【摘要】 目的 探討同種異基因骨髓間充質干細胞（bone mesenchamal stem cells，BMSC）靜脈輸注對大鼠到小鼠胰島移植物的功能保護和小鼠糖尿病狀態改善。 方法 全骨髓培養法獲得C57BL/6小鼠BMSC。不連續梯度離心法分離純化Sprague-Dawley（SD）大鼠胰島，將300胰島當量的胰島單獨或與BMSC聯合移植入鏈脲菌素誘導的糖尿病BALB/c小鼠腎包膜下，并通過尾靜脈在移植后0、3和5 d注射CM-DiI標記的BMSC 5×105/只，對照組給于磷酸鹽緩沖溶液。移植后監測血糖，第9天處死小鼠，取肝、脾、胸腺、淋巴結和移植胰島的腎臟，冰凍切片，熒光顯微鏡觀察CM-DiI標記細胞的組織分布；免疫熒光法觀察移植物中胰島素和胰高血糖素表達，評價胰島的功能。 結果 BMSC靜脈輸注后主要分布于胸腺，其次是脾臟和淋巴結，腎和肝組織中未觀察到BMSC；BMSC聯合胰島移植組血糖控制水平優于其他組，且在第7天的口服糖耐量實驗優于單純胰島移植組。 結論 與胰島聯合移植的BMSC對受者免疫器官和組織有明顯的趨向性，且對胰島細胞的體內存活有一定保護作用。【Abstract】 Objective To research on the protection function by the allogeneic rat bone mesenchymal stem cells (BMSC) on rat to mouse islet transplantation and the improvement of diabetic state in mouse. Methods BMSC were prepared from C57BL/6 mouse bone marrow cells and identified by flow cytometry (FCM). Islets were isolated from Sprague-Dawley (SD) rats with Ficoll discontinuous centrifugation. CM-DiI labeled BMSC at 5×105 for one mouse were intravenously infused into STZ induced diabetic BALB/c mice after rat to mouse islet transplantation at day 0, 3 and 5. Mice with PBS intravenously infused after islet transplantation were set as the negative controls. Blood glucose was monitored every day at the first 3 days after transplantation, and then monitored every two days. At day 9 after transplantation, spleen, thymus, lymph nods, liver and islets recipient kidney were harvested. Ice slices were prepared and CM-DiI labeled cells were investigated with fluorescence microscope. Results CM-DiI-labeled BMSC were mainly distributed in thymus followed by spleen and lymph nodes. In liver and kidney, there was no red fluorescence observed. The blood sugar control for combined BMSC infusion group was superior to other groups, and the control level of islet combined BMSC infusion group were better than single islet transplantation group in OGTT at day 7. Conclusion Allogeneic BMSC can sustain the insulin secretion of islets in vivo and tend to distribute in immune organs or adenoid tissues after infusion.
目的 建立恒河猴外周血CD4+CD25+調節性T淋巴細胞（regulatory T cells，Tregs）快速有效的分離純化方法。 方法4～5歲健康恒河猴10只，雌性4只，雄性6只，體重5～8 kg；于大隱靜脈抽取外周血，每只抽取8 mL。密度梯度離心法分離外周血單個核細胞（peripheral blood mononuclear cell，PBMC），分別采用非人靈長類Tregs分離試劑盒的生物素標記的混合抗體和磁珠標記的抗生物素抗體陰性分選，以及大鼠抗人CD4-活化蛋白C（activated protein C，APC）和抗APC多選試劑盒中的磁珠標記的抗APC抗體陽性分選出 CD4+ T淋巴細胞，比較兩種方法獲得細胞的得率、活性及純度；選擇得率、活性和純度較高的分選方法獲得的CD4+ T淋巴細胞，用磁珠標記的抗人CD25抗體進行陽性分選，獲得 CD4+CD25+ Tregs，流式細胞儀檢測純度、活性及FoxP3表達水平，以及Tregs對刀豆蛋白（concanavalin A，ConA）刺激的自體CD4+CD25-效應性T淋巴細胞（effective T cells，Teffs）增殖的抑制功能。 結果CD4+ T淋巴細胞陽性分選和陰性分選后，細胞活性均達95%左右，差異無統計學意義（P gt; 0.05），但陽性分選后CD4+ T淋巴細胞得率和純度均顯著高于陰性分選（P lt; 0.05）。后續CD4+CD25+ Tregs分選采用陽性分選法獲得的CD4+ T淋巴細胞。經雙陽性分選收集的目的細胞中CD4+CD25+ Tregs占76.2% ± 8.6%，活細胞比例為93.3% ± 4.7%，FoxP3陽性細胞比例為74.2% ± 6.9%。混合培養后Tregs均對ConA刺激的Teffs的增殖有抑制作用。 結論免疫磁珠雙陽性分選法能有效分選出恒河猴外周血中有功能的CD4+CD25+ Tregs。