CN1317031C - 增强实体肿瘤细胞化疗敏感性的高效混合制剂 - Google Patents
增强实体肿瘤细胞化疗敏感性的高效混合制剂 Download PDFInfo
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Abstract
本发明涉及一种能增强实体肿瘤细胞化疗敏感性的高效混合制剂，该混合制剂是将一种影响实体肿瘤细胞周期调控的药物和一种周期特异性化疗药物按照一定的浓度比例混合制得。其中影响实体肿瘤细胞周期调控的药物是施他宁，周期特异性化疗药物为健择。通过使用施他宁和健择的混合制剂后，杀伤效果明显较单用化疗药物提高。
Description
技术领域
本发明涉及生物医学领域，尤其涉及一种能增强实体肿瘤细胞化疗敏感性的高效混合制剂。
背景技术
实体肿瘤包括乳房癌、胃癌、胆囊癌、前列腺癌等身体多个系统的多种恶性肿瘤，是危害人民健康、引发病人死亡的三大病种之一。虽然手术切除仍旧是治疗手段的首选，但对于根治性手术后的患者，及时有效的化疗是配合手术治疗的重要手段。对于丧失手术机会的晚期患者，化疗更是延长患者生存期的关键所在。现有的化疗，就是发挥药物在细胞周期不同时相的阻断作用杀伤癌细胞。同样的化疗药物在不同癌肿的治疗中表现出不同的预后，提示不同肿瘤在化疗敏感性方面存在差异，但其机制目前尚不清楚。同时，化疗药物无选择性地对人体正常组织所产生细胞毒性作用，即化疗副作用，限制了以提高化疗药物浓度来提高其疗效的做法。因此，虽然临床上通过建立不同化疗药物的组合、改变化疗药物的给药途径、研发高效低毒的新化疗药物等方法，结果仍旧不甚满意，对于化疗敏感性极差的实体肿瘤，如胆囊癌，目前临床上至今尚未找到有效的治疗方法。
因此，对化疗的敏感与否和化疗副作用的发生程度是影响患者生存时间和生存质量的关键环节。寻找有效的药物组合以提高实体肿瘤细胞的化疗敏感性，降低化疗药物用量以减少副反应发生，是提高实体肿瘤患者生存率以及生存质量的重要手段，也是我们本发明的目的所在。
发明内容
本发明的目的在于提供一种能增强实体肿瘤细胞化疗敏感性的高效混合制剂。
本发明的目的是通过以下技术方法来实现的：能增强实体肿瘤细胞化疗敏感性的高效混合制剂是将一种影响实体肿瘤细胞周期调控的药物和一种周期特异性化疗药物按照一定的浓度比例混合制得。其中影响实体肿瘤细胞周期调控的药物为施他宁(简称S)，周期特异性化疗药物为健择(简称GZ、Z)。
通过实验发现，使用化疗药物施他宁和健择的混合制剂后，对癌细胞的杀伤效果明显较单用化疗药物提高。
增强实体肿瘤细胞化疗敏感性的混合制剂在制备治疗实体肿瘤细胞药中的应用。实体肿瘤细胞是胆囊癌细胞。
附图说明
图1为药物影响细胞生长周期的实验结果。
图2为施他宁协同健择发挥对癌细胞活性抑制作用的实验结果。
图3为施他宁协同健择增加诱导细胞凋亡的实验结果。
图4为施他宁增加细胞内健择浓度作用的实验结果。
具体实施方式
本发明增强实体肿瘤细胞化疗敏感性的高效混合制剂是将一种影响实体肿瘤细胞周期调控的药物和一种周期特异性化疗药物按照一定的浓度比例混合制得。本发明的实验中采用的是施他宁，周期特异性化疗药物选用健择。临床应用中，两种成份的浓度范围是健择不高于3.5mg/ml，施他宁不高于150μg/ml。健择和施他宁的优选浓度为：健择0.5～2.5mg/ml，施他宁62.5～150μg/ml。
本发明中胆囊癌细胞GBC-SD购于中国科学院上海细胞生物学研究所细胞库。细胞用含15％小牛血清的RPMI1640培养基，培养于37℃，5％CO2恒温恒湿培养箱。
下面结合实施例对本发明作进一步描述。
实施例1MTT法对细胞活性的测定
将对数生长期的GBC-SD细胞以5×104cell/ml的浓度接种于96孔培养板，每孔0.1ml。对照组加入不含药的培养基，健择组分别加入含不同浓度健择的培养基处理，联合用药组分别加入含不同浓度(0.5mg/ml、1.0mg/ml、1.5mg/ml、2.0mg/ml、2.5mg/ml、3.0mg/ml、3.5mg/ml)健择和62.5μg/ml施他宁的培养基处理，每组浓度平行5孔。置于37℃，5％CO2恒温恒湿培养箱培养3天，实验终止前4小时每孔加入5mg/mlMTT 10μl，培养结束后每孔加入0.04N DMSO(二甲基亚砜)，每孔100μl，振荡10min，使MTT还原产物完全溶解，用BioRad 550型酶标仪，以550nm为实验波长，655nm为参照波长测定其吸收度。以药物的不同浓度和细胞的存活率作图。通过这种方法可以准确有效的测定不同浓度的药物以及药物联合作用对胆囊癌细胞活性的影响。从图1可以看出，用药组处于S期的细胞数要明显高于阴性对照组，说明药物对细胞的生长周期有一定的影响。从图2可以看出，健择在较高浓度下对胆囊癌细胞的活性有一定的抑制作用，但当与62.5μg/ml的施他宁联合作用后，这种抑制作用被明显增强。在施他宁的协同作用下，0.2mg/ml的健择对胆囊癌细胞的抑制作用与3.2mg/ml的健择单独作用的效果相当。而且联合用药组的抑制效果要明显好于两种药物单独作用的效果。由此可见，施他宁可以协同健择发挥对癌细胞活性的抑制作用。
实施例2流式细胞分析(FCAS)
将对数生长期的GBC-SD细胞以105cell·ml-1的浓度接种于3.5cm培养皿，每皿5ml，对照组加入不含药的培养基，健择组加入含1mg/ml健择的培养基处理，联合用药组加入含1mg/ml健择和150μg/ml施他宁的培养基处理，每浓度平行3皿。置37℃，5％CO2的恒温恒湿培养箱培养3天，细胞用胰蛋白酶消化后稀释到1×106/ml，用PBS离心洗涤两遍，用90％甲醇PBS溶液4℃下固定1h。固定后，细胞经1100g离心5min，用冰PBS洗涤两遍，RNA酶处理10min。随后，细胞冰冻10min，用溴化丙啶(PI)染色1.5min后，用流式细胞仪测定细胞凋亡和细胞周期。
凋亡指数(AI)＝凋亡细胞数/总细胞数
不同浓度的S对细胞内GZ浓度的影响
从流式细胞分析的结果可以看出1mg/ml健择可以明显的诱导胆囊癌细胞的凋亡。与150μg/ml施他宁协同作用后，这种诱导凋亡的作用被明显增强，是健择单独作用的1.8倍。见图3。
实施例3细胞内药物浓度测定
细胞的药物处理过程同流式细胞分析，待药物处理结束后，用胰蛋白酶消化处理，1000g离心10min收集细胞，再用PBS离心洗涤两遍，每组加0.5ml双蒸水超声处理30min，14000g离心10min取上清用高效液相法(HPLC)检测细胞内的药物浓度。用Sb-C18反向柱测定，流动相配比条件为5％甲醇和95％水，检测466nm下的特异吸收峰，并以其出峰面积为纵坐标作图。从HPLC的结果可以看出联合用药组细胞内健择的浓度明显高于健择单独用药组，这表明施他宁可以促进健择向细胞内的转运过程。
见图4。
Claims (5)
1.一种增强实体肿瘤细胞化疗敏感性的混合制剂，其特征在于：该混合制剂是将一种影响实体肿瘤细胞周期调控的药物和一种周期特异性化疗药物按照一定的浓度比例混合制得，其中影响实体肿瘤细胞周期调控的药物是施他宁，周期特异性化疗药物为健择。
2.如权利要求1所述的混合制剂，其特征在于：施他宁、健择溶解于生理盐水，健择浓度不高于3.5mg/ml，施他宁浓度不高于150μg/ml。
3.如权利要求2所述的混合制剂，其特征在于：健择和施他宁的浓度分别为，健择0.5～2.5mg/ml，施他宁62.5～150μg/ml。
4.如权利要求1或2或3所述的混合制剂在制备治疗实体肿瘤细胞药中的应用。
5.如权利要求4所述的应用，其特征在于：实体肿瘤细胞是胆囊癌细胞。
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| WO1997005167A1 (en) * | 1995-07-28 | 1997-02-13 | Romano Deghenghi | Somatostatin-analogous cyclic peptides with inhibitory activity on growth hormone |
| CN1531441A (zh) * | 2001-01-12 | 2004-09-22 | �о���Ӧ�ÿ�ѧЭ��ɷ�����˾ | 抑制血管增殖的药物组合物及其使用方法 |
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| WO1997005167A1 (en) * | 1995-07-28 | 1997-02-13 | Romano Deghenghi | Somatostatin-analogous cyclic peptides with inhibitory activity on growth hormone |
| CN1531441A (zh) * | 2001-01-12 | 2004-09-22 | �о���Ӧ�ÿ�ѧЭ��ɷ�����˾ | 抑制血管增殖的药物组合物及其使用方法 |
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| Title |
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| 健择为主联合化疗治疗晚期恶性肿瘤的临床观察 仲琴，实用肿瘤学杂志，第15卷第3期 2001 * |
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