綠藻對BALB/c 小鼠全身性及腸道免疫之影響
Effects of Chlorella on systemic and intestinal immune response in BALB/c mice
中文摘要
綠藻是一種單細胞藻類，含有豐富的營養成分，常被作為營養補充之保健食品。
目前國內對綠藻保健功能的研究尚侷限於抗氧化、降低血壓、膽固醇及減重代餐 等。而國外雖陸續有實驗發現口服綠藻熱水抽出物可有效的增強實驗動物對抗腫 瘤、消滅病毒和細胞內寄生細菌的功能，但是這些研究都是利用感染實驗動物模 式來進行，尚未有文獻發表以健康的動物進行實驗來評估綠藻的免疫調節功能。
為了能深入瞭解、分析綠藻何種成分具有免疫調節作用，本實驗以BALB/c 小白 鼠為實驗動物，將其分為對照組、飼料中添加1% 或 10% 的綠藻粉，以及每日 灌食0.5ml 綠藻生長因子 （Chlorella growth factor, CGF）等四組，整體 性的評估綠藻對宿主非特異性、特異性免疫、及腸道免疫反應的影響。並進而針 對具有Biological Response Modifiers(BRM)功效的 CGF，在試管中評估其 對 (1)小鼠腹腔巨噬細胞 (2) 巨噬細胞株 RAW 264.7 (3)血液內顆粒性白血球 和單核球細胞吞噬作用的影響。同時並測量CGF 刺激脾臟細胞的增生反應和細 胞激素的分泌，以探討綠藻及CGF 具免疫調節功效的可能作用機制。
動物實驗結果顯示：餵食一週後，10%綠藻組及 CGF 組老鼠之顆粒性白血球 吞噬能力較對照組明顯升高(p<0.05)，且 CGF 組的單核球吞噬能力也較對照 組高；第三週後1%、10%綠藻組及 CGF 組的單核球吞噬能力和對照組有顯著 差異，而CGF 組的顆粒性白血球吞噬能力也顯著提高。在體外實驗中，外加 CGF 亦可使腹腔巨噬細胞、RAW 264.7 細胞株及全血中吞噬細胞的吞噬能力提 升。餵食五週後，犧牲老鼠取脾臟細胞，分析不同比例的自然殺手細胞毒殺標靶 細胞能力，以及淋巴細胞增生和細胞激素的分泌量，結果顯示10%綠藻組 、 CGF 組的自然殺手細胞毒殺能力明顯增加，其中又以 10%綠藻組增加最為顯 著(p<0.01)。而淋巴細胞的增生反應，四組間並無顯著差異，但在試管中將 CGF 加入脾臟細胞培養液中，則可促進淋巴細胞增生。特異性免疫反應的評估 是對四組老鼠採用腹腔注射OVA 或口服投予 OVA、霍亂毒素（Cholera toxin, CT），每週抽眼窩血或收集腸道灌洗液，利用 ELISA 的方法，測量各組老鼠 週邊血中專一性抗體IgM、IgG 與腸道 Total sIgA 和專一性 sIgA 抗體濃度的變 化。結果顯示1% 綠藻組老鼠血清中專一性抗體 IgM、IgG 分泌量較高，10%綠 藻組和CGF 組則較低；而四組老鼠腸道中 Total sIgA 和專一性 sIgA 的分泌則 無顯著差異。綜合評估，不論是採飼料添加綠藻粉或以胃管灌食綠藻生長因子，
綠藻的確可以明顯增強小鼠的吞噬作用和自然殺手細胞毒殺能力等非特異性免 疫能力，但對免疫後小鼠血液中專一性抗體IgM、IgG 和腸道 sIgA 的產生則無 明顯的影響。
英文摘要
Chlorella vulgaris, a unicellular green algae, contains rich nutrients and has been used widely as a health food supplement. Studies on Chlorella’s health benefits have been mainly focused on its effects as an antioxidant and a weight loss meal- replacement, and on the improvement of hypertension and
hyperlipidemia.Currently, it has been also demonstrated that hot water extracts of Chlorella vulgaris can boost host’s defenses against tumors, and viral and
intracellular bacterial infections. However, those studies are mostly done on the infected or immunocompromised animals, and there is much to be evaluated on their effects on the immunity of healthy animals. Thus, the specific aim of this dissertation is to evaluate the effects of Chlorella vulgaris and Chlorella growth factor（CGF）which was from the hot water extracts of Chlorella vulgaris, on the innate, acquired, and intestinal mucosal immunity of healthy BALB/c mice. To accomplish this goal, animals were divided into four study groups. They were the control group and three test groups of feeding with 1% or 10% addition of
Chlorella vulgaris powder in the food or forced feeding with 0.5ml CGF daily.The in vitro biological effect of Chlorella vulgaris was also evaluated on the capacity of phagocytosis of isolated peritoneal macrophage, RAW264.7 macrophage cell line, granulocytes and monocytes.
The phagocytic activity of granulocytes was determined to be higher (p<0.05) in the groups that fed with 10% Chlorella powder and CGF after one week,
meanwhile, that of monocytes derived from the CGF group was also higher than the control group. At week 3, the phagocytic activity of monocytes derived from all three test groups was higher than that of the control group. In vitro studies, the addition of CGF in the assay can also promote the phagocytosis of peritoneal macrophage, phagocytes from the whole blood, and RAW264.7 cell lines. At week 5, splenocytes were harvested and analyzed for natural killer cell（NK）
cytotoxicity in different effector/target cell ratio, lymphocyte proliferation, and cytokine secretion in the presence of mitogen. NK cytotoxicity was shown to be higher in both 10% Chlorella and CGF groups (p<0.01). While CGF could promote lymphocyte proliferation in vitro, the same effect has not been detected in all three test groups.
To study the effect of Chlorella vulgaris on adaptive immunity, all four groups have been immunized with ovalbumin(OVA) by intraperitoneal (IP) injection and by oral administration of OVA or cholera toxin（CT）separately for the detection of specific antibodies in the sera or intestinal lavage. Our results showed that the anti-OVA specific IgM、IgG titer are higher in group 1% Chlorella, but not in both 10% Chlorella and CGF groups.There was no significant difference among the four groups on anti-CT and anti-OVA specific sIgA. In summary, no matter feeding mice
with Chlorella diet or oral administration with CGF, our results suggested that Chlorella may modulate the innate immunity, both on phagocytosis and NK cytotoxicity. However, the stimulating effect of Chlorella on specific IgM, IgG and intestinal sIgA production may not be significant
