自體免疫疾病的致病機轉到目前為止仍然是很清楚，而凋亡小體無法被正確的辨及清除一直被認為是造成自體免疫疾病的主要原因。因此，研究自體抗原的產生以及分析影響吞噬作用及清除凋亡細胞的因子，是本實驗主要的研究動機。Transglutaminase 2 (TG2)是一種多功能酵素，能結催化仰賴鈣子的反應而造成蛋白質轉譯後修飾，最近的研究發現，TG2是與細胞凋亡的一個重要成員，甚至影響巨噬細胞辨及清除凋亡小體。
實驗中用UVB 1650 J/m2照射人角質細胞株 (HaCaT) 製備凋亡小體，觀察細胞形態，發現細胞質皺縮，細胞表面出現小泡 (blebs) ，隨時間點增加細胞呈現懸浮態，由DNA ladder的實驗發現UVB 1650 J/m2作用12小時後，染色質出現DNA片段化現象，以氏細胞儀分析UVB作用後24小時發現有56.2 % 細胞進細胞凋亡。TG2的mRNA則在UVB 1650J/m2處後1小時減少，之後隨時間增加，在12小時達到最高點，於24小時後再減少。進一步分析吞噬過程中巨噬細胞吞噬能和TG2之表現，結果發現，吞噬螢光乳膠微珠 (latex-beads) 時吞噬指 (phagocytosis index ; PI) 和吞噬指 (phagocytosis ratio ; PR) 在各時間點皆小於5 %，且TG2 mRNA無顯著差;吞噬apoptotic cells時，phagocytosis index於4小時達到最高(PI=43.5%)，phagocytosis ratio則隨著時間增加直到6小時(PR=43.5%)，且TG2 mRNA於4小時達到最高點，顯示凋亡小體會激巨噬細胞中TG2的表現。Recombinant TGF-β (rTGF-β1) 與吞噬作用關係，研究結果發現 (1) rTGF-β1於0~20ng/ml劑下會造成細胞凋亡，(2) rTGF-β1會增加巨噬細胞中TG2 mRNA表現，其表現並與rTGF-β1劑成正比 (p<0.05)， (3) rTGF-β1的劑會增加phagocytosis ratio (p<0.05)，但rTGF-β1對於吞噬作用時TG2 mRNA的表現無顯著差。我們的結認為凋亡小體會激巨噬細胞中TG2的表現。另外rTGF-b1可增加TG2的表現且增加phagocytosis ratio，並可能抑制凋亡小體誘發TG2產生的生物功能，全身性紅斑性瘡的致病機轉可能是其巨噬細胞對apoptotic cells無反應，使得TG2沒有增加而吞噬能下，導致清除凋亡細胞的能下。
The mechanism of autoimmune disease remains unclear. Moreover, the deficiency of phagocytic cells to recognize and clear apoptotic body is a main factor of the induction of autoimmune responses. Therefore, it becomes important to study the mechanism involved in the production, recognition and clearance of apoptotic cells. Transglutaminase 2 (TG2) is a multifunctional enzyme with the function of cross-linking Ca2+-dependent reactions which result in post-translational modification of substrate proteins. Results from several independent laboratories indicated that TG2 plays a potential role in cell apoptosis and also in the clearance of apoptotic body. In this study, we investigated the role of TG2 in the clearance of apoptotic cells in RAW264.7. After keratinocytes were irradiated with UVB 1650 J/m2, cell apoptosis was analyzed by light microscopy, DNA fragmentation and flow cytometry. We observed the structure of small surface blebs in keratinocytes, and condensation of nuclear chromatin. DNA fragmentation was observed at 12h and 24h after keratinocytes irradiation. The cells at sub-G1 phase (56.2%) were detected at 24h by FACS. After irradiation, TG2 mRNA level was declined at 1hr, and increased at later time points (6-12h), but was repressed after 12 h. In the experiment of phagocytosis, it was found that RAW264.7 had higher ability to ingest apoptotic cells than latex-beads. TG2 mRNA level was also higher in phagocytosis with apoptotic cells than with latex-beads. The roles between TGF-β1 and TG2 expression in phagocytosis were also further studies. Our results showed that rTGF-β1 did not induce apoptosis of RAW264.7 at concentration of 0-20 ng/ml,and rTGF-β1 was able to increase the level of TG2 expression in a dose -dependent way in RAW264.7 cells. Overall, our results demonstrated that apoptotic cells and rTGF-β1 could induce TG2 expression and increase phagocytosis with apoptotic cells in RAW264.7 cells. The defect response of apoptotic cells to phagocytic cells following impairment of phagocytosis and clearance of apoptotic cells may play a role in the pathogenesis of systemic lupus erythematosus.
