第二军医大学学报  2019, Vol. 40 Issue (1): 20-24   PDF    
甲状腺结节微波消融治疗后6个月消融区细胞活性的酶组织化学检测
闫磊1,2Δ, 章建全1,3Δ, 陈红琼3, 吴震中1, 程杰3     
1. 海军军医大学(第二军医大学)长征医院超声诊疗科, 上海 200003;
2. 解放军100医院超声科, 苏州 215007;
3. 上海国际医学中心, 上海 201318
摘要: 目的 探讨甲状腺结节微波消融治疗术后6个月消融区组织内细胞活性情况。方法 选择2017年12月至2018年9月甲状腺结节微波消融治疗术后6个月进行消融区粗针穿刺活组织检查病理评估的患者20例24个消融区。使用切割式活检针分别对消融区中央域及边缘域进行粗针穿刺活组织检查,取得标本条置入液氮制成冰冻切片。采用酶组织化学染色法检测细胞内琥珀酸脱氢酶(SDH)和还原型烟酰胺腺嘌呤二核苷酸磷酸黄递酶(NADPH-d)的活性,并与常规病理切片H-E染色观察到的细胞形态及组织结构对比。结果 微波消融术后6个月24个消融区的中央域及边缘域均成功取材。消融区中央域SDH及NADPH-d酶组织化学染色情况一致性好,阴性率均为95.83%(23/24)。消融区边缘域SDH及NADPH-d酶组织化学染色情况一致性好,阴性率均为91.67%(22/24)。23个中央域及22个边缘域的H-E染色切片均显示为红染无结构的大片坏死组织;1个中央域、2个边缘域H-E染色切片显示部分为坏死结构、部分为纤维组织增生,纤维组织增生处与酶组织化学染色阳性区域位置一致。结论 微波消融术后6个月甲状腺结节消融区组织符合凝固性坏死改变,仍处于失活状态,酶组织化学染色结合H-E染色能够对陈旧消融区做出较为客观的评价。
关键词: 甲状腺结节     热消融术     酶组织化学染色     琥珀酸脱氢酶     还原型烟酰胺腺嘌呤二核苷酸磷酸黄递酶    
Enzyme histochemical detection of cell viability in ablation area of thyroid nodules at 6 months after microwave ablation
YAN Lei1,2Δ, ZHANG Jian-quan1,3Δ, CHEN Hong-qiong3, WU Zhen-zhong1, CHENG Jie3     
1. Department of Ultrasound, Changzheng Hospital, Naval Medical University(Second Military Medical University), Shanghai 200003, China;
2. Department of Ultrasound, No. 100 Hospital of PLA, Suzhou 215007, Jiangsu, China;
3. Shanghai International Medical Center, Shanghai 201318, China
Supported by National Natural Science Foundation of China (81171436).
Abstract: Objective To explore the cell viability in the ablation area of thyroid nodules at 6 months after microwave ablation by enzyme histochemical staining. Methods Twenty-four ablation areas of thyroid nodules were selected from 20 patients who underwent histopathological assessment of the ablation area by core needle biopsy at 6 months after microwave ablation between Dec. 2017 and Sep. 2018. Core needle biopsy was performed at the central and marginal regions of the ablation area with a cutting biopsy needle. The specimens were obtained and placed in liquid nitrogen to make frozen sections. Enzyme histochemical staining was used to detect the activities of succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), and the difference of cell morphology and histological structure was compared with H-E staining results. Results The specimens of the central and marginal regions of 24 ablation areas were successfully obtained. The histochemical staining of SDH and NADPH-d in the central region of ablation area had good consistency, and the negative rates were both 95.83% (23/24). The histochemical staining of SDH and NADPH-d in the marginal region of ablation area also had good consistency, and the negative rates were both 91.67% (22/24). H-E staining of 23 central regions and 22 marginal regions showed pink amorphous mass of necrosis. H-E staining of 1 central region and 2 marginal regions showed partly necrotic and fibrous tissue hyperplasia. The location of fibrous tissue hyperplasia was consistent with the location of the positive region of enzyme histochemical staining. Conclusion At 6 months after microwave ablation, the tissue in the ablation area of thyroid nodules is consistent with coagulative necrosis, and is still inactivated. SDH or NADPH-d enzyme histochemical staining combined with H-E staining can objectively evaluate the old ablation area.
Key words: thyroid nodule     thermal ablation     enzyme histochemical staining     succinate dehydrogenase     nicotinamide adenine dinucleotide phosphate diaphorase    

超声引导下经皮微波消融术是治疗甲状腺结节的新兴方法,近年来凭借出血少、微创伤、无瘢痕、保护甲状腺功能等优势在临床上逐渐受到重视[1-2]。超声引导下经皮热消融治疗的本质在于通过射频、微波、激光等手段使病灶受热发生凝固性坏死,坏死组织经患者自身免疫系统逐渐吞噬吸收、直至消失。研究证实,虽然甲状腺结节热消融术后即刻消融区组织结构仍维持原态,但受热细胞已失去活性[3-4]。由于患者病灶的大小、位置、周围血供及患者的自身免疫状态等存在个体差异,消融区坏死组织吸收的速度不一,消失的时间也从数月至数年不等[5],在此期间,消融区组织结构是否瓦解、是否存在活性备受关注。微波消融术后6个月是消融区病理变化的关键点,此时消融区内多呈大片坏死组织,偶可见少量细胞,但此期细胞活性状态如何尚未见文献报道。本研究通过观察微波消融术后6个月甲状腺结节消融区的细胞活性关键酶琥珀酸脱氢酶(succinate dehydrogenase,SDH)和还原型烟酰胺腺嘌呤二核苷酸磷酸黄递酶(nicotinamide adenine dinucleotide phosphate diaphorase,NADPH-d)的活性,探讨消融区是否会出现凝固变性细胞未完全坏死甚至复活的情况。

1 资料和方法 1.1 一般资料

选择自2017年12月至2018年9月于海军军医大学(第二军医大学)长征医院及上海国际医学中心复诊的甲状腺结节微波消融治疗术后6个月的患者20例,其中女性15例、男性5例,年龄为18~65岁,中位年龄为45岁。20例复诊患者中单发甲状腺结节者16例16枚结节,多发结节者4例共8枚结节。纳入标准:(1)经微波消融治疗甲状腺结节术后6个月对消融区进行病理学评估的患者;(2)甲状腺结节微波消融区各径线长度均>10 mm,满足粗针穿刺活组织检查的要求且取材区域均位于消融区内;(3)消融区位置有安全穿刺路径。排除标准:(1)有严重出血倾向或凝血障碍的患者;(2)服用抗凝药物且停药不足2周的患者;(3)穿刺路径有无法避开的粗大血管或其他重要结构的患者。本研究经海军军医大学(第二军医大学)长征医院伦理委员会及上海国际医学中心伦理委员会审批,所有患者均签署知情同意书。

1.2 仪器设备与试剂

使用意大利百胜MyLabTM Alpha便携式彩色超声诊断系统(高频探头频率为7×106~14×106 Hz)行超声引导下穿刺。使用意大利Presia HS半自动切割式活检针(针长100 mm、外径18 G、切割槽10 mm/20 mm可调)行组织学标本取材。琥珀酸脱氢钠购自美国Sigma公司,氯化硝基四氮唑蓝(nitro blue tetrazolium chloride,NBT)、Triton X-100及β-NADPH均购自生工生物工程(上海)股份有限公司。

1.3 取材与检测方法

患者取仰卧位、颈部略后伸,常规消毒铺洞巾,局部浸润麻醉皮肤穿刺点及穿刺路径,于超声实时引导下对消融区进行粗针穿刺活组织检查,根据消融区取材部位径线调整活检针切割槽长度。取材部位:(1)消融区中央域,即消融区中央部位;(2)消融区边缘域,即消融区内距离边缘5 mm以内的部位。边缘域取材部位不满足活检针切割槽范围时,则仅对中央域取材。所得组织条置入冻存管放入液氮罐冷冻,制作冰冻切片,切片厚度为7~10 μm,然后分别使用琥珀酸脱氢钠和NBT行酶组织化学染色检测SDH及NADPH-d活性,并行常规H-E染色后观察组织结构和细胞形态。

1.4 酶组织化学染色结果判断及H-E染色

镜下观察SDH及NADPH-d酶组织化学染色阳性均为紫黑色,表示SDH及NADPH-d有活性。任选5个高倍镜(×400)视野观察,根据紫黑色颗粒的数量和范围将每张切片的染色强度分为3个等级:(1)阴性,无紫黑色颗粒;(2)弱阳性,散在少量紫黑色颗粒;(3)阳性,5个高倍镜视野中有3个或3个以上视野为大量或满视野紫黑色颗粒。镜下观察H-E染色石蜡切片中消融区组织结构和细胞形态情况,判断凝固性坏死程度。

1.5 统计学处理

采用SPSS 20.0软件进行数据处理。计数资料以例数和百分数表示。比较各区域SDH与NADPH-d酶组织化学染色情况,判断二者染色结果是否一致并与H-E染色结果对照分析。两种染色结果一致性分析采用Kappa检验,Kappa值≥0.75表示一致性较好,Kappa值<0.4表示一致性较差,Kappa值为0.4~0.74表示一致性一般。采用Fisher确切概率法比较同一区域SDH与NADPH-d阴性率。检验水准(α)为0.05。

2 结果 2.1 甲状腺结节微波消融术后6个月消融区粗针穿刺活组织检查取材

本组20例患者共24个消融区。对所有消融区中央域及边缘域均成功穿刺取材,共获得48个组织条,标本条长度为10~20 mm,直径约1 mm,颜色苍白或淡红,连续性好。

2.2 甲状腺结节微波消融术后6个月SDH及NADPH-d酶组织化学染色结果

镜下观察23个(95.83%)消融区中央域SDH及NADPH-d染色呈阴性,镜下均未见紫黑色颗粒(图 1A1B);1个消融区中央域SDH及NADPH-d染色呈阳性,镜下见紫黑色颗粒(图 2A2B);消融区中央域SDH与NADPH-d酶组织化学染色阴性率比较差异无统计学意义(P>0.05)。镜下观察22个(91.67%)消融区边缘域SDH及NADPH-d染色呈阴性,镜下均未见紫黑色颗粒(图 3A3B);2个消融区边缘域SDH及NADPH-d染色呈阳性,镜下均见紫黑色颗粒(图 4A4B);消融区边缘域SDH与NADPH-d酶组织化学染色阴性率比较差异无统计学意义(P>0.05)。48条标本制成酶组织染色切片,镜下观察SDH染色与NADPH-d染色结果均一致,消融区两种染色在中央域及边缘域一致性均较好,Kappa值均为1。

图 1 甲状腺结节微波消融术后6个月消融区中央域SDH(A)、NADPH-d(B)酶组织化学染色阴性结果 Fig 1 Negative enzyme histochemical staining of SDH (A)and NADPH-d (B) in central ablation region of thyroid nodules at 6 months after microwave ablation SDH: Succinate dehydrogenase; NADPH-d: Nicotinamide adenine dinucleotide phosphate diaphorase. Original magnification: ×200

图 2 甲状腺结节微波消融术后6个月消融区中央域SDH、NADPH-d酶组织化学染色阳性结果 Fig 2 Positive enzyme histochemical staining of SDH and NADPH-d in central ablation region of thyroid nodules at 6 months after microwave ablation A: Enzyme histochemical staining of SDH showed that purple-black particles were visible in some areas of the central ablation region, suggesting that SDH was active; B: Enzyme histochemical staining of NADPH-d showed that purple-black particles were visible in some areas of the central ablation region, suggesting that NADPH-d was active. SDH: Succinate dehydrogenase; NADPH-d: Nicotinamide adenine dinucleotide phosphate diaphorase. Original magnification: ×100

图 3 甲状腺结节微波消融术后6个月消融区边缘域SDH(A)、NADPH-d(B)酶组织化学染色阴性结果 Fig 3 Negative enzyme histochemical staining of SDH (A) and NADPH-d (B) in the marginal ablation region of thyroid nodules at 6 months after microwave ablation SDH: Succinate dehydrogenase; NADPH-d: Nicotinamide adenine dinucleotide phosphate diaphorase. Original magnification: ×200

图 4 甲状腺结节微波消融术后6个月消融区边缘域SDH、NADPH-d酶组织化学染色阳性结果 Fig 4 Positive enzyme histochemical staining of SDH and NADPH-d in the marginal ablation region of thyroid nodules at 6 months after microwave ablation A: Enzyme histochemical staining of SDH showed that purple-black particles were visible in some areas of the marginal ablation region, suggesting that SDH was active; B: Enzyme histochemical staining of NADPH-d showed that pale purple-black particles were visible in some areas of the marginal ablation region, suggesting that NADPH-d was active. SDH: Succinate dehydrogenase; NADPH-d: Nicotinamide adenine dinucleotide phosphate diaphorase. Original magnification: ×100

2.3 H-E染色结果与SDH、NADPH-d活性区域的对比

H-E染色镜下观察结果显示23个消融区中央域及22个消融区边缘域均呈现大片凝固性坏死组织,红染无结构(图 5A5B),其对应的SDH及NADPH-d活性均为阴性。1个消融区中央域及2个消融区边缘域H-E染色镜下显示部分为片状坏死结构,部分为纤维组织增生,片状坏死处相应部位的SDH及NADPH-d染色均呈阴性,纤维组织增生处(图 5C5D)对应SDH及NADHP-d为阳性。

图 5 甲状腺结节微波消融术后6个月消融区中央域和边缘域H-E染色 Fig 5 H-E staining in central and marginal ablation regions of thyroid nodules at 6 months after microwave ablation A: H-E staining in the central ablation region showed pink amorphous mass of necrotic material; B: H-E staining in the marginal ablation region showed pink amorphous mass of necrotic material; C: H-E staining showed necrotic material and proliferation of fibrous tissue in the central ablation region, and enzyme histochemical staining of SDH and NADPH-d was both positive in hyperplastic fibrous tissue; D: H-E staining showed necrotic material and proliferation of fibrous tissues in the marginal ablation region, and enzyme histochemical staining of SDH and NADPH-d was both positive in hyperplastic fibrous tissues. SDH: Succinate dehydrogenase; NADPH-d: Nicotinamide adenine dinucleotide phosphate diaphorase. Original magnification: ×200 (A, B); ×100 (C, D)

3 讨论 3.1 SDH与NADPH-d酶组织化学染色评价细胞活性情况的价值

细胞内SDH和NADPH-d是细胞呼吸的关键酶,研究人员通常使用酶组织化学染色法检测SDH和NADPH-d的活性并以此判断组织细胞的活性[6-12]。本团队既往研究发现,SDH及NADPH-d广泛存在于甲状腺组织及甲状腺结节细胞中,并具有活性;当甲状腺结节接受微波热消融治疗时,这2种酶瞬间受热失去活性,酶组织化学染色呈阴性,提示经微波消融治疗后甲状腺结节内细胞迅速进入失活状态[13]。本研究也采用酶组织化学染色法证实SDH与NADPH-d的活性状态具有高度一致性。因此,SDH、NADPH-d酶组织化学染色能够同时真实反映组织中细胞的活性状态。

3.2 微波消融术后6个月消融区组织病理变化

研究证实微波消融治疗使组织受热凝固,术后即刻在光学显微镜下观察到受热组织的结构、细胞外形和排列、细胞核形态暂不会发生明显变化,此时组织处于彻底坏死前的热凝固状态,之后随着时间推移消融区组织才会逐渐溶解、吸收、消失[1]。虽然人体消融区组织随时间推移其病理演变过程不易获得,但可推测这一过程应与病理学上凝固性坏死的转归一致[14]。文献报道,个别患者热消融术后1周、1个月进行手术切除,消融区病理切片除可见坏死区外,亦可见结构形态完整的肿瘤细胞[15-16],说明这时消融区组织仍未进入完全坏死阶段。本研究发现,甲状腺结节微波消融术后6个月时消融区几乎均呈现大片红染坏死无结构样,提示此时消融区坏死组织结构已经完全崩塌溶解,在此时检测酶活性能更全面地了解陈旧消融区组织的活性状态。

3.3 微波消融术后6个月消融区组织细胞的活性情况

本研究发现微波消融术后6个月多数消融区中央域SDH及NADPH-d酶组织化学染色法检测结果均一致,呈阴性,结合H-E染色镜下病理观察到的大片红染无结构,提示此区域已进入完全坏死状态,在形态和功能两个方面都不再是病灶,无需担心消融区的存在会对患者造成不良影响。1个消融区中央域、2个消融区边缘域酶活性染色部分区域为阳性,其对应H-E染色区域恰为纤维组织增生的区域。在失活的坏死消融区中央或边缘出现具有活性的纤维组织细胞,正符合组织损伤修复过程中的改变。

本研究对甲状腺结节微波消融术后6个月消融区细胞活性状态进行了初步探讨,SDH、NADPH-d酶组织化学染色与H-E染色组织学方法结合可较为客观全面地评估消融区组织的活性状态。本研究也存在一些不足:(1)仅对消融术后6个月甲状腺消融区进行评估,对术后1、3、12个月时的消融区细胞活性情况还需进一步研究;(2)纳入的病例数较少,尚待更多数据进一步验证本研究结论。

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