榄香烯联合VEGF多克隆抗体对大鼠脑胶质瘤脑组织PCNA、T1MP-1表达的影响

【摘要】 目的 探讨榄香烯联合血管内皮生长因子（VEGF）多克隆抗体对大鼠脑胶质瘤脑组织增殖细胞核抗原（PCNA）、组织金属蛋白酶抑制剂-1（TIMP-1）表达的影响。方法 采用脑内注射C6胶质瘤细胞建立大鼠胶质瘤模型，选取建模成功48只大鼠随机分为模型组、榄香烯组、VEGF多克隆抗体组、榄香烯联合VEGF多克隆抗体组各12只。模型组腹腔注射0.5ml生理盐水，榄香烯组腹腔注射0.5ml榄香烯注射液，VEGF多克隆抗体组腹腔注射0.5mlVEGF多克隆抗体，榄香烯联合VEGF多克隆抗体组腹腔注射0.5ml榄香烯注射液、0.5mlVEGF多克隆抗体。隔日给药，连续7次。比较各组大鼠瘤体体积与抑瘤率、脑组织PCNA与TIMP-1表达情况。结果 （1）瘤体体积与抑瘤率：模型组、榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组大鼠瘤体体积分别为（68.54±8.12、37.65±6.24、39.12±6.41、30.12±5.24）mm3；香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组抑瘤率明显为（45.07±6.45、42.92±6.52、56.05±7.21）%。榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组大鼠瘤体体积明显小于对照组（t=10.449, 9.851, 13.772,P<0.01）；榄香烯联合VEGF多克隆组大鼠瘤体体积明显小于榄香烯组、VEGF多克隆组（t=3.201, 3.766, P<0.05），抑瘤率明显高于榄香烯组、VEGF多克隆组（t=3.932, 4.679, P<0.05）。（2）脑组织PCNA与TIMP-1表达：模型组、榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组次瘤体组织PCNA指数分别为（73.12±10.21、44.24±6.42、47.36±7.12、37.45±5.24）；TIMP-1指数分别为（45.24±6.45、62.45±8.15、59.45±7.32、78.65±11.24）。榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组瘤体组织PCNA指数明显低于模型组（t=8.295, 7.447, 10.767, P<0.05, P<0.01），TIMP-1指数明显高于模型组（t=5.736, 5.401, 8.931, P<0.05, P<0.01）；榄香烯联合VEGF多克隆组瘤体组织PCNA指数明显低于榄香烯组、VEGF多克隆组（t=2.838, 3.491, P<0.05），TIMP-1指数明显高于榄香烯组、VEGF多克隆组（t=4.042, 4.700, P<0.05）。结论 榄香烯联合VEGF多克隆抗体有助于抑制脑胶质瘤体组织生长。

【关键词】 脑胶质瘤；榄香烯；VEGF多克隆抗体；增殖细胞抗原；组织金属蛋白酶抑制剂-1

【中图分类号】 R739.4 【文献标识码】 A

The Effect of Elemene in Combination with Vascular Endothelial Growth Factor (VEGF) Polyclonal Antibody on the Expression of Proliferating Cell Nuclear Antigen (PCNA) and Tissue Inhibitor of Metalloproteinase-1 (T1MP-1) in Brain Glioma Tissue of Rats

Zhou Jiangbo 1 Wang Dong 1 Ma Jingji 2 Zhang Jianjun 1*

1 Neurosurgery Department, fourth Central Hospital of Tianjin 300140

2 Neurosurgery Department of Tianjin No. 1 Central Hospital 300192

Corresponding author: Zhang Jianjun, Email：

Foundation project:Tianjin Natural Science Foundation (No. 17 JCYBJC27300)

Abstract: Objective: To investigate the effect of elemene in combination with VEGF polyclonal antibody on the expression of PCNA and TIMP-1 in brain glioma tissue of rats. Methods: The rat glioma models were built by intracerebral injection of C6 glioma cells. Forty-eight successful rat models were selected and randomly divided into model group, elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group, with 12 rats in each group. Rats in model group were performed intraperitoneal injection of 0.5 ml normal saline, rats in elemene group were performed intraperitoneal injection of 0.5 ml elemene injection, rats in VEGF polyclonal antibody group were performed intraperitoneal injection of 0.5 ml VEGF polyclonal antibody, and rats in elemene in combination with VEGF polyclonal antibody group were performed intraperitoneal injection of 0.5 ml elemene injection and 0.5 ml VEGF polyclonal antibody. The drug was administered on alternate days for 7 consecutive times. The tumour volume, tumor inhibition rate and the expression of PCNA and TIMP-1 in brain tissue of the rats between the groups were compared. Results: (1) Tumor volume and tumor inhibition rate: The tumor volumes in model group, elemene group, polyclonal group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (68.54±8.12, 37.65±6.24, 39.12±6.41, 30.12±5.24 mm3), respectively; the tumor inhibition rates in elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (445.07±6.45, 42.92±6.52, 56.05±7.21) %, respectively. The tumor volume in elemene in combination with VEGF polyclonal antibody group was significantly smaller than that in control group (t=10.449, 9.851, 13.772,P<0.01); the tumor volume in elemene in combination with VEGF polyclonal antibody group was significantly smaller than that in elemene group and VEGF polyclonal antibody group (t=3.201, 3.766, P<0.05), and the tumor inhibition rate was significantly higher than that in elemene group and VEGF polyclonal antibody group (t=3.932, 4.679, P<0.05). (2) As to the expression of PCNA and TIMP-1 in brain tissue: the PCNA indexes of tumors in model group, elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (73.12±10.21, 44.24±6.42, 47.36±7.12, 37.45±5.24), respectively, and the TIMP-1 indexes were (45.24±6.45, 62.45±8.15, 59.45±7.32, 78.65±11.24), respectively. The PCNA indexes of tumors in elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were significantly lower than that in model group (t=8.295, 7.447, 10.767, P<0.05, P<0.01), the TIMP-1 indexes were significantly higher than that in model group (t=5.736, 5.401, 8.931, P<0.05, P<0.01); the PCNA index of tumor in elemene in combination with VEGF polyclonal antibody group was significantly lower than that in elemene group and VEGF polyclonal antibody group (t=2.838, 3.491, P<0.05), and the TIMP-1 index was significantly higher than that in elemene group and VEGF polyclonal antibody group (t=4.042, 4.700, P<0.05). Conclusion: Elemene in combination with VEGF polyclonal antibody can help to inhibit the growth of brain glioma tissue.

Key words: Brain glioma; Elemene; VEGF polyclonal antibody; PCNA; TIMP-1

手术切除是目前脑胶质瘤主要治疗手段，但术后易复发，目前多采用手术联合放化疗治疗。血管内皮生长因子（VEGF）多克隆抗体具有阻断VEGF信号通路、抑制新生血管形成的作用[1]。榄香烯提取物具有提高机体免疫能力、杀伤肿瘤细胞、促进细胞凋亡的作用[2]。增殖细胞核抗原（PCNA）是存在于细胞核内的酸性蛋白，其过度表达参与了胶质瘤的恶性进展[3]；组织金属蛋白酶抑制剂-1（TIMP-1）为基质金属蛋白酶-9（MMP-9）特异性抑制剂[4]。本文分析榄香烯联合VEGF多克隆抗体对大鼠脑胶质瘤模型脑瘤组织中PCNA、TIMP-1表达的影响。

材料与方法

1材料

实验动物 60只SD雄性大鼠，体重220-250（225.23±13.12）g；由天津医科大学动物实验中心提供。生产许可证号SCXK(津)2017-0315，使用许可证号SYXK(津)2017-0610。

1.2 药物与试剂 榄香烯乳注射液：大连华立金港药业有限公司，国药准字H10960114，规格0.1g/20ml；VEGF多克隆抗体：武汉博士德生物工程有限公司；免疫组化PCNA试剂盒：美国Santa Cruz公司；免疫组化T1MP-1试剂盒：福新迈新技术开发公司。

1.3 主要仪器 倒置显微镜：日本奥林马斯公司CKX41型；全自动酶标仪：美国BIO-TEK公司ELX-800型；切片机：德国LEICA RM2245型。

2 方法

2.1 细胞培养与处理 C6大鼠脑胶质瘤细胞株复苏成功后，使用普通培养液置于培养箱（温度370C，相对湿度95%，50%CO2）常规培养。当细胞处于对数生长期时，使用0.25%的胰蛋白酶适量消化3-5min，弃消化液。使用PBS液制成细胞悬液并调整至1×1010/l细胞浓度。

2.2 建模 大鼠尾静脉注射1%戊巴比妥钠（40mg/kg），于大鼠头顶双侧眼裂线后约1.5cm横向切开头皮，自前窗中点1.5cm矢状旁3cm处钻一骨孔，抽取10μl C6细胞悬液，经原骨孔处垂直进针硬膜下6mm，后退1mm，以1μl/min速度注入，骨蜡封闭骨孔，缝合切口。接种14d抽取5只大鼠，取其脑组织，显微镜下显示细胞呈梭形排列，可见细胞核分裂，肿瘤中心伴明显出血及栅栏状坏死，肿瘤微血管丰富，说明造模成功。

2.3 分组干预 选取48只建模成功荷瘤大鼠，随机分为模型组、榄香烯组、VEGF多克隆抗体组、榄香烯联合VEGF多克隆抗体组各12只。榄香烯剂配制成1%浓度，VEGF多克隆抗体稀释成1mg:1000ml比例。模型组腹腔注射0.5ml生理盐水，榄香烯组腹腔注射0.5ml榄香烯注射液（20mg/kg），VEGF多克隆抗体组腹腔注射0.5mlVEGF多克隆抗体（50μg/只），榄香烯联合VEGF多克隆抗体组腹腔注射0.5ml榄香烯注射液、0.5mlVEGF多克隆抗体。均隔日给药，连续7次。

2.4 观察指标

2.4.1 瘤体体积及抑瘤率 术后15d，麻醉后处死大鼠，断头取脑，4%多聚甲醛固定1周，沿脑表接种穿刺点行冠状切口，测量肿瘤长宽高，计算肿瘤体积、抑瘤率。抑瘤率=（模型组-实验组）肿瘤体积/对照组肿瘤体积×100%。

2.4.2 PCNA与T1MP-1表达 取肿瘤组织，石蜡包埋切片（4μm），常规HE染色确定病理诊断。石蜡切片脱蜡水化，加50μl 50%过氧化氢，PBS冲洗，室温下孵育。分别加入PCNA单抗、T1MP-1单抗，4C过夜。PBS冲洗，加链霉素抗生素-过氧化物酶溶液，DAB显色。常规复染、冲洗、干燥、封片。正常组织为对照组，以胞核或胞质中出现棕色颗粒为阳性，高倍视野下（400倍）阳性细胞数占总肿瘤细胞百分率为PCNA、TIMP-1阳性指数。

2.5 统计学方法 用SPSS21.0软件统计，计量资料用（ ±s）表示，多组间比较采用单因素方差分析，两组间比较采用t检验。

结 果

1 瘤体体积与抑瘤率比较 榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组大鼠瘤体体积明显小于对照组（P<0.01）；榄香烯联合VEGF多克隆组大鼠瘤体体积明显小于榄香烯组、VEGF多克隆组（P<0.05），抑瘤率明显高于榄香烯组、VEGF多克隆组（P<0.05）。榄香烯组、VEGF多克隆组两组瘤体体积、抑瘤率比较无差异（P>0.05）。见表1。

表1 4组脑胶瘤大鼠瘤体体积、抑瘤率比较（±s）

组别

例数

瘤体体积（mm3）

抑瘤率（%）

模型组

12

68.54±8.12

　

榄香烯组

12

37.65±6.24b

45.07±6.45

VEGF多克隆组

12

39.12±6.41b

42.92±6.52

榄香烯联合VEGF多克隆组

12

30.12±5.24bc

56.05±7.21c

F

8.654

4.365

P

0.000

0.025

注：干预组与模型组比较，瘤体体积：t=10.449, 9.851, 13.772, bP<0.01；榄香烯联合VEGF组与榄香烯组、VEGF多克隆组比较，瘤体体积：t=3.201, 3.766, cP<0.05, 抑瘤率：t=3.932, 4.679, cP<0.05。

2 PCNA与T1MP-1表达比较 榄香烯组、VEGF多克隆组、榄香烯联合VEGF多克隆组瘤体组织PCNA指数明显低于模型组，TIMP-1指数明显高于模型组（P<0.05, P<0.01）；榄香烯联合VEGF多克隆组瘤体组织PCNA指数明显低于榄香烯组、VEGF多克隆组（P<0.05），TIMP-1指数明显高于榄香烯组、VEGF多克隆组（P<0.05）。见表2。

表2 4组脑胶瘤大鼠瘤体组织PCNA、TIMP-1表达比较（±s）

组别

例数

PCNA指数

TIMP-1指数

模型组

12

73.12±10.21

45.24±6.45

榄香烯组

12

44.24±6.42b

62.45±8.15a

VEGF多克隆组

12

47.36±7.12a

59.45±7.32a

榄香烯联合VEGF多克隆组

12

37.45±5.24bc

78.65±11.24bc

F

8.435

5.362

P

0.001

0.014

注：干预组与模型组比较，PCNA指数：t=8.295, 7.447, 10.767, aP<0.05, bP<0.01, TIMP-1指数：t=5.736, 5.401, 8.931, aP<0.05, bP<0.01；榄香烯联合VEGF组与榄香烯组、VEGF多克隆组比较，PCNA指数：t=2.838, 3.491, cP<0.05, TIMP-1指数：t=4.042, 4.700, cP<0.05。

讨 论

肿瘤细胞凋亡与血管形成明显相关，通过阻断肿瘤血管形成可促进肿瘤细胞凋亡。VEGF抗体能竞争性结合循环VEGF，阻断VEGF信号通路，抑制内皮细胞迁移与增殖、血管形成[5]。榄香烯易通过血脑屏障，可抑制DNA、RNA合成，VEGF多克隆抗体主要作用是拮抗肿瘤组织血管形成。本研究中，榄香烯联合VEGF多克隆组大鼠瘤体体积明显小于榄香烯组、VEGF多克隆组，抑瘤率明显高于榄香烯组、VEGF多克隆组，说明榄香烯联合VEGF多克隆抗体抑制大鼠脑胶质瘤生长效果肯定。

PCNA在G0-G1期细胞无明显表达，S期达到峰值，G2-M期明显降低[3]。TIMP-1是判断脑胶质瘤恶性程度的重要指标。榄香烯能明显抑制体外培养人肝癌MMP-2、MMP-9表达水平[6]。本研究中，榄香烯联合VEGF多克隆组瘤体组织PCNA指数明显低于榄香烯组、VEGF多克隆组，TIMP-1指数明显高于榄香烯组、VEGF多克隆组，这也可能是联合治疗能够抑制脑胶质瘤增殖的主要原因。

利益冲突  所有作者均声明不存在利益冲突

作者贡献声明  周江波、王东、张建军实验操作、论文撰写、数据整理、统计学分析；马景鑑实验指导、论文修改。

参 考 文 献

Huang Y, Carbone DP. Mechanisms of and strategies for overcoming resistance to anti-vascular endothelial growth factor therapy in non-small cell lung cancer [J]. Biochim Biophys Acta, 2015, 1855(2): 193-201. DOI：10.1016/j.bbcan.2015.01.003

郭立彬，董秋峰，付宏亮，等. β榄香烯对人胶质瘤SHG44细胞增殖及凋亡的影响[J].现代生物医学进展，2016,16(6):1039-1042.DOI：10.13241/j.cnki.pmb.2016.06.010

GUO Li-bin,DONG Qiu-feng,FU Hong-liang,et al.Effects of β-Elemene on the Proliferation and Apoptosis of SHG44 Human Glioma Cells[J] .Progress in Modern Biomedicine，2016,16(6):1039-1042.DOI：10.13241/j.cnki.pmb.2016.06.010.

Lv Q, Zhang J, Yi Y, et al. Proliferating Cell Nuclear Antigen Has an Association with Prognosis and Risks Factors of Cancer Patients: a Systematic Review [J]. Mol Neurobiol, 2016, 53(9): 6209-6217.DOI： 10.1007/s12035-015-9525-3

刘晋川.TIMP-1、CXCL9在脑胶质瘤患者中的表达及意义分析[J].标记免疫分析与临床，2017,24(10):1112-1114,1141.DOI：10.11748/bjmy.issn.1006-1703.2017.10.009

LIU Jing-chuan.Expression and Significance of TIMP-1 and CXCL9 in Patients with Glioma[J].Labeled Immunoassays and Clinical Medicine，2017,24(10):1112-1114,1141.DOI：10.11748/bjmy.issn.1006-1703.2017.10.009

[5]罗文杰，刘明华，罗小莉，等.贝伐珠单抗注射液治疗晚期结肠癌的临床研究[J].中国临床药理学杂志，2018,34(12):1407-1409,1419.DOI：CNKI:SUN:GLYZ.0.2018-12-005

LUO Wen-jie,LIU Ming-hua,LUO Xiao-li,et al.Clinical trial of bevacizumab injection on advanced colon cancer[J]. The Chinese Journal of Clinical Pharmacology,2018,34(12):1407-1409,1419.DOI： CNKI:SUN:GLYZ.0.2018-12-005

[6]毛雨秋. β-榄香烯注射液对人肝癌HepG-2细胞侵袭、迁移的作用及其相关机制[J].实用癌症杂志，2018,33(5):700-703.DOI：CNKI:SUN:SYAZ.0.2018-05-002

MAO Yuqiu.Study of β-elemenum Injection on the Invasion,Metastasis of Human Hepatic Carcinoma HepG-2 Cell and the Related Mechanism[J]. The Practical Journal of Cancer，2018,33(5):700-703.DOI：CNKI:SUN:SYAZ.0.2018-05-002