纯化后海藻硫酸多糖蛋白复合物抗肿瘤作用研究
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摘要
目的：针对海藻硫酸多糖蛋白复合物（SPPC）的有效活性成分的构成特点，对SPPC进一步纯化处理，提高其有效活性成分含量；并研究纯化后海藻多糖蛋白复合物（PSPPC）在体内、外的抗肿瘤活性，探讨PSPPC的抗肿瘤作用机制。
材料与方法：1.将海藻硫酸多糖蛋白复合物（SPPC）经乙醇分级，Sevage法去杂蛋白，再经活性炭脱色处理后得纯化后海藻硫酸多糖蛋白复合物（PSPPC）样品。2.以人肝癌细胞株（SMMC-7721）作为实验靶细胞，采用体外细胞培养技术，通过MTT法、流式细胞术等方法观察了不同浓度PSPPC对SMMC-7721细胞的增殖抑制作用；并以小鼠荷H22肝癌移植瘤模型为研究对象，通过腹腔给药，观察不同浓度PSPPC对小鼠肿瘤生长情况的影响和肿瘤组织病理形态学变化，探讨PSPPC是否也能在体内有效地抑制肿瘤细胞的生长。3.研究确证PSPPC的体内调节抗氧化酶活性和体外直接清除自由基作用。4.探讨PSPPC体外诱导人肝癌细胞（SMMC-7721）凋亡作用。5.在小鼠荷H22肝癌移植瘤模型上，进一步探讨PSPPC对荷瘤小鼠免疫功能的影响：称量各组小鼠胸腺和脾脏的重量，计算胸腺指数和脾脏指数；分离荷瘤小鼠外周血淋巴细胞（PBLC），测定PSPPC对荷瘤小鼠T淋巴细胞体外增殖作用的影响；ELISA法检测PSPPC治疗小鼠血清中IL-2和IL-12的活性水平。
结果：1.经有效纯化处理后可使PSPPC有效成分含量明显提高，总糖、硫酸根和蛋白质含量分别为37.63%（P﹤0.01），26.47%（P﹤0.01）和2.84%（P﹤0.01）。2. MTT法观察不同浓度PSPPC对SMMC-7721肝癌细胞体外增殖作用，结果表明SMMC-7721肝癌细胞增殖受到抑制，并且具有时间和剂量的依赖性。流式细胞术检测发现，0.4、2.0和10.0mg/ml PSPPC作用SMMC-7721肝癌细胞48h后均可使G0/G1期细胞增多，S期和G2/M期细胞减少，细胞阻滞于G1→S期，阻止细胞进行有丝分裂，从而抑制细胞的增殖。PSPPC体内抑瘤实验结果显示：低剂量组与高剂量组PSPPC的抑瘤率分别为：30.48％和33.38%，PSPPC组与阴性对照组相比差异显著（P <0.01）。小鼠肿瘤组织切片后HE染色结果显示：PSPPC作用H22荷瘤小鼠后，镜下可见H22肿瘤细胞大片坏死，且坏死区周围可见凋亡小体存在。3. PSPPC在体外具有很强的清除超氧阴离子自由基（O2·）和羟基自由基（·OH）-的作用。PSPPC能够调节体内抗氧化酶的活性，抑制脂质过氧化产物的生成，即其具有一定的体内抗氧化作用。4.PSPPC能诱导SMMC-7721细胞发生凋亡。经PSPPC处理后，SMMC-7721细胞内凋亡相关蛋白procaspase-3有不同程度的表达降低；DNA凝胶电泳法检测到DNA ladder现象；AnnexinⅤ法检测到0.4、2.0和10.0 mg/ml PSPPC处理48h后的SMMC-7721细胞出现了早期凋亡现象，凋亡细胞百分率分别为11.13％，26.05％和29.56％，PSPPC处理组与对照组比较有显著性差异（P <0.01）。5.经PSPPC处理后，荷瘤小鼠免疫功能低下状态得到改善。PSPPC能够一定程度的增强NK细胞杀伤活性，并且能够提高淋巴细胞增殖功能。
结论：海藻硫酸多糖蛋白复合物在一定程度上是复杂的复合物，有效纯化、分析海藻硫酸多糖蛋白复合物的生物活性十分必要。经有效纯化处理后可使其有效成分含量明显提高。游离杂蛋白含量显著降低的同时保护了多糖分子上结合的多肽或蛋白质分子，从而提高了有效成分的生物活性。体外抗肿瘤实验发现，PSPPC对SMMC-7721肝癌细胞的体外增殖具有明显的抑制作用。本研究建立了H22荷瘤小鼠模型，通过腹腔注射给药PSPPC，结果发现PSPPC具有一定的抑瘤作用。本研究还对PSPPC的抗肿瘤作用机制进行了初步探讨。体外实验证明，PSPPC能够在一定程度上清除超氧阴离子自由基（O2·）和羟基自由基（·OH），说明PSPPC能够直接发挥体外抗氧化作用。体内实验-
结果显示，PSPPC具有一定的体内抗氧化作用，能够调节抗氧化酶的活性，抑制脂质过氧化产物的生成等。PSPPC同时可影响肿瘤细胞中凋亡相关蛋白的表达，诱导肿瘤细胞凋亡, PSPPC诱导SMMC-7721肝癌细胞凋亡的机制可能是通过caspase-3的活化来实现的。本研究进一步证明，PSPPC还可以改善肿瘤所致的小鼠免疫功能低下状态。根据本项研究获得的各项实验结果，我们认为PSPPC的抗肿瘤作用的几种可能机制为：诱导肿瘤细胞凋亡；调节机体抗氧化酶活性；直接清除活性氧自由基；提高机体自身的免疫机能。
Objective: To investigate the inhibitory effect of PSPPC on tumor growth in H22tumor-bearing mice in vivo and on human hepatoma SMMC-7721 cells growth in vitro, and explore its possible mechanisms of antitumor activity.
Materials and methods: 1.SPPC was purificated by ethanol fractionation, taking off the dissociative protein, and decoloring with charcoal. 2. Taking the human hepatoma SMMC-7721 cells as our experimental object, the SMMC-7721 cell’s proliferation inhibitiontreated with PSPPC was observed by the way of MTT-colorimetric assay and Flow cytometric analysis. Hepatocellular carcinoma cells H22 were subcutaneously injected into mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The tumor growth were measured, tumor growth inhibition rate was calculated, and the alterations of histomorphologism of H22 tumor were taken and examined by H&E staining to assess the inhibitory effects of PSPPC on tumor growth. 3. In order to explore the possible mechanisms of antitumor activity of PSPPC, further bioactivities were detected, which included: In vitro, free radical scavenging activities of PSPPC; In vivo, the functions of modulating the activities of antioxidase and inhibiting the production of MDA of PSPPC were detected. 4. To study the apoptosis effects of PSPPC on human hepatoma SMMC-7721 cells.5.The effects of PSPPC onthe H22 tumor-bearing mice immunofunction were also observed in vivo. Based on the same H22 xenograft tumor models, the effects of PSPPC on the immune function were surveyed in mice from the following aspects: the thymus glands and spleens in the H22 tumor-bearing micewere excised and weighted and the thymus gland index and spleen index were caculated, respectively, which help to indicate whether PSPPC can affect the immune organs’function or not; the effects of PSPPC on the proliferation of peripheral blood lymphocyte (PBLC)separated from spleen of the H22 tumor-bearing mice in vitro evaluated using MTT assay; the alternations of concentration of murine serum IL-2 and IL-12 were examined by ELISA.
Results: 1. After purification, the total available components of PSPPC were obviously increased, the contents of sulfate, total polysaccharide and protein in PSPPC were 26.47%, 37.63% and 2.84% respectively. 2. PSPPC exhibited a suppressive effect on SMMC-7721 cells growth in concentration and time-dependent manners. PSPPC at concentrations (0.4,2.0 and 10.0 mg·ml-1) dose-dependently increased the cell number at G1 phase and decreased the number at S phase and G/M phase in SMMC-7721 cells,that means, PSPPC could disrupt celldivision by blocking cell cycle at the checkpoint G1à2/M, ( which resulting in the inhibitionof cell proliferation. Marked inhibitory effect of PSPPC on the transplanted hepatocellularcarcinoma H22 was observed in the tumor-bearing mice. The inhibitory rates were 33.38% and30.48％in the groups treated with high and low dosage of PSPPC, respectively (P <0.01 vs.control group). Histopathological examination revealed widespread necrosis in the tumors.Numerous apoptotic bodies were observed in the tumors under the electron microscope.3.Theresults from the free radical-scavenging systems (Superoxide Anion and Hydroxyl Radical)revealed that PSPPC had significant free radical-scavenging activity in vitro. PSPPC couldmodulate the activities of antioxidase and inhibite the production of MDA, that means, PSPPCshowed significant antioxidant activity. 4. PSPPC can induce the apoptosis of SMMC-7721cells: PSPPC dose-dependently decreased the expression of the relative apoptotic proteins(procaspase-3); The treatment of PSPPC resulted in the induction of DNA fragmentation;Annexin assay discovered , after being treated with 0.4,2.0 and 10.0 mg·ml-1 PSPPC for 48h,the cells’early stage apoptosis rate were 11.13％,26.05％and 29.56％, respectively, higherthan that of control(P <0.01).5. Moreover, PSPPC could improve the immune function intumor-bearing mice. The NK activity and proliferation ability of T lymphocytes were increasedin tumor-bearing mice.
Conclusion: The components of sulfated polysaccharide-protein complex (SPPC) arecomplicated. It is undoubtedly a promising and singnificant work to purificate the SPPC andexplore the bioactivities of PSPPC. After purification, the total available components of thesamples were obviously increased. Collectively, the bioactivities of PSPPC were also enhanced.Our research had proved that PSPPC could significantly inhibit the proliferation of the humanhepatoma SMMC-7721 cells. Hepatocellular carcinoma cells H22 were subcutaneously injectedinto mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The experiment inH22 tumor-bearing mice documented PSPPC had obvious effects on tumor inhibition. PSPPCshowed significant free radical-scavenging activity in vitro, and could modulate the activitiesof antioxidase and inhibite the production of MDA In vivo. PSPPC could induce apoptosis ofSMMC-7721 cells with a dose-dependent manner. Moreover, PSPPC-induced apoptosis waspartially attributed to the activation of caspase-3. Further investigation proves that PSPPCcould improve the immune function in tumor-bearing mice. Based on the conclusionsmentioned above, some possible mechanisms of antitumor activity of PSPPC can be proposedas: Inducing the apoptosis of tumor cells; scavenging free radical; modulating the activities ofantioxidases; improving the immune function.
材料与方法：1.将海藻硫酸多糖蛋白复合物（SPPC）经乙醇分级，Sevage法去杂蛋白，再经活性炭脱色处理后得纯化后海藻硫酸多糖蛋白复合物（PSPPC）样品。2.以人肝癌细胞株（SMMC-7721）作为实验靶细胞，采用体外细胞培养技术，通过MTT法、流式细胞术等方法观察了不同浓度PSPPC对SMMC-7721细胞的增殖抑制作用；并以小鼠荷H22肝癌移植瘤模型为研究对象，通过腹腔给药，观察不同浓度PSPPC对小鼠肿瘤生长情况的影响和肿瘤组织病理形态学变化，探讨PSPPC是否也能在体内有效地抑制肿瘤细胞的生长。3.研究确证PSPPC的体内调节抗氧化酶活性和体外直接清除自由基作用。4.探讨PSPPC体外诱导人肝癌细胞（SMMC-7721）凋亡作用。5.在小鼠荷H22肝癌移植瘤模型上，进一步探讨PSPPC对荷瘤小鼠免疫功能的影响：称量各组小鼠胸腺和脾脏的重量，计算胸腺指数和脾脏指数；分离荷瘤小鼠外周血淋巴细胞（PBLC），测定PSPPC对荷瘤小鼠T淋巴细胞体外增殖作用的影响；ELISA法检测PSPPC治疗小鼠血清中IL-2和IL-12的活性水平。
结果：1.经有效纯化处理后可使PSPPC有效成分含量明显提高，总糖、硫酸根和蛋白质含量分别为37.63%（P﹤0.01），26.47%（P﹤0.01）和2.84%（P﹤0.01）。2. MTT法观察不同浓度PSPPC对SMMC-7721肝癌细胞体外增殖作用，结果表明SMMC-7721肝癌细胞增殖受到抑制，并且具有时间和剂量的依赖性。流式细胞术检测发现，0.4、2.0和10.0mg/ml PSPPC作用SMMC-7721肝癌细胞48h后均可使G0/G1期细胞增多，S期和G2/M期细胞减少，细胞阻滞于G1→S期，阻止细胞进行有丝分裂，从而抑制细胞的增殖。PSPPC体内抑瘤实验结果显示：低剂量组与高剂量组PSPPC的抑瘤率分别为：30.48％和33.38%，PSPPC组与阴性对照组相比差异显著（P <0.01）。小鼠肿瘤组织切片后HE染色结果显示：PSPPC作用H22荷瘤小鼠后，镜下可见H22肿瘤细胞大片坏死，且坏死区周围可见凋亡小体存在。3. PSPPC在体外具有很强的清除超氧阴离子自由基（O2·）和羟基自由基（·OH）-的作用。PSPPC能够调节体内抗氧化酶的活性，抑制脂质过氧化产物的生成，即其具有一定的体内抗氧化作用。4.PSPPC能诱导SMMC-7721细胞发生凋亡。经PSPPC处理后，SMMC-7721细胞内凋亡相关蛋白procaspase-3有不同程度的表达降低；DNA凝胶电泳法检测到DNA ladder现象；AnnexinⅤ法检测到0.4、2.0和10.0 mg/ml PSPPC处理48h后的SMMC-7721细胞出现了早期凋亡现象，凋亡细胞百分率分别为11.13％，26.05％和29.56％，PSPPC处理组与对照组比较有显著性差异（P <0.01）。5.经PSPPC处理后，荷瘤小鼠免疫功能低下状态得到改善。PSPPC能够一定程度的增强NK细胞杀伤活性，并且能够提高淋巴细胞增殖功能。
结论：海藻硫酸多糖蛋白复合物在一定程度上是复杂的复合物，有效纯化、分析海藻硫酸多糖蛋白复合物的生物活性十分必要。经有效纯化处理后可使其有效成分含量明显提高。游离杂蛋白含量显著降低的同时保护了多糖分子上结合的多肽或蛋白质分子，从而提高了有效成分的生物活性。体外抗肿瘤实验发现，PSPPC对SMMC-7721肝癌细胞的体外增殖具有明显的抑制作用。本研究建立了H22荷瘤小鼠模型，通过腹腔注射给药PSPPC，结果发现PSPPC具有一定的抑瘤作用。本研究还对PSPPC的抗肿瘤作用机制进行了初步探讨。体外实验证明，PSPPC能够在一定程度上清除超氧阴离子自由基（O2·）和羟基自由基（·OH），说明PSPPC能够直接发挥体外抗氧化作用。体内实验-
结果显示，PSPPC具有一定的体内抗氧化作用，能够调节抗氧化酶的活性，抑制脂质过氧化产物的生成等。PSPPC同时可影响肿瘤细胞中凋亡相关蛋白的表达，诱导肿瘤细胞凋亡, PSPPC诱导SMMC-7721肝癌细胞凋亡的机制可能是通过caspase-3的活化来实现的。本研究进一步证明，PSPPC还可以改善肿瘤所致的小鼠免疫功能低下状态。根据本项研究获得的各项实验结果，我们认为PSPPC的抗肿瘤作用的几种可能机制为：诱导肿瘤细胞凋亡；调节机体抗氧化酶活性；直接清除活性氧自由基；提高机体自身的免疫机能。
Objective: To investigate the inhibitory effect of PSPPC on tumor growth in H22tumor-bearing mice in vivo and on human hepatoma SMMC-7721 cells growth in vitro, and explore its possible mechanisms of antitumor activity.
Materials and methods: 1.SPPC was purificated by ethanol fractionation, taking off the dissociative protein, and decoloring with charcoal. 2. Taking the human hepatoma SMMC-7721 cells as our experimental object, the SMMC-7721 cell’s proliferation inhibitiontreated with PSPPC was observed by the way of MTT-colorimetric assay and Flow cytometric analysis. Hepatocellular carcinoma cells H22 were subcutaneously injected into mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The tumor growth were measured, tumor growth inhibition rate was calculated, and the alterations of histomorphologism of H22 tumor were taken and examined by H&E staining to assess the inhibitory effects of PSPPC on tumor growth. 3. In order to explore the possible mechanisms of antitumor activity of PSPPC, further bioactivities were detected, which included: In vitro, free radical scavenging activities of PSPPC; In vivo, the functions of modulating the activities of antioxidase and inhibiting the production of MDA of PSPPC were detected. 4. To study the apoptosis effects of PSPPC on human hepatoma SMMC-7721 cells.5.The effects of PSPPC onthe H22 tumor-bearing mice immunofunction were also observed in vivo. Based on the same H22 xenograft tumor models, the effects of PSPPC on the immune function were surveyed in mice from the following aspects: the thymus glands and spleens in the H22 tumor-bearing micewere excised and weighted and the thymus gland index and spleen index were caculated, respectively, which help to indicate whether PSPPC can affect the immune organs’function or not; the effects of PSPPC on the proliferation of peripheral blood lymphocyte (PBLC)separated from spleen of the H22 tumor-bearing mice in vitro evaluated using MTT assay; the alternations of concentration of murine serum IL-2 and IL-12 were examined by ELISA.
Results: 1. After purification, the total available components of PSPPC were obviously increased, the contents of sulfate, total polysaccharide and protein in PSPPC were 26.47%, 37.63% and 2.84% respectively. 2. PSPPC exhibited a suppressive effect on SMMC-7721 cells growth in concentration and time-dependent manners. PSPPC at concentrations (0.4,2.0 and 10.0 mg·ml-1) dose-dependently increased the cell number at G1 phase and decreased the number at S phase and G/M phase in SMMC-7721 cells,that means, PSPPC could disrupt celldivision by blocking cell cycle at the checkpoint G1à2/M, ( which resulting in the inhibitionof cell proliferation. Marked inhibitory effect of PSPPC on the transplanted hepatocellularcarcinoma H22 was observed in the tumor-bearing mice. The inhibitory rates were 33.38% and30.48％in the groups treated with high and low dosage of PSPPC, respectively (P <0.01 vs.control group). Histopathological examination revealed widespread necrosis in the tumors.Numerous apoptotic bodies were observed in the tumors under the electron microscope.3.Theresults from the free radical-scavenging systems (Superoxide Anion and Hydroxyl Radical)revealed that PSPPC had significant free radical-scavenging activity in vitro. PSPPC couldmodulate the activities of antioxidase and inhibite the production of MDA, that means, PSPPCshowed significant antioxidant activity. 4. PSPPC can induce the apoptosis of SMMC-7721cells: PSPPC dose-dependently decreased the expression of the relative apoptotic proteins(procaspase-3); The treatment of PSPPC resulted in the induction of DNA fragmentation;Annexin assay discovered , after being treated with 0.4,2.0 and 10.0 mg·ml-1 PSPPC for 48h,the cells’early stage apoptosis rate were 11.13％,26.05％and 29.56％, respectively, higherthan that of control(P <0.01).5. Moreover, PSPPC could improve the immune function intumor-bearing mice. The NK activity and proliferation ability of T lymphocytes were increasedin tumor-bearing mice.
Conclusion: The components of sulfated polysaccharide-protein complex (SPPC) arecomplicated. It is undoubtedly a promising and singnificant work to purificate the SPPC andexplore the bioactivities of PSPPC. After purification, the total available components of thesamples were obviously increased. Collectively, the bioactivities of PSPPC were also enhanced.Our research had proved that PSPPC could significantly inhibit the proliferation of the humanhepatoma SMMC-7721 cells. Hepatocellular carcinoma cells H22 were subcutaneously injectedinto mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The experiment inH22 tumor-bearing mice documented PSPPC had obvious effects on tumor inhibition. PSPPCshowed significant free radical-scavenging activity in vitro, and could modulate the activitiesof antioxidase and inhibite the production of MDA In vivo. PSPPC could induce apoptosis ofSMMC-7721 cells with a dose-dependent manner. Moreover, PSPPC-induced apoptosis waspartially attributed to the activation of caspase-3. Further investigation proves that PSPPCcould improve the immune function in tumor-bearing mice. Based on the conclusionsmentioned above, some possible mechanisms of antitumor activity of PSPPC can be proposedas: Inducing the apoptosis of tumor cells; scavenging free radical; modulating the activities ofantioxidases; improving the immune function.
引文
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[1] Ruperez P, Ahrazem O, Leal JA. Potential antioxidant capacity of sulfated olysaccharidesfrom the edible marine brown seaweed Fucus vesiculosus. J. Agic. Food Chem. 2002,50(4):840~845.
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[8] 魏文青，王明霞，丛建波等.海藻硫酸多糖抗乙型肝炎病毒的实验研究.中华肝脏病杂志.10(2):112.
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