表阿霉素—活性炭混悬液对乳腺癌腋窝转移淋巴结化疗效果的研究
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摘要
目的 探讨活性炭微粒-表阿霉素(charcoal activated absorbing epirubicin,Epi-CH)混悬液对乳腺癌腋窝转移淋巴结中癌细胞凋亡和增殖的作用。
方法 60例临床检查或B超探查腋窝淋巴结肿大的乳腺癌病人随机分为两组，治疗组40例，对照组20例。在改良根治术前48-72h治疗组于瘤床或肿瘤周围注射Epi-CH 10mg，对照组注射表阿霉素水溶液10mg，术后清点清除的腋窝淋巴结总数和黑染淋巴结数。用高效液相色谱法(外标法)测定治疗组黑染和未黑染淋巴结中表阿霉素的含量。用末端转移酶标记法(Tunel)检测腋窝淋巴结凋亡指数(apoptosis index,AI)，用免疫组化S-P法检测腋窝淋巴结增殖指数(proliferation index,PI)及凋亡相关蛋白bc1-2、bax、C-myc、Fas、Fas-L的表达。用SPSS10.0软件包进行统计学处理。
结果 治疗组共清扫腋淋巴结650个，平均每例15.20±1.22个；对照组共清扫232个，平均11.16±1.43个，治疗组平均数多于对照组(p＜0.01)。治疗组中腋窝淋巴结的黑染率为88.8％(577/650)；黑染淋巴结癌转移率为37.9％(219/577)，未黑染淋巴结转移率为24.7％(18/73)，黑染淋巴结癌转移率明显增高(p＜0.05)；直径≤1.0cm淋巴结黑染率为91.60％(475/519)，直径＞1.0cm淋巴结黑染率为77.9％(102/131)，前者高于后者(p＜0.01)。治疗组注射Epi-CH后48—72h内黑染淋巴结中表阿霉素的浓度平均为443.0±
广西医科大学2001级硕士研究生学位论文
123.Ing/g，未黑染组淋巴结浓度平均为31，8士11 .gng/g，前者是后者的14.3
倍，有显著性差异印<0.01)。治疗组35例腋窝淋巴结阳性病人癌巢丰富的黑
染癌转移淋巴结48个与对照组巧例病人阳性淋巴结24个癌细胞的Al、Pl
和凋亡相关蛋白bcl一2、bax、c一yc、Fas、Fas一比较，治疗组Al明显升高印
<0.01)，pl明显下降(P<0.05)、bel一2、 bax、Fas一L蛋白表达上升(p<0.05)，
而C劝yc、Fas蛋白表达无明显变(P>0.05)。
结论应用自行研制的活性炭一表阿霉素混悬液进行乳腺癌淋巴化疗，可以增
加手术清除的腋窝淋巴结数目，提高腋窝淋巴结清除率;活性炭能够携带表
阿霉素并停留在腋窝淋巴结内，显著增加表阿霉素在局部淋巴结中的浓度，
并诱导淋巴结内癌细胞凋亡增加和增殖减少;活性炭一表阿霉素混悬液的淋巴
化疗作用可能是通过上调bax、Fas一L蛋白表达来完成。活性炭一表阿霉素混悬
液对乳腺癌的淋巴化疗具有化疗效果肯定、化疗药物用量少、毒副作用低、
临床使用方便的优点，值得临床推广应用。
Objectives To investigate the chemotherapeutic effect of charcoal activated absorbing epirubicin suspension (Epi-CH) on the axillary metastasis lymph nodes in patients with breast cancer.
Methods 60 patients with breast cancer were divided into two groups randomly. 40 patients (Epi-CH group) were injected Epi-CH 10mg into the region around the tumor or biopsy excision. 20 patients (control group) were injected epirubicin lOmg into the same region. After 48-72 hours, the modified radical mastectomy was operated. The axillary lymph nodes and the staining lymph nodes were calculated. The concentration of epiribicin in lymph nodes was measured by high performance liquid chorography (HPLC). The apoptosis index (AI) was detected by terminal deoxynucleotidy trans ferase mediated digoxigenin-dUTP nick end labeling (Tunel). The proliferation index (PI) and the expressions of apoptosis related gene proteins(including bcl-2, bax, C-myc, Fas, Fas-L) were detected by S-P. Statistical analysis was performed using SPSS 10.0. Results The average number of dissected lymph nodes in Epi-CH group was 15.20\1.22, and that in control group was 11.10\1.43, The average number of
dissected lymph nodes in Epi-CH group was markedly higher (p < 0.01) .In Epi-CH group, the staining rate of axillary lymph nodes was 88.8% (577/650). The metastasis rate of staining lymph nodes (219/577) was significantly higher than that of non-staining lymph nodes (18/73) (p < 0.05). The proportion of the staining lymph nodes with diameter > 1.0cm(102/131) was lower than that with diameter ≤ 1.0cm (475/519). In Epi-CH group, The concentration of epirubicin in staining lymph nodes (443.0 ± 123.1ng/g) was higher significantly than that of non-blackened (31.8±11.9ng/g) (p < 0.01). AI of blackened lymph nodes in Epi-CH group (9.5% ±2.1%) was increased remarkable than that in control(3.8% ±1.4%) (p < 0.01). PI of blackened lymph nodes in Epi-CH group (60.1 % ± 12.1 %) was decreased significantly than that in control group(73.5%±12.4%) (p < 0.05). The expressions of bcl-2, bax, Fas-L gene protein in Epi-CH group were up-regulated(p < 0.05), but those of C-myc and Fas was not different between two groups (p > 0.0
5).
Conclusions The study indicates that lymphatic chemotherapy using Epi-CH can increase the dissected lymph nodes during modified mastectomy, rise the concentration of epirubicin in axillary lymph nodes, enhance the cancer cell apoptosis and reduce the cancer cell proliferation. The lymphatic chemotherapy effects of Epi-CH result from up-regulation of expressions of bax and Fas-L gene
proteins.
方法 60例临床检查或B超探查腋窝淋巴结肿大的乳腺癌病人随机分为两组，治疗组40例，对照组20例。在改良根治术前48-72h治疗组于瘤床或肿瘤周围注射Epi-CH 10mg，对照组注射表阿霉素水溶液10mg，术后清点清除的腋窝淋巴结总数和黑染淋巴结数。用高效液相色谱法(外标法)测定治疗组黑染和未黑染淋巴结中表阿霉素的含量。用末端转移酶标记法(Tunel)检测腋窝淋巴结凋亡指数(apoptosis index,AI)，用免疫组化S-P法检测腋窝淋巴结增殖指数(proliferation index,PI)及凋亡相关蛋白bc1-2、bax、C-myc、Fas、Fas-L的表达。用SPSS10.0软件包进行统计学处理。
结果 治疗组共清扫腋淋巴结650个，平均每例15.20±1.22个；对照组共清扫232个，平均11.16±1.43个，治疗组平均数多于对照组(p＜0.01)。治疗组中腋窝淋巴结的黑染率为88.8％(577/650)；黑染淋巴结癌转移率为37.9％(219/577)，未黑染淋巴结转移率为24.7％(18/73)，黑染淋巴结癌转移率明显增高(p＜0.05)；直径≤1.0cm淋巴结黑染率为91.60％(475/519)，直径＞1.0cm淋巴结黑染率为77.9％(102/131)，前者高于后者(p＜0.01)。治疗组注射Epi-CH后48—72h内黑染淋巴结中表阿霉素的浓度平均为443.0±
广西医科大学2001级硕士研究生学位论文
123.Ing/g，未黑染组淋巴结浓度平均为31，8士11 .gng/g，前者是后者的14.3
倍，有显著性差异印<0.01)。治疗组35例腋窝淋巴结阳性病人癌巢丰富的黑
染癌转移淋巴结48个与对照组巧例病人阳性淋巴结24个癌细胞的Al、Pl
和凋亡相关蛋白bcl一2、bax、c一yc、Fas、Fas一比较，治疗组Al明显升高印
<0.01)，pl明显下降(P<0.05)、bel一2、 bax、Fas一L蛋白表达上升(p<0.05)，
而C劝yc、Fas蛋白表达无明显变(P>0.05)。
结论应用自行研制的活性炭一表阿霉素混悬液进行乳腺癌淋巴化疗，可以增
加手术清除的腋窝淋巴结数目，提高腋窝淋巴结清除率;活性炭能够携带表
阿霉素并停留在腋窝淋巴结内，显著增加表阿霉素在局部淋巴结中的浓度，
并诱导淋巴结内癌细胞凋亡增加和增殖减少;活性炭一表阿霉素混悬液的淋巴
化疗作用可能是通过上调bax、Fas一L蛋白表达来完成。活性炭一表阿霉素混悬
液对乳腺癌的淋巴化疗具有化疗效果肯定、化疗药物用量少、毒副作用低、
临床使用方便的优点，值得临床推广应用。
Objectives To investigate the chemotherapeutic effect of charcoal activated absorbing epirubicin suspension (Epi-CH) on the axillary metastasis lymph nodes in patients with breast cancer.
Methods 60 patients with breast cancer were divided into two groups randomly. 40 patients (Epi-CH group) were injected Epi-CH 10mg into the region around the tumor or biopsy excision. 20 patients (control group) were injected epirubicin lOmg into the same region. After 48-72 hours, the modified radical mastectomy was operated. The axillary lymph nodes and the staining lymph nodes were calculated. The concentration of epiribicin in lymph nodes was measured by high performance liquid chorography (HPLC). The apoptosis index (AI) was detected by terminal deoxynucleotidy trans ferase mediated digoxigenin-dUTP nick end labeling (Tunel). The proliferation index (PI) and the expressions of apoptosis related gene proteins(including bcl-2, bax, C-myc, Fas, Fas-L) were detected by S-P. Statistical analysis was performed using SPSS 10.0. Results The average number of dissected lymph nodes in Epi-CH group was 15.20\1.22, and that in control group was 11.10\1.43, The average number of
dissected lymph nodes in Epi-CH group was markedly higher (p < 0.01) .In Epi-CH group, the staining rate of axillary lymph nodes was 88.8% (577/650). The metastasis rate of staining lymph nodes (219/577) was significantly higher than that of non-staining lymph nodes (18/73) (p < 0.05). The proportion of the staining lymph nodes with diameter > 1.0cm(102/131) was lower than that with diameter ≤ 1.0cm (475/519). In Epi-CH group, The concentration of epirubicin in staining lymph nodes (443.0 ± 123.1ng/g) was higher significantly than that of non-blackened (31.8±11.9ng/g) (p < 0.01). AI of blackened lymph nodes in Epi-CH group (9.5% ±2.1%) was increased remarkable than that in control(3.8% ±1.4%) (p < 0.01). PI of blackened lymph nodes in Epi-CH group (60.1 % ± 12.1 %) was decreased significantly than that in control group(73.5%±12.4%) (p < 0.05). The expressions of bcl-2, bax, Fas-L gene protein in Epi-CH group were up-regulated(p < 0.05), but those of C-myc and Fas was not different between two groups (p > 0.0
5).
Conclusions The study indicates that lymphatic chemotherapy using Epi-CH can increase the dissected lymph nodes during modified mastectomy, rise the concentration of epirubicin in axillary lymph nodes, enhance the cancer cell apoptosis and reduce the cancer cell proliferation. The lymphatic chemotherapy effects of Epi-CH result from up-regulation of expressions of bax and Fas-L gene
proteins.
引文
1．沈镇宙，韩企夏，邵志敏，等．乳腺肿瘤体积、淋巴结情况与预后的关系．中华外科杂志，1991；29(9)：554-557
2. Donegan WL.Staging and primary therapy. In Donegan WL.Spratt TS(Editors) Cancer of the Breast.Philadelphia WB.Saunders,1988:385
3. Krag D, Weaver D, Ashikaga T, et al.The sentinel node in breast cancer--a multicenter validation study. N Engi J Med. 1998 Oct 1;339(14):941-6.
4. Robert E. Coleman, M.D. High dose chemotherapy Rationale and results in breast carcinoma. Cancer 2000 Jun 15; 88(12 suppl): 3059-64
5. Cart J, Dreher B, Carr I. Lymphatic metastasis; lymphangiochemotherapy of mammary cancer: ascitic form of rat mammary adenocarcinoma 13762. Clin Exp Metastasis. 1983 Jan-Mar, 1(1):29-38.
6. Hashida M, Liao MH, Muranishi S,et al.Dosage form characteristics of microsphere-in-oil emulsion. Ⅱ: Examination of some factors affocting lymphotropicity.Chem Phann Bull 1997;25(9):2410-2414
7. Akamo Y, Mizuno I, Takeyama H, et al. Chemotherapy targeting regional lymph nodes by gastric submucosal injection of liposomal adriamycin in patients with gastric cancer.Gan To Kagaku Ryoho. 1997 Sep;24(12): 1712-4
8. Takahashi T, Hagiwara A, Shimotsuma M, et al. Prophylaxis and treatment of peritoneal carcinomatosis: intraperitoneal chemotherapy with mitomycin C bound to activated carbon particles. World J Surg. 1995 Jul-Aug;19(4):565-9.
9. Kobayashi O, Onodera S, Rino Y, et al. Evaluation of medicinal lymph node dissection in advanced gastric cancer. Nippon Geka Gakkai Zasshi. 1994 Dec;95(12):860-5
10．许良中．实验肿瘤病理方法学．上海医科大学出版社．1997：128
11. Akeo Hagiwara,Toshio Takahashi, Bikko Lee,et al. Lymphatic high distribution of small sized activated carbon particle adsorbing mitomycin C [J] Akita J,Med 1985; 11:581-585
12. Sawai K, Hagiwara A, Shimotsuma M,et al. Rationale of lymph node dissection for breast cancer-from the viewpoint of analysis of axillary lymphatic flow using activated carbon particle CH40. Gan To Kagaku Ryoho. 1996 Mar;23 Suppl 1:30-5
13. Imanishi T, Hagiwara A, Sawai K, et al. Visualising lymph nodes by aclarubicin bound to activated carbon particles in breast cancer surgery. Gan To Kagaku Ryoho. 1997 Sep;24(12):1796-8
14. Yokota T, Saito T, Narushima Y, et al. Lymph-node staining with activated carbon CH40: a new method for axillary lymph-node dissection in breast cancer. Can J Surg. 2000 Jun;43(3): 191-6.
15. Akamo Y, Mizuno I, Takeyama H, et al. Chemotherapy targeting regional lymph nodes by gastric submucosal injection of liposomal adriamycin in patients with gastric cancer.Gan To Kagaku Ryoho. 1997 Sep;24(12):1712-4.
16. Natsngoe S, Shimada M, Kumanohoso T, et al. Enhanced efficacy of bleomycin adsorbed on silica particles against lymph node metastasis in patients with esophageal cancer: a pilot study. Surgery. 1995 Jun;l17(6):636-41
17．邱存平，王昌美，温玉明等．活性炭吸附卡铂在口腔癌及其颈淋巴结转移中的初步
临床应用．实用口腔医学杂志．2001；17(1)：9-11
18．詹庆友，徐立，孙小卫，等．新辅助化疗对乳腺癌细胞凋亡和增殖的影响．癌症，2002;21(2)：186-188
19．邵志敏，李俊，吴昊，等．术前化疗诱导乳腺癌细胞凋亡的临床意义．中国肿瘤杂志，2000；22(4)：295-7)
20．范宇，傅西林，王颖，等．细胞凋亡及其相关基因宇与乳腺癌预后关系的研究．中国肿瘤临床，2000；27(7)：524-528
21. Ellis PA, Smith IE, McCarthy K, et al. Preoperative chemotherapy induces apoptosis in early breast cancer. Lancet. 1997 Mar 22;349(9055):849
22. Suzuki K, Kazui T, Yoshida M, et al. Drug-induced apoptosis and p53, BCL-2 and BAX expression in breast cancer tissues in vivo and in fibroblast cells in Vitro. Jpn J Clin Oncol. 1999 Jul;29(7):323-31.
23．廖泉，赵玉沛，蔡力行，等．化疗药物诱导胰腺癌细胞凋亡过程中p53和bcl-2基因表达量的变化．中华肝胆外科杂志，2000；6(2)：105-7
24. Ellis PA, Smith IE, Detre S, et al. Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy. Breast Cancer Res Treat. 1998 Mar;48(2): 107-16
25. Hartitnez-Arribas F, Nunez-Villar MJ, et al. Immunofluorometric study of Bcl-2 and Bax expression in clinical fresh tumor samples from breast cancer patients. Anticancer Res.2003 Jan-Feb;23(1B):565-8
26. Carroll JS, Swarbrick A, Musgrove EA, et al. Mechanisms of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer cells: implications for the antiproliferative effects of antiestrogens. Cancer Res. 2002 Jun 1 ;62(11):3126-31
27．彭志海，刑同海，裘国强．，等．结肠癌细胞珠Fas／Fas-1的表达与凋亡的关系．中国癌症杂志2001：11(1)：25-9
2. Donegan WL.Staging and primary therapy. In Donegan WL.Spratt TS(Editors) Cancer of the Breast.Philadelphia WB.Saunders,1988:385
3. Krag D, Weaver D, Ashikaga T, et al.The sentinel node in breast cancer--a multicenter validation study. N Engi J Med. 1998 Oct 1;339(14):941-6.
4. Robert E. Coleman, M.D. High dose chemotherapy Rationale and results in breast carcinoma. Cancer 2000 Jun 15; 88(12 suppl): 3059-64
5. Cart J, Dreher B, Carr I. Lymphatic metastasis; lymphangiochemotherapy of mammary cancer: ascitic form of rat mammary adenocarcinoma 13762. Clin Exp Metastasis. 1983 Jan-Mar, 1(1):29-38.
6. Hashida M, Liao MH, Muranishi S,et al.Dosage form characteristics of microsphere-in-oil emulsion. Ⅱ: Examination of some factors affocting lymphotropicity.Chem Phann Bull 1997;25(9):2410-2414
7. Akamo Y, Mizuno I, Takeyama H, et al. Chemotherapy targeting regional lymph nodes by gastric submucosal injection of liposomal adriamycin in patients with gastric cancer.Gan To Kagaku Ryoho. 1997 Sep;24(12): 1712-4
8. Takahashi T, Hagiwara A, Shimotsuma M, et al. Prophylaxis and treatment of peritoneal carcinomatosis: intraperitoneal chemotherapy with mitomycin C bound to activated carbon particles. World J Surg. 1995 Jul-Aug;19(4):565-9.
9. Kobayashi O, Onodera S, Rino Y, et al. Evaluation of medicinal lymph node dissection in advanced gastric cancer. Nippon Geka Gakkai Zasshi. 1994 Dec;95(12):860-5
10．许良中．实验肿瘤病理方法学．上海医科大学出版社．1997：128
11. Akeo Hagiwara,Toshio Takahashi, Bikko Lee,et al. Lymphatic high distribution of small sized activated carbon particle adsorbing mitomycin C [J] Akita J,Med 1985; 11:581-585
12. Sawai K, Hagiwara A, Shimotsuma M,et al. Rationale of lymph node dissection for breast cancer-from the viewpoint of analysis of axillary lymphatic flow using activated carbon particle CH40. Gan To Kagaku Ryoho. 1996 Mar;23 Suppl 1:30-5
13. Imanishi T, Hagiwara A, Sawai K, et al. Visualising lymph nodes by aclarubicin bound to activated carbon particles in breast cancer surgery. Gan To Kagaku Ryoho. 1997 Sep;24(12):1796-8
14. Yokota T, Saito T, Narushima Y, et al. Lymph-node staining with activated carbon CH40: a new method for axillary lymph-node dissection in breast cancer. Can J Surg. 2000 Jun;43(3): 191-6.
15. Akamo Y, Mizuno I, Takeyama H, et al. Chemotherapy targeting regional lymph nodes by gastric submucosal injection of liposomal adriamycin in patients with gastric cancer.Gan To Kagaku Ryoho. 1997 Sep;24(12):1712-4.
16. Natsngoe S, Shimada M, Kumanohoso T, et al. Enhanced efficacy of bleomycin adsorbed on silica particles against lymph node metastasis in patients with esophageal cancer: a pilot study. Surgery. 1995 Jun;l17(6):636-41
17．邱存平，王昌美，温玉明等．活性炭吸附卡铂在口腔癌及其颈淋巴结转移中的初步
临床应用．实用口腔医学杂志．2001；17(1)：9-11
18．詹庆友，徐立，孙小卫，等．新辅助化疗对乳腺癌细胞凋亡和增殖的影响．癌症，2002;21(2)：186-188
19．邵志敏，李俊，吴昊，等．术前化疗诱导乳腺癌细胞凋亡的临床意义．中国肿瘤杂志，2000；22(4)：295-7)
20．范宇，傅西林，王颖，等．细胞凋亡及其相关基因宇与乳腺癌预后关系的研究．中国肿瘤临床，2000；27(7)：524-528
21. Ellis PA, Smith IE, McCarthy K, et al. Preoperative chemotherapy induces apoptosis in early breast cancer. Lancet. 1997 Mar 22;349(9055):849
22. Suzuki K, Kazui T, Yoshida M, et al. Drug-induced apoptosis and p53, BCL-2 and BAX expression in breast cancer tissues in vivo and in fibroblast cells in Vitro. Jpn J Clin Oncol. 1999 Jul;29(7):323-31.
23．廖泉，赵玉沛，蔡力行，等．化疗药物诱导胰腺癌细胞凋亡过程中p53和bcl-2基因表达量的变化．中华肝胆外科杂志，2000；6(2)：105-7
24. Ellis PA, Smith IE, Detre S, et al. Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy. Breast Cancer Res Treat. 1998 Mar;48(2): 107-16
25. Hartitnez-Arribas F, Nunez-Villar MJ, et al. Immunofluorometric study of Bcl-2 and Bax expression in clinical fresh tumor samples from breast cancer patients. Anticancer Res.2003 Jan-Feb;23(1B):565-8
26. Carroll JS, Swarbrick A, Musgrove EA, et al. Mechanisms of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer cells: implications for the antiproliferative effects of antiestrogens. Cancer Res. 2002 Jun 1 ;62(11):3126-31
27．彭志海，刑同海，裘国强．，等．结肠癌细胞珠Fas／Fas-1的表达与凋亡的关系．中国癌症杂志2001：11(1)：25-9