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白细胞介素-12互补脱氧核糖核酸直接注射:在小鼠肝转移模型中单次注射的抗转移作用

IL-12 cDNA direct injection: antimetastatic effect from a single injection in a murine hepatic metastases model.

作者信息

Weber Sharon M, Qi Chen, Neal Zane, Sondel Paul, Mahvi David M

机构信息

Department of Surgery, University of Wisconsin Hospital, Madison 53792, USA.

出版信息

J Surg Res. 2004 Dec;122(2):210-7. doi: 10.1016/j.jss.2004.04.021.

Abstract

BACKGROUND

Interleukin 12 (IL-12) gene therapy is an effective antitumor agent in local and metastatic murine tumor models. We sought to evaluate the antimetastatic effect of IL-12 cDNA in a liver metastases model.

MATERIALS AND METHODS

A liver metastases model was induced by creating a "primary" splenic tumor through inoculation of 1 x 10(5) TS/A adenocarcinoma cells directly into the inferior pole of the spleen in female BALB/c mice. On day 4, 50 microg of IL-12 cDNA or control plasmid DNA was injected into splenic tumor, followed by splenectomy on day 8. Mice were sacrificed on day 25 to assess liver tumor burden. IL-12 mRNA and mIL-12 and IFN-gamma protein levels were assessed after IL-12 injection. Peripheral blood CD4+, CD8+, and NK cells were quantified on day 14 using FACS. To determine the significance of site of cytokine DNA injection, IL-12 cDNA was injected on day 4 into splenic tumor or into the non-involved spleen after isolation of the inferior and superior portions of the spleen, respectively, with surgical clips. Splenectomy was performed on day 8 and sacrifice was performed on day 25.

RESULTS

IL-12 mRNA was detected in the liver 8 h after injection, with a peak at 24 h. After splenic injection, protein levels of IL-12 and IFN-gamma were detectable in the liver and spleen 24 h after treatment. IL-12 and IFN-gamma were not detectable in control animals. In the peripheral blood, there was a marked increase in NK cells (13% of total lymphocytes versus 4%, control) and in the CD4+/CD8+ ratio (5.5 versus 1.9). At day 25, there was a marked antimetastatic effect after IL-12 injection into either splenic tumor [liver:body weight, 6.2 versus 10.9 (control), P = 0.007] or non-involved spleen (6.8 g versus 10.7 g, P = 0.005). There was no difference in the antimetastatic effect between animals injected into splenic tumor or non-involved spleen (P = 0.3).

CONCLUSION

Injection with a single dose of IL-12 cDNA into splenic tumor or non-involved spleen resulted in a profound antimetastatic effect. Splenic IL-12 injection results in mRNA expression in the liver, protein expression in the liver and spleen, and a marked increase in NK cells and the CD4+/CD8+ ratio in peripheral blood.

摘要

背景

白细胞介素12(IL-12)基因疗法在局部和转移性小鼠肿瘤模型中是一种有效的抗肿瘤药物。我们试图评估IL-12 cDNA在肝转移模型中的抗转移作用。

材料与方法

通过将1×10⁵个TS/A腺癌细胞直接接种到雌性BALB/c小鼠脾脏下极来建立“原发性”脾脏肿瘤,从而诱导肝转移模型。在第4天,将50μg的IL-12 cDNA或对照质粒DNA注射到脾脏肿瘤中,然后在第8天进行脾切除术。在第25天处死小鼠以评估肝脏肿瘤负荷。在注射IL-12后评估IL-12 mRNA、mIL-12和IFN-γ蛋白水平。在第14天使用流式细胞术对外周血CD4⁺、CD8⁺和NK细胞进行定量。为了确定细胞因子DNA注射部位的重要性,在第4天分别在脾脏上下部分分离后,将IL-12 cDNA注射到脾脏肿瘤或未受累的脾脏中,然后用手术夹夹住。在第8天进行脾切除术,并在第25天处死。

结果

注射后8小时在肝脏中检测到IL-12 mRNA,在24小时达到峰值。脾内注射后,治疗后24小时在肝脏和脾脏中可检测到IL-12和IFN-γ蛋白水平。在对照动物中未检测到IL-12和IFN-γ。在外周血中,NK细胞显著增加(占总淋巴细胞的13%,而对照组为4%),CD4⁺/CD8⁺比值也增加(5.5比1.9)。在第25天,将IL-12注射到脾脏肿瘤[肝脏:体重,6.2比10.9(对照),P = 0.007]或未受累的脾脏中(6.8 g比10.7 g,P = 0.005)后,均有显著的抗转移作用。将IL-12注射到脾脏肿瘤或未受累脾脏的动物之间的抗转移作用没有差异(P = 0.3)。

结论

向脾脏肿瘤或未受累的脾脏注射单剂量的IL-12 cDNA可产生显著的抗转移作用。脾内注射IL-12导致肝脏中mRNA表达、肝脏和脾脏中蛋白表达,以及外周血中NK细胞和CD4⁺/CD8⁺比值显著增加。

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