CAJ | 학술논문

目的体外研究利拉鲁肽诱导骨髓间充质干细胞(BM-MSCs)分化为胰岛素分泌细胞(IPCs),并在体内进一步观察IPCs移植对1型糖尿病(T1DM)大鼠的治疗作用.方法 (1)体外采用密度梯度离心联合差壁培养法分离、纯化大鼠BM-MSCs,进一步分为未诱导组、高糖+尼克酰胺诱导组、胰高血糖素样肽1(GLP-1)诱导组和利拉鲁肽诱导组；(2)倒置显微镜下观察各组细胞形态变化,双硫腙染色鉴定诱导后细胞,荧光定量PCR检测巢蛋白(Nestin)、胰十二指肠同源盒1(PDX-1)、葡萄糖转运蛋白2(Glut-2)、葡萄糖激酶(GK)、胰岛素和胰高血糖素等基因,细胞免疫荧光检测胰岛素和胰高血糖素等蛋白；(3)将180 ～ 220 g的30只雄性SD大鼠以60 mg/kg剂量腹腔注射链脲佐菌素制备T1DM模型,造模成功后按随机数字表法分为对照组(T1DM组,n=8)、未诱导的BM-MSCs移植组(BM-MSCs组,n=9)和经利拉鲁肽诱导的BM-MSCs移植组(LIRA+ BM-MSCs组,n=9),给予相应干预8周,待血糖基本稳定后,选取4只正常、同龄、雄性SD大鼠作为对照,行腹腔注射的葡萄糖耐量试验(IPGTT)进一步观察移植后细胞对高糖刺激的反应性.结果 (1)利拉鲁肽诱导后BM-MSCs形态逐渐变圆,呈明显的聚集性生长状态,双硫腙染色为阳性；与高糖+尼克酰胺诱导组比较,利拉鲁肽诱导组细胞Nestin mRNA表达下调(0.003 8±0.000 4比0.007 5±0.003 0,P＜0.05),胰岛素(0.000 20±0.000 03比0.000 08±0.000 02)和胰高血糖素(0.001 1±0.0004比0.000 7±0.000 1)等mRNA表达上调(F=7.26、10.06、4.92,均P＜0.05),PDX-1、Glut-2、GK mRNA表达亦上调；利拉鲁肽诱导组和GLP-1诱导组细胞胰岛素或胰高血糖素蛋白表达均呈阳性.(2)体内实验示,与T1DM组比较,LIRA+ BM-MSCs组和BM-MSCs组大鼠8周末血糖均明显降低[分别为(28.0±1.2)、(8.9±1.1)、(14.5±0.9)mmol/L,F=719.61,均P＜0.05]；IPGTT提示移植IPCs后的大鼠血糖在30 min时升至峰值,150 min时降至空腹水平,血糖变化曲线与正常组类似.结论体外利拉鲁肽可以在一定程度上促进BM-MSCs分化成为IPCs,且移植后的IPCs能够在体内进一步发挥降糖作用. 目的體外研究利拉魯肽誘導骨髓間充質榦細胞(BM-MSCs)分化為胰島素分泌細胞(IPCs),併在體內進一步觀察IPCs移植對1型糖尿病(T1DM)大鼠的治療作用.方法 (1)體外採用密度梯度離心聯閤差壁培養法分離、純化大鼠BM-MSCs,進一步分為未誘導組、高糖+尼剋酰胺誘導組、胰高血糖素樣肽1(GLP-1)誘導組和利拉魯肽誘導組；(2)倒置顯微鏡下觀察各組細胞形態變化,雙硫腙染色鑒定誘導後細胞,熒光定量PCR檢測巢蛋白(Nestin)、胰十二指腸同源盒1(PDX-1)、葡萄糖轉運蛋白2(Glut-2)、葡萄糖激酶(GK)、胰島素和胰高血糖素等基因,細胞免疫熒光檢測胰島素和胰高血糖素等蛋白；(3)將180 ～ 220 g的30隻雄性SD大鼠以60 mg/kg劑量腹腔註射鏈脲佐菌素製備T1DM模型,造模成功後按隨機數字錶法分為對照組(T1DM組,n=8)、未誘導的BM-MSCs移植組(BM-MSCs組,n=9)和經利拉魯肽誘導的BM-MSCs移植組(LIRA+ BM-MSCs組,n=9),給予相應榦預8週,待血糖基本穩定後,選取4隻正常、同齡、雄性SD大鼠作為對照,行腹腔註射的葡萄糖耐量試驗(IPGTT)進一步觀察移植後細胞對高糖刺激的反應性.結果 (1)利拉魯肽誘導後BM-MSCs形態逐漸變圓,呈明顯的聚集性生長狀態,雙硫腙染色為暘性；與高糖+尼剋酰胺誘導組比較,利拉魯肽誘導組細胞Nestin mRNA錶達下調(0.003 8±0.000 4比0.007 5±0.003 0,P＜0.05),胰島素(0.000 20±0.000 03比0.000 08±0.000 02)和胰高血糖素(0.001 1±0.0004比0.000 7±0.000 1)等mRNA錶達上調(F=7.26、10.06、4.92,均P＜0.05),PDX-1、Glut-2、GK mRNA錶達亦上調；利拉魯肽誘導組和GLP-1誘導組細胞胰島素或胰高血糖素蛋白錶達均呈暘性.(2)體內實驗示,與T1DM組比較,LIRA+ BM-MSCs組和BM-MSCs組大鼠8週末血糖均明顯降低[分彆為(28.0±1.2)、(8.9±1.1)、(14.5±0.9)mmol/L,F=719.61,均P＜0.05]；IPGTT提示移植IPCs後的大鼠血糖在30 min時升至峰值,150 min時降至空腹水平,血糖變化麯線與正常組類似.結論體外利拉魯肽可以在一定程度上促進BM-MSCs分化成為IPCs,且移植後的IPCs能夠在體內進一步髮揮降糖作用.
목적 체외연구리랍로태유도골수간충질간세포(BM-MSCs)분화위이도소분비세포(IPCs),병재체내진일보관찰IPCs이식대1형당뇨병(T1DM)대서적치료작용.방법 (1)체외채용밀도제도리심연합차벽배양법분리、순화대서BM-MSCs,진일보분위미유도조、고당+니극선알유도조、이고혈당소양태1(GLP-1)유도조화리랍로태유도조；(2)도치현미경하관찰각조세포형태변화,쌍류종염색감정유도후세포,형광정량PCR검측소단백(Nestin)、이십이지장동원합1(PDX-1)、포도당전운단백2(Glut-2)、포도당격매(GK)、이도소화이고혈당소등기인,세포면역형광검측이도소화이고혈당소등단백；(3)장180 ～ 220 g적30지웅성SD대서이60 mg/kg제량복강주사련뇨좌균소제비T1DM모형,조모성공후안수궤수자표법분위대조조(T1DM조,n=8)、미유도적BM-MSCs이식조(BM-MSCs조,n=9)화경리랍로태유도적BM-MSCs이식조(LIRA+ BM-MSCs조,n=9),급여상응간예8주,대혈당기본은정후,선취4지정상、동령、웅성SD대서작위대조,행복강주사적포도당내량시험(IPGTT)진일보관찰이식후세포대고당자격적반응성.결과 (1)리랍로태유도후BM-MSCs형태축점변원,정명현적취집성생장상태,쌍류종염색위양성；여고당+니극선알유도조비교,리랍로태유도조세포Nestin mRNA표체하조(0.003 8±0.000 4비0.007 5±0.003 0,P＜0.05),이도소(0.000 20±0.000 03비0.000 08±0.000 02)화이고혈당소(0.001 1±0.0004비0.000 7±0.000 1)등mRNA표체상조(F=7.26、10.06、4.92,균P＜0.05),PDX-1、Glut-2、GK mRNA표체역상조；리랍로태유도조화GLP-1유도조세포이도소혹이고혈당소단백표체균정양성.(2)체내실험시,여T1DM조비교,LIRA+ BM-MSCs조화BM-MSCs조대서8주말혈당균명현강저[분별위(28.0±1.2)、(8.9±1.1)、(14.5±0.9)mmol/L,F=719.61,균P＜0.05]；IPGTT제시이식IPCs후적대서혈당재30 min시승지봉치,150 min시강지공복수평,혈당변화곡선여정상조유사.결론 체외리랍로태가이재일정정도상촉진BM-MSCs분화성위IPCs,차이식후적IPCs능구재체내진일보발휘강당작용.
Objective To investigate the differentiation effect of liraglutide on bone marrow mesenchymal stem cells (BM-MSCs) into insulin producing cells (IPCs) in vitro,and further to observe the therapeutic action of IPCs on type 1 diabetes (T1DM) rats.Methods BM-MSCs were separated and purified by density gradient centrifugation combined with attachment culture method in vitro,and they were divided into control group,high glucose with nicotinamide induction group,glucagon-like peptide 1 (GLP-1) induction group and liraglutide induction group.The morphological change of cells was observed by inverted microscope during induction,and they were confirmed by dithizone (DTZ) staining.Gene measurement of Nestin,pancreatic and duodenal homeobox 1 (PDX-1),glucokinase (GK),glucose transporter-2(Glut-2),insulin and glucagon were tested by Real-time PCR.Protein expression of insulin and glucagon were detected by cell immunofluorescence.Thirty SD rats (weight 180-220 g) were injected 60 mg/kg of streptozotocin to establish the type 1 diabetes model,then they were randomly divided into control group (group T1DM,n =8),BM-MSCs transplantation group (group BM-MSCs,n =9) and BM-MSCs induced by liraglutide transplantation group (group LIRA + BM-MSCs,n =9).Random blood glucose was monitored regularly during the treatment for 8 weeks.In addition,the intraperitoneal injection of glucose tolerance test (IPGTT) was carried out after the blood glucose was stable,by which another 4 normal rats were selected as control.Results (1) In vitro,BM-MSCs showed distinct tendency to form clumps at confluence when liraglutide was added,and dithizone staining was positive.Compared to high glucose with nicotinamide induction group,the mRNA expression of Nestin was lower in liraglutide induction group (0.003 8 ±0.000 4 vs 0.007 5 ±0.003 0),while the level of insulin(0.000 20 ±0.000 03 vs 0.000 08 ± 0.000 02) and glucagon(0.001 1 ±0.000 4 vs 0.000 7 ±0.000 1,F =7.26,10.06,4.92,all P ＜0.05) was higher.The mRNA expression of PDX-1,Glut-2,GK was also increased.The expressions of insulin and glucagon protein were positive in liraglutide and GLP-1 induction group.(2) In vivo,compared to group T1DM,the concentration of randomly blood glucose was significantly decreased both in group LIRA + BM-MSCs and group BM-MSCs ((28.0 ± 1.2),(8.9 ± 1.1),(14.5 ± 0.9) mmol/L,respectively,F =719.61,all P ＜ 0.05).IPGTT showed blood glucose reached to a peak at 30th minutes and dropped to fasting glucose at 150th minutes in group LIRA + BM-MSCs,which was similar to normal.Conclusions BM-MSCs could be enhanced to differentiate into IPCs by liraglutide in vitro,and further to make effect by IPCs in vivo.