Wang Hao, Wang Jun, Shi Xiaolei, Ding Yitao
Department of Hepatobiliary Surgery, Drum Tower Clinical Medical College of Nanjing Medical University, Nanjing,China.
J BUON. 2017 Nov-Dec;22(6):1517-1524.
One of the major challenges in delivering cytokines for the treatment of hepatocellular carcinoma (HCC) is the mode of delivery. This study hypothesized that genetically engineered bone marrow derived mesenchymal stem cells (BMSCs) co-expressing IFN-γ and IL-10 can serve as a potential therapeutic strategy in the treatment of HCC by inhibiting cell proliferation.
Male Sprague-Dawley rats (n=5, 200-250 g) for BMSCs isolation and Nude/SCID mice (n=35,12-20g) to develop liver cancer xenograft model were used. Mice were subcutaneously injected HepG2 cell suspension on left flank. BMSCs were genetically engineered with the recombinant lentiviral vectors expressing IFN-γ and IL-10. The experiments were performed in 5 groups (phosphate buffered saline/PBS, BMSCs, BMSC-IFN-γ, BMSC-IL-10 and BMSC-IFN-γ-IL-10) and the genetically engineered BMSCs were transplanted into HCC mice. Cell viability was measured by MTT assay followed by the evaluation of the effect of cell-cycle regulators (p21, p27, cyclin D1 and Rb). Protein expression of p38, ERK and JNK was assessed by immunohistochemistry using the cell proliferation marker Ki67.
The combination of two cytokines (IFN-γ and IL- 10) engineered into BMSCs resulted in a significant reduction in HepG2 cell viability (*p<0.05 vs PBS-treated and #p<0.05 vs BMSC-treated group). Significantly increased expression of cell cycle inhibitors p21 and p27 in parallel with reduced cyclin D1 expression were observed. Reduced phosphorylation of Rb demonstrated the repression of G1/S progression. BMSC-IFN-γ-IL-10 treatment significantly reduced the tumor growth at the end of 36 days compared to the group treated with PBS or BMSCs alone. This effect was accompanied with the modulation of MAPK pathway with the activation of p38 and JNK, and inactivation of ERK.
The co-expression of IFN-γ and IL-10 in BMSCs inhibits HCC in vitro and in vivo by modulating cell cycle regulators and MAPK pathway.
细胞因子递送用于治疗肝细胞癌（HCC）的主要挑战之一是递送方式。本研究假设，共表达干扰素-γ（IFN-γ）和白细胞介素-10（IL-10）的基因工程骨髓间充质干细胞（BMSCs）可通过抑制细胞增殖作为治疗HCC的一种潜在治疗策略。
使用雄性Sprague-Dawley大鼠（n = 5，体重200 - 250 g）分离BMSCs，并使用裸鼠/重症联合免疫缺陷（Nude/SCID）小鼠（n = 35，体重12 - 20 g）建立肝癌异种移植模型。小鼠左腹皮下注射HepG2细胞悬液。用表达IFN-γ和IL-10的重组慢病毒载体对BMSCs进行基因工程改造。实验分为5组（磷酸盐缓冲液/PBS、BMSCs、BMSC-IFN-γ、BMSC-IL-10和BMSC-IFN-γ-IL-10），将基因工程改造的BMSCs移植到HCC小鼠体内。通过MTT法测定细胞活力，随后评估细胞周期调节因子（p21、p27、细胞周期蛋白D1和视网膜母细胞瘤蛋白（Rb））的作用。使用细胞增殖标志物Ki67，通过免疫组织化学评估p38、细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNK）的蛋白表达。
将两种细胞因子（IFN-γ和IL-10）导入BMSCs后，HepG2细胞活力显著降低（*与PBS处理组相比，p < 0.05；与BMSCs处理组相比，#p < 0.05）。观察到细胞周期抑制剂p21和p27的表达显著增加，同时细胞周期蛋白D1的表达降低。Rb磷酸化的降低表明G1/S期进程受到抑制。与单独用PBS或BMSCs处理的组相比，表示在36天结束时，BMSC-IFN-γ-IL-10处理显著降低了肿瘤生长。这种作用伴随着丝裂原活化蛋白激酶（MAPK）通路的调节，p38和JNK激活，ERK失活。
BMSCs中IFN-γ和IL-10的共表达通过调节细胞周期调节因子和MAPK通路在体外和体内抑制HCC。
