复合多糖抗辐射和抗肿瘤作用及其机制研究
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摘要
近年来，恶性肿瘤已经成为严重危害人类健康、导致人类死亡的第一大恶疾。恶性肿瘤的治疗目前仍以手术治疗、放疗、化疗为主要方法。然而放射治疗和化学治疗存在着特异性较差，且能够对机体免疫功能造成损伤的弊端。因此研制毒性低、疗效好的抗癌活性成分且可保护和提高肿瘤患者免疫功能，尤其是提高患者的细胞免疫功能，进而减轻放、化疗的毒副作用的制剂，对于预防肿瘤的发生，提高肿瘤的治疗效果具有重要意义。大量的研究表明，多糖具有增强免疫功能、抗肿瘤作用，不同来源的植物多糖其发挥作用的机制不同，为此我们提出这样的假设，把不同的多糖按一定的比例配制成混合制剂，进入机体后将通过不同的途径提高机体的抗肿瘤和抗辐射作用，并探讨其作用机制，为肿瘤患者的临床治疗提供重要的实验依据。本课题从以下三个部分展开研究：
第一部分复合多糖的抗肿瘤活性及作用机制探讨。
采用MTT法分别检测人参多糖、松茸多糖和香菇多糖三种多糖对B16和H22肿瘤细胞的生长抑制作用，结果显示三种多糖对肿瘤细胞的生长抑制作用不显著；采用体外淋巴细胞增殖实验检测三种多糖对免疫细胞的影响，结果表明香菇多糖和松茸多糖能够促进淋巴细胞增殖，CTL实验进一步证实三种多糖能够通过增强淋巴细胞的增殖能力进而抑制肿瘤细胞的生长。每种多糖选择三个有效作用剂量通过正交试验找到复合多糖最佳的配比方案。
分别建立B16和H22荷瘤小鼠模型，给予不同剂量治疗，观察复合多糖的抗肿瘤作用及其对化疗药物抗肿瘤作用的影响。测定各组荷瘤小鼠肿瘤的重量、体积，脾淋巴细胞CD4+和CD8+亚群数，自然杀伤细胞(NK)和细胞毒性T淋巴细胞(CTL)活性以及肿瘤坏死因子-α (TNF-α)、白介素-2(IL-2)、白介素-4(IL-4)和干扰素-γ (IFN-γ)等细胞因子水平。结果显示，与模型组、复合多糖单独治疗组、化疗药物单独治疗组比较，复合多糖联合化疗药物治疗组荷瘤小鼠肿瘤重量和体积显著降低；与化疗药物单独治疗组比较，复合多糖治疗组和复合多糖联合化疗药物治疗组血清中TNF-α、IL-2和IFN-γ水平显著升高、IL-4水平显著降低，脾淋巴细胞CD4+和CD8+亚群数显著升高，NK和CTL细胞活性显著升高；结果表明复合多糖与化疗药物联用使用的抗肿瘤效果显著优于复合多糖或化疗药物的单独使用。复合多糖增强化疗药物抑制肿瘤细胞生长的作用机制为多糖能够增强NK和CTL细胞活性，刺激肿瘤细胞死亡相关细胞因子（TNF-α、IL-2和IFN-γ等）的分泌，维持机体淋巴细胞CD4+和CD8+亚群数。
第二部分复合多糖的体内抗辐射实验研究
建立γ射线小鼠辐射损伤模型，观察复合多糖对小鼠辐射损伤的影响。将实验动物随机分成5组，即空白对照组、辐射对照组、复合多糖低剂量组、复合多糖中剂量组和复合多糖高剂量组，照射前预防性连续给药14天，然后除空白对照组外其余组均接受4.0Gy的γ射线全身均匀照射一次，于照射后第1、3、7天取材，检测各组小鼠的脾指数、胸腺指数、脾淋巴细胞增殖能力、外周血白细胞数、骨髓DNA含量、骨髓有核细胞数、骨髓嗜多染红细胞微核数、血清中MDA含量、SOD活性、CAT活性和GSH-Px活性等指标。结果显示，与辐射对照组相比，复合多糖组小鼠的脾指数、胸腺指数、脾淋巴细胞增殖能力、外周血白细胞数、骨髓DNA含量、骨髓有核细胞数、以及血清中SOD活性、CAT活性和GSH-Px活性均显著升高，骨髓嗜多染红细胞微核数和MDA含量显著下降。结果表明，复合多糖可以从增强机体免疫功能、保护造血系统和调节氧化-还原平衡系统三方面来拮抗辐射对机体的损伤。
第三部分复合多糖的毒性实验研究
采用最大耐受量实验和骨髓嗜多染红细胞微核实验检测复合多糖的毒性。最大耐受量试验可知实验组小鼠给予30g/kg·BW复合多糖后，观察期14天内小鼠无死亡，状态良好，未见异常症状；观察期结束后对小鼠进行解剖，肉眼观察小鼠的肝脏、肾脏、心脏、脾脏、肺脏的主要器官未见异常改变；与对照组相比，未发现病变和异常。通过实验测得小鼠经口灌胃给予复合多糖最大给药剂量大于30g/kg·BW。小鼠骨髓嗜多染红细胞微核实验表明，与阴性对照组相比，复合多糖实验组小鼠骨髓细胞嗜多染红细胞微核率无显著变化，复合多糖不会对机体染色体产生损伤。
综上所述，本课题成功的研制出由松茸多糖、香菇多糖和人参多糖组成的复合多糖，它可以通过增强NK和CTL细胞活性，刺激肿瘤细胞死亡相关细胞因子的分泌以及维持机体淋巴细胞CD4+和CD8+亚群数等途径增强化疗药物抑制肿瘤细胞生长的作用；还可以从增强机体免疫功能、保护造血系统和调节氧化-还原平衡系统三方面来拮抗辐射对机体的损伤。
Cancer is the first cause of death among the adult population. The strategiesemployed in clinical practice are to remove the tumor through operation which isfollowed by chemotherapy. The disadvantages of chemotherapy are that theirspecificity is not satisfying and lymphocytes which play pivotal role in fightingtumors are destroyed. Moreover, immunosuppression is detected in patients withcancers. Therefore, how to improve immune response of cancer patients is critical incancer treatment. Substantial studies report that polysaccharides haveimmunomodulatory or antitumor activities. Reports show that polysaccharides fromdifferent herbs have different activities. Thus, we hypothesize that combination ofpolysaccharides from different herbs will produce better efficacy in inhibiting tumorcell growth and antagonize radiation damage.
Part Ⅰ The anti-tumor effects of the mixture of three polysaccharide
MTT assay results show that PLE, PTM and PG can produce no significanteffects on the growth of B16and H22cells, which suggest that PLE, PTM and PG donot have the capacities to kill tumor cells, and PLE and PTM can produce effects onthe growth lymphocytes. CTL activity was determined by lactate dehydrogenase(LDH) release assay. The results showed that PLE, PTM and PG were able toincrease the activities of CTL. The dosage proportions of mixture of threepolysaccharides were optimized by orthogonal test.
Mice were injected with H22or B16cell and then treated by chemotherapydrugs (5-fluorouracil or cyclophosphamide), mixture of polysaccharides (PM), orchemotherapy drugs+PM. The tumor weight, tumor volume, the subgroups of CD4+and CD8+in spleen, the natural killer (NK) cell and cytotoxic T lymphocyte (CTL)activities of splenocytes and the levels of Tumor Necrosis Factor-α (TNF-α),Interleukin-2(IL-2), Interleukin-4(IL-4), and Interferon-γ (IFN-γ) were determined. Compared with mice from model, chemotherapy drugs, PM groups, the mice treatedwith chemotherapy drugs+PM showed: significantly smaller of tumor weight andvolume. Compared with mice from chemotherapy drugs, the mice treated with PMand chemotherapy drugs+PM showed:(1) significantly increased NK and CTLactivities of splenocytes;(2) significantly increased subgroups of CD4+and CD8+inspleen (3) signficantly higher levels of TNF-α, IL-2, and IFN-γ in serum;(4)significantly lower levels of IL-4in serum; Combination of polysaccharides fromlentinus edodes, tricholoma matsutake and ginseng can help chemotherapy drugsproduce more effective inhibition of H22and B16cell growth. One of themechanisms by which PM strengthened the efficacy of chemotherapy drugs ininhibiting H22and B16cells growth was that PM can increase the activities of NKand CTL. Another mechanism through which PM helped inhibit cancer cell growth isthat they can stimulate the secretion of cytokines related to tumor cell death. Theother mechanism that PM enhanced chemotherapy drugs inhibiting H22and B16cells growth was that PM can increase the percentage of subgroups of CD4+andCD8+.
Part Ⅱ The anti-radiation effects of the mixture of three polysaccharide
Establish γ-ray radiation damage models of mice and observe the anti-radiationeffects of PM. The mice were divided randomly into five groups: Normal controlgroup (NC), Irradiation control group (IC), Low-dose PM group (PML),Medium-dose PM group (PMM), High-dose PM group (PMH). PM was administeredto the mice for14days. After the14days, the mice in all groups were irradiated with4Gy γ-ray except NC group. The spleen index, thymus index, the prolife reaction ofsplenocytes to ConA, white blood cell count, the content of bone marrow DNA, the number ofnucleated bone marrow cells, the micronucleus rate of bonemarrow polychromatic erythrocyte,MDA content, SOD activity, CAT activity and GSH-Px activity were determined in1st,3rd,7thday after radiation. Compared with mice from IC group, the mice treatedwith PM showed:(1) significantly increased spleen index, thymus index and the prolifereaction of splenocytes to ConA;(2) significantly increased white blood cell count, thecontent of bone marrow DNA and the number of nucleated bone marrow cells;(3) significantly decreased the micronucleus rate of bone marrow polychromaticerythrocyte;(4) significantly increased SOD activity, CAT and GSH-Px activities inserum;(5) signficantly decreased MDA content in serum. PM had strong anti-γ-rayradiation activity.
Part Ⅲ Toxicological studies of the mixture of three polysaccharide
The toxicity of PM was determined by maximum tolerance experiment and bonemarrow micronucleus test. The maximum tolerance test results showed: maximumtolerance dose (MTD)>30g/kg·BW. Micronucleus test in mice showed that therewere no significant differences between the PM group and the negative group. PMwas non-toxic and did not induce the micronuclear rates.
In conclusion, combination of polysaccharides from PLE, PTM and PG werenon-toxic and can help produce a better efficacy of tumor inhibition together withchemotherapy drugs. PM can antagonize the adverse effects, such as the low functionalimmune status, oxidative and hematopoietic system damage, caused by γ-ray radiation.
第一部分复合多糖的抗肿瘤活性及作用机制探讨。
采用MTT法分别检测人参多糖、松茸多糖和香菇多糖三种多糖对B16和H22肿瘤细胞的生长抑制作用，结果显示三种多糖对肿瘤细胞的生长抑制作用不显著；采用体外淋巴细胞增殖实验检测三种多糖对免疫细胞的影响，结果表明香菇多糖和松茸多糖能够促进淋巴细胞增殖，CTL实验进一步证实三种多糖能够通过增强淋巴细胞的增殖能力进而抑制肿瘤细胞的生长。每种多糖选择三个有效作用剂量通过正交试验找到复合多糖最佳的配比方案。
分别建立B16和H22荷瘤小鼠模型，给予不同剂量治疗，观察复合多糖的抗肿瘤作用及其对化疗药物抗肿瘤作用的影响。测定各组荷瘤小鼠肿瘤的重量、体积，脾淋巴细胞CD4+和CD8+亚群数，自然杀伤细胞(NK)和细胞毒性T淋巴细胞(CTL)活性以及肿瘤坏死因子-α (TNF-α)、白介素-2(IL-2)、白介素-4(IL-4)和干扰素-γ (IFN-γ)等细胞因子水平。结果显示，与模型组、复合多糖单独治疗组、化疗药物单独治疗组比较，复合多糖联合化疗药物治疗组荷瘤小鼠肿瘤重量和体积显著降低；与化疗药物单独治疗组比较，复合多糖治疗组和复合多糖联合化疗药物治疗组血清中TNF-α、IL-2和IFN-γ水平显著升高、IL-4水平显著降低，脾淋巴细胞CD4+和CD8+亚群数显著升高，NK和CTL细胞活性显著升高；结果表明复合多糖与化疗药物联用使用的抗肿瘤效果显著优于复合多糖或化疗药物的单独使用。复合多糖增强化疗药物抑制肿瘤细胞生长的作用机制为多糖能够增强NK和CTL细胞活性，刺激肿瘤细胞死亡相关细胞因子（TNF-α、IL-2和IFN-γ等）的分泌，维持机体淋巴细胞CD4+和CD8+亚群数。
第二部分复合多糖的体内抗辐射实验研究
建立γ射线小鼠辐射损伤模型，观察复合多糖对小鼠辐射损伤的影响。将实验动物随机分成5组，即空白对照组、辐射对照组、复合多糖低剂量组、复合多糖中剂量组和复合多糖高剂量组，照射前预防性连续给药14天，然后除空白对照组外其余组均接受4.0Gy的γ射线全身均匀照射一次，于照射后第1、3、7天取材，检测各组小鼠的脾指数、胸腺指数、脾淋巴细胞增殖能力、外周血白细胞数、骨髓DNA含量、骨髓有核细胞数、骨髓嗜多染红细胞微核数、血清中MDA含量、SOD活性、CAT活性和GSH-Px活性等指标。结果显示，与辐射对照组相比，复合多糖组小鼠的脾指数、胸腺指数、脾淋巴细胞增殖能力、外周血白细胞数、骨髓DNA含量、骨髓有核细胞数、以及血清中SOD活性、CAT活性和GSH-Px活性均显著升高，骨髓嗜多染红细胞微核数和MDA含量显著下降。结果表明，复合多糖可以从增强机体免疫功能、保护造血系统和调节氧化-还原平衡系统三方面来拮抗辐射对机体的损伤。
第三部分复合多糖的毒性实验研究
采用最大耐受量实验和骨髓嗜多染红细胞微核实验检测复合多糖的毒性。最大耐受量试验可知实验组小鼠给予30g/kg·BW复合多糖后，观察期14天内小鼠无死亡，状态良好，未见异常症状；观察期结束后对小鼠进行解剖，肉眼观察小鼠的肝脏、肾脏、心脏、脾脏、肺脏的主要器官未见异常改变；与对照组相比，未发现病变和异常。通过实验测得小鼠经口灌胃给予复合多糖最大给药剂量大于30g/kg·BW。小鼠骨髓嗜多染红细胞微核实验表明，与阴性对照组相比，复合多糖实验组小鼠骨髓细胞嗜多染红细胞微核率无显著变化，复合多糖不会对机体染色体产生损伤。
综上所述，本课题成功的研制出由松茸多糖、香菇多糖和人参多糖组成的复合多糖，它可以通过增强NK和CTL细胞活性，刺激肿瘤细胞死亡相关细胞因子的分泌以及维持机体淋巴细胞CD4+和CD8+亚群数等途径增强化疗药物抑制肿瘤细胞生长的作用；还可以从增强机体免疫功能、保护造血系统和调节氧化-还原平衡系统三方面来拮抗辐射对机体的损伤。
Cancer is the first cause of death among the adult population. The strategiesemployed in clinical practice are to remove the tumor through operation which isfollowed by chemotherapy. The disadvantages of chemotherapy are that theirspecificity is not satisfying and lymphocytes which play pivotal role in fightingtumors are destroyed. Moreover, immunosuppression is detected in patients withcancers. Therefore, how to improve immune response of cancer patients is critical incancer treatment. Substantial studies report that polysaccharides haveimmunomodulatory or antitumor activities. Reports show that polysaccharides fromdifferent herbs have different activities. Thus, we hypothesize that combination ofpolysaccharides from different herbs will produce better efficacy in inhibiting tumorcell growth and antagonize radiation damage.
Part Ⅰ The anti-tumor effects of the mixture of three polysaccharide
MTT assay results show that PLE, PTM and PG can produce no significanteffects on the growth of B16and H22cells, which suggest that PLE, PTM and PG donot have the capacities to kill tumor cells, and PLE and PTM can produce effects onthe growth lymphocytes. CTL activity was determined by lactate dehydrogenase(LDH) release assay. The results showed that PLE, PTM and PG were able toincrease the activities of CTL. The dosage proportions of mixture of threepolysaccharides were optimized by orthogonal test.
Mice were injected with H22or B16cell and then treated by chemotherapydrugs (5-fluorouracil or cyclophosphamide), mixture of polysaccharides (PM), orchemotherapy drugs+PM. The tumor weight, tumor volume, the subgroups of CD4+and CD8+in spleen, the natural killer (NK) cell and cytotoxic T lymphocyte (CTL)activities of splenocytes and the levels of Tumor Necrosis Factor-α (TNF-α),Interleukin-2(IL-2), Interleukin-4(IL-4), and Interferon-γ (IFN-γ) were determined. Compared with mice from model, chemotherapy drugs, PM groups, the mice treatedwith chemotherapy drugs+PM showed: significantly smaller of tumor weight andvolume. Compared with mice from chemotherapy drugs, the mice treated with PMand chemotherapy drugs+PM showed:(1) significantly increased NK and CTLactivities of splenocytes;(2) significantly increased subgroups of CD4+and CD8+inspleen (3) signficantly higher levels of TNF-α, IL-2, and IFN-γ in serum;(4)significantly lower levels of IL-4in serum; Combination of polysaccharides fromlentinus edodes, tricholoma matsutake and ginseng can help chemotherapy drugsproduce more effective inhibition of H22and B16cell growth. One of themechanisms by which PM strengthened the efficacy of chemotherapy drugs ininhibiting H22and B16cells growth was that PM can increase the activities of NKand CTL. Another mechanism through which PM helped inhibit cancer cell growth isthat they can stimulate the secretion of cytokines related to tumor cell death. Theother mechanism that PM enhanced chemotherapy drugs inhibiting H22and B16cells growth was that PM can increase the percentage of subgroups of CD4+andCD8+.
Part Ⅱ The anti-radiation effects of the mixture of three polysaccharide
Establish γ-ray radiation damage models of mice and observe the anti-radiationeffects of PM. The mice were divided randomly into five groups: Normal controlgroup (NC), Irradiation control group (IC), Low-dose PM group (PML),Medium-dose PM group (PMM), High-dose PM group (PMH). PM was administeredto the mice for14days. After the14days, the mice in all groups were irradiated with4Gy γ-ray except NC group. The spleen index, thymus index, the prolife reaction ofsplenocytes to ConA, white blood cell count, the content of bone marrow DNA, the number ofnucleated bone marrow cells, the micronucleus rate of bonemarrow polychromatic erythrocyte,MDA content, SOD activity, CAT activity and GSH-Px activity were determined in1st,3rd,7thday after radiation. Compared with mice from IC group, the mice treatedwith PM showed:(1) significantly increased spleen index, thymus index and the prolifereaction of splenocytes to ConA;(2) significantly increased white blood cell count, thecontent of bone marrow DNA and the number of nucleated bone marrow cells;(3) significantly decreased the micronucleus rate of bone marrow polychromaticerythrocyte;(4) significantly increased SOD activity, CAT and GSH-Px activities inserum;(5) signficantly decreased MDA content in serum. PM had strong anti-γ-rayradiation activity.
Part Ⅲ Toxicological studies of the mixture of three polysaccharide
The toxicity of PM was determined by maximum tolerance experiment and bonemarrow micronucleus test. The maximum tolerance test results showed: maximumtolerance dose (MTD)>30g/kg·BW. Micronucleus test in mice showed that therewere no significant differences between the PM group and the negative group. PMwas non-toxic and did not induce the micronuclear rates.
In conclusion, combination of polysaccharides from PLE, PTM and PG werenon-toxic and can help produce a better efficacy of tumor inhibition together withchemotherapy drugs. PM can antagonize the adverse effects, such as the low functionalimmune status, oxidative and hematopoietic system damage, caused by γ-ray radiation.
引文
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