转导人TIMP-2基因抑制胃癌浸润转移的实验研究

Experimental study on inhibition of invasion and metastasis of gastric cancer by transducting human TIMP-2 Gene

目的外源性人ＴＩＭＰ－２基因在人ＬＡＫ细胞中的表达及其对胃癌浸润转移的抑制作用。方法通过磷酸钙－ＤＮＡ共沉淀的方法将ＴＩＭＰ－２重组真核表达质粒ｐＬ（ＴＩＭＰ－２）ＳＮ转染兼性包装细胞ＰＡ３１７包装形成病毒颗粒。用病毒液感染人ＬＡＫ细胞，再以Ｎｏｒｔｈｅｒｎ杂交、ＳＤＳ－ＰＡＧＥ及反向酶谱试验进行表达及活性分析。用转基因ＬＡＫ细胞（ＬＡＫ／ＬＴ）２×１０６给胃癌转移模型腹腔内注射。结果ＴＩＭＰ－２在ＬＡＫ／ＬＴ中ｍＲＮＡ高度表达，在ＬＡＫ中弱表达。ＬＡＫ／ＬＴ细胞中有相对分子质量２１０００大小的蛋白质表达，具抑制Ⅳ型胶原酶降解明胶的活性。肿瘤种植后１０周，对照组肿瘤体积为（４．００±１．７０）ｃｍ３，肝转移率和腹膜转移率均达１００％，脾脏转移率为７０％（ｎ＝１０）；单用ＬＡＫ细胞治疗组肿瘤体积为（２．７２±１．２０）ｃｍ３，肝及腹膜转移率分别为６０％和７０％；ＴＩＭＰ－２高表达的ＬＡＫ／ＬＴ细胞治疗组肿瘤体积仅（１．２１±１．１６）ｃｍ３，肝转移率与腹膜转移率仅分别为２０％和３０％。肿瘤生长抑制率达７０％，肝及腹膜转移抑制率分别为８０％和７０％。结论ＴＩＭＰ－２对胃癌生长及转移具有抑制作用。

Objective To study the expression of the human tissue inhibitor of metalloproteinase-2 (TIMP-2) gene transducted into human LAK cells and the inhibition effect on the invasion and metastasis of gastric cancer in vivo. Methods The recombinant plasmid of retroviral vector with TIMP-2 expression were transfected into PA317 packing cells with calcium phosphate-DNA coprecipitation. The transfected PA317 packing cells were selected by adding 600g/ml of G418 in culture medium. Human LAK cells were infected with virus stocks. The anti-G418 clone was amplified. Total cellular RNA of the LAK and LAK/LT cells were isolated. Northern blots were hybridized with TIMP-2 cDNA probes. SDS-PAGE and reverse zymogram analysis were performed on the culture medium. The SCID mice model, which constructed by orthotopic implantation into the gastric wall with histologically intact tumor tissue, were treated with LAK and LAK/LT cells by injecting i.p. once a week for four weeks, with PBS was injected i.p. in controls. The mice were sacrificed ten weeks after implantation. Results TIMP-2 mRNA were expressed at high level in LAK/LT cells and an increase in the amount of functional TIMP-2 secreted. The mean tumor volume in the control, LAK and LAK/LT groups were 4.001.70, (2.721. 20)cm3 and (1.211.16)cm3, respectively. Metastases to the liver were 100%(n=10), 60%(n=10), and 20%(n=10), respectively, and metastases to the peritoneum were 100%, 70%, and 30%, respectively in the three groups. Conclusion These results confirmed that TIMP-2 did possess inhibitory effect on tumor growth and metastasis of gastric cancer.