長期血液透析病人T細胞分化與體內維他命D濃度有關

Abstract

1. 緒論 在末期腎衰竭須長期透析的病人中，心血管疾病、感染與惡性腫瘤分居這群病人死亡率的前三名；而在這些疾病裡，免疫系統均扮演重要的角色。其中，以感染為例，因為細菌感染引發的敗血性休克，在長期血液透析病人身上，死亡率是一般人的一百到三百倍之多，即使在經過種族、年齡、性別或本身有糖尿病校正的前提下，死亡率仍高於一般病人的五十倍之多。因此了解血液透析病人免疫的改變與如何改善血液透析病人的免疫功能成為腎臟科醫師共同努力的目標。根據之前許多的研究指出，活性維他命D在一般病人身上可作為免疫系統調節劑；而維他命D目前是治療透析病人因為鈣磷不平衡所引發次發性高副甲狀腺血症最主要的藥物之ㄧ；不僅如此，血液透析病患體內維他命D的濃度常常不足，甚至嚴重缺乏。由於目前現有資料的不足，所以本研究主要在探討: 1. 長期血液透析病人在體內維他命D不足的情況之下，病人T細胞的分化是否會因維他命D的濃度而有所改變，且T細胞的分化是否與其他因子相關? 2. 在給予活性維他命D治療次發性高副甲狀腺血症後，T細胞分化與細胞激素是否有所改變? 3. 最後評估病患預後是否與體內維他命D濃度或T細胞分化情形有關? 2. 研究設計與方法 (1) 研究設計 研究期間自2009年1月1日起至2010年6月30日止，總共收集了57位在天主教耕莘醫院新店總院與永和分院長期血液透析的病患 (定義為血液透析至少超過3個月)。當中主要納入條件為年齡介於18到75歲，且三個月內並沒有接觸過活性維他命D的藥物或其衍生藥物；而包括已知現行感染或腫瘤，目前正在服用會影響免疫功能的藥物，如類固醇或免疫抑制劑，及愛滋病毒感染或未治療的免疫疾病病患，均予以排除。 (2) 研究方法 在臨床收案的基礎期，利用週中透析尚未接觸管路之前，抽取病患約10ml全血，利用實驗技術將周邊血液單核球與血清分離。接下來將單核球與刺激原接觸培養，並利用多重螢光染色的方式，分別染上表面CD4抗原與不同的細胞內激素；最後利用細胞流式儀來區分T細胞的分化。另外，將血清與細胞培養的上清液分別用ELISA的方式來檢測不同的細胞激素；同時，也採樣病患的生化值與血液值；利用統計分析的技術來檢測T細胞的分化，及血清與培養上清液的細胞激素；看看是否與體內維他命D的濃度相關或其他血液或生化檢查值有關。接下來，再根據病患的血清副甲狀腺值，依照美國國家腎臟基金會的臨床指引 (NKF K/DOQI guideline)，給予活性維他命D不同劑量三個月，三個月後再重複基礎期的實驗加以評估。最後，依據病患臨床預後來檢測體內維他命D濃度與T細胞之間的關係。 3. 結果 在57位長期血液透析病患中，有61%病人體內血液為維他命D缺乏 (定義為 血清25(OH)D濃度小於20 ng/ml)。將所有收案病患根據體內的維他命D濃度分為維他命D缺乏 (35人) 與不缺乏 (22人) 兩組。結果發現: 體內的維他命D濃度並不會與臨床上檢測的血紅素，血比容，透析前尿素氮，肌酸酐，白蛋白、全鈣離子、磷離子、或血清中副甲狀腺素有關。其次，在維他命D缺乏組中發現: 在血清中的Th2細胞激素(介白素-4與介白素-5)較低，而在細胞培養上清液中的Th1細胞激素(介白素-2與干擾素-γ) 較高。另外，血液透析病患體內T細胞在經過刺激原的刺激後，維他命D缺乏組則大多數都以Th1表現為主，而非維他命D缺乏組則多半以Th2表現為主。在治療組方面，因次發性高副甲狀腺血症的病人，給予活性維他命D之後，雖然統計上血清副甲狀腺素與體內25(OH)D濃度並沒有明顯改變，但包括血清與細胞培養上清液中的Th1及Th2細胞激素都有明顯統計上的差異性，且Th2分化也明顯上升。最後，在病人預後上，死亡的病患均屬於維他命D缺乏組，且T細胞分化以Th1為主，不過由於發生死亡的病人較少，且研究期間較短，並沒有辦法直接解釋體內維他命D與預後的關連性。 4. 結論 由此看來，給予血液透析病患維他命D，不僅僅可以治療因鈣磷不平衡所造成的次發性高副甲狀腺血症，也可以降低Th1分化與提高Th2分化比例。目前許多研究報告顯示Th2細胞具有抗發炎的效果。不過，由於收案病人較少且研究期間較短，是否能真正看出血液透析病患體內維他命D濃度的改變與臨床預後有關，值得未來更進一步的研究。

Background The cellular and humoral immune responses in patients on chronic hemodialysis (HD) are impaired. To achieve an adequate immune response, the na&iuml;ve T-cells will be differentiated to T helper cell and then to Th1 and Th2 cells after they are stimulated by pathogen. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the activated form vitamin D is widely used in HD patients with secondary hyperparathyroidism (SHPT) and also is a well known immunomodulatory agent. Here we investigated the T cells differentiation and cytokines expression in different serum 25-hydroxyvitamin D (25(OH)D) levels patients, the T cell differentiation and cytokine results after treatment with activated vitamin D in the SHPT patients, and patient’s clinical manifestation. Material and methods: 57 patients on chronic HD over 3 months were enrolled during January 1st, 2009 to June 30th, 2010 in Cardinal Tien Hospital and Yung-Ho Branch. Patients with systemic infection, malignancy, taking immunosuppressive medication and who took activated vitamin D or analogues in the past 3 months were all excluded. The peripheral blood mononuclear cells (PBMCs) and sera were collected while mid-week predialysis. The PBMCs were cultured and stimulated by mitogens in different time point. Then the T cells were triplely stained by surface and intracellular cytokine markers and the differentiation was analyzed by flow cytometry. The 25(OH)D level in the sera was detected by enzyme-linked immunosorbent assay (ELISA). The Th1 (interleukin (IL)-2 and interferon (IFN)-γ) and Th2 (IL-4 and IL-5) cytokine levels in the sera and culture supernatants were also evaluated by enzyme-linked immunosorbent assay (ELISA) methods. In the SHPT patients, we prescribed the different dosage of activated vitamin D (Calcijex&reg;, Abbott.) according to the NKF K/DOQI guideline for 3 months. Repeated previous cell culture and ELISA exam were done after 3 months later. Patient’s outcome and clinical condition would be followed and analyzed during study period. Results We divided the patients into 2 groups (vitamin D deficiency (VDD): <20 ng/ml in vitamin D and non-vitamin D deficiency (NVDD): ≧20 ng/ml in vitamin D) according to their 25(OH)D level. In the VDD group, the Th2 cytokine (IL-4 and IL-5) were lower in the sera, and the Th1 cytokine (IL-2 and IFN-γ) were higher and Th2 cytokine (IL-4 and IL-5) were lower in the culture supernatant. Besides, the T cell differentiation was towards to Th1 type in the VDD group, but Th2 type in the NVDD group. The T cell differentiation was not influenced by biochemistry examinations, such as albumin, hematocrit, calcium, phosphate, creatinine or dialysis vintage. After treatment with activated vitamin D in the SHPT patients, the serum iPTH and 25(OH)D level were not significant difference. However, the level in Th1 cytokines was decreased and that of Th2 cytokines were both increased in the sera and the culture supernatant. The T cell differentiation was also more towards to Th2 phenotype than Th1 phenotype. The mortality cases were found with prevalent Th1 cell differentiation and vitamin D deficiency. Conclusion Our finding indicated that the T-cell differentiation is only correlated with serum 25(OH)D level. The higher vitamin D in the sera, the more prevalent in Th2 cytokines and Th2 differentiation was found. Treatment with activated vitamin D also influenced the T cell differentiation and cytokines expression in the SHPT patients. Because Th2 cell has the anti-inflammatory effect, activated vitamin D treatment may not only have therapeutic potential for secondary hyperparathyroidism, but also can improve the immune response in the chronic HD patients.