复方中药99-克星超声介入治疗肝癌裸鼠移植瘤凋亡与增生

摘要

目的

探讨超声引导瘤内注射复方中药99-克星治疗肝癌SMMC-7721裸鼠移植瘤的抗癌作用.

方法

取人肝癌SMMC-7721裸鼠皮下移植模型28只, 随机分为99-克星中药治疗组14只, 无水乙醇与生理盐水治疗组各7只. 全部裸鼠在接种10 d后每隔5 d向肿瘤内注入治疗药物共4次, 治疗20 d后处死. 应用免疫组织化学方法检测移植瘤细胞Ki-67抗原的表达并行流式细胞仪(FCM) DNA分析以及采用TUNEL 方法对移植瘤细胞凋亡现象进行观察.

结果

中药99克星组与乙醇组的Ki-67指数分别为17±9 ％、21±11 ％ , 均显著低于盐水组的32±13 ％ (P <0.05); 克星组凋亡指数高达49±5 %, 显著高于乙醇组的12±2 ％和盐水组的10±4 ％ (均P <0.01); FCM DNA分析显示盐水组的异倍体峰出现率高达57 %, 明显高于克星组的7 %与乙醇组的0 % (P 均<0.05), 而克星组的凋亡峰出现率高达93 %, 明显高于乙醇组的14 %与盐水组的0 % (P 均<0.01).

结论

复方中药99克星能显著抑制裸鼠移植瘤细胞的增生, 并诱发其凋亡. 研究结果表明复方中药99克星具有强大的破坏与抑制肿瘤细胞生长的作用, 其疗效不亚于无水乙醇, 其主要机制可能有: (1)抑制癌细胞的增生, (2)诱导癌细胞凋亡. 复方中药99-克星抗肿瘤的作用机制不同于无水乙醇, 尤其以抑制癌细胞增生和诱导癌细胞凋亡的作用机制在抗肿瘤中具有重要的意义. 本研究为传统中药治疗肝癌提供了一个新的给药途径与理论依据, 其在肿瘤治疗研究中具有重要的意义和良好的发展前景.

关键词: N/A

Apoptosis and proliferation of hepatocellular carcinoma in nude mice with percutaneous intratumor injection of Chinese herbal medicine "Star-99"

Xiao-Dong Lin, Li-Wu Lin, Yi-Mi He, Shang-Da Gao, Fa-Duang Yang, En-Sheng Xue

Xiao-Dong Lin, Li-Wu Lin, Yi-Mi He, Shang-Da Gao, En-Sheng Xue, Department of Ultrasound, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China

Fa-Duang Yang, Department of Pathology, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China

Supported by the Research Programs of Science and Technology Commission Foundation of Fujian Province, No. 2000Z138

Correspondence to: Li-Wu Lin, Department of Ultrasound, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China. lxdghl@163.net

Received: January 15, 2003
Revised: January 25, 2003
Accepted: February 19, 2003
Published online: September 15, 2003

Abstract

AIM

To probe the anti-cancer effect of ultrasound-guided local injection with Chinese traditional medicine "Star-99" in the treatment of hepatocellular carcinoma (HCC).

METHODS

Twenty-eight human hepatocellular carcinoma SMMC-7721 transplantation nude mice were randomly divided into Star-99 group containing (14 patterns), ethanol group (7 patients) and saline group (7 patterns ). Ten days after transplantation, the medicine was injected into the tumors of all the nude mice every 5 days. On day 20 after the first injection, the nude mice were killed. Immunohistochemistry assay was adopted to detect the expression of Ki-67 antigen in HCC. The tumor tissues were sent for flow cytometry (FCM) and DNA analysis. Apoptotic cells were visualized by TUNEL assay. All the experiments were carried out under double blind.

RESULTS

The Ki-67 Labeling index in Star-99 group (17±9 %) and ethanol group (21±11 %) was significantly lower than that in saline group (32±13 %) (P <0.05). The mean apoptotic index (AI: percentage of TUNEL signal positive cells) in Star-99 group (49±5 %) was significantly higher than that in ethanol group (12±2 %) and the saline group (10±4 %) (P <0.01). FCM and DNA analysis showed that the appearance rate of the apoptosis peak in Srar-99 group was 93 %, markedly higher than that in ethanol group (14 %) and saline group (0 %) (P <0.01).

CONCLUSION

Star-99 can inhibit cancer cells proliferation remarkably and induce the cancer cell apoptosis. It has strong effects of inhibiting and destructing cancer cells. Its curative effect is as good as the ethanol. Its major mechanisms can be as follows: (1) It can inhibit cancer cell proliferation. (2) It can induce cancer cell apoptosis. The mechanism of Star-99 is different from that of ethanol.The mechanisms of inhibiting cancer cell proliferation and inducing cell apoptosis is of great significance. This study will provide a new pathway for drug administration and an experimental basis for the treatment of HCC with traditional Chinese herbal. The study of Star-99 in the treatment of tumor is of profound significance and has good prospects.

Key Words: N/A

0 引言

我国肝癌常见超声引导介入治疗肝癌的方法依然存在一定的局限性[1-10]. 复方中药99-克星有强大的抑制肿瘤生长的作用, 并且其作用机制明显不同于无水乙醇[11,12]. 为进一步探讨超声引导经皮瘤内注射复方中药99克星的疗效机制, 我们通过应用免疫组织化学方法检测移植瘤细胞Ki-67抗原的表达并行流式细胞仪(FCM) DNA分析、TUNEL 方法对细胞凋亡现象进行观察以评价99克星抑制肝癌细胞增生与诱导肝癌细胞凋亡的生物效应.

1 材料和方法

1.1 材料

实验动物由厦门大学抗癌中心医学实验动物室(合格证号: 闽医动字第23-007号)提供的5-8周龄BALB/CA裸鼠(SPF级, 雌雄兼用), 质量18.0±2.1 g, 在无病原体的层流架内饲养, 无菌操作下定期更换笼具、垫料、饮用水和标准饲料(由上海必凯公司提供). 将人肝癌SMMC-7721细胞培养悬液离心后去上清, 再加细胞培养液配成5×10¹⁰ /L, 在裸鼠背部皮下注射0.2 mL. 当肿瘤长至1 cm左右时, 无菌条件下取下部分瘤块, 切成0.2×0.2× 0.2 cm瘤块, 用套管针移植到另一只裸鼠背部皮下, 形成皮下移植并传代.

1.2 方法

接种人肝癌移植瘤裸鼠28只, 随机分3组:中药99-克星组 (下称克星组, 主要成分为天南星、丹参与黄芪等)14只, 无水乙醇组(下称乙醇组)7只, 生理盐水组(下称盐水组)7只. 实验动物接种后10 d, 采用高频超声(日本ALOKA-5500超声仪, 探头频率10 MHz)测量肿瘤最大长径、宽径与厚径, 并计算体积. 以5号针头刺入肿瘤中心, 缓缓注入各组药物0.1 ml, 每隔5 d注射1次, 共4次, 在末次注射5 d后再超声测量肿瘤三径后将裸鼠处死. 应用免疫组织化学方法检测Ki-67抗原的表达. 石蜡标本4 mm厚连续切片, 免疫组化采用SP法. 鼠抗人Ki-67单克隆抗体MIB1(即用型)、S-P免疫组化试剂盒购自福州迈新公司, 参照说明书及文献操作. Ki-67抗原阳性产物定位于细胞核内, 呈褐色或黄褐色颗粒状, 胞质一般不着色. 部分细胞核分裂时显示出整个细胞的阳性反应. 采用北京航天航空大学CMIAS 医学图像分析系统, 每个标本在低倍镜(×100)下观察并确定有代表性的Ki-67染色阳性视野, 连续观察10个高倍视野(×400), 计算每个高倍视野瘤细胞中的阳性数. 计算肿瘤Ki-67指数 (Ki-67 Labeling index Ki-67 LI), Ki-67 LI＝阳性细胞总和／计数细胞总数 ×100 %. 采用末端转移酶介导的dUTP切口末端标记方法(TUNEL)检测凋亡细胞. 试剂盒(POD法)购自福州迈新公司(德国Boehringer Mannheim公司生产), 参照说明书及文献操作. TUNEL染色以细胞核有明显棕黄色颗粒为阳性, 采用北京航天航空大学CMIAS 医学图像分析系统, 每张切片观察10个高倍视野(×400), 随机计数不少于1 000个细胞的阳性细胞数. 计算肿瘤细胞凋亡指数(Apoptotic index, AI), AI＝凋亡细胞数／计数细胞总数×100 ％. 取新鲜肿瘤组织制备细胞混悬液行FCM DNA分析, FCM分析DNA的异倍体峰与凋亡峰的出现率.

统计学处理 实验数据经 SPSS for Windows 8.0软件包行单因素方差分析和x²检验统计学处理, 以P <0.05为差异有显著性意义.

2 结果

99克星组与乙醇组的Ki-67指数分别为17±9 ％, 21±11 ％, 均显著低于盐水组的32±13 ％ (P <0.05) (图1). 克星组凋亡指数AI为49±5 %, 明显高于乙醇组的12±2 %和盐水组的10±4 ％ (P <0.01) (图2). 盐水组中57％(4/7)出现异倍体峰(图3), 显著高于未出现异倍体峰的乙醇组0 % (0/7)与克星组的7 % (1/14)(P <0.05). 另一明显特点是克星组有93 % (13/14)见亚G1期细胞形成的凋亡峰(图4), 显著高于乙醇组的14％ (1/7)(P <0.01), 而盐水组无1例出现凋亡峰.

图1 裸鼠肝癌组织的KI-67标记×400(细胞核有明显棕黄色颗粒为阳性). A: 99-克星组; B: 盐水组.

图2 裸鼠肝癌组织的TUNEL标记×400(细胞核有明显棕黄色颗粒为阳性). A: 99-克星组; B: 盐水组.

图3 注射生理盐水裸鼠肝癌组织FCM DNA分析出现异倍体峰(图中黄色峰).

图4 注射99-克星裸鼠肝癌组织FCM DNA分析出现凋亡峰(图中蓝色峰).

3 讨论

临床上已有多种超声引导介入治疗肝癌的方法, 近年来国内外相继推出激光、微波、射频以及高能聚焦超声等在超声引导下介入治疗肝癌, 其中有的疗效甚至可与外科手术切除相媲美[2-5]. 但是以上多种方法依然存在一定的局限性[6-10]. 我们发现99-克星有强大的抑制肿瘤生长的作用, 其疗效不亚于无水乙醇[11,12]. 病理细胞学和组织学观察也显示, 克星组肿瘤细胞出现肿胀、变性坏死与程序性坏死为主的特征性改变, 与乙醇组肿瘤组织呈凝固性坏死为主的改变明显不同. 更为独特的是, 克星组所有的变性肿胀肿瘤组织中同时出现许多淋巴细胞, 甚至形成淋巴细胞团, 而乙醇治疗组未见到明显的淋巴细胞. 这一发现提示复方中药99-克星能够促进机体局部组织炎症反应和淋巴细胞活跃, 其抑制或杀伤肿瘤细胞的作用很可能与细胞免疫功能增强机制有关, 他与无水乙醇单纯杀灭肿瘤细胞而缺乏组织反应的作用机制显然不同[11,12]. 因此值得进一步深入研究其作用机制.

Ki-67是一种敏感而特异的增生期细胞标记物, 他可能是一类新的细胞核非组织蛋白[13-15], 与细胞周期密切相关. Ki-67抗原与细胞DNA半保留复制相藕连, 在有丝分裂的G1, G2, M, S期都表达, 其中G2, M期表达最强, G0期不表达[16,17]. 因其半衰期短, 细胞脱离增生周期后迅速降解, 检测结果的可靠性明显优于目前常用的半衰期长的增生期细胞标记物即增生细胞核抗原(PCNA), 肿瘤细胞增生越活跃, 肿瘤细胞ki-67表达水平亦相应增高, 因此被视为较可靠的全面反映细胞群体增生水平的指标[18-20]. 本实验结果显示99克星组与乙醇组 的Ki-67指数分别为17±9 ％ 、21±11 ％ , 均显著低于盐水组的32±13 ％ (P <0.05). 99克星组与乙醇组移植瘤细胞ki-67表达明显减弱, 表明大量的癌细胞处于细胞增生周期的静止期, 提示99克星与乙醇一样能阻止癌细胞进入分裂期, 从而抑制肿瘤的生长. 这表明99克星与乙醇一样能显著抑制肝癌细胞的增生, 从而抑制裸鼠SMMC-7721移植瘤生长.

调控细胞凋亡成为目前肿瘤治疗研究的一个新热点[21-25]. 我们应用TUNEL法检测发现克星组的凋亡指数高达49 ±5 ％, 显著高于乙醇组的12±2 ％和盐水组的10± 4 ％ (P <0.01), 这与FCM 的DNA分析显示99-克星组中93 % (13/14)出现典型的亚二倍体的凋亡峰, 乙醇组仅1例出现低值的凋亡峰, 而盐水组无1例出现凋亡峰的结果相一致. 值得提出的是从DNA分析与TUNEL检测以及Ki-67抗原表达检测结果均显示乙醇组虽然肿瘤细胞生长也受抑制, 但并不表现为以凋亡为主的改变. 相反克星组DNA分析以及TUNEL检测结果均表明其抑制肿瘤生长的主要机制之一是诱导肝癌细胞凋亡, 二者有明显差别.本结果表明复方中药99-克星具有强大的抑制与破坏肿瘤生长的作用. 除了能直接杀灭肿瘤细胞以及增强细胞免疫功能外, 还能抑制肝癌细胞的增生并且诱导肝癌细胞凋亡, 明显不同于无水乙醇, 尤其以抑制肝癌细胞的增生及诱导肝癌细胞凋亡的作用机制具有重要意义, 因此其在肿瘤的治疗研究中具有重要的意义和良好的发展前景.

备注

$[Footnotes]

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