tumor grows in two different stages, ie, a slow growth stage with avascularity and a fast growth stage with vascularity. In the slow growth stage, tumor’s growth relies on the diffusion ventilation, with the maximum diffusion diameter of 150μm. When the diameter of the tumor exceeds 1-2mm, the further growth needs blood supply. It is reported that most of the solid tumors short of blood vessels grew rather slowly but developed fast with blood vessels. Doubling generation time of vascular endothelial cells (VECs) of health persons is about one year, but that of tumor is only four days! Therefore, blocking the blood supply of tumor to inhibit its growth is one of the hot spots of current research.
Hepatic metastasis of malignant cancers is accomplished mainly by diffusion metastasis, lymphatic metastasis, blood route metastasis and implantation metastasis caused by malignant cell shedding. The liver is the largest parenchymatous organ of the human body, and is considered the major location of many malignant cancer metastasis because of its double blood supply. Meanwhile, hepatic metastasis is commonly seen in the blood route metastasis of colon cancer. It was reported that for the patients with colorectal cancer, about 10%-25% had been found with hepatic metastasis when they underwent primary operation, another 25% were found with hepatic metastasis during follow-up and over 50% finally had hepatic metastasis, which proved that the hepatic metastasis was the leading cause for death of patients with colon cancer. Therefore, inhibition of metastatic hepatic carcinoma is the key for the improvement of survival rate.
Recently, with the development of ultrasound contrast and the application of new ultrasonic contrast agent, the problems caused by the biological effects of microbubbles contrast agents have gradually aroused a great concern. Ultrasound contrast has been extensively employed in the diagnosis of liver diseases and it’s playing an important role. Physical characteristic and biological effects (heat effect, cavatition erosion and mechanical effect) of ultrasound have been widely used in many medical fields including health care, disease prevention, disease treatment (e.g. vitro macadam, hemostasis and ablation of tumor) and biotechnology (e.g.gene transfection). As a kind of artificial cavitation nuclei, ultrasonic contrast agent can not only degrade cavitation threshold and accentruate cavatition but also diminish the ultrasonic energy for cavatition. Furthermore, ultrasonic cavatition itself exerts kinds of biological effect on peripheral histiocytes, for instance, damaging cellular membrane and DNA, breaking DNA chain, inhibiting cellular proliferation, accelerating cell apoptosis, and depressing metastasis and adhesiveness of cells by recombination of cystoskeletons as well as altering distribution of carcinoma cell cycle, which are helpful in many aspects of clinical treatment including suppression of growth and metastasis of tumor and facilitation of tissue repair. Accordingly, intermission irradiation on the primary foci with high mechanical index (MI) ultrasound contrast can enhance ultrasound cavatition erosion and inhibit hepatic metastasis of colon cancer.
Objective:
In the study, based on an animal model of micrometastasis hepatoma, we employed high MI ultrasound contrast of intermission irradiation on primary foci to cause damage to tumor tissues and peripheral microvessels via cavatition erosion with accentruated microbubbles. In the meantime, the effect of high MI ultrasound contrast on locomotory movement, proliferation, apoptosis as well as on cystoskeletons of microfilament and microtube of carcinoma were investigated to further discuss if the high MI ultrasound contrast enhancing cavatition erosion can lead to the changes of the number and size of hepatic metastasis of colon cancers so as to explore a new way for the inhibition of hepatic metastasis of colon cancers.
Methods:
1.In vivo study of high MI ultrasound contrast in rats
(1) Establishment of rat model of metastatic hepatic carcinoma: Lovo cells of colon cancer were cultured in DMEM medium containing 10% inactivated fetal bovine serum, when the cells at exponential phase of growth were selected for experimental study, which involved 62 male SD rats weighing 200-250g, of which 10 rats were injected with colon cancer Lovo cells via the spleen and sacrificed at days 2, 4 and 6 respectively to dissect the liver for observing the number and size of metastasis so as to determine optimal bombardment time.
(2) Experimental groups: all rats were divided randomly into four groups, ie, control group, microbubbles plus ultrasound irradiation group, simple ultrasound group and simple microbubbles group, 12 rats in each group. At day 4 after inoculation with colon cancer Lovo cells, the rats were brought into the experiment. was given to depilate The abdomen of rats were depilated with 8% sodium sulfide and the they were anesthetized with the injection (40mg/kg) of 1% pentobarbitone sodium before operation. The rats were fixed in dorsal position, the high-frequency probe was used to find the spleen location and shifted to phased array (at the frequency of 1.5MHz, MI1.7, imaging depth of 4cm). The probe was put onto the spleen of SD rats. Ultrasound contrast SonoVue (1ml/kg) was injected quickly via caudal vein, followed by 1ml saline injection to wash the tube. Ultrasound radiation was performed 20 times with the intermision of 6 seconds.
(3) The liver and spleen were processed with HE staining, histopathologic examination and transmission electron microscope to observe their ultrastructural changes.
2.In vitro study of high MI ultrasound contrast in rats
(1) Colon cancer Lovo cells were cultured in DMEM medium containing 10%inactivated newborn calf serum, then the cells at exponential phase of growth were selected for observing the refraction density and divided into four groups including control group, microbubbles plus ultrasound irradiation group, simple ultrasound group and simple microbubbles group, of which microbubbles plus ultrasound irradiation group was treated with type Vivid Five color Doppler, with wave frequency 1.5MHz, mechanical index 1.7,visualization depth 4cm,irradiation 6S,intermission 6S,total 20 times.
(2) Transwell method and laser confocal technique were used to detect the cell migration ability and its effect on microfilament and microtube of the cells.
(3) MTT method was used to observe the proliferative changes of carcinoma cells.
(4) Flow cytometry was used to detect the cell cycle and the apoptotic change and observe the cell proportion at stages G1, S and G2/G1.
Results:
1.Results of high MI ultrasound contrast on hepatic metastasis of colon cancer:
(1) In the 10 rats that were injected with colon cancer Lovo cells via the spleen and sacrificed and was dissected at days 2, 4 and 6 respectively, white nodosity metastasis with diameter of 1-2mm was observed at day 4 after inoculation, so it could be selected as the optimal exposure time. In the other 52 SD rats, 4 died of anesthetic accident, 41 were found with 1-5mm white nodosity metastasis shown in the dissection of the liver 14 days after inoculation, with success rate of 85.4%.
(2) Ten days after treatment in different groups, the SD rats were sacrificed, with focal lyard micrometastases and leion for 2-5mm was found in the liver in control group.The tumor micrometastases for 1-2mm was found in microbubbles plus ultrasound wave irradiation group. Compared with control group, either the number or the size of carcinoma were decreased more significantly in microbubbles plus ultrasound wave irradiation group (P<0.01). There was insignificant statistical difference in aspects of number and size of carcinoma in ultrasound radiation group and simple microbubbles group in comparison with control group (P>0.05).
(3) The rats in all groups were sacrificed at the 10th day and the tissues from the liver and spleen were removed for pathological examination. Tumor grew in the liver tissue, mostly round or ova in shape, and was distributed in mass. However, there could be seen remarkable decrease of tumor cells, massive hemorrhagic necrosis and thrombogenesis in the spleen of microbubbles plus ultrasound wave irradiation group. Meanwhile, the number of thrombus in the spleen tissue of microbubbles plus ultrasound was obviously more than that in the other Groups.
(4) Ultrastructural changes of the spleen tissues shown by transmission electron microscope. In simple control group, tumor cells had large cellular nuclei, low ratio of kytoplasm to nucelus and more mitochondria with no swelling, while endothelial cells had regular structure with tight conjunction and normal mitochondria. In microbubbles plus ultrasound group, transmission electron microscope showed obvious damaged ultrastructure and that the tumor cells had swelled and globular mitochondria, which was different from simple control group. At the same time, endothelial cells were varied in morphous, with swelled mitochondria and disjunction. There was no significant difference among ultrasound radiation group and simple microbubbles group in comparison with simple control group.
2.Effect of high mechanical index ultrasound contrast on proliferation, apoptosis and metastasis of colon carcinoma Lovo cells:
(1) Millicell method showed marked metastasis of Lovo cells in control group. While after microbubbles plus ultrasound wave irradiation, Lovo cells were found with significant decrease of metastasis (P<0.01), with large quantity of float and adiaphanous dead cells. There was no statistical difference among metastasis number in simple ultrasound radiation and simple microbubbles compared with control group (P>0.05). The metastatic cells in control group, simple ultrasound and simple microbubbles group were more than those in microbubbles plus ultrasound group.
(2) Laser confocal microscopic detection of expression of microfilamin F-actin in VSMC in each group showed that microtubule staining manifested compact network structure extending towards margins in control group; no statistical difference was found in simple ultrasound group and simple ultrasound microbubbles group compared with control group. Compared with control group, expression of microtubule was weaker and sparser, network structure aligning along cell macroaxis in microbubbles plus ultrasound group. In control group, the cell microfilament staining manifested as compact filament network structure protruding many trifling and short sentus ecphyma, with obvious drawing-off character and directivity. There was no statistical difference between simple ultrasound group plus simple ultrasound microbubbles group and control group. In microbubbles plus ultrasound group, there could be seen marked decrease of network-like cells with dull fluorescence and decrease of trifling and short sentus ecphyma in kytoplasm of Lovo cells.
(3) The results of cell proliferation by MTT method showed that the cell proliferation in control group was more than that in microbubbles plus ultrasound group (P<0.01), but no statistical difference was found in aspect of cell proliferation between simple ultrasound group and simple microbubbles group compared with control group (P>0.05), indicating remarkable inhibitive effect on cell proliferation in microbubbles plus ultrasound group.
(4) Results of flow cytometry detecting cell cycle and apoptosis of colon carcinoma Lovo cells in all groups showed that cell ratio was decreased markedly at phase G1 (P<0.01),but increased at phase S (P<0.01). Compared with control group, there was no statistical difference upon cell cycle in simple ultrasound group and simple microbubbles group (P>0.05). In comparison with control group, cell ratio at phase G1 was elevated (P<0.01), but decreased at phase S, and apoptosis peak was added in microvesicle plus ultrasound group (P<0.01). These results indicated that high mechanical index ultrasound contrast can bring about cell cycle change, induce apoptosis of colon carcinoma cells and that microbubbles agents could markedly accentruate the effect of ultrasound inducing apoptosis.
Conclusions:
(1) High MI ultrasound contrast can decrease the number and size of metastatic hepatic carcinoma and inhibit hepatic metastasis of cancer.
(2) High MI ultrasound contrast can induce the massive apoptosis of tumor, thrombogenesis of interior and peripheral tissues and hence result in ultrastructural changes of the tumor cells.
(3) High MI ultrasound contrast can inhibit the metastasis and proliferation of colon cancer Lovo cells and induce the changes of the cystoskeleton.
(4) High MI ultrasound contrast can bring about the change of colon cancer Lovo cell cycle and induce the apoptosis but inhibit the proliferation of colon cancer Lovo cells.
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