CN1911440A - 一种用于形成纤维蛋白膜的试剂盒及其应用 - Google Patents
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Abstract
本发明涉及一种用于形成纤维蛋白膜的试剂盒,它包括浓度为50-100毫克/毫升的纤维蛋白原、浓度为100-1000国际单位/毫升的凝血酶和浓度为20-60毫摩尔/升的氯化钙。本发明也涉及所述试剂盒在制备在临床肿瘤手术中阻断组织外游离癌细胞的扩散的试剂中的应用。将所述的纤维蛋白原和凝血酶交替涂布,形成一层网络状致密的纤维蛋白膜,阻断在肿瘤病人手术过程中由于机械创伤或切口所造成的游离肿瘤细胞的扩散,从而达到降低和减少肿瘤病人术后“病灶”复发和转移的风险,提高病人的存活时间和几率。
Description
技术领域
本发明涉及一种用于形成在临床肿瘤手术中可阻断组织外游离癌细胞扩散的纤维蛋白膜的试剂盒;本发明也涉及所述试剂盒的应用。
背景技术
肿瘤病人手术过程中由于机械创伤或切口所造成的游离肿瘤细胞的扩散,会增加肿瘤病人术后“病灶”复发和转移的风险,降低病人的存活时间和几率。纤维蛋白原和凝血酶等形成的复合物,可用于减少根治性乳房切除术和肿块切除术后血清肿的形成;目前临床上使用较多的纤维蛋白胶主要用于止血,上述产品都不能阻断游离肿瘤细胞的扩散。
因此,本发明提供的纤维蛋白膜试剂盒能将网状致密的纤维蛋白膜用于阻断游离癌细胞的扩散中。
发明内容
本发明的目的就是提供一种用于形成纤维蛋白膜的试剂盒;
本发明的另一个目的是提供所述试剂盒的用途。
本发明提供一种用于形成纤维蛋白膜的试剂盒,它包括浓度为50-100毫克/毫升的纤维蛋白原、浓度为100-1000国际单位/毫升的凝血酶和浓度为20-60毫摩尔/升的氯化钙;所述的纤维蛋白原优选浓度为60-80毫克/毫升,更优选70毫克/毫升,所述的凝血酶优选浓度为300-800国际单位/毫升,更优选400-600国际单位/毫升,所述的氯化钙优选浓度为30-50毫摩尔/升,更优选40毫摩尔/升;所述的凝血酶和氯化钙组合成为凝血酶的氯化钙溶液;所述的纤维蛋白原和凝血酶为人血液来源;所述的试剂盒还包括说明书。
本发明还提供所述的试剂盒在制备纤维蛋白膜中的应用,及其在制备阻断临床肿瘤手术中游离肿瘤细胞扩散的试剂中的应用。
本发明在临床肿瘤手术中可以阻断手术创伤和切割所引起的游离肿瘤细胞的扩散,提高肿瘤患者术后的生存率,有很好的生物相容性,并且使用方便。
附图说明
图1和图2分别是用本发明试剂盒所形成的纤维蛋白膜在其不同位点处的电子扫描显微镜照片。
图3A1、图3A2是对照组细胞侵染性实验结果的显微镜照片
图3B1、图3B2是实验组去掉纤维蛋白膜后细胞侵染性实验结果的显微镜照片
图3C1、图3C2是从实验组去掉的纤维蛋白膜的显微镜照片
具体实施方式
人体血液凝固过程已被研究即阐明,纤维蛋白原和凝血酶在其中的作用也为人们所熟知,而且也有许多利用该原理的产品在使用,但都用于防止术后血块形成或用于止血。本发明人经过广泛而深入的研究,发现通过凝血酶对纤维蛋白原的激活作用,使纤维蛋白原逐渐聚合后形成的一层网络状致密的纤维蛋白膜能阻断肿瘤细胞穿透扩散,因而提供了一种用于形成纤维蛋白膜的试剂盒,其中的纤维蛋白原和凝血酶优选的为人血液来源。
本发明试剂盒中的纤维蛋白原的浓度为50-100毫克/毫克,优选的为60-80毫克/毫升,最好是70毫克/毫升;凝血酶的浓度为100-1000国际单位/毫升,优选的为300-800国际单位/毫升,最好是400-600国际单位/毫升;氯化钙的浓度为20-60毫摩尔/升,优选的为30-50毫摩尔/升,最好是40毫摩尔/升。
本发明的试剂盒优选的还包括说明书。
这样使用本发明的试剂盒:将试剂盒中的纤维蛋白原和凝血酶交替涂布2-4次,形成的纤维蛋白膜孔径大于0微米小于10微米,更优选的是小于0.6微米,而人体肿瘤细胞一般直径是大于10-100微米,因此,用本发明的试剂盒所形成的纤维蛋白膜能完全能阻断所述人体肿瘤细胞的扩散。
具体地说,纤维蛋白原和凝血酶交替涂布在肿瘤组织或“病灶”表面形成纤维蛋白膜,形成一层能阻断肿瘤细胞穿透扩散的网络状致密的纤维蛋白膜,阻断在肿瘤病人手术过程中由于机械创伤或切口所造成的游离肿瘤细胞的扩散,从而达到降低和减少肿瘤病人术后“病灶”复发和转移的风险,提高病人的存活时间和几率。
本发明的优点在于:
(1)应用于临床肿瘤手术中阻断手术创伤和切割所引起的游离肿瘤细胞的扩散,提高肿瘤患者术后的生存率;
(2)有很好的生物相容性;
(3)使用方便。
下面结合实施例说明本发明,但并不意味着以任何方式限制本发明。
实施例1 纤维蛋白膜的形成
(1)在0.8×0.8厘米的玻璃板上交替涂布3层70毫克/毫升的纤维蛋白原和凝血酶(由450国际单位/毫升凝血酶和40毫摩尔/升氯化钙溶液组成);
(2)待涂膜凝固后,用2.5%的戊二醛固定液4℃固定2小时(固定后可贮存数周,对超微结构无明显不良影响);
(3)用1%的锇酸固定30分钟后,脱水并临界点干燥,装样上机观测。
结果见图1、图2所示的两张电镜照片,它们在纤维蛋白膜不同位点处拍摄得到,放大倍数为5,000倍的视野。可以看出,所形成的纤维蛋白膜没有出现明显的孔状结构。由照片放大比例尺估计可知,蛋白膜所形成的孔径大小是小于0.6μm的,而人体肿瘤细胞一般直径大小为10-100μm,因此,形成的纤维蛋白膜是可以阻断肿瘤细胞扩散。
实施例2 纤维蛋白膜试剂盒用于阻断组织外游离癌细胞扩散
(1)用85毫克/毫升的纤维蛋白原和凝血酶(由700国际单位/毫升凝血酶和40毫摩尔/升氯化钙溶液组成)交替涂布24孔板中的测试培养皿(inserts)的底部外表面3次,形成一层致密光滑的纤维蛋白膜;
(2)用无血清培养基溶胀测试培养皿(inserts)底膜后,以0.5~1.0×106接种密度接入300微升的细胞悬浮液,其中包括胃癌细胞株MKN45和AGS,结肠癌细胞株Ls174T,以及乳腺癌细胞株MDA-MB-231,进行培养,培养48小时。
(3)移去培养液,用棉签轻轻擦拭没有透过测试培养皿(inserts)底膜的细胞,染色20分钟,晾干;
(4)对照组不涂纤维蛋白原和凝血酶;
(5)纤维拍照,记录试验结果。
结果见图3A1、图3A2、图3B1、图3B2、图3C1、图3C2。
从中清楚地看出,与对照组相比,交替涂布纤维蛋白原和凝血酶形成的纤维蛋白膜将肿瘤细胞完全阻断,从而使得细胞全部阻留在测试培养皿(inserts)底膜上而没有穿透纤维蛋白膜。因此,说明纤维蛋白膜是可以完全阻断游离肿瘤细胞的侵袭扩散的。
通过体外实验来模拟由纤维蛋白原和凝血酶交替涂布在肿瘤组织或“病灶”表面形成纤维蛋白膜,阻断在肿瘤病人手术过程中由于机械创伤或切口所造成的游离肿瘤细胞的扩散,从而达到降低和减少肿瘤病人术后“病灶”复发和转移的风险,提高病人的存活时间和几率。
Claims (8)
1.一种用于形成纤维蛋白膜的试剂盒,其特征在于,包括浓度为50-100毫克/毫升的纤维蛋白原、浓度为100-1000国际单位/毫升的凝血酶和浓度为20-60毫摩尔/升的氯化钙。
2.如权利要求1所述的试剂盒,其特征在于,所述的纤维蛋白原浓度为60-80毫克/毫升,凝血酶浓度为300-800国际单位/毫升,氯化钙浓度为30-50毫摩尔/升。
3.如权利要求1所述的试剂盒,其特征在于,所述的纤维蛋白原浓度为70毫克/毫升,凝血酶浓度为400-600国际单位/毫升,氯化钙浓度为40毫摩尔/升。
4.如权利要求1-3任一所述的试剂盒,其特征在于,所述的凝血酶和氯化钙组合成为凝血酶的氯化钙溶液。
5.如权利要求1-3任一所述的试剂盒,其特征在于,所述的纤维蛋白原和凝血酶为人血液来源。
6.如权利要求1-3任一所述的试剂盒,其特征在于,还包括说明书。
7.权利要求1-3任一所述的试剂盒在制备纤维蛋白膜中的应用。
8.权利要求1-3任一所述的用于形成纤维蛋白膜的试剂盒在制备阻断临床肿瘤手术中游离肿瘤细胞扩散的试剂中的应用。
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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CNA2005100285774A CN1911440A (zh) | 2005-08-08 | 2005-08-08 | 一种用于形成纤维蛋白膜的试剂盒及其应用 |
JP2008526012A JP2009504643A (ja) | 2005-08-08 | 2006-07-11 | フィブリン膜を合成するために使用する凍結乾燥トロンビンおよび凍結乾燥フィブリノーゲンのキット、ならびにその適用 |
CA002618666A CA2618666A1 (en) | 2005-08-08 | 2006-07-11 | A kit of lyophilized thrombin and lyophilized fibrinogen used to compound fibrin membrane, and its application |
AU2006276769A AU2006276769A1 (en) | 2005-08-08 | 2006-07-11 | A kit of lyophilized thrombin and lyophilized fibrinogen used to compound fibrin membrane, and its application |
PCT/US2006/026739 WO2007018894A2 (en) | 2005-08-08 | 2006-07-11 | A kit of lyophilized thrombin and lyophilized fibrinogen used to compound fibrin membrane, and its application |
EP06786779A EP1924278A4 (en) | 2005-08-08 | 2006-07-11 | KIT OF LYOPHILIZED THROMBIN AND LYOPHILIZED FIBRINOGEN FOR THE REINFORCEMENT OF THE FIBRIN MEMBRANE AND ITS APPLICATION |
RU2008108806/15A RU2008108806A (ru) | 2005-08-08 | 2006-07-11 | Набор лиофилизированного тромбина и лиофилизированного фибриногена, используемый для приготовления фибриновой мембраны, и его применение |
US11/990,203 US20090232790A1 (en) | 2005-08-08 | 2006-07-11 | Kit of Lyophilized Thrombin and Lyophilized Fibrinogen Used to Compound Fibrin Membrane, and Its Application |
KR1020087004884A KR20080044844A (ko) | 2005-08-08 | 2006-07-11 | 피브린 막을 합성하는데 이용되는 동결 건조된 트롬빈과동결 건조된 피브리노겐의 키트 및 이의 용도 |
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CNA2005100285774A CN1911440A (zh) | 2005-08-08 | 2005-08-08 | 一种用于形成纤维蛋白膜的试剂盒及其应用 |
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CN1911440A true CN1911440A (zh) | 2007-02-14 |
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CNA2005100285774A Pending CN1911440A (zh) | 2005-08-08 | 2005-08-08 | 一种用于形成纤维蛋白膜的试剂盒及其应用 |
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US (1) | US20090232790A1 (zh) |
EP (1) | EP1924278A4 (zh) |
JP (1) | JP2009504643A (zh) |
KR (1) | KR20080044844A (zh) |
CN (1) | CN1911440A (zh) |
AU (1) | AU2006276769A1 (zh) |
CA (1) | CA2618666A1 (zh) |
RU (1) | RU2008108806A (zh) |
WO (1) | WO2007018894A2 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108220233A (zh) * | 2016-12-21 | 2018-06-29 | 上海透景诊断科技有限公司 | 细胞分离器具表面处理方法、相关器具、外周血稀有细胞或循环肿瘤细胞快速高效分离方法 |
CN113209390A (zh) * | 2021-05-06 | 2021-08-06 | 中山大学孙逸仙纪念医院 | 一种用于阻断卵巢上皮性肿瘤扩散的薄膜材料及其制备方法 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120177610A1 (en) * | 2007-09-19 | 2012-07-12 | Kieu Hoang | Manufacturing and Purification Processes of Complex Protein found in Fraction IV to make a separated Apo, Transferrin , and Alpha 1 Anti strepsin (A1AT) or A combined Transferrin / Apo/Human Albumin/A1AT and all new found proteins |
EP2556842A1 (en) * | 2011-08-11 | 2013-02-13 | Bioftalmik, S.L. | Composition in the form of film comprising fibrinogen and a fibrinogen activator and the applications thereof |
CN112043834B (zh) * | 2020-06-24 | 2022-06-24 | 四川大学华西医院 | 一种负载顺铂的纤维蛋白胶复合体系 |
CN113509547A (zh) * | 2020-12-09 | 2021-10-19 | 四川大学华西医院 | 凝血酶在预防或治疗癌症中的应用 |
CN116115817A (zh) * | 2022-07-01 | 2023-05-16 | 南方医科大学 | 一种纤维蛋白生物医用胶的研制 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5395923A (en) * | 1993-02-23 | 1995-03-07 | Haemacure-Biotech, Inc. | Process for the obtention of a biological adhesive made of concentrated coagulation factors by "salting-out" |
DE19617369A1 (de) * | 1996-04-30 | 1997-11-06 | Immuno Ag | Lagerstabile Fibrinogen-Präparate |
US20030021777A1 (en) * | 1998-02-20 | 2003-01-30 | Roy Harris | Product comprising bound fibrinogen and thrombin for use in wound therapy or surgical repair |
ITMI20022501A1 (it) * | 2002-11-26 | 2004-05-27 | Dorin Olimpiu Petrescu | Medicazione organica cicatrizzante ed emostatica. |
-
2005
- 2005-08-08 CN CNA2005100285774A patent/CN1911440A/zh active Pending
-
2006
- 2006-07-11 CA CA002618666A patent/CA2618666A1/en not_active Abandoned
- 2006-07-11 RU RU2008108806/15A patent/RU2008108806A/ru unknown
- 2006-07-11 EP EP06786779A patent/EP1924278A4/en not_active Withdrawn
- 2006-07-11 WO PCT/US2006/026739 patent/WO2007018894A2/en active Application Filing
- 2006-07-11 AU AU2006276769A patent/AU2006276769A1/en not_active Abandoned
- 2006-07-11 KR KR1020087004884A patent/KR20080044844A/ko not_active Application Discontinuation
- 2006-07-11 JP JP2008526012A patent/JP2009504643A/ja not_active Withdrawn
- 2006-07-11 US US11/990,203 patent/US20090232790A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108220233A (zh) * | 2016-12-21 | 2018-06-29 | 上海透景诊断科技有限公司 | 细胞分离器具表面处理方法、相关器具、外周血稀有细胞或循环肿瘤细胞快速高效分离方法 |
CN108220233B (zh) * | 2016-12-21 | 2021-07-09 | 上海透景诊断科技有限公司 | 细胞分离器具表面处理方法、相关器具、外周血稀有细胞或循环肿瘤细胞快速高效分离方法 |
CN113209390A (zh) * | 2021-05-06 | 2021-08-06 | 中山大学孙逸仙纪念医院 | 一种用于阻断卵巢上皮性肿瘤扩散的薄膜材料及其制备方法 |
Also Published As
Publication number | Publication date |
---|---|
WO2007018894A2 (en) | 2007-02-15 |
US20090232790A1 (en) | 2009-09-17 |
KR20080044844A (ko) | 2008-05-21 |
JP2009504643A (ja) | 2009-02-05 |
WO2007018894A3 (en) | 2009-01-08 |
CA2618666A1 (en) | 2007-02-15 |
AU2006276769A1 (en) | 2007-02-15 |
EP1924278A4 (en) | 2009-08-12 |
RU2008108806A (ru) | 2009-09-20 |
EP1924278A2 (en) | 2008-05-28 |
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