綠色螢光小鼠與豬之骨髓間葉幹細胞轉分化成胰島素分泌細胞之研究

第一型糖尿病為自體免疫缺失性疾病，導致病患胰臟之 β 細胞逐漸被自體抗體攻擊殆盡而導致高血糖，因而終生需仰賴外源胰島素治療。迄今，一般認為胰島細胞移植乃最符合生理之療法，但需面對胰島細胞來源缺乏與異體移植可能導致之免疫排斥問題，因此如何解決胰島細胞來源與免疫排斥，即成為當今研究需克服之目標。近期之研究證實骨髓間葉幹細胞（mesenchymal stem cells, MSCs）可於體外成功轉分化為胰島細胞，體外分化約需 4 個月，且僅 10% 之分化率，效率仍低。曾有文獻指出細胞外基質（extracellular matrix, ECM）可促使細胞具有較好之遷移（migrate）及形成三級結構（three-dimensional structure）之能力，鑑此，如何利用細胞外基質與分化前之細胞結構單一細胞或團塊（single cell or clumps），建立有利於提高 MSCs 分化為胰島素分泌細胞（insulin producing cells, IPCs）之效率，已成為本研究之重點目標。　　本研究使用之螢光小鼠與豬之骨髓間葉幹細胞（EGFP-mMSCs/EGFP-pMSCs）乃分離自攜帶 β-actin 啟動子之綠色螢光蛋白質（enhanced green fluorescent protein, EGFP）轉基因小鼠與豬之骨髓液。初期，本試驗利用不同濃度之葡萄糖（17.5, 23.0, 28.5, 33, 39.5 mM）添加於分化培養基中，擬建立 EGFP-pMSCs 與 EGFP-mMSCs 分化為 EGFP-pIPCs 與EGFP-mIPCs之誘導分化培養基。結果顯示 EGFP-pMSCs 於含 28.5 mM 葡萄糖誘導培養基誘導培養 18 天後，可獲得最佳之 EGFP-pIPCs 分化效率（0.25 cluster/cm2），（P<0.01）。另於EGFP-mMSCs的分化誘導試驗中利用28.5 mM葡萄糖誘導培養 EGFP-mMSCs 18 天後亦得到較多之 EGFP-mIPCs （0.14 cluster/cm2），（P<0.01）。此外，本試驗利用不同分子量（A組: MW > 120000, B組: MW > 300000, C組: MW > 500000）之 poly-D-lysine 作為 ECM ，以磷酸緩衝液為控制組，探討其對於 EGFP-pMSCs 分化效率之影響。結果顯示 B 組於誘導 18 天後所得之 EGFP-pIPCs 分化效率較其他組別為高（5.56 clusters/cm2）（P<0.05）。本試驗亦探討 MSCs 之細胞結構對於 EGFP-mMSCs 與 EGFP-pMSCs 分化為 EGFP-mIPCs/EGFP-pIPCs 之影響，本試驗以 EGFP-mMSCs 與 EGFP-pMSCs 為單細胞組（single cells）或細胞團（clumps）之細胞結構培養，結果顯示以細胞團結構之EGFP-mMSCs誘導5天後即可分化至 EGFP-mIPCs，而細胞團細胞結構之 EGFP-pMSCs 於誘導 9 天後亦可較獲得較多（6.54 clusters/cm2）之EGFP-pIPCs，（P<0.05），顯示立體細胞團塊之 MSCs 相較於單細胞組具有較高之 IPCs 分化效率。　　對於分化後 EGFP-pIPCs 之分子特徵則以逆轉錄聚合酶連鎖反應技術分析之，針對胰臟特異性表現基因與轉錄因子包括：Insulin, Glucagon, Somatostatin, Glucokinase, Pax6, Nkx6.1, Glut2 等進行檢測，結果證實 EGFP-pIPCs 皆可表現前述基因。另進行即時定量聚合酶連鎖反應（Real-Time PCR）分析亦顯示 Insulin, Islet1, Nkx6.1, Glut2 基因表現量隨 EGFP-pMSCs 之分化天數（第0天、第3天、第6天）而逐漸增加。藉由細胞免疫染色技術亦證實分化後之 EGFP-mIPCs 與EGFP-pIPCs 皆可表現 Insulin 與Ｃ-peptide 蛋白質。而 EGFP-pIPCs 亦於高糖（28.5 mM）之誘導下，於培養基中偵測到分泌之豬胰島素蛋白質，分泌濃度約 8.94±0.9 ng/ml。最後，以 PBS （200 μl）、EGFP-pMSCs （6╳106）、分化後之 EGFP-pIPCs （6╳106）移植於 NOD/LtJ 第一型糖尿病小鼠皮下，結果顯示接受 EGFP-pIPCs 移植之 NOD/LtJ 小鼠血糖值下降幅度顯著高於其他組（6╳106 MSCs, PBS, control），且 20 天內之存活率為 100%，他組則於 16 至 18 天死亡。　　由以上結果顯示葡萄糖、細胞外基質與立體 MSCs 結構皆對於綠色螢光小鼠與豬之骨髓間葉幹細胞轉分化（trans-differentiation）為胰島素分泌細胞具有貢獻，並能改善其分化效率。再者，分化後之螢光豬胰島素分泌細胞經證實可分泌胰島素且異種移植至 NOD/LtJ 糖尿病小鼠皮下亦可降低其體內之高血糖。綜合上述，帶有螢光基因之骨髓間葉幹細胞可有效率的轉分化為胰島素分泌細胞，未來可進一步應用至大型糖尿病動物模式或人類臨床治療研究。

關鍵字

糖尿病；胰島素分泌細胞；幹細胞

並列摘要

Type I diabetes is an autoimmune disorder characterized by the progressing destructively loss of pancreatic β- cell. Efforts toward routine islet cell transplantation as a means for reversing type I diabetes have been hampered by islet availability as well as allograft rejection. Several studies have confirmed mesenchymal stem cells (MSCs) could trans-differentiate into insulin producing cells (IPCs), however, it took a long period (4 months) at a low frequency (10%). It was also reported that cells migration in three-dimensional extra-cellular matrix (ECM) plays a key role of cluster formation. Therefore, using ECM and cell mass structure to generate an ameliorated protocol for preparing a renewable and abundant transplantable islet clusters is demanded. Marrow-derived MSCs isolating from the femur of transgenic porcine and mice (EGFP-pMSCs/EGFP-mMSCs) harboring with β-actin-EGFP (enhanced green fluorescent protein) gene were used in the studies. The first experiment, to generate an appropriate induction conditions that EGFP-pMSCs and EGFP-mMSCs differentiate into islet cells, different concentration (17.5, 23.0, 28.5, 33, 39.5 mM) glucose was added to induction medium to determine the effect of dose dependent. The result shows EGFP-pMSCs inducted with 28.5 mM glucose for 18 days, the formation ratio of islet-like cluster (0.25 cluster/cm2) is extremely higher than other groups (P<0.01). In the result of EGFP-mMSCs inducted to islet-like cluster (0.14 cluster/cm2) is similar to the tendency of EGFP-pMSCs induction by using 33 mM glucose. The second experiment, poly-D-lysine with three different molecular weight (MW) (MW > 120,000, MW > 300,000, MW > 500,000) were allocated to treatment groups (A, B and C) as ECM, respectively. The results indicated that islet-like cluster formation ratio (5.56 clusters/cm2) of IPCs in group B was significantly higher than other treated groups (P<0.05). Furthermore, EGFP-mMSCs/pMSCs with colonies and single cells were simultaneously seeded and cultured in induction medium to determine whether three-dimensional structures would effect IPCs induction. Experimental results revealed EGFP-mMSCs/pMSCs seeded by colonies exhibiting the largest number of IPCs (6.54 clusters/cm2) within 9 days (P<0.05), clarified the importance of colony formation. In addition, multiple genes include: Insulin, Glucagon, Somatostatin, Glucokinase, Pax6, Nkx6.1, Glut2, expression on inducted islet-like cells were determined by RT-PCR assay. Genes (Insulin, Islet1, Nkx6.1, Glut2) expression profile of time dependent (day0、day3、day6) were determined by Real-Time PCR. Immunocytochemistry was also used to confirm that EGFP-mIPCs/pIPCs are able to synthesize C-peptide and insulin. Insulin secretion level (8.94±0.9 ng/ml) in high glucose (28.5 mM) conditioned medium was quantitated by EIA. Further study regarding the hyperglycemia in NOD (non-obesity diabetes) mice were alleviated significantly after subcutaneous xenograft of 6×106 EGFP-pIPCs and the survival rate of transplanted mice within 20 days was 100%, but NOD mice treated with PBS, EGFP-pMSCs and control group survive up to 18 day. According to above results indicate glucose concentration containing in induction medium and ECM play a key role of cluster formation in EGFP-mMSCs/pMSCs induction, and improve their efficiency trans-differentiating into functional islet cell clusters even secret insulin to reverse the hyperglycemia of NOD mice via subcutaneous xenograft of EGFP-pIPCs. These observations propose that MSCs expressing GFP is capable of trans-differentiating into functional insulin-producing cells and is possible to extend to the studies of pig models of humans diabetes as a reference means to alleviate limitations of surrounding islet cell transplantation.

並列關鍵字

diabetes ； insulin producing cells ； stem cells

參考文獻

Abraham, E. J., C. A. Leech, J. C. Lin, H. Zulewski and J. F. Habener. 2002. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology 143: 3152-3161.

Baharvand, H., H. Jafary, M. Massumi and S. K. Ashtiani. 2006. Generation of insulin-secreting cells from human embryonic stem cells. Dev. Growth Differ. 48: 323-32.

Barry F. P. 2003. Biology and clinical applications of mesenchymal stem cells. Birth Defects Res. 69: 250-256.

Bernard, B. A. 2008. Human skin stem cells. J. Soc. Biol. 202: 3-6.

Blotard, C., M. C. Villa, C. Becourt, H. P. Gia, C. Huc, P. Sempe, M. M. Protier. 1992. Peripherin: An islet antigen that is cross-reactive with nonobese diabetic

國際替代計量

綠色螢光小鼠與豬之骨髓間葉幹細胞轉分化成胰島素分泌細胞之研究

主題瀏覽