探讨大鼠羊膜间充质干细胞（R-AMSCs）对1型糖尿病（T1D）模型大鼠血糖和胰岛的影响。

培养妊娠17 ~ 19 d大鼠胎盘来源的羊膜间充质干细胞（AMSCs），流式细胞检测干细胞表面标志物CD29、CD34、CD45和CD73，免疫荧光鉴定波形蛋白（Vimentin），三系分化（成骨、成脂、成软骨）实验鉴定其多向分化潜能。24只成年雄性SD大鼠随机分为对照组、T1D （模型）组和T1D+R-AMSCs （干预）组，每组8只。采用腹腔注射65 mg/kg链脲佐菌素诱导建立T1D模型，建模成功1周后，干预组经尾静脉注射5 × 10⁶ R-AMSCs （每周1次，持续3周），对照组、模型组注射等体积PBS。干预结束2周后，异氟烷麻醉处死，收集血液及胰腺组织。检测各组大鼠血糖水平、血清胰岛素和C肽含量、胰腺组织病理变化、神经元素3、胰腺十二指肠同源盒因子1的mRNA和蛋白表达水平，分析R-AMSCs对T1D的治疗效果。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。重复测量设计定量资料采用重复测量设计方差分析，两两比较采用Bonferroni事后检验。

采用酶消化法成功分离R-AMSCs，流式细胞、免疫荧光（波形蛋白Vimentin阳性）及三系分化诱导实验结果证实其具有干细胞特性。血糖检测结果显示，与模型组相比，干预组大鼠血糖水平降低（P < 0.05）。与对照组相比，模型组血清胰岛素水平[（5.48 ± 1.51）比（11.18 ± 2.42）mU/L]及C肽含量水平[（149.88 ± 14.19）比（199.35 ± 22.10）pg/mL]均降低（P均< 0.05），而与模型组相比，干预组血清胰岛素水平[（7.67 ± 1.33）比（5.48 ± 1.51） mU/L]及C肽含量水平[（176.04 ± 17.88）比（149.88 ± 14.19） pg/mL]升高（P均< 0.05）。胰腺组织HE染色显示，干预组胰腺形态结构和胰岛素表达较模型组改善。与对照组相比，模型组Ngn3 mRNA （0.57 ± 0.15比1.00 ± 0.18）及蛋白水平（0.80 ± 0.04比1.11 ± 0.08）、PDX mRNA （0.47 ± 0.08比1.00 ± 0.11）及蛋白水平（0.84 ± 0.13比1.19 ± 0.06）降低（P均< 0.05）。与模型组相比，干预组Ngn3 mRNA （0.87 ± 0.06比0.57 ± 0.15）及蛋白表达（1.03 ± 0.11比0.80 ± 0.04）、PDX-1 mRNA （0.71 ± 0.14比0.47 ± 0.08）及蛋白表达（1.12 ± 0.11比0.84 ± 0.13）增加（P均< 0.05）。

R-AMSCs治疗3周可有效降低T1D模型大鼠血糖水平，改善胰腺病理形态并上调胰腺关键转录因子Ngn3和PDX-1表达以及血清胰岛素、C肽水平，表明其对T1D具有一定的治疗作用。

To investigate the effects of rat amniotic mesenchymal stem cells (R-AMSCs) on blood glucose and pancreatic tissue in type 1 diabetes (T1D) model rats.

Amniotic mesenchymal stem cells (AMSCs) were isolated from the placentas of pregnant rats at 17-19 days of gestation. Flow cytometry was used to detect the expressions of stem cell surface markers (CD29, CD34, CD45, CD73), immunofluorescence was performed to identify vimentin expression, and trilineage differentiation (osteogenic, adipogenic, chondrogenic) was induced to evaluate the multipotency. Twenty-four adult male SD rats were randomly divided into three groups (n = 8 per group), which named control, T1D (model), and T1D + R-AMSCs (intervention) respectively. The T1D model was established by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). One week after successful modeling, the rats in intervention group were received tail vein injections of 5 × 10⁶ R-AMSCs (once per week for 3 weeks), while the control and model groups received equal volumes of PBS. 2 weeks after the intervention, blood and pancreatic tissue were collected after anesthesia with isoflurane. The expression levels of blood glucose, serum insulin and C-peptide, the pathological changes of pancreatic tissue, as well as the expression levels of mRNA and protein of Neurogenin 3 (Ngn3) and pancreatic and duodenal homeobox 1 (PDX-1) were measured to evaluate the therapeutic effects of R-AMSCs. The differences among multiple groups were compared with ANOVA test, and the LSD-t test was used for pairwise comparisons between groups. Repeated measures design quantitative data were analyzed using repeated measures design analysis of variance, and pairwise comparisons were conducted using Bonferroni post hoc tests.

R-AMSCs were successfully isolated using enzymatic digestion. Flow cytometry, immunofluorescence (vimentin-positive), and trilineage differentiation confirmed their stem cell characteristics. Blood glucose levels were significantly lower in the intervention group compared to the model group (P < 0.05). Compared with the control group, the level of serum insulin [ (5.48 ± 1.51) vs (11.18 ± 2.42) mU/L] and C-peptide levels in model group were decreased significantly [ (149.88 ± 14.19) vs (199.35 ± 22.10) pg/mL] (P < 0.05). In contrast, the levels of serum insulin [ (7.67 ± 1.33) vs (5.48 ± 1.51) mU/L] and C-peptide levels [ (176.04 ± 17.88) vs (149.88 ± 14.19) pg/mL] in the intervention group were significant higher compared to the model group (P < 0.05). HE staining of pancreatic tissue showed that the pancreatic morphologtical structure and insulin expression in the intervention group were improved compared to the model group. Compared with the control group, the expression level of Ngn3 mRNA (0.57 ± 0.15 vs 1.00 ± 0.18), protein (0.80 ± 0.04 vs 1.11 ± 0.08) as well as PDX mRNA (0.47 ± 0.08 vs 1.00 ± 0.11), protein (0.84 ± 0.13 vs 1.19 ± 0.06), mRNA and protein were significant lower than those in model group (all P < 0.05). Compared with the model group, the expression levels of Ngn3 mRNA (0.87 ± 0.06 vs 0.57 ± 0.15) and protein (1.03 ± 0.11 vs 0.80 ± 0.04), as well as PDX-1 mRNA (0.71 ± 0.14 vs 0.47 ± 0.08) and protein (1.12 ± 0.11 vs 0.84 ± 0.13) in the intervention group were increased (all P < 0.05) .

Three weeks of R-AMSCs treatment could reduce the blood glucose levels effectively, improve the pathological morphology of the pancreas, and up-regulate the expression of key pancreatic transcription factors (Ngn3 and PDX-1) as well as serum insulin and C-peptide levels in T1D model rats, indicating a potential therapeutic effect of R-AMSCs on T1D.