抗IV型胶原酶单抗3G11与力达霉素偶联物的抗肿瘤作用

目的 观察抗IV型胶原酶单抗 3G11与力达霉素 (LDM)偶联物的抗肿瘤作用。方法 用MTT法测定其对肿瘤细胞的增殖抑制作用 ;用小鼠移植性肝癌H2 2观察体内抗肿瘤作用。结果 3G11 LDM偶联物保留了单抗3G11与IV型胶原酶和靶细胞H2 2细胞的结合能力 ,体外试验H2 2细胞显示比游离LDM更强的细胞增殖抑制作用。体内 3G11 LDM偶联物 0 0 5和 0 10mg·kg- 1 对小鼠移植性肝癌H2 2的抑瘤率分别为 87 8%和 97 2 % ,而游离LDM0 0 5mg·kg- 1 的抑瘤率为 6 7 1% ,且 3G11 LDM偶联物组小鼠的中位生存时间比LDM组明显延长。结论 3G11 LDM偶联物对小鼠移植性肝癌H2 2的抑瘤作用比LDM强 ,可能成为抗肿瘤靶向药物。

胶原酶Ⅳ预处理对足三里穴注射庆大霉素胃电的影响

目的 :探讨穴位注药后初始药效发生中传递药物信息的物质基础。方法 :应用胶原酶Ⅳ在足三里穴上方 6mm同经 (足阳明胃经 )上预处理 ,30min后足三里穴注射庆大霉素 ,观察给药后庆大霉素对胃电的影响 ,并与等量胶原酶Ⅳ在足三里穴上方 6mm经旁 (足阳明胃经旁开 4mm)预处理组和生理盐水足三里穴上方同经预处理组相比较。结果 :胶原酶Ⅳ足三里穴上方同经上预处理能完全阻断足三里穴注射庆大霉素的初始抑制胃电的作用 ,而足阳明胃经旁预处理组和生理盐水预处理组无此阻断作用。结论 :庆大霉素足三里注射可明显抑制胃电活动。胶原Ⅳ在足三里穴注射庆大霉素后初始药效中具有传递药物信息的作用。

金荞麦提取物抑制肿瘤细胞侵袭、转移和HT-1080细胞产生Ⅳ型胶原酶的研究

目的探讨金荞麦提取物对肿瘤细胞侵袭和转移的影响。方法以人工重组基底膜及小鼠黑色素瘤高转移株自发性肺转移模型观察了金荞麦提取物对Ｂ１６－ＢＬ６细胞的体外抗侵袭活性和体内抗转移作用；用聚丙烯酰胺凝胶电泳法进一步观察了其对人纤维肉瘤ＨＴ－１０８０细胞Ⅳ型胶原酶的产生及活性的影响；同时用ＷＳＴ法观察了该药的细胞毒性。结果金荞麦提取物在１００ｍｇＬ－１剂量下能明显抑制Ｂ１６－ＢＬ６细胞侵袭；在２００ｍｇｋｇ－１剂量下能有效抑制Ｂ１６－ＢＬ６黑色素瘤细胞在Ｃ５７／ＢＬ６小鼠体内自发性肺转移。该药对Ｂ１６－ＢＬ６和ＨＴ－１０８０细胞无明显细胞毒作用。该药能抑制ＨＴ－１０８０细胞Ⅳ胶原酶的产生，但对酶的活性无明显影响。结论金荞麦提取物具有明显的抗癌侵袭和转移的作用。

Ⅳ型胶原酶与动脉粥样硬化

Ⅳ型胶原酶作为基质金属蛋白酶家族成员之一，可降解基底膜的主要结构成分-Ⅳ型胶原，在血管平滑肌细胞穿过细胞外基质从中膜向内膜迁移过程中起重要作用。本文介绍Ⅳ型胶原酶的结构、生理功能、基因表达和活性调节，通过分析生长因子和细胞因子对该酶基因转录的影响，探讨血管内膜损伤引发细胞外基质结构重建的分子机制，并讨论该酶在动脉粥样硬化和血管再狭窄发生中的意义。

Detection of type Ⅳ collagenase activity in malignant ascites

AIM: Type IV collagenase participates in invasion and metastasis of cancer cells. Malignant asdtes is a manifestation of advanced malignant disease that is associated with invasion and metastasis of the peritoneal cavity. Thus, it is reasonable to hypothesize that type IV collagenase is linked to malignant ascites. The purpose of our study was to detect type IV collagenase activity in malignant ascites so as to provide the scientific basis for clinic diagnosis and treatment of malignant ascites. METHODS: Cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=-23) from patients with gastric cancer (n=6), colon cancer (n=5), ovarian cancer (n=8) and other cancers (n=4), including 2 hepatocellular cancers, 1 pancreatic cancer, 1 primary peritoneal carcinoma were collected by paracentesis. The ascites were made cellfree by centrifugation and stored frozen at -70℃ before determination. Type IV collagenase activity was determined by gelatin zymography. RESULTS: The activity of matrix metalloproteinases-2 and -9 could not be detected in ascites of hepatic cirrhosis and tuberculous peritonitis but could be detected in 20 and 18 out of 23 malignant ascites respectively. The positive rate of type IV collagenase (MMP-2, 87.0% and MMP-9, 78.3%) was higher than that by routine ascites tests (P<0.01) in malignant ascites. Furthermore, the activity of MMP-2 was higher than that of MMP-9 (P=0.022<0.05). CONCLUSION: Type IV collagenase is positive in malignant ascites. Detection of type IV collagenase activity is useful in qualitative diagnosis of ascites. Type IV collagenase may play an important role in malignant ascites formation.

Antitumor activity of anti-type IV collagenase monoclonal antibody and its lidamycin conjugate against colon carcinoma

AIM： Type IV collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for rnAb-directed therapy. The irnrnunoreactivity of rnAb 3G11, a rnAb directed against type Ⅳ collagenase in human colorectal carcinomas, was studied by irnrnuno-histochernical （IHC） staining, rnAb 3G11 was conjugated to an antiturnor antibiotic lidarnycin （LDM）. The antiturnor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice. METHODS： ELISA, gelatin zyrnography, and Western blot assay were used for the biological characterization of rnAb 3G11. The irnrnunoreactivity of rnAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice. RESULTS： As shown in ELISA, mAb 3Gll reacted specifically with type IV collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay, mAb 3G11 showed positive irnrnunoreactivity in most cases of colorectal carcinoma, and negative irnrnunoreactivity in the adjacent non-malignant tissues. By gelatin zyrnography, the inhibition effect of rnAb 3G11 on the secretion activity of type IV collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10 000-fold more potent than that of rnitornycin C （MMC） and adriarnycin （ADM）. 3G11- LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an IC50 value of 5.6× 10^-19 mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively. CONCLUSION： mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.

作用于基质金属蛋白酶的抗生素

基质金属蛋白酶 (matrix metalloproteinases,MMPs)是在与组织重塑有关的生理过程和以细胞外基质过度破坏为主要特征的病理过程中起重要作用的一类锌离子依赖性内肽酶。近些年来 ,MMPs在恶性肿瘤侵袭、生长和转移过程中的作用日益受到重视。我们以在肿瘤细胞破坏和穿过基底膜发生侵袭和转移过程中起重要作用的 MMPs家族成员 IV型胶原酶为研究对象 ,利用抗 IV型胶原酶单克隆抗体 3D6为工具 ,采用间接 EL ISA方法研究了四种抗生素对 IV型胶原酶或能够分泌 IV型胶原酶的 PG细胞的作用。结果表明 ,geldanamycin和博安霉素均能竞争性地抑制 3D6与 IV型胶原酶或 PG细胞的结合 ,说明其作用于 IV型胶原酶并可能对其活性产生抑制作用 ;而米诺环素在实验浓度下仅对 3D6和 PG细胞的结合表现出抑制作用。另外 ,我们发现地红霉素亦能够抑制单抗 3D6与 IV型胶原酶或 PG细胞的结合。结果表明 ,这四种抗生素可能作用于基质金属蛋白酶。

乳腺癌基质降解产物对MCF-7细胞系增殖和侵袭的影响

目的观察乳腺癌基质降解产物(DECM)对人乳腺癌细胞株MCF-7增殖和侵袭的影响,探讨DECM对肿瘤增殖和浸润的作用,及对MMP-7蛋白与SDF-1蛋白表达的影响。方法采用胰酶消化法,分别分离来自人正常乳腺组织、乳腺癌旁组织及乳腺癌组织切取物中细胞和基质,提取基质成分,洗涤基质得到3种母液,分别为正常乳腺组织母液、癌旁组织母液、癌组织母液,再以IV型胶原酶消化基质,得到3种DECM,分别为乳腺组织DECM、癌旁组织DECM、癌组织DECM,分别用3种母液和3种DECM作用于人乳腺癌细胞株MCF-7,设置空白对照,通过MTT法、软琼脂克隆形成实验观察癌细胞增殖的变化,用Tran-swell实验观察细胞侵袭能力变化,通过Western Blot检测人乳腺癌细胞株MCF-7细胞中MMP-7和SDF-1蛋白的表达变化。结果 IV型胶原酶作用人乳腺不同组织来源的基质得到的DECM均可以使MCF-7细胞株增殖和侵袭能力增强,与空白组及相应母液组比较差异有显著性(P<0.01);DECM可以上调MCF-7中MMP-7和SDF-1蛋白的表达,与空白组及相应母液组比较差异有显著性(P<0.01)。结论 DECM具有促进肿瘤细胞增殖及侵袭能力的生物学活性;该能力可能与上调肿瘤细胞MMP-7和SDF-1蛋白表达有关。

单链抗体与力达霉素辅基蛋白融合蛋白制备过程的优化

优化以包涵体形式表达的抗Ⅳ型胶原酶单链抗体与力达霉素辅基蛋白融合蛋白Fv-LDP制备过程。通过单因素或正交设计优化其包涵体制备与溶解、固定化金属螯合层析和分步透析复性过程,Sephadex G-75层析精细纯化蛋白,基于流式细胞术的饱和结合实验检测其抗原结合活性。优化后,Fv-LDP包涵体纯度为63.9%,溶解度为95.7%;纯化后Fv-LDP纯度达95%以上,复性后单体含量占60%,精细纯化后提升为85%,比初始复性条件提高5.6倍,能够与人肺腺癌PAa细胞和肝癌BEL-7402细胞结合。Fv-LDP制备过程被成功优化,为其生产和研发奠定了实验基础,也为其他以包涵体形式表达的基于单链抗体的小型化抗体药物制备提供了比较实用的方法。