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Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-04-15; 11(4): 415-418
在线出版日期: 2003-04-15. doi: 10.11569/wcjd.v11.i4.415
热休克蛋白70与IL-2对小鼠肝癌移植模型的治疗比较
傅庆国, 沈晓东, 孟凡东, 郭仁宣
傅庆国, 沈晓东, 孟凡东, 郭仁宣, 中国医科大学附属第一医院普外二科 辽宁省沈阳市 110001
傅庆国, 男, 1966-01-10生, 辽宁省沈阳市人, 汉族, 1989年锦州医学院本科毕业, 学士, 1999年于中国医科大学获博士学位, 副教授, 副主任医师, 主要从事肿瘤生物治疗方面研究. 已发表相关论文16篇.
通讯作者: 傅庆国, 110001, 辽宁省沈阳市, 中国医科大学附属第一医院普外二科. qingguofu@hotmail.com.
电话: 024-23256666-6237
收稿日期: 2002-07-16
修回日期: 2002-11-15
接受日期: 2002-12-01
在线出版日期: 2003-04-15

目的

比较分析肿瘤热休克蛋白70(HSP70)与IL-2对小鼠肝癌移植模型的治疗作用, 评价HSP70的疗效, 为应用HSP70治疗人类恶性肿瘤提供有重要参考价值的实验依据.

方法

细胞培养、蛋白质纯化技术、电泳技术、Western-blot法、动物实验等.

结果

IL-2及HSP70对小鼠肝癌移植模型均有治疗作用, 其中IL-2 10万U、HSP70 10 μg效果最佳, IL-2 5万U及HSP70 5 μg有部分治疗作用, 10万U的IL-2与5 μg HSP70治疗效果相当, HSP70 10 μg 治疗后有40%小鼠移植肿瘤全部消退, 长期生存790 d, 与对照组及其他各组相比, 差异具有显著性(P<0.01, P<0.05).

结论

HSP70具有良好的抗肿瘤免疫活性, 其疗效明显优于IL-2, 本研究对于应用HSP70治疗人类的恶性肿瘤有十分重要的借鉴意义.

关键词: N/A
引文著录: 傅庆国, 沈晓东, 孟凡东, 郭仁宣. 热休克蛋白70与IL-2对小鼠肝癌移植模型的治疗比较. 世界华人消化杂志 2003; 11(4): 415-418
Comparison of therapeutic efficacy between tumor-derived heat shock protein 70 and interleukine-2
Qing-Guo Fu, Xiao-Dong Shen, Fan-Dong Meng, Ren-Xuan Guo
Qing-Guo Fu, Xiao-Dong Shen, Fan-Dong Meng, Ren-Xuan Guo, Second General Surgery Department, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China
Correspondence to: Qing-Guo Fu, The Second General Surgery Department, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning Province, China. qingguofu@hotmail.com
Received: July 16, 2002
Revised: November 15, 2002
Accepted: December 1, 2002
Published online: April 15, 2003

AIM

To compare and analyze the therapeutic effect of tumor-derived heat shock protein 70 and globally accepted interleukin-2, to evaluate the anti-tumor capacity of HSP70, and to provide significant information for HSP70 administration to treat human cancers.

METHODS

Cell Culture, techniques for protein extraction and purification, SDS-PAGE, Western-blot and animal experiment were used in this study.

RESULTS

Both IL-2 and HSP70 showed therapeutic effect in tumor-bearing mice. The best effect was observed in 100 000 U IL-2 and 10 μg HSP70 administrations, and partial efficacy was found in 50000 U IL-2 and 5 μg HSP70 administrations. The effect of 100000 U IL-2 was nearly as good as that of 5 μg HSP70. About 40 % mice receiving HSP70 10 μg administration survived over 90 days, the average survival period of this group was over 56.8 days, whereas the control group was 17.3 days, IL-2 50 000 group, 26.3 days, IL-2 100 000 group, 36.6 days, and 5 μg HSP70 group, 27.7 days. Significant difference was found (P<0.05) when compared with the HSP70 10 μg group and control group.

CONCLUSION

HSP70 has a specific anti-tumor effect and obviously exceeded IL-2 .Those data provide significant information for the treatment of human cancers.

Key Words: N/A
0 引言

恶性肿瘤的免疫疗法已经成为继手术、放化疗之后的又一重要的肿瘤综合治疗手段[1-8], 随着特异性肿瘤抗原的发现和研究的发展, 研究应用肿瘤特异性抗原, 进行人类恶性肿瘤的治疗已成为一个重要的方向[9-18]. 国内外对肿瘤热休克蛋白70(Heat Shock Protein 70, HSP70)进行了深入的研究, 发现HSP70确有明显的特异性的抗肿瘤作用[19-22], 不但能够使健康小鼠获得100%的保护性作用, 而且能使荷瘤小鼠的肿瘤生长受到明显的抑制, 部分动物长期生存、肿瘤全部消退, 而IL-2经过多年的实验与临床研究证实[23-25], 其中治疗有效率在20 %左右, 为评价HSP70的治疗作用, 作者以近交系615系小鼠对HSP70及IL-2的治疗作用进行动物实验对照, 以此为HSP70的应用提供有实用意义的参考依据.

1 材料和方法
1.1 材料

将5×109个HcaF细胞置于40 ml低渗缓冲液(30 mmol/L NaHCO3, 0.5 mmol/L苯甲基酰磺酰氟, pH7.2)中, 以超声粉碎机破碎, 4 ℃ 100000 g超速离心2 h, 取上清过 ConA-Sepharose柱, 收集未结合组分, 4 ℃缓冲液A (20 mmol/L Tris-HC1, 20 mmol/L NaC1, 15 mmol/L β-疏基乙醇, 3 mmol/L MgCl2, pH7.5)中透析过夜, 然后将样品加到 ADP-agarose柱上, 结合部分用含3 mmol/L ADP的缓冲液A进行洗脱, 将洗脱液加于 Mono Q柱, 用 FPLC系统进行分离, 200-500 mmol/L梯度NaC1洗脱, 分别收集各洗脱峰蛋白, 4 ℃缓冲液B(10 mmol/L Tris-HC1, pH7.5)中透析过夜. 将各色谱柱分离的样品及Mono Q柱的各洗脱峰样品在100 g/L SDS-PAGE上进行分子量及纯度测定, 银染色, 再转印至硝酸纤维素膜上, 以抗HSP70单抗为一抗, 碱性磷酸酶标记的羊抗小鼠IgG为二抗, 进行免疫印迹分析. 提取的HSP70以(动物实验)来鉴定其抗肿瘤活性.

1.2 方法

动物分为对照组、 HSP70 5 μg组2个、HSP70 10 mg组2个, IL-2 5万U组2个、IL-2 10万U组2个, "IL-2化"组分别用于观察治疗作用. 治疗方法: 接种HcaF细胞1×106个/只, 7 d成瘤后, HSP70 : 第1个月1次/wk连4次; 第2个月10 d/次连3次; 以后15 d/次至实验结束. IL-2: 5万U、10万U1次/d, 连5 d皮下注射; 2 000U 1次/d, 连10 d皮下注射; 2 000U 2次/wk皮下注射, 至实验结束."IL-2化"组, 在对照组全部死亡后, 取5只健康小鼠先于1 wk连用IL-2 10万U/d, 连5 d, 作者称其为"IL-2化"(Interleukine-2 Preparation). 于第8天接种HcaF细胞1×106个/只, (同时)再取一组IL-2 10万U和HSP70 10 μg治疗组小鼠于不同于上次接种部位与IL-2化组同时再次接种HcaF细胞1×106个/只(活细胞率>96%), 同时停止治疗, 观察各组小鼠的肿瘤生长情况.

2 结果

经过二次亲合层析和Mono Q柱的分离后, 得到纯度较高的分子质量约为70 kD的蛋白质, 经免疫印记分析证实该蛋白质即为HSP70(图1). 经毛细管电泳分析纯度达100 %.

图1
图1 对照组、I5、I10、H5、和H10组小鼠的肿瘤切除标本(从左至右).

IL-2与HSP70对小鼠肿瘤均有较明显的抑制作用, 应用免疫治疗后, 小鼠的生存期均有延长, 与对照组相比差异显著(P<0.01或0.05). 但HSP70较IL-2具有更显著的治疗作用, IL-2治疗组无一长期生存, 而HSP70 10 μg 组有40%的小鼠经治疗后获长期生存790 d, 其肿瘤最终全部消退(表1)(图2).

表1 IL-2及HSP70治疗结果.
分组肿瘤大小(cm)肿瘤质量(g)腹水量(g)腹水出现率(%)生存天数(d)长期生存率(%)
对照组1.92±0.433.89±1.5710.8±2.310017.3±5.6-
I5组11.86±1.783.74±0.936.4±4.86026.3±3.1-
I10组11.93±2.183.69±0.471.0±2.02036.6±4.8-
H5组11.63±0.343.54±0.643.0±0.84027.7±5.6-
H10组10.36±0.110.56±0.15-->56.8±32.440
图2
图2 对照组、I5、I10、IL-2化和I10组2、H10组2小鼠的肿瘤切除标本(从左至右).

为明确HSP70与IL-2治疗作用的不同, 我们设一IL-2化组, 对比观察再接种后肿瘤的再发率及对生存期、生存率的影响(表2). IL-2 10万U治疗后及预先应用IL-2均不能抑制肿瘤的发展, 而应用HSP70 10 mg后无一小鼠出现肿瘤, "IL-2化"组的生存期与对照组无差异显著性(P>0.25), 肿瘤大小及肿瘤重量之间亦无显著性差异(P>0.25), 但应用IL-2治疗后未出现腹水(图3).

表2 HcaF细胞再接种结果.
分组肿瘤大小(cm)肿瘤质量(g)腹水量(g)生存天数(d)腹水出现率(%)长期生存率(%)
对照组192±0.433.89±1.5710.8±2.317.3±5.6100-
IL-2治1.96±0.383.62±0.88-29.1±8.7--
疗组2(n =4)
"IL-2化"组1.95±0.643.31±0.861.0±2.018.2±3.720-
H10治疗组2--->58.7±28.9-40
图3
图3 HSP70的SDS-PAGE和Western-blot结果.
3 讨论

HSP70在很多恶性肿瘤组织中均有较高的表达, 实验研究证明肿瘤组织来源的HSP70是一种肿瘤特异性抗原, 诱导体内CD8+T淋巴细胞成为肿瘤特异性的CTL并使Th1型细胞因子升高[28], 具有特异的抗肿瘤作用[29-34], HSP70具有良好的应用前景, 研究其治疗作用, 评价其疗效有重要的实用意义. IL-2是由活化的T淋巴细胞产生的具有调节免疫功能和抗肿瘤作用的重要的细胞因子, 已证明其对肿瘤的有效率为20 %左右, IL-2通过增强LAK、NK、TIL等促进其他细胞因子的表达等作用, 调节机体的免疫功能, 发挥抗肿瘤免疫效应[35-40].

本研究证实, IL-2及HSP70均具有抑制肿瘤的作用, 通过比较分析, 表明应用10万UIL-2与HSP70 5 mg的治疗作用相当, 而应用10 mg HSP70显示了显著的治疗及抑瘤作用, 该组小鼠不但肿瘤显著缩小, 而且40%获长期生存, 肿瘤完全消退, 充分证明HSP70的抗肿瘤作用明显优于IL-2.

为进一步研究HSP70与IL-2在治疗作用上的差异, 作者进行了再接种实验, 结果表明, 应用IL-2治疗后或预先应用IL-2都不能有效地抑制肿瘤的进展, 提示IL-2的应用要足量、连续, 而停用IL-2后抗肿瘤免疫功能亦减弱, IL-2不能使机体维持长期的、主动的抗肿瘤免疫效应, 这正是由于IL-2不具特异性抗肿瘤作用的表现. 但应用HSP70治疗后再接种HcaF细胞, 无一小鼠再发生肿瘤, 且生存率及生存期限未受到影响, 证明HSP70能够诱导机体产生特异性的抗肿瘤免疫效应, 这种效应在治疗停止后可在相对较长时间内维持, 有助于延缓肿瘤的复发, 更加证明HSP70具有IL-2不可比拟的优越性.我们以IL-2作为已为大多学者所认可的治疗作用为参照, 研究了HSP70的治疗作用, 结果显示, HSP70较IL-2具有显著的抑瘤和治疗作用, 更主要的是HSP70诱导的抗肿瘤免疫在停止治疗后仍可在较长时间内维持, 这对防止肿瘤复发、延缓肿瘤进展, 有重要意义, 本研究对于研究以HSP70治疗人类的恶性肿瘤有重要的参考价值.

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