基于诱导细胞凋亡与自噬机制的灰树花多糖D组分联合维生素C体内抗肿瘤作用研究

Medicinal and edible products of Grifola frondosa have many biological activities such as anti-tumor、immune regulation and hypoglycemic and so on, which has always been a treasure in our country's traditional Chinese herbal medicine research. The D-Fraction of Grifola frondosa polysaccharide (DFP) which been extracted from its fruiting body has definite structure and the extraction technology of it is mature. It can exert anti-tumor effect by inducing tumor cell apoptosis in vitro experiments and regulating immunity in vivo experiments. However, most of study results suggest that when using DFP separately to treat tumor cells, the dosage is large and the inhibition effect is not obvious. And in vivo experiments, it has not been reported that the Grifola frondosa polysaccharide has a direct inhibition of tumor growth. Our previous in vitro experimental results showed that the combination of low-dose DFP and vitamin C (VC) significantly inhibited the proliferation of hepatocellular carcinoma SMMC-7721 and HepG2 cells, and the combined treatment induced tumor cell apoptosis through the mitochondrial pathway. At the same time, the autophagy activity of tumor cells also increased significantly. This is not reported in the current research at home and abroad. This research intends to study the direct anti-tumor effect of the combination therapy on tumor-bearing mice in vivo based on the results of the in vitro studies , and to explore the molecular mechanism from two aspects of inducing cell apoptosis and autophagy. Simultaneously, to investigate the toxicity and the indirect effect of combination therapy on immune function in mice. This study will provide an experimental basis for the clinical practice of liver cancer treatment for the combination of Grifola frondosa polysaccharide and vitamin C .

药食同源产物灰树花具有抗肿瘤、免疫调节和降血糖血压等多种生物活性,一直是我国传统中草药研究中的瑰宝。从其子实体中提取出的灰树花多糖D组分(DFP)结构明确,提取技术成熟,可在体外实验中通过诱导肿瘤细胞凋亡,和在体内实验中调节机体免疫功能来发挥抗肿瘤作用。但多数体外实验研究结果提示单用DFP处理肿瘤细胞时用药量大且抑制作用不明显,而在体内抗肿瘤实验中,未报道灰树花多糖具有直接抑制肿瘤生长的作用。我们前期体外实验结果表明,联合应用小剂量DFP和维生素C(VC)比单独用药可显著抑制肝癌SMMC-7721和HepG2细胞增殖,且联合用药可通过线粒体途径诱导肿瘤细胞发生凋亡,同时肿瘤细胞的自噬活性也大幅度提高。这在目前国内外的研究中均未见报道。本课题拟在体外研究结果的基础上，进一步对联合用药在荷瘤鼠体内开展抗肿瘤的实验研究,从诱导细胞凋亡和细胞自噬两方面探讨联合用药的直接抗肿瘤分子机制;同时检测联合用药对正常小鼠的毒性及对免疫调节功能的影响,从免疫调节方面探讨联合用药的间接抗肿瘤效应。本研究为灰树花多糖和维生素C联合用药在肝癌治疗的临床实践上提供实验基础。

灰树花是一种具有抗肿瘤、调节免疫功能等多种生物活性的药食同源产物。前期体外实验结果提示联合应用灰树花多糖D组分（GFP）和维生素C（VC）对肝癌SMMC-7721细胞具有显著的抑制作用。本项目研究从循证医学及体内实验研究两方面进一步验证联合用药的抗肿瘤效应。体内实验中，构建出小鼠H22肝癌肿瘤疾病模型，并分别给予单用GFP、GFP/VC联合用药干预。三周后的实验结果显示，联合用药组比对照组具有显著的抑制肿瘤生长效应，抑瘤率分别为57.92%（联合用药口服组）和69.58%（联合用药腹腔注射组）;免疫组化（IHC）和WB结果中，肿瘤组织中凋亡相关蛋白（PRAP、Caspase-3）和自噬相关蛋白（Beclin-1、LC-3）表达均增高，提示抗肿瘤作用机制为同时诱导肿瘤发生细胞凋亡和自噬；联合用药还可增强小鼠体内的细胞免疫功能（CD4+、CD8+T细胞、B细胞和Treg细胞百分比均增高）和体液免疫功能（免疫强化细胞因子IFN-γ、TNF-α、IL-2和IL-12p70分泌量增高，同时免疫抑制细胞因子GM-CSF、MIP-3a/CCL20、IF-4和IF-10分泌量降低）；此外联合用药对荷瘤鼠肝脏和肾脏的损害较少，提示药物的毒性较小。以上实验结果已发表学术论文多篇，均为国内外首次报道，为灰树花的产业转化及灰树花多糖和维生素C联合用药在肝癌治疗的临床实践上提供理论和实验基础。