- 學位論文
營養性葉酸缺乏與自體免疫關係之研究
Relationship between nutritional folate deficiency and autoimmunity
摘要
第一章 葉酸代謝與需求及葉酸缺乏對人類淋球細胞激素與抗體的影響 中文摘要 葉酸 (folic acid or folate) 為 B 群水溶性維生素，其主要功能為單碳的代謝，包括胺基酸的代謝、嘌呤嘧啶及 s-adenosylmethionine (SAM) 的生合成，決定細胞的正常生長和繁殖。在免疫系統的研究方面，大部針對動物作研究，且大部份在較早時期 (1960-1970)，只知葉酸缺乏會減少老鼠淋巴細胞數目及增加感染率，但對於人類免疫系統的研究郤很少。 因此我們將正常人週邊血液淋巴細胞 (peripheral blood lymphocytes, PBLs) 培養於控制組與葉酸缺乏組培養液下，並加 PHA 刺激 3 天。結果顯示葉酸缺乏的人類淋巴細胞內葉酸濃度只有最初的 44%，細胞因缺乏葉酸而降低了 thymidine 的 de novo synthesis，比控制組攝取了更多的 [3H]-deoxythymidine，而減少外加 [6-3H] deoxyuridine 的利用。受 PHA 刺激的葉酸缺乏細胞其生長增殖率從培養的第 1 天到第 3 天皆明顯低於控制組的細胞 (p<0.05)，細胞生長週期在培養的第 2 天 G0-G1 phase 發生延遲，而導致 S 及 G2-M phase 明顯少於控制組的細胞。對於 IL-2、 IL-4、 IL-6、 IL-10 及 IFN-γ 等細胞激素的 mRNA 表現，葉酸缺乏的細胞皆少於控制組，並且在分泌上也明顯低於控制組的細胞 (p<0.05)。並發現葉酸缺乏組對於免疫球蛋白 IgG 與 IgM 製造也明顯低於控制組 (p<0.05)。 由以上的結果，我們可知營養性葉酸缺乏會延緩人類淋巴細胞對 PHA 的刺激，減少細胞激素及抗體的分泌製造，延遲細胞生長週期，而影響淋巴細胞的生長。 第二章 葉酸缺乏與淋巴細胞程式凋亡、低甲基化及抗雙股去氧核糖核苷酸抗體間之影響 中文摘要 葉酸因其複雜的生化功能，而決定了細胞的正常生長和繁殖，有研究指出葉酸的缺乏會導致細胞的程式凋亡 (apoptosis)，但對於其引起細胞程式凋亡的機轉並不清楚。另外因葉酸其中一個重要的功能在使 homocysteine 接受一個甲基而成為 methionine，而 methionine 再進一步的合成細胞內甲基主要的提供者 SAM，因此缺乏葉酸會導致 DNA 的低甲基化。而文獻報告指出淋巴細胞 DNA 的低甲基化與自體免疫疾病的發生有關，然而卻沒有針對葉酸缺乏是否增加自體免疫疾病的發生作研究。因此本章目地在探討葉酸缺乏所引發的人類淋巴細胞程式凋亡是影響何種蛋白、並測定淋巴細胞 DNA 的甲基化及是否引起抗 dsDNA 抗體的產生。 我們將人類淋巴細胞培養於控制組與葉酸缺乏的培養液，培養 3 到 9 天，在收集細胞的前 2 或 3 天加入PHA 刺激 48 或 72 小時。葉酸缺乏的人類淋巴細胞其生長率於第 4 天以後就明顯低於控制組的細胞，未受 PHA 刺激的細胞，控制組於第 7 天以後會自動進入細胞生長週期，葉酸缺乏的細胞則否。而受PHA 刺激 48 和 72 小時的細胞，葉酸缺乏組的細胞則於第 5 天與第 4 天開始明顯在 S phase 發生停滯 (p<0.05)，並於第 4 天開始發生細胞程式凋亡。在培養的第 3 天，葉酸缺乏組的細胞所測得的IL-2、 bcl-2 及 Fas 的 mRNA 少於控制組，於第 6 天所測得的 bcl-2 及 Fas 蛋白表現少於控制組，並測得細胞質中有 cytochrome c 與 caspase 3。在第 5 天所測得的 p53 蛋白，發現葉酸缺乏組的細胞明顯的低於控制組。 收集第3 天與第 9 天的培養液，發現葉酸缺乏組培養液中所產生的 homocysteine 明顯高於控制組的培養液(p<0.05)，而第 9 天的葉酸缺乏組培養液中所含的 homocysteine 則更高出第 3 天的葉酸缺乏組培養液(p<0.05)。在第 3 天就測得葉酸缺乏組的人類淋巴細胞 DNA 呈現低甲基化。令人驚訝得是PBLs 培養於未受 PHA 刺激的培養液中8 天，竟在葉酸缺乏組的培養液中測得抗 dsDNA 抗體。 由上以的結果，我們推測營養性葉酸缺乏造成人類淋巴細胞發生細胞程式凋亡，是因細胞在 S phase 發生停滯，且並不依賴 p53 及 Fas 蛋白，是因生長因子 IL-2 及保護細胞免於程式凋亡的 bcl-2 蛋白減少所造成，並可能造成自體免疫的發生。 第三章 葉酸缺乏與自體免疫關係－動物實驗 中文摘要 Lipotropes 包括葉酸、methionine、 choline、 維生素 B12 等營養素，主要的功能在提供甲基，如 one-carbon metabolism 及 homocysteine homeostasis。而當葉酸缺乏時，所有與單碳代謝有關的反應都會受影響。葉酸缺乏會增加 homocysteine，已有多篇研究指出 hyperhomocysteine 與心血管疾病的發生有密切關係。而高的 homocysteine 會降低SAM/SAH 的比例，造成 DNA 的低甲基化。另有研究指出好發於女性的 SLE，其週邊血液單核球細胞 (peripheral blood monocytes ) 的 DNA 比正常人較呈現低甲基化。 Richardson 研究群的研究證明於老鼠 CD4+T 細胞加入 DNA methyltransferase inhibitors，可使 DNA 呈現低甲基化狀態，且會引起自體免疫現象，但卻從未有報導指出營養性葉酸的缺乏是否與自體免疫的發生有關。而我們 in vitro 的研究中，發現葉酸缺乏的人類淋巴細胞會產生抗 dsDNA 抗體。因此本章的研究目的是由 in vivo 的動物實驗來觀察營養性葉酸缺乏是否引起老鼠自體免疫現象，並在給予自體免疫的小老鼠10倍葉酸補充，觀察是否可改善其自體免疫的現象。 我們的結果顯示控制組老鼠血漿中的葉酸濃度平均在 30 到 40 ng/mL, 而葉酸缺乏組老鼠的葉酸濃度則在 3.5 ng/mL 以下，並且葉酸缺乏的老鼠脾臟細胞培養於葉酸缺乏培養液中比控制組脾臟細胞於葉酸缺乏培養液中會攝入更多外來的 [H3]-deoxythymidine 及更少的 [6-3H] deoxyuridine。在飼養第 10 週，葉酸缺乏組的脾臟細胞以 PHA 刺激，其生長增殖率並不明顯。而葉酸缺乏的老鼠其體重則明顯低於控制組老鼠 (p<0.05)，第 11 週，心臟、肺臟及腎臟對體重的比例皆明顯變大 (p<0.05)，死亡率在第 11 週則高達 78%，在第 4 週以後 葉酸缺乏組的老鼠脾臟細胞 DNA 呈現低甲基化。且發現在第 6 週以後，葉酸缺乏組的老鼠脾臟細胞 CD11a 表現高於控制組的脾臟細胞 (p<0.05)。在第 8 週以後，葉酸缺乏組的老鼠脾臟細胞 CD40L表現高於控制組的脾臟細胞 (p<0.05)。 第 4 週的葉酸缺乏組的老鼠脾臟細胞 DNA 發生斷裂，第 8 週的脾臟與腎臟組織切片皆出現較多的 apoptotic bodies，由 flow cytometry 偵測 apoptosis 的比例，在第 6 週以後，葉酸缺乏組的老鼠脾臟細胞發生 apoptosis 的比例明顯高出控制組的老鼠脾臟細胞 (p<0.05)。第 10 週葉酸缺乏組老鼠血漿中明顯出現抗 dsDNA 抗體 (p<0.05)，而在第 6 週以後，葉酸缺乏組老鼠尿中蛋白質濃度高於控制組老鼠，第10 週的老鼠腎小球環間膜細胞出現增生 (mesangium proliferation or mesangium matrix increased)。 在 10 倍葉酸補充的自體免疫老鼠實驗中，在第 12 週10 倍葉酸補充組的小鼠脾臟細胞 DNA 比控制組的小鼠脾臟細胞有更多的甲基化 (p<0.05)，也明顯比控制組的小鼠脾臟細胞少產生 apoptotic bodies 。在第 12 與 25 週，發現10 倍葉酸補充組小鼠的血漿中抗 dsDNA 抗體明顯少於控制組的小鼠，且腎絲球環間膜細胞沒有明顯增生。第 20 週，10 倍葉酸補充組小鼠的尿中蛋白質濃度明顯少於控制組的小鼠 (p<0.05)。 由以上的結果，我們推測葉酸缺乏會增加自體免疫現象的發生，10倍葉酸的補充可保護自體免疫老鼠出現自體免疫現象。
並列摘要
Chpater 1 Folic acid is a water-soluble vitamin and belongs to the B-vitamin group. The main function of folic acid is one-carbon metabolism, including amino acid metabolism, purine and pyrimidine metabolism and compound of s-adenosylmethionine (SAM). Folate plays an important role in cell growth and proliferation. Most past studies, focused on animal immune system and the result revealed that the lymphocyte number of folate-deficient rat was reduced and the infection rate of folate-deficient rat was obviously increased. However, there are fewer studies on folate deficiency in human immune system. Therefore, in this study we used normal person’s peripheral blood lymphocytes (PBLs) and cultured them in control and folate-deficient media with PHA stimulation for three days. The result revealed that the intracellular folate of folate-deficient PBLs in day 3 had reduced to 44% of initial PBLs. The PBLs lacking folate showed reduced thymidine de novo synthesis and uptook more [3H]-deoxythymidine than the control group, and then exogenous [6-3H] deoxyuridine utilization became reduced. The growth rate of folate-deficient PBLs with PHA stimulation was lower than that of control group from day 1 to day 3 (p<0.05). The cell cycle of folate-deficient PBLs delayed G0-G1 phase in day 2, and this resulted in S and G2-M phases being significantly lower than the control group (p<0.05). Regarding IL-2, IL-4, IL-6, IL-10 and IFN-γ mRNA expression, folate-deficient PBLs expressed these cytokines less than the control group did. The secretion of these cytokines in folate-deficient PBLs was significantly lower than that of the control group (p<0.05). The IgG and IgM production of folate-deficient PBLs was significantly lower than that of the control group (p<0.05). Taken together, we found out that folate deficiency would delay the PHA stimulation to PBLs, decrease the secretion of cytokines and antibody production, delay the cell cycle progression, and influence the growth of PBLs. Chapter 2 Folate determines the normal growth and proliferation of cells because of its complex biochemistry. Some studies suggested that folate deficiency could cause cell apoptosis, but how this was caused was not clear. The other important function of folate is helping homocysteine to accept a methyl group to become methionine; and in further synthesis, methionine becomes SAM, the main methyl donor in cell. Thus, folate deficiency would cause DNA hypomethylation. The literatures show that DNA hypomethylation of lymphocyte is related to raise the incidence of autoimmune diseases. However, there has never been a study about whether folate deficiency raises the incidence of autoimmune diseases or not. Therefore, the purpose of this chapter is to discuss what kind of the protein would affect apoptosis of PBLs when folic acid is lacking, to determine DNA hypomethylation of PBLs and anti-dsDNA antibodies production. In this study, we cultured PBLs in control and folate-deficient media for 3 to 9 days and added PHA for the final 48 and 72 h prior to harvesting the cells. The growth rate of PBLs in folate-deficient medium was significantly lower than that in control medium from day 4 (p<0.05). PBLs without PHA stimulation were automatically entered into the cell cycle on day 7 in the control group but not in the folate-deficient group. After stimulation by PHA for 48 hours and 72 hours, folate-deficient PBLs arrested obviously in S phase on day 5 and day 4 (p<0.05) and the apoptotic rate of folate-deficient PBLs was higher than that of control group after day 4 (p<0.05). On day 3, the IL-2, bcl-2 and Fas mRNA expression of folate-deficient PBLs were less than those of the control group. On day 6, the bcl-2 and Fas protein in folate-deficient PBLs were less determined than those in the control group. The cytochrome c and caspase 3 of folate-deficient PBLs were increased in cytosol. On day 5, folate-deficient PBLs expressed the p53 protein less than control group did. We collected the culture media on day 3 and day 9, and we found that the homocysteine level from folate-deficient medium was significantly higher than that from control medium (P<0.05). Therefore, the homocysteine level on day 9 was higher than that on day 3 in folate-deficient medium (p<0.05). On day 3, DNA of PBLs revealed hypomethylation in folate-deficient group. To our surprise, the anti-dsDNA antibodies were determined to be higher in folate-deficient medium from PBLs cultured without PHA stimulation for 8 d than those in control medium. From above results, we suggest that folate deficiency causes apoptosis in PBLs because of S phase arrest and also that it didn’t rely on p53 and Fas protein, instead of reducing growth factor, IL-2, and bcl-2 protein which protects cell from apoptosis, and it may increase the incidence of autoimmune diseases. Chapter 3 Lipotropes include folate, methionine, choline and vitamin B12, and the main function is a methyl donor, for example, one-carbon metabolism and homocysteine homeostasis. In folate deficiency, all of the reactions which relate to one-carbon metabolism are affected. Folate deficiency would increase homocysteine level. Many papers indicate that cardiovascular disease has been associated with hyperhomocysteine. High homocysteine level would decrease the SAM/SAH ratio and cause DNA hypomethylation. Some studies also suggested that the DNA of peripheral blood monocytes of SLE patients presented a higher degree of hypomethylation than that of healthy normal controls. Richardson’s group proved that affected treatment of CD4+T cells from rat with DNA methyltransferase inhibitors, the cells presented DNA hypomethylation and increased autoimmune phenomenon. However, no literature indicates whether autoimmune relates to folate deficiency. Our in vitro study demonstrated that human lymphocytes cultured in folate-deficient medium produced anti-dsDNA antibodies. The purpose of this chapter is to present this in vivo, observe whether rat fed with folate-deficient diet presents autoimmune disease and treat the autoimmune mouse with a ten-fold concentration of folate as supplement, and then to observe whether the autoimmune phenomenon is improved or not. The results revealed that the average of control rat plasma folate concentration was from 30 to 40 ng/mL and folate concentraion of folate-deficient rat plasma was 3.5 ng/mL. Furthermore, the spleen cells of folate-deficient rat cultured in folate-deficient medium uptook more [H3]-deoxythymidine and less [6-3H] deoxyuridine than those of control rat cultured in the same circumstances. In the tenth week, the growth rate of the spleen cells of folate-deficient rats with stimulated by PHA was not obviously different. The body weight of folate-deficient rat was significantly lower than that of control group (p<0.05). In the eleventh week, the proportions of heart, lung and kidney to body weight of folate-deficient rat were significantly heavier than those of control group (p<0.05) and the death rate reached 78 %. After the fourth week, spleen DNA of folate-deficient rat presented hypomethylation. After the sixth week, CD11a expression of spleen cells of folate-deficient rat was higher than that of control group (p<0.05). After the eighth week, CD40L expression of spleen cells of folate-deficient rat was higher than that of control group (p<0.05). In the fourth week, the spleen DNA of folate-deficient rat has became fragmented. In the eighth week, the sections of spleen and kidney tissue of folate-deficient rat appeared to contain apoptotic bodies. In the sixth week, the apoptotic rate of spleen cells of folate-deficient rat was significantly higher than that of control group (p<0.05) from flow cytometry determination. In the tenth week, the plasma of folate-deficient rat displayed anti-dsDNA antibodies (p<0.05). After the sixth week, the urine protein concentration of folate-deficient rat was significantly higher than that of control group. In the tenth week, the glomerule of folate-deficient rat presented mesangium proliferation. From treating a ten-fold concentration of folate in autoimmune mouse study, the spleen DNA of mice fed in a ten-fold concentration of folate diet had more methylation and also less apoptotic bodies than those of mice fed in nornal control diet in the twelfth week. In the twelfth and twenty-fifth week, anti-dsDNA antibodies in plasma of mice fed in a ten-fold concentration of folate diet was significantly less than that of control group and glomerule of mice fed 10 times folae diet was not proliferated. In the twentieth week, urine protein concentration of mice fed in a ten-fold concentration of folate diet was significantly lower than that of control group (p<0.05). From above data, we suggest that folate deficiency would increase autoimmune development and ten-fold concentration of folate supplement would prevent autoimmune mice developing autoimmune symptoms.