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1、 苦瓜蛋白抑制肿瘤基质金属蛋白酶的活性及表达 作者：熊术道，尹丽慧，李景荣，叶爱芳，章圣辉，温博贵【摘要】 目的 探讨苦瓜蛋白对H22荷瘤小鼠基质金属蛋白酶表达及其活性的影响。方法 将清洁级ICR雄性小鼠随机分成5组，建立肝癌H22移植瘤模型，分为阴性对照组、高剂量组(T3)、中剂量组(T2)、低剂量组(T1)及阳性对照组（CTX,环磷酰胺处理组）。在普通饲料饲喂的基础上采用腹腔注射给药法分别予阴性对照组生理盐水；高、中、低剂量组分别给以不同剂量苦瓜蛋白；阳性对照组予环磷酰胺。观察苦瓜蛋白
2、对抑瘤率的影响，明胶酶谱法检测荷瘤小鼠血清基质金属蛋白酶MMP-2、MMP-9的活性及其变化。结果 苦瓜蛋白组低、中、高剂量组及阳性对照组抑瘤率分别为52.05%、66.89%、85.81及65.10；各苦瓜蛋白干预组与阴性对照组相比，MMP-2、MMP-9的活性降低，酶原表达下调(P<0.01)，上述作用与苦瓜蛋白剂量相关。结论 苦瓜蛋白具有抑制基质金属蛋白酶表达及其活性的作用，其抗肿瘤作用可能与苦瓜蛋白具有抑制MMPs的活性及其表达有关。 【关键词】 苦瓜蛋白移植性肿瘤抗肿瘤作用基质金属蛋白酶 Abstract: Purpose To e
3、xplore effect of Momordin on expression and activity of matrix metalloprotease in murine transplanted tumor(H22). Methods Male mice of ICR species were randomly divided into 5 groups and then constructed the transplantation tumor model of liver cancer H22. The negative control group were treated by
4、normal saline; T1, T2 and T3 group by Momordin with low, middle and high dose respectively and positive control group by cytoxan (CTX group). All the mice were feeded ordinarily with intraperitoneal drugs administration. Tumor inhibitive rate of Momordin was measured. Activity of MMP-2 and MMP-9 in
5、serum was detected by Gelatin Zymography. Results Tumor inhibition rate in low, middle and high dose of Momordin and CTX group were 52.05%, 66.89%, 85.81% and 65.10%, respectively. The activity of MMP-2 and MMP-9 significantly decreased in all Momordin treated groups compared to the negative control
6、 group (P<0.01). The above function correlated to the dose of Momordin. Conclusion Momordin plays a role of inhibitory effect on the expression and activity of matrix metalloprotease and its anti-tumor activity may be involved in regulating the expression and activity of matrix metalloprotease.&n
7、bsp; Key words: Momordin; transplanted tumor; anti-tumor activity; matrix metalloprotease. 苦瓜(momordica charantia) 属葫芦科植物，中医认为其具有消暑涤热、明目、解毒之功效。苦瓜蛋白(momordin)是从苦瓜籽中分离出来的一种分子量约为30kD的碱性糖蛋白质，属于型核糖体失活蛋白(ribosome inhibiting protein，RIP)。近年来研究表明RIP具有明显的抗肿瘤、抗病毒作用，具有极高的药用价值1，2。本
8、文在前人分离纯化RIP的工作基础上，采用强阳离子交换层析SP Sepharose High Performance为分离介质分离纯化得到了苦瓜籽蛋白，我们前期的研究表明苦瓜RIP可以明显诱导K562细胞产生细胞凋亡3，4。为进一步研究苦瓜RIP对动物移植性肿瘤的作用（in vivo）的分子机制，我们研究了苦瓜蛋白对移植性肝癌H22荷瘤小鼠抑瘤作用及基质金属蛋白酶的抑制作用，以进一步阐明苦瓜蛋白抗肿瘤作用的机制，为促进苦瓜蛋白应用于抗肿瘤治疗提供理论和实验基础。 1 材料与方法 1.1 &n
9、bsp; 材 料 主要仪器：Image master凝胶成像系统（GE health），垂直电泳仪(Bio-Rad mini-protein)。 主要试剂：丙稀酰胺、甲叉双丙稀酰胺、过硫酸铵及TEMED等电泳试剂购置于上海生工生物工程有限公司。 实验动物与细胞株：清洁级ICR雄性小鼠，体重18g22g，温州医学院实验动物中心提供。ICR小鼠H22腹水瘤细胞由浙江中医学院生命科学系惠赠。 1.2 方&nbs
10、p; 法 1.2.1 苦瓜蛋白分离与纯化5 1 预冷梯度丙酮沉淀得到的苦瓜籽粗蛋白经SP Sepharose High Performance 阳离子交换层析柱在GE-Health中压层析系统中分离纯化得到苦瓜蛋白。 1.2.2 荷瘤小鼠模型的建立 无菌条件下抽取肝癌小鼠腹水中的H22细胞，用无菌生理盐水将收集到的肿瘤细胞浓度调整为1106/ml，于ICR小鼠左前肢腋下行无菌皮下接种，每只小鼠接
11、种0.2ml细胞悬液，细胞总数为2105。 1.2.3 实验动物分组及处理 小鼠接种H22细胞后24h分组（每组12只），随机分为低剂量苦瓜蛋白给药组（T1，25mg/kg），中剂量苦瓜蛋白给药组(T2，50mg/kg)和高剂量苦瓜蛋白给药组(T3，100mg/kg)；另设阴性对照组(con，给以等量生理盐水)；阳性对照组（CTX，40mg/kg）。各组均腹腔注射给药，给药体积为每只0.2 ml，每天1次，连续12天。停药后次日对小鼠进行眼静脉穿刺取血，离心分离血清-20冻存备用。 &nb
12、sp; 1.2.4 苦瓜蛋白对荷瘤小鼠肿瘤抑制作用 停药后次日处死小鼠，剥离瘤体称其质量，计算肿瘤生长抑制率(抑瘤率)。抑瘤率=(对照组平均瘤重量-治疗组平均瘤重量)100对照组平均瘤重量。 1.2.5 MMP酶谱检测6 制备7.5丙稀酰胺凝胶（含0.1明胶），血清120稀释后加入等量的无还原剂2SDS上样缓冲液，进行聚丙烯酰胺凝胶电泳，电泳完毕后将胶置于2.5% Triton X-100溶液中室温摇洗2次，每次15min；加入
13、反应液（50mM Tris-HCl (pH 7.5)，150mM NaCl，10mM CaCl2，0.02%NaN3）37孵育过夜。取出凝胶进行考马斯亮蓝染色并脱色，结果用Iamgemaster进行拍照，结果分析采用Quantity one 软件进行密度扫描分析。其中：MMP活性表现为深色背景中的白色或浅色条带。其中相对分子量92kD为MMP-9酶原相应条带，84kD为其活化形式；72kD为MMP-2酶原相应条带，64kD为其活化形式相应的条带，凝胶照相后进行灰度分析以估计各形式MMP的活性。 1.3 统计学处理
14、; 用One-way ANOVE 统计方法进行student t检验，P<0.05者即具有显著性差异。 2 结 果 2.1 苦瓜蛋白对荷瘤小鼠肿瘤抑制作用的影响 不同剂量苦瓜蛋白及CTX对ICR小鼠H22实体瘤均有明显抑制作用。25、50及100mg/kg苦瓜蛋白组抑制率分别为52.05%、66.89%及85.81；CTX阳性对照组肿瘤抑制率为65.10%，与阴性对照组比较差异有显著性(P<
15、001)（见表1）。 2.2 苦瓜蛋白对荷瘤小鼠血清MMPs作用的影响 不同剂量苦瓜蛋白及CTX对H22荷瘤ICR小鼠血清MMPs酶原表达及活性均有明显的下调作用（与阴性对照组比较，P<001）；不同剂量苦瓜蛋白组间差异有显著性(P<0.05)，MMPs表达及活性下调与苦瓜蛋白处理的浓度的高低呈正相关，以高剂量组变化最明显（见表1，图1）。 3 讨 论 近年来，从祖国传
16、统医药宝库中发掘的抗肿瘤药物用于肿瘤治疗已经成为一个新的研究和发展方向，并在临床应用中取得了可喜进展。苦瓜作为药食同源的植物，苦瓜蛋白药理活性显著，毒副作用小，具有抗肿瘤、抗病毒等作用。其中苦瓜蛋白属于型核糖体失活蛋白，是从苦瓜种仁中分离出来的一类能够使核糖体失活、抑制细胞蛋白质合成的碱性糖蛋白。近年来研究发现某些植物来源的型RIP能够诱导肿瘤细胞产生凋亡13，而对正常的小鼠上皮培养细胞(Smac-13)无抑制作用7。另外，我们分离得到的苦瓜蛋白经实验证实可以明显诱导K562细胞产生细胞凋亡3，同时细胞黏附分子CD54表达上调与苦瓜RIP诱导肿瘤细胞凋亡相关4。 &nbs
17、p; MMPs 作为一组Zn2依赖性具有相关结构的内肽酶，可水解细胞外基质。它们在生理状态下主要参与组织发育、伤口愈合、骨生长及血管生成等参与组织的重建等。在病理情况下，MMPs则能促进许多疾病的发生、发展，如参与肿瘤的侵袭和转移以及促进新生血管的形成等病理过程，在肝癌等肿瘤中高表达MMP-2及MMP-9，因此阻断细胞外基质降解及抑制MMPs活性已逐渐成为肿瘤治疗的新靶点8，9。本研究结果表明，苦瓜蛋白对小鼠移植性肿瘤H22的生长有显著的抑制作用；苦瓜蛋白对MMP-2及MMP-9的酶原及其活性具有明显的抑制作用，与苦瓜蛋白作用剂量呈正相关，其中，以MMP-9活性降低最为明显。目前已知MMPs体
18、内的主要抑制物是组织抑制剂(TIMPs)，其中TIMP-2分布最为广泛，不仅可以与已活化的多种MMPs结合并抑制其活性，还可与酶原结合，抑制酶原的活化10。另外，RECK ( reversion inducing cysteine rich protein with kazalmotifs)基因是近年来发现的新型MMP抑制剂，可在转录后水平抑制多种MMP的表达，从而抑制MMP参与的肿瘤侵袭及转移和炎症的进展6，11。因此，苦瓜蛋白可能通过通过上述机制来抑制MMPs的合成，特别可能是抑制其活性等机制来发挥抗肿瘤生长、侵袭和转移作用。但苦瓜蛋白抑制MMPs的具体机制尚需下一步研究加以证实。&nbs
19、p; 综上所述，苦瓜蛋白具有明显抑制基质金属蛋白酶的活性；其抗肿瘤作用可能和苦瓜蛋白具有抑制MMPs的活性及其表达等因素有关。【参考文献】 1 Lee-Huang S, Huang PL, Sun Y, et al. Inhibition of MDA-MB-231 human breast tumor xenogarfts and HER2 expression by antitumor agents GAP31 and MAP30J. Anticancer Res, 2000, 20(2A):653-659.2 郎海滨, 糜漫天, 程天民 苦瓜核糖体失活
20、蛋白的纯化及其诱导HL-60细胞凋亡的研究J 营养学报, 2004, 26(2):139-143.3 熊术道, 尹丽慧, 李景荣, 等. 苦瓜蛋白诱导K562细胞凋亡及其对Bcl-2、PCNA蛋白表达的影响J. 中国免疫学杂志, 2006, 22(11):1002-1005.4 熊术道, 尹丽慧, 李景荣, 等. 粘附分子CD54、CD44在苦瓜蛋白诱导K562细胞凋亡中的作用J. 中国临床药理学与治疗学, 2005, 10（12）:1403-1407.5 Tsang KY, Ng TB. Isolation and characterization of a new ribosome ina
21、ctivating protein momorgrosvin, from seeds of the monks fruit Momordica grosvenoriiJ. Life Sci, 2001, 68(7):773-784. 6 Liu LT, Chang HC, Chiang LC, et al. Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasionJ. Cancer Res, 2003, 63(12):3069-3072. 7 Ling
22、 J, Liu WY. Cytotoxicity of two new ribosome-inactivating protein, cinnamomin and camphorin, to carcinoma cellsJ. Cell Biochem Funct, 1996, 14(3):157161.8 Ogasawara S, Yano H, Momosaki S, et al. Expression of matrix metalloproteinases (MMPs) in cultured hepatocellular carcinoma (HCC) cells and surgi
23、cally resected HCC tissuesJ. Oncol Rep, 2005, 13(6):1043-1048. 9 Kruger A, Arlt MJ, Gerg M, et al. Antimetastatic activity of a novel mechanism-based gelatinase inhibitorJ. Cancer Res, 2005, 65(9):3523-3526. 10 Woo CH, Lim JH, Kim JH. Lipopolysaccharide induces matrix metalloproteinase-9 expression
24、via a mitochondrial reactive oxygen species-p38 kinase-activator protein-1 pathway in Raw 264.7 cellsJ. J Immunol, 2004,173(11):/Porganization system and working mode, the selection of a number of municipal strengthening group to build innovation and strengthen the organization of the basic level or
25、ganizations advanced model.(seven) the manual work of the Communist Youth League Organization published and League branch of manual work, to promote the use of two manuals in the citys organizations at various levels. The county committee, directly under the group (work) is under the jurisdiction of
26、 grassroots League and group (total) using branch ratio shall not be less than 70%.Three, to further strengthen the groups leadership and team building(a) to strengthen the leagues leadership at all levels of construction. Continue to do a good job of municipal Party Committee Organization Departmen
27、t on the strengthening of the Communist Youth League cadres management opinions implementation. To promote the group counties (autonomous counties, cities) Commission and city directly under the group (workers) appointed to carry out the Five team building activities. Improve the League Association
28、Department working mechanism. Conscientiously fulfill the duties of CO, combined with the city group directly under (the) Committee of the general election and the adjustment, actively jointly with the unit where the party organization department good group leadership selection with the work. Do a g
29、ood job in municipal Party committee assistant committee leadership and leading cadres of the annual assessment work, adhere to and improve the assistant secretary of the Communist Youth League talk system group To weave individual conversation and collective conversation, to strengthen the committe
30、es of all levels, the Standing Committee of the Standing Committee, the establishment of a sound system of central group learning, the system of investigation and study, the system of important issues and the reporting system.(two) to strengthen the work of education and training of cadres to consci
31、entiously implement the Central Committee. On the implementation of grassroots cadres training project and opinions on carrying out the mission municipal CYL Cadres training project implementation opinions, full implementation of the project of the City Youth League cadres training education. Focus
32、on the county grassroots cadres and workers focused on training, the new school system vocational training League cadres, for the Municipal Organization Department jointly held the third session of the Communist Youth League system training cadres in party. Focusing on the construction of new social
33、ist countryside, strengthening village cadres and rural construction team group member of the young team, the county organizations at various levels throughout the year is expected to train 100 00 rural grassroots cadres, plans to train 1000 village secretary of the village Party branch.(three) acti
34、vely promote grassroots cadres election work. Under the unified leadership of the party organization, to strengthen grassroots organizations direct work, standardize the election work rules and procedures, in the designated candidate, open competition to democratically elected branch secretary at th
35、e same time, actively and steadily the development of Township, streets and other grassroots organizations in the direct election of the pilot work, on the basis of summarizing the experience and gradually expand the scope of the pilot, and gradually formed a set of measures and cadres election syst
36、em.(four) to strengthen the cadres testing exercise. To study the development of XX city from 2006 to 2008 cadres testing exercise planning. To select outstanding cadres to the central mission in Beijing, Shanghai, Guangzhou and other central organs and sending work in developed areas. With the Cent
37、ral Committee the implementation of the western region and ethnic minorities cadres training plan . To carry out the eighth batch of municipal organs attachment cadre management, selection, training work. The outstanding cadres to start the National Development Bank XX branch testing exercise.(five)
38、 to strengthen the group construction. The main position to play the role of Youth League cadre education and training as the city, in accordance with the requirements of post training, further curriculum scientifically and rationally, adopt arrangement menu course, basic theory includes group work,
39、 focus, hot and difficult issues, to enhance the content and practice; to strengthen the construction of teaching staff, the integration of teachers, establish a quality teacher training team, establish and implement course evaluation system; take delivery of training to the countryside, on-site tra
40、ining, and increase the rural grassroots cadres training efforts.Four, efforts to strengthen the work of young talent(a) to tenth XX young 54 medal in recognition of the publicity, and actively explore the XX youth service. 54 Medal of institutionalization, standardization and socialization mechanis
41、m. Give full play to the XX award in recognition of 54 youth medal Association, Youth Award winner in the 54 chapter, investigation activities. Who won the Youth Service medal, dedication to the society to create conditions, build the stage. During the 54, named in recognition of the tenth XX young
42、54 medal. The association of XX editing of young talent , the exchange of information and communication platform for the XX of outstanding young talents.(two) the establishment of municipal Party committee of young talent information database. In accordance with the feasible, effective, classificati
43、on management, implement step by step principle, the establishment of municipal Party committee of youth talent database; guiding organizations at various levels generally established the young talent database, construction of city, county (directly under the Communist Youth League), grassroots orga
44、nizations three young talent information database the relevant information collection system, and gradually establish the citys young people, the Communist Youth League youth talent information network, provide information platform for young talent resource development.Five, take effective measures
45、to strengthen the mission management(a) to strengthen the mission management work. Strengthen the dues collection and management, continue to adhere to the dues collection regularly reporting system, the annual comprehensive award in recognition of the tour and the tour one vote veto system audit sy
46、stem. Do statistical work in the group. The group service award in recognition of the advanced units, standardization, promoting group management work institutionalized.(two) to strengthen the organization of research and information exchange efforts. Grassroots carry out research, efforts to form a
47、 number of theoretical results. Make full use of the network carrier, to explore the establishment of electronic group service system, promote the work of Communist Youth League information.Attachment 4:XX municipal Party Committee Propaganda Department of the Communist Youth League work pointsIn 20
48、06, the Communist Youth League propaganda ideological work XX to Deng Xiaoping theory and the important thought of Three Represents as guidance, study and implement the partys sixteen and the spirit of the the fifth Plenary Session of the 16th CPC Central Committee implementation of the eight plenary session of the two committee, the citys cultural work conference, the Propaganda Department of the meeting and the the fourth Plenary Session of the 15th CPC Central Committee permanent mission spirit, closely around