(human telomerase reverse transcriptase htert) doi: /j.issn

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1 Chinese Journal of Tissue Engineering Research September 3, 2015 Vol.19, No.36 ( ) human telomerase reverse transcriptase htert (human telomerase reverse transcriptase htert) htert 50 SD mg/kg 36 SD htert 12 htert htert 4 htert (P < 0.05) htert (P > 0.05) 6 htert(p < 0.05) (P < 0.05) htert(p < 0.05) htert :R394.2 :B : (2015) [J] (36): doi: /j.issn Telomerase reverse transcriptase gene transfection of human amniotic electrical mesenchymal stem cell transplantation for treatment of diabetes mellitus Fu Jian-ru (Department of Internal Medicine, Xianshuigu Hospital of Jinnan District, Tianjin , China) Abstract BACKGROUND: The amniotic membrane is the rejected material after birth. Amniotic mesenchymal stem cells are characterized as easy harvesting, strong proliferation ability, no ethical controversy, and low immunogenicity. OBJECTIVE: To electrotransfer human telomerase reverse transcriptase (htert) gene into amniotic mesenchymal stem cells transplanted into diabetic rats and to explore its effect on diabetic rats. METHODS: Human amniotic mesenchymal stem cells were isolated, cultured and electrotransferred by htert gene. Ten of 50 Sprague-Dawley rats were randomized selected as controls, and the remaining rats were used to establish diabetic models through injection of 45 mg/kg streptozotocin. Thirty-six model rats were randomized into model group, cell transplantation group and htert-transfected cell transplantation group, with 12 rats in each group. In the latter two groups, human amniotic mesenchymal stem cells and htert-transfected amniotic mesenchymal stem cells were injected via sublingual veins, respectively. After transplantation, blood glucose levels were monitored dynamically, and plasma insulin concentration was detected every week. Pancreas tissues were taken and cut into sections for histological observation using hematoxylin-eosin staining. RESULTS AND CONCLUSION: At 4 weeks after transplantation, the blood glucose levels were significantly lower in the two cell transplantation groups than the model group (P < 0.05), and especially in the htert-transfected cell transplantation group, the blood glucose level was close to the normal value (P > 0.05). However, the model group still Fu Jian-ru, Master, Physician, Department of Internal Medicine, Xianshuigu Hospital of Jinnan District, Tianjin , China Corresponding author: Fu Jian-ru, Department of Internal Medicine, Xianshuigu Hospital of Jinnan District, Tianjin , China Accepted: ISSN CN /R CODEN: ZLKHAH 5827

2 . had a higher blood glucose level. At 6 weeks after transplantation, compared with the model group, the plasma insulin concentration was significantly increased in the two cell transplantation group (P < 0.05), and the severity of pancreatic injury was also eased in these two groups (P < 0.05), especially in the htert-transfected cell transplantation group (P < 0.05). These findings indicate that htert-transfected amniotic mesenchymal stem cell transplantation can dramatically decrease blood glucose level and relieve pancreatic injury in diabetic rats, which is an effective method for treatment of mellitus diabetes in rats. Subject headings: Stem Cells; Tissue Engineering; Blood Glucose; Diabetes Mellitus Fu JR. Telomerase reverse transcriptase gene transfection of human amniotic electrical mesenchymal stem cell transplantation for treatment of diabetes mellitus. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(36): Introduction [1-3] 3 [4-6] [7-8] (human telomerase reverse transcriptase htert) [9-10] [11] [12-13] htert 1 Materials and methods Becon Dickinson DMEM 0.25% Gibco htert β-actin BD MBP mrna PCR KPL ELISA Excell() AMV Eukare SD g SCXK( ) [16] D-Hank s 0.25% r/min 10 min 20.01% DNA r/min 1 h 10%FBS DMEM 25 cm %CO 2 40 h 90% 0.25% P3 PE FITC CD9 CD13 CD19 CD29 CD34 CD44 CD45 CD46 CD htert 37 5%CO 2 24 h PBS 800 g 5 min 3 20 µg DNA 30 min 350 V/cm 25 µf T 0.9 ms htert 30 min DMEM 10% 1% 37 5%CO SD SD12 h P.O. Box 10002, Shenyang

3 . 45 mg/kg (ph 4.5) 12 2 d mmol/l htert 12 htert / htert L RT-PCR TRIzolRNA RNA PCR 94 5 min s s s min 4 PCR 3 PCR 5 ml β-actin GDSS000 Image-Pro Plus 8.0hTERTβ-actin mrna htert 5 -CTGAAGTGTCACAGCCTGTTT-3' 5 -CACACATGCGTGAAACCTGTA-3' 111 bp 111 bp β-actin 5 -TATCGGACGCCTGGTTAC-3' 5 -CTCAGCCTTGACTGTGCC-3' 151 bp 151 bp htert %(5 ml/kg) 20 ELISA g/l SPSS 15.0 x _ ±s t α= Results ( 1A)2 8 h 15 µm 1B 2.3 htert RT-PCR htert 48 h 2RNA RNA A 260 nm A 280 nm A 260 nm/280 nm = RNA PCR htert 750 bp htert mrna mmol/l mmol/l htert 7.01 mmol/l mmol/l htert (P < 0.05) 1 6 htert htert (P < 0.05) htert 6 htert htert (P < 0.05) 3 ISSN CN /R CODEN: ZLKHAH 5829

4 . A B 1 ( ) Figure 1 Morphology of amniotic mesenchymal stem cells cultured under inverted microscope A ( 40) B 2 ( 100) bp (htert) Figure 2 htert expression in amniotic mesenchymal stem cells after transfection 1 4 Marker htert htert 750 bp mrna A B C D 3 ( 100) Figure 3 Histological changes of the pancreas islet of rats (hematoxylin-eosin staining, 100) A B C D hterthtert 1 (x _ ±s mmol/l) Table 1 Variation of blood glucose levels in rats at different time after amniotic mesenchymal stem cell transplantation via sublingual veins n 5 d 14 d htert ±0.05 a 22.35±0.06 ab 20.03±0.32 ab 17.01±0.12 b 4.02±0.01 a 22.42±0.04 ab 19.68±0.01 ab 14.27±0.01 b n 21 d 28 d ±0.04 a 4.02±0.01 a ±0.07 ab 26.21±0.08 ab ±0.03 ab 12.03±1.08 ab htert ±0.02 b 7.02 ±0.07 b 2 (x _ ±s µg/l) Table 2 Variation of fasting insulin levels in rats at different time after amniotic mesenchymal stem cell transplantation via sublingual veins ±5.32 ab 15.41±3.02 ab 0.33± ± ±0.03 ab 9.02±0.11 ab htert 0.29±0.10 a 12.38±0.32 a a P < 0.05 htert b P < 0.05 htert htert a P < 0.05 b P < 0.05 htert 5830 P.O. Box 10002, Shenyang

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