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目的观察纤连蛋白重组多肽CH50对小鼠肝癌细胞侵袭和肿瘤血管形成能力的影响,探讨CH50多肽治疗肿瘤可能的分子机制。方法接种H22肝癌细胞建立小鼠肿瘤模型,采用裸DNA-尾静脉注射方法在小鼠体内表达CH50多肽,进行基因治疗。HE染色观察治疗后肿瘤细胞侵袭能力和血管形成的改变；逆转录-聚合酶链反应(RT-PCR)和明胶电泳检测肿瘤组织边缘基质金属蛋白酶-9(MMP-9)mRNA和蛋白表达及活性；体外培养H22细胞,检测CH50多肽对相关基因表达和细胞黏附能力的影响。结果小鼠体内非定位转染表达CH50多肽,对肿瘤生长具有明显的抑制作用,接种后第16天,治疗组瘤重为(0．78±0．18)g,低于质粒对照组和空白对照组(P＜0．05)；从组织水平观察,CH50多肽治疗导致肿瘤细胞大量坏死,可抑制肿瘤细胞侵袭生长和肿瘤血管生成,抑制肿瘤血管建立侧支循环。pCH510治疗组的肿瘤边缘组织中,活性型MMP-9的含量为3．2±0．7,显著低于空白对照组和质粒对照组,且活性型MMP-9占总MMP-9的比例也显著降低,是空白对照组和质粒对照组的1/4左右,但对MMP-9 mRNA表达没有明显影响。CH50多肽可下调H22细胞的αv、β3、cdc2基因的表达水平,降低H22细胞整合素αvβ3的活性。结论CH50多肽可以通过影响MMP-9和整合素αvβ3的功能起到抑制肿瘤生长、侵袭和肿瘤血管形成的作用。
Objective To investigate the effect of recombinant human fibronectin CH50 on the invasion and tumor angiogenesis in mouse hepatocellular carcinoma (HCC) and to explore the possible molecular mechanism of CH50 polypeptide in tumorigenesis. Methods H22 hepatocarcinoma cells were inoculated to establish mouse tumor model. The naked mice were injected with tail vein to express CH50 peptide in mice for gene therapy. The invasiveness and angiogenesis of tumor cells were observed by HE staining. The expression of MMP-9 mRNA and protein and the activity of MMP-9 at the edge of tumor tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) and gelatin electrophoresis H22 cells were cultured in vitro to detect the effect of CH50 on the expression of related genes and cell adhesion. Results The expression of CH50 polypeptide was inhibited by non-localized transfection in vivo and the tumor growth was significantly inhibited. On the 16th day after inoculation, the tumor weight in the treatment group was (0.78 ± 0.18) g, which was lower than that in the plasmid control group and blank Control group (P <0.05). From the tissue level observation, treatment with CH50 peptide resulted in a large number of tumor cells necrosis, which could inhibit the invasion and growth of tumor cells and tumor angiogenesis, and inhibit the collateral circulation of tumor vessels. The content of active MMP-9 in pCH510-treated group was 3.2 ± 0.7, which was significantly lower than that in blank control group and plasmid control group, and the ratio of active MMP-9 to total MMP-9 Significantly decreased, which was about 1/4 of the blank control group and plasmid control group, but had no obvious effect on the expression of MMP-9 mRNA. CH50 could down-regulate the expression of αv, β3 and cdc2 genes in H22 cells and decrease the activity of integrin αvβ3 in H22 cells. Conclusion The CH50 polypeptide can inhibit tumor growth, invasion and tumor angiogenesis by affecting the function of MMP-9 and integrin αvβ3.